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2018-04-03T01:21:36.116Z | {
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} | s2 | Some properties of starches of grain amaranths and several millets.
Starch granules were prepared from seeds of Amaranthus hypochondriacus L., A. caudatus L., proso millet, Japanese barnyard millet and foxtail millet. Amylose contents and the distribution of alpha-1,4 linked chain of amylopectin were determined by gel filtration of isoamylase-debranched starches. Some physical and chemical properties of the starches were also examined by scanning electron microscopy, X-ray diffractometry, photopastegraphy, and differential scanning calorimetry, together with starch-granule susceptibility to amylases. The existence of both normal and waxy types in the same species of a grain amaranth, A. hypochondriacus L., was confirmed. A. caudatas starches were identified to consist of mainly typical amylopectin and 5-7% amylose. The starches have some unique properties, namely, high starch-granule susceptibility values to amylases as well as those of A. hypochondriacus and unique pasting properties.
Both waxy (wx) and glutinous (gl) mutants and their normal or non-glutinous counterparts have been identified in each one of the species of barley (Hordeum vulgare L.), foxtail millet (Setaria italica Beauv.), maize (Zea mays L.), proso millet (Panicum miliaceum L.), rice (Oryza sativa L.) and sorghum (Sorghum bicolor (L.) Moench) among cultivated Gramineae (1). These mutants produce starch granules in the endsperm and the pollen which stain red with iodine and which contain nearly (2). Seeds of proso millet SGK-110 were collected in Afghanistan in 1978, and proso millet #76373 and 76382 were collected in Nara Prefecture, Japan in 1976, by Sakamoto. These seeds were grown in a glass-house of the Plant Germ-plasm Institute, Kyoto University, in 1976. Seeds of Japanese barnyard millet and two strains of glutinous foxtail millet, one having black seed coat and the other yellow, cultivated at a home farm of Oto-son, Nara in 1980 were kindly provided by Mr. and Mrs. Osamu Ebisudani, Shinohara, Oto-son, Nara Prefecture, Japan.
Starch granules. Starch granules were isolated from the seeds by a modification of the method of MacMasters et al. (7) combined with Schoch's method (8) as described earlier (2). About 10g each of the seeds of grain amaranths and proso millet except #76382 (about 2g of seeds) and about 20g each of the seeds of the other millets were used for preparation of starch granules. Normal maize starch granules were a commercial product and kindly provided by Dr. Taizo X-ray diffraction diagrams X-ray diffraction diagrams obtained from starches of the grain amaranths corresponded to A-type crystalline structure which is typical of starches usually found in grains ( Fig. 1b-1f). The diagrams for the millet starches were also of A type similar to that of the normal maize starch, although the diagrams are omitted.
DSC thermograms of starches Figure 6 shows the DSC thermograms of the grain amaranths and cereal starches. The gelatinization characteristics of starch in a DSC thermogram can be presented by various temperatures: the onset temperature (To); the peak temperature(s) (Tp); and the conclusion temperature (Tc). These temperatures and heat of gelatinization are summarized in Table 2. Linear regression analysis revealed that significant correlation between To values and values of temperature for initiation of gelatinization estimated from photopastegrams (r=0.781, n=10, p<0.01). Starch-granule susceptibility to amylases Table 3 shows susceptibility values to pancreatin of starch granules of proso millet SGK-110 and #76373, Japanese barnyard millet, foxtail millet and normal maize as a control. The waxy-type starch granules of cereals, in general, tended to be digested faster than the normal-type ones by amylases. This is also the case for starch granules shown in Table 3. Exhaustive degradation of starch granules by the crystalline glucoamylase of R. niveus revealed that starches of the grain amaranths, waxy proso and foxtail millets were hydrolyzed nearly 100% by the end of 24-48hr (Fig. 7). The hydrolysis of the normal-type starch granules of proso and Japanese barnyard millets proceeded at a comparatively slow rate under these conditions and the starches achieved only about 60% degradation after 120-hr incubation, as reported previously for the normal maize starch granules (21). DISCUSSION We confirmed that there are both normal and waxy-types in the same species of Amaranthaceae, A. hypochondriacus L., by the investigation of properties of starches isolated from the seeds (Table 1). Starch granules of A. hypochondriacus #76338-2 consist of nearly 100% typical amylopectin the same as those of #76343-1 reported previously (2). The widespread distribution of starches staining red with iodine has been recognized for the past century (1). However, their properties have not been fully investigated except those from the cereals such as maize, rice, sorghum and barley. Moreover, the existence of both normal-and waxy-types in the same species of cultivated grain crops which belong to a plant family other than Gramineae is an infrequent case. Amylose content (22%) of the normal-type starch of A, hypochondriacus #76345-5 was higher than that (14%) of #76339-2 described earlier (2). The difference in apparent amylose content may involve both cultivation and environmental effects. We identified that A. caudatus starches consist mainly of typical amylopectin and small amounts (5-7%) of amylose (Table 1). Starch granules from maize, sorghum and rice kernels homozygous for wx have been reported to have from 0 to 6% of amylose (22). This apparent amylose content may be due to the measurement technique used, to the effect of non-waxy starch granules from maternal tissue, to differences in the degree of branching, or to the presence of some linear materials as described by Shannon and Garwood (22). A small percentage of amylose usually was present in samples of commercially milled waxy maize starch (2), and this was due to contamination by normal starch, as was readily observed by light micros copy (1). When iodine was added, the presence of a few deep-blue granules among those staining reddish-brown was observed. However, the amylose content of A. caudatus starches was not due to contamination. None of the granules stained deep blue or deep brown, but all appeared to be stained reddish-brown and contain small amounts of high molecular-weight linear materials, i.e. amylose (Table 1 and Figs. 3c-3e). These results suggest that A. caudatus may have a wx perisperm gene similar to the wxa endosperm gene which is found in Argentine waxy maize and reported to deposit starch having 2-5% amylose (23,24).
Recently, the seeds of Amaranthus edulis, which is considered to be of the same species as A. caudatus, have been brought to attention, because very favorable essential amino acid composition of the Australian stock of A. edulis seeds was appraised by chemical analysis and in feeding experiments with chick and rats (25,26). Now, we revealed some unique properties of A . caudatus starch, namely, higher starch-granule susceptibility values to amylases as well as those of A. hypochondriacus ( Fig. 7 and Table 2 in Ref. 2) and pasting properties ( Fig. 5 and Table 2). These results may indicate that seeds of the grain amaranths are potentially valuable sources for food, feed and fuel. Several millets, for example, foxtail, Japanese barnyard and proso millets, and fi nger millets (Eleusine coracana Gaertn.), are grain crops under relict cultivation in Japan. They have been cultivated by diverse and scattered peoples since prehistoric times and were especially important grain crops under barnyard or shifting cultivation. Foxtail and proso millets have been reported to have both glutinous and non-glutinous endosperm and sometimes intermediate ones, while finger and Japanese barnyard millets have just one type, non-glutinous endosperm indicated using the iodine-staining method of the tissue. However, properties of their starches have not been fully investigated in the light of the present knowledge of starch chemistry and biochemistry.
We identified that amylose contents of proso millet starches are 28% of the normal, 0% of the waxy and 1% of the intermediate type and all of the starches have typical amylopectin (Fig. 4 and Table 1). Thus, cereals, especially the so-called intermediate types of cereal grains by the iodine-staining method may have to be re examined for properties of their starches. | v3-fos |
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} | s2 | Cadmium, lead and zinc in growing rats fed corn leaf tissue grown on soil amended with sewage sludge or heavy metal salts.
Rats were fed, from weaning through 11 weeks of age, dried leaf tissue of corn plants grown on soil amended with regular NPK fertilizer (150-22-62), with 33.6 ad 67.2 metric ton/ha of sewage sludge or with salts of cadmium (10kg/ha), lead (25kg/ha), and/or zinc (50kg/ha). A very high proportion of the cadmium (cd) consumed was eliminated in feces. Only in rats fed diets containing leaf tissue from plants grown on soil to which CdCl2 salt or the high level of sludge had been added did the metal accumulate in significantly greater quantity than in rats fed a standard diet without leaf tissue. Most of the carcass accumulation of Cd could be accounted for by that in the liver and kidneys. The proportion of dietary zinc (Zn) that was excreted in feces was less than that for Cd, indicating that more Zn was absorbed into the body. There was no correlation between intake and accumulation of Zn in the tissues, however, so that much of the absorbed Zn must have been eliminated in some way. Fecal elimination did not serve as a way to rid the body of excessive intake of lead (Pb). However, with intakes ranging from 2 to 11 mg total in this study, the carcass load did not exceed 1.1 mg of Pb. Thus absorbed Pb, like Zn, must also be eliminated efficiently. No gross signs of toxicity or of physiological impairment were observed in rats fed any of the plant tissue samples.
Introduction
Treatment and disposal of municipal wastewater and sewage sludge are major environmental problems. The need to find safe ways of disposal, and anticipation of some benefit to be gained, resulted in numerous studies involving sewage sludge as a soil treatment on agricultural lands. The potential hazard of certain heavy metals accumulating in soils from applied sludge and subsequent transfer of these metals through the food chain to man is recognized (1,2). Hinesly et al. (3) reported that furrow irrigation of corn plots with heated, anaerobically digested sludge led to increases in cadmium and zinc in leaf and grain tissues that were related *Departments of Food Science and Agronomy, University of Georgia Agricultural Experiment Station, Georgia Station, Experiment,Georgia 30212. to rate of application. Sludge treatment of stripmine soil increased yields of corn without causing significant differences in heavy metal content of the grain (4). In subsequent studies from the same laboratory, Garcia et al. (5) investigated translocation and accumulation of heavy metals in various tissues of corn plants. Generally, the highest concentrations of metals were found in roots and leaves and the lowest in grain and cob. Increases in zinc, copper, chromium and cadmium in leaf tissue occurred as a result of sludge application, with the increase in cadmium being greatest. Furr et al. (6) grew plants representing major classes of edible garden crops on sludge amended soil in pots. Of the 42 elements determined, 12 were found at higher concentrations in sludge treated versus untreated areas with at least three of the December 1981 197 crops grown; beans, cabbage, carrots, millet, potatoes, and tomatoes. Cadmium and zinc were found at particularly high concentrations in cabbage, millet and onions. Chaney (7) and Page (2) have reviewed the effects of soil characteristics on the extent of uptake of such elements by plants.
We fed grains of corn, sorghum, and soybeans that were grown on soils treated with sewage sludge to weanling rats (8). Differences in metal content of liver and kidney tissues associated with type of soil treatment were found only for Mn, Cu and Fe and even for these elements all values were within normal ranges. Cd, Ni and Pb were not present at detectable levels in the grains or rat tissues. Melsted et al. (9) used heavier applications of sludge than we did and found Cd in corn grain and in liver, kidney and duodenum tissues of pheasants fed the grain. Cd load in plant and bird tissues increased with increasing rates of sewage sludge applied to the land.
Leaf tissue of plants grown on sludge amended soil accumulate higher concentrations of heavy metals than do the grains or fruits of the plants. Transfer of these metals, especially cadmium and zinc, to mammals fed the crop components has been reported. Cadmium was increased in kidneys of guinea pigs fed Swiss chard (10) but composting the sludge seemed to reduce the rate of transfer of metal to the plant (11). The concentration of cadmium and zinc in lettuce was closely correlated with the element levels in the respective municipal sludges on which they were grown. Cadmium was significantly higher in kidneys of mice fed the sludge grown lettuce than in those fed lettuce grown on soil alone (12). In rats fed turnip greens grown on soil treated with sewage sludge, cadmium level of liver and kidney was increased in proportion to the rate of application of sludge to the soil (13). Heffron et al. (14) fed silage corn grown on soil amended with municipal sludge to sheep. Cadmium was higher in liver, kidney, spleen and muscle, and zinc was higher in muscle of the animals fed the sludge grown corn than in those fed the control corn.
In the experiment reported here, transfer of heavy metals through plant to animal from a municipal sewage sludge was compared with that of metals derived from inorganic salts applied to the soil in the field. Leaves of corn plants grown on field plots treated with sludge and salts of cadmium, lead and zinc were fed to rats for eight weeks. Determination of the amount of the elements in the feces indicated the extent of absorption of the metals. Accumulation in total carcass and specific tissues was also assessed.
Materials and Methods
Corn (Zea mays L.) "Pioneer 3009" was grown on a Cecil sandy loam (clayey, kaolinitic, thermic, Typic Hapludult) soil which had received two rates of sewage sludge applied to the soil and incorporated. The sewage sludge was from an industrial and a residential municipal, secondary treatment digested sewage sludge plants. The rates were 33.6 and 67.2 metric ton/ha applied over a 4-year period. Cadmium, lead or zinc was applied as a sidedress solution (500 mg/6.1 m row) to the soil surface along the row when corn plants were approximately 1.5 m high. Cadmium was applied at the rate of 10 kg/ha as CdC12, lead at the rate of 25 kg/ha as Pb(CH3COO)2 and Zn at the rate of 50 kg/ha as ZnSO4.
Whole plant samples (above ground) were taken from the sewage sludge and heavy metal treated plots and divided into leaves, stalk and ear components when the plants had reached the "dent" stage of maturity for the corn grain. Samples were dried at 70°C in a forced-air oven, ground to pass a 0.5 mm mesh screen and samples stored in plastic containers for analysis and feeding trails.
Dried corn leaf tissue was incorporated as 15% of the total weight of rat diets (Table 1). Casein (supplemented with methionine), a complete vitamin mixture, and a complete mineral mixture were added at the same concentration as that used in the standard diet so that essential nutrients were provided at required levels without reliance upon those contained in the corn leaf tissue. Soybean oil was added at 20% of the diets to compensate for the nondigestible carbohydrate and ash of the plant material by increasing the caloric density of dSulkafloc.
Environmental Health Perspectives the diet. Leaf material was replaced in the standard diet with 5% each of cellulose, sucrose, and corn starch. Weanling rats were maintained on the standard diet for the initial 48 hours after arriving in the laboratory and then sorted into eight groups of ten animals each on the basis of body weight. One group was fed the standard diet, and one was fed the diet containing each lot of corn leaves for eight weeks. The rats were housed individually in stainless steel cages with wire mesh floors and provided with diet and deionized water ad libitum. Feces were collected each morning and stored at -5°C for subsequent analysis. Feed intake was monitored for the entire feeding trial and rats were weighed at weekly intervals.
At the end of the feeding period, half of the rats in each group were given a lethal dose of pentobarbital intraperitoneally. After death of the rat, food particles and other debris were removed from the fur by vacuuming. The peritoneal cavity was opened and the GI tract and its contents, from the proximal end of the esophagus to the anus, were removed, taking care that no blood loss occurred. The remaining part of the rat, hereafter referred to as carcass, was disintegrated by digestion in nitric acid. The fat, which rose to the top of the digestion vessel and solidified after cooling, was discarded, and the aqueous portion was made to volume from which aliquots were taken for mineral analysis.
The other rats in each diet group were anesthetized with pentobarbital and exsanguinated by severing the abdominal aorta. The liver, kidneys, heart and testes were removed and freeze-dried for subsequent analysis.
Diets, leaf tissue and animal tissues were digested with nitric and perchloric acids and inorganic elements were determined by flame atomic absorption, except for Cd which was determined by flameless atomic absorption. Data were subjected to analyses of variance and differences among means were evaluated by Duncan's (15) multiple range test where appropriate.
Results and Discussion
Slight burn was evident on the lower leaves of the corn on the Cdand Zn-treated plots, but no visual effects were noted for the Pb-treated plots or the sewage sludge-treated plots. The burn effects on the Cdand Zn-treated plots did not appear to influence subsequent plant growth or physiological development.
The data of Table 2 show growth performance and gross body composition of rats fed the diets containing the corn leaf tissues and those of rats fed a standard diet of purified ingredients. Growth and feed efficiency (weight gained/feed consumed) were both reduced by incorporation of the plant material into the diets. These effects were probably due to the nature of the corn leaf itself rather than to the prior soil treatment since none of the sludge or salt treatments gave effects different from that of the normal NPK fertilizer treatment.
The weight of dry fecal material produced was markedly increased by addition of the plant tissue to the diets. The increase in fecal mass from rats fed the diets containing corn tissue over that from rats fed the standard diet accounts for about 40% of the weight of leaf tissue ingested.
Organ weights and proportions of body weight were all within normal ranges for liver, kidney, heart and testes of rats fed the diets containing corn leaf tissue. Liver and kidney percentages of body weight tended to be somewhat higher for rats fed diets with leaf material from corn grown on plots treated with the high rate of sewage sludge than those fed loaf tissue produced with NPK fertilizer.
The relationships between dietary intake and fecal output of the elements cadmium, zinc, and lead are shown in Figure 1A, 1B and 1C, respectively. A very high proportion of the cadmium ingested passed out of the body without being absorbed. This is indicated by a regression coefficient and a correlation coefficient each of which approaches a value of one. Furthermore, although all rats fed diets containing corn leaf tissue consumed 15 ,ug or more of cadmium during the 8-week study, only those fed tissue from plants grown on plots treated with cadmium salt accumulated total carcass loads of more than 1.5 pug of cadmium (Table 3). For the rats fed diets containing leaves from corn grown on plots amended with the salt mix or with cadmium alone, the difference between intake and fecal excretion of cadmium amounted to about 120 and 360 ,ug of cadmium, respectively. However, the increase in carcass load of the element over that of rats fed the corn grown on the plots treated with NPK fertilizer was only 2.4 and 4 jig, respectively. Thus, in these cases in which a significant amount of dietary cadmium was not accounted for by that in the feces, much of what was not absorbed must have been eliminated through the kidneys, or been present in the tissues of the GI tract which were not included in the portion of the carcass analyzed. The data of Table 3 showing cadmium content of total carcass and of selected rat tissues are listed in descending order of level of dietary intake of the element. These data clearly show the strong tendency of liver and kidney to accumulated cadmium with increasing dietary intake. In fact, a large proportion of the total carcass accumulation associated with cadmium additions to the plots on which the corn plants were grown is accounted for by the excess of cadmium in these two organs. Although total carcass load of rats fed leaf tissue grown on HSS treated plots was not significantly greater than that of rats fed NPK produced leaves, cadmium level of kidney was more than four times and of liver three times greater in the former than in the latter group bValues in a column not followed by a common superscript letter are significantly different at p -0.05. and testes of rats fed the diets with corn leaf from plants produced with the single cadmium salt treatment also display a burden of excess cadmium accumulation.
The correlation between dietary intake and fecal output of zinc is highly significant, also (Fig. 1B). The regression coefficient is somewhat lower than that obtained from the intake-output relationship for cadmium, indicating that more of the dietary zinc is absorbed into the body. Unlike cadmium, there are well established physiological needs for zinc. Except for the diet containing corn leaves from plants grown on the soil to which sludge was applied at the high rate, dietary zinc concentration was only two or three times the published requirement for the rat of 12 mg/kg (16). There were no significant differences in zinc content of total carcass or of liver, kidney, heart or testes tissues among rats fed diets containing the various samples of corn leaves or between any of these and the rats fed the standard diet.
The relationship between fecal output and di-etary intake of lead was quite different from that of the other two elements considered above. For rats fed the six diets containing corn leaves from plants grown on plots without lead salt additions, correlation between intake and output was not significant. Over an intake range of about 2 to 6 mg of lead, the fecal output was about 2 mg of lead. For those rats fed the diets with leaf tissue from plants produced on plots to which the lead salt was applied, fecal output was increased but was still less than 50% of intake. The correlation between lead intake during the 8-week feeding trial and total lead content in the carcass was not significant ( Table 4). The mean intake for the eight diets fed varied from 2 to 11 mg of lead, but the carcass load of the element at the end of the study ranged only from 0.7 to 1.1 mg of lead. These data suggest that, though significant amounts of lead were absorbed into the body by rats fed some of these diets, most of the absorbed lead must have been eliminated in some way. Differences among diet means for lead content bValues in a column not followed by a common superscript letter are significantly different at p s 0.05.
201
December 1981 of kidney, heart, and testes were statistically significant but were poorly correlated with level of intake or total carcass content of the element.
Liver contained about 35 jig lead and the sum of this element in liver, kidney, heart and testes was less than 7% of the total in the carcass. Under normal circumstances, the Sprague-Dawley rat will achieve more than 90% of his maximum body size by 11 weeks of age. Thus this feeding trial, which occurred during the first 8 weeks after weaning at 21 days, took place during the period of rapid growth when nutrient supply to the body is most critical. There were no gross signs of physiological impairment or physical distress in the rats fed corn leaf tissue produced on plots amended with sludge or the inorganic salts. The accumulation of heavy metals in the corn leaves caused no apparent change in the growth performance of the rats fed these plant tissues during the short, but crucial, period following weaning. The overall reduction in growth rate of rats fed corn leaf tissue, including that grown on plots treated with the usual NPK fertilizer, was due to the indigestible nature of the material itself. The rat has a fairly large caecum but cannot utilize as high dietary concentrations of cellulose as a ruminant can.
The accumulation of cadmium in soft tissues, especially liver and kidney, could result in chronic toxicity in the animal fed tissues from plants grown on sludge amended soil. It could also be a source of cadmium ingestion by man if the plant material were fed to meat animals. Before the potential hazard to man of applying municipal sewage sludge to agricultural land can be assessed, some carefully planned, long-range studies must be made. The objectives of these studies should focus primarily on preventing accumulation of toxic elements in plants grown on the treated land since the alternative of stopping their transferral to man would be far more complex. Rates of sludge application to land that are economically feasible in terms of both crop production and waste disposal should be determined. Chaney (7) has shown that a high ratio of zinc to cadmium in the soil 'as well as maintaining a soil pH near neutrality will reduce the accumulation of the latter element in plants. Other ways to decrease uptake of this and other heavy metals by plants should be investigated. | v3-fos |
2017-07-29T04:25:17.278Z | {
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} | s2 | Carcass measurements of naked neck and normal chicks
From a cross between two heterozygous parents (Nalna+) for the naked neck gene, 10 birds were chosen at random within each sex and each of the two genotypes, Na/na+ (naked neck) and na+lna+ (normal plumage). They were slaughtered at 8 weeks of age. Morphological traits and weights of various parts of the carcass were measured. Plumage in p. 100 of body weight was highly significantly less for naked neck birds in both sexes. Conversely, boneless meat p. 100 and meat/bone ratio in half carcass were significantly higher (respectively at 5 p. 100 and at 1 p. 100 level) for the Nalna+ genotype than for na+/na+. Weight of head (absolute and p. 100 of body weight) was lower for the first genotype (at 5 p. 100 level). The same appeared for heart, but with a genotype X sex interaction. The main suggested conclusion is, on the whole, a better meat yield for naked neck chicken. Significant effects of sex, on the other hand, were found on various traits independently of genotype at Na locus. The naked neck gene (Na) has been found associated with lower chick mortality in response to high sublethal temperature (SMITH & L EE , 1977) and with better growth rate at room temperature kept above 30 °C till the age of 10 weeks (B ORDAS et al., 1978 ; M ONNET et al., 1979). In a comparison done in Egypt at two hatching seasons, it was found (Z EIN-E L-D EIN et al., 1981) that heterozygous chicks for the naked neck gene were at an advantage for growth in the hotter season. Concerning effects associated with this gene on body composition, an important reduction of plumage weight, in absolute terms and in p. 100 of live weight, has been shown (BO RDAS et al., 1978 ; M ONNET et al., 1979). These authors did not find significant differences for other carcass components. However, it appeared interesting to do a similar comparison in our local conditions and with measurements partly different from those included in the previous works.
Chicks were produced by crossing heterozygous Na/na + males and females (Na being the allele responsible for the naked neck phenotype, na+ the normal allele : H UTT , 1949 ;S OMES ,1980). These parents were progeny of birds sent as day-old chicks in october 1978 from Laboratoire de Génétique Factorielle, LN.R.A., Jouy-en-Josas (France).
In june 1979, in a hatch including about 100 day-old chicks, 10 birds were chosen at random within each sex and each of the two genotypes, Na/na + (naked neck) and na + /na+ (normal plumage). The Na/Na genotype was not considered because two low numbers were available. These birds, raised in flood pens and fed ad libitum, were slaughtered at 8 weeks age. Morphological traits and weights of various parts were measured on carcasses. The different variables are mentioned in table 1. Additional comments can be given as follows : -weight of feathers is obtained after dry plucking ; -dressed carcass is the difference between plucked carcass and the sum of giblets, viscera, head, neck and shanks + feat ; edible portion is the sum of dressed carcass and total giblets ; -dressend carcass is divided into two halves and within one half, the total amount of meat, bone and skin is determined, so that « boneless meat p. 100 » is the proportion of muscle in this half carcass. Table 1 gives the mean values of measurements and carcass components, for each sex and genotype. Weights are expressed in abwiute values and in per cent of live body weight. Table 2 shows for each trait the F values and significance corresponding to the analysis of variance with genotype at Na locus and sex as sources of variation. As concerns organ weights only variance analysis on absolute value is presented when significance or absence of significance is the same for the trait expressed in absolute value and in per cent of body weight. However, variance analysis is also presented on percentage for feathers and dressed carcass, where significant effects appear only in this case, and for boneless mat and meat/bone ratio for the same reason.
The aim of the present work was to evaluate the effect of genotype at Na locus on body composition of chicks. These first results are to be considered as a preliminary indication, in view of the limited numbers available.
The mean live body weights within each sex are higher for the Na/na + genotype, as found by Z EIN -E L -Ð EIN et al. (1981) in this season, but on the present sample, owing to the small numbers, the difference is not significant.
On organ weights expressed in absolute values, our results show a significant effect of genotype (p [ 0.05) for heart, head and femur, which are lighter on average in naked neck birds. As mean live weight of these birds is superior (not significantly in this small sample but significantly in the larger sample studied by Z EIN -E L -D EIN et al., 1981), these differences would be relatively more important on these variables expressed in per cent of body weight.
The difference between genotypes for feather weight, in absolute value, does not reach significance, but when expressed in p. 100 of live weight, the decrease of plumage weight associated with the Na/na + genotype becomes highly significant. Two other traits, expressed respectively as a percentage and as a ratio, show a significant effect of genotype : boneless meat as per cent of eviscerated carcass, and the ratio of meat to bone weight in this carcass. This ratio is significantly (p ! 0.01) higher for naked neck chicks.
The effect of sex is significant or highly significant on blood, liver, viscera, head, legs (higher mean for males) and per cent plumage and dressing percentage (higher mean value for females). The sex x genotype interaction is significant for blood, heart and dressing percentage. However, it may be noticed that in this limited sample the sex dimorphism for live body weight is unexpectedly low, so that the observed effects of sex and sex x genotype interaction on carcass measurements should be verified on additional data. Among differences associated with genotype at Na locus, that concerning weight of plumage in per cent of body weight is in line with results obtained by B ORDAS et al. (1978) and MotvrrET et al. (1979). Conversely, the data of B ORDAS et al. (1978) do not show differences between males of different genotypes at the Na locus for relative weight of the heart, nor for per cent of bone and muscle, but these percentages concerned only the thigh, while in the present work they relate to the whole carcass.
It will be interesting to confirm this last point, as it is connected with meat yield and thus may be of practical value and suggest some additional advantage of naked neck birds in specific environmental conditions. Reçu pour publication en octobre 1981. | v3-fos |
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"year": 1981
} | s2 | Archaeological Investigations at the San Pedro Aceqnia San Antonio, Texas
The irrigation system in San Antonio was an integral part of its early history. Within the San Antonio area irrigation was a necessity to extend the planting season. An extended planting season would allow for diversified crops with different harvesting periods. The first municipal irrigation system was the San Pedro Acequia. It was constructed to serve the town with fresh water for household use and agricultural purposes. As the population increased and technology improved, the acequias changed in both appearance and function. In time, the San Pedro Acequia system became part of San Antonio's forgotten past. Recently, excavations on the Government Service Administration (GSA) property in downtown San Antonio unearthed portions of the San Pedro Acequia. Investigations revealed the Acequia underwent three construction phases within the (GSA) property. Results of the archaeological investigations and literature research gives a vivid picture of the history of the San Pedro Acequia.
Earlier Phase I investigations of this GSA property were carried out in June 1979 (Valdez and Eaton 1979) in order to determine whether or not the Spanish Colonial San Pedro Acequia ran through the property. Two portions of the acequia were uncovered by these investigations. As a result of these tests, additional study was recommended. The current work, Phase II of the investigations, was initiated in order to more precisely document the routing of the acequia prior to future development of the property by the GSA. The Phase II field work was done by Augustine Frkuska, Jr., with assistance of Waynne Cox and Harvey Smith, Jr. Dr. Thomas R. Hester, Center Director, and Jack D. Eaton, Associate Director, provided overall supervision of the project. In addition to the field work, extensive literature research was done for the study area. This research is by no means exhaustive, although enough information was collected to document and form interpretations of the archaeological work in Phases I and II. A study of the San Pedro Acequia has presented a clearer picture of what once was an integral part of the history of San Antonio.
SETTING
The General Services Administration property is located between two major drainages. The San Pedro Creek flows south and lies to the west of the GSA property, and the San Antonio River, which also flows south, lies to the east of the property. Across the GSA property, the land is flat with a gentle slope toward the south. At present, the area is parkland covered with grass and a large number of pecan trees are dispersed throughout the park, along with a few live oaks, elms, and palms. Buildings once stood along the west side of the property fronting S. Flores Street. The locations of these buildings are marked by concrete and tile slabs which posed a problem for locating test trenches along the predicted route of the acequia.
The city blocks that were investigated by the CAR include NCB 2547, 2548, 2553 5 2552, and 2969. The GSA property lies south of the Bexar County Courthouse and the old U.S. Arsenal, and between S. Flores Street and Main Street. Sheridan Street forms the northern property while W. Guenther Street forms the southern boundary.
HISTORICAL SKETCH
In 1718 Spanish missionaries and soldiers arrived at San Pedro Springs to begin the first significant European settlement, which later became known as San Antonio. Among their major concerns was the availability of water in a semiarid environment. This obstacle was overcome by a plan to construct a system of acequias (water ditches) to irrigate their farm lands (Castaneda 1936:96).
Historic information is sparse about which acequia was dug first or its exact location. Valdez and Eaton (1979:1) suggest that the acequia known as the San Pedro Acequia originated from a series of channels constructed at San Pedro Springs by a few early settlers prior to 1718. However, the first major acequia constructed is conceded to be the Acequia Madre for the Mission of San Antonio de Valero. A historical sketch of missionaries and soldiers in relationship to the early San Antonio settlement can be reviewed in Fox, Bass, and Hester (1976).
The Canary Islanders arrived in March of 1731 and found five missions established in the area. Three of these missions were inaugurated just four days before the Islanders arrived (Holmes 1962:12). The Islanders, who were given the title of Hidalgos, saw the natives as a cheap work force for field work and irrigation projects. The Franciscans in the established missions thought of the Islanders as unwanted competition as well as a disturbing element for the natives. The friction between the two groups on a number of issues is well documented throughout the Bexar County Archives (Buck 1949: Bolton 1915. The settlers were commissioned to create a municipal government and to be assigned land with water rights (Buck 1949:94). The land and water rights were to be made permanent after an irrigation canal was dug. The Presidio de Bexar (tvlilitary Plaza) and San Fernando Church (Plaza de las Islas) were to become the center for the newly founded municipality (Cox 1899:217;Eaton n.d.). A description of houses and ownerships around "Plaza de Armas ll can be reviewed in Chabot (1931). The acequia was to provide water to the town as well as to individuals' property along the way. Unfortunately, the construction of the acequia was delayed, since building homes and planting crops consumed a great deal of the Islanders' time. Glick (1972:27) states that as few as seven of the group knew the fundamentals of irrigation. Antonio Rodriguez of Gran Canaria Island apparently was one of the knowledgeable members. Father Benito Fernandez de Santa Ana, head of the newly inaugurated missions, asked Rodriguez to supervise work on the construction of the Concepcion Acequia (Holmes 1962:23;Glick 1972:40;Buck 1949:177). It is possible that Antonio Rodriguez was the only irrigation engineer in the settlement, and the eventual removal of Rodriguez from the municipal work force could have been an additional reason for the long delay in the construction of the San Pedro Acequia.
The actual digging of the San Pedro Acequia did not start until 1738 (Arneson 1921 :124). Buck (1949:179) writes about the large celebration at San Pedro Springs where the acequia was to draw its waters. The acequia would then extend to the Presidio, running in front of San Fernando Church, and on to the labon~ (fields). Records of when the water came through the Villa de San Fernando are vague. The lands north of the Villa were to have been watered by 1740 (ibid.). Glick (1972:40) recites an early document where the working water system of Villa de San Fernando in 1750 is used as an argument for the development of irrigation on the San Xavier River. The exact time the acequia reached the lands south of the Villa is still speculation. When the acequia was completed it was said to have been six feet wide, two feet deep and four miles long, and irrigated some 400 acres (Arneson 1921 :124).
The years 1740 through 1760 were a time of devastating Indian raids by the Apaches and Comanches (Ramsdell 1959:22;Tunnell and Newcomb 1969:157). The upkeep of the acequia became a precarious affair, especially the areas away from the protection of the Presidio. This can be seen in the early laws governing the upkeep and the usage of the acequia system. These first laws were included in a document called "Laws of the Indies. 1I They simply stated that each user pledged to keep his part of the acequ;a clean, repair the gates, and be in readiness to fight off any of the King's enemies (Arneson 1921 :128;Buck 1949:179). Very few other ordinances were made by the municipal government. This can be attributed to the solidarity of the users of the canal, and this solidarity can be directly attributed to the needs of the times. For instance, every February was the time set aside to have all the farmers clean out the acequias (Arneson 1921 :129). The settlers, no doubt, found strength in numbers while in the fields during the cleaning period.
Orders from Spain were issued in May of 1773 to close the presidios and missions of East Texas. Families from Los Adaes and Los Ais were relocated to the Villa de San Fernando (Bolton 1915:385 Secularization of the mission lands came in the last quarter of the eighteenth century. Each mission had its own farmland and irrigation system that would come under the control of the municipality. The lands were divided between the Indians of the missions, the soldiers of the Presidio, and the settlers of the town (Broussard 1967:5). The authorities that once governed the San Pedro Acequia and Upper Labor Ditch now had miles of irrigation ditches on both sides of the San Antonio River to regulate. Apparently. the distance between the authority and the mission acequias was too great. Dobkins (1959:121) writes that the farmlands south of the city once cultivated by the missions were in wilderness as early as 1840.
The beginning of the nineteenth century brought outside influence to the Spanish town. The Louisiana Purchase of 1803 brought westward movement of English speaking people and pressure towards independence. In September of 1810, Father Miguel Hidalgo started a revolutionary movement in Mexico that brought refugees into the northern part of Mexico (Ramsdell 1959:27). These refugees also brought stories of independence. However, the taste of independence in San Antonio was bitter. The next few decades were devastating to the San Antonio area. The unstable political climate was to have a long term effect on the acequias in general, and many of the town's prominent people were forced to flee. This depopulation of the town and the severe flood of 1819 brought the San Pedro Acequia watering efficiency to its lowest level since its construction (Ramsdell 1959:30). This is shown through an official complaint issued to the city council from the "Labors de Abajo" in 1822. The complaint stated that the city was not doing its share in the upkeep of the San Pedro Acequia (Glick 1972:46).
Texas, under newly independent Mexico, began to grow through the efforts of Moses Austin in his quest to colonize Texas with Americans. The colonization was rapid and Mexico became weary of its changing frontier. The question of independence was brought forth again and San Antonio would be the main recipient of the hardships in the struggle. Very little is said about the actual role the acequias had during the struggle for the independence of Texas. However, recent excavations at the Alamo by the Center for Archaeological Research may provide needed information for this time period. The Mexican military in the early 1840s under Vasquez and Woll drained San Antonio of some of its citizens and city records (James 1938:19;Broussard 1967:27). Taken along with the city records were land titles with their water rights. The population dropped from 800 in 1839 to 600 in 1843 (Broussard 1967:27 In 1843 French and German immigrants began arrlvlng in San Antonio. Soon problems arose between the immigrants and the townspeople concerning land ownership and water rights. As a result, the city council was moved to translate all laws regarding acequias and their operation from Spanish and English into German and French. These translations were to be placed in public places by 1844 (Broussard 1967:33). The unfamiliarity with the laws and customs that governed the acequias caused great alarm in the city council. On May 15, 1847, the council used its power to insure a water supply for its own use by setting aside every fifth day for city use (City Council Minutes, Journal A, 1847).
The German and American populations grew, spreading their influence throughout the city and consequently much of the architecture and the municipal government took on an Anglo flavor. The first American ditch commissioner was Capt. J. H. Beck, appointed in 1850 (Corner 1890:50). In 1852 the state legislature placed the construction and operation of irrigation works owned jointly by two or more different persons under the control of the county courts. The courts were authorized to order meetings for election of commissioners and to assess and collect fines (Hutchins 1928:269). On August 17, 1852 a committee was appointed by the court to draft rules and regulations for civil and police administration of irrigation property in Bexar County (Commissioners Court Minutes, Vol. A-l:81). It is interesting to note that the charter provisions made both at this time and later reflected Spanish control methods for irrigation.
Holmes (1962:83) contends that the acequias we~e permitted to fall into a state of disrepair due to a cycle of wet weather starting in 18550 At this time a traveler through Texas noted: The system of aqueducts for artificial irrigation extends for many miles iround San Antonio. 0 . . These watercourses still retain their old Spanish name "Acequia." A large part of them are abandoned but in the immediate neighborhood of the city they are still in use, so that every garden patch may be flowed at will (Holmes 1962:83). 5 In 1856 the corporate bounds of the city of San Antonio extended six miles from the San Fernando Cathedral (Huesinger 1951:-26). The expansion of the city created problems with the acequias. Among the problems were those concerning all road or pedestrian bridges crossing over the acequias. Holmes (1962:94) gives the accounts of city newspapers concerning this public problem; it is also well documented throughout the city council minutes. The city charter of 1857 empowered the mayor and the city council to reopen the old irrigation ditches, within or beyond the city limits of that time, and to regulate all matters pertaining to the operation and the distribution of water for irrigation. The charter also made it an offense, punishable by fine or imprisonment, to defile the San Pedro Ditch by washing clothes, watering horses, or any other means.
In 1859 the U.S. Army acquired a large section of land, south of downtown San Antonio, for an arsenal (Huesinger 1951:27). The San Pedro Ditch ran through the property and was still in operation at the time the arsenal was built (Fox 1978a: 3).
The 1860s brought little change to the San Antonio acequia system. The war between the states brought confusion and problems to the controlling authorities, and the economy of San Antonio was at a low ebb. Ramsdell (1959:45) quotes Vinton James: The trash and garbage was thrown into the backyards. Drinking water was obtained from shallow wells and irrigation ditches, and this water became contaminated from outhouses, and typhoid fever and malaria were prevalent.
In 1866 a cholera epidemic broke out in San Antonio, which was probably the incentive the city needed to force a cleanup.
In the 1870s prosperity once again came to San Antonio. The ranches brought capital to San Antonio as a result of the high prices paid for wool and cattle. The railroad came through in 1877 and boosted the prosperity even higher. The San Antonio Water Company was organized the same year; it drew its water from the headwaters of the San Antonio River (Corner 1890:55). The neglected acequias began to be refurbished, and the city council passed a resolution in 1879 to clean the acequias: . . . sum of $690.00 or as much as to be necessary to clean San Pedro, Upper Labor, and Alazan in accordance to city charter (City Council Minutes, Vol. E, December 5, 1879).
To keep the acequias clean, an ordinance against defiling the water in the ditches was passed November 16,1880 (City Council Minutes, Vol. E, 1880).
The 1880s brought a revival in caretaking and usage control of the acequias. It appears that any subsequent changes made to the acequias were to be approved by the city council. On April 18, 1882 the city council approved a measure to put a cover over the San Pedro Acequia from Story's Stable to Houston Street, and Houston Street to Simon Fest's property. On February 21, 1882 the city council was petitioned to straighten the ditch that ran through the U.S. Arsenal and to build a bridge over the acequia at Arsenal Street. On April 17, 1883 a proposal to stone line San Pedro Ditch through San Pedro Park, a distance of 300 to 400 feet, was brought before the council. A short time after completion, a public improvement project was initiated to raise the park's acequia west wall 12 more inches for the entire length of the wall. As late as 1887 the citizens petitioned the city council for protection from overflow of the acequia. On September 19, 1883 an estimation of $200.00 was made for 200 yards of erected stone wall at the place of the overflow.
The drilling of artesian wells,· as well as the founding of the San Antonio Water Company, helped eliminate the need of the acequia for a clean water supply. The drilling of the wells into the lower rock stratum was said to have a diminishing effect on the water issuing from the springs (James 1938:149).
Since the San Pedro Acequia took its water from such springs, it was soon to become an unreliable source of clean water. The acequias of San Antonio then began to take on a new role, that of collecting run-off water. Arneson (1921: 129) writes: .
Small watercourses, carrying storm'waters, were frequently allowed to empty directly into the canal. When this was done,.however, a masonry wall or wasteway, was provided in the lower bank of sufficient size to permit all the storm water to spillover at that point, thereby not endangering the canal with wholesale washouts.
Holmes (1962:117) also quotes Andrew Morrison on the subject of a need for a more modern drainage system for the increased population. The population in San Antonio was 12,256 in 1870 and increased to 37,673 in 1890(Ramsdell 1959. The city streets were being improved by macadamizing or surfacing with mesquite blocks. These improvements, along with building constructions, virtually eliminated the natural ground where water could be absorbed, thus creating a larger amount of run-off water during wet weather.
The acequias soon became less important. Petitions began to filter into the city council meetings for closing portions of the acequias. Pros and cons were heard ,by the council; reports by physicians and engineers were he~rd, and the evidence was weighed. Despite the increasingly negative attitude toward the acequias, it was the general concensus of the city council to keep the channels open and to continue improvements wherever needed. In 1891 a resolution was passed to build storm and trash gates on the San Pedro Acequia 200 yards north of W. Houston Street.
Trash and stagnant water were always present in the acequia and the city was worried about this as a serious health problem. Mayor George Paschal reported to the city council in 1893:
7
He also reported to the council the necessity for clean water, indicating that one in every 57 people dying yearly from unclean water in San Antonio was cause for concern. Perhaps this was the beginning of the end for the acequias running through the city. The city government abolished the ditch commissioner's office in 1899. It was not until 1906 that the water is said to have ceased flowing in the San Pedro Acequia (Schuchard 1951 :30). Six years later, on September 3, 1912, an ordinance was passed to have the San Pedro Acequia officially closed (City Council Minutes, Vol. V., September 3,1912).
PREVIOUS ARCHAEOLOGICAL RESEARCH
There are literally miles of acequias (water ditches) criss-crossing and latticing the metropolitan area of San Antonio and lands to the south ( Fig. 1 :Locations 1-11). There have been comparatively few detailed archaeological investigations and publications dealing with these facts of pioneer engineering.
The first series of excavations was made by the Texas Historical Survey Committee, in collaboration with the Witte Museum, at the Acequia Madre in Hemisfair Plaza (Location 1) and the Zilker property just north of the Downtowner Motel (Location 2). Both excavations revealed ditches lined with walls of limestone block (Sorrow 1972). In 1969 the Texas Archeological Salvage Project excavated part of the Acequia Madre (Location 3) just north of the Daughters of the Republic of Texas Library on the Alamo grounds (Sorrow 1972).
During 1973 to 1980, the CAR-UTSA conducted the following field investigations of the acequia system. In 1973, Thomas R. Hester excavated a small portion of the same acequia on the Alamo grounds (Location 4) just behind the souvenir shop (Adams and Hester 1973). In 1977 portions of the San Pedro Acequia on the old U.S. Arsenal property, just east of the commander's house (Location 6) were uncovered. This excavation revealed the acequia to have limestone block walls (Fox 1978a). During the same year, portions of the Alazan acequia just southwest of San Pedro Springs Park were excavated (~b~d.). Excavations under the field direction of Paul Katz uncovered what is believed to be an unlined acequia near Hemisfair Plaza (Location 8) in the Arciniega Street area (Katz 1978). In 1978 the Center conducted investigations at the Gresser House (Location 5), where an unlined ditch, perhaps a QO~tadoh (lateral or branch) of the Conception Acequia was uncovered (Ivey 1978b). In 1979, the Center investigated the Alamo grounds along the north wall (Cavalry Courtyard), where an earthen ditch northwest of the museum was uncovered (Location 10); the identification of this ditch is still being considered (Fox and Ivey, report in preparation). In addition, field work has just been completed by the Center on the Alamo Plaza-River Walk Linkage project on the west side of Alamo Plaza. Here a branch of the Acequia Madre was uncovered (Location 9), and it was found to be unlined (Ivey and Fox, report in preparation This will be followed by descriptions of test trenches A-R which were dug under the supervision of Augustine Frkuska during Phase II of the investigations. Figure 2 indicates the locations of these trenches and the actual routing of the San Pedro Acequia through the GSA properties.
Observation Phase I
TJte.n.c.h 1
This trench was excavated parallel to W. Sheridan Street where the San Pedro Acequia was estimated to enter the GSA property from the north (Fig. 2). The trench, which began 96 feet west of the corner of Sheridan and S. Main, was about 98 feet in length and 40 inches in width. The soil stratigraphy of the south wall of the excavated trench indicated that the acequia was dug through 12 inches of black alluvium or loam followed by 24 inches of brown clayey loam. Below this was 19 inches of brown sandy loam which lay upon basal caliche.
The distinctive profile of a shallow ditch, beginning at the surface and extending 51 inches down to the caliche base, was noted ( Fig. 3). The width of the ditch measured approximately 9 feet (the base of the ditch was at caliche bedrock as noted). The fill consisted of two major levels. The earliest deposit was a clayey ash and charcoal loam approximately 23 inches in depth. On the east side of the ditch (acequia) was a vertical wooden post. This post is possibly a remnant of a retaining wall structure built to slow the erosion on the east bank. The second major deposit was approximately 29 inches in depth and consisted of a silt fill with charcoal flecks. It was within this second layer that the majority of artifacts were found.
TJte.n.c.h 2
This trench was located about 25 feet from W. Johnson Street (Fig. 2). The trench was dug parallel with the street to a length of 98 feet and a depth of 2 feet. The trench profile revealed 16 inches of black topsoil covering 12 inches of a dark brown clayey loam. Below this was 8 inches of brown sandy loam which rested upon basal caliche. No ditch profile was noted in the trench wa 11 s.
TJte.n.c.h 3
This trench was located 31 feet south of Trench 1 (Fig. 2). The trench was dug to a length of 28 feet and a depth of 45 inches. A profile of a ditch was noted and recorded in the south wall of this trench (Fig. 3). The trench was cut through the same stratigraphy as noted in Trench 1. However, the widths of the deposits varied in this southern wall profile.
The ditch profile appears to represent a disturbance in the acequia's east bank. A small pipe was placed parallel to the ditch, probably utilizing the previously excavated acequia when it was open. The present ditch measures almost 13 feet in width and 38 inches in depth. However, the actual width of San Pedro Acequia would be closer to 10 feet. The oblique angle in which the trench was cut gives an appearance of a wide acequia channel. The acequia fill consisted of four major deposits. The earliest deposit was 12 inches of an ash and charcoal lens containing very few artifacts. A dark silt deposit 10 to 24 inches in thickness rested upon the ash and charcoal lenso Cultural debris was also noted in this layer. The latest deposit is approximately 10 inches thick and consists of mixed topsoil.
T~eneh~ 4, 5, and 6 These three trenches were placed north of W. Johnson Street running parallel to the Calvert Street extension shown in Figure 2. Each trench measured 72 feet in length. Trench 4 lay to the east of Trench 5. Trench 5 and Trench 6 ran north and south along a common line. Soil profiles revealed natural stratigraphy with some surface modifications. The first 8-12 inches of surface material contained gravel and brick fragments mixed with soil. Below this was 8-12 inches of dark clay, 16 inches of dark brown clay, and 8 inches of light brown soil with caliche mixed in. This rested upon basal caliche.
T~eneh 7 This trench began 10 m east of Calvert Street and approximately 220 feet south of W. Sheridan (Fig. 2). The trench was dug west to east for a length of 148 feet. The north wall of the trench displayed a profile of a ditch 9 feet in width and 29 inches in depth (Fig. 3). The ditch had been dug through 8 inches of alluvial topsoil followed by approximately 24 inches of dark brown clay loam. Below lay 12 inches of yellowish tan clay loam which covered basal caliche.
The ditch profile displayed three major layers of deposits. The bottom layer consisted of a 5-inch charcoal lens with a silt mixed in. Upon this rested a 6-inch burned ash lens with cultural debris. The top deposit consisted of a gray silt with cultural debris 16 inches thick at its widest point.
T~enehe.6 8 and 9 These two trenches were located north of Trench 7 and parallel with W. Sheridan (Fig. 2). The profiles revealed the basic natural soil stratigraphy as described in previous trench profiles. Trench 8 was dug to a length of 25 feet and Trench 9 to a length of 50 feet. No acequia outline was noted in either of the trenches.
T~eneh 10
This trench was placed west of Trench 2 and parallel with W. Sheridan (Fig. 2). The west end of the trench was located 65 feet from S. Flores Street. The purpose was to extend the testing in that area to expose the acequia.
The soil stratigraphy differs from what was found in Trench 7. In the eastern part of the trench the topsoil was a gray-brown clay about 43 inches in depth. Below this was 16 inches of brown clay loam which graded into caliche gravels. The west side of the trench displayed dark brown topsoil overlying large grain sand to caliche bedrock. No sign of an acequia was found in this trench. However, the evidence of disturbance in the area is obviouso T~enQh 11 This trench was located 33 feet north of Trench 2 and 145 feet east of S. Flores Street (Fig. 2). The trench was dug south to north for a length of 15 feet. The trench exposed two parallel walls of cut limestone block running east and west.
The walls of the acequia are 20-22 inches in thickness and spaced 35 inches apart (Fig. 3). The walls are vertical and measure 39 inches from the top to the unlined bottom. The limestone blocks appear to be faced with a pink colored sandy lime mortar. Chinking was used to fill spaces and to help set the stone upon the caliche bedrock.
The interior fill of the acequia is represented by two deposits. The first is a 6-inch layer of dark gray clay containing charcoal flecks and a few cultural artifacts. The remaining fill is a caliche and soil mix with an abundant amount of cultural debris. It would appear that this fill was intentionally deposited to fill the acequia. On top of the acequia is an 8-inch layer of asphalt. This asphalt covered any surface indications of the stone-lined acequia below.
T~enQh 12 This trench was located 58 feet west of Trench 11 and 80 feet north of W. Johnson Street (Fig. 2). The trench was dug east to west to a length of 10 feet. The trench was placed at this location to expose the acequia which appeared to turn in a southerly direction as indicated by surface anomalies. A portion of the limestone block wall was uncovered. The direction of the acequia appeared to angle toward the corner of S. Flores Street and W. Johnson Street.
The south wall of the trench revealed the stone-lined acequia as described in Trench 11 (Fig. 3). However, the stone walls at this location were covered with 8 inches of dark topsoil, and there were three layers of deposits within the acequia walls. The bottom layer was 12 inches of dark gray clay of the same description as found in Trench 11. Upon the gray clay lies a 5-inch layer of sandy silt with snail shells throughout the level. The remaining 22 inches of fill was a caliche soil mix containing the majority of the cultural material collected. The acequia at this location did not rest upon the caliche bedrock.
T~enQh 13
This trench was dug east to west. The west end of the trench was 26 feet east of Calvert Street and 46 feet south of W. Sheridan Street. The length of the trench was approximately 73 feet. The purpose of the trench was to see if the stone-lined acequia was in the area, as suggested by its existence in Trenches 11 and 12. However, no acequia, lined or unlined, was found in the area. Soil deposition in the trench profile consisted of four distinct layers. The topsoil was approximately 16 inches of black alluvial loam followed by 12 inches of dark brown clay. Below this was 8 inches of brown sandy loam mixed with the caliche from the caliche layer below. Trench A was positioned on the north side of W. Johnson Street just inside the sidewalk (Fig. 2). The trench was approximately 12 feet in length and 45 feet from the corner of S. Flores. The stone-lined acequia was encountered when the backhoe grazed the top of the limestone blocks. Excavation was continued by hand uncovering two parallel limestone blocks approximately 3 feet apart running in a north-south direction. The south end of the acequia continued beneath W. Johnson Street in which an iron pipe about 20 inches in diameter carried the water beneath the road surface. The limestone blocks were 1 foot below the present surface. They averaged 18 inches in width and the faces of the blocks contained chisel marks. The interior fill of the acequia contained caliche gravels and caliche soil mixed. No cultural artifacts were observed in Trench A.
T~eneh B
Trench B was placed on an improved extension of Calvert Street. The trench was oriented north and south, in anticipation of the acequia's path between Trenches 7 and 11 (Fig. 2). The south end of Trench B was begun 95 feet from W. Johnson Street and excavated to a length of 48 feet. The trench was dug to a depth that revealed natural caliche. Both walls of the backhoe trench were cleaned to observe the characteristics of the acequia (Fig. 4). The acequia is approximately 8 feet in width with a depth of 3 feet. A cedar post was noted on the north half of the profile, and a cedar plank was noted on the south half of the profile. The cedar post and board suggested the past construction of a retaining wall along both banks of the acequia.
The acequia appears to have been dug through two layers of natural occurring soils. The top layer is 24 inches thick and grades from a black to a dark brown clay loam. This is overlying a lighter brown clay loam about 12 inches thick. As noted in the earlier trenches of Phase I, the thicknesses vary due to the gradation of the soils. The brown clayey loam overlies a white caliche layer on which the acequia bottom also rests. The interior of the acequia is surfaced by a caliche fill with limestone chips associated with the gravel road. Beneath this layer lies what appears to be intentional fill of yellow-brown gravels, with a maximum thickness of 18 inches. Beneath this layer was a dark clay loam with numerous pieces of charcoal and cultural materials. The maximum thickness of this cultural layer was about 12 inches. The deposit that lies beneath the cultural zone consisted of a silty clay loam with gravels. No cultural debris was noted in this level.
T~eneh C Trench C was located approximately 18 feet from Trench B (Fig. 2). The southernmost point of Trench C is 145 feet north of W. Johnson Street. The trench was oriented north and south with a total length of 45 feet. The west wall profile was cleaned and recorded (Fig. 5). The acequia in the profile was approximately 30 feet wide; however, this large width was due to the oblique angle in which the trench was excavated into the acequia's path. At the north end of the trench was a large amount of fractured limestone and a few pieces of these stones contained chisel marks on one face. This might suggest that in this area a junction was made of the stone-lined portion of the acequia and the original unlined portion.
The acequia at this point was excavated through two clayey loam deposits. These loam deposits are described in Trench B and represent the natural stratigraphy throughout the investigated area, unless otherwise mentioned. The acequia1s interior fill consisted of caliche and limestone chips in the top layer. Beneath this layer in the northern portion is a black ashy clay loam with fragments of limestone block and other cultural materials. A large concrete pad superficially separated the two acequia sections. The unlined portion of the acequia underlies the road surface with 12 inches of black clayey soil containing chunks and pieces of asphalt. This layer overlies and intrudes into a yellow-brown soil with caliche gravels. This 6-inch gravel layer rests upon a dark brown to black clay loam with charcoal pieces and some cultural artifacts. The oblique angle of the cut gives the appearance of a very thick layer containing cultural material. It is possible that the profile represents a thinner cultural layer that was deposited on the bank of the acequia. The bank was later excavated by the backhoe. Beneath this cultural zone was a layer of dark soils with a heavy concentration of charcoal pieces and land snail shells. No cultural materials were observed in this layer.
T~enQh V Trench 0 was located on the east side of Calvert Street approximately 50 feet east of Trench C. The southern end of Trench 0 is approximately 160 feet north of W. Johnson Street. The trench was oriented north and south and was excavated to a depth of 3 feet and a length of 25 feet. The acequia had cut through two clay loam layers (Fig. 6), and the bottom layer rested upon the caliche bedrock. The width of the acequia measures 13 feet. Again, the angle of the trench cut made the profiled width wider than the actual ditch width. The interior stratigraphy contained ten distinct layers of fill. The surface layer was a reddish brown gravel occasionally topped with a thin lens of topsoil and grass. Beneath this initial layer was 12 inches of varying gravels. The gravel layers consisted of a yellowish brown gravel, a layer of black clay and gravel mix, and a similar layer of yellowish brown gravel. Beneath these gravel layers was a layer of dark soil with charcoal and snail shells. Within this 18-inch layer, household artifacts were recovered. Beneath this layer was a thin deposit of silt and rusted iron. The final two layers, or the first two depositional layers, are a black clay with caliche pebbles and a 3-inch layer of black silt with a heavy concentration of charcoal and land snail shells. The charcoal and shell concentration is a fairly common occurrence on the bottom of the acequia. A cedar plank found seems to divide two of the largest acequia deposits. The deposit on the north side was described above. The 12-inch deposit on the south side is a gray-black ashy soil. This layer rests upon the charcoal and snail shell concentration level that runs continuous beneath the cedar plank. Across from the cedar plank, a post hole was encountered on the north bank of the acequia. The cedar plank with a distinct soil deposit on each side indicates that a retaining wall was in the acequia at this point. The post hole along with the plank suggests a structure contemporary with a similar feature located in Trench B. Trench E was excavated 10 feet east of Trench 11. The trench was placed on the outside edge of the north wall of the acequia (Fig. 2). The south wall of the backhoe trench was cleaned to expose the exterior block work of the acequia. The north wall of the backhoe trench was inspected for traces of the original unlined acequia. The unlined acequia route was projected from the profiles in Trenches B, C and D to intercept the stone-lined portion in this area. The exterior face of the acequia was roughly cut and unlike the dressed face of the interior. The north wall of the backhoe trench showed no sign of a ditch profile. The artifacts from this trench were found to occur along the outside fill of the stone-lined acequia.
T~eneh F 19 Trench F was excavated inside the stone-lined acequia parallel with Trench E (Fig. 2). The length of the trench measures 10 feet from Trench 11 to a concrete building foundation under which the acequia continues. Portions of the trench were excavated to basal caliche at approximately 3 feet in depth (Figs. 7, ll,a). The interior face of the acequia was brushed clean and it was noted that the face was chiseled from top to bottom with a light brown sandy mortar between the blocks. The acequia interior fill was a homogeneous caliche soil with cultural artifacts. A thin layer of gray silt with a few snail shells and traces of charcoal lie beneath. Noted within this caliche fill were large fragments of limestone slabs. These slabs suggested that the stone-lined acequia was covered at one time. The deposit suggests that the acequia was intentionally filled with caliche and debris.
T~enehe~ G and H
Trenches G and H were placed along the south and east perimeter of the concrete building foundation (Fig. 2). These trenches were approximately 2-1/2 to 3 feet in length. The objective was to verify the stone-lined acequia route, but the backhoe trenches east of Calvert Street showed no signs of the stone-lined acequia continuing in an east and west direction. Therefore, the stone-lined acequia route must continue north along the Calvert Street extension and make a 90 0 turn to the east where it was observed in Trench C. No artifacts were recovered from the two short exploratory trenches.
Trench I was located approximately 3 feet from the west end of Trench E (Fig. 2). The trench was oriented north and south with the southern end beginning on the north wall of the stone-lined acequia. The objective was to locate the unlined portion, but the negative results in Trench E might indicate that the unlined portion is probably farther north. The trench was excavated to a length of 26 feet and a depth of 4-1/2 feet (Fig. 8). The center of the unlined acequia was found to be 15 feet 8 inches from the stone-lined portion. The profile of Trench I depicts two separate ditching operations within the original acequia. The acequia was dug through two natural clay loam levels and the bottom of the acequia rested upon the caliche bedrock. Above the acequia was a 6-8 inch layer of yellow caliche underneath a 4-6 inch layer of yellow gravels.
These layers were surfaced by a thin layer of asphalt and topsoil. The interior stratigraphy of the acequia consisted of five layers. The bottom layer was a thin deposit of silt with a concentration of charcoal and snail shells. Above the silt layer was a thin layer of caliche similar to the basal caliche. Rested upon the caliche layer was a 16-inch layer of a grayish brown soil. This layer contained few artifacts. Above the grayish brown soil was a 13-inch layer of gray-black clay soil void of any artifacts. Resting upon the clay was a thin layer of fine grain caliche similar to the basal caliche. Before this thin layer of caliche was placed upon the gray-black clay soil. a ditch approximately 30 inches deep was dug on the north bank of the acequia. The soil inside the ditch consisted of a gray clay with pockets of charred refuse and charcoal. Artifacts observed in this layer date from the middle twentieth century. Above this ditch a shallow trench 1-1/2 feet in depth was dug after the layer of yellow caliche was laid down. This shallow trench was filled with a 7-inch layer of pea-sized gravel topped with a layer of brown soil mixed with rubbish. The small drain uncovered just to the north suggests that the shallow trench was used as a lateral to disperse water underground.
Tn~nQh ]
Trench J is an east-west backhoe trench. The trench was located between Trench 9 and Trench 3 (Fig. 2). The east end of the trench is approximately 120 feet west of Main Avenue, and 105 feet south of W. Sheridan Street. The trench was dug to a length of 123 feet and an average depth of 4 feet. The center of the acequia was located 33 feet from the east end of the trench (Fig. 8). The acequia was dug through two layers of clay loam ranging in color from a dark brown at the surface to a medium brown next to the basal caliche. The acequia measures an unusual 15 feet in width and approximately 30 inches deep. The unusual width is probably due to erosion of the banks and the angle of the trench cut.
The soil stratigraphy inside of the acequia consisted of three major layers of deposits. The bottom layer was a brown soil with a molded gray coloriDq mixed in. Fragments of land snail shells were noted close to the bottom of the level. A 15-inch gray clayey soil layer with pieces of charcoal rested upon the base deposit. The majority of the artifacts observed were collected from within this gray clay. Three large fragments of limestone containing chisel marks were also observed in this layer. Above this layer was a deposit of gray-black clay soil approximately 7 inches thick. This layer contained no cultural artifacts. Above this was a 4-inch layer of black clay loam, a thin layer of yellow gravels, and a surface layer of topsoil with grass.
Tn~nQh K Trench K was located on the south edge of W. Johnson Street and 25 feet east of S. Flores Street (Fig. 2). This trench was excavated to a length of 25 feet and a depth of approximately 10 inches. The probability of utility lines in the area determined the trench depth. A concrete cap was encountered 46 feet from S. Flores Street. The cap appeared to cover the stone-lined acequia and was poured into a wooden form approximately 3 feet wide. The length was not determined because of W. Johnson Street to the north and a sidewalk and building foundation to the south.
Tne.nc.h L
Trench L was designed to transect the acequia route east and west. The east end of Trench L was located 62 feet east of S. Flores Street and 86 feet from W. Johnson Street (Fig. 2). Trench L was excavated to a length of 8 feet, terminating against a brick foundation and the foundation was found to continue 3 feet below the surface. The trench was located between two large concrete slabs. The materials unearthed included mortar and brick.
Tne.nc.h M
Trench M was located east of Trench L approximately 82 feet south of W. Johnson Street and was parallel to a gravel alley (Fig. 2). The trench was excavated north to south for a distance of 20 feet, with a depth of 3 feet. The acequia's center was located approximately 98 feet south of W. Johnson. The acequia measured 8 feet in width and approximately 2 feet in depth (Fig. 9). The route of the acequia at this point ran east-west. The acequia was dug through two layers of clayey loam ranging in color from a black to a brown before the basal caliche was encountered. It appears that the natural soil layers observed north of W. Johnson Street holds true for the soils on the south side of W. Johnson Street .
. There were three principal layers of deposition in the acequia. The primary deposit which rests upon basal caliche was composed of a silty loam a few inches thick with a concentration of charcoal and snail shells. Deposited on top of the bottom layer was 18 inches of dark soils intermixed with traces of charcoal and snail shells. It was within this layer that the majority of the cultural debris was observed. The final deposit consisted of three inches of caliche. A brown topsoil covers the area excavated by the trench.
Tne.nc.h N
Trench N was located between Trench M and Trench L in order to verify the acequia's location (Fig. 2). The trench was excavated to a length of 26 feet and a depth of approximately 4-1/2 feet. Unlike the previous trench, the acequia did not rest upon the caliche bedrock. It was, however, dug through the two familiar layers of clay loam. The acequia measured 10-1/2 feet in width and 24 inches in depth (Fig. 9).
The soils inside the acequia were made up of three major layers. The bottom and first layer measured 13 inches. This layer consisted of a dark soil intermixed with charcoal and snail shell. A gray ashy soil 10 inches thick rested upon the previous layer and together these first two layers held the few artifacts observed in this trench. The third layer consisted of a black clay. An 8-inch layer of yellow gravels and a thin layer of brown topsoil made up the
BX 337
remaining strata overlying the acequia. A shallow trench to the north was profiled, but no artifacts were observed.
TILe.nc..h 0
Trench 0 was located south of Trench M (Fig. 2). This trench was located 196 feet south of W. Johnson Street and measured 20 feet in length and ca. 39 inches in depth. The profile of the acequia within the trench was 6-1/2 feet wide and 29 inches deep (Fig. 10). The acequia had been dug through the natural strata mentioned in Trench M; the bottom of the acequia rested upon caliche bedrock.
The interior fill of the acequia consisted of three deposits. The bottom deposit was a thin concentration of snail shells and charcoal mixed with dark brown clay. A very thin layer of rusted metal covered this bottom layer. A loose layer of black loam 26 inches thick rested upon the bottom layer. Within this loose fill there were cultural artifacts, limestone rock, and small pockets of caliche. A thin layer of yellow gravels rested over the cultural layer. Four inches of grayish black clay, another 4 inches of loose sandy soil and mortar, and a thin layer of topsoil with grass made up the remaining accumulations overlying the acequia.
TILe.nc..h P Trench P was located southwest of Trench 0 (Fig. 2) and was excavated in a west to east direction. If Trenches M, Nand 0 represented a rerouting ditch, then Trench P should cut across the original acequia. The trench was excavated to a length of 38 feet and a depth of 70 inches. The acequia was uncovered on the east end of the trench and was located approximately 77 feet north of W. Guenther Street and 110 feet east of S. Flores Street. The acequia profile in the trench measures 18 feet in width and 23 inches in depth (Fig. 10). The oblique angle of the trench cut to the acequia route, and the badly eroded banks caused the appearance of a large width. The acequia was cut through two layers of clay loam that rested upon a basal caliche. The description of the soils were detailed in earlier trenches.
The stratigraphy inside of the acequia represents five distinguished layers of deposition. The initial deposit that lies on the bottom was a thin layer of charcoal and snail shells. Above this layer was an 8-inch layer of dark soil containing cultural artifacts. The soil was a dark clay loam with a loose consistency, and small snail shell fragments could be seen within this level. Upon this layer were two caliche layers that measured a total of 3 inches. A grayto-black clayey soil level that measured 10 inches in depth and a 31-inch loose sandy soil level were the final two layers of soil accumulation over the acequia.
TILe.nc..h 12 Trench Q was located inside the stone-lined acequia between Trenches 11 and 12 (Fig. 2) (Fig. ll,b), and it was noted that the stone masonry was a different style. A collection of small limestone blocks with occasional clay bricks were mortared together in different localities along the top; this later work could suggest that the wall height was raised to accommodate a higher water level.
The surface overburden consisted of a layer of asphalt over a caliche fill.
Other portions of the trench were covered with topsoil and grass. The west end of Trench Q uncovered a disturbed area. This disturbance displaced some of the limestone block on both sides of the acequia. It is very likely that a sewer line was trenched through the acequia walls at this point. Artifacts that were selected from the surface indicated that the surface had not been exposed for any great length of time before a covering was placed over it.
TfLC!-VLC.h R Trench R was located at the stone-lined portion south from Trench 12 to a distance of 20 feet (Fig. 2). The trench area was covered with a layer of asphalt and caliche and is presently used as a parking lot. The backhoe was used to clear this heavy overburden which averaged 9 inches in thickness. The remaining few inches above the acequia stones were cleaned by hand and the surfaces of the acequia walls were brushed clean and photographed. The masonry work consisted of large limestone blocks and each block contained chisel marks on the face. The few artifacts collected were contemporary in date with those found on the surface in Trench Q.
THE ARTIFACTS
All of the materials recovered in the field from Phase I and Phase II excavations were removed to the laboratory for analysis. The specimens were cleaned, identified, described, classified and cataloged. The artifacts are presented by categories (ceramic, glass, metal, etc.) and the quantities collected are indicated. They are listed according to the trench in which they were found. Selected artifacts are illustrated in Figures 12 to 18. All materials are stored at the Center for Archaeological Research laboratoryo One of the most useful tools the archaeologist has for dating historic sites is the pottery recovered during the investigations. In addition to the form, decoration and paste, a potter's mark (Fig. 17) is very useful for identification and dating. Many of the identified pottery wares described below have potter's marks to indicate that they were made here or were imported from Europe, mostly from England.
Other artifacts used by the archaeologists for dating are glass bottles. Soda water bottles of the late 19th century, for example, frequently had the name and address of the bottle maker embossed on the side or bottom ( Fig. 12 and Fig. 16a,b). By using an old city directory, the approximate dates of when the business was established and when it was terminated can often be determined. A bottle found in an old refuse deposit, which can be identified by the markings, was probably discarded during the time the bottle maker was in business, thus giving an approximate date for the bottle and the associated debris. Other bottle indicators useful for dating include the color of the glass and the seam marks. The earliest bottles were hand-blown. Later the bottles were formed in a mold and the neck and mouth applied by hand. Eventually the entire bottle was molded in one operation. With the development of the molds, seam marks changed in length through time, providing additional indicators which are datable.
There is a great deal of literature on the subject of old pottery and glass available for the archaeologist to refer to during analysis, and many of the artifacts described here were identified by referring to the literature. Much of the pottery and glass, and many other artifacts, have been identified by Anne A. Fox, who has kindly introduced the author to the subject literature. Metal 1 jug base fragment with Bristol glaze exterior and Albany slip interior 1 rim sherd Rockingham glaze exterior 1 jug base fragment with salt glaze exterior and brown glaze interior 1 circular base of a stemmed glass 2 green bottl es with "wittl e marks II and embossed 1 etteri ng on the base. SAXLEHNERS / BITTERQUELLE / HUNYADI / JANOS / (Fig. 13,d) and (Fig. l6,c). Andres Saxlehner was an interesting figure in history whom the bitter bottle was made to honor. Toulouse (1971:257) gives the dates 1863 to ca. 1900 when these bottles were made. (Munsey 1970:135). Edwards (1971 :103) wrote that ink bottles of J. Bourne were made from 1833-1861.
CONCLUSIONS
In the course of archaeological investigations, both in Phases I and II, a total of 31 trenches were dug on GSA property. Of the 31 trenches, 21 were found to contain traces of the San Pedro Acequia. The profiles described in this report suggest that three major periods of construction or modification are represented.
The first period of acequia construction is represented by an unlined ditch. This is supported by the profiles observed in Trenches 1, 3, J, 7, D, C, B, I, and P. These trenches trace the acequia as it enters the GSA property at W. The majority of support in determining the periods of construction was derived from historical documents, ethnographic accounts, and maps or photographs. The artifactual material representing the earliest age of the acequia is very limited. The lack of 18th century artifacts can possibly be explained by the early historical accounts. We know that the area in the 18th century consisted of open fields, located away from the main area of population and activity. Additionally, it is known that the acequias were periodically cleaned.
When the population pushed south of Main Plaza, the need for control over the. water of the acequia became greater. Improvements on the acequia were continuous. Small feeder channels to irrigate an individual's land were added when needed. The improvements for a more maintenance-free water system can be seen by the cedar post and plank support structures. These structures were probably placed throughout the acequia wherever they were needed to slow down the erosion of its banks. As the population intensified, the need for flood control and reclamation of the land became more important. The construction of limestone block channel walls improved the water distribution. The block construction is represented by the evidence uncovered in Trenches A, C, E, F, G, H, K, Q, and 12. The route of the stone-lined portion appears to begin in Trench C, where it joins with the unlined portion. The stone-lined portion runs east to the west side of the Calvert Street extension. At this point it appears to make a 90 0 turn east and travels ca. 85 feet, where it begins to make a southeast curve to a point 45 feet west of S. Flores Street and W. Johnson Street. The stone-lined portion is probably occupying the same trench dug for the original acequia. Trench K on the south side of W. Johnson Street reveals what looks like the south end of the limestone block construction.
The path of the stone-lined portion of the acequia gives the appearance of following certain property lines. This would suggest that the rerouting of the acequia was done to develop the area for residential or business occupation. This can be observed by the remains of concrete building foundations and asphalt paving in the area. Artifacts usually associated with the household (i.e., kitchen refuse) represented the vast majority of the items recovered. An advertisement sign for used cars nearby and the numerous old automobile parts observed in the acequia indicate that a used car business occupied this area at one time. The occurrence of what appears to be large fragments of limestone slabs suggests that they were -~----u seaasacover, ng-o verThe-sIone-1 fnedpo rt i 6n-:~~-H i-sto rTc-af!'.)', we~know--tha t -'the--~'~--'~--'~' acequia was covered in the areas of heavy pedestrian traffic. This covering would prevent the type of artifact deposition which would occur in an open ditch. In other words, at the termination of the acequia1s usage, the open acequia would accumulate soil and refuse first. The covered portion would stay protected until eventually or intentionally the covering was removed and the void filled. Tentatively, the artifacts support the sequence of the acequia modifications in this area. The artifacts from the trenches representing the reclaimed land are late 19th century, whereas the artifacts from the stone-lined portion suggest an early 20th century continuous deposit.
The final construction period is represented by the trenching activity south of W. Johnson Street. This acequia is a dirt-lined construction rerouted from the original acequia route. Trenches M, N, and 0 reveal the existence of the rerouted portion. The concrete foundations suggest that the land was reclaimed for building purposes. The rerouted ditch appears to intersect the original acequia 90 feet south of W. Johnson Street. From this point the main acequia travels east for ca. 100 feet. The ditch made a 90 0 turn south and paralleled an unmarked alley for ca. 240 feet, where it rejoined the original path of the San Pedro Acequia. The original acequia route, which was filled to reclaim the land, should contain artifacts older than those accumulated in the rerouted portion. Unfortunately, the quantity of artifacts observed and collected in this area is insufficient to determine any more than a tenuous sequence of dates for the two activities.
The time span represented by the acequia modifications is relatively short. The stone-lined construction is estimated to have been built about the same time as the rerouted unlined ditch south of W. Johnson Street. These constructions or modifications are estimated to have been finished at the turn of the 19th century. The San Pedro Acequia was closed in 1912 and was filled in about 10 years later. Specifically, the exact time each was filled was determined by the current necessity.
The San Pedro Acequia in the area of S. Flores Street and W. Johnson Street is documented to have a lateral, or branch. This branch is to have routed water to fields east of the San Pedro Creek and west of the main acequia. No evidence of this lateral was uncovered in the course of investigations. The actual junction is suspected to lie underneath W. Johnson Street. The possibility of any such evidence south of W. Johnson Street is slim, since that portion was probably eliminated by the water main trenching paralleling S. Flores Street.
RECOMMENDATIONS
Historical research and archaeological excavations revealed that the original 18th century San Pedro Acequia traversed the GSA property ( Fig .. 2), and it has been recognized to have undergone two additional periods of modifications. The original route of the acequia is the most difficult segment to identify on the property since the width of the original ditch was found to be inconsistent due to its earthen construction and many subsequent alterations through maintenance.
The first period of acequia modification was the added stone-lined portion which was constructed to divert water from the original route. The dressed limestone block masonry reflects a construction tradition that was typical of the work done throughout San Antonio in the late 19th and early 20th centuries. This portion of the acequia is relatively well preserved; however, there are a few disturbed areas.
The second modification was the rerouting of the acequia below W. Johnson Street (Fig. 2). This rerouting is estimated to have been done about the same time as the installation of the stone-lined portion. The difference is that the southern rerouted portion is of earthen construction and was exposed for a relatively shorter time. Therefore, the route indicated in Figure 2 serves as the location of the archaeologically sensitive area along this portion. Any future land development should allow for adequate clearance on each side of the acequia.
As mentioned in the text, the GSA property is partly covered with concrete building foundations and asphalt. Some of the structural remains appear to have no basements and the removal of the foundations should be done with caution so as not to destroy the acequia that may lie beneath. It would be advantageous to the contractor if an archaeologist were to collaborate when the foundations were removed. In addition, any ground disturbance outside the sensitive areas of the acequia should be examined by an archaeologist. The possibility exists for small feeder channels or laterals in addition to late 18th century house remains that might still exist for this area.
After reaching the end of its long and productive use, the San Pedro Acequia was buried. This has preserved the acequia's physical features, along with the cultural artifacts of its time, and a wealth of archaeological information still remains stored within the accumulation of soils inside the acequia.
ACKNOWLEDGMENTS
Numerous people helped to bring this project to a successful conclusion. The author expresses his thanks to the following persons. Dr. Thomas R. Hester, Director of the UTSA Center for Archaeological Research, served as Principal Investigator. Jack Eaton, Associate Director and Co-Principal Investigator, provided general supervision and aid in assembling the manuscript. Anne Fox and Jake Ivey provided assistance and advice during the artifact analysis and manuscript preparation. Carmen Perry of the John Peace Library at The University of Texas at San Antonio assisted in locating out-of-print books valuable for historical research. Guadalupe Gonzales shared his valuable time in showing me old surveying notes and plates, volumes of translated manuscrtpts, and many other archival documents. The staff working in the City Clerk's room helped in locating volumes of city council minutes. Manuel Cantu, of Cantu's Hauling Service, provided a transit and stadia free of charge, for locating and mapping all excavations and acequia positions. Blackie's Backhoe Service provided a backhoe with an operator whose precision made a hand-held shovel look slow and clumsy. | v3-fos |
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} | s2 | Serum and liver cholesterol levels of rats and mice fed soy-bean protein or casein.
Rats and mice were fed soy-bean protein or casein diets for 10 and 50 weeks, respectively, during which terms their serum cholesterol levels were analyzed periodically. Rats fed high-cholesterol diets containing soy protein or the amino acid mixture simulating soy protein produced lower levels of serum cholesterol throughout the experiments, as compared with those on the corresponding casein-type diets. Feeding soy protein resulted in a significant decrease in serum apoA-I and apoB, but the relative concentration of high-density lipoprotein-cholesterol was kept at the higher level. The concentration of liver cholesterol was also lower in rats fed the plant protein. In mice fed a cholesterol-free diet, the cholesterol-lowering effect of soy protein was noticeable at an early stage of the feeding periods, by 20 weeks. The extent of lipid peroxidation in rats and mice determined as TBA-reactive substances in serum was found to be the same when protein diets were given, while it was significantly higher when an amino acid mixture of the soy protein type was fed to rats. The results confirm that soy protein exhibits its hypocholesterolemic effect even when a diet rich in cholesterol is fed. The cholesterol-lowering effect of soy proteins appears to be a phenomenon common to rodents.
lower levels of serum cholesterol throughout the experiments, as com pared with those on the corresponding casein-type diets. Feeding soy protein resulted in a significant decrease in serum apoA-I and apoB, but the relative concentration of high-density lipoprotein-cholesterol was kept at the higher level. The concentration of liver cholesterol was also lower in rats fed the plant protein.
In mice fed a cholesterol-free diet, the cholesterol-lowering effect of soy protein was noticeable at an early stage of the feeding periods, by 20 weeks. The extent of lipid peroxidation in rats and mice determined as TBA-reactive substances in serum was found to be the same when protein diets were given, while it was significantly higher when an amino acid mixture of the soy protein type was fed to rats. The results confirm that soy protein exhibits its hypocholesterolemic effect even when a diet rich in cholesterol is fed. The cholesterol-lowering effect of soy proteins appears to be a phenomenon common to rodents. Key Words soy-bean protein, casein, serum cholesterol, apolipoproteins, lipid peroxidation Dietary protein has been found to have a significant effect on serum cholesterol levels in humans as well as experimental animals. In numerous studies that provided protein-dependent changes in serum cholesterol, comparisons have been made exclusively between animal and plant proteins, particularly casein and soy-bean protein, respectively (1)(2)(3)(4)(5)(6)(7)(8)(9)(10). In general, soy protein produces lower levels of serum cholesterol than casein.
The antihypercholesterolemic effect of soy protein, however, appears to be modified by the level and type of dietary fats (6,11). As reported previously (6), the cholesterol-lowering effect of soy protein in rats as compared with casein was most marked when cholesterol-free low-fat diets were fed. Yadav and Liener (10) showed, utilizing high-cholesterol diets, that in rats soy protein produced serum cholesterol levels which were significantly lower than when casein was fed, but the evaluation of this result was indeed restricted due to an insufficient supply of essential fatty acids in their diets. In our preceding trials with rats (6), addition of cholesterol to the diet markedly obscured the response of serum cholesterol to dietary regimens. Kim et al. (8,9) fed swine a high-fat, high-cholesterol diet and observed that animals on soy protein had a lower serum cholesterol as compared to those on casein. Sirtori et al. (3) also provided evidence that in type II hyperlipoproteinemic patients vegetable protein exerted a cholesterol-lowering effect relatively inde pendent of the content of fats and cholesterol in diets.
The aim of the present study was to confirm the hypocholesterolemic action of soy protein in rats in the presence of dietary cholesterol. In addition, in order to know the species-specific difference in response to the type of dietary proteins, the effects of soy-bean protein and casein on the serum cholesterol level of mice was investigated. In the course of these studies, the concentration of serum apolipo proteins and the extent of lipoperoxidation were also measured.
RESULTS
Experiments with rats on cholesterol-enriched diets Table 1 shows weight gain, food intake, liver weight and the concentration of serum and liver glycerolipids. In each experiment there were no differences found in growth and food intake in relation to the type of dietary proteins or amino acid mixtures. The liver weight was significantly heavier on feeding casein than on soy protein. Serum phospholipid and liver triglyceride were significantly higher in rats fed casein, while no differences were observed when the amino acid mixture diets were fed.
The concentration of serum cholesterol in the course of 10 weeks of feeding is illustrated in Fig. 1. When intact protein was used as a nitrogen source (experiment 1, Fig. la), the casein group showed significantly higher levels of serum cholesterol throughout the experiments. The difference was evident even after 2 weeks of feeding. In contrast to the moderate progressive decrease in serum cholesterol of rats fed soy protein with time, the time course of serum cholesterol in rats fed casein varied considerably at higher levels. In experiment 2, where amino acid mixtures replaced intact proteins, serum levels of cholesterol of rats given the mixture simulating soy-bean protein was virtually invariable and was slightly but con sistently lower than those of the animals fed the casein-type amino acid mixture (Fig. lb). However, a significant difference was observed only after 10 weeks.
The serum concentrations of cholesterol in lipoproteins and of apolipoproteins are summarized in Table 2 together with those of TBA-reactive substances. Although the concentration of HDL-cholesterol remained unchanged after feeding different proteins, the ratio of HDL to total cholesterol was significantly higher in the soy protein group than in the casein group, since soy protein reduced serum cholesterol levels markedly. Similar, but somewhat moderate responses were demonstrated when the amino acid mixture diet was fed. The difference in type of dietary proteins greatly influenced the content of serum apoA-I and apoB. Feeding soy protein caused significantly lower levels of these apolipoproteins. The extent of lipoperoxidation was apparently the same when protein diets were fed, but it was more prominent when the amino acid mixture of the soy protein-type was the die tary source of nitrogen. Table 3 shows the concentration of cholesterol in the liver and adipose tissue. Casein and its amino acid mixture caused a significantly greater accumulation of cholesterol in the liver as compared with the corresponding soy protein groups. There was no marked difference in the cholesterol content of adipose tissue between the two dietary regimens.
Experiment with mice on cholesterol free diets
As shown in Table 4, food intake, weight gain and liver weight were essentially the same for mice fed a low-fat cholesterol-free casein or soy protein diet. The concentration of liver cholesterol was significantly higher in mice fed casein than in those fed soy-bean protein.
The changes in serum cholesterol and TBA-reactive substances are illustrated in Fig. 2 as a function of feeding periods. Casein caused a significant elevation of serum cholesterol as compared with soy protein during the initial 20 weeks, while at
DISCUSSION
Although our previous experiments with rats fed a 1 % cholesterol diet failed to demonstrate a definite hypocholesterolemic effect of soy-bean protein and its amino acid mixture as compared with the corresponding casein diets (6), the present study where the dietary level of cholesterol was reduced to one-half that of the previous trials, 0.5%, clearly showed a dietary protein-dependent difference in serum cholesterol, thus soy protein exhibiting its cholesterol-lowering effect as in the case of cholesterol-free diets. It is therefore likely that the large excess of dietary cholesterol mitigates the effect of dietary protein on serum cholesterol. Kim et al. (8,9) and Forsythe et al. (20) showed the hypocholesterolemic action of soy protein in swine fed a high-cholesterol diet. The difference in serum cholesterol level was more marked with the intact protein diet (experiment 1) than with the amino acid mixture diet (experiment 2), indicating that the cholesterol-lowering action may not be solely ascribed to the difference in amino acid composition, but partially relates to non-proteinous materials occurring (less than 10%) in the soy-bean protein preparation (21). However, the difference in the rate of absorption of individual amino acids when proteins or amino acid mixtures were fed should be taken into consideration.
When diets free of cholesterol were given (7), both VLDL-plus LDL -cholesterol and HDL-cholesterol decreased proportionally in rats on the soy protein diet, as compared with those on the casein diet, whereas, as observed in the present study, addition of cholesterol to the diet reduced VLDL-plus LDL-cholesterol but not the HDL-counterpart. Thus, the ratio of HDL-to total cholesterol was markedly higher in those on soy protein, showing the reduction of the so-called "atherosclerosis index ." This was in essence comparable with the result obtained after feeding diets free of cholesterol; the ratio remained unchanged or increased moderately (7). These changes in the distribution of cholesterol in relation to dietary proteins obviously differed from those in humans (3) and rabbits (22), probably due to the difference in the distribution of cholesterol in serum lipoproteins in rats and these species. The mechanism responsible for causing these changes in the distribution of cholesterol in serum lipoproteins is not apparent. Kritchevsky and his colleague directed their attention to the role of arginine in the level of serum cholesterol (23). Eklund and Sjoblom (24) recently showed a significant negative correlation between the level of dietary arginine and serum VLDL-plus LDL-cholesterol levels in female rats fed a cholesterol-free diet. Soy protein contains approximately twice as much arginine than casein. However, the situation seems not to be so simple as to be attributable to the difference in the content of a single amino acid, arginine in this case, since the responsibility of this amino acid was not fully supported in male rats (7). Katan et al. (25) suggested the role of glycine in the hypocholesterolemic action of soy protein. Huff and Carroll (26) claimed the importance of interaction between essential and non-essential amino acids in determining plasma cholesterol.
The concentration of serum apoA-I and apoB was also altered. When rats were fed a soy protein diet free of cholesterol, serum apoA-I decreased significantly but apoB increased (6,7). Feeding a soy protein diet containing high cholesterol resulted in the decreased level of both apoA-I and apoB. Although the decrease in apoB when cholesterol was included in the diet well paralleled that of VLDL plus LDL cholesterol, the reduction of apoA-I was not consistent with the response of HDL -cholesterol which was essentially unchanged by soy protein. The reduction of circulating apoA-I on cholesterol-free soy protein diets at least partly results from a decreased production of this apolipoprotein in the intestine (unpublished obser vation), while the rise of apoB is perhaps due to increased hepatic production. Since cholesterol feeding causes a significant reduction of hepatic cholesterogenesis, different responses are possible in the levels of circulating apoB in rats fed diets with or without cholesterol. The antihypercholesterolemic effect of soy-bean protein was for the first time demonstrated in mice. However, the effect of dietary protein on serum cholesterol was restricted only to the relatively younger stage and it was not clear when mice became older. This age-related response to dietary protein seems to be partly relevant to the failure to observe the rise in serum cholesterol with age, particularly in rats fed casein.
Recently, the peroxidation reaction has attracted attention in relation to several prevalent diseases as well as aging (27). Though the lipoperoxide level continued to rise with age, the quality of dietary protein showed no different effect in mice. The same was apparently true for rats fed protein diets, while there was a moderate increase in the concentration of TBA-reactive substances in the serum of rats fed the soy protein-type amino acid mixture diet. Of course we measured only at one selected interval with rats, but this observation did not agree with that found in rats fed a cholesterol-free diet, where no protein-dependent difference could be seen (7). Harman (28) suggested that the type of dietary proteins might have a close connection to the level of free radical reaction which is responsible for lipid peroxidation. Anyhow, a more detailed study needs to be done in this respect.
In conclusion, soy protein, as compared with casein, was less hypercholestero lemic to rats even when a high-cholesterol diet was fed. With a low-fat cholesterol free diet, the cholesterol-lowering effect of soy protein was also evident in mice. Available information strongly suggests that the hypocholesterolemic effect of soy protein is the phenomenon commonly observable in the rodents so far examined, i.e., rabbits, rats and mice.
The authors are grateful for the skilful technical assistance of Mr. N. Ishiwaki and Mr.
T. Zaizen. Amino acids and soy protein isolate were the generous gift of Ajinomoto Co., Tokyo and Fuji Oil Co., Osaka, respectively. This work was supported by a Grant-in-Aid for Special Project Research (No. 412008) from the Ministry of Education, Science and Culture of Japan. | v3-fos |
2018-04-03T04:38:09.791Z | {
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} | 0 | [] | 1981-06-01T00:00:00.000Z | 39749223 | {
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} | s2 | Analysis of inorganic fiber concentrations in biological samples by scanning electron microscopy.
in by scanning electron microscopy. j work 7 (1981) 101-108. Analyzing fibers with electron microscopic techniques involves several preparation steps, especially during the analysis of fibers in human tissue. The influ ence of these steps on the analytical result was studied in detail. Fiber number was unaffected by the mild sonication of fiber suspensions analyzed with scanning electron microscopy. Significant fiber losses appeared during the filtration of fiber suspensions through 0.8-flm pore-size Nuclepore membranes. Shrinkage of the tissue during dry ashing broke long fibers and consequently increased the fiber concentration and affected the fiber length distribution. Dry ashing, however, removed more of the organic debris than wet ashing; thus specimens prepared with dry ashing were more suitable for scanning electron microscopic analysis. A fairly good correlation was demonstrated for the analysis of fibers with scanning and transmission electron micro scopy.
The assessment of airborne inorganic fiber concentration has tradi1ti.onally been performed by optical phase-contrast microscopy. For a Ibetter identification and quantification Of inorganic fibers, electron microscopy (EM), inclUlding scanning (SEM), transmission (TEM), and se,anningi transmission (STEM) electron microscopes fi'libed with X-ray and electron microanalytical equipment, has recently been introduced.
These techniques are also used for the analysis of inorganic fibers in human autopsy matedal, especially !lung tissue. Different microscopk methods often reqmre different preparation of the tissue samples. In a recent paper by Morgan & Holmes (16) different aspects of the preparation and analysis of lung tissue are discussed. They emphasize tha't drying, 1 low-temperature ashing of the tissue and ultrasonic dispersion of the ash are contramdicated. Such teclmiques have, on the o1fuer hand, previlously -been used by other authors (3,4,7). Different preparation techniques have, however, not been thoroughly compared.
In the present investigation we have studied 'the influeIliCe of different preparation steps on inorganic fiber concentration as measured by SEM.
Material and methods
The different prepamtion steps which may lead to serious ftber losses have been tested on Union Internationale Contre Le Oancer (UICC) standard crocidoli'te as-beStos and on fibers from autopsy material of subjects who died of asbestos-related diseases.
All fibers with an aspect ratio equal to or greater than 3:1 were counted. Fiber numbers were evaluated at a magnifioation of 4,500 X iJn a Jeol J'SM-35 SEM, and in each specimen either 100 fibers or the 0355-3140/81/020101-8 fibers in 200 view fields were counted. The calculation of :relative standard deviations (RSD) were /based on four samples.
All liquids ·used in 1Jhe analyses were filtered, and the SEM samples were goldcoarted before analysis. The resolution of the SEM was -tested on standard UICC ohrysotile on a Nuelepore fnter with a pore size of 0.8 pm. The TEM analyses were peflformed in a Philips 301 TEM at a magnification of 5,000 and 20,000 X.
Sample preparation
Stock solutions of the standard UICC samples were prepared by dispersing 1 mg of asbestos in a 100-ml water/ethanol solution :in ,an ultrasonic bath. Ethanol was added Ito avoid hydrophobic areas on the Nudepore membranes during the fHtration process.
The stock solution was diluted 1:100 with distiHed water. Different volumes of this solution were filtered through the membranes iby means of a water suction pump. Particle ag.glomeration and adhesion to ithe glass walls occurred during storage, and ,a new stock solution was prepared for each series of :analyses.
Tissue samples were cut from lungs preserved in formaLdehyde and either dried to 'Constant weight at 80°C or weighed ilIl the wet 'Conditbion. For .the determination of the dry-to-wet weight ratio, adjacent tissu'e samples were dried as described pre-Vli'ously.
Digestion of tissue
The following three procedures were used to remove oI1ganic material from the tissue samples: 1. Wet digestion (WD) in 1 N sodium hypochlorite in 0.1 N sodium hydroxide. Sodium 'hypochlorite (5-10 ml) was added to -the tissue in cel1ltrifuge glasses thart were kept at 60-70°C untilla complete reaction was obtained. A mixture of the sodium hypochlor1te solution and di~yl ether (5 ml) was thoroughly shaken and thereafter centrifuged at 4,000 rlmin for 20 min. Each time the procedure was repeated 'twice, the €'ther layer being removed with a pipeMe. The ether fraction was filtered through Celas silver membranes and analyzed for fibers wiJtJh SEM. The un-reacted sodium hypochlorite was neutralized by 1 N hydrochloric acid and sonioated. In one series of analyses the ether extraotion was omitted, and the WD samples were filltered directly through the Nudepore membranes.
3. Low-temperature ashing (LTA) at temperatures below 200°C in a Tracedab model 505 LTA. Generally 50 W of forward power wi'1ih an oxygen flow of 225 ml/min was USled. Some samples were also ashed at high power LTA, ie, at an energy input greater than 150 W of forward power at an oxygen flow of 150 ml/min. A oomplete ashing was obtained for 10-40 mg of dry tissue wiJthin 2-5 h, depending on .the size of 1fue tissue pieces. 'I1he ash remaining a:liter LTA was dispersed iJn 0.5 N hydrochloric acid, a few milli!lilters of etbhanol being added, and kept in the ultrasonic bath for 5 min. The tissue salts were then rapidly dissolved by the hydrochloric acid.
Filtration
Due to 1fu.eir high filtration efficiency, porous membranes are normally used for the ,air filtra'1Jion of mkrofibers. Compared to tlle structure of plain Nudepore membranes, the porous structure of the membranes is a poor ba~kground for SEM work, hut penetra1ion of the Nuclepore may occur depending on the pore sire (4,8).
In this study we have compared 0.2-and 0.8-pm Nuclepore membranes versus 0.8-Fm Gelman Metricel polyvinyl chloride (PVC) filters using standard UICC croci-doUte prepared from water suspensions. The PVC filters were prepared for SEM according to the method published by LeGuenet al (5). Samples for the TEM analy..ses were prepared with a modified Jaffe Wick washing teohnique (2).
Ultrasonic treatment
The samples weI'e 'treated for 5 min in an U1trasonic bath. VigoTO<uS sonication may spM fibers and should be avoided. This possibility was investigated by comparing the direct :£iltration of standard solutions whi~h had been mildly sonicated in a bath (less than 0.2 Wlml) to solutions v~gorously sonicated with a probe with a power owtput greater 1Jhan 3 WlmI.
Results
Larg,e initer-and :intra:laboratory variations may occur in nber counbing ras H is based on subjective observations of the partrcles with the use of different equipment. In most papers an estimation of the coeffident of variation of the method is not ind:iJcated. For the standard UICC samples analyzed in SEM at 4,500 X a relative standard deviation (RSD) of 10 0 10 was found for repeated counts on the same samp1e. CounHng different samples prepared from >the same stock solution gave an RSD for high nber loadi'ngs (ie, grearter than 10,000 fiJbers/mm 2 ) of 16010 fOrI' tlhe Gelman fi1teI1sand 10010 for the Nuclepore membranes. For lower fiber 10ading r s the precision was even bet/trer fo,rtlhe Nuclepor,e membranes. The RSD for the tissue samples was better than 20 010, includimg intra-and interlobar fi/ber density variation (4). A rrelationship between the magnificaltion ,employed and fiber numbers has previously been demonstrated by Gylseth et a:l (4). In table 1 the results from the comparison of the different filter types are given, and a significant higher filteration efficiency is indicated for Nuclepore 0.2-pm and PVC 0.8-pm than for Nuclepore 0.8-jlm membranes.
Tarb'les 2 and 3 show the fiber counts in two dirHererut series with different agitation meihods. There was no significant in- crease in fiber concentratton roter the samples had been treated in an ultrasonic ba!th. Table 3 partly be avoided either by ultrasonication or by the use of a "rubber policeman" before filtration (table 3). A comparison of the different digestion procedures against direct fHtra!mon of standard UICC cI1oddolite suspensions gave a mean recovery of 85 and 90 % for LTAand WD/e1Jher extraction, respectively. HTA at 600°C ror 1 h gave a re- cO\l1ery of less than 20 0/0. When the temperature was reduced to 500°C, 'the recovery increased to 55 0/0. Samplesashed at high-power LTA and filtered on 0. 8-,um Nu'Clepore membr.anes were compared to adjacent samples ashed at 'low-power LTA and filtered on 0.2-,um NU'clepore membranes {table 4). On the average the latter technique gave six times higher filber concentrations <than the former. WDand LTA were compared from adja'cent tissue samples (table 5). After Table 6. Fiber concentrations in adjacent tissue samples after low-temperature ashing (samples 1, 2, 3) and wet digestion (1', 2', 3') followed by two ether extractions. As pointed out by Ascroft & HeppleS'ton (1) and Morg,an & Holmes (6), fiber losses or even an iJncrease in fiber nurrib'€r may oocur during the digestion {WD) of tissue sample for quanti,ta'tive fiber estimation. Errors may also ,be iJntroduced by LTA (6).
We have employed LTA for some yea,rs, and ourexp'€rienoe is til,at, under carefully Discussion complete WD, the solutions were directly filtered onto the membranes. WD gave, on the average, 35 % lower fiber concentrations than LTA. LTA treatment of the wet-digested and SEM-mounted Nudepore membranes did not improve the resu]ts. In fact, the LTA treatment of the Nuclepore membranes gav'€ a filter residue which could be misinterpreted as fibers.
In 'a $'€Cond series of analyses of adjaC'€TIt tLssue s'amples from another lung with the ethere~traction procedure, the data given 1n table 6 were obmined; fuey gave concentrations that averag'ed 37 Ofo low'€r than those for LTA. For two corresponding samples of the same lung, the diameter and lengJth of 100 !tbers were ev,aIluated in each sample. No difference appeared between 'the Itwo 'as r,eg,ards the d1ameter diStribution, Whereas WD, in comparison to LTA gave a significantly larger proportion of Ilonger f1bers (fig 7).
A comparison of the LTA samples with SEM and TEM analysis has been per-fOl'IIled employing a magnification of 4,500 X for the SEM and 5,000 X and 20,000 X !for the TEM (table 7). The results for SEM/4,500 X versus TEMI 20,000 X ar'€ plotted on a log-log scale in Sample oontrolled conditions, it is an effective and gentle method for the removal of organic materi,al. It entaHs low fiber losses and reprodUcible results. High plasma energies (ie, power input greater than 150 W) produce local overheating in the tissue pieces and ;introduce filber losses of significant degree. As pointed out, these effects may he avotded with low plasma energies (ie, power inpwt of less than 60 W) at an increased oxygen How (ie, approx 225 ml/min). T'he data show that LTA gives a slighitly increased fiber count when compared to WD, a finding indicating that fibers Ibreak during tissue shrinkage. The difference may, however, partly be caused by the short and ,thin fibers being covered by the organic debris left after WD. As expected, the diameter distrilbution is not affected, whereas the WD method, when compared to LTA, gives a length distribution sl,ightly biased towards longer fibers.
The whole ashed sample can be filtered with LTA, and thus any extractions introdudng fiber losses can be avoided. For SEM fiber counting i't~lso gives a better background. Furthermore, the most precise tissue mass indicator is dry tissue weight. W1henthe wet tissue is cut, weighing errors are introduced due 'to 'liquid loss. Fo,rmaldehyde also evaporates fairly rapidly, and therefore contributes to the error. Therefore the samples should be weighed in airtight beakers. Lungs which are intratracheally :£ixed contain a significant amount of liquid and have a low dry-towet weight ratio. Other lungs are infiltrated with tumors or consist of pneumonic areas of highly different density and liquid content, and therefore their weigJhtlweight ratios vary. The dry-to-wet bssue weight ratios variled from 6 to 15 0/0 in our <Study. If dry weight is used, paraffin-embedded material from retrospective studies may be analyzed; thus a comparison of data from retrospective and prospective Sltudies becomes possible.
With direct filtmtion of the suspension, fi:ber losses are Teduced when compared to those of 'the other pl'etreating s'teps. It is 'important to use membranes with a sufficiently smaU pore size to prevent fiber penetration. Our data show 1:hat 0.2-,urrn Nuclepore membI"anes give higher fiher counts than O.8-pm porous PVC membranes. The difference demonstrated may be due to -the etching of the PVC membranes necessary for the release of the fibers. Nuclepore membranes also give a better image for counting in SEM than the etched PVC membranes. A comparison of frber size distributions for 0.2-and 0.8-,um Nuclepore membranes showed that the 0.2-,um membranes contained both shorter and thinner fi'bers than the 8-,um ones. Therefore, the filtration of samples on Nuiclepore membranes should only be done with filters wtth a pore size of 0.2 ,urn or less; a compromise between filtration speed and efficiency must be made.
The use of sonication for the homogenization of fi;ber suspensions has also been discussed 'by Morgan & Holmes (6). There are two mai'n features, a homogeneous dispersion ,and a rapid dissolution of the tissue salits. Our data, along with those (jf Ohatfield et al (2) and Spumy et al (9), show that homogenization of fiber suspensions may well be performed !by ultrasonics if a sufficiently low power input is used (ie, 0.2 W/rdl or less). High-power sonication may significantly increase fiber number.
Before TEM analysis the Nudepore memlbranes ·a,re cut and transferred to TEM grids; thereafter the membrane is removed by dissolution in chlorororm. fiber losses may occur during these steps. These losses are iIndicated by the lower counts obtained faa: the TEM analysfus at a magnJificatton of 5,000 X when it is com-paTed to ,the SEM analysis a,t 4,500 X. If the TEM magnification is increased to 20,000 X, the loss is near:ly compensated for. At the same ,time the effec,ts of iner,eased magnification on the fiber number is demonstrated.
The dHferentpreparatilon methods may affect asbestos bodies differently than naked fibers. A study dealing with ,these problems is at present in progress in our laboratory. | v3-fos |
2014-10-01T00:00:00.000Z | {
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} | 0 | [] | 1981-01-01T00:00:00.000Z | 1535747 | {
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"Biology",
"Medicine"
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} | s2 | Germinal cell mutagenesis in specially designed maize genotypes.
We have used three inbreds of Zea mays in our in situ and laboratory studies in environmental mutagenesis. Inbred W22 plants homozygous for wx-C were used in a study to detect the possible mutagenic properties of 32 pesticides or combination of pesticides under modern agricultural conditions. The large numbers of pollen grains analyzed and the ease in detecting mutant pollen grains enabled us to treat the experimental plants with field recommended rates of pesticides. In a current study we are evaluating the possible mutagenicity of Chicago municipal sewage sludge. We are measuring the frequency of mutant pollen grains in inbred M14 at both the wx-C and wx-90 heteroalleles. These plants were exposed to various concentrations of municipal sewage sludge under field conditions. We have inbred Early-Early Synthetic for five generations and tested this inbred with known mutagens. Early-Early Synthetic is a rapidly maturing inbred growing from kernel to anthesis in approximately 4 weeks and attaining a height of approximately 50 cm. Plants of this inbred have been chronically treated with ethylmethanesulfonate (EMS) or maleic hydrazide (MH) under laboratory conditions and forward mutation at the wx locus was measured in the pollen grains. EMS and MH were mutagenic at concentrations of 1 microM and 10 nM, respectively. The concentrations of EMS and MH were calibrated in Early-Early Synthetic to a linear increase in the frequency of forward mutant pollen grains. The construction of a maize monitor for environmental mutagens is currently in progress. This assay will measure forward or reverse mutation at the wx locus in pollen grains, point mutation in somatic cells and will incorporate a cytogenetic endpoint in root-tip cells. ImagesFIGURE 2.FIGURE 3.FIGURE 5.
Introduction
Historically, plants have been. used as indicator organisms in studies on mutagenesis in higher eukaryotes. Plant systems have a variety of well defined genetic endpoints including alterations in ploidy, chromosome and chromatid aberrations, micronuclei, sister chromatid exchanges, and specific locus and multilocus assays in both nuclear and cytoplasmic genomes. Plant genetic assays have inherent advantages as indicators of environmental mutagens and are the only systems currently in use as in situ monitors of polluted air (1-3), polluted water (4), and agricultural pesticides (5,6). An excellent description of the advantages of plant systems as monitors of environmental mutagens has been written by Nilan (7).
An environmental mutagen is a physical or chemical agent released into the environment that can alter the genome or the proper functioning of the January 1981 genome. The presence of such genotoxic agents in the environment is a serious threat to the public health (8)(9)(10)(11)(12)(13). Depending upon the ontogenetic stage of an organism an environmental mutagen may exert teratogenic effects, affect the aging process, induce mutations that involve germinal cells (14)(15)(16), precipitate coronary disease (17) or cause mutations that lead to the neoplastic transformation of somatic cells (12,(18)(19)(20)(21)(22). Genetic assays have been developed to determine the mutagenic properties of chemicals (23). The approach to reduce the impact of environmental mutagens upon the public health is simply to reduce the exposure of people to such agents. The role of regulating the release of toxic chemicals into the environment is a responsibility of the U.S. Environmental Protection Agency. The enforcement powers of such regulation are derived from federal statutes such as the Toxic Substances Control Act, the Resource Conservation and Recovery Act and the Clean Air and Clean Water Acts. Environmental mutagens are defined as toxins (genotoxins) and thus are under the control of several federal and state agencies. However, a battery of good and quantitative genetic assays must be used to adequately determine the genotoxic properties of chemicals, combinations of chemicals and complex environmental pollutants. The work that we present in this paper is an attempt to devise and calibrate an assay in a higher eukaryote that would be useful in a battery of genetic assays to detect the presence of mutagens in the environment.
A series of problems arises when working with higher eukaryote species. The resolution of a genetic endpoint is usually lowered when one proceeds from prokaryotes (bacteria) to lower eukaryotes (fungi) to higher eukaryotes (rodents, humans, angiosperms). As the complexity of the genetic information and the architecture of the chromosome increases, the size of the population routinely analyzed for each assay decreases. Thus the concentration of the test chemical or complex mixture must be increased and problems due to the degree of tolerance for the test agent may arise. Another difficulty is the treatment protocol. Most genetic assays involve acute exposure; however, people are usually exposed to environmental mutagens under chronic conditions.
The objectives of this study are: to define and test a point mutation assay that would have a high degree of genetic resolution based on a higher eukaryote (Zea mays), to investigate the use of this assay under both laboratory and in situ conditions, to calibrate a genetic endpoint with known mutagens under acute and chronic exposure regimens, and to construct special lines of maize that carry a number of different genetic endpoints for use as monitors for environmental mutagens.
The waxy Locus of Z. mays
We chose mutation at the waxy (wx) locus in maize as the genetic endpoint because it is well defined and it is easy to detect mutations in both the kernel and pollen grain. The use of pollen grains as genetic indicator organisms provides a high degree of genetic resolution. Also a tassel growing within a sporophyte may be considered analogous to a suspension culture growing with time. Therefore this system is suitable for tests involving chronic treatment of sporophytes with mutagens. Finally the tassels can be stored and the wx locus assayed for mutation in the pollen grains at a time removed from the end of the treatment protocol or anthesis. This advantage allows for the collection of large numbers of samples during a growing season and the analysis of pollen grains at any convenient time.
Near the beginning of the twentieth century an 62 altered kernel characteristic in maize was introduced into the United States from China that was different from the American varieties of floury, sweet, flint, or pop. This novel variety was termed "waxy" because the endosperm had the appearance of hard wax (24). Genetic studies confirmed that the waxy allele was recessive to starchy (Wx) and wx segregated in the F2 generation as a Mendelian monohybrid (25). The waxy characteristic was due to a mutation that altered the composition of the starch in the endosperm of the kernel. In waxy kernels, the starch of the endosperm contains only amylopectin, while in kernels carrying the dominant allele,Wx, the endosperms contain starch composed of a mixture of amylopectin and amylose (26,27). Because of the presence of amylose the endosperms of kernels carrying the Wx allele stain a blue-black color when reacted with iodine. When an iodine solution is reacted with endosperms of kernels homozygous for wx a red color is produced.
The enzymatic differences inherent in the endosperms of homozygous wx maize kernels as compared to those kernels carrying the dominant Wx allele were investigated by Nelson and Rines (28). Homozygous wx kernels contain the same amount of starch as starchy kernels. However, the waxy kernels do not have the uridine diphosphate glucose transferase system. Since the starch in waxy kernels is entirely composed of amylopectin (27), this carbohydrate must be synthesized by a different biochemical pathway than the branching of amylose formed via uridine diphosphate glucose transferase (28).
The use of the iodine test provided an early, rapid and accurate chemical assay for a genetic characteristic. It was soon discovered that the wx phenotype could be detected by the iodine test in the microgametophytes (pollen grains). Pollen grains are functional haploids and in a heterozygous plant both alleles segregate according to Mendel's first law (29,30). Furthermore, the data indicate that a single gene and its alleles can be similarly expressed in both the sporophytic and gametophytic generations.
Since the wx allele can be detected in single pollen grains it was suggested that this system be used in the study of the genetic fine structure of a locus in a higher eukaryote (31). These suggestions were based on the then recent discoveries that redefined the classical structure of the gene (32). The increase in genetic resolution, however, required very large populations (33) and a population in excess of 10 is usually impossible to analyze in higher eukaryotes. Maize pollen grains were suited to the problem of population size because great numbers could be analyzed rapidly. Also, at that Environmental Health Perspectives time a few independently occu (heteroalleles) were collected for cis-trans tests (34). A m illustrating the position of foi wx-H21, wx-C, wx-90, and wxis presented in Figure 1.
The starch type of a pollen g the genetic constitution of that the parental sporophyte. Thus, of wx to Wx can be detected I grains from plants that are I that stain a dark blue-black cob an iodine test (34,36). Intraj between two different mutati can be analyzed by scoring fc from plants that are intercrosses the different heteroalleles (34, To date, 31 different hete: mapped at the wx locus (35). tions are controlling element alli wx-m-8, wx-B3, and wx-B4. Th of interest because they pos alleles that are prevented fron controlling element (37). Final of gene conversion (nonrecip: combination) has been report (38).
The studies cited in this pap of the wx locus as an indicator analysis of dose-response relati ies in which the primary obje4 tion of new wx alleles and their not discussed.
In maize both acute and chi chemical and physical mutage This classification is justifiab sense as well as in terms of the treatment because of the diffe: at risk, the number of cell X morphogenetic processes inv irring wx mutations conducted by Eriksson and others (39-44) on acute and were available and chronic exposure of maize to radiation will be wap of the wx locus used as examples of the differences inherent in the ur mutational sites, treatment regimes. Forward mutation at the wx -B from Nelson, (35) locus in pollen grains was the genetic endpoint.
In experiments that involve acute exposures to a rTain is controlled by mutagen two approaches are usually used, constant pollen grain, not by time duration at varying doses, and constant dose a genetic reversion for various durations. Eriksson (39) treated maize )y scoring for pollen plants that were at the microsporocyte stage of iomozygous wx and development. Most of the developing meiocytes of r when subjected to the plants were at prophase I. The plants were genic recombination exposed to rates of y-radiation of 1.5 to 200 R/hr. ons at the wx locus The radiation exposures ranged from 0 (control) to )r Wx pollen grains 400 R during a 2 hr period. All the tassels were 3 of lines representing collected and fixed on the same day following treat-36). ment and it was assumed that all of the anthers roalleles have been were in the same stage of development when treated. Five of these muta-The control frequency of forward mutant pollen eles, wx-ml, wx-m-6, grains was 3.7 x 10-4 while an exposure of 3 R tese heteroalleles are increased the frequency of mutants to 5.7 x 10-4 ;sess functional Wx and 400 R increased it to 6.0 x 10-3. The frequency n functioning by the of mutant pollen grains increased in a linear manner lly, the phenomenon for exposures through 200 R. rocal intragenic re-Under a chronic treatment regimen the sporo-;ed at the wx locus phyte is subjected to mutagenic treatment during a major portion of its life cycle. Each cell generation ier deal with the use is exposed to a limited quantity of the mutagen and of mutation and the it is assumed that a constant rate of mutagen enters ionships. Other studthe target cells (i.e., the anther). A number of other ctive was the inducfactors exists that can influence the genetic regenetic analysis are sponse when the plant is chronically treated with a mutagen. A mutational event may occur at any ronic treatments by ontogenetic stage and yield mutant pollen grain ens have been used. clusters that vary in size inversely related to the ile in the biological development of the plant. The number of germline duration of mutagen cells varies at different stages during ontogeny. rent cell populations The probability of inducing a scorable mutation at {enerations and the the wx locus in a germline cell increases as a plant olved. Experiments develops; however, the number of mutant pollen grains that result from such a mutational event WCbm4 decreases. Finally, the frequency of the induction , of mutation varies as a function of the dose of a mutagen. Under chronic conditions, Eriksson (39) treated maize plants in a gamma field for 50 days until anthesis. The rates of exposure ranged from 5 to 200 R/day. The control frequency of forward mutant wx pollen grains was 2.5 x 10-4 . At the lowest exposure rate, 5 R/day, the frequency of mutant pollen grains increased to 7.9 x 10 . A linear dose-response curve was observed for expowx-C wx-B x, sure rates of 5 to 87 R/day. The frequency of mutant pollen grains increased much more rapidly at the WX-9OX higher exposure rates, 130 to 0 R/day. and four heteroalleles on The general interpretation of linear dose-response Nelson (35)].
curves at low mutagen exposures is that such a response indicates one-hit events or point mutations or minute deletions. An increased slope due to higher doses is interpreted as due to two-hit events or chromosome aberrations. The above studies on forward mutation at the wx locus have demonstrated that acute and chronic gamma radiation induces a linear increase in mutation frequency with increased dose. The effect of x-radiation on reversion frequencies of three wx heteroalleles, wx-H21, wx-C, and wx-90 , and on the frequency of intragenic recombination among three different heteroallelic combinations has been reported by Briggs and Smith (45). They found an increase in the reversion frequency of the wx-90 heteroallele and a significant decrease in intragenic recombination between the wx-C and wx-90 heteroalleles. Bianchi (46) analyzed the effect of increased x-radiation (0 to 1680 R) on plants heteroallelic for the following genotypes: wx-H211 wx-90, wx-CIwx-H21, and wx-90/wx-C. The induction of Wx pollen grains was scored. Only the wx-H211wx-90 heteroallelic combination showed a significant increase in the frequency of Wx pollen grains. However, a direct dose-dependent response for the induction of aborted pollen grains was observed in all genotypes. Of great interest was the fact that in homoallelic plants for wx-C or wx-90, an increase in revertant Wx pollen grains over the spontaneous frequency was demonstrated. For wx-C the exposure of 0, 800 R, and 1600 R of x-rays gave frequencies of revertant pollen grains of 1.2, 7.5, and 5.0 x 10-5, respectively. For wx-90 the same radiation doses induced frequencies of revertant pollen grains of 2.3, 5.6, and 11.5 x 10-5, respectively. Thus wx-C and wx-90 can revert to the dominant allele after exposure to a mutagen.
The analysis of maize pollen grains for forward or reverse mutation at the wx locus is based on the fact that a pollen grain carrying a Wx allele synthesizes the carbohydrate amylose while a pollen grain carrying the wx allele does not (27,28). A pollen grain carrying a Wx allele will turn black with the gelatin-iodine stain while a pollen grain carrying a wx allele will stain a tan color. Slides of pollen grains that were used in the following studies were prepared by a procedure developed by Nelson (34) and modified by Plewa (47).
In the reverse mutation tests plants homozygous for a specific wx allele were used. After treatment the tassels were harvested and the pollen grains analyzed. In a field of pollen grains carrying a wx allele exceptional pollen grains that carry the dominant Wx allele were scored. As illustrated in Figure 2 a black-staining pollen grain (Wx) is indicated among numerous tan-staining pollen grains (wx). It is assumed that a pollen grain from a homozygous 64 wx sporophyte that has acquired the ability to synthesize amylose resulted from a reverse mutation at the wx locus. However, suppressor mutations may also produce phenotypically "revertant" pollen grains. The number of revertant pollen grains and an estimate of the total number of viable pollen grains were determined. In the forward mutation test plants homozygous for the dominant allele, Wx, were used. After treatment, harvesting and slide preparation the slides were analyzed for forward wx mutants. The specific genetic alteration that caused the inability of a pollen grain to synthesize amylose may have been a point mutation within the wx cistron, a deletion in or of the wx cistron, a chromosome aberration that resulted in a deficiency that included the wx locus or a chromosome aberration that induced a position effect that repressed the expression of the Wx allele. Finally, the possibility exists that a mutation resulted in a regulatory gene involved in the control of amylose synthesis. In this assay, tan-staining wx mutants were scored among a field of black-staining Wx pollen grains (Fig. 3). Although the forward mutation assay is not as specific as a reversion test, it provides a measurement of genetic damage in the sense that it involves a single locus.
In both the reverse and forward test procedures, the frequency of clear, collapsed, aborted pollen grains was also determined. The frequency of mutant pollen grains was determined by first scanning the entire microscope slide with a stereomicroscope at a magnification of 40 x and counting every exceptional pollen grain. The number of viable and aborted pollen grains on the slide was estimated by counting pollen grains within 20 randomly distri- buted 1 mm2 areas and multiplying by an appropriate factor. Thus, the data from each slide included the number of mutant pollen grains, the estimated number of viable pollen grains and the estimated number of aborted pollen grains.
Statistical Tests
Statistical tests were used to describe the distribution of the frequencies of mutant pollen grains and to determine the level of significance among the groups within an experimental design. Parametric statistics such as the mean, standard error of the mean, and the analysis of variance were used to detect significant differences between the frequencies of mutant pollen grains in control and experimental (i.e., treated) plants. The frequency of mutant pollen grains was determined for each plant by dividing the total number of mutant pollen grains by the estimated number of viable pollen grains derived from the slides of a single tassel. The population of pollen grains analyzed was kept approximately the same for each tassel. The mean frequency of mutant pollen grains was determined as the average of the frequencies of mutants of each tassel within a control or treatment group. The standard error of the mean for the tassels was determined. The standard error was defined as SD/VN, where SD is the standard deviation and N is the total number of viable pollen grains (48). An analysis of variance was conducted to determine if a significant difference existed among the mean frequency of mutant pollen grains in the control and the mean frequency of mutants in the various January 1981 treatment groups. We regarded the mean frequency of mutant pollen grains in the control groups as representative of the spontaneous frequency of mutants.
Another test statistic (4)) that we used to determine if a treatment induced a significant increase in the frequency of mutant pollen grains was developed by Katz (49). He adapted the 4) statistic for use in the analysis of mutation data from pollen grains (50). The test is defined as:
In Situ Tests Evaluation of Pesticides
We have conducted a comprehensive evaluation of the mutagenic properties is 32 pesticides or combination of pesticides used in commercial corn production. The tests were conducted in situ under modern agricultural conditions and in the laboratory using both field grade and technical grade formulations. The laboratory studies included the evaluation of each pesticide or combination for the induction of mutation in Salmonella typhimurium and gene conversion in Saccharomyces cerevisiae. Each compound was assayed directly, with S9 in vitro mammalian microsome activation and with maize 1S in vivo plant activation protocols (6,51,52). In this paper we shall only discuss the in situ experimental design where reversion at the wx-C locus in maize pollen grains was measured to determine the presence of an environmental mutagen. The purpose of this discussion is to outline procedures that insure an adequate experimental design for in situ evaluations of environmental mutagens.
A separate herbicide test plot and an insecticide test plot were constructed. The test plots were divided into subplots with the dimensions of 10 m in length and 3 m in width. Control subplots were distributed within each test plot. The middle row of each subplot was planted with seven kernels of inbred W22 homozygous for the wx-C allele. The outer rows of each subplot were planted with commercial hybrid corn to simulate field conditions. The appropriate pesticide or combination of pesticides was applied separately to each subplot prior to emergence of the seedlings. After the plants reached early anthesis, tassels from each wx-CIwx-C plant were harvested, labeled and stored in 70% ethanol. The tassels were agitated several times in clean ethanol to wash away contaminant Wx pollen from the commercial varieties of corn. To insure that only pollen from inbred W22 was analyzed, unopened florets were removed from the tassels and rinsed in clean ethanol. The anthers were dissected from these florets, the pollen grains were suspended in the gelatin-iodine stain and slides were prepared for analysis.
Data from the in situ pesticide tests are presented in Tables 1, 2, and 3. The control data are from subplots in the herbicide test plots, subplots in the insecticide test plot and a control plot on virgin uncultivated soil. The summed control values based on three years of studies indicate a frequency of revertant pollen grains of 4.64 x 10-5. However, we compared the control values for each test plot with the values for each pesticide treatment within the same test plot. Several pesticides induced mutation where at least a doubling in the frequency of revertants over control values was observed. In Table 1 the data indicate that most treatments with s-triazine herbicides increased the frequency of revertant pollen grains. The herbicides that induced a mutagenic response were cyanazine, SD50093 (a formulation of atrazine + cyanazine) and procyazine + Dual. Slightly higher frequencies of revertants were observed in the treatments of cyanazine + Lasso and propachlor + cyanazine. The datafromthe insecticide test plot indicate that chlordane, Dyfonate and heptachlor induced higher frequencies of revertants. In Tables 2 and 3, data from additional in situ studies on the s-triazine herbicides are presented. The 4+ statistic was used to test for significant differences between the control frequency of mutant pollen grains and the revertant frequencies from plants treated with simazine, atrazine, cyanazine, and SD50093. All four s-triazine herbicides indicated a positive mutagenic response with (53). From these studies we concluded that the wx locus in maize pollen grains was a useful and sensitive assay for environmental mutagens under in situ conditions. The advantages of the assay were that the plants were able to chronically monitor a discrete environment for the presence of mutagens, the tassels could be stored indefinitely after the treatment and analyzed when convenient, the plants were easily incorporated into an agricultural setting, the assay was relatively rapid for a higher eukaryote and the large populations of pollen grains analyzed provided a good basis for the statistical interpretation of the data.
Evaluation of Municipal Sewage Sludge
We have begun a second in situ study and have improved our methods of environmental monitoring by virtue of our experience with the pesticide test plots. An attractive method of disposal of sludge resulting from the treatment of municipal sewage is the application of this organic matter to agricultural lands or its use in reclaiming land disturbed by surface mining. We are assaying sludge from the Calumet and Southwest plants of the Metropolitan Sanitary District of Chicago for the presence of mutagenic agents. This study has a variety of prokaryote and eukaryote genetic endpoints, however, in this paper we shall only present the experimental design for the in situ tests involving maize. A test plot was constructed on the NW900 plots at the University of Illinois Agronomy Research Center near Elwood, Illinois. A history of the use of these plots is presented in Table 4. It is important to know the past conditions of the area that is to be monitored. We were especially interested to know of the past use of pesticides on the test plots. The NW900 plots had four levels of sludge application in 1979 (Fig. 4). The control plot, Number 13, received no sludge application and adequate nitrogen was provided by the application of chemical fertilizer. The maximum treatment plot, Number 5, re- No sludge applied; plots in spinach 1977 No sludge applied; plots in spinach 1978 No sludge applied; plots in spinach 1979 Plots Numbers 5, 13, 14, and 15 used in the maize itwx locus assay ceived 17.8 cm of liquid sludge which was approximately 21.0 mt/ha of dry material. The one-half treatment plot, Number 15, received 8.9 cm of liquid sludge which was approximately 10.5 mt/ha of dry material. The one-fourth maximum treatment plot, Number 14, received 4.5 cm of liquid sludge which was approximately 5.0 mt/ha of dry material. The construction of the NW900 test plots and the application of the sludge was supervised by Dr. T. Hinesly of the Department of Agronomy at the University of Illinois.
Two lines of the maize inbred M14 were used, and the genotype of each line was wx-C/wx-C and wx-9OIwx-90. Thirty kernels of each line were planted in each plot and the sporophytes grown to early anthesis at which time the tassels were harvested. The pollen was analyzed by Dr. S. Wood of the Institute for Environmental Studies at the University of Illinois. Although the data are currently being evaluated there appears to be a direct dosedependent effect with an increased percentage of aborted pollen grains in the lines homozygous for the wx-C allele in the sludge treated plots. However, no increased frequency of revertant pollen grains over the control frequency was noted in any treatment group. A different response was observed in the lines that were homozygous for wx-90. There was no significant difference in pollen abortion among the control and treatment groups, however, tassels from the maximum treatment plot had a higher frequency of revertant pollen grains than the control. Additional tests are in progress.
The examples outlined in this section were presented to indicate the experimental designs that have been conducted to evaluate the practical use of maize as an environmental monitor. The requirements to make an adequate in situ assay are: (1) the knowledge of the history of the area to be assayed, (2) the construction of test plots with 68 adequate controls, (3) the use of defined inbreds with specific alleles and a sufficient population of plants per treatment group, (4) personnel to tend the test plots and harvest the tassels, (5) the careful and competent analysis of the pollen grains, and (6) proper statistical evaluation of the data.
Inbred Early-Early Synthetic
During the last few years we evaluated a number of rapidly maturing maize varieties for use in studies on environmental mutagenesis. A useful variety, Early-Early Synthetic was developed by Dr. D. E. Alexander of the Department of Agronomy at the University of Illinois. We have inbred this variety for five generations and have tested the plants under acute and chronic exposure to mutagens under laboratory conditions. Early-Early Synthetic is a rapidly maturing inbred that develops from kernel to tassel emergence in approximately 4 weeks in a plant growth chamber. The plant reaches a height of approximately 50 cm and can be grown in 10 cm diameter plastic pots. In a 0.92 m plant growth chamber we easily accommodated 40 plants to maturity. A mature Early-Early Synthetic plant is illustrated in Figure 5.
We evaluated Early-Early Synthetic for its response to chronic exposure of low concentrations of ethyl methanesulfonate (EMS) or maleic hydrazide (MH). The genetic endpoint was forward mutation at the wx locus in pollen grains. An experimental design included 20 sealed and locked plant growth chamber in an annex to our laboratory. The chamber that contained the treatment groups was kept at a time schedule that was advanced 12 hr from real time so the administration of mutagen solutions was conducted under darkened conditions. A plant growth chamber for the control groups was in our laboratory. All the plants were given identical amounts of water and fertilizer. The only variable was the concentration of mutagen in the treatment groups. The chemical mutagen, EMS or MH, was administered to the plants in an aqueous solution. The mutagen solution was prepared immediately prior to the treatment of the plants. A 50-ml portion of a known molar concentration was poured into the soil of each pot three times a week. The control plants received water only. The data from the EMS experiments are presented in Table 5. This table is in a form that complies with the suggestions generated by the Plants Working Group of the USEPA Gene-Tox Program (54). Table 5 provides information on the molar concentration of EMS for each treatment group, the number of treatments or treatment days and the total exposure of EMS in moles per plant. The number of mutant pollen grains and the estimated number of viable pollen grains analyzed are presented as the frequency of mutant pollen grains. The percentage of pollen abortion was determined by estimating the number of aborted pollen grains and dividing by the sum of the viable plus aborted pollen grains. The percentage of pollen abortion is a measurement of gametophytic death and may have genetic as well as nongenetic causes.
In the four experiments the molar concentration of the EMS solutions ranged from 1 FLM to 10 mM, and the total amount of EMS administered to individual plants ranged from 6.5 x 10-7 to 4.5 x 10-3 mole. Plants exposed to 6.0 x 10'5 mole during their sporophytic generation produced only aborted (Fig. 6). The total chronic exposure of EMS in experiment 4141 ranged from 1.1 x 10-6 moles to 6.8 x 10-6 mole ( Table 5). The frequency of mutant pollen grains in every treatment group was significantly different (p < 0.001) from the control group and dose-dependent. Figure 7 illustrates the data from experiment 4141. The frequency of mutant pollen grains increased in a linear manner as a function of dose (coefficient of determination,r2 = 0.93). However, there was no correlation between the exposure of EMS and the percentage of aborted pollen grains (coefficient of correlation, r = 0.22). Thus, EMS is a potent mutagen to Early-Early Synthetic when chronically administered to the soil. In Table 6 we present data which demonstrate chronic exposure of Early-Early Synthetic to maleic hydrazide (MH) induces forward mutation at the wx locus in pollen grains. The experimental design was the same as for the EMS treatments. The total chronic exposure of MH in experiment 4143 ranged from 5.0 x 10-9 to 5.0 x 10-7 mole. The frequency of mutant pollen grains ranged from 4.59 x 10-5 for the control group to 5.54 x 10-4 for the plants exposed to the highest concentration of MH. A statistically significant increase in the frequency of mutant pollen grains was observed in all treatment groups. This increase was dose-dependent and linear, r2 = 0.99 (Fig. 8) The percentage of pollen abortion increased directly with the dose of MH (r = 0.96). These data clearly indicate that MH is an exceedingly potent mutagen in maize.
We chose MH as a test agent because this compound is a potent inducer of chromosome aberrations in many plant systems. However, MH is either ineffective or weakly active in bacterial mutation assays and in mammalian genetic and cytogenetic assays, reviewed by Haley (55); and Swietlinska and Zuk (56). MH is a potent mutagen in maize (Table 6), and preliminary data suggests that this agent may be a plant promutagen that can be activated by a maize tissue homogenate into a form mutagenic in S. typhimuir-um strain TA98 (57).
Future Studies
For future studies we have incorporated the yellow green-2 (yg-2) and dwarf (d) alleles into Early-Early Synthetic. Plants that are heterozygous for yg-2 can be used to detect mutation in seedling leaves (58). These plants could be used to measure germinal cell mutation in the pollen grains and somatic cell mutation in the leaves of the same sporophyte. Early-Early Synthetic plants homozygous for d would be very small, rapidly-maturing plants that would probably achieve a height of only 15-25 cm. These plants would be especially useful in laboratory studies or for in situ monitoring of enclosed areas. We shall investigate root-tip chromosomes for aberrations and sister chromatid exchanges as genetic endpoints for environmental monitoring. A procedure for sister chromatid exchange analysis in maize root-tip chromosomes has been developed by Chou and Weber (59). Finally, we shall continue the calibration of mutation induc-tion in Early-Early Synthetic with a variety of chemical and physical mutagens.
In conclusion, the information presented demonstrates that Z. mays is a useful and sensitive genetic indicator organism. The plant has a number of genetic endpoints and can be used under laboratory or in situ conditions for studies in environmental mutagenesis. | v3-fos |
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} | s2 | The effect of temperature and genotype on growth traits, plasma glucose and uric acid in Dwarf and Normal White Leghorn females
Day old White Leghorn female chicks of 7 sire families and two genotypes at the sex-linked dwarfism locus (Dw and dw) were reared on deep litter under normal (around 15-20 °C after 4 weeks) and hot temperature (around 25-30 °C) from 0-18 weeks of age. and uric acid levels at 18 weeks were measured. Results are as follows : 1) A very highly significant difference between genotypes was obtained in plasma uric acid level at 18 weeks, dwarfs having almost the double values than normals. As expected body weight and shank length differences were very highly significant. A difference for plasma glucose was found significant at 5 p. 100 level only with pooled environments. 2) Plasma uric acid was found to be appreciably elevated in the hot temperature group (increase being relatively more in normals than in dwarfs). Hot temperature had a very highly significant depressent effect on 8 and 12 weeks body weight. The effect was observed to be less significant at 4 weeks while it was non-significant for 18 weeks body weight and for plasma glucose level. 3) Effect of sire family was found to be significant for all the traits measured. No trait showed any genotype X family interaction. 4) Very highly significant interaction between genotype at Dw locus and temperature treatment was observed for body weights at 8, 12, 18 weeks and plasma uric acid level, these traits being less affected at high temperature for dwarfs. Four week body weight and shank length were also found to show significant interaction whereas such effect was non-significant for plasma glucose level.
1) A very highly significant difference between genotypes was obtained in plasma uric acid level at 18 weeks, dwarfs having almost the double values than normals. As expected body weight and shank length differences were very highly significant. A difference for plasma glucose was found significant at 5 p. 100 level only with pooled environments.
2) Plasma uric acid was found to be appreciably elevated in the hot temperature group (increase being relatively more in normals than in dwarfs). Hot temperature had a very highly significant depressent effect on 8 and 12 weeks body weight. The effect was observed to be less significant at 4 weeks while it was non-significant for 18 weeks body weight and for plasma glucose level. 3) Effect of sire family was found to be significant for all the traits measured. No trait showed any genotype X family interaction. 4) Very highly significant interaction between genotype at Dw locus and temperature treatment was observed for body weights at 8, 12, 18 weeks and plasma uric acid level, these traits being less affected at high temperature for dwarfs.
Four week body weight and shank length were also found to show significant interaction whereas such effect was non-significant for plasma glucose level.
-Introduction
A considerable amount of research has been carried out on the dwarfing effect of the dw gene after it was first described by H UTT (1949) RE , 1974 ;HORST & PETERSE N , 1978) and a tendency to lower mortality (B ERNIER & A RSCOTT , 1972). But, it appears that very little research has been conducted to study the performance of dwarf layers, particularly dwarf Leghorns, reared from the beginning under hot environmental conditions simulating the tropical climate.
In view of the above, a study on dwarf and normal White Leghorn, reared under normal and hot temperature, has been undertaken and is in progress (results up to growth stage are presented in this paper), which may answer the possibility of utilizing the dwarf gene in layer populations, especially in the context of tropical conditions.
-Material and methods
Dwarf and normal White Leghorn female chicks were obtained from heterozygous sires Dwdw mated with dwarf females in two hatches at an interval of two weeks, during the month of september 1980. The chicks were reared on deep litter from 0 to 18 weeks of age in two temperature groups, the first hatch under normal temperature (around 15-20 °C) without heating after 4 weeks of age, and the second under hot temperature (around 25 to 30 °C) with additional heating. Temperature fluctuations occurred for both groups due to imperfect insulation of the pens.
Data on diurnal temperature variations, body weights at 4, 8, 12 and 18 weeks of age, shank lengths at 12 weeks and plasma glucose and uric acid at 18 weeks were obtained from a total of 247 individuals, out of which half (122) were dwarfs.
The two blood parameters were chosen as indicators of metabolic activity, and, for the second, of protein catabolism, in view of possible effects associated with the dwarfing gene.
Birds were distributed at random into the two temperature groups within each sire family, although the representation of each sire family and genotype could not be exactly equal due to the variations in the number of each genotype hatched under each sire family.
The plasma glucose and uric acid were measured immediately after the collections of blood samples (on the morning, birds being without food from 17 h the day before) at 18 weeks of age by a « Beckman Glucose Analyser apparatus.
For each trait analysis of variance was done separately for the two environments and then for environments combined with genotype at the Dw locus as the controlled source of variation. Phenotypic correlation coefficients among different traits were also calculated within environment and genotype. The pooled estimates of correlation coefficients were done after testing the correlations for homogeneity. Covariance analysis on plasma uric acid was carried out, to test the effect of genotype after removing the effect of body weight. Table I gives weekly averages of daily maximum and minimum temperature in &dquo;C under normal and hot temperature on deep litter from 4th to 18th week of growth. Table 2 presents the mean, standard deviation (S.D.) and coefficient of variation (C.V.) for the body weights, shank length, plasma glucose and uric acid under the two temperatures with genotypes separate and combined. Also presented in table 2 is the per cent of dwarf to normal genotypes (dw/Dw X 100) for the two treatments. Tables 3 and 4 give the analysis of variance for the characters with environments separate and combined respectively. Table 5 presents the phenotypic correlations of plasma glucose and uric acid with body weights and shank length under separate genotype and environment whereas table 6 gives all the correlations pooled for both genotypes and environments on a within-group basis. Table 7 shows the covariance analysis between genotypes for plasma uric acid with effect of body weight on plasma uric acid removed.
-Discussion
Body weights As mentioned in table I maximum and minimum temperature of 30 &dquo;C and above under hot environment was achieved only during 7th-8th week. Corresponding to this it is observed that maximum reduction of body weight of 41.37 gm due to temperature was also achieved during this period. Although the maximum and minimum temperature at 4 weeks was also near 30 &dquo;C, it appears that its effect on body weight was carried over later and there was very little difference in body weight (7.0 gm) between normal and hot temperature at the 4 week stage. From tables 1 and 2 it is also observed that although the difference in temperature between normal and hot group was 10 °C or more during 7th-8th weeks, l2th-l3th weeks, 13th-14th weeks, 16th-17th weeks and 17th-18th weeks, the body weight difference between the normal and hot groups reduced after 8 weeks stage to the extent that at 18 weeks the difference was non-significant. Thus in this present experiment the difference of temperature of 10 &dquo;C or above between normal and hot group had a bearing on body weight only when the maximum or minimum temperature in hot group was above 30 &dquo;C. But this effect may also be due to the stage of growth and age of the birds. From the percent values of dwarf to normals under normal and hot temperature group in table 2 it can be easily observed that dwarfs have relatively less reduction in body weight under hot temperature compared to normals. This effect was also maximum (dwarfs being 67.3 p. 100 of normals in the control group v 72.0 p. 100 in the hot group) at the 8 week stage when the maximum and minimum temperature in the hot temperature group was around 30 °C or above.
The relatively better growth of the dwarf genotype under hot temperature can be seen from table 4 where the effect due to genotype X treatment interaction is highly significant for body weights.
The effect due to treatment X family interaction was also found to be highly significant for body weight and shank lengths indicating thereby that within genotype there exists familial differences for tolerance of heat. M ERAT et al. (1974) using two different environmental temperatures (20 °C v 28-34 °C) also observed a better resistance to heat in dwarf birds that decreased less their laying performances and food intake and increased less their water intake. MATTER & A HMAD (1971) found a less increase in body temperature in dwarfs following abrupt change of environmental temperature from 22 to 40 &dquo;C. S ELV nRn.rnH (1974) suggests that dwarf hens will be at an advantage in hot climates. H ORST & P ETERSEN (1978) tested dwarf and normal White Leghorns under moderate (20 °C) and high (32 °C) temperature and observed significant interaction favouring the lighter body weight types for egg production and body weight at the higher temperature. However, no special resistance to high temperature of dwarf chickens reared at 40 °C v 22 °C was found by A HU tn D et al. (1971,1974). G UILLAUME (1976) suggests that the better performance of dwarfs under hot temperature may be related to the size of the bird and to lower thyroid activity.
Glucose
From table 3 it can be observed that levels of plasma glucose in dwarfs and normals were not significantly different within each environment. However at both temperatures this parameter is slightly lower for dw birds and table 4 shows that when environments are combined the difference between genotypes is significant at 5 p. 100 level. This suggests a slightly different regulation mechanism of plasma glucose in dw and Dw genotypes in accordance with results of G UILLAUME (1972). On the other hand, there exists a significant sire family effect on plasma glucose. The temperature apparently does not affect the plasma glucose level. WARD & P ETERSON (1973) also did not find a change in plasma glucose level in broilers exposed to 33-35 &dquo;C for 4 hours. However, the average plasma glucose level reported in the present study is somewhat lower than the value of 233 mg/100 ml observed by T APPER & K ARE (1960) in White Leghorn hens. This difference may be attributed to age, strain or environmental differences.
Phenotypic correlations between different characters under separate genotype and environment (table 5) were tested for heterogeneity and found to be non-significant. Pooled correlation estimates were found (table 6) to be non significant with all characters except plasma uric acid indicating that plasma glucose level was independent of body weights and shank length and slightly negatively associated with plasma uric acid level.
Uric acid
The mean values obtained (table 2) are in the range of values considered as normal : see for instance BELL & FREEMAN (1971).
From table 2, 3 and 4 it can be observed that plasma uric acid level is highly significantly affected by genotype (almost double in dwarfs as compared to normals : 36.25 mg v 19.90 mg/1) and treatment, with a significant genotype X treatment interaction. The level increases with temperature in both dwarfs and normals, the proportional increase being more in normals than in dwarfs (33.20 v 53.30 mg/1). WARD & P ETERSON (1973) also obtained significantly higher plasma uric acid level in broilers exposed for 4 hours to 33-35 &dquo;C. The possible reason attributed to such higher level was cellular damage as a direct consequence of heat. If this explaination holds good it again leads to the conclusion that dwarfs sustain hot temperature better as concerns such cellular damage.
Phenotypic correlations of plasma uric acid with all other traits, with genotype and environment separate, were all non significant and low (table 5). But the pooled estimates (table 6) were found to be significantly negative at 5 p. 100 level with 8 week, 12 week body weight and plasma glucose and at 1 p. 100 level with 18 week body weight. The covariance analysis removing the effect of body weight on plasma uric acid shows that the effect of genotype on plasma uric acid remains highly significant, indicating a strong and specific effect of the dwarf gene, independent of its size reducing effect, on plasma uric acid.
Studies on uric acid clearance by the kidney tubules indicates (BELL & FREEMAN, 1971) that as the plasma level of uric acid increases, the amount filtered continues to increase, until at a very high plasma level the ability of the tubules to secrete uric acid declines. Impaired renal clearance of uric acid of hereditary origin in chicken has also been reported by A USTIC & COLE (1972) where the renal clearance of high uric acid strain was markedly less than that of the normal strain. It is also reported that when an organism cannot, by dietary means, obtain sufficient amounts of an essential amino acid, it catabolizes body protein, to obtain this amino acid. This process naturally leads to an increased excretion of nitrogen, in birds mostly as uric acid.
Scanning through the literature it appears that there is no report yet on the measure of either plasma uric acid or its clearance in dwarfs. However, WOOD et al. (1971) demonstrated that protein metabolism of dwarf hens differed from that of normal siblings. G UILLAUME (1972) also demonstrated that the level of free amino acids is lower in dwarf birds. G UILLAUME & L ARBIER (1974) and G UILLA U ME (1975) also obtained an estimation of protein anabolism per g of tissue higher in dwarf chicks, which was similar to the findings of BROWN et al. (1972). Since the dwarf chick has a smaller protein retention (G UILLAUME , 1969) this higher anabolism (per unit body weight) is necessarily bound to a faster catabolism.
Thus the higher plasma uric acid level in dwarfs, reported in the present study, may be due to an hereditary impaired renal clearance associated with the dw gene or to the result of higher protein catabolism as reported by G UILLAUME (1975). Also the dwarfs may not be able to obtain, by dietary means, sufficient amount of some essential amino acids, resulting into higher protein catabolism, thereby higher plasma uric acid level. These various possibilities for higher uric acid level in plasma of dwarfs need to be verified. | v3-fos |
2018-12-18T07:50:31.490Z | {
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} | s2 | Determination of Animal Fat in Margarine by High Pressure Liquid Chromatography of the Sterols
The utility of high pressure liquid chromatography as a routine method to detect animal fats in vegetable margarines on the basis of sterol content was investigated. Cholesterol, which makes up almost 100% of the total sterol in animal fats, constitutes only 0-4% of the total sterols in vegeta• ble oils. The major phytosterols in vegetable oils currently used for margarine production are B-sitosterol, stigmasterol and campesterol. Brassicasterol is less conunon but in rapeseed oil it is present in high proportion. The minor phytosterols are: ~ 5-avenasterol, ~ -stiqmasterol, 7 ~ -avenasterol, 24-methyl cholest-7-enol and cholesterol. A uBondapak c18 column and a yariable wavelength ultraviolet detector along with a variety of mobile phases, have been used throughout this investigation. A method was developed for isolating, extracting and derivatizing sterols from biological material. Optimum chromatographic conditions are described. Lard, soy bean, corn, cottonseed, safflower, sunflower, olive, walnut and rapeseed oils were analyzed. A good separation was obtained for free sterols from vegetable o·ils. Free cholesterol could be separated from the major phytosterols but not from the whole group and in a
The major phytosterols in vegetable oils currently used for margarine production are B-sitosterol, stigmasterol and campesterol. Brassicasterol is less conunon but in rapeseed oil it is present in high proportion. The minor phytosterols are: ~5 -avenasterol, ~7-stiqmasterol, A uBondapak c 18 column and a yariable wavelength ultraviolet detector along with a variety of mobile phases, have been used throughout this investigation. A method was developed for isolating, extracting and derivatizing sterols from biological material. Optimum chromatographic conditions are described. Lard, soy bean, corn, cottonseed, safflower, sunflower, olive, walnut and rapeseed oils were analyzed. A good separation was obtained for free sterols from vegetable o·ils. Free cholesterol could be separated from the major phytosterols but not from the whole group and in a ii blend of 25% lard and 75% corn oil, cholesterol did not show up clearly.
With benzoate derivatives a good separation was obtained for cholesterol from major phytosterols, but brassicasterol from rapeseed oil and some minor phytosterols from other oils interfered with the cholesterol peak. Because of this interference, the detectable ratio of animal fat in vegetable margarine is dependent on the raw material.· Fifty percent of animal fat can be easily detected in all kinds of margarines.
In soy bean oil marqarine, 25% animal fat can be easily detected. The method is reproducible, rapid, and worth further investigation to find a column and solvent system capable of separating cholesterol completely from all phytosterols, so that very low levels of animal fat could be detected in vegetable oil.
iii ACKNOWLEDGEMENT · I wish
INTRODUCTION
Ghee is the name of butter fat obtained from cow's or sheep's milk, to which some flavors are added. That was the multipurpose butter in our country (Saudi Arabia) • Then, _./ the margarine expansion overwhelmed our market and consumers preferred the margarine for its convenience. Most of the import"ed margarine was labeled as "Substitute Ghee." Some labels specified that it had a vegetable and/or animal origin and under the name of "Substitute Ghee" a lot of animal fat was dumped on the country, especially when the prices of vegetable oils were high. To prevent such commercial fraud, to avoid the health problems of cholesterol, and due to the fact that lard is not allowed to enter the country on religious grounds, the Saudi Arabian government has prohibited the import of dripping fat and permits only margarine of vegetable origin. Usually, the margarine manufacturer has pre-set criteria for the finished fats and oils he uses CAnderson andWilliams, 1969 andMassiello, 1978), and different formulas geared to the cost of the oils which are the major ingredient cost in margarine. Prices of oils and fats change a good deal, like all commodity prices. The premium or special margarines are not subject to such adjustments.
The table following shows the fluctuations in lard use as an ingredient of margarine (Anderson and Williams, 1969 (Riepma, 1970).
14 15
Total 1500 1535 1720 Due to the fact that every imported margarine must be checked for the presence of animal fats, our Food Quality contrql Laboratory is in need of a quick routine method for the detection of animal fat in vegetable margarine.
Measuring cholesterol, a sterol formed predominantly in animals, is one of the most frequently performed assays in the laboratory, for which a wide variety of methods are available and used. The classical method is colorimetric assay based on the photometric measurement of the color formed when cholesterol reacts with Lewis acid (Abell et al., 1958).
However, because of the hazards associated with using the strong acid medium in which the color is formed, alternate methodologies have been and are being developed. Most of the newer methods are based on enzymatic hydrolysis and oxidation (Gray et al., 1977 andAnonymous, 1979), or on chromatographic analysis (Driscoll et al., 1971). The enzymatic reactions are followed by colorimetric or electrochemical analysis.
By gas chromatography alone, the determination of steroids in biological samples cannot usually be achieved. It is necessary to employ other procedures to separate crude fractions and isolate sterols by preparative methods. The isolation of such sterols from a biological material is commonly established by fractionation of lipid extracts by column chromatography or thin layer chromatography (TLC) and in many cases by silver nitrate-silica gel TLC of either the free sterols or their acetates lRees, 1976). Although liquid chromatography (LC) has been used in analysis of lipids in general (Aitzetmuller, 1975), its specific application in cholesterol methodology has been limited because • cholesterol and related compounds absorb very little UV radiation in the relatively high wave length in which most UV detectors used · in LC operate. Consequently, LC has been used in assays in which column chromatography is followed by chemical analysis of collected chromatographic fractions (Shin, 1963) or in which the eluate is monitored on-line with other types of detectors. These detectors include the refractive index detector (Werthessen et ai., 1970), the moving-wire flame ionization detector (Worth and MacLeod, 1969), and a laser infrared detector (Freeman, 1974 (Dam •. 1958 Steroids of some kind are apparently present in all living organisms, with the possible exception of bacteria. They include sterols, certain sapogenins, alkaloids, heart poisons and hormones. Each steroid in common possesses a characteristic tetracyclic carbon skeleton, namely, the skeleton of the perhydrocyclopentanophenanthrene molecule ( Figure 1) (perhydro = fully hydrogenated (Dence, 1980).
In completely saturated steroids, rings A, B, ~nd C are cyclohexane rings, and ring D is a cyclopentane ring. Cyclohexane rings are not planar, for if they were, the c-c-c bond angles would be 120° instead of the observed values which lie much closer to the tetrahedral angle of 109-110°.
Aromatic rings are planar.
In naming steroids, we begin by orienting the molecule as shown in Figure 2 for cholestane, a saturated C27 steroid. The numbering ' is as indicated. According to the International Union of Pure and Applied chemistry-International Union of Biochemistry (IUPAC-IUB) 1967, the use of a steroid name implies that groups at positions 8, 9, 10, 13, and 14 are oriented in the BB, 9a, lOB, 13B and 14a configurations, respectively, unless stated to the col'ltrary. We then need only to specify in the name the configuration at position 5 and 17 and any other positions of asymmetry. By long standing tradition, the terms a and Bare also applied as shown in structure of Figure 3. Many steroids have common or trivial names such as cholesterol.
Systematic names, however, are based on certain parent hydrocarbon systems. Elaboration is described using principles from organic nomenclature and expressing this by means of prefixes and suffixes. An example for sterols is given in Figure 4 (Dence, 1980). frequently used solvents. One very unusual solubility property of steroids has been known for a long time. This is the fact that cholesterol and other 3B-hydroxy steroids are precipitated from solution .by digitonin, itself a glycoside of a 3B-hydroxy-spirostan, whereas 3a-hydroxy steroids are not so precipitated. The 3B-hydroxy steroid is precipitated as a complex of the glycoside. Other saponins will work also but digitonin was found to give nearly quantitative precipitation (Cook, 1958) . For ultraviolet and visible spec-• troscopy, the C=C and C=O bonds are the ones most important in steroids of natural origin. The C=N also plays an important role in derivatives used for analysis. The commonly measurable region is from 210 to 800nm of which the region from 400 to 800 is regarded as the visible and lower wavelength as the near ultraviolet region. Pure cholesterol, for instance, has no significant absorption from 210 to 800 nm. On the other hand. 5,6-dehydrocholestanol (cholesterol) 5 exhibits end-absorption because of a peak from the ~ bond near 200nm (Nes and McKean, 1977) • That is due to the fact that only certain transitions are allowed and they are dependent on the environment, extent of conjugation, degree of substitution and nature of the atoms joined by pi electrons (Pomeranz and Meloan, 1971 for it to appear consequentially in ,their tissues. There is a strong suggestion that this discrimination against sterols with modified side chains begins with the vertebrates. In many sub. sequent investigations cholesterol was found to be the dominant sterol of all higher vertebrates. Except for the gastrointestinal tract, mammalian sterol is nearly but not completely pure cholesterol. Vertebrate tissues usually not only contain no sterols with side chains which have been alkylated, shortened or enlarged, but also no 6 22sterols (Nes and McKean, 1977). However, the sterols of vertebrates are by no means always absolutely pure cholesterol. Conunon animal sterols include small amounts of the 6°and 6 7 -analogs (cholestanol and lathosterol), the 6 24 -derivative "(demosterol), 65 17 -derivative (7-dehydrocholesterol) and others (Nes and McKean, 1977 and plants but also between particular classes of plants (Goad, 1977) .
Cholesterol is the major sterol of animals. The typical plant sterols are: campesterol, sitosterol and stigmasterol ( Figure 7). The addition of a C at C24 of the side chain distinguishes phytosterols f:t0m.animal sterols. It is now recognized that cholesterol (cholest-5-en-3B-ol} is widespread in the plant kingdom, but it usualLy constitutes only a few percent of the sterol mixture (Goad, 1977). Weinrauch (Riepma, 1970) .
~pes of margarines
First and most typical are margarines composed of vegetable oils (Massiello, 1978). A second is blend margarines which combine animal and vegetable fats in one F'°partion or another. They are commonly referred to as "AV" or "VA" products, depending on the predominance of animal ("A") or vegetable { "V") fat. The "AV" types are required by the standards to be between 50 and 90 percent animal fat.
A typical "AV" weight composition would be 90 percent pure lard and 10 percent soybean oil. The third is that of . margarines composed wholly of animal fats. Usually the fat content is sl ightly hardened pure lard {Riepma, 1970) . Today there are ten different types of margarine produced. There • are regular, ·::whipped and polyunsaturated margarines in both stick and soft forms. There are diet margarines, liquid aargarines and new 60% vegetable oil spreads. These products cater to the needs of many different segments of the population (Massiello, 1978). In recent years, medical research findings have suggested the advisability of increasing the ~roportion of polyunsaturated fatty acids in the diet and reduci.ng the intake of cholesterol. Parodi (1975)
Raw materials
The grease is collected from slaughtered animals. Then ) it is washed, minced and put into autoclaves with double jackets. Steam is blown in at normal pressure. The fat is drawn off, while the residue or greaves are collected on the screens of the autoclave. The fat is then crystallized slowly at about 30°C. The crystallized product, which is a granular mass, is filtered and pressed yielding about 60 percent liquid with melting point between 28 and 34° c.
The vegetable oils are obtained from oil-bearing seeds or fruits. The seed is crushed, heated moistened with a little steam and then pressed. The oil after that is refined, degurnrned, deodorized and hydrogenated. Hydrogenation consists of adding very pure hydrogen to oil, with a finely divided nickel catalyst (Van Stuyvenberg, 1969).
llll?tarine manufacture
The fats and oils in proper proportions are heated to 30 oc or more and oil soluble ingredients (emulsifiers, coloring agents, flavoring agents and vitamins) added. An aqueous phase containing milk or dried protein and water is prepared separately. After pasteurizing and cooling, salt and preservatives are added. The two phases are blended and the resulting emulsion is rapidly chilled in heat exchangers and allowed to stand in crystallizing chambers for 2 minutes until the product is sufficiently stable to be extruded and packaged. For soft tub margarine, the fluid melt is agitated during chilling to prevent fat crystals from growing into a firm network. The emulsifying agents (mono and diglycerides, sometimes lecithin) help to keep the water dispersed as fine droplets within the oil phase. Whipped margarines are produced by introducing nitrogen gas just prior to chilling and ~intaining vigorous agitation during the crystallizing step.
The low fat spreads with a higher water content require more emulsifying agents and more careful control of production conditions in order to maintain the water-in-oil emulsion (Brekke, 1980).
IJ!!lysis of sterols
Measuring cholesterol is one of the assays that have passed through several laboratory investigations and critical discussions since it was discovered and until now.
fenerally, any method for sterol determination involves one or more of the following stages: 1 Both free sterols and esterif ied sterols will be ex-23 tracted from biological tissue by common lipid solvents such as ethylether and chlorof~rm-methanol (Bligh and Dyer) and the alcohol and ester forms can be separated to some extent from other neutral lipids by silicic acid column chromatography and thin layer chromatography (Kates, 1972 andHeftman, 1975
Isolation of sterols
On saponifying the lipids with an alkali in alcoholic solution, the esterified fatty acids of the lipid, including the steryl esters, form alkali salts (soaps) and the liberated alcohols constitute the neutral fraction. After dilution with water, the higher alcohols and sterols are separated from the hydrolyzed material by extraction with an organic solvent such as petroleum ether (Kates, 1972) or ethyl ether (AOAC, 1980) which is immiscible in water.
The material recovered from the solvent forms the unsaponifiable matter. known as cholesterol digitonide (Cook, 1958). Digitonin is used frequently for the separation of 3B-hydroxy (precipi- After the precipitation is complete, it is necessary to split the digitonide into its components and to recover the The evaluation and criticism of digitonide formation method is as follows: A) The advantages are: (l) The low solubility of the complex makes the test very sensitive (2) The reaction has been used directly as a granimetric method for the determination of choleste·rol.
B) The disadvantages are: (1) The formation of insoluble complex is not restricted to digitonin but saponins, tiyonin and digitonin and the alkaloid formation give precipitates w·ith cholesterol.
Commercial digitonin frequently contains gitonin as impurity.
(2) Variable amounts of water of crystallization of the digitonide may affect the gravimetric results.
(3) Digitonin forms molecular complexes with many nonsteroid substances, e.g. phenols and terpene alcohols. After that, digitonin solution was added to precipitate the free cholesteroL. The dried precipitate of cholesterol diqitonide was dissolved in acetic acid. Next, the temperature of the water bath was adjusted to 25° and acetic anhydride was added followed by concentrated sulfuric acid.
The intervals between the addition of reagents was so timed that not less than 27 minutes nor more than 37 minutes elapsa:i between the addition of H2S0 4 and reading the color. -Once immobilized, an enzyme is of ten stable for weeks • or even months.
-It is easy to separate the insoluble enzyme derivative from reactants and/or products for reuse.
-The derivative can be packed in a column similar to those used in liquid chromatography or it can be used in a stirred tank with the product.
-The enzyme derivative has different catalytic properties from the enzyme and these properties can be controlled by immobilization conditions. Bottle (2) It should be noted that the procedure is not specific for tholesterol since cholesterol oxidase oxidizes any sterols in which the hydroxyl . group at carbon 3 is in the B-position.
'rherefore , phytosterols, such as stigmasterol and sitosterol, also react in the assay.
This is no longer used · in steroid chromatography, except for some polar steroid conj~gates. Other chromatographic sheets have also lost the race, because they cannot resist the aggressive reagents and solvents used for sterols.
Glass-fiber paper is unsuitable because its coarse texture produces excessive diffusion of chromatographic zones llllr layer chromatography.
Thin layer chromatography of steroids has reached a state of perfection where one can hardly expect to devise major improvements in developing and detection methods.
Evaluation of chromatography by computer methods is simply a device for exploiting the wealth of information provided by TLC (Johnson, 1973 Gas cnromatography is a process by which a mixture is separated into its constituents by a moving gas phase passing over a sorbent. It has produced sweeping changes in many fields of research since its introduction in 1952.
Basically, it is a separation process capable of extremely high resolving power. Its direct applicability is limited to compounds which can be voiatilized without decomposition, such as therinally stable, nonionic compounds with a maximum ~lecular weight of around 400-500. This powerful analytical method has contributed significantly to advances in many fields -for example, catalyst research, biochemical studies and flavor analysis. Scaled-up in size, gas chroma-mwraphytography also offers a method for preparing very pure compounds.
38
Grunwald (1970) Therefore, it is clear that different retention data demonstrate nonidentity, but identical data may be ambiguous. This is best shown for the case of sterols epimeric at C-24, because so far all analysis of such epimeric pairs of compounds by GLC have produced identical retention data for both isomers (Ikekawa, 19681 Although high pressure liquid chromatography has been used in analysis of lipids in general (Aitzetmuller, 1975} and in reference sterols separation (Colin ~ al., 1979}, its specific application in sterols methodology has been limited because cholesterol and related compounds absorb 4 _ Rees, et al. (1976) applied the HPLC to sterols separation. They recommended that the reversed phase separation of sterols on uBondapak c 18 c~n be applied to the preparative separation of sterols mixtures isolated from biological ~aterials by the conventional techniques of columns and thin layer chromatography. Colin, et al. (1979) have described a method for separating standard free sterols by high pressure liquid chromatography, using pyrocarbon modified silica gel column. Due to the limitation of their detector, they could not measure cholesterol at wavelength lower than 254nm, which is quite higher than cholesterol maximum absorption region for UV radiation. However, they recommended using this detector rather than Refractive Index detector. Newkirk, et al. (1981) have used high pressure liquid chromatography for determining cholesterol in foods.
-
They eluted cholesterol as its benzoate ester on uBondapak c 18 column. Detection was carried out with variable wavelength detector. When they compared their results with results obtained from gas chromatographic analysis, it was found that HPLC is a favorable technique. (2) Columns: uBondapak c 18 , 3.9mm x 30cm, and u-Porasil, 3.9mm x 30cm, Waters Associates.
Reagents
(1) HPLC solvents; acetonitrile, methanol, isopropyl alcohol, dichloromethane, and hexane were either Nanograde Dlallinckrodt) or Distilled in Glass (Burdick and Jackson) • UV-grade acetonitrile and isopropanol were used in some experiments. All solvents, including distilled water, were prefiltered through millipore or glass fiber filters.
(S) Petroleum ether and dichloromethane were Nanograde (Mallinckrodt) . l!terials (l} Beef fat, pork fat, vegetable oil and margarines were purchased at local supermarkets. Rapes~ed oil was extracted from crushed rapeseed. (2) Cholesterol, stigmasterol, campesterol and a plant aterol mixture were purchased from Applied Science, Supelco and steraloids.
IJlpo·nif ica tion
One gram of oil and/or margarine was weighed into a 50 ml glass tube equipped with Teflon-lined cap and 15 ml Of l.S M KOH in methanol was added. The tube was then sealed, kept in a water bath at l00°C for 20 minutes and allowed to· cool to room temperature. After cooling, 10 ml water and 1 5 ml petroleum ether were added. The tube was •haken for one minute and put in a centrifuge for three l'f.nutes at 2000 rpm to separate the PE layer from the soap.
The PE layer was then pipetted to a small beaker and evaporated to near dryness on a water bath and then under a stream of nitrogen.
lll"C of free sterols The residue of sterols obtained from PE evaporation was dissolved in 5 ml dichloromethane {DCM) , dried over anhydrous sodium sulphate and concentrated to 1 ml, from which 10 ul was injected into the LC.
Acety la tion • The sterol residue was dissolved in 1.2 ml acetic anhydride:pyridine, 2:1 (v/v) and transferred to a glass tube equipped with a Teflon-lined cap. The reaction solution was kept on a water bath at 37°C for 15 minutes and then on an ice bath for 2 minutes. After that, 0.6 ml PE, 0.6 ml acetone and 0.6 ml water were added. The tube was capped, shaken and put again in the 37°C water bath for 5 minutes.
Then the reaction solution was transferred to a small separatory funnel and washed four times with 2 ml water each.
The PE layer containing the sterols acetates was transferred from the separatory funnel, dried over anhydrous sulphate and concentrated to 1 ml, from which 10 ul was injected.
J!nzoylation
The sterol residue was dissolved in 1 ml acetone and transferred to a 20 ml qlass tube equipped with Teflonlined cap and 10 ml of 7.5 M sodium hydroxide in water and 0.2 ml benzoyl chloride were added. The tube was capped and shaken vigorously on a mixer for , 3 minutes. The reaction •ixture was then transferred to a separatory funnel containing 25 ml water and extracted two times with 15 ml DCM each.
The DCM extract was collected in another separatory funnel and washed two times with 20 ml of saturated sodium carbonate and one more time with 20 ml water. The DCM layer was then dried over anhydrous sodium sulphate and concentrated to 1 ml. Benzoates from standards were made by benzoylating 5 mg of the free sterol and diluting the final DCM extract to 10 ml . (4) The majority of runs were made with an attenuator ~etting of 0.1 and recorder chart speed of 0.5 cm/min.
1Pitial trials
To choose the column for this work both uPorasil and uaondapak c 18 , the two most common HPLC columns were tested for the resolution of free sterols and their benzoates. A variety of solvent systems were employed. All sterols showed sood elution individually with mixtures of isopropanol, acetonitrile, methanol, hexane and/or dichloromethane, but the separation of mixtures of sterols was better in the uBondapak c 18 column as shown later. Figure 9 shows the best separation obtained for free sterols on uPorasil.
Peaks 1 and 2 are solvent peaks, while Peaks 3 and 4 are unresolved sterols. Colin et al. (1979) also tried the nor--·mal phase for free sterols and found that lanosterol eluted first, then a whole group of mono-and di-unsaturated sterols and finally ergosterol which was well separated from others. The resolution of sterol benzoates on uPorasil was also poor {Figure 10). Accordingly, the use of such a column is not effective for routine analysis and the UBondapak c 18 (Figure 14) was the choice for this work.
The determination of sterols in unsaponified oil was attempted by dissolving 0.5 g of rapeseed oil or corn oil in 10 ml dichloromethane and injecting 10 ul into the column. Many peaks · followed this injection and so the chromatogram was confusing, as shown in Figure 11. Not only th e small amounts of free or esterif ied sterols (1% of are the total lipid) masked by other lipid components but injection of crude oil is impractical because it would require extensive analysis time to allow elution of all peaks before injecting another sample. Therefore, saponification was adopted for sterol determination.
Direct injection of a diluted (isopropanoll saponification mixture did not give good results due to the large • solvent peak, as shown in Figure 12. To prepare mobile phase (b} acetonitrile was added l+l to solvent system (a}. Figure 14 showed a good separation for corn oil sterols but stigmasterol unfortunately Still had a retention time similar to cholesterol. up clearlv, perhaps due to the fact that the proportion of. • • I phytosterols in corn oil (1%} is much higher than that of cholesterol in lard lO .1%} (USDA, 1979) . In Figure 17, 25% lard in corn oil could barely be detected. Figure 19. Acetates of cholesterol and stigrnasterol were successfully prepared by heating cholesterol and stigmasterol for 15 minutes in a water bath with acetic anhydride alone, but extracts of saponified oils did not chromatograph well a fter acylation. This approach was not pursued further for the following reasons: (1) The acetates of sterols • absorb UV radiation at very low wavelength (Grasselli and Ritchey, 1975) and were less sensitive than the benzoate derivatives, which could be analyzed at 230 nm, a more convenient wavelength, and (2) cholesteryl acetate could not be separated from some phytosteryl acetates. Rees et al. (1979 ) a sensitive detection at wavelength. 230 run and the benzoylation was reproducible. Fitzpatrick and Siggia (1973) used pyridine as solvent for the benzoylation reaction, while Blau and King (1978) describe a procedure using sodium hydroxide. Both procedures were attempted and use of sodium hydroxide was adopted for the following reasons: (1) Pyridine can form a compound with benzoyl chloride that might interfere in the chromatogram, (2) a lot of washings especially with hydrochloric acid are needed when pyridine is there. These are laborious and may cause loss of sample, and Gardner (1978). The agreement with the theoretical is reasonably good. Unfortunately, the peak of major interest, the "cholesterol peak," shows the most discrepancies. However, the patterns in different samples for the same kind of oil were shown to be reproducible ( Figure 26 and 27). Table 3 shows the results after analysis of several oil samples • .Analysis of mixtures of vegetable oils and lard 75 Cholesterol constitutes around 95% of the lard sterol content as shown in Figure 28 and in the literature (Dence, 1980) . Cholesterol is present in vegetable oils but at small aI\lOUnts, 0-4% of the totaL sterol (Weihrauch andGardner, 1978 andAppelqvist andOhlson, 1972). Vegetable sterols are not present in lard according to Figure 28 and Nes and McKean (1977). For the purpose of detecting animal fat in vegetable margarines, mixtures of lard and vegetable margarines were prepared at the ratios of 0, 25, SO, 75, and 100% lard. The sterols were extracted, benzoylated and analyzed by HPLC. Figures 29 and 30 and Tables 4 and 5 show the trend of cholesteryl benzoate peak to increase in accordance with the amount of lard added to soya bean oil margarine or corn oil margarine. From these results we can deduce that the cholesterol value is a good indication for the presence of lard in a vegetable margarine. If the peak exceeds 15%, it is highly likely that the margarine contains more than 25% animal fat. If the cholesterol is higher than 30%, the sample certainly contains over 50% animal fat. lard ana 75% margarine, tel 50% lard am 50% margarine, CD> 75% lard am 25% margarine, am (E) 100% lard. uBomapak c 18 column with nd:>ile phase acetoni trile 100% at flow-rate 2 ml/min am detection at 230 rm. (2) each.
• Two different margarines. Single determination of
Conclusion
The results obtained through this work have proved that high pressure liquid chromatography is a good tool for the analysis of sterols. Although, the separation was diff icult between cholesterol and some minor phytosterols, the detection of animal fat was possible. The utility of HPLC is practical when the fraudulation is more than 25% and critical at less proportions, especially if rapeseed oil were present in the margarine. However, HPLC performs separation' and detection at the same time without any preparative procedures such as thin layer chromatography or digitonin precipitation, which is a good factor for obtaining more accurate results. It is convenient for such routine analysis and time-savinq. Of its economical disadvantages, the deuterium lamp of the detector is expensive and it has only short life. What is recommended for achieving better results for this work is a column that can separate cholesterol from the entire vegetable oil sterol group.
Sterols standards were not abundantly available and their purity was often poor. So, it is recommended to have more advanced preparative procedures for obtaining less expens ive pure sterols, a task in which HPLC also could be a good tool. | v3-fos |
2018-12-13T06:55:46.285Z | {
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} | s2 | Seed Germination of North American Orchids. I. Native California and Related Species of Calypso, Epipactis, Goodyera, Piperia, and Platanthera
Seeds of native California and related orchids were germinated in vitro on five basal media and 43 modifications. Germination periods extended from several weeks to almost 2 yr. Seeds from immature capsules germinated faster and in higher proportions than those from mature fruits. The most suitable media for orchid germination are those devised for the culture of barley embryos (Norstog) and a modified Curtis medium containing urea and calcium carbonate instead of ammonium nitrate and calcium nitrate.
Introduction
Attempts to germinate seeds of terrestrial orchids have been partially successful (DOWNIE 1940(DOWNIE , 1949KNUDSON 1941, STOUTAMIRE 1965HADLEY 1970;HARVAIS 1974;FAST 1976;CLEMENTS and ELLYARD 1979). Methods successful for one species are not always applicable to others, and procedures for orchids from one region may not be suitable for those from another. Because of this and since they are much more dependent on their mycorrhizal fungi for germination than epiphytic species (STOUT-AMIRE 1974;WARCUP 1975), seeds of terrestrial orchids are difficult to germinate in vitro. Since the available information is mostly empirical, very little is known about the germination and development of terrestrial orchids, especially those from North Temperate regions (reviews by WITHNER ized by immersion in filtered calcium hypochlorite solution (7 g/100 ml distilled water) for 10 min and opened under sterile conditions; their contents were scraped and placed on the agar surface. Mature seeds were sterilized by soaking in the sterilizing solution for 10 min. Short glass tubes (cut from disposable pipettes) stuffed with cotton at both ends and repipetting bulbs were used to sterilize the seeds, to wash them with sterile distilled water, and to dispense them into culture flasks (HARRISON 1970;HARRISON and ARDITTI 1970).
Modifications of the Curtis medium (CURTIS 1936(CURTIS , 1943 were used for the germination of all species (tables 1, 2). Additional media were employed for Epipactis gigantea and Calypso bulbosa (tables 1, 2). The fungus, Rhizoctonia repens (provided by Dr. GEOFFREY HADLEY, University of Aberdeen) was cultured on a shaker (60 oscillation/min) in a defined medium (POWELL and ARDITTI 1975) for 1 mo, and the liquid was freed of hyphae by filtration. The filtrate was sterilized by Millipore filtration (pore size 0.22 j.m) and incorporated into culture media at 100 ml/liter. Seeds were germinated and seedlings maintained under several combinations of illumination, pH, and concentration and composition of culture media (tables 1-7).
Results
Seeds in these experiments germinated by first forming protocorms, which is typical for orchids (ARDITTI 1967(ARDITTI , 1979. In each species ca. 90%o of the protocorms were initially white, even under illumination, but turned green with time (tables 2-4, 6, 7). Some protocorms of Calypso bulbosa and Platanthera saccata were green from the outset (tables 3, 7). In Epipactis, Goodyera, Piperia, and Platanthera (the last two were at one time part of the same genus, Habenaria), rhizome formation generally occurred after protocorms appeared (tables 4, 6, 7). No rhizomes formed in Calypso (table 3) developed shoots before the appearance of rhizomes (tables 4, 6, 7). Roots appeared following the shoots in all species (tables 3, 4, 6, 7), after which the seedlings developed into small plantlets.
Mature seeds of C. bulbosa germinated only on the Norstog medium and on a modification of the Curtis solution. Immature seeds germinated on both fulland half-strength Curtis medium (tables 2, 3).
Epipactis gigantea seeds from immature capsules germinated better than those from ripe fruits (table 4). Ethrel at 5-20 mg/liter improved germination only marginally, and the solvent, 20 ml/liter of 70%7 ethanol, had a similar effect (table 4). There was no germination on media containing 2.5 g/ liter of Ethrel or 2 g/liter of graphite. Germination does not seem to have been affected by changes in pH ranging from 5 to 7.5 (table 4). Seeds of E. gigantea germinated well on the Norstog medium; those of E. atrorubens did not.
In the dark, protocorms were always white, and most turned green on being moved to light. However, some protocorms produced by immature seeds of E. gigantea (tables 4, 5) developed white rhizomes, roots, and leaves and formed plantlets in small bushy clumps ca. 3 cm high. These clumps increased in diameter and number of shoots but not in height, and some have not turned green after 2 yr of culture Curtis (1936Curtis ( , 1943, HC = half-strength Curtis (1936Curtis ( , 1943; MC = modified Curtis (1936Curtis ( , 1943, same medium as full-strength Curtis except that CaNOs and NH4NOs were replaced with 250 mg/liter urea and 148 mg/liter CaCOa; N = Norstog (1973); RE4 = Robert Ernst medium minus peptone plus vitamins and hormones; RE6 = Robert Ernst, personal communication; vit = vitamins; WS = Wolter-Skoog (1966). The listing of two or more media (separated by commas) is an indication of similar results. The temperature was 22 + 2 C for both light and dark cultures; light intensity was ca. 0.8 mw/cm2; photoperiods were 16 h. For details see tables 4-7.
c Seeds from immature capsules. d Seeds from mature capsules.
(tables 4, 5). The addition of naphthaleneacetic acid (0.1 mg/liter) and benzyladenine (1 mg/liter) separately and together or only kinetin (1 mg/liter) had no visible effects on color and growth of these seedlings (table 5). Irrespective of medium, some of them spontaneously produced green shoots and roots after ca. 18 mo of culture. Seed lots of G. oblongifolia differed in percentage of germination. The addition of cytokinins or vitamins improved the germination of some seeds. On fulland half-strength Curtis medium plus cytokinin, germination was better in the dark. Goodyera tesselata formed plantlets faster than G. oblongifolia (table 6).
Except in one instance, seeds of Platanthera saccata germinated poorly within 6 mo (tables 2, 7). Percentage of germination on fulland half-strength Curtis medium was not affected by illumination. On a modified Curtis medium, germination was better under illumination than in the dark. Addition of Rhizoctonia repens filtrate did not enhance germination in either the dark or light (table 7). Platanthera hyperborea and Piperia maritima germinated on the Norstog medium while P. saccata did not; P. hyperborea germinated rapidly but at a low percentage on the modified Curtis medium. Piperia elegans produced only white protocorms on a vitamin-enriched Curtis solution (table 7).
Several times, seeds of P. saccata on a Curtis medium under illumination formed protocorms which developed enlarged roots, 6-8 cm long, with numerous absorbing hairs. Green shoots remained 1 cm long or less or were entirely absent (table 7). Seeds in one culture (of two) from a single batch germinated underneath the agar surface and de-veloped into white protocorms. The protocorms enlarged but did not differentiate further or produce chlorophyll until they were transferred to the surface of fresh medium (table 7). Enlarged roots with very small shoots also developed when P. saccata was cultured on half-strength Curtis medium or full-strength Curtis solution with vitamins (table 7). Seeds on the vitamin-enriched solution germinated in the dark and were subsequently moved to the light. Piperia and Platanthera protocorms also developed in a manner similar to that of other orchids.
Discussion
Orchid seed germination differs from that of other seeds because of the absence of an endosperm, radicle, and leaf rudiments. Swelling of the embryo is followed by the formation of a round, top-shaped body called a protocorm. Other organs subsequently appear (see ARDITTI 1967ARDITTI , 1979. We have defined germination (tables 2-7) as the formation of green or white protocorms. Development is discussed in terms of the appearance of chlorophyll, absorbing hairs, rhizomes (in some species), shoots, and roots (tables 2-7).
Calypso bulbosa seeds from ripe capsules germinated very poorly or not at all (table 3). However, the germination of immature seed was as high as 80% (table 3). Differences were also evident in the germination of ripe and unripe seeds of Epipactis gigantea (table 4). This suggests that seeds of these species become dormant as they mature, which is in line with previous reports regarding orchid seed dormancy, suggesting it may exist only in some sp ecies (CURTIS 1936;VERMEULEN 1947;KNUDSON Cd - ; Z4 1950;LIDDELL 1953;MCINTIRE, VEITCH, and WRIGLEY 1972;WILDHABER 1974;STOUTAMIRE 1974;WRIGLEY 1976;ARDITTI 1979). Another possibility is simply a decrease in viability with maturation. Illumination reduced germination in C. bulbosa and E. gigantea and had a varied effect on Goodyera oblongifolia, G. tesselata, and all species of Piperia and Platanthera (tables 2-4, 6, 7). Seeds of epiphytic orchids germinate in both the light and dark but require illumination for further development (AR-DITTI 1967, 1979, which is also true for some terrestrial species. Other terrestrials germinate best in the dark or are inhibited by light (ARDITTI 1967(ARDITTI , 1979STOUTAMIRE 1974). Thus, generalizations regarding the effects of light on orchid seed germination are not possible because of insufficient information. Our findings may require a reexamination of the view that the inhibitory effect of light on the germination of seeds of North Temperate terrestrial orchids is a protective mechanism (STOUTAMIRE 1974). If it is, two obvious questions are why (a) seeds of epiphytic and tropical terrestrial orchids, which can also be subject to drying, do not have a similar mechanism and (b) only some terrestrial species germinated as part of this project seem to be inhibited by light. An answer to both seems to be that, if a protective mechanism exists, it is not universal even among terrestrial species of North Temperate origin.
The inconsistent effect of vitamins on the germination of G. oblongifolia, Platanthera saccata, and Piperia elegans (table 6) is in line with previous reports regarding other orchids (WITHNER 1959(WITHNER , 1974ARDITTI 1967ARDITTI , 1977ARDITTI , 1979STOUTAMIRE 1974). Also, like other orchid seeds (ARDITTI 1967(ARDITTI , 1979, (table 4). Plant hormone effects on the germination of orchid seeds (especially terrestrial species) differ (WITHNER 1959(WITHNER , 1974ARDITTI 1967ARDITTI , 1979HADLEY 1970;HARVAIS 1973;STOUTAMIRE 1974;TAMANAHA, SHIMIzu, and ARDITTI 1979). Our results are similar: Auxins, cytokinins, or Ethrel seems to hinder the germination of E. gigantea and enhance that of G. oblongifolia in the dark (tables 4, 6). In E. gigantea the effects of Ethrel are marginal and similar to those of the ethanol used as solvent (table 4). If the slightly enhanced germination is an ethanol effect, it confirms previous reports (THURSTON, SPENCER, and ARDITTI 1979). However, our findings should be treated with caution since the germination rates were very low and because 2.5 mg/liter of Ethrel had no effect at all.
Albino seedlings of nonsaprophytic orchids (VAJRABHAYA 1977) usually do not survive for long. Therefore, the development of white seedlings of E. gigantea is not unusual, but their survival is. If they are genetic albinos, it is also not surprising that the addition of hormones and complex additives to the culture media failed to induce chlorophyll formation (table 5). The spontaneous appearance of green shoots suggests that at least some of these seedlings are not genetic albinos. In nature a certain proportion of Epipactis are chlorophyll free and either perish or are parasitic on their endophytes. Furthermore, it is possible that some or all of these seedlings turn green as a result of a stimulus provided by the fungus.
Disproportionally large roots and very small shoots, like those of P. saccata seedlings, have occasionally been observed in cultures of epiphytic genera. Therefore, it is possible that this growth pattern is not typical for the species.
Seeds of some terrestrial orchids are difficult to germinate asymbiotically (ARDITTI 1967(ARDITTI , 1979STOUTAMIRE 1974) and others are not (HADLEY 1970;HARVAIS 1973;FAST 1976;CLEMENTS and ELLYARD 1979). Therefore, our findings suggest that procedures for the germination of different species can be developed. | v3-fos |
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} | s2 | Serum-free growth of normal and transformed fibroblasts in milk: differential requirements for fibronectin.
Bovine milk may be used as a supplement for the serum-free growth of certain fibroblastic cells in culture. The growth properties of three representative cell types in milk-supplemented medium were examined; fibroblastic cell strains, fibroblastic cell lines, and transformed fibroblasts. Transformed fibroblasts, which included RNA and DNA tumor virus-transformed cells and carcinogen-transformed cells, grew in milk. Instead of growing attached to the culture dishes, as they normally do in serum, transformed fibroblasts grew in milk as large clusters in suspension. In contrast, nontransformed fibroblastic cell strains and cell lines did not grow in milk-supplemented medium. Fibroblasts transformed by a temperature-sensitive transformation mutant of Rous sarcoma virus were temperature-sensitive for growth in milk. The failure of cells to adhere to the substratum in milk-supplemented medium suggested that milk might be deficient in attachment factors for fibroblasts. When the attachment of fibroblastic cells in milk- supplemented medium was facilitated by pretreating culture dishes with fibronectin, (a) transformed cells grew attached rather than in suspension, (b) normal cell lines attached and grew to confluence, and (c) normal cell strains adhered and survived but did not exhibit appreciable cell proliferation.
The physiological function of milk is to provide substances important to the nutrition, growth, and development of the newborn. In addition, mammalian milk contains viable cells (1, 2) . Both human and bovine milks contain up to 4 X 10 6 cells/ml, primarily macrophages, T lymphocytes, and B lymphocytes. In previous studies, we demonstrated that both human (3,4) and bovine (5) milks contain polypeptide growth factors that stimulate DNA synthesis and cell division of confluent cultures of fibroblasts. The presence of mitogens and viable cells in milk prompted us to evaluate milk as a replacement for serum in cell culture. In an initial report, we demonstrated that milk could support the long-term growth of epithelial cells in culture (6). Only colostrum, a highly enriched milk produced by mammals in the first day or two after birth of the newborn, was effective in supporting epithelial cell proliferation. In addition, colostrum was selective, supporting the growth of epithelial cells but not fibroblasts. When plated in either colostrum or regular milk, fibroblasts adhered very poorly to the substratum, suggesting that milk lacks factors that are necessary for the attachment of fibroblasts. This ob-294 servation prompted us to investigate whether milk would be selective for the growth of cells that do not require attachment to the substratum to proliferate. In this report, we show that transformed fibroblasts grow readily in milk-supplemented medium as suspension cultures . On the other hand, normal fibroblasts do not grow in milk unless the attachment of cells is mediated by an adhesion-promoting factor, such as fibronectin .
Cell Culture
Rat embryo cultures (LRI) prepared from Lewis rat embryos and three Schmidt-Ruppin D (SRD) Rous Sarcoma virus (RSV) transformants of Lewis rat embryo cells (LR3/1, LR3/2, and LR3/3) were described previously (7). F2408 cells, which are in established cell line derived from Fischer rat embryos (8), were provided by Dr . C. Basilico, New York University School of Medicine, New York. FRD-4 is an SRD transformant of F2408 cells infected and cloned as described previously (7). FR24D-1 cells were obtained by transforming F2408 cells by a subgroup D recombinant of LA24, a temperature-sensitive transformation mutant of RSV (see below) . Kirsten Sarcoma virus-transformed NIH 3T3 cells (KNIH) and SV40-transformed 3T3 cells (SV29) were gifts of Dr . C. Scher, THE JOURNAL OF CELL BIOLOGY " VOLUME 88 FEBRUARY 1981 294-300 Sidney Farber Cancer Institute, Boston, Mass . Mouse sarcoma 180 cells were purchased from American Type Culture Collection, Rockville, Md.
Avian cells and viruses were propagated by standard techniques (9). Virus strains and avian cells have been previously described (7,10). The procedures for host range studies, interference assays, and virus cloning were as described previously (l0) .
The subgroup D recombinant of LA24 was constructed by infecting C/E chick embryo cells (CEC) with LA24 and the subgroup D leukosis virus, RAV-50. 48 h later, the virus produced by these cultures was harvested and used to infect C/AE CEC. Harvests from C/AE CEC were collected and cloned repeatedly on C/AE CEC. After six cycles of cloning, stocks of virus were grown up on C/E CEC and the subgroup of the virus was examined by host range and interference assays . Stocks of this recombinant, designated LA24D, exhibited only subgroup D properties. F240ß cells were transformed by LA24D, cloned . and grown into cultures by the method described previously (7).
Stock cultures of all cells were routinely cultured in Dulbecco's modified Eagle's medium (DMEM, Grand Island Biological Company [GIBCO], Grand Island, N. Y.) containing glucose (4 .5 g/liter), penicillin (50 U/ml), and streptomycin (501rg/ml) and supplemented with 10% calf serum (Colorado Serum Co ., Denver, Colo .) . Cells were subcultured after treatment with 0.25% trypsin, 0.2% EDTA (TE, GIBCO) . Transformed cells were subcultured twice weekly and normal cells (both strains and lines) every 3-4 d as needed, with care to see that the cultures never grew to confluence. Stocks of the temperature-sensitive transformed cell line (FR24D-1) were cultured at the permissive temperature (35°C) . All of the other cells were routinely grown at 37°C.
Preparation of Milk for Cell Culture
Milk and colostrum were prepared for use as a medium supplement for cell culture as described previously (5,6) . Colostrum was obtained within 24 h after birth of the calf, whereas regular milk was obtained randomly at various stages in the lactation period . For cell culture, fresh bovine milk was obtained immediately after milking. Within several hours, the milk was centrifuged at 12,000 g for 30 min to remove cellular debris (pellet) and fat (floating on the top) . The defatted milk was frozen at -20°C. For cell culture, milk was thawed, diluted in DMEM (20% vol/vol, or less), and filtered through 0.45-lam Nalgene filter units (Nalge Co., Nalgene Lobware Division, Rochester, N. Y.) for sterilization .
Growth Studies
Cells were detached from culture dishes by incubation with l ml of trypsin-EDTA (TE). An equal volume of soybean trypsin inhibitor (Type 1-S, Sigma Chemical Co., St . Louis, Mo., 0.25% in phosphate-buffered saline) was added to stop the reaction . The cells were diluted to 10 ml in unsupplemented DMEM, and the cell suspension was centrifuged at 800 rpm in an International IEC centrifuge (International Equipment Co ., Needham Heights, Mass.) at room temperature. The pellet was resuspended in DMEM, and the cell concentration was determined with a Coulter model Zf electronic particle counter (Coulter Electronics, Inc., Hialeah, Fla.) . Aliquots of cell suspension were added directly to medium containing the desired supplement (milk, colostrum, serum, or unsupplemented) to give a final cell concentration of 2 x 10" cells/ml . The cell suspension (0 .5 ml) was placed into 24-well microtiter plates (16 mm diameter, Costar, Data Packaging, Cambridge, Mass.) to give an initial cell density of 1 x 10°cells/well (5 x 103 cells/cm) . Because cells remained in suspension in milk, cultures were fed (every 3-4 d) by adding 0.5 ml of fresh medium to each well without replacing the supernates, except where indicated otherwise.
For cell counting, the culture supernates were transferred to Coulter counting vials containing an equal volume of TE. To each well, 1 ml of TE was added to dissociate the remaining cells in the well. The plates and the counting vials were incubated at 37°C for --10 min. At the end of the incubation period, 0.5 ml of calf serum was added to each well, the cell suspension was vigorously pipetted with a pasteur pipette, and the suspension pooled with the supernate-TE mixture in the Coulter vials . The cell suspension was diluted to 10 ml with Isoton II (Curtin Matheson Scientific Inc., Woburn, Mass .), and cell counts were determined by Coulter counting.
Passaging of Cells in Milk
Transformed cells growing in suspension in milk-supplemented medium were mixed with an equal volume of TE and incubated at 37°C for 15-20 min. Singlecell suspensions were o~jained by vigorously pipetting thecultures with a pasteur pipette. The cells were centrifuged from this suspension and resuspended in medium supplemented with 10% milk or 10% calf serum to give a final concentration of 1 x 10°cells/well in Costar 24-well microtiter plates.
Fibronectin Treatment of Culture Dishes
Human plasma fibronectin (Collaborative Research, Inc., Waltham, Mass.) was suspended in DMEM (25 lag/ml) and 0.4 ml was added to each well of 24well microtiter plates (final concentration -5 lag/cm) . The plates were incubated at room temperature for a minimum of 30 min, the supernates were aspirated, and the cell suspension was added immediately.
Histology and Photography
Clusters of cells growing in milk were fixed in glutaraldehyde (2%), postfixed in OsO,, and embedded in Epon-Araldite resin with dimethylaminomethyl phenol-30 (Ladd Research Industries, Inc., Burlington, Vt.). Thick sections were cut and stained with Azure II-methylene blue.
Cells were photographed under phase in a Nikon model MS inverted-phase microscope.
Growth of Cells in Milk
The growth properties of a fibroblastic cell strain (LR1), a fibroblastic cell line (F240ß), and a Rous sarcoma virus-transformed fibroblastic cell line (LR3/1) in medium supplemented with various concentrations (0-20%) of milk, colostrum, and serum were examined ( Fig . 1) . RSV-transformed cells grew in all three media . The cell density at the optimal concentration of colostrum was -r30% of that observed in serum . In milk, the transformed cells grew to a density -12% of that achieved in serum . Furthermore, the final cell number in colostrum represented a 50-fold increase and in milk represented a 20-fold increase over the initial plating value . On the other hand, the cell strain and cell line grew in serum but not in milk or colostrum at any of the concentrations tested. The approximate generation times for RSV-transformed cells growing in milk, colostrum, and serum were 31, 22, and 14 h, respectively (not shown).
Growth Properties of Temperature-sensitive RSV-transformed Cells in Milk
In general, cells transformed by temperature-sensitive transformation mutants of RSV are temperature-sensitive for a variety of properties considered to be in vitro markers for cellular transformation (11) . If growth in milk and colostrum is a phenotype associated with cellular transformation, then it should also be temperature-sensitive. F2408 cells transformed by a subgroup D recombinant of a temperature-sensitive transformation mutant of Rous sarcoma virus were cultured in various concentrations of milk, colostrum, and serum at 35°C (permissive temperature) and 39°C (nonpermissive temperature) (Fig . 2) . At 35°C, the temperature-sensitive cells (FR24D-1) grew well in all three media, consistent with the growth response observed for wild type virus-transformed cells. At 39°C, the temperature-sensitive transformed cells exhibited growth behavior similar, but not identical, to that of the normal parental cell line (F2408). They grew well in serum, not at all in milk, but, unlike the parental cells, they showed some growth in the higher concentrations of colostrum at 39°C .
Growth curves of the temperature-sensitive cells in 10% milk, colostrum, and serum are shown in Fig. 3. These cells grew well at the permissive temperature in serum, colostrum, and milk . The generation times at 35°C in serum, colostrum, and milk were 15, 22, and 29 h, respectively . At 39°C, these cells grew in serum with a doubling time of 18 h but did not grow at all in colostrum or milk . The lack of growth in milk at 39°C was not caused by cell death. Cultures incubated at 39°C in milk for various periods of time, up to 7 d, were shifted down to 35°C . In every case, growth resumed following a lag period of several days at a rate comparable to that observed with cultures incubated at 35°C for the entire experiment. In contrast, temperature-sensitive cells incubated in unsupplemented medium at 39°C failed to survive (data not shown) .
Long-Term Passage of Transformed Cells in Milk
The temperature-sensitive transformed cells can be subcultured at the permissive temperature in medium supplemented with milk. In one series of experiments, cells were subcultured 12 times over 5 mo (-60 population doublings) before the CONCENTRATION (%vollvol) study was terminated . Growth kinetics were examined periodically and the generation times in milk remained constant (-30 h) . In addition, these cells remained temperature-sensitive for growth in milk and displayed the characteristic temperaturesensitive properties when transferred to medium containing serum (not shown) .
Phase contrast photomicrographs of the growth patterns of RSV-transformed cells in serum, milk, and colostrum are presented in Fig. 4. In serum-supplemented medium, transformed cells grew attached to the dish (Fig. 4 a) . However, in colostrum (Fig. 4b) and milk (Fig . 4 c and d), transformed cells grew as large clusters in suspension . There was no attachment of transformed cells to the culture dishes in regular milk at concentrations ranging from 0 to 30%. In colostrum, there was no attachment of transformed cells at concentrations from 0 to 10% but some attachment (<10% of the cells) occurred at higher concentrations . In milk-supplemented medium, the size of the cell clusters increased with time in culture. Relatively small clusters were seen in the first 5 d (Fig . 4 c) . By 10 d, some of the clusters approached 1 mm in diameter (Fig . 4d). When serum was added to the transformed cells growing in milk, the clusters attached to the dish, spread, and the cells grew out from the clusters to form confluent monolayers (Fig . 4e). There was no evidence of necrosis at the center of large clusters, as shown in the histological section (Fig. 4f).
A comparison of the growth responses of a variety of transformed cells in medium supplemented with serum, colostrum, and milk are presented in Table I. Cell lines transformed by RSV, Kirsten sarcoma virus, SV40, and by a carcinogen were tested . Transformed cells all grew well in colostrum, reaching saturation densities ranging from 15 to 50% of that attained in serum. These transformed cells also grew in milk, but not so well as in colostrum. There was no growth of any of these cell lines in unsupplemented medium . In milk and colostrum, all of these transformed cell lines grew in suspension as large cell aggregates, but in serum they grew attached to the dish . FIGURE 4 Comparison of the morphology of RSV-transformed cells growing in serum, colostrum, and milk . RSV-transformed cells growing in the various media were photographed after 10 d in culture, except where indicated otherwise. a, 10% calf serum (LR3/ 1) ; b, 10% colostrum (LR3/1); c, 10% milk, day 5 (LR3/1) ; d, 10% milk (LR3/1) ; e, FR24D-1 cells were grown at 35°C in 10% milk for 7 d followed by the addition of calf serum and photographed 3 d later; f, histology of a cluster of LR3/1 cells growing in 10% milk .
Effects of Fibronectin on the Growth of Cells in Milk
Transformed cells grow in suspension in medium supplemented with milk or colostrum but grow attached to the substratum when cultured in medium supplemented with serum. This may be caused by the presence in serum of attachment factors that are absent from milk . One factor present in serum that has been found to mediate the adhesion of a variety of cell types to either plastic or collagen substrata is fibronectin (12,13). The fibronectin content of milk and colostrum was analyzed . Fibronectin like material was detected in colostrum but not in older milk . However, the amount of fibronectin in colostrum was considerably less than in serum (Steimer et al ., manuscript in preparation) . The apparent lack of fibronectin in milk prompted us to test the effects of adding plasma fibronectin to milk and colostrum-supplemented cultures.
Tissue culture dishes were coated with 5 tttg/cm 2 of human plasma fibronectin, also referred to as cold-insoluble globulin (CIG) (14,15). Temperature-sensitive RSV-transformed cells were plated at the permissive and nonpermissive temperature in the presence and absence of CIG. In serum, these cells grew attached to the dish at both temperatures (Fig . 5 a and b) . The coating of culture dishes with CIG had a dramatic effect on the morphology and growth response of cells in milk-supplemented medium. At 35°C in the absence of CIG, these cells grew in suspension and formed clusters (Fig . 5 c) . At 39°C, the temperature-sensitive cells remained in suspension without growing (Fig . 5 d) . When plasma fibronectin was added to the temperature-sensitive transformed cells cultured in milk at 35°C, the cells grew attached to the substratum rather than in suspension (Fig . 5 e) and assumed a transformed morphology similar to that in serum. At 39°C, CIG promoted the attachment of the temperature-sensitive transformed cells in milk (Fig . 5f). The cultures grew to confluence and the morphology of the cells was similar to that of normal cells growing in serum.
The results of growth studies of the temperature-sensitive transformed cells in the presence and absence of CIG are shown in Table II . In serum, the temperature-sensitive transformed cells grew at both temperatures . The addition of CIG had no effect on cell growth. In milk, there was growth at 35°C in either the presence or absence of CIG. At 39°C, there was growth only when CIG was present. There was some attachment of the temperature-sensitive transformed cells at 39*C in colostrum without exogenous CIG. These attached cells proliferated, resulting in a final cell number -10% of that found in colostrum in the presence of CIG. Fibronectin alone was not sufficient for cell growth because cells did not grow in unsupplemented medium on CIG precoated dishes .
Normal cell lines and cell strains did not grow in medium supplemented with milk (Fig . 1) . The growth behavior of these cells in milk with CIG was examined . The cell line F2408 and the cell strain LRI were cultured in the presence and absence of CIG in unsupplemented medium and medium supplemented with either serum, milk, or colostrum (Table III) . In serum, both cell types grew equally well whether or not CIG was added. However, in milk-or in colostrum-supplemented medium, the normal cells grew only in the presence of CIG. The addition of CIG increased the saturation density of F2408 Cells of each cell type (1 x 10°) were plated in 24 well microtiter plates in medium supplemented with 5, 10, or 20% of either milk, serum, or colostrum . The cultures were incubated for 10 d with feeding every 3 d. On day 10, all cultures were counted. Each value represents the average of duplicate wells. ND, Not determined . ' The optimal serum concentration was 10%. $ The optimal milk concentration was 20%, except SV29 which required 30% . § The optimal colostrum concentration was 10%, except LR5/1 and KNIH which required 5% . 298 THE JOURNAL OF CELL BIOLOGY " VOLUME 88, 1981 cells by 75-80-fold in either milk or colostrum. In the presence of CIG, F2408 cells grew with a generation time of 29 h in milk as compared with a generation time of 24 h in serum (not shown) . Although LRI cells required fibronectin to attach and survive in milk or colostrum, growth was minimal (Table III) . The cell number, as compared with the initial plating density, represented only 40 and 60% increase in colostrum and milk, respectively .
DISCUSSION
Milk is a source of growth factor activity (3)(4)(5) and can be used as a supplement for growing certain cell types in culture (6). However, the growth response of a variety of cell types is much different from that observed in serum. The growth properties of rat cells representing three cell types were examined: fibroblastic cell strains, fibroblastic cell lines, and transformed fibroblasts. There were several outstanding differences between the growth behavior of these cells in milk-and that in serumsupplemented medium. (a) Unlike serum, milk is selective and only allows transformed cells to grow. (b) In milk, transformed cells grow in suspension rather than attached to the culture dish as they do in serum. (c) Normal cells grow in milk only when attachment factors are provided .
The differences in the growth behavior of fibroblasts in milk and in serum may, in part, be attributed to differences in fibronectin content. Fibronectin like material can be detected in colostrum but not in older milk . However, the amount of fibronectin in colostrum is only 4-5% of that found in serum (Steimer et al ., manuscript in preparation) . This deficiency of fibronectin does not prevent the growth of transformed cells. These cells will grow in milk as suspension cultures . However, normal cells must attach to the substratum to proliferate. Attachment factors, such as fibronectin, must be added for normal fibroblasts to grow in milk-supplemented medium . Milk and fibronectin are all that are required to replace serum for the growth of a normal fibroblast cell line . However, fibroblast cell strains are more fastidious and require additional factors for growth that are absent from milk . Experiments in progress indicate that the addition of various factors, such as transferrin, epidermal growth factor, or insulin, will improve the growth of cell strains in milk .
Colostrum is a specialized milk produced in the first days after birth. Colostrum is highly enriched in protein and fat (16) . In addition, the growth factor activity is much greater than that of milk produced later in the lactation period . For example, colostrum at a concentration of 2% contains more growth factor activity than regular milk at a concentration of 20% (5). In general, colostrum is a better growth supplement for cell culture than regular milk. Transformed cells grow at a faster rate and reach a higher density in colostrum-supplemented medium . In addition, normal cell lines cultured on fibronectin-coated dishes in colostrum-supplemented medium reach a higher saturation density than in milk . Colostrum contains a small, but detectable amount of fibronectinlike protein. The presence of fibronectin may explain why there is a limited amount of attachment and growth of normal fibroblast lines in colostrum in the absence of exogenous fibronectin .
The fibronectin-free property of milk may provide several advantages for studying the growth of cells in culture. Milk as a supplement may be useful for assaying the ability of attachment factors to promote adhesion of various cell types. For example, the effects of fibronectin on the adhesion and proliferation of nonfibroblastic cells, such as epithelial cells, may be studied . In addition, the effectiveness of other attachment factors, such as chondronectin (17) or laminin (18), on the attachment and proliferation of homologous and heterologous cell types can be assayed. Such experiments cannot be done with serum supplementation without first removing the fibronectin. The fibronectin-free characteristic of milk may be used to select for the growth of certain cells in culture . For example, transformed fibroblasts that are anchorage independent (19) grow in milk but normal cells that are anchorage dependent fail to grow . In a mixed population of normal and transformed cells, it may be possible with the use of milk to preferentially select for the transformed cells . In addition, milk may be useful for culturing other anchorage-independent cell types, such as chondrocytes (20) . Milk is an inexpensive, readily obtainable growth supplement for cells in culture whose potential uses should be explored . | v3-fos |
2018-04-03T02:49:23.484Z | {
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} | s2 | Characteristics of fall-out plutonium in soil.
Two kinds of samples have been tested to investigate the adsorption of plutonium in soil under natural conditions. In a soil sample containing only about 5% of organic materials, no significant leaching of plutonium was observed with three kinds of extractants, namely, 1N-ammonium acetate, 5%-EDTA and 0.1N-sodium citrate, while some plutonium was leached with 0.1N-citric acid. The results of solubility tests made with natural soil organic acids, namely, humic and fulvic acids, showed that solubilization of plutonium by these acids is unlikely to occur in this soil sample. On the other hand, in another soil sample rich in organic materials (about 50%), significant leaching of plutonium was observed with all the extractants tested. The leaching of plutonium was especially very high with 0.1N-sodium citrate (about 60%) ; sodium citrate showing an initial pH of 8.4 probably solubilized some of organic materials present. Experiments made on extraction of organic materials with alkali solution also suggested that as much as 60-70% of plutonium in this soil sample was associated with some organic acids, whose carbon content was assumed to be 30-40% of the total organic carbon in the soil sample. This finding is very important from a view point of the uptake of plutonium by plant because plutonium associated with such organic acids is presumed to exist abundantly in cultivated soils which are quite rich in organic materials.
characterization study over soil plutonium is now intensively desired to predict its dis tribution and biological availability. This paper describes the characteristics of fall-out plutonium in soil under natural conditions.
Soil samples accumulating rather high levels of fall-out radionuclides were collected in the coastal area of Japan Sea where much snow-fall occurs in winter sea son, and solubilities of plutonium in these samples with natural organic acids, mineral acids and chelate agents were examined.
Furthermore, the fraction of plutonium associated with organic materials in soil samples was estimated.
Sample
Surface soil samples were collected at two places where relatively high levels of fall-out radionuclides were accumulated.
One sample was collected from the ground surface under the eaves of temple at Kohno (35°30'N, 135°29'E) in southern part of Fukui Pref. Snow and rain water from the large roof must pass over the ground sur face. Another sample was collected from a moderately flat ridge about 1100m above sea level at Mt. Kanmuri (35°46'N, 136°25'E) in Fukui Pref. The soil from Kohno con tains only about 5% organic materials, and against this, the soil from Mt. Kanmuri has an exceptionally high concentration of organic materials, reaching about 50%. Besides these soil samples, a sea sediment sample was collected from the sea floor of Nyu Bay (35°42'N, 135°58'E) in Fukui Pref. All of these samples are the same as those described in previous report", in which are shown the depth profiles and the particle size depen dences of several fall-out radionuclides, such as ....... Pu, "'Am and 137 Cs. After large rocks and plant frogments were removed, the soil samples were air-dried and then were sieved through a 500-rim screen. Kohno sample was furthermore sieved through a 250-tm screen to remove finer pebbles. As for sea sediment, the fraction below 37 Pm was used for study because previous work') had shown that plutonium was concentrated in the smaller size fractions.
Each specimen was blended for 24 hr by a ball-mill, and the following contents of plutonium (239.24.Pu) were found respectively for each sample, that is, 0.108 pCi/g. dry for Kohno sample, 0.495 pCi/g. dry for Mt. Kanmuri sample and 0.188 pCi/g. dry for Nyu Bay sample. The humic and fulvic acids used for the leaching tests of plutonium in soil sample were extracted from the two kinds of soils. The method of extraction and purification was reported in detail elsewhere"'.
Elementary compositions of humic and fulvic acids and their ash contents are shown in Table 1. Infrared spectra of these organic acids were measured. They were similar to those reported by Kumada and Aizawa11,12, General Experimental Method 1) Solubility of soil plutonium : Solubility of soil plutonium with several selected reagents were tested in the following way. Each soil or sediment sample of 10g was suspended in the reagent solution of 100 and/or 300 ml in a polyethylene bottle. The bottle was sealed, and then was continuously shaken for 20 hr (72 and 300 hr partly) being kept at 27±1°C by a thermostat.
Alter this procedure, the suspended soil was separated by centrifugation at 36,000 rpm for 60 min. This ultra-centrifugation makes even colloidal clay particle precipitate.
Since the plutonium content in the leachate was expected to be extremely low, the sedimentary residue obtained was subjected to a radiochemical analysis for 239° 240Pu In the leaching experiments with soil organic acids, each 10 g of the Khono sample was suspended in the organic acids solution of 100 ml or 150 ml in the polyethylene bottle. The bottle was sealed, and then was continuously shaken for 20 to 330 hr being kept at 27±1°C by a thermostat.
The sedimentary residue obtained by centrifugation was subjected to a radiochemical analysis for 239,240Pu. The content of organic acid in the leachate was determined spectrophotometrically, and pH determination was also made.
2) Plutonium associated with soil organic acid : To ascertain the presence of plu tonium in organic fraction, soil samples were treated with alkali to extract organic acids. Both 0.1 N-sodium hydroxide solution and mixed solution of 0.1 N-sodium hydro xide and 0.1 N-sodium pyrophosphate (expressed as mixed solution hereinafter) were used as reference extractant for organic acids. The flow scheme of this experiment is shown in Fig. 1. The resultant three fractions, namely, the sedimentary residue, humic and fulvic acid fractions, were subjected to a radiochemical analysis for 239, 240Pu and the determination of the amount of total organic carbon as follows.
3) Analyses of ....... Pu and total organic carbon in each fraction : The following procedure of the radiochemical analysis of plutonium is almost the same as that des cribed in a previous reports'. Plutonium in the sedimentary residue was leached out by hot concentrated nitric acid after adding the known amount of 236Pu as a yield tracer, and then isolated from chemical elements other than plutonium by means of an anion exchange resin in the nitrate form. The plutonium are electroplated onto a stainless steel disc cathode. As for the solutions of humic and fulvic acid fractions, these solu tions were heated almost to dryness, and then decomposed by nitric acid and hydrogen peroxide.
The resultant residue was treated with the same manner as the sedimentary residue.
RESULTS AND DISCUSSION
1) Solubility of plutonium in soil and sediment For the purpose of clarifying the nature of plutonium adsorbed in soil or sediment matrices, a series of extraction experiments were carried out using several leaching reagents.
The leaching experiments with 1 N-ammonium acetate would provide infor mation about plutonium taking an exchangeable form. On the other hand, since organic reagents such as EDTA (Ethylenediaminetetraacetic acid) and citric acid make a organic complex with plutonium strongly, the leaching experiments with these reagents would provide information about whether the solubilization of plutonium is caused or not by similar complexing materials present in soil or sediment.
As sodium citrate solution at For Kohno sample, no significant leaching of plutonium was found in the extrac tions with ammonium acetate, EDTA and sodium citrate.
The results obtained with 100 ml extractant were quite similar to those obtained with 300 ml extractant. The extraction with citric acid showed that about 30°% of plutonium was leached by 72 hr extraction, and no increase was observed in further 300 hr extraction.
These results suggest that plutonium in Kohno sample doesn't take exchangeable form but is likely to be in carbonate matrices.
The slight solubility of plutonium with citric acid may suggest the presence of plutonium in the form solubilized with such a similar complexing materials present in soil.
On the other hand, in the case of Mt. Kanmuri sample rich in organic materials, leaching of plutonium was higher for all the reagents tested, especially much higher for sodium citrate, than in the case of Kohno sample. In the case of the extraction with sodium citrate, pH values in the suspended solutions from Kohno sample (pH: 7.6) and Mt. Kanmuri sample (pH: 5.6) were different.
The supernatant solutions obtained with ammonium acetate, EDTA and citric acid were colored light brown, while that with sodium citrate was colored dark brown. The prominently low pH value may be due to the fact that sodium citrate in an initial pH of 8.4 solubilizes some of the organic materials. From the observation that as much as 60% of plutonium was leached with sodium citrate by shaking for a short time (20 hr), it is considered that a part of pluto nium present in this sample was associated with organic materials.
This problem is discussed further in the following section.
For the sea sediment from Nyu Bay, ammonium acetate didn't leach any plutonium. This fact suggests that plutonium in the sediment doesn't take exchangeable form. The complexing with EDTA or citric acid leaches about 40-50 of plutonium from the sediment, but sodium citrate doesn't leach plutonium.
Significantly different results were obtained for the extractions with citric acid and with sodium citrate.
This suggests that some plutonium is in carbonate matrices.
The results obtained by complexing reagents suggest that the solubilization of plutonium adsorbed on sediment may occur with similar complexing materials present in the marine environment.
In all cases, plutonium in soil and sediment is unlikely to be present in the simple exchangeable form. The nature of fall-out plutonium adsorbed on soil surface is influ enced largely by the physico-chemical properties of soil, and, furthermore, chemical and biochemical changes taking place in the environment. On the basis of the data from two kinds of soils, one might conclude that amount of organic materials in soil have an important influence on the nature of plutonium in soil under natural conditions.
2) Leaching of plutonium with natural organic acids
In contrast with the data as mentioned above, the data on the leaching experiments made with natural soil organic acids seems to be very interesting to study the migra tion of plutonium in soil. For this experiments, Kohno sample containing only a little organic materials was used, because plutonium originally bound to the organic materials might be leached if the experiments is made with Mt. Kanmuri sample rich in organic materials.
The results of a series of leaching experiments with 100 ppm fulvic acid are summarized in Fig. 2 along with those with 1000 ppm humic and fulvic acids.
As seen in Fig. 2, the fulvic acid doesn't leach the plutonium in soil under the condition studied. The value of pH in suspension didn't change so much because of the buffering action of soil. As shown in the lower part of this Fig., the fulvic acid con tent in supernatant solution obtained by ultra-centrifugation decreased to 30% of the added fulvic acid after 20 hr. And then the content decreased slightly and reached about 10% of the original value after 330 hr. The similar decreases were also observed by the leaching experiments with 1000 ppm humic and fulvic acids for 160 hr period. Since humic and fulvic acids are adsorbed scarecely on polyethylene bottle, these de creases of organic acids are mainly due to the coagulation and the sorption on the soil. Therefore, even if plutonium in soil should form soluble complex with organic acids and may be dissolved into the solution, such complex seems to be removed soon from the solution by coagulation and sorption on soil. These results are consistent with 3) Plutonium associated with natural organic acids From the above mentioned point of view, it is important to ascertain whether plu tonium exists in organic form or not under natural conditions. The two kinds of soil samples were treated with both 0.1N-sodium hydroxide solution and the mixed solution to extract organic acids. As seen in Fig. 3, showing the percentages of plutonium and total organic carbon in each fraction, plutonium was detected in both humic and fulvic fractions of two kinds of soil samples. For Kohno sample by leaching with sodium hydroxide or the mixed solution, 4.3% or 6.8% of the total plutonium was found respec tively for the organic fractions, which corresponded to 35% or 39% of the total organic carbon in original soil. On the other hand, for Mt. Kanmuri sample by leaching with sodium hydroxide or the mixed solution, as much as 72% or 61% of the total plutonium was found respectively for the organic fractions, which corresponded to 33% or 41% of the total organic carbon in original soil. Though the large difference isn't clearly observed in leaching of plutonium and organic acids between with sodium hydroxide and with the mixed solution, the leaching with the mixed solution tends to increase the plutonium amount in fulvic fraction, and against this, to decrease the plutonium amount in humic fraction. This may be explained by the fact that plutonium is liable to form complexes with phosphate ions in the mixed solution. Taking into account the results of these leaching experiments, it may be concluded that these experiments show the presence of plutonium bound to organic acids in soil, especially in Mt. Kanmuri sample. For the sediment from Nyu Bay, 6.5% of plutonium was found in the leaching fraction with 0.5%-sodium hydroxide solution, although the leaching of plutonium wasn't observed with 30%-hydrogen peroxide"' As for the presence of plutonium-organic complex in soil, there are some reports" by using solid chelating resin (Chelex 100) and DTPA (Diethylenetriaminepentaacetic acid). Such studies have also shown the evidence that a part of plutonium present in soil was in organic form. However, it has not yet been clarified how much fraction of pluronium is associated with the organic materials. Therefore, studies on this point must be made furthermore.
In this aspect, it is particularly interesting that as much as 60-70/00 of plutonium in Mt. Kanmuri sample is associated with organic acids. Studies for cultivated soils rich in organic materials seem to be necessary to understand how much organic form of plutonium become available to plant with time. | v3-fos |
2014-10-01T00:00:00.000Z | {
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} | s2 | Flow system for automated analysis of maize pollen.
Pollen grains are haploid gametes of uniform shape and size, and can be obtained in large quantity. If appropriate traits are used, they can be an excellent material for investigation of rare but important biological events like intracistronic recombinations or mutations induced by very low level of mutagens. This advantage will be further improved, if the laborious counting and examination can be made automatically. For automation of pollen analysis, techniques of flow analysis and image analysis would be applicable. Flow analysis with a optical detector was tested using maize pollen. Pollen grains were transported by gentle suction through a glass capillary which was placed under a microscope. Interruptions of the light path by pollen grains were detected by a silicon photocell after optical magnification and converted into electric pulses. The frequency distribution of pulse height was examined by a multichannel pulse height analyzer. 10(6) pollen grains would be counted and classified within about 30 min for a pollen suspension dilute enough for separation of each pulse. The flow system tested seems promising for detection of Wx mutant pollen in a wx pollen population after iodine staining if illumination of sample particles is improved. ImagesFIGURE 1.
Introduction
Pollens of higher plants are haploid and can be handled in large numbers. If appropriate genetic markers can be used, they seem to be good material for investigation of rare but important biological events like intracistronic recombination (1-3) or mutation (4)(5)(6). Since pollen grains are uniform particles in size and shape, analysis by electronic instruments would seem possible. The advantages of use of pollen would be further improved if the laborious and time-consuming procedure of scoring of pollen could be replaced by automated analytical instruments. Such instrumental analysis would be useful both in counting very large numbers of pollen and in objective classification of characters of pollen grains.
Two types of measurement can be expected, i.e., flow system and image analysis. Both systems may be used as total examination or as sampling analysis. In the flow system, however, basically all the particles are counted and examined, one by one, when they pass through the detector. The simplest * Department of Induced Mutation, National Institute of Genetics, 1,111, Yata, Misima, Sizuoka, Japan 411. January 1981 instrumentation may be a combination of particle detector and counter. If the characteristics of the particle can be classified into groups by the nature of the signals which were generated at the detector when the particle passes, addition of counters and signal analyzer will complete a basic measuring system. One such system was assembled to examine pollen grains of maize (Fig. 1). The present paper reports the preliminary results of an automated flow analysis system and makes practical evaluation for further improvements.
Materials and Methods
System Set-up The flow system used is shown schematically in Figure 2. Pollen samples were suspended in water and agitated by a small stirring blade. They were transported through a glass capillary (about 0.8 mm diameter) by gentle suction of air using a microtube pump. The capillary was fixed between a slide glass and cover slip, and the spaces around the capillary were filled with balsam. The capillary was positioned precisely in the light path of a microscope. Interruptions of the light path by pollen grains 165 FIGURE 1. Pollen of waxy maize stained with iodine. In this material, the darkly stained pollen grain at the center is a wild type (Wx) recombinant. Phenotypic revertant towardl non-waxy may be comparable to this dark pollen grain.
were detected by a silicon photocell (Hamamatsu TV, S876-16BR) after optical magnification of about x 10. Electric pulses from the silicon photocell were amplified and sent to a multichannel pulse height analyzer (Canberra, Series 30). The multichannel analyzer (MCA) used was capable of storage of data up to 106_1 counts in each of its 1024 channels. It also had regions of interest (ROI) function with integrated data readout. Each input pulse was assorted to corresponding channel according to its pulse height. The number of assorted pulses was recorded in counters of each channel. Counts of neighboring channels could be pooled by ROI function by designating the region of interest. The frequency distribution of pulse height could be displayed on a CRT screen of the MCA while collecting and analyzing the data. This display could be transferred onto paper by use of an X-Y plotter. Numerical data could be printed out by a line printer. The X-Y plotter and line printer complete the instrumental set up. Extra care was taken to stabilize the light source to illuminate the capillary. Mechanical fixation, soldered leader wires, and an electronic voltage regulator were used.
Sample Preparation
The present experiments were intended to test the flow type automatic analysis for detection of phenotypic reverse mutation of waxy (wx) pollen to non-waxy (Wx) pollen. For this purpose, mature maize pollen was used to test the flow system and to obtain optimal flow rate and other operational data. 166 Mature maize pollen had been harvested in the field and stored in 70% ethanol. Some younger pollen usually used in fine structure analyses of wx locus, was also tested after iodine staining (Fig. 1). In both cases, pollen grains were washed clean with 70% ethanol a few times to eliminate small debris in the sample suspension.
Pulse Shape Analysis
Shapes of the electric pulses generated by interruptions of light by pollen grains were brought to a standstill for observation on a CRT screen of a dual channel synchroscope (Iwatsu Electric, Synchroscope SS-5050) using digitized memory (Kawasaki Electronica, Transient Memory TM-1410). The Transient Memory had outputs for an X-Y plotter to make a paper copy of pulse shape. To the second channel of the Synchroscope, either calibration voltage for pulse height or timing pulses for pulse width could be applied for measurements.
Measuring Procedure
After a 1 hr warmup to stabilize the instruments, the sample vial and exhaust vial were set in position. The agitator of the sample vial and microtube pump for transportation were turned on. When the grains start moving through the capillary, electric pulses were confirmed on a monitor Environmental Health Perspectives synchroscope (Matsushita Communication Industrial, VP-5102A). MCA was activated for data collection by turning on the "collect" switch. When most of the sample suspension was transported, and before air bubbles came up, data collection by MCA was terminated by turning off the "collect" switch. ROI was set after data collection to include the major part of the peak of frequency distribution of pulse height. The X-Y plotter and digital printer recorded results of analysis. January 1981
Flow System
Although the capillary used for maize pollen grain was quite large, particles flowed at the center of the capillary when suspension was transported at the rate of 2 ml/min or 4 m/min. This centering of pollen grains in flow was an unexpected advantage of the present system and made focusing of pollen images on the photocell easy. Clogging in the flow system could be avoided by use of a thick capillary and cleaned pollen suspension. However, the cleaning of the pollen suspension eliminated both small debris and empty shells of abortive pollen. The latter fraction would be biologically important in some experiments, but in the present experiments only fully grown pollen grains were analyzed.
Pulse Shape
The silicon photocell used had a narrow lightsensitive area of 1.2 mm x 6 mm, and was placed to transverse the image of the capillary. This gave a sharp pulse and high resolution. Pulse shapes were uniform with little variation in pulse height when a diluted pollen suspension was used. Examples of the pulse shapes are shown in Figure 3. The count rate for this concentration was 500 pulses/sec or 3 x 104 pulses/min. Clustering or clumping of the particles occurred when the concentration of suspension was higher. An example of such piled up pulses is shown in Figure 4.
Frequency Distribution of Pulse Height
Electric pulses were generated by interruption of the light path by pollen grains. The height of each pulse reflects the characteristics of the pollen, the optical density, which might relate to size and color of pollen grain. The size of pollen grain was also expressed as width of each pulse. In the present experiments, the size of pollen grains did not vary very much by visual examination. The frequency distribution of pulse height was analyzed by MCA. An example of the frequency distribution of pulse height of unstained mature pollen of maize is shown in Figure 5 together with ROI readout data. In case of piled up pulses like those in Figure 4, the peak of the frequency distribution of pulse height was broad. A mixture of younger pollen was also examined after iodine staining, but, as discussed later, pulse heights did not differentiate between Wx and wx as far as transmitting light was used.
Discussion
Automation of the pollen analysis may be worthwhile both for quick and accurate counting of the total number of pollen grains and in objective classification of the character of pollen. If well cleaned maize pollen were to be used, counting of the total number of pollen grains would be easy with the flow system reported here and a low cost electronic counter. Counting of pulses which exceeded in their height the predetermined threshold level could be made by using a discriminator or comparator circuit. Recent advancements in the electronic industry have developed a compact and relatively low cost multichannel pulse height analyzer (MCA). The MCA used here had 1024 counters, each capable of 106_1 counts, and was able to sort pulse to the corresponding counter according to pulse height. This could be used as main analyzer of the flow system.
Theoretically, four classes of pulses, each differing in height, would be expected in reversion experiments of waxy maize pollen if optical detection of particles was used. They would be, in order of increasing height, (1) small debris and noises inherent to the elements used, (2) abortive pollen, (3) normal pollen, and (4) mutant Wx pollen. As described before, the height of the pulse might reflect optical thickness and color of the pollen grain. The size of pollen grains did not vary significantly, but as maize pollen grains were eggshaped and the directions of their axes might vary randomly in flow, the optical thickness as sensed by the photodetector might deviate considerably. However, the difference in color of typical Wx and wx pollen after iodine staining was unmistakably clear. Wx pollen stains dark blue-black and wx light brown. For objective classification of pollen character, use of an appropriate color filter will help to differentiate mutant Wx pollen from parental wx pollen.
Illumination of pollen grains was important. In the case of wx-Wx pollen experiments, Wx pollen can be detected best with reflecting light after iodine staining. However, if only reflecting illumi-nation were used with a dark background, darkly stained Wx pollen would produce lower electric pulses than pulses produced by light colored wx pollen. This would make distinguishing between darkly stained mutant Wx pollen and empty shells of abortive pollen difficult. Improvement is possible by adopting both types of illuminations. Transmitting illumination would produce an electric pulse which corresponds to each pollen grain, and reflecting illumination would permit subtraction of the height according to lightness of color. This would leave pulses from dark Wx pollen less affected and maintaining their height highest among the four classes of pulses described before. An additional red color filter would help in differentiating mutant Wx pollen pulses from background wx pollen pulses.
It should be mentioned that in the present set-up, sample and exhaust vials were exchangeable and pollen was intact after examination. Pollen could be counted or examined repeatedly for the total number and presence of mutant pollen grains or could be spread on a large slide glass for visual examination. | v3-fos |
2014-10-01T00:00:00.000Z | {
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} | s2 | Pollen genetic markers for detection of mutagens in the environment.
To utilize and exploit pollen for in situ mutagen monitoring, screening and toxicology, the range of genetic traits in pollen must be identified and analyzed. Traits that can be considered include ornamentation, shape and form, male sterility viability, intraspecific incompatibility, proteins and starch deposition. To be useful for the development of mutagen detection systems proteins should be: (1) activity stainable or immunologically identifiable in the pollen, (2) the products of one to three loci, and (3) gametophytic and nuclear in origin. Several proteins including alcohol dehydrogenase in maize, which meet those criteria will be discussed. The waxy locus in barley and maize which controls starch deposition has been characterized genetically and methods have been developed for pollen screening and mutant detection. At Washington State University a waxy pollen system is being developed in barley for in situ mutagen monitoring. The basis is an improved method for staining and scoring waxy pollen mutants. Specific base substitution, frameshift, and deletion mutant lines are being developed to provide information about the nature of the mutations induced by environmental mutagens. Thirty waxy mutant lines, induced by sodium azide and gamma-rays have been selected and are being characterized for spontaneous and induced reversion frequencies, allelism, karyotype, amylose content, and UDP glucose glucosyltransferase (waxy gene product) activity. Twelve mutant alleles are being mapped by recombinant frequencies.
Introduction
Appropriate systems to monitor i7 situf the genetic hazards of environmental pollutants to man, animals, and plants, are very rare. Indeed, among higher eukaryotes only two systems, both plants, have been used with any success (1). This paper is concerned with improving the capabilitv of plants for in situ monitoring of genetic effects of the environment.
In developing an in situ monitoring system it is first necessary to examine the criteria for such a system. An appropriate system should inclu(le: chromosome organization and molecular biology similar to that of the human organism, i.e., a eukaryote; well-defined and simply inherited genetic markers or traits; ability to provide reliable mutation frequency data from different stages in the life cycle at very low doses of environmental January 1981 mutagens; capability for measuring chronic exposures; a means for providing data on the nature of mutations, especially at the DNA level; short time between mutation induction and mutant screening; relatively low cost of culture and cost an(d time of training technicians to score genetic endpoints following mutagen treatment; small space requirement for mobile atmospheric testing laboratories and growth controlled chambers; and ability to grow under a wide range of atmospheric and soil conditions. Plants meet many of these criteria and have a number of advantages over mammalian and(i prokaryotic systems for environmental mutagen monitoring (1). In addition to the advantages over mammalian systems, plants possess a pollen genetic system.
Pollen has several attributes for measuring biological damage when used as a monitor for air or soil pollution. It is haploid so that the genetic changes occurring in plants up to the time of complete development and final mitosis can be 19 scored directly. This permits chronic exposure of the sporophyte to a mutagenic agent x-hile observXing the genetic effects of such exposure in the haploid germinal cell. In additioin, pollen systems provide a highly sensitive means for screeninig and(i monitoring environmental mutagens since inereases in genetic effects of low levels of mutagens canl be recorded. Mutant traits can be quicklv tietecte(l from among millions of pollen grains. This genetic resolution, unique among higher eukaryotes ani(i paralleling that of prokaryotes, permits the scoring of intragenic events-thus providing an undlerstanding of the nature of inducedl mutations in higher plants. Further advantages include low cost and ability to measure genetic damage at tlifferent stages of pollen development in terms of reduction of pollen germination, chromosome aberrations firagments and dicentrics in pollen mitosis, micronuclei in tetrads (2) and nondisjunction (3)1 antI mutants of morphological and biochemical traits. Such traits or markers will now be described.
Potential Pollen Genetic Markers
To fully utilize and exploit pollen in mutageni monitoring, screening and toxicology, the range of genetic traits in pollen must be identifiedl anti analyzed. A reasonably extensive review of the literature has determined that there are currently available very few pollen genetic markers that could be developed for mutagen monitoring svstems. This is in part due to the lack of genetic an(d molecular biological knowledge of many potential traits. Furthermore, the useful traits are limite(d to the gametophytic or haploid portion of the pollen grains. The following summarizes the status of some potential markers. They includle outer wall ornamentation, pollen shape and form, male sterility, viability, incompatibility, protein variation, an(i starch deposition. Ornamentation Sculpture or ornamentation of the outer layer (tectum) of the outer wall (exine) which is readily observed under a light microscope might be considered an obviously useful genetic trait. It involves the distribution and dimension of spines, ridges, perforations, papillae and patterns of spinulas on ridges. However, it is generally recognized (4) that it is difficult to distinguish ornamentation patterns between species and even genera, and to use such patterns for taxonomic classification. This suggests that ornamentation does not involve simple genetic control. Furthermore, origin of the exine is sporophytic (5), and the ornamental sculpturing is 20 probably of no value in a mutagen monitor where mutants expressedl in the haploid state are required.
Shape and Form
Distinct shapes and forms of the pollen ar e known but genetic analysis is lacking. Moreover. it is likely that the structures involved in shape and(I form are sporophytic in origin anti that allometrv is not a useful trait for measuring effects of mutagenis. However, pine (Pinus) pollen may have some potential for the screening of environmental mutagens. This pollen normally has two attache(i bladdlers: however, pollen with three anti four bladileres have been observed (R. Mack, personal communicationi). If simply inherited anti gametophytic in origin, this trait may prove useful.
Male Sterility
Genetic male sterility will leati to increase(d number of aborted pollen. Genes for male sterility have been identified in many plants. For instanice, in barley 16 loci are known (6,7). In such planits, forward mutations to male sterility could be itlentifie(l. However, since cytoplasmic male sterilitv also exists and leads to aborted pollen, scoring for forward mutations could be complicateti. On the other hand, reverse mutation in a homozvgous genetic male sterile line could lead to normal pollen (detected by fluorescein diacetate) anti seeti set, both easily scored.
Viability
Viability is probably controlled by a number of genes. This could be a useful multilocus system for mutagen monitoring. Stains for revealing viable or nonviable pollen are available to quickly record mutations at these loci. Mulcahy (8) has suggested an approach for recording such mutations.
Intraspecific Incompatibility
Mutations for genes controlling incompatibility, such as the S locus in Oenothera organensis can be readily detected and represent a potentially powerful monitoring system. Since this subject is covered elsewhere in this symposium (9) it is not further described here.
Protein Markers
Most of the apparent useful heritable traits identified in the pollen of various plant species are Environmental Health Perspectives proteins. However, pollen proteins have been identified and analyzed for reasons other than utilization in a mutagen monitoring and screening system, such as to determine allergen effects, incompatibility relationships ("recognition substances"), various aspects of beekeeping, and to identify varieties and subvarieties by isozvme patterns (10). Numerous proteins, mostly enzymes, have been identified in pollen. Most were described by Brewbaker (11) and all are listed in Table 1. Most are incorporated in the pollen wall and a major problem in analysis is to determine if a giveni protein is in the sporophytic or gametophvtic portion of the pollen (5). Proteins incorporate(d within the inner wall or intine are gametophvtic in or'iginl, while those associated with the outer wall or exinie are sporophytic in origin (5). The cell wall proteins, consisting of antigens as well as enzymes, are freely diffusible with the sporophytic proteins being released first (5). The time of synthesis and incorporation of pro- (11) teins in the pollen is important in relation to mutation induction and detection. It appears that proteins are synthesized and incorporated soon after the release of microspores frorm the tetrads (5,17).
Most proteins have not been characterized genetically nor localized within the pollen grains. Therefore, much more work is necessary before their potential use in mutagen monitoring systems can be evaluated. Nevertheless, several protein pollen markers as described below have some promise. These markers are: activity stainable or immunologically identifiable in intact pollen grains, the product of one to three genetic loci, and gametophytic and nuclear in origin. Such characteristics facilitate detection and screening of genetic variants.
The substrates naphthol S-Bi or AS-TR phosphates can also be used; however, the reaction is not as rapid (25). In addition, Fast Garnet GBC can be used as a coupler to yield a purple-black color and has been shown to specifically inhibit AP3 (19).
Three AP1 isozymes occur in maize pollen (20). The enzyme appears to be a dimer and is synthesized after meiosis since pollen from heterozygous plants lack hybrid enzyme. Segregation ratios of AP1 isozyme activity levels showed that the isozymes are controlled by a single gene.
Leucine aminopeptidase is widely distributed in angiosperm pollen (10). In maize endosperm there are apparently four genes involved (27). However,in pollen it appears that only one to three are expressed for a given species (10,11). Genetic polymorphisms occur in maize pollen for the LP2 locus (11). Electrophoretically separated proteins are activity stained by using L-leucyl-3-naphthylamide HCI as a substrate and Black K salt, a diazonium dye, as a coupler, to yield a deep purple color. Such a procedure might be useful for pollen staining, but as yet the enzyme has not been localized.
a-Amylase (Amy) is apparently under the control of a single gene in maize (28) and barley (29,30). In barley there are at least three alleles at the Amy locus which give rise to the three isozymes, a-type 1, a-type 2, and a-type 3. Jones and Chen (31) used immunofluorescent antibodies to localize a-amylase in barley aleurone cells. It should be possible to use a similar method for detecting a-amylase in pollen. Amylase has been shown to be readily diffusible 22 from birch pollen after 1 min, indicating it may be sporophytic in origin (32). However, Knox and Heslop-Harrison (25) report that the activity was localized in the intine. Further work to localize the enzyme accurately is needed.
The alcohol dehydrogenase (Adh) locus in maize (33)(34)(35) is controlled by two unlinked genes, Adhi and Adh2. The functional enzyme can be found electrophoretically as either a homodimer or a heterodimer of the gene products of the Adhi and Adh2 loci. The maize alcohol dehydrogenase has been purified and partially characterized (35) and numerous mutant enzymes have been electrophoretically characterized by Schwartz (33,36). Only Adhi is expressed in the pollen grain, and most of the Adhi polypeptides are synthesized after anaphase II of meiosis (37). The use of maize Adhi as a monitor of environmental mutagens has been discussed by Freeling (35) and is reviewed further in this publication (38).
There are several pollen wall antigens which may be useful for mutagen monitoring. Of these, antigen E is perhaps the best characterized. Immunofluorescent antibodies (39) revealed antigen E of ragweed (Ambrosia) to be localized to the intine. Antigen E was localized to the intine and exine of the pollen of Cosmos (40). With fresh pollen, much of the antigenic material was discharged into the medium; therefore, it may be necessary to use frozen pollen for effective immunofluorescent detection and screening.
Starch Deposition (Waxy)
The waxy trait has been observed in over 15 genera of angiosperms and is characterized by failure of amylose deposition in the endosperm and pollen grain starch (41). Starch granules in the cereal pollen grain begin to appear just after the second pollen mitosis (42) and are gametophytically controlled (41). Waxy and nonwaxy pollen stain differentially with iodine, blue for pollen with normal starch and amylose content and reddishbrown for pollen with amylopectin and no amylose. All of the early genetic research on waxy pollen has been reviewed by Eriksson (41). Genetic studies in maize, barley, rice, and sorghum concluded that the waxy character is inherited as a monofactorial recessive.
The waxy locus offers several distinct advantages for a mutagen monitoring system. Because mutants are identified by iodine staining, the procedure is relatively simple and not as time consuming as procedures requiring cumbersome electrophoretic detection methods. In addition, the pollen screening procedure is much less costly than those Environmental Health Perspectives requiring complex reagents or immunochemicals for mutant identification. Some knowledge of the biochemistry of the waxy gene product is available. Waxy mutants in maize completely lack starch granule-bound uridine diphosphoglucose (UDPG) gluocosyltransferase activity (43) and enzyme activity is linearly proportional to the number of starchy (Wx) alleles present in the endosperm (44). These results support the suggestion that the waxy locus is the structural gene for the UDPG glucosyltransferase.
The pollen waxy locus of maize has been used to detect the genetic effects of herbicides and is being developed into a mutagen monitoring system (45,46). This system is described in detail in these proceedings (47).
Development of a Waxy Pollen System in Barley
The development of a waxy pollen system in barley for mutagen monitoring (both frequencies and nature of events) has been underway at Washington State University for several years. Included in the overall objective is to develop a genetic system that is highly sensitive to low levels of mutagens and that can measure the frequencies and kinds of changes at the DNA level. The attributes of the pollen system have been described above.
Barley was selected because it: (1) is genetically well characterized, (2) is a self-pollinated diploid, (3) has seven pairs of relatively large chromosomes, (4) is easy to culture and grows under a wide range of climate and soil conditions, (5) is small in stature and many plants can be confined to laboratories and controlled growth chambers, (6) exhibits some meiotic synchrony, and (7) is the foremost of all plants in terms of studies on experimental mutagenesis (48)(49)(50). The synchrony of meiosis permits treatment of microsporocytes and scoring for waxy mutants and chromosome aberration in pollen at precise times. It is also adaptable for in situ monitoring of genetic activity in the environment in and around laboratories, factories, nuclear reactors and waste chemical dump sites, and of chemicals involved in agricultural practices. Mutations at the waxy locus in barley were used to detect the mutagenic effect of ethylene oxide in air around Stockholm (51).
A key to the development of this system is a rapid mass-screening technique for analyzing waxy mutants of pollen (52). It permits the analysis by one recorder of one million pollen grains from 15 different spikes in an 8-hr day and has led to reliable data for forward mutations and mutant allele revertants and recombinants.
Mutant lines induced by base substitution (sodium azide, ethyl methanesulfonate), frameshift (acridine, proflavin), and deletion (ethyl methanesulfonate, y-rays) mutagens are being selected and developed to provide information about the nature of the mutations induced by environmental mutagens and to provide a broad base for detecting different mutagens.
To date, about 30 mutant lines induced primarily by sodium azide and gamma rays have been selected. These are being characterized for spontaneous reversion rates, amylose content, enzyme activity, and karyotype. They will also be characterized as to reversion frequencies induced by the different mutagen types.
Sodium azide is probably the best candidate in barley for a base substitution mutagen. It does not induce chromosome breaks in barley nor sister chromatid exchanges in mammalian cells (53). However, it induces very high frequencies of chlorophyll-deficient mutations in barley and has been shown to be a base substitution mutagen in Salmonella (54). It is a major environmental mutagen as it is used in medicine, agriculture, auto air bags, and airplane escape chutes. Spontaneous frequencies of some waxy mutant lines induced by sodium azide are shown in Table 2. For the most part, the reversion frequencies are high but stable from year to year. Among these mutant lines, Az 24 and Az 44 appear to have the most stable baseline reversion frequency since little difference in reversion frequency is observed between plants within these mutant lines.
Spontaneous reversion frequencies from some lines induced by y-irradiation are shown in Table 3.
These frequencies range from 6.2 x 10-5 to 31.6 x 10-5. The mutant line gamma 5 appears to have the most stable baseline reversion frequency and is the most promising putative deletion line thus far available.
Chemical and karyotype characterization of the waxy mutant lines has made considerable progress.
Twenty-one azide-induced and seven y-ray-induced waxy mutants were analyzed for amylose content and none contained this compound. In addition, six y-induced and two azide-induced waxy mutants were analyzed for UDPglucose glucosyltransferase activity. One azide-induced mutant showed substantial activity (36% of normal); however, this might possibly be due to the presence of contaminating nonendosperm tissue in the starch extract.
Examinations of chromosome morphology during somatic metaphase and meiosis have revealed no major change in several azide-induced mutants, but some possible chromosome changes in two y-ray induced mutants.
Six mutant waxy alleles were preliminarily mapped in 1979 from allelic recombination frequencies (55). Recombination frequencies from additional crosses among these plus six other alleles are now being obtained for more comprehensive mapping of the locus. | v3-fos |
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} | s2 | Storage fungi of onion and their control
Botrytis allii Munn caused total onion damages of 1 5—20 % during storage in 1975 1979, and was present on 80—90 % of the spoilt onions. The proportion of damage caused by Fusarium oxysporum Schl. was o—s % and was present in o—lo % of the spoilt onions. The early weight losses during storage of the onions were mostly due to storage pathogens which spread via the onion sets used as propagation material. This can be prevented very effectively by soaking the sets in benomyl solution before planting out. The unusually high fungus content of the sets resulted in a reduced yield. Spraying with fungicide early on in the growing season and applying different amounts of nitrogen fertilizer had no significant effect on the number of storage pathogens. A low stroge temperature did not inhibit the development of storage pathogens, it merely slowed it down.
Introduction
During the last few years, 500-600 ha have usually been under onion cultivation in Finland, Since the Finnish growing season is short, onion sets, which are almost completely (about 85 %) of foreign origin, have to be used for onion cultivation. Holland is the most important supplier. Onions are grown from seed almost exclusively in the Aland Islands over an area of about 130 ha. In countries to the south of Finland, onions to be stored are grown from seed and hence the occurrence and spread of storage pathogens in Finland is likely to be different from that elsewhere.
Studies have been carried out earlier in Finland on onion storage (jAMALAINEN 1962, AURA 1963. However, multiplier onions were almost exclusively used at that time and the low storage temperatures used today had not become common practice. No storage studies have been carried out since onion sets came into common use. According to information provided by onion growers, storage pathogens have caused total onion losses of as high as 50-60 % in modern cold stores. For this reason, storage pathogens are considered to be one of the most serious problems in onion growing. Right up until a few years ago, Botrytis allii Munn, which is the most important storage pathogen of onion, was reported to infect onions in the field, usually close to harvesting time or during harvesting in Finland (jAMALAINEN 1962) and also elsewhere (HEINZE 1974). However, a number of studies have shown that B. allii spreads to onions via the seeds and is dormant during the growing season (MAUDE and PRESLY 1977 a).
Fusarium oxysporum Schl. is also a fungus which causes spoilage of onions (WALKER 1952, HEINZE 1974. However, the damage it causes is only slight (BÖTTCHER 1973). This fungus is known to spread via the seeds and sets (PARKINSON andCLARKE 1964, NOBLE andRICHARDSON 1968).
The aim of this study was to determine the most common and most important storage pathogens of onions, their transfer via onion sets, and possible control by means of fungicide treatment of both the sets and the onion plants. The growth capacity of the most important storage pathogens at different temperatures, and the effect of nitrogen fertilization on the number of storage pathogens has also been examined.
Material and methods
Fungal determinations carried out on the onions The onions in 10 commercial growers' stores in the Turku, Joensuu and Turenki area during the period 1975-78 were studied in order to determine which pathogenic fungi affect onions in store. In addition, onions were grown at Viikki from sets of differing origin in 1976-79, in order to determine what type of fungi arc to be found on onions. A total of about 110 000 onions and the storage pathogens growing on them were examined.
In the first year of the experiment, the infected onions were divided into disease classes on the basis of their symptoms. Samples were taken from the demarcation line between diseased and healthy onion tissue and then surface sterilised in 1 % sodium hypochlorite. The pieces of plant tissue were then transferred to PDA medium and also to moist filter paper in petri dishes. In subsequent years, onions spoiled by Botrytis allii, Fusarium oxysporum, Aspergillus niger and Fenicillium spp. were classified directly by eye or through examination under a stereo-microscope. In all uncertain cases, however, pieces of onion tissue were removed from the border between diseased and healthy tissue for incubation on damp filter paper followed by examination under the microscope.
Fungal determinations carried out on the onion sets The fungi present on onion sets intended for field trials, as well as the degree of infection, were determined by incubating bisected onion sets, which had first been surface sterilised in 1 % sodium hypochlorite, on filter paper in petri dishes (o=l4 cm) for 10 days. The fungi were identified under a stereo-microscope and, whenever necessary, also under an ordinary research microscope. The degree of infection by Aspergillus niger, Fusarium oxysporum and Penicillium spp. was estimated using a o-3 classification, in which 0 = healthy and 3 = fungus covering 1/2 of the cut surface of the set. 60 sets were examined from each lot. Altogether 67 onion lots were checked in 1977 and 16 in 1978, the sets being selected from the onion lots shown in Figs. 1, 3 and 4. Preliminary experiments were carried out in 1976 to determine the best methods to be used and the type of fungi to be found on the sets (TAHVONEN and RIIKONEN 1977, RIIKONEN 1978. The results obtained in these preliminary experiments arc not presented here because they arc in agreement with those obtained in 1977-78. The onion lots depicted in Figs. 3 and 4 have been grown from domestic and foreign 'Stuttgarter Riesen' sets, 1 5-22 mm in size. The onion lots shown in Fig. 1 also include some other planting sizes and varieties.
Control and nitrogen fertilisation experiments
The sets used in the storage pathogen control experiments were soaked for 15 minutes in a benomylsolution (0,2 % Benlate preparation). Five different fungicides were used in spraying the onion plants; benomyl (Benlate, 1.2 kg/ha), thiophenate methyl (Topsin M, 1.4 kg/ha), tolylfluanide (Euparen M, 5.0 kg/ha), captaphol (Difolatan 80 WP, 1.6 kg/ha) and vinclozolin (Ronilan, 1.5 kg/ha) mixed in 2000 1 water/ha. Spring sprayings were carried out when the shoots were 10-1 5 cm high and the second spraying after one week. Autumn sprayings were carried out 2 weeks and 2 + 1 weeks before harvesting. Sprayings carried out in the other experiments arc shown in the tables.
The effect of nitrogen fertilization on the storage pathogens of the onion crop in onion set and onion cultivation was followed by fertilizing with 1000 or 11 50 kg chloride-free mixed fertilizer (N;P 2 0 5 :K 2 0 = 7:24:14)/ha. The highest nitrogen levels were obtained by adding the required amounts of nitrogen as calcium nitrate. The sets grown in the onion set experiment were graded after storage into size classes of 10-15 mm and 15-22 mm, and then planted in a normally-fertilized (see other experiments) field without any fungicide treatment. From 1200-1400 kg/ha chloride-free mixed fertilizer was used in the other experiments. All the fertilizers, including nitrogen, were applied as row fertilization before planting out. The nitrogen fertilizer and control experiments (POHTO 1979) carried out in 1976, which were pilot experiments for those carried out in 1977-78 and whose results and methods were the same as those used in 1977-79, will not be dealt with here. The onion plants used in the spraying experiments carried out in Viikki in 1977 (Tables 3 and 4) were inoculated five times, starting from the middle of July, at intervals of two weeks, by spraying them with a suspension of Botrytis allii at a level of 600 1/ha (one 9 cm-petri dish containing B. allii on PDA medium /I H 2 O).
The variety, 'Stuttgarter Riesen' was used in all the control and nitrogen experiments apart from those carried out in 1975 using variety 'Superbunt'.
The temperature experiments
In the temperature experiments, storage at I°C and + I°C was carried out in a mechanically cooled store where the temperature variation vas ± O.5°C. Storage at 2-B°C was done in a store cooled by means of outside air and that at 20°C in and ordinary room where the temperature variation vas ± 2°C. The onion sets to be grown in the temperature experiment were artificially inoculated before planting with a suspension of Botrytis allii and Fusarium oxysporum in order to ensure that the crop would be infected.
The growing and storing methods of onions
The experiments carried out at Viikki were planted by hand. At Turenki, where the experiments were carried out in the fields and store of a commercial grower, planting was done by machine. Planting was carried out in different years during the period 15.-25. 5. and the crops harvested during 18.-25. 8. All the onions from the experiment were placed, after harvesting, into net bags without removing the stalks and then mechanically dried in circulating air at 25 -3O°C for s-lo days. During storage the air was continuously drawn over the onions by means of a fan in order to remove excess moisture. An effort was made to replicate, as far as possible, the conditions prevailing in normal onion stores, where 500-700 m } air nr'hr 1 are used in drying and 100-200 m 5 tn^'h -1 in storing.
Statistical methods
The results of the control and nitrogen fertilization experiments have been tested by means of variance analysis or the t-test. The effect of the degree of fungal infection on the sets, which consists of the summed values for Aspergillus niger, Fusarium oxysporum and Penicillium spp., on the yield, and the effect of the Botrytis allii and Fusarium oxysporum levels of the stored onions on the weight loss has been depicted and tested by means of the regression equations, y= bx + aory = a ej?x and the correlation coefficients.
Storage fungi of onions
Botrytis allii Munn B. allii was usually found in 80-90 % of the completely spoilt onions (Table 1).
The proportion of the fungus on spoilt onions in the different onion lots varied from 56 to 100 %. The total losses caused by this fungus in the different onion lots varied, in the absence of the control treatment, from 5 to 30 %, usually being 15-20 %. The frequency of occurrence of the fungus in the commercial grower's stores was of the same order of magnitude.
The losses in weight of the onions during storage, caused by evaporation throught cell respiration and the effect of the storage pathogens, was explained very well by the degree of infection by B. allii alone. In 1977, the correlation between the B. allii -% and the percentage weight loss was 0.958 xxx and in 1978, 0.966 xxx . When the Fusarium oxysporum content was included, the weight losses caused by factors other than these two pathogens was, during long-term storage, less than 10 % (Fig. I). A degree of overall infection of 40 % even caused weight losses of about 30 %.
B. allii was also the most common fungus found in the onion sets. The fungus was found in 15 %of the set lots in 197 7 and in69%in 1978. The fungus contents varied from 2 to 74 %. However, it was difficult to identify B. allit on the onion sets because the presence of many other species of fungi on the sets inhibited or made identification of B. allit difficult. When sets were artificially infected with B. allii for the temperature experiments, the fungus was not even found on non-surfacesterilised sets and it was not until the onions had been stored at the end of the growing season was the fungus found. Surface sterilisation of the sets with sodium hy- (RIIKONEN 1977). B. allit was not found to have spread from infected onions to healthy ones during storage when moisture was removed effectively by means of ventilation. In field trials in which the distance between the sample plots was 0.8 m, the fungus was not found to have spread significantly in a single case during the growing season from an infected, unsoaked onion lot to a healthy or benomyl-soaked lot (Tables 1 and 2). (1977) The symptoms produced on the onions by the pathogen (Fig. 2) and also the microscopic characteristics of the pathogen were the same as those described in the literature ( ELLIS and WALLER 1974).
The proportion of F. oxysporum on completely spoiled onions varied from o-4B %. However, it was usually below 10 %. The total losses during storage of onions not treated with the control measures varied from 0-15%, generally being less than 5 % (Fig. 3). The damage percentages in the commercial grower's stores were similar to those obtained in the experiments at Viikki. The fungus was not found to have spread from infected onion lots to healthy ones during the growing season or during storage.
F. oxysporum was exceedingly common in the onion set lots studied, the degree of infection varying from 1.7 to 100 %. The proportion of heavily infected (class 3) sets was, however, mostly under 20%. There was clear positive correlation between the number of heavily infected sets and the F. oxysporum -% of the stored onion crop (Fig. 3). Severe Fusarium infection on the sets, together with severe infection by Aspergillus niger and Penicillium, brought about a decrease in the onion yield (Fig. 4). Spoilage caused by F. oxysporum started as rotting of the short and flattened stem of the onion, which continued to spread upwards along individual scale leaves. The dry outer scale leaves were more reddish-brown than usual. The infected tissue of bisected onions initially appeared waterish and later desiccated and shrunken starting from the base. Light coloured mycelia were often abundant in the region where the leaves joined the stem (Fig. 5).
The pathogen produced exaedy the same symptoms on the sets as on the large onions. When storage of onion sets at a temperature of 25-38°C was terminated, the infected sets were mummifieded and became separated out during the cleaning and grading of the crop. The sets at this stage usually appeared to be healthy. When the sets were grown in the field, the pathogen caused premature death of the roots and yellowing of the leaves when the seeds had been inoculated with a suspension of F. oxysporum prior to sowing.
The microscopic appearance of the fungus was the same as that described in the literature ( BOOTH 1971).
Other fungi Aspergillus niger v. Tiegh. was common (21-29 % of infected lots) in one of the joint stores of the commercial grower. However, it was only found occasionally in the other stores, where it grew as black mold on the outermost leaves without completely spoiling the onions (Fig. 6). A. niger was common on the sets (83 % of the lots), usually occurring on the surface of the sets and in some lots as an exceedingly abundant systemic pathogen. In such cases it had, together with other fungi, a reducing effect on the size of the crop (Fig. 4).
Penicillium spp. occurred widely on the surface of onions, usually reducing, however, the quality only. Only in a few individual cases did it completely spoil the onions. Penicillium fungi were also common in all the set lots. The degree of infection showed considerable variation; the fungus occurred only on the surface of the sets or penetrated systemically throughout the inside of the sets, in which case spoilage was rapid during inspection and the yield of the sets was smaller than normal.
Control of storage fungi of onions
Effect of soaking onion sets in benomyl on storage fungi Soaking onion sets in benomyl alone before planting out, almost completely inhibited onion spoilage during storage throughout the course of the experiments (Tables 1 and 2). The degree of B. allii infection on the soaked onion sets was always less than 5 % at the end of the storage period apart from two lots of onions in 1978 when treatment reduced the B. allii-% from 25.7. to 7.5 and 31.6 to 9.6. Soaking in benomyl almost completely protected the onions against Fusarium rot. The fungus was not found at all in six treated lots in 1977 and in the other four lots at levels of less than 2 %. Fusarium rot was not found at all in the 1978 material (Table 1).
Effect of fungicide treatment in the field on Botrytis allii Spraying the onion plants during the growing season, in the spring or the autumn, did not reduce damage caused by B. allii during storage. The reduction in the B. allii-% was only of the order of I-3 %-units and in these cases only when the onion sets were healthy or soaked before planting in benomyl (Tables 2,3, 4 and 5). When the sets were severely infected by B. allii or else B. allii had been spread over the plants a number of times, treatment of the plants with fungicide reduced the B. allii-% of the stored onions (Tables 3 and 6). Spreading B. allii a number of times increased the difference between the B. allii-% of the untreated sets and those soaked in benomyl by only 4 %-units, although the plants were kept continuously in a moist condition by means of irrigation. There was no difference between the B. allii-% of onion sets soaked only in benomyl and those which were not soaked but sprayed with fungicide a number of times (Table 3). When onion sets were being grown, spraying the plants with fungicide in spring and in autumn did not reduce the B. allii-% of the following year's crop. Onions grown from the smallest onion sets were healthier than those grown from the largest ( Table 7). Effect of nitrogen fertilisation and storage temperature on storage fungi Application of nitrogen fertilizer to the sets and onions had no effect at all on the storage fungi of onions throughout the course of the experiments (Tables 5, 6 and 7). A high storage temperature of 20°C increased the proportion of F. oxysporum among the storage fungi. Spoilage of onions caused by B. allii during storage at temperatures below O°C occurred at a slightly slower rate and at a lower level than at the highest temperature (Fig. 7). In the preliminary trials carried out in 1976, B. allii inoculated on healthy onions also grew at temperatures below O°C, but F. oxysporum only caused onion spoilage at temperatures above O°C (POHTO 1979).
Discussion
The results of these experiments show that Botrytis allit, which was clearly the most serious storage pathogen of onions, primarily spreads to the onion crop via the sets. This is in good agreement with recent studies in which B. allit was shown to be seed-borne and cause latent infection that breaks out as the disease during storage (TICHELAAR 1971, BRÄUTIGAM 1977, MAUDE and PRESLY 1977a, b, BOCHOW and BÖTTCHER 1978, BOCHOW and EL-MOSALLAMY 1979. When the two-year cultivation method was used, the sets were latently infected from infected seed during the first growing season. Infection by B. allii docs not become apparent during storage of the sets because they are presumably too young physiologically for the fungus to develop (cf. BOCHOW and EL-MOSALLAMY 1979).
In contrast to previously-held beliefs (JAM ALAINEN 1962, HEINZE 1974, B. allii was not found to infect onions to any significant degree during the growing season, despite the fact that the plants were artificially infected with the fungus and irrigated in order to maintain a high degree of moisture. Furthermore, according to MAUDE and PRESLY (1977 b), infection during the growing season is only slight. The coloured outer scale leaves of onions have been found to stimulate Aspergillus niger and to inhibit B. allii ( HATFIELD et ai. 1948). The young tissue of onions (the middle leaves) are more resistant than the older leaves lying under the coloured outer leaves (BOCHOW and EL-MOSALLAMY 1979). Both these feature well explain why the onions were not infected by the end of the growing season and not even when artificially infected in the field. B. allii infection did not occur during harvesting on any untopped onions which were dried immediately after being lifted, al- though there were large numbers of the pathogen's conidia on the surface of the plants.
The possibilities of infection taking place from the soil and the effect of late harvesting or mechanical damage, such as mechanical harvesting, on infection by B. allii were not investigated in this study. They will be dealt with in further studies.
The other storage fungi studied in this investigation are of only slight importance in Finland. Aspergillus niger and Penicillium fungi are mainly pathogens which only reduce the quality of the onion crop. Fusarium oxysporum, however, may cause a certain amount of damage in stores in isolated cases. The fungus is presumably only set-borne in Finland, because the fungus requires much higher temperatures than arc to be found in Finland to infect onions from the soil (ABAWI and LORBEER 1972).
The reduction in the size of the crop caused by set-borne Aspergillus, Fusarium and Penicillium, when present in large numbers, is presumably of more importance than the storage diseases caused by these pathogens. In favourable conditions, F. oxysporum causes damping-off (ABAWI andLORBEER 1971, 1972) and reaches the stem via the roots (PARKINSON and CLARKE 1964), thus reducing onion growth by killing off the roots prematurely. The fungus can even spoil the onions in the field (WALKER 1952). Penicillium has been found to weaken the growth of garlic (SMALLEY 1954), the growing of which resembles that of onion sets. According to the experiments carried out in this study, the high degree of moldincss on the sets is the cause or the result of some other factor producing the poor yield of the sets. For this reason, attention is also nowadays paid to these fungi in Finland in the quality control of sets, the levels of these fungi on severely infected sets not being allowed to exceed 10 %.
The most effective and only suitable method for controlling storage fungi of onions was found to be soaking the sets in fungicide before planting. Benomyl and thiophanatemethyl preparations have been approved in Finland for this purpose. The effectiveness of soaking the sets in fungicide appears to be the same for seed dusting in annual cultivation (MAUDE and PRESLY 1977b, BOCHOW and BÖTTCHER 1978, WARD 1979. When the storage fungi of onions are controlled by carefully soaking the sets, a healthy crop can, according to the results of this study, easily be stored in a wellventilated store from one growing season to the next. Instead of using expensive, mechanically refrigerated stores, cheaper means of storage which employ the outside air for cooling purposes can be used since infected onions will also spoil in cold stores at the lower recommended storage temperatures (cf. AURA 1963). The storability of the onions can be estimated already in the autumn by determining the B. allii content of a sample of onions taken from the crop. This study has given very clear hints about this: the main storage losses of onions are caused by B. allit, the fungus passes to the store from the field in onions which are already infected and no longer spreads during storage. The preliminary experiments which have been carried out upto now on the estimation of storage capacity have given rather reliable results. Further studies will be carried out by developing methods which the grower himself can easily and surely perform to determine the state of health of the onion crop. The most suitable storage time can then be estimated. sonen, Tauno Koivunen and Pentti Heinänen, from the Department of Plant Pathology, and students Arja Pohto and Marja-Lecna Lahdenperä have participated in the practical side of this study. Mr. Erkki Hiidenkari and Mr. Jouko Mäkinen have provided much assistance and advice in carrying out the experiments at Turenki. I would like to express my sincere thanks to all who have given me assistance and advice. | v3-fos |
2014-10-01T00:00:00.000Z | {
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} | s2 | Identification of the ingredients in curna, kvatha curna lehya and rasayana - a simple microscopic method.
Triphala Curna, Triphatladi Kvatha Curna, Inji Rasayanam and Manibhadra Lehya of Indian System of Medicine were examined microscopically and the methods of identifying their ingredients were reported as one of the quality control standards.
Powder analysis of the above crude drugs, which were produced in the local market, was carried out individually as suggested by Trease and Evans (1966) and Johanson (1939) and their salient anatomical features were recorded using the microscope and camera lucida. Then the compound formulations were prepared as in Vaidya Yoga Ratnavali and Siddha Pharmacopoeia of IMPCOPS and they were observed under the microscope.
OBSERVATION & RESULTS:
Powder Analysis of Crude drugs: Individual crude drugs were powdered to 40-80 mesh and the powder was mounted on a slide in suitable reagent and it was examined under the microscope and their salient anatomical features were recorded as follows.
II. Irregular masses of parenchyma with numerous prismatic crystals of 28-56 microns.
III. Laticifers of 1120 micron long and 12-20 micron diameter were found mostly in single or rarely in groups and then they were slightly bifurcated near the tips.
III. Fibres quite long, non septate, often found in bands and unstained with phlorogucinol.
III.
Numerous stone cells.
VI. Stone cells 308 microns long 28 micron broad. III. Calcium oxalate crystals were 28 microns in size.
V. Fibres 1120 microns long and 14 microns in dia.
II.
Resin bodies yellow or orange coloured found inside or outside the lacticifers.
III.
Rosette of calcium oxalate crystals in 16 -28 micron size found abundantly.
V. Fibres frequently found in groups each with single row of pits. Upper epidermis of leaf, wavy walled with graminaceous stomata. II.
Lower epidermis wavy walled with circular silica deposits. III.
Leaf sheath with numerous unicellular and rigid trichomes of 98 micron length, all pointing upwards. IV.
Fragments of leaf with parallel variation.
II. POWDER ANALYSIS OF COMPOUND FORMULATIONS:
After examining the salient anatomical features of the individual powdered crude drugs, the compound formulations Triphala Curna Triphaladi Kvatha Curna, Inji Rasayanam and Manibhadra Lehya were examined to detect the presence of their ingredients.
(a) Triphala Curna: This Ayurvedic Curna is a fine powder of the following three ingredients, all in equal proportion by weight and it is used as a stringent, laxative and antibacterial and it also relieves constipation.
Its ingredients are: 1. Bibitaki. 2. Haritaki and 3. Amali. This Curna was observed under the microscope and the following were observed.
i. Unicellular unbranched and clothing tyrichomes of 68 microns long with globular base containing tannin, angular parenchymatous epicarp with coloured bases of trichomes and thin walled parenchyma with numerous starch granules confirmed the presence of Bibitaki.
ii. Articulated and pitted laticifers confirmed the presence of Haritagi.
iii. Irregular masses of parenchyma with numerous prismatic crystals and individual laticifers occasionally bifurcated at ends confirmed the presence of Amalaki.
Thus the inclusion of Three ingredients in this compound formulation could be justified.
(b) Triphala Kvatha Curna:
This Ayurvedic Kvatha Curna is the coarse powder of the following eight ingredients, all in equal proportion by weight and its decoction is a mild laxative, decreases the amount of urine in diabetes insipidus and it is need as a vehicle for other medicines in all kinds of urinary disorders. v. Upper epidermis with wavy walled parenchyma and graminaceous stomata, lower epidermis with circular silica deposits, leaf sheath tissues with rigid trichomes and, of all small, green leaf bits with parallel renation which were microscopically visible indicated the presence of Vamsapatra.
vi. Spherical pollen grains of 56 micron trichomes of 364 -390 microns yellow coloured petal tissues and of all small fragments of petals and stamens which were visible to naked eye confirmed the presence of Hemapuspa.
Thus the inclusion of all the eight ingredients in the Kvatha Curna could be identified.
(c) Manibhadra Lehya: This Ayurvedic Lehya is the Linctuse, prepared by adding the powder of the following ingredients in Guda Syrup. It is a laxative, very good anthelmintic and blood purifier. It is also useful in piles, chronic skin diseases and habitual constipation. Its ingredients are Guda 1200gms. Vidanga 100gms. Amlaki 100gms. Haritaki 100gms. and Trivrt 300gms.
The Lehya was dissolved in excess amount of water and filtered. The residue was observed under microscope and the following results were noticed.
i. Black fragments of pericarp. Its columnar stone cells in a single row in the sizes of 140 × 280 microns and orange colored membranous testa, confirmed the presence of Vidanga.
ii. Starch granules in tetrads, spherical resin bodies. Rosette of Calcium oxalate crystals, alternately pitted vessels and bands of fibres with single row of bits confirmed the presence of Trivrt.
ii. Irregular masses of parenchyma with numerous Prismatic crystals, laticifers mostly found solitary and slightly bifurcated at ends confirmed the presence of Amlaki.
iv. Articulated laticifers with simple pits confirmed the presence Haritaki.
Thus all the 5 ingredients of the Lehya could be identified.
Discussion:
The microscopic method is found to be very simple and inexpensive and it could successfully be applied to the compound drugs which contain not more than eight ingredients. This method could also be tried with compound drugs of several ingredients if projection microscope is used. Moreover the number of ingredients in the medicine is not the limiting factor but the diversity of ingredients in the medicine is important so that the contrasting anatomical features of the ingredients could easily be traced. | v3-fos |
2014-10-01T00:00:00.000Z | {
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} | s2 | Genetic and biochemical characterization of waxy mutants in cereals.
During fine structure analyses of artificially induced waxy mutants in maize by use of Nelson's pollen analysis methods, it was noticed that some mutants showed a phenotype intermediate between waxy and normal nonwaxy. Such intermediate or leaky waxy mutants were frequent among mutants induced by a chemical mutagen, EMS. They seemed to be located evenly within the waxy locus. This would suggest that they might have been missense mutations, which would produce full-sized but partially inactivated enzymes, rather than deletions or frameshift mutations. To confirm the intermediate phenotype, a rapid measuring system was developed to measure waxiness of endosperm quantitatively. It was based on the blue value method and is applicable to a single grain of rice. Among the 27 waxy mutant lines of maize, including 11 EMS-induced, two of the EMS-induced mutants were clearly intermediate. Eighteen EMS induced wx mutants of rice were also examined, and nine were intermediate. ImagesFIGURE 1.
Introduction
Pollens of higher plants have many advantages in genetical analyses. They are haploid germ cells and can be examined in very large population if appropriate genetic trait is used. Fine structure analyses of waxy (wx)(1-3) and amylose extender (ae) (4) in maize as well as glutinous (gl: comparable to wx) in rice (5) are examples of successful analyses utilizing the merits of pollen analysis. Intralocus mapping of mutant alleles would be a good means to see possibilities of the presence of point mutations in higher plants. To obtain materials for such analyses, many wx mutants have been induced in maize by using ionizing radiations, germicidal ultraviolet light, or chemical mutagens (6)(7)(8). In these experiments, wx mutants were detected after a test cross of the treated materials with a standard tester wx stock. In the case of ultraviolet light, mutants were detected after self-pollination, but later they were crossed to the tester wx to confirm allelism. Therefore all of these waxy mutants belonged to the wx *Department of Induced Mutation, National Institute of Genetics, Misima, Sizuoka-ken, Japan. January 1981 locus on chromosome 9. During fine structure analyses of these wx mutants by using Nelson's pollen analysis method (2), it was noticed that some mutants showed intermediate phenotype between wx and normal nonwaxy (Wx) (9). An example of such an intermediate wx mutant is shown in Figure 1. Here the intermediate wx mutant had been crossed to one of the standard wx mutant (wx90), and segregating pollen were stained with iodine. The dark pollen at the center is considered to be a nonwaxy recombinant which may serve as Wx color standard in the same preparation. Other pollen grains segregated into light-colored wx and intermediate-colored mutant pollen in a theoretical one to one ratio. Those intermediate wx mutants were stable in their degree of waxyness. Such intermediate or leaky waxy phenotypes were inherent in some of the mutants induced by a chemical mutagen, ethyl methanesulfonate (EMS). They seemed to be located evenly within the waxy locus (9). They might have been missense mutations which might produce protein or enzyme with normal length but its function partially inactivated. To confirm intermediate phenotypes, a rapid measuring system was developed to measure waxyness of _ *s~~~. -1 !^ws. W.. endosperm starch quantitatively. It was based oil the blue value method to measure amylose fraction of starch (10). Results of measurements of wvx mutant lines in maize and also in rice are reported here. Distribution characteristics of the waxyness indices were also discussed.
Plant Materials
Normal nonwaxy (Wx) strain of maize (6311R) used was a multiple dominant genetic stock with purple plant color. Standard waxy (wx) mutant strain of maize (639) was a multiple recessive genetic stock with C sh1 and bz marker genes. These stocks were originally obtained from Dr. H. H. Smith, Brookhaven National Laboratory. Other genetic stocks were kindly supplied by Dr. R. W. Briggs (Funk Seeds International), Dr. 0. E. Nelson (University of Wisconsin), and Maize Genetics Cooperative. Other waxy mutants were induced in the strain 6311R by seed treatment with radiations or EMS, or pollen irradiation by germicidal ultraviolet light. Most of the materials were used with 6311R background.
The standard waxy starch mutant of rice, 504T65wx, was obtained from Dr. H. Morishima (National Institute of Genetics). Other wx mutants in rice examined were induced by EMS treatment of seeds of a normal nonwaxy Japonica variety, Norin No. 8 (11, 12). These materials were selfpollinated and selected for normal growth for a few generations. 36
Preparation of Starch Solution
Measurements were done on a kernel basis. Each whole maize kernel was crushed to coarse meal. The kernel was placed in a plastic cylinder on a piece of iron plate. A short iron rod was inserted into the cylinder and was hit with a hammer. The meal was transferred to a test tube. A 5-ml portion of distilled water was added to each tube. For rice, hulls were removed, and each grain was crushed with pliers and put into a test tube. A 3-ml portion of water per tube was used for rice. The test tubes were autoclaved at 1 atm for 15 min. After stirring for several seconds while hot, they were left overnight for cooling and sedimentation of debris. The supernatant fluid was used in the measurement.
Iodine Reagent and Staining
Half strength of Nelson's 12-KI solution (2) was used to stain the starch solution. The recipe adopted was 450 mg iodine and 2.5 g KI in 500 ml of water. To reduce the effect of color of excess iodine reagent, staining was done in excess of starch. A 1-ml portion of sample starch solution was mixed with 0.1 ml of iodine reagent and 2 ml of water in a 10 mm x 10 mm glass absorption cell. The cell was immediately set in the colorimeter. The measurement was completed within 1 min.
Colorimetry
The colorimetric apparatus was designed to measure with two wavelengths (430 nm and 660 nm) simultaneously (13,14). As shown in Figure 2, it FIGURE 2. Schematic of colorimeter. Absorption cell is equipped with inlet, outlet tubes and a stirring blade for dilution. Light beam was divided by mirrors and measured by silicon photocells behind interference filters.
Environmental Health Perspectives consisted of an incandescent lamp, an absorption cell, mirrors to divide the light beams, interference filters, and silicon photocells of photometry grade (Hamamatsu TV, S780-8BK). A small mixing blade and inlet and outlet capillaries were inserted into absorption cell so that continuous dilution could be done while concentration was monitored photometrically. When the sample solution was diluted to 50% transmittance for 430 nm, an electric signal was produced to command a digital printer to print out the transmittance value for 660 nm. This transmittance value for 660 nm was used as index to express waxy phenotype. Monochromatic lights obtained by using interference filters were used for high reproducibility.
ranged from 63 to 70. In rice, the values were a little higher both in Wx and wx lines. Figures 3 and 5 show distribution of wx indices of each kernel of the wx mutants of maize and rice respectively. High reproducibility of the measurements is demonstrated in strain 6311R (Wx) in Figure 3. M14, another Wx inbred line, showed wider distribution of the waxy indices probably inherent to this line. In Figures 4 and 6, wx mutants were placed in orders of mean wx indices of each wx mutant line.
In maize, most of the 27 wx mutant lines examined were complete wx mutants (Fig. 4). Eight wx mutant lines were genetic stocks of translocation or other phenotypes. Those wx genes might be presumably of the same origin. 1J2, R, A39, 1M2, 90
Results
The rationale of the photometry at two wavelengths is to measure amylose which is deleted in the wx phenotype, by the blue value method at a certain concentration of starch solution which was determined photometrically. Amylopectin is a major and common fraction in both types of starches. In Wx starch, amylopectin amounted to about 75% and in wx starch it reached almost 100% (15)(16)(17). In solution, amylopectin is stained reddish purple by iodine (18). To bring the concentration of sample solution to a predetermined level, the iodinestained solution was diluted continuously while monitoring the concentration of iodine-amylopectin complex by 430 nm blue light. When the concentration reached to 50% transmission at 430 nm, the percent transmission (%T) value at 660 nm red light was measured and recorded by a digital printer. This %T value at 660 nm represents the concentration of the blue colored amylose-iodine complex.
For preparation of the starch solution, alkaline dispersion was also tested. Starch could be dispersed well from the clushed endosperm by 1N KOH solution. Howe ver, it was not used in the present measurements for two reasons: first, it requires neutralization of each sample solution before iodine staining; second, it reduced the difference of indices between Wx and wx. Compared to alkaline dispersion, autoclaving was convenient and good for uniform preparation of many sample solutions. The time of autoclaving was varied, and no significant difference was found from 5 to 30 min. A period of 15 min was adopted because it is the same condition as routine sterilization procedure in our laboratory. Measurements of waxy phenotype were made for each kernel. and H21 are known to be mutants of independent origins. 75UVwxl and 75UVwx2 were mutants induced by irradiation of pollen grains with germicidal ultraviolet light. Mutants with prefixes 71. 72, and 74 were EMS-induced mutants. Of 11 EMSinduced wx mutants, two were clearly intermediate (Fig. 3) and another two were next to them (Fig. 4). In rice, some wx mutants were very close to normal Wx (Figs. 5 and 6). The mutant 74wx3 had been found as doubtful wx mutants. Examination of waxy indices on a kernel basis revealed that the distribution of the indices was extended to higher region compared to normal Wx, indicating that amylose content was affected to some extent, though the mean value of the indices did not differ significantly. Here, the line 74wx3 was considered very leaky wx mutant. Among the 18 EMS-induced wx mutant lines, nine could be classified as intermediate wx mutants.
Compared to maize, in which wx mutants were detected after a test cross with a standard wx line, wx mutant in rice were detected in M2 endosperms 38
Discussion
The nature of mutation in higher plants has been studied extensively by many investigators in related fields. After the suggestion by Stadler and Roman (19) 8 74wx3 75wx3 74wx5 74wx8 76wx3 75wx2 74wx2 75wxl 74wx7 76wx2 74wxl 75wx5 504 T 65 wx 76wx5 74wx9 wx73-1 76wxl 74wx6 However, induction of a point mutation in its strict definition, singje base-pair substitution, and its confirmation became possible by the recent developments of studies on chemical mutagenesis (20)(21)(22). Detailed analyses of mutant characters and knowledge of biochemistry concerning both in chemical reactions in mutagenesis (23)(24)(25) and gene function (26) would support this view. Intralocus fine structure analysis developed by Nelson (1) would have limited resolution in measuring the size of genetic alteration which caused the mutation. Immunological detection of CRM and electrophoretic detection of modified protein might be a good means to show that the mutation concerned was a missense type base pair substitution. These methods were successfully applied to sh1 locus mutants in maize (27).
CRM and modified proteins were detected in some EMS-induced sh1 mutants. Intermediate or leaky phenotypes reported here would be another examples to indicate, though indirectly, the presence of full size protein molecules which is affected its function only partially. The intermediate phenotypes reported here in maize were seemed to be inherent to certain EMS induced wx mutant lines. These wx mutant genes were allelic to other wx mutants suggesting that they were in the same wx cistron.
A modifier of Wx-wx phenotype, amylose extender (ae), is known on a different chromosome. It is a recessive gene and increases the amylose or amyloselike fraction in starch both in Wx or wx January 1981 Distribution of induced wx mutants as placed by Wx pollen grain frequencies in hybrids between tester wx and the mutants. Arbitrary grading of iodine stain was made between (1) no background segregation, and (4) half of the wx pollen stained so dark that scoring of Wx needed extra care in staining [from Amano (9)].
phenotypes (15,16,28). But the present examples of the intermediate wx mutant line are not of this case. There was no phenotypic expression of ae-wx double mutations (15), and no ae-Ae segregation was observed. It can be also noticed from Figure 7 that there are too many intermediate wx mutants induced by EMS to permit them to be ascribed to simultaneous mutation at ae locus. In Figure 7, besides the recombinational distances from the standard wx stock, 639, expressions of wx phenotype as observed after iodine staining of pollen grains were classified into four groups. The 39 * X * -classifications were made between colors of standard wx and normal Wx recombinant pollen grains in the same slide preparation. Figure 6. Mean wx indices for each wx mutant line were distributed rather continuously from the Wx region (74wx3)to fully wx region (74wx7). Since in these analyses all viable wx mutants were analyzed, it might be comparable to the results shown in Figure 7, as for sampling of materials. In both materials, EMS treatments were made under conditions favorable for less chromosome aberration (29). Simply, EMS was dissolved in distilled, deionized water without use of any buffering salts. The solution was acidic and the pH was around 3. High efficiency in induction of mutation and good survival of the induced mutant (7,9) would support the explanation of intermediate wx phenotype by missense mutation. Intermediate phenotypes were also observed in some of the non-CI (9) or sh1 mutations. These facts would suggest that an intermediate phenotype might be a rather common phenomenon where point mutations could occur.
In conclusion, it is advised that the monitoring system for mutagenic activity of environmental pollutants be prepared also for intermediate mutant phenotypes if point mutation inducers are expected. | v3-fos |
2018-12-06T21:42:19.480Z | {
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} | s2 | Controlling Weeds in Wheat Stubble in Extreme Western Kansas
This report is brought to you for free and open access by New Prairie Press. It has been accepted for inclusion in Kansas Agricultural Experiment Station Research Reports by an authorized administrator of New Prairie Press. Copyright 1981 Kansas State University Agricultural Experiment Station and Cooperative Extension Service.
Average rainfall between wheat harve's't and frost at Tribune, Kansas, is about 6.5 inches-30 percent of the total expected during the entire fallow period in a fallow-wheat system. Much of this late summer and fall moisture is wasted if weeds and volunteer wheat are allowed to grow undisturbed. Since moisture is usually the limiting factor in crop production, preserving additional water during fallow should increase crop yields. Or, the fallow period might be shortened. For example, fallow-wheat-sorghum (FWS) involves two crops in three years with two fallow periods of about 11 months each. Whereas, fallow-wheat or falldw-sorghum systems produce two crops in four years.
The study reported here compares several cropping systems, involving fallow, wheat, and sorghum (Table 1 ). The tests will run for several more years, so these results are only preliminary. Thus far we have harvested sorghum four years, 1972years, , 1973years, , 1974years, , and 1975years, , and wheat three years, 1973years, , 197 4, and 1975years, . Sorghum yielded well in 1972years, , 1973years, , and 1975, but an early fall freeze in 197 4 markedly reduced yields. Aver-age wheat yields were high all three years, 31 to 42 bu/a (Table 2).
When conventional tillage was used, wheat after sorghum (FWS) yielded the same as wheat after conventional fallow (fW), 33 bu/a. In the same FWS system, sorghum yielded 46 bu/ a compared with 53 bu/ a in the fallow-sorghum rotation (FS). But, two~ crops (wheat and sorghum) were produced in three years rather than two crops in four years. The conventional systems did not include complete weed control in the wheat stubble following harvest.
Inthe two experimental systems, FWS+ and FW+, blading plus a residual herbicide (an 80% atrazine product) controlled weeds in the wheat stubble. Equivalent late summer and fall weed control could have been accomplished with additional tillage in lieu of the herbicide. But the atrazine aided in weed control the next spring and, in the FWS+, in the sorghum crop, although in some years spring tillage was needed to control grass weeds. More moisture was stored in the soil in the FWS+ system than in the co~J_ventional FWS system, probably largely because the usual system did not control after-harvest weeds. The increased moisture contributed to increased yields. The FWS+ plots had 4.1 inches of soil moisture at planting time, compared w ith 2.9 for the conventional system, an increase of 41 percent. Likewise, sorghum yields increased from 46 bu/ a to 53 bu/ a, a 17 percent increase. Whe· at yields in these systems averaged 38 bu/ a in the FWS+ and 33 bu/a for FWS. However, the difference was from only 1975 yields. Wheat yields under the two systems did not differ in 1973 and 1974 (Table 2).
In the widely used fallow-wheat system, wheat stubble is often left untouched until the spring after ·harvest. Our work shows weed control in wheat stubble is as important in FW as in FWS. We used the same treatment described in the FWS+ system, except the atrazine rate was reduced from 2.5 to 1.5 lb/ a product. Average soil moisture at wheat planting in-'reased 32 percent (from 4.4 to 5.8 inches), and average wheat yields increased 21 percent (33 to 40 bu/ a) .
Fall weed control was not advantageous every year; the benefit depended on amount and distribution of rainfall. Figure 1 gives the amount and distribution of precipitation for 1971-1975. Note the wide variation and that the average was less than long time figures. When moisture after harvest was low, weed growth was limited even without control measures. When fall rain was above average, 1972, and 1973, fa.ll weed control led to increased crop yields (FW wheat yields in 1974and 1975, and FWS sorghum yields in 1973and 1974). Fall weed control in 1971 and 1974 (when rainfall was low) increased neither moisture nor yields. However, fall weed control has never depressed yields.
Atrazine persists relatively long in the soil. Its rate of breakdown depends on many factors, including temperature, moisture, organic mat~ ter, and application rate. It is commonly usf for weed control in corn, sorghum, and s6m-.. fallow systems. However, it is not registered for use in wheat-fallow system in western Kansas, and its use is not recommended.
Our results show the importance of controlling fall weeds. Until atrazine or some other herbicide is registered and found effective, wheat stubble s·hould be bladed to control weeds and volunteer wheat.
Yields from the various cropping systems are sometimes difficult to compare directly. We converted grain yields to pounds per acre per year <lb/a/ yr, Table 2). Highest yield was from FWS+, 1768 lb/ a/ yr. That was 249 lb/ a/ yr or 16 percent more than conventiona I FWS at 1519 lb/ a/ yr. Both FWS and FWS+ yielded substantially more than FW (990 lb/ a/yr) and FW+ (1200 lb/ a/ yr). Again the advantage of fall weed control is readily apparent when the FWS+ and FW+ are compared, respectively, with FWS and FW.
Ultimately net return, not total yield, will decide acceptance or rejection of a practice. When two crops, wheat and sorghum, are involved relative prices and price changes make accurate comparisons difficult. Possible additional costs for herbicides, tillage, seed, harvesting, and orher items such as interest must also be considered. Gross returns can be easily calculated from yields given in Table 2. Use current or expected commodity prices. In any event and with virtually any realistic price structure, it seems that FWS+ (1768 lb grain/ a/yr) will net substantially more per acre than will FW (990 lb/ a/ yr). This is a progress report of continuing research at the Tribune Branch Experiment Station. We will continue to work and continue to bring results to farmers, producers, and other interested persons.
Publications and public meetings by the Kansas Agricultural Experiment Station are available a nd open to the public regardless of race, color, national origin, sex, o r religion.
This publication from Kansas State University Agricultural Experiment Station and Cooperative Extension Service has been archived. Current information: http://www.ksre.ksu.edu. | v3-fos |
2018-04-03T06:06:55.071Z | {
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} | s2 | Control and induction of ovulation in cattle.
The control and induction of ovulation in cattle are discussed with particular reference to use of progesterone-impregnated coils in heifers and beef cows. Progesterone treatment for 14 days was required to obtain precise onset of oestrus. With 7, 9 or 12 days of progesterone treatment a luteolytic agent in the form of a prostaglandin (PG) or oestradiol benzoate had to be used. Fertility was normal after treatment durations of 7, 9 or 12 days, but fertility after 14-day treatment requires further testing. The progesterone coil was not effective in maintaining luteal-phase levels of progesterone throughout a 12-day treatment and increasing the concentration of progesterone in the coil from 4 to 20% was not effective in elevating the progesterone concentrations in blood. When progesterone concentrations dropped below approximately 1.5 ng/ml the basal level of LH began to rise before removal of the coil. A 2-fold rise in the basal level of LH was observed following the removal of the progesterone coil. This early rise in LH was absent in cows which did not ovulate after they were given a 12-day progesterone treatment and GnRH 24-36 h after removal of coils to induce the main LH peak. Absence of this early rise suggests that frequency and amplitude of episodic LH release were inadequate in the post-partum anovulatory period. Ovariectomy in the early post-partum period was not followed by an abrupt LH release.
Introduction
Beef cows suckling calves return to oestrus within 30-120 days following parturition. The length of this post-partum anoestrus is affected by breed, plane of nutrition and age, but primarily by the suckling stimulus (Inskeep & Lishman, 1979). Increasing the calf crop by 5 % and advancing the calving date by 6 days could increase the estimated annual output of beef cattle in the United States of America by $797 million (Gerrits, Blosser, Purchase, Terrill & Warwick, 1979). Thus, small improvements in reproductive performance could result in large economic gains.
Attempts to synchronize oestrus in beef cows suckling calves have been hampered by the problem of prolonged post-partum anoestrus (Hafs, Manns & Drew, 1975;Roche, 1976a). Various methods have been used to shorten post-partum anoestrus and to control oestrus. The use of prostaglandin (PG) F-2a requires the presence of a corpus luteum, while the use of progesterone alone or in conjuction with pregnant mares' serum gonadotrophin (PMSG), gonadotrophin-releasing hormone (GnRH), and the temporary or early weaning of calves does not. Since cows with active and inactive ovaries are likely to be present in a herd of cows, a successful regimen must be capable of inducing and controlling a fertile ovulation in both circumstances. Development of such methods requires a better understanding of the endocrine control of follicular growth and ovulation. This paper deals with methods to control and induce ovulation in post-partum cattle.
Control of ovulation
The aim in controlling ovulation in cattle is to have over 90% of treated animals ovulating within a 24-60-h period, so that one or two inseminations at pre-arranged times can be used. This means that treatments must synchronize the regression of the corpus luteum or delay periovulatory endocrine events until the corpora lutea have regressed. The synchronous termination of the luteal phase can be brought about by injection of luteolytic doses of PGF-2a or one of its analogues. This approach is not effective in beef cows suckling calves because 30-50% of the cows are in anoestrus (Roche, 1976a;Chupin, Pelot, Alonso de Miguel & Thimonier, 1976). The alternative method of delaying periovulatory events by administration of progesterone or synthetic progestagens is preferred because it facilitates the induction of ovulation (Roche, 1976a;Mulvehill & Sreenan, 1977;Petit, M'Baye & Palin, 1979). A major problem in using progesterone to control oestrus is the large daily dose of 20-30 mg required to suppress ovulation. One method of administering sufficient progesterone for this purpose is the use of a progesterone-releasing intravaginal coil (PRID; Abbott Laboratories, U.S.A.), which is inserted into the vagina by means of a speculum (Roche, 1976b). A second problem is low fertility at the synchronized oestrus following the 18-21 days of progesterone administration. This duration of progesterone treatment is necessary to give precise control of ovulation when progesterone or progestagens are used without a luteolytic agent. However, limiting the duration of treatment to between 9 and 12 days (Wiltbank & Kasson, 1968;Mauleon, 1974;Roche, 1974) overcomes the problems of low fertility.
Progesterone treatment for 12 days
Progesterone has no effect on the lifespan of the corpus luteum after Day 3 of the oestrous cycle (Ginther, 1970). A luteolytic agent is required to obtain precise onset of oestrus after 9-12 days of progestagen treatment. The luteolytic agent initially used was 5 mg oestradiol valerate (Wiltbank & Kasson, 1968) injected at the start of treatment. Even when oestradiol benzoate was used at the start of a 12-day treatment, the onset of the synchronized oestrus was delayed in 10-20% of treated animals (Roche, 1974;Roche, 1976c). In heifers this delay in onset of oestrus is related to progesterone concentrations at the time of removal of the PRID. Animals with progesterone concentrations greater than 2 ng/ml had a delayed onset of oestrus when compared to animals with lower levels (Mauer, Webel & Brown, 1975;Roche & Gosling, 1977). An experiment (J. F. Roche, unpublished) was carried out on lactating dairy cows, 5 or more weeks post partum, to determine whether the delay in onset of oestrus after coil removal was due to poor control of luteal function or to variation in follicular growth. Injection of PMSG at the time of coil removal did not reduce the variation in onset of oestrus (Table 1) indicating that delayed follicular growth was unlikely to be responsible for the variation in onset of oestrus. There was a significant correlation, however, between the level of progesterone in milk 24 h after coil removal and subsequent onset of oestrus (P < 0.01). Cows in oestrus within 2 days of coil removal had a milk progesterone concentration of 1.1 + 0.19 ng/ml, compared with 4.96 + 1.07 ng/ml for cows in oestrus on Day 4 and 14.3 + 4.1 ng/ml for those in oestrus between Days 5 and 7 after coil removal. It can therefore be concluded that the major reason for variation in onset of oestrus in cattle following the 12-day progesterone treatment is poor control of luteal function.
The stage of the oestrous cycle at the start of a 12-day progesterone treatment affects the subsequent response. Animals treated between Days 0 and 5 of the oestrous cycle are most likely to have a delayed oestrus (Mauleon, 1974;Roche, 1974;Wiltbank & Gonzalez-Padilla, 1975). However, Woods, First & Pope (1967) reported that progesterone shortened the oestrous cycle in animals treated between Days 0 and 3. Ginther (1970) showed that progesterone was less effective in shortening the oestrous cycle as the interval between injection and the previous oestrus increased and no effect occurred when progesterone was given after Day 3. Oestrogens can shorten the oestrous cycle (Wiltbank, 1966;Lemon, 1975) and oestradiol or oestradiolprogesterone combinations can function as a luteolytic complex at the start of a 9-12-day progesterone treatment. The need to use progesterone in the luteolytic complex is determined by the speed of the initial rise in progesterone. It is important to get the concentration of progesterone close to mid-luteal phase concentrations (3-6 ng/ml) as soon as possible to prevent the oestrogen inducing ovulation in animals in the follicular phase (Roche, 1974) and to produce a maximum luteolytic effect in animals that have recently ovulated (Ginther, 1970). Therefore when progestagen implants are used extra progesterone is required at the start of treatment (Roche, 1974;Wiltbank & Gonzalez-Padilla, 1975), but with the Silastic coil the initial release rate is sufficient (Mauer et al., 1975;Roche & Gosling, 1977).
If a treatment period of less than 12 days is used, the onset of oestrus is more variable. Significantly more heifers and dairy cows were observed in oestrus within 3 days of coil removal after a 12-day treatment compared to a 9-day treatment period (Roche, 1978). Fertility in animals bred at oestrus after 9 or 12 days of treatment was similar to that obtained in control cows.
Progesterone treatment for 14 days
Some countries will not allow routine use of oestrogens in cattle, and there is a need to develop alternative treatments. It has been shown that there is an interaction between the requirement for oestrogen and the length of progesterone treatment (Roche, 1978). When the treatment period is extended to 14 days oestrogen is not required to produce synchronized oestrus. However, in field trials using dairy cows, fertility following 14 days of progesterone treatment was significantly lower than fertility in control cows or in cows which had had a 12-day progesterone treatment with oestrogen given at the start of treatment. This lower fertility was due mainly to a low conception rate following a single insemination 56 h after treatment (table 2). Other work with dairy cows and heifers (Ellicott, Thompson & Hill, 1977;O'Farrell, 1978) has shown no reduction in fertility following one or two fixed-time inseminations after 12or 14-day treatment periods. Fertility following a 14-day treatment period appears to be normal, particularly when two inseminations at 56 and 72 h have been used. However, this treatment regimen needs to be tested in large-scale experiments.
Progesterone in combination with prostaglandin
An alternative treatment is to use a 7-or 9-day treatment period with progesterone and an injection of PGF-2a, or one of its analogues, at the end, or 1 or 2 days before the end, of the progesterone treatment. This eliminates the need for oestrogen and it is claimed that by giving the prostaglandin 1 or 2 days before the end of the progesterone treatment, the onset of oestrus is more precise (Thimonier, Chupin & Pelot, 1975;Hansel & Beal, 1979). Fertility following the 7-day progesterone treatment with prostaglandins given at the end (Roche, 1976d) or 1 day before coil removal (Hansel & Beal, 1979) is normal. Thus, there are advantages in using a progesterone-PGF-2a treatment in comparison to other treatment regimens.
The optimum concentration of progesterone in blood during a synchronization treatment has not been clearly defined. The importance of this question has become apparent since it has been demonstrated that progesterone has a negative feedback effect on basal LH levels in the ewe (Hauger, Karsch & Foster, 1977). A concentration of progesterone in blood sufficient to block oestrus may not suppress the LH and FSH concentrations to the basal values found during the luteal phase of the oestrous cycle.
Concentration of progesterone while coil was in vagina
In attempts to evaluate the relationships between progesterone and LH during a synchronized oestrous cycle, two experiments have been carried out with heifers. In the first experiment, 40 heifers, in the luteal phase (Days 8-10) or follicular phase of the oestrous cycle (Days 17-18), received intravaginal coils containing either 2% or 6.75% progesterone for 7 days. All animals received an injection of PGF-2a the day before the coils were removed. The blood concentrations of progesterone and LH before insertions of coils, and during treatment are shown in Text- fig. 1. In the second experiment, 24 of the heifers, following a normal oestrous cycle, were allocated at random to the following treatments for 12 days: (i) 4% progesterone coil with oestrogen capsule; (ii) 4% progesterone coil + injection of PG at coil insertion; (iii) as in (ii) but 20% coil used. Progesterone concentrations at time of coil insertion, during and after treatment are shown in Text- fig. 2.
The coil did not maintain luteal-phase concentrations of progesterone throughout the treatment period (Text-figs 1 and 2). Increasing the concentration of progesterone from 4 to 20% in Exp. 2 did not result in a significant increase in blood concentrations of progesterone, indicating that this is not an effective way to increase blood concentrations during the last half of a 12-day treatment. Decreasing the percentage of progesterone from 6.75 to 2% in Exp. 1 did result in a significant decrease in progesterone concentrations in heifers treated for a 7-day period during the follicular, but not the luteal phase of the cycle. It is also apparent that the shorter the duration of treatment, the higher the concentrations of progesterone in blood during treatment (Text-figs 1 and 2). Therefore, a 7-day treatment period has the advantage of maintaining blood levels of progesterone at normal luteal-phase levels during the treatment period.
In Exp. 1, declining concentrations of progesterone in follicular-phase heifers given 2% Coil inserted (days) Text- fig. 1. Mean concentrations (+ s.e.m.) of progesterone and LH in serum of follicular or luteal-phase heifers before and during a 7-day treatment with progesterone coils. Coils contained 2 or 6.75% progesterone and all animals were injected with PGF-2a 1 day before removal of coils. There were 10 heifers per treatment.
progesterone coils were associated with increases in daily LH concentrations and an earlier onset of LH and FSH peaks after treatment. This suggests that progesterone, as in the ewe (Hauger et al., 1977), has a negative-feedback effect on basal LH concentrations in heifers. The time course of the decline in progesterone and increase in LH concentrations following removal of coils is shown in Table 3. Within 4 h of removal of coils, progesterone concentrations had dropped by 58% while LH concentrations had increased by 86%. The optimum concentration of progesterone required to control oestrus may be that which maintains plasma LH at concentrations found during the luteal phase of the oestrous cycle, rather than the lower concentrations of progesterone required to block oestrus during treatment. The inter-relationships between ovarian steroids, pituitary gonadotrophins and follicular growth need to be more clearly defined so that the correct concentration of progesterone to maintain normal pituitary-ovarian function during a synchronizing treatment can be determined. Presently available delivery systems may have to be refined in order to maintain higher levels of progesterone in blood during the second half of a 12-or 14-day treatment period.
Conclusions
Because of the ensuing low fertility, the 18-21-day progestagen treatments have been superseded by treatment periods varying from 7 to 14 days. With a 14-day treatment period, a Coil inserted (days) Days after removal Text- fig. 2. Mean concentrations of progesterone (+ s.e.m.) before, during and after a 12-day treatment with progesterone.
There were 8 heifers per treatment. Coils contained 4 or 20% progesterone and heifers in two treatments received an injection of PGF-2a at time of insertion of coils while the animals in the other group received 4% coils and a gelatin capsule containing 10 mg oestradiol benzoate. luteolytic agent is not required in heifers to get a precise onset of oestrus. Fertility appears normal in most trials but this treatment regimen has not been as extensively tested as a 12-day treatment period. With a 12-day period, a luteolytic agent is required and an injection of 5 mg oestradiol benzoate or a gelatin capsule containing 10 mg oestradiol adhered to the coil is used. With a 7-day treatment period, an injection of prostaglandin is given at the end or 1 day before the end of treatment. This results in a precise onset of oestrus, particularly when prostaglandin is given 1 day before end of treatment, and fertility is normal (Roche, 1976d;Hansel & Beal, 1979). The 7-day treatment with a progesterone coil also has the advantage of maintaining high levels of progesterone, but the coil is not an effective method of maintaining luteal-phase levels of progesterone for more than 5-7 days. Preliminary evidence on the interaction between concentration of progesterone in blood and basal LH suggests that low progesterone levels may be associated with an increase in basal LH concentrations towards the end of treatment. A re-evaluation of presently available delivery systems for administering progesterone is required.
Induction of ovulation in beef cows
The factors responsible for initiation of follicular growth in cattle after parturition are not clearly understood. It is generally accepted that basal levels of gonadotrophins are required to maintain follicular growth. During the oestrous cycle of cattle at least 2 or 3 waves of follicular growth occur (Rajakoski, 1960;Ireland, Coulson & Murphree, 1979). This indicates that follicular growth may be continual despite high levels of plasma steroids. The factors that initiate growth and development of the follicle in the post-partum period are obscure. A study of 60 adult milked dairy cows has shown that the average size of the largest follicle increased from 9.6 mm at Day 7 post partum to 11•3 mm at Day 14 and 13.0 mm at Day 30. There were no differences in size of follicles between cows milked twice daily and suckling cows at Day 30 post partum (Wagner & Hansel, 1969). However, suckling delayed the appearance of follicles greater than 10 mm at 9-16 days after parturition (Wagner & Oxenreider, 1971). More information is available on concentrations of hormones in blood during the post-partum period in beef cows. There is an increase in basal LH concentrations due to an increase in amplitude and frequency of episodic LH pulses beginning about 4 weeks before ovulation (Humphreys, 1977;Lamming, 1978). An LH peak occurs which may be responsible for a small transient rise in progesterone (Corah, Quealy, Dunn & Kaltenbach, 1974;Arije, Wiltbank & Hopwood, 1974;Lamming, 1978). This transient rise in progesterone lasts for 2-4 days and is generally followed by a normal ovulatory LH peak and a functional corpus luteum.
In spite of the lack of detailed factual information on initiation of growth and development of the follicle in the post-partum period, many attempts have been made to induce ovulation by hormonal therapy. The original impetus for this came from Casida, Meyer, McShan & Wisnicky (1943), who showed that the ovary of the post-partum cow was sensitive to exogenous gonadotrophins before first ovulation. Early attempts to induce ovulation in the post-partum cow using steroids gave rise to confusing results, with some authors (Foote, Hauser & Casida, 1960;Fosgate, Cameron & McLeod, 1962) reporting a delay in first ovulation following progesterone administration, while others (Ulberg & Lindley, 1960;Foote & Hunter, 1964;Saiduddin, Quevedo & Foote, 1968) reported that progestagen, particularly in combination with oestrogen, advanced the time of ovulation. Fertility was generally variable or low. Mulvehill & Sreenan (1977) reported that injection of 750 i.u. PMSG at the end of a 9-day progesterone treatment was an effective method of inducing a fertile ovulation in beef cows. This small dose of PMSG resulted in 15% of twin births with the greatest frequency in cows treated after 60 days post partum.
Use of GnRH or oestradiol to induce ovulation
Post-partum beef cows were injected with 100 lig GnRH or 400 [tg oestradiol benzoate at Day 15 and again on Day 30 after calving and the number of animals with LH peaks (Mawhinney, Roche & Gosling, 1979) was determined by taking blood samples every hour for 30 h after injection. Daily blood samples for progesterone assay (Gosling, Parker & Fottrell, 1975) were also taken from 10 to 60 days after calving. Following treatment, 7/20 cows had an LH surge after oestradiol benzoate and 6/22 had an LH surge after GnRH compared to a 100% response obtained in follicular-phase cows. Both compounds failed to induce consistently an LH surge or ovulation. These results suggest that the hypothalamo-hypophysial axis was not capable of normal response to GnRH or oestradiol within the first 30 days of calving.
Use of progesterone coil to induce ovulation
Although effectiveness of progesterone treatment on induction of ovulation in the post-partum cow is not clear, there are sufficient data (Roche, 1976a;Mulvehill & Sreenan, 1977) to suggest that it has some beneficial effects. In a series of 4 experiments (S. Mawhinney & J. F. Roche, unpublished) we examined the effect of a 12-day progesterone treatment followed by injections of LH and FSH, GnRH or oestradiol benzoate on ovulation. Mainly first calving Hereford cross beef cows suckling calves were used. In all experiments, animals were grouped according to lactation number and post-partum interval and randomized within the various treatments.
Ovarian activity before and after treatment was assessed by measurement of progesterone (Gosling et al., 1975) in blood samples collected three times weekly. Animals with 3 consecutive values above 1.5 ng/ml were deemed to have ovulated. To determine whether LH peaks had occurred, jugular vein blood samples were obtained from a cannula every 4 h for 48 h beginning 24 h after removal of coils. LH was measured as previously described (Mawhinney et al., 1979). Animal numbers in some experiments were small because some cows had reached normal reproductive activity before treatment and were excluded.
Experiment 1. Twenty-six anoestrous cows were randomly allocated to a control group or to a group receiving progesterone coils containing 6.75% progesterone for 12 days and had a 10 mg oestradiol benzoate capsule attached. The 12-day progesterone treatment did not affect the number of anoestrous cows ovulating (11/15) within a 4-week period after treatment when compared with the number of control cows ovulating (10/14).
Experiment 2.
In this experiment 18 cows were allocated at random to 3 treatments: (i) control, (ii) progesterone coils and capsule as in Exp. 1, and (iii) as in (ii) plus 10 injections of 1 mg LH (NIH-LH-B9) and 1 mg FSH (NIH-FSH-B1) every 6 h beginning 60 h before removal of the coils. The number of cows that showed oestrus and that ovulated after the 3 treatments were 0/7, 1/6 and 4/5, and 1/7, 4/6 and 5/5 respectively. Large supplies of bovine LH and FSH were unavailable and so there were too few animals treated with LH and FSH for statistical analysis to be meaningful. However, the results suggest that silent ovulations were more frequent after the progesterone treatment than after progesterone-gonadotrophin treatment, and that the latter treatment resulted in more cows showing overt oestrus after removal of the coils than in the case of cows treated with coils alone. Experiment 3. Cows ranging from 15 to 50 days post partum were allocated at random to the following treatments: (i) control, (ii) 6.75% progesterone coils, and (iii) 6.75% progesterone coils and an intramuscular injection of 400 ag oestradiol benzoate at time of removal of coils. Based on progesterone concentrations in plasma, all animals were acyclic at the start of the experiment. Following removal of coils, LH peaks were observed in 1/9 cows receiving the coils and in 3/9 cows receiving coils and oestradiol benzoate; 3/4 of the cows with LH peaks ovulated. Failure of the animals to have an ovulatory LH peak was the major cause for the low number of treated cows that ovulated after removal of the coils. This suggested inadequate follicular development during and after progesterone treatment.
Experiment 4. In this experiment, 14 anoestrous cows received progesterone coils for 12 days (6.75% progesterone) and were then allocated at random to receive (i) no further treatment, (ii) injection of 400 ag oestradiol benzoate at time of coil removal, and (iii) injection of 10014 LH-releasing hormone (GnRH) 36 h after removal of the coils. The number of cows that had LH peaks after treatment were 1/3, 3/5 and 6/6 for the 3 treatments, respectively. However, only 6/9 cows that had LH peaks in response to oestradiol benzoate or GnRH ovulated. Again, these results suggest that induction of an LH peak following progesterone is not always an effective method of increasing the number of animals ovulating.
The results of these experiments suggest that the progesterone coil on its own is only partly effective in inducing ovulation in the post-partum beef cow suckling calves (17/33 ovulated after coil removal in the 4 experiments). Failure of cows to ovulate after progesterone treatment can be attributed to a failure to have an LH peak or to ovulate in response to an induced LH peak.
Characteristics of the LH peak in cattle
The foregoing observations on failure of progesterone treatment to induce ovulation prompts the question as to what are the characteristics of the LH peak required to induce ovulation in cattle consistently. The characteristics of the events leading up to the LH peak and the LH peak itself can be observed (Text- fig. 3) for animals given 6.75% progesterone coils for 7 days from Days 17 or 18 of the cycle. During the 7-day treatment with progesterone, basal blood concentrations of LH doubled within 4 h and remained high up to the time of the LH peak. Following the peak, LH concentrations returned to base-line values, similar to those found before and during progesterone treatment. This 2-fold rise in LH in heifers is similar to that reported recently in the ewe (Hauger et al., 1977). The importance of this rise in LH was not appreciated until concentrations of LH before and after the LH peak in the post-partum cow were examined in detail. In those cows in which an LH peak and ovulation occurred (indicated by a rise in progesterone), the concentrations of LH before the peak were significantly higher (4-8 + 0.3 ng/ml) than after the peak (2.8 + 0.3 ng/ml). Values were similar before (2.6 + 0.1 ng/ml) and after (2.1 + 0.1 ng/ml) the peak in cows that had no progesterone rise (Text- fig. 4). The absence of a rise in LH before the main peak appears to be associated with the failure to ovulate and this could indicate inadequate follicular stimulation. This rise in LH before the peak probably reflects increased amplitude and/or frequency of episodic LH secretion.
Control of LH secretion in the post-partum cows
The absence of the early rise in LH described leads to the question of the nature of the control mechanisms governing LH secretion in the post-partum period. Ovariectomy of cyclic heifers results in an increase in LH (Hobson & Hansel, 1972;Beck, Smith, Seguin & Convey, 1976), suggesting that ovarian steroids have a negative feedback effect on LH. Replacement therapy showed that both progesterone and oestradiol were required to maintain LH at concentrations similar to those found in intact cyclic heifers (Beck et al., 1976). Several workers have reported that the pituitary of the post-partum beef cow is refractory to GnRH. It is not clear whether the low levels of LH in the post-partum period are due to negative feedback from the ovary or to refractoriness of the hypothalamo-hypophysial system. To test the hypothesis that 50 - unpublished). The linear regression of LH values against time was determined for each animal and the mean slopes and constants of the regression lines were compared between groups of animals ovariectomized at different times (Table 4). In contrast to the abrupt increase in concentrations of LH in ovariectomized heifers (Hobson & Hansel, 1972;Beck et al., 1976), there was only a gradual increase in plasma LH concentrations for cows at the 3 different post-partum intervals. The mean constants and slopes were similar, showing no differences in LH secretion following ovariectomy at Days 25, 45 and 65 post partum. These results suggest that the ovary, through negative feedback effects, is not responsible for maintaining the low levels of LH in post-partum cows shortly after calving.
Conclusions
The gradual but small increase in LH secretion after ovariectomy of the post-partum beef cow, the variable period of refractoriness of the hypothalamo-hypophysial axis to release LH in response to GnRH or oestradiol (Webb et al., 1977;Radford et al., 1978; Mawhinney et al., 1979), and the decreased LH release in vitro from pituitary extracts of suckling cows (Carruthers, Convey & Hafs, 1980), all suggest that the endocrine cause of anoestrus in the beef cow lies with the hypothalamo-hypophysial axis. Suckling does not affect basal FSH, prolactin, oestradiol or glucocorticoid concentrations (Humphreys, 1977;Carruthers et al., 1980).
Pituitary concentrations of LH and FSH and hypothalamic concentrations of GnRH are not altered by suckling (Carruthers et al., 1980), although the frequency and amplitude of episodic LH release were reduced. In addition, pretreatment with progesterone and subsequent injection of GnRH resulted in all cows having LH peaks but not all cows ovulated. In those cows that had an LH peak without ovulation, the rise in LH before the main peak was not observed. This absence could relate to a decreased frequency and amplitude of episodic LH release, indicating that GnRH synthesis and release may be inadequate during the anovulatory period. The key to understanding the endocrine cause of anovulation in the beef cow may well depend on a greater understanding of the intricacies of the brain, the physiology of GnRH synthesis and release from the hypothalamus, and the factors that modulate the effects of GnRH on the pituitary gland. | v3-fos |
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} | s2 | Role of gastrointestinal microflora in nitrogen and mineral balances in young mice fed on autoclaved and irradiated diets.
Male germ-free (GF) and conventional (CV) mice were fed on steam-sterilized (autoclaved for 30 min at 121 degrees C) and gamma irradiation-sterilized (5 Mrad with 60Co) diets for a one-week adjustment period from 4 weeks of age. During the subsequent week, the amounts of feed eaten were determined, and the feces and urine were collected eaily. The effects of the mode of diet sterilization and intestinal microflora on the feed consumption, body weight gain, feed efficiency (body weight gain/feed consumption), feed N efficiency (body weight gain/feed N consumption), and the excretion, absorption and retention of N, Ca, Mg and P and the bone deposits of these minerals were investigated. 1) The method of sterilization of the diet did not appreciably influence these parameters, except for the apparent digestibility of N and ratio of N retention. Compared to autoclaved diet-fed mice, irradiated diet-fed mice showed a higher apparent digestibility of of N and lower ratio of N retention. 2) Feed consumption in both GF and CV mice showed no major differences. Body weight gain, feed efficiency and feed N efficiency were higher in GF mice than in CV mice. 3) GF conditions increased the apparent digestibility of N, Ca, Mg and P. Lower total excretion of N, Ca and P in the feces and urine, a higher retention of Ca and P, and higher ratios of retention of N and these minerals were observed in GF mice. 4) The results in GF mice indicated a higher weight of moisture- and fat-free bone and a higher Ca, Mg and P concentration in the bones (femurs and tibias with fibulas) with respect to the body weight minus the weight of the digesta.
3) GF conditions increased the apparent digestibility of N, Ca, Mg and P. Lower total excretion of N, Ca and P in the feces and urine, a higher retention of Ca and P, and higher ratios of retention of N and these minerals were observed in GF mice. 4) The results in GF mice indicated a higher weight of moisture and fat free bone and a higher Ca, Mg and P concentration in the bones (femurs and tibias with fibulas) with respect to the body weight minus the weight of the digesta. The effect of treatment of germ-free (GF) animal diets by gamma irradiation to eliminate their bacterial load has received considerable attention in recent years, particularly in relation to heat sterilization.
It has been observed that gamma irradiation has a minimal influence on protein quality in commercial rat diet and that autoclaving reduces protein quality (1)(2)(3). However, no comparison has been made concerning the digestibility and retention of N in mice after treatment of diet by autoclaving or gamma irradiation. Moreover, no work has been done to compare the effects of the two kinds of sterilization on the mineral balances in any experimental animals except rats (4).
Pleasants et al. (S) observed urinary calculi and soft tissue calcification in GF mice on a particular diet while the conventional (CV) controls remained healthy. This observation indicates the possibility of basic differences in the metabolism of minerals, including Ca, Mg and P, which could lead to pathological conditions in GF mice.
The previous study showed that GFadult rabbits excreted a higher percentage of ingested Ca and P in the urine than CV rabbits (6). The absence of viable microflora in the digestive tract was associated with an increase in the retention of Ca, Mg and P in young Japanese quails (7). Reddy et al. (8,9) extended this finding to adult rats, showing that GF rats had higher retentions and bone concentrations of Ca, Mg and P than CV animals. At the same time, Gamier and Sacquet (10) found that GF rats have higher retentions of Ca and P. On the other hand, no comparisons of the retention of Ca, Mg and P in GF and CV mice have been reported.
Therefore, the studies reported here were designed for young mice to elucidate the effect of the mode of sterilization and the intestinal microflora of (a) the absorption and retention of N, Ca, Mg and P, and (b) the bone distribution of Ca, Mg and P. (14). Total P was analyzed by the Allen method (15) after wet ashing of the samples with HClO4.
The data were analyzed statistically by the analysis of variance. The difference between the means was considered significant if p<0.05.
RESULTS
In the present experiment, with the exception of N balance, there were no significant differences in the results with respect to the process of sterilization of the diet. Therefore, except for N balance study, no comparison was made between the autoclaved diet-fed group and the irradiated diet-fed group. Almost the same N intake was observed between the groups fed on the autoclaved and irradiated diets, but less fecal N excretion was shown in the group The intakes of N, Ca, Mg and P were approximately comparable in the GF and CV groups, and the fecal excretion of N, Ca, Mg and P was less in GF mice than in CV mice. Therefore, the apparent digestibilities of N and the three minerals were higher in the GF group. The urinary excretion of these minerals was greater, and total excretion of N, Ca and P from the feces and urine was less in GF mice. The retention of Ca and P, and the ratio of retention of N and these minerals were greater in GF mice than in CV mice.
Tables 6 shows the bone (the femur and tibia with fibula) weight and the contents of ash, Ca, Mg and P in the bones of GF and CV mice at 44days of age. There was no substantial difference between these weights and contents in GF and CV mice except for Mg % and Pmg.
The distention of the cecum is one of the major abnormalities seen in GF rodents including mice (16). Therefore, the bone weight and ash, Ca, Mg and P contents in the bone with respect to the body weight minus the weight of the digesta were calculated from Table 6, and are shown in Fig. 1. The weights of moisture and fat-free bone and levels of ash, Ca, Mg and P contents in the bone were affected by the microbial status of the animals, and were higher in GF mice than in CV animals, although the bone ash content did not reveal any statistically significant difference between the GF and CV groups.
DISCUSSION
Autoclaving of diets has been reported by several workers to reduce the N digestibility in rodents. Irradiation of diets, on the other hand, was shown to have a negligible influence (1,2) or an advantageous effect (3) on the N digestibility. Compared with irradiation, autoclaving decreased the apparent digestibility of N in this study. The decrease in the N digestibility after autoclaving might have been caused by modification of the proteins (2).
In spite of the lower apparent digestibility of N, a higher ratio of N retention was observed after autoclaving than after irradiation. Further studies concerning the utilization of N compounds should be undertaken when mice are fed on diets sterilized by different procedures.
Andrieux et al. (4) observed that body weight gain and the apparent digesti bility of P were greater in rats fed on an autoclaved diet than in those fed on an irradiated diet. This disagreement with our result in which there was no difference due to the diet sterilization process may be attributed either to the use of a different animal species or a different diet from ours.
The findings for feed consumption in Table 3, in which no difference was obtained between GF and CV mice, agree with reports for guinea-pigs by Newton and De Witt (17), for rats by Luckey (18), and for rabbits by Yoshida et al. (6). On the other hand, the result was different from that of Yamanaka et al. (19) who obtained a lower body weight gain in the GF group of mice. It is possible that this difference may be due to the use of different strains of mice or different diets.
While our results, which showed that the feed efficiency, feed N efficiency, apparent digestibility of N and ratio of N retention in GF mice were higher than in CV mice, were in agreement with those of Yamanaka et al. (20) on N digestibility of mice and those of Forbes and Park (21) on the feed efficiency of chicks, they were contrary to the results obtained from experiments on N digestibility in rats (22)(23)(24)(25) and on feed efficiency and feed N efficiency in Japanese quails (7). This may indicate that there are species differences in the effects of the intestinal microflora on feed efficiency, feed N efficiency and N digestibility.
Cardiac output in young adult GF rats was found to be approximately 30 lower than in CV controls (26,27), and adult GF rats (27) and mice (28) had O2 consumption values lower by 24% and 12% respectively than those found in the comparable CV animals. Since GF rodents have a lower metabolic rate than their CV counterparts, it is quite possible that GF mice have a higher feed efficiency, feed N efficiency and N retention than CV mice.
In addition to the apparent digestibility of N, the fact that GF conditions in mice increased the apparent digestibility and net retention of Ca and P, which has already been observed in Japanese quails (7) and rats (8)(9)(10), is shown in Table 4. The increase in retention of these minerals was reflected in increased deposition of T. YOSHIDA, S . SHINODA, T. DRANO, and K. MAEJIMA the minerals in mice bones (Fig. 1) as was shown for rats (8).
Since the intestines of GF animals are thinner than those of their CV counterparts (29), it can be expected that the thinner GF gut may transport N, Ca and P more rapidly so that less remains in the gut (30).
Most workers agree that trypsin survives longer in the gut of GF animals (31). It seems possible, therefore, that GF mice improved N digestibility.
Another factor has been shown to increase the absorption and retention of Ca and P. It is considered that enhanced levels of intestinal brush border Cat + stimulated ATPase [EC 3.6.1.3] and alkaline phosphatase [EC 3.1.3.1] together with mucosal calcium-binding protein (32) may be responsible for the increased Ca and P absorption observed in GF mice.
A third possibility is the effect of steroid compounds on the Ca absorptive system. GF rats secrete and retain bile acids mostly as conjugates (33), whereas in CV animals bile acids are present mainly in the unconjugated form (34). Conjugated bile salts, such as taurocholate or taurodeoxycholate, have been reported to enhance the absorption of Ca (35,36), in addition to their possible influence on vitamin D uptake. Moreover, it is conceivable that enteric microflora might alter the vitamin D structure as observed with other steroid compounds.
In conclusion, this investigation indicates that the absence of viable intestinal microflora in mice plays an important role in the absorption and retention of N, Ca and P, and the bone concentrations of these minerals. It is expected that future studies of this nature will be directed towards establishing optimal concentrations of N and these minerals in the diets fed to GF animals. It will also be of value to understand more clearly the mechanism of the increased absorption of N and these minerals in GF animals. | v3-fos |
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} | s2 | Antibodies to the major insoluble milk fat globule membrane-associated protein: specific location in apical regions of lactating epithelial cells.
Milk lipid globules of various species are surrounded by a membrane structure that is separated from the triglyceride core of the globule by a densely staining fuzzy coat layer of 10- to 50-nm thickness. This internal coat structure remains attached to the membrane during isolation and extraction with low- and high-salt buffers, is insoluble in nondenaturing detergents, and is enriched in an acidic glycoprotein (butyrophilin) with an apparent Mr of 67,000. Guinea pig antibodies against this protein, which show cross-reaction with the corresponding protein in some (goat) but not other (human, rat) species, have been used for localization of butyrophilin on frozen sections of various tissues from cow by immunofluorescence and electron microscopy. Significant reaction is found only in milk-secreting epithelial cells and not in other cell types of mammary gland and various epithelial tissues. In milk-secreting cells, the staining is restricted to the apical cell surface, including budding milk lipid globules, and to the periphery of the milk lipid globules contained in the alveolar lumina. These findings indicate that butyrophilin, which is constitutively secreted by surface budding in coordination with milk lipid production, is located at the apical surface and is not detected at basolateral surfaces, in endoplasmic reticulum, and in Golgi apparatus. This protein structure represents an example of a cell type-specific cytoskeletal component in a cell apex. It is suggested that this antigen provides a specific marker for the apical surface of milk-secreting cells and that butyrophilin is involved in the vectorial discharge of milk lipid globules.
Lactating mammary gland has received increasing attention as a model for studies of secretory mechanisms and membrane dynamics. Two major secretory mechanisms operate in milksecreting mammary epithelial cells. Lactose and milk proteins such as a-lactalbumin and caseins appear to be packaged together in Golgi apparatus-derived secretory vesicles and exit from the cell by the process of exocytotic fusion of secretory vesicle membrane with the apical plasma membrane (see references 13, 28, and 41, and references cited therein) . In contrast, morphological and biochemical evidence indicates that milk lipid globules are enveloped in apical plasma membrane during their extrusion from the cells (2; for reviews, see references 26, THE JOURNAL OF CELL BIOLOGY " VOLUME 89 JUNE 1981 485-494 ©The Rockefeller University Press " 0021-9525/81/06/0485/10$1 .00 and 37) . Thus, formation of milk fat globule membrane (MFGM) provides an opportunity to study selective budding of regions of apical surface membrane and selective accumulation or exclusion of membrane proteins from regions of membrane that participate in envelopment of a cytoplasmic component, here the lipid droplet (26,37,38,49).
On electrophoretic separation, MFGM from different species have been found to contain a limited number of size classes of major polypeptides (20,31,32) . Two of these polypeptides (band 3, apparent Mr 155,000, containing xanthine oxidase [cf. reference 331 ; and band 12, apparent Mr 67,000) have been found to be enriched in the salt-and water-insoluble material Electron micrograph of a section through a pellet of isolated and washed bovine milk fat globule membranes (MFGM), showing membrane sheets with typical "unit-membrane" appearance and densely stained fuzzy coat on one surface, i .e ., the previously internal side . Bar, 0 .2 ,am . x 100,000.
of the dense coat tightly associated with the inner face of MFGM (20). This coat material can be obtained as an insoluble fraction resistant to extraction of MFGM with high-salt buffers, various detergents, or chaotropic agents (20). The predominant polypeptide of this coat material (band 12, Mr 67,000) has been partially characterized and has been found to be an acidic glycoprotein that tenaciously retains small amounts of membrane phospholipids (20,31) . This protein therefore appears to present the unusual example of a glycoprotein that is insoluble in detergents, is associated with a structure located on the cytoplasmic face of the surface membrane, and may function in budding of milk lipid globules . Because it is continually lost from the milk-secreting cell, it must be synthesized at relatively high rates, in coordination with the production of the milk lipids. In this communication we present evidence by immunolocalization that the band 12 protein, for which the name butyrophilin' is proposed, is a specific marker for the apical cell cortex of milk secreting epithelial cells .
Materials
Mammary tissue from lactating Holstein cows, obtained from a local slaughterhouse, was immediately frozen in isopentane cooled with liquid nitrogen and stored at -70°C until processing for immunofluorescence microscopy (cf. reference 14). Liver, muzzle, and small intestine tissues were also obtained and rapidly frozen . Calf thymus was similarly processed. MFGM were prepared as in previous studies (20,25,32), and coat fractions were obtained from MFGM by extraction with 1% Triton X-100 in 10 mM Tris-HCI, pH 7.4 (20) . For comparison, MFGM fractions from other species (human, goat, rat) were similarly prepared (20) .
Antibodies
Band 12 protein was obtained by electrophoresis in slab gels containing SDS and was recovered from gel slices as described (3,14,(18)(19)(20) . Antibodies directed against this protein were elicited in guinea pigs following an immunization schedule essentially as described (18,20). The IgG fraction was isolated from this serum by ammonium sulfate precipitation and DEAE cellulose chromatography (14) . In addition, rabbit and mouse antibodies against band 12 protein described previously (19,20) were used . Specific rabbit antibodies against cytochrome bs, ' Meaning "associated with milk fat" as well as "having affmity to milk fat" ; from the Greek words ro Bo6Tvpov for butter, and 4)iaos, meaning: "liking something, being liked, belonging to, being related to." 48 6 THE JOURNAL OF CELL BIOLOGY " VOLUME 89, 1981 against actin, and against the total prekeratin fraction of desmosome-attached tonofilaments from bovine muzzle were as described (8,14). For controls, preimmune sera and IgG fractions were used .
Immunofluorescence Microscopy
Procedures for indirect immunofluorescence microscopy on cryostat sections were as described (15) . Sections were air-dried and/or fixed in -20°C cold acetone or, alternatively, fixed with 2% formaldehyde freshly prepared from paraformaldehyde in phosphate-buffered saline (PBS). In some experiments, special precautions such as short incubations in PBS solutions or in PBS containing 100 MM MgC1 2 (14, 30) were taken in order to minimize artificial losses during incubation . Fluorescein isothiocyanate (FITC)-conjugated goat antibodies against rabbit IgG and FITC-conjugated rabbit antibodies against guinea pig IgG or murine IgG were used as second antibodies (Miles-Ueda, Rehovot, Israel).
Electron Microscopy
Isolated MFGM and coat fractions made therefrom were fixed and processed for electron microscope examination of ultrathin sections as in previous studies (13,20,25).
For immunoperoxidase localization studies, sections of frozen tissues prepared as described above were air-dried and briefly (5 min) treated with cold (-20°C) acetone . The dried sections were incubated with antibodies ("50 pg/ml purified IgG) for 45 min at room temperature. After three washes with PBS, each for 5 min, the sections were allowed to react with peroxidase-conjugated rabbit immunoglobulins against guinea pig IgG (Cappel Labs., Cochranville, Pa .) for another 45 min at room temperature. After three washes in PBS the samples were fixed for 10 minwith either 2% formaldehyde (freshly made from paraformaldehyde) or 2.5% glutaraldehyde, both in PBS. Fixed samples were then washed with 50 mM Tris-HCl buffer (pH 7.6) and allowed to react for 10 min with a solution of 3,3'-diaminobenzidine tetrahydrochloride (DAB ; Serva, Heidelberg, Germany; 5 mg of DAB in 10 ml of 50 mM Tris-HCI, pH 7.6) to which 5 pl of 30% solution of H202 had been added immediately before use. After two washes with distilled water, the samples were treated wtih 1% aqueous osmium tetroxide for 30 min. After being washed with distilled water, the sections were dehydrated, embedded, and processed as described for flat-embedding of tissue culture cells grown on cover slips (12) . Ultrathin sections obtained from such preparations were either "double-stained" with uranyf acetate and lead citrate or were not stained at all. For evaluation of the significance of immunoperoxidase staining only unstained ultrathin sections were used.
Electrophoresis and Immunological Detection of Proteins
Polyacrylamide gel electrophoresis in the presence of SDS was as described (16,20). Two-dimensional gelelectrophoresis was performedessentially according to O'Farrell (35) . Samples were solubilized in lysis buffer (35) directly or after treatment with low concentrations of SDS as described by Kelly and Cotman (29) . Alternatively, samples were dissolved by boiling for 7 min in 10 mM sodium-potassium-phosphate buffer (pH 7.5) containing 5% SDS and 10%o 2mercaptoethanol, cooled to room temperature, and cleared by centrifugation at 18,000 g. The supernate was precipitated with 9 vol of acetone at -20°C, and the precipitate was washed, by resuspension and centrifugation first with -20°C cold acetone-water (9 :1, vol/vol), and then with -20°C cold 96% acetone . The final pellets were dried under Nz and kept dry until solution in lysis buffer (35) .
Gels were stained with Coomassie Blue or, for detection of glycoproteins, with the periodic acid-Schiff (PAS) reaction (cf. reference 20).
Immunoreplicae using agarose gel overlays were made as described (14,16,19). Alternatively, proteins separated by gel electrophoresis were transferred to nitrocellulose paper sheets by blotting for 24 h at room temperature essentially according to Towbin et al. (43) . The sheets were soaked in 1% bovine serum albumin (BSA) in PBS for 12 h at room temperature, rinsed three times with PBS, and incubated for 1 h at room temperature with the specific solution of guinea pig antibodies diluted 1 :100 with PBS containing 2% BSA. Thereafter, the sheets were washed five times with PBS, followed by one wash in 10 mM Tris-HCl (pH 7.4) containing 0.5 M NaCl, and one wash in 10 mM Tris-HCI (pH 7.4) containing 0.15 M NaCl. They were then incubated for 2 h at room temperature with "'I-labeled protein A in PBS containing 2% BSA (total radioactivity per sheet: 0.5-1 .0 tLCi) . To remove unbound protein A, the sheets were washed first five times with 0.5% Triton X-100 in 10 mM Tris-buffer (pH 7.4) for I h at room temperature, then in 10 mM Tris-buffer containing 0.5 M NaCl, and finally in 10 mM Tris-buffer containing 0.15 M NaCl. The washed sheets were dried between filter paper at 80°C for 15 min and were exposed to Kodak X-Omat R film at -70°C.
Gel Electrophoresis of MFGM and Characterization of Antibodies
Intact milk lipid globules are surrounded by a membrane that has a typical unit membrane appearance and is derived from the apical plasma membrane . Usually, a thick coat (10-50 nm) of densely staining material is observed between the inner face of the MFGM and the outer shell of the lipid droplet (13, 20,37,45) . This internal coat material is maintained on MFGM during isolation and extensive washing ( (20). These two polypeptide bands are also prevalent in MFGM from other species (illustrated for goat in Fig . 2 and human milk in Fig . 3 ; see also references 20 and 34) . On two-dimensional gel electrophoresis of total MFGM, of residual MFGM coat material extracted with high-salt buffer and Triton X-100, or of purified band 12 protein obtained by excision from gels, elution and acetone treatment (see Materials and Methods), butyrophilin appears as an acidic protein (cf. reference 31) and, in cow's milk, reveals at least four distinct isoelectric variants focusing at approximate pH values of 5 .30, 5 .27 (major component), 5 .24, and 5 .22 ( Fig. 4a and b). No other protein of Mr 67,000 is seen on two-dimensional gel electrophoresis of whole MFGM or band 12 protein preparations excised from SDS polyacrylamide gels (Fig. 4a) . When such gels are stained using the PAS reaction, the whole series of isoelectric variants is positively stained (Fig. 4 c), confirming and extending previous findings from our laboratories (20) and from Mather (31) that butyrophilin is glycosylated. By contrast, xanthine oxidase (Fig. 4 a), contained in "band 3 protein" of MFGM, is not significantly stained with the PAS reaction (20, 31), even after overloading (not shown here; this protein has several variants with apparent isoelectric pH values in the range from 7 .3 to 7 .7 ; cf. reference 31) .
Guinea pig antibodies to bovine band 12 protein eluted from SDS polyacrylamide gels were found to be positive by immunodiffusion analysis using the procedure described by Yen et al . (47) for solubilization of antigen (data not shown) . Antisera and IgG fractions were also characterized by the immunoreplica and "immunoblot" methods . The antibodies reacted strongly and specifically with the Mr 67,000 polypeptide present FIGURE 2 SDS polyacrylamide gel (7 .50) electrophoresis of whole MFGM proteins (a) from goat (slot 2) and cow (slot 3), in comparison with reference proteins (bars in slot 1, from top to bottom : phosphorylase a, glutamate dehydrogenase, and chymotrypsinogen) . Note the predominance of proteins of band 3 (asterisk) containing xanthine oxidase and band 12 (triangle) containing butyrophilin . The purified band 12 protein used as antigen is shown in slot 2 of b, in comparison with reference proteins (slot 1 in b; bars denote, from top to bottom : myosin, fi-galactosidase, transferrin, BSA, glutamate dehydrogenase, actin, and chymotrypsinogen) . in bovine MFGM (Figs. 3 and 4d) and, after two-dimensional gel electrophoresis, it was shown that the antibodies reacted with the whole range of isoelectric variants (Fig . 4d) . The corresponding butyrophilin in goat MFGM also reacted with the antibodies but the Mr 67,000 polypeptide of human MFGM did not react (Fig. 3) . Likewise, no cross-reaction was seen between the Mr 67,000 polypeptides of bovine and rat MFGM (not shown; see also references 20 and 34).
Light Microscopy
When frozen sections of lactating bovine mammary gland were examined by immunofluorescence microscopy using antibodies to butyrophilin, strong staining was observed over regions of epithelial cells that bordered alveolar lumina (Fig. 5) . In addition, structures within lumina were also stained, which allowed positive identification of milk lipid globules extruded from cells (Figs. 5 a and 6 a and b) . The inset in Fig. 5 a shows a putative lipid globule fixed during extrusion into the lumen to be stained entirely with antibodies to butyrophilin . Positive staining was also seen in the most distal parts of budding milk lipid globules (Fig . 6 a) . In addition, "punctate" fluorescence in small particles was observed within the alveolar lumina and might be attributed to MFGM fragments sloughed from milk fat globules during "aging" of secreted globules or during fixation (for electron microscopy indicating delamination of MFGM material, see, e.g ., reference 45) . Punctate discontinuities of fluorescence were also seen around some of the milk fat globules, nascent or postsecretory, and might reflect either sloughing of parts of the MFGM material (cf. reference 43) or natural small discontinuities of the internal coat layer as described by electron microscopy (e.g ., reference 20; see also Fig. 9 c) . Intracellular lipid droplets, which lack a continual membrane envelope (e .g ., references 2, 13, 37, and 45), did not appear to be surrounded by the antigen (Fig. 5 b and c) . Myoepithelial cells and cells within the lamina propria were not stained with these antibodies . Although apical regions of epithelial cells were intensely stained (Figs. 5 and 6 a and b), we never observed staining over basal and lateral regions of epithelial cells, suggesting that butyrophilin is selectively located in apical regions of epithelial cells and on luminal lipid globules . Similar immunofluorescence staining was found after the various modifications of incubation with antibodies mentioned in Materials and Methods, indicating that little, if any, of this antigen was lost or inactivated during the preparation . Rabbit and mouse antibodies against bovine butyrophilin gave practically identical results, showing strong staining of the apical rim of milk-secreting cells (not shown here ; an example for murine antibodies has been presented in reference 19). Control experiments in which preimmune serum or IgG were used as first antibodies showed no significant fluorescence (Fig. 6 c) .
Staining patterns observed with antibodies to butyrophilin were different from those observed with antibodies to other apically enriched proteins such as tonofilamentous cytokeratin and actin (Fig. 6 d-f) . Antibodies against prekeratin and actin also did not stain structures within alveolar lumina (Fig . 6 d and f) . Prekeratin antibodies gave intense fluorescence in myoepithelial cells (Fig . 6 d; see also references 14 and 42) and in lactating epithelial cells, where they appeared to be especially enriched at small foci of fluorescence coincident with desmosomal contacts (arrows in Fig. 6d). These foci of fluorescence most probably represent the short tufts of desmosome-attached tonofilaments that, in the cow but not in rodents, are seen in 488 THE JOURNAL OF CELL BIOLOGY " VOLUME 89, 1981 these regions (for electron microscope evidence, see reference 48). Similarly, antibodies to actin intensely stained the myoepithelial cells (Fig. 6e and f) as described previously (14) but also, in agreement with our earlier study (14), apical regions of epithelial cells. It is in this region that actin-containing micro-FIGURE 4 Two-dimensional gel electrophoretic separation of polypeptides of total washed bovine MFGM showing that butyrophilin (B), which occurs in four major isoelectric variants (short arrows in a, brackets designated B in b-d), is the by far predominant acidic protein present in this structure. Arrows in the upper right indicate direction of isoelectric focusing (IEF) and electrophoresis in the presence of SDS in the second dimension. A corresponding parallel IEF gel (first dimension) is shown separately at the top margin (bracket denotes the diameter of the gel cylinder); a one-dimensional separation of total MFGM polypeptides (same sample) has been run, together with the second dimension of the two-dimensional gel electrophoresis, on the left margin (denoted in a by the bracket with arrows and the heading "SIDS"). Asterisks denote the position of "band 3 protein" containing xanthine oxidase activity . The large arrowhead on the left gel track denotes the position of band 12 protein containing butyrophilin . The small arrowheads denote isoelectric variants of "band 13 protein" that have been immunologically identified as breakdown products of butyrophilin, probably by proteolysis (not shown here ; see also reference 20) . The appearance of butyrophilin ( B) on two-dimensional gel electrophoresis, relative to co-electrophoresed acidic reference proteins, is shown in b. In addition to co-electrophoresed bovine serum albumin (BSA) (apparent isoelectric pH range of the three major isoelectric variants : 6.2-6 .4 ; for similar values, see references 1 and 10) and murine vimentin ( V) (isoelectric pH of major unphosphorylated variant in several mammalian species, cow included : 5.30; cf . references 17, 22, and 40), the relative position of a-actin (A) from rabbit skeletal muscle as seen in a parallel electrophoresis is indicated. a and b have been stained with Coomassie Blue . For comparison, the PAS reaction of a gel electrophoretic separation similar to that shown in a is presented in c, illustrating the carbohydrate content of the whole range of isoelectric variants of butyrophilin ( B) . Correspondingly, the reaction of the series of isoelectric variants of butyrophilin, seen after two-dimensional gel electrophoresis (a-c), with the guinea pig antibodies used in this study is demonstrated in the radioautograph of a nitrocellulose blot-immunoreaction using '25 1-labeled protein A for detection of immunoglobulins (d) .
FIGURE 5 Immunofluorescence microscopy of frozen sections through lactating cow's udder after staining with antibodies to butyrophilin (band 12 protein) from bovine MFGM, seen with epifluorescence (a and c) or phase-contrast (b) optics . Note specific staining of alveolar epithelial cells and certain particles, probably milk fat globules, present in the alveolar lumina ( L) . The inset shows, in a slightly oblique section, staining of apical regions and of a budding milk fat globule (arrow) . The restriction of immunofluorescence staining to the apical region is especially well seen, at higher magnification, in oblique sections (b and c) . Note generally high phase contrast, i .e ., dry mass concentration, in apical regions (e .g ., b), probably related to high density of proteinaceous structures in this region (cf . reference 13) . Bars : a, 100 pm ; inset (in a), b, and c, 50lim .
filaments are most commonly observed (13). Staining with antibodies to actin was not seen over lipid globules or over luminally disposed areas of budding lipid globules (Fig. 6f), in agreement with the demonstrated absence of considerable amounts of actin in MFGM (27). However, regions just under budding lipid globules bound antibodies to actin (Fig. 6f, inset), in agreement with our electron microscope observation that microfilament bundles can occur in basal ("stalk") regions of budding lipid globules in arrays reminiscent of microfila-ment organization in cleavage furrows .2 By contrast, antibodies to cytochrome ba showed strong staining over the whole cytoplasm (Fig. 7), as previously shown for rat mammary gland (8).
Several other bovine tissues, such as liver, muzzle epidermis, small intestine, and thymus, that were examined by immuno- Immunofluorescence microscopy of frozen section through lactating bovine mammary gland as seen after staining with antibodies to cytochrome bs . Note that the whole cytoplasm is intensely decorated by these antibodies, indicating that the endomembranes are very accessible to the IgGs . a, Phase contrast ; b, epifluorescence . L, alveolar lumina . Bars, 50 Ltm .
fluorescence microscopy after application of antibodies to butyrophilin were observed to be negative. The absence of positive localization in apical regions of other polarized epithelial cells such as small intestine (Fig . 8) deserves comment. Distribution of actin microfilaments and various associated proteins present in microvilli and terminal webs of intestinal epithelium has been intensively studied (e .g ., references 5, 6, 9, 21) . In particular, a major apical cytoskeleton protein of Mr 68,000, for which the name fimbrin has been proposed (7), has been shown to be localized in brush borders of small intestine. Our observation that butyrophilin antibodies do not significantly stain intestinal epithelial cells distinguishes this protein from fimbrin and further illustrates the specificity of butyrophilin for the lactating mammary epithelial cell . Both ultrastructure and immunolocalization also distiguish butyrophilin of mammary gland from other insoluble apical proteins such as the desmosome-tonofilament meshwork of the "apical skeletal disk" described in intestinal epithelium (11) .
Electron Microscopy
The reaction of butyrophilin antibodies with components of lactating mammary gland cells of the cow's udder was also examined at the electron microscope level, employing the immunoperoxidase method on frozen sections that had been dried and processed in a manner similar to that for immunofluorescence microscopy (see Materials and Methods) . Light microscope controls demonstrated that the immunoperoxidase procedure gave the same results as the immunofluorescence technique, showing enrichment, if not exclusive localization, of the antigen in apical regions of milk-secreting epithelial cells (Fig . 9 a) . As indicated by the inset to Fig . 9 a, DAB reaction within this zone can be found on the apical side of budding milk fat globules . This distribution of staining was also found on electron microscope examinations of such preparations, using ultrathin sections without any additional staining (Fig . 9 6 and c) : strong staining was only seen on apical plasma membrane including surfaces of nascent milk fat globules ( Fig. 9 b) . No significant staining was detected on basolateral plasma membrane regions, in all other intracellular membranes, or in nuclei ( Fig. 9 b) . Negative reaction of butyrophilin antibodies was also observed in myoepithelial cells (Fig . 9 b) and in all cell types present in the lamina propria and blood capillaries (not shown here) . Higher magnification of the luminal surface region of budding milk fat globules clearly showed that the entire 10to 50-nm-thick cytoplasmic coat layer underneath the surface membrane was positively stained by DAB reaction product, except for some occasional small discontinuities Immunofluorescence microscopy of frozen sections through lactating mammary gland of cow after treatment with antibodies to butyrophilin from bovine MFGM (a and b), with guinea pig preimmune serum (c), with antibodies to prekeratin from desmosome-attached tonofilaments of bovine muzzle (d; antibody preparation GP,9BET), and with rabbit antibodies to actin (e and f) . Note that antibodies to butyrophilin stain only apical regions of milk-secreting cells, including the surfaces of milk lipid globules that are in the process of budding (arrow in a) or detached (arrow in b) in the alveolar lumina (L) . No staining is seen with control IgG (c) . The staining pattern of antibodies to prekeratin is different and shows preference for myoepithelial cells (14) and desmosome-tonofilamen t complexes at cell boundaries (arrows in d) . Antibodies to actin also stain, with special intensity, myoepithelial cells (e .g ., ME in insets of e and f) and certain apical regions (e .g., arrows in e and f) ; they do not stain the surfaces of milk fat globules ( FG) (e, phase contrast ; f, epifluorescence) but often show strong reaction at basal regions of milk fat globule buds (e .g ., arrows in insets of e and f) . Bars, 25 gm (a, b, d, and insets in e and f) and 50 Pm (c, e, and f) . known to occur on milk fat globule surfaces from various species, human included (20). DISCUSSION We have identified, by immunofluorescence and electron microscopy, an antigen that is located in the apical region of milk-secreting epithelial cells and around the lipid globules found in alveolar lumina of mammary gland and is recognized by antibodies to butyrophilin from bovine MFGM. From our observations it is apparent that the constituent recognized by these antibodies does not occur, in amounts detectable by this technique, in any other cells present in mammary gland . Within the secretory epithelial cells this antigen is concentrated, if not exclusively located, in apical regions of the cell, prominently in a dense coat covering the cytoplasmic aspect of the plasma membrane . The visualization, by immunofluorescence microscopy, of such a structure of a thickness (10-50 nm) much below the resolution of the light microscope, as a relatively wide, intensely fluorescent rim is not surprising in view of the visualization of even thinner structures such as individual microtubules (see, e .g., reference 36) that have been converted to "self-emitting" fluorescent objects by binding of FITC-conjugated IgG. Because the butyrophilin antigen is also present around extracellular lipid globules, it is most probably a constituent of the apical plasma membrane and/or the dense cytoplasmic coat associated with it. Detectable amounts of the antigen are not present in basal or lateral regions of these cells or in the supranuclear cytoplasm where the Golgi apparatus is located and endoplasmic reticulum is particularly abundant (2, 4, 13, 23). However, the observation of an absence of reaction in intracellular membranes does not necessarily provide proof that the protein is completely absent from these membranes: it could be present in very small amounts, or the specific deter- Immunofluorescence microscopy of frozen sections through bovine small intestine as seen in oblique (a) and cross (b) sections of intestinal villi after reaction with antibodies to band 12 protein of bovine MFGM . Note absence of significant staining . Bars, 25 gm . 49 2 THE JOURNAL OF CELL BIOLOGY " VOLUME 89, 1981 minant(s) could be lacking or masked in a special mode . On the other hand, we have shown that endoplasmic reticulum membranes, for example, are accessible to immunoglobulins when mammary gland tissue is prepared as in this study, as shown by the strong staining with antibodies to cytochrome b5 ( Fig. 7 ; see also reference 8).
It has been suggested or implied by other workers that MFGM originates, at least to a considerable degree, directly from secretory vesicles or Golgi apparatus without first being integrated into apical plasma membrane (24,39,44,46) . Although our results do not exclude the possibility of some contribution from other cell membranes to the formation of MFGM, they appear to strongly favor the concept that apical plasma membrane is the major source of the membrane complex enveloping the milk lipid globules . Especially interesting in this context is our observation that butyrophilin is localized nearly over the entire apical surface and is not restricted to regions engaged in budding . This indicates that the structure contributing this insoluble apical protein is associated with the surface membrane and is not only formed as the result of a local interaction of the apical membrane with a milk lipid droplet .
That this antigen has not been observed in other tissues or cell types suggests that butyrophilin is a marker specific for the apical plasma membrane of lactating mammary epithelial cells. A wide tissue survey for this protein coupled with immunolocalization will be necessary to test this concept. The biological function of butyrophilin is not known. Nevertheless, it is tempting to speculate that this protein, which is so abundant in MFGM, is a plasma membrane-associated cytoskeletal protein functionally involved in the recognition, budding, and vectorial discharge of milk lipid globules at the cell apex .
As mentioned above, butyrophilin has some unusual properties : (a) It is enriched in "cytoskeletal" fractions obtained after extraction with high-and low-salt buffers and nondenaturing detergents (20). (b) Unlike other cytoskeletal proteins, however, it contains some carbohydrates, notably mannose, glucosamine, and galactose . Our data indicate that a considerable proportion, if not all, of this protein, or at least the antigenic determinant recognized, is contained in the dense coat covering the internal side of the MFGM. At present, however, we do not know how the carbohydrate residues of butyrophilin are oriented, relative to the plasma membrane surface . The molecular location of the carbohydrate residues and the antigenic determinants in relation to the MFGM coat also remains to be determined . The simplest interpretation, that butyrophilin might be a glycosylated protein associated with the cytoplasmic side of apical plasma membrane, is in obvious conflict with current concepts of biosynthesis and location of glycoproteins . However, there are several other possible ways by which this protein could be formed and organized : For example, butyrophilin could be an integral transmembrane protein that is glycosylated in the part of the molecule exposed on the external surface while the other, probably larger, part of the molecule is exposed on the cytoplasmic side and is a constituent of the coat . On the other hand, the localization of butyrophilin in the cytoskeletal coat structure on the inner aspect of the apical plasma membrane could as well reflect an unusual rearrangement of membrane glycoprotein(s) in a special locally restricted structure, or the unusual glycosylation of a cytoskeletal protein in a special structural complex . Future experiments on the biosynthesis of butyrophilin are necessary to the understanding of the nature and origin of this remarkable protein . | v3-fos |
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} | s2 | Gene frequencies in the blood group systems of the Cuban Charolais
A total of 223 dam-son pairs of the Cuban Charolais breed were used to establish gene frequencies in the following eleven blood group systems : systems A, B, C, F, J, L, M, S, Z, R’, T’. In the F system the population was found to be in Hardy-Weinberg equilibrium. Fifty eight alleles were found in the B system and forty three alleles in the C system. The ten most frequent alleles in the Cuban Charolais are not encountered in the Holstein, Santa Gertrudis, Criollo or Zebu breeds found in Cuba. The allele Su" (U’ without U’ 2) not found in European breeds was present in the S system. It was concluded that the Cuban Charolais showed a high variability and was decisively different from other breeds of cattle found in Cuba. The presence of the Su" allele would seem to indicate that Bos indicus genes were present in Cuban Charolais Cattle.
A total of 223 dam-son pairs of the Cuban Charolais breed were used to establish gene frequencies in the following eleven blood group systems : systems A, B, C, F, J, L, M, S, Z, R', T'. In the F system the population was found to be in Hardy-Weinberg equilibrium. Fifty eight alleles were found in the B system and forty three alleles in the C system. The ten most frequent alleles in the Cuban Charolais are not encountered in the Holstein, Santa Gertrudis, Criollo or Zebu breeds found in Cuba. The allele S u " (U' without U' 2 ) not found in European breeds was present in the S system. It was concluded that the Cuban Charolais showed a high variability and was decisively different from other breeds of cattle found in Cuba. The presence of the S u " allele would seem to indicate that Bos indicus genes were present in Cuban Charolais Cattle. (1975) reported that the Cuban Charolais breed, which was well adapted to the climate and was found mainly in the « Manuel Fajardo Genetic Centre in Jiguani, Granma Province, had its origins in animals imported from France at the beginning of the century. As RESTON , 1970) of the Cuban Charolais were good, however, they did not have the calving difficulties associated with the French breed. This indicated certain differences in the genetic constitution of the breed which might be analysed with the aid of blood groups.
The objective of the present work was to establish the gene frequencies of eleven blood group systems in Cuban Charolais Cattle. Approximately 10 ml of blood was taken from the jugular vein of each animal. Samples were collected with the following anticoagulant solution : sodium citrate : 20 g ; sodium chloride : 5 g ; sodium cyanide : 0,4 g ; distilled water to 1 000 ml.
The reagents Fl to F20 were experimental reagents produced by the Jouy-en-Josas Laboratory.
In the F system the gene frequencies were estimated by directly counting the three genotypes. The square root method (C OTTERMAN , 1954) was used for the simple systems, while in the complex systems the iterative method was applied (C EPPELINI et a l ., 1956 ;N EIMANN -S ORENSEN , 1956).
III. -Results and d1scussion
A. -Test for genetic equilibrium To test whether the population appeared to be in genetic equilibrium the observed numbers of the three genotypes of the F system were compared with the expected numbers (table 1). The population was found to be in equilibrium. Because anti-S' was not available in the R' system, and because factor M 1 was not found, all the above systems, except the F system, behave like one factor, two allele systems. It should be noted that the frequency of the M factor was extremely low (0.004 : only two animals were found to be positive for this factor). The gene frequencies obtained in the Cuban Charolais appear in table 2.
The results agreed with those previously obtained by RoNnn et al. (1971) with the exception of the Z allele which had an increased frequency. This increase may be as much due to the sampling effect as to any possible selection effect caused by greater use of certain bulls (R ENDEL , 1963).
C. -A svstem
Although only two reagents were used in the A system, thereby differentiating three phenogroups A, AZ' and a, this was treated as a complex system, using the iterative method for gene frequency calculations. Results are shown in table 3. They are very similar to those reported by R ONDA et al. (1971) in the same breed.
D. -B system Fifty eight phenogroups of the B system were observed. They are presented in table 4. It should be noted that 1 p. 100 of the alleles present in the breed were not identified due to their low frequencies.
Results presented in this paper differ somewhat from those reported by R IBAS & M ITAT (1975) and R ONDA et al. (1971). This may be due to different specificities of the reagents used.
A comparison of at least 9 of the most frequent phenogroups of this breed with the phenogroups found in five other breeds in Cuba (Hosltein, Charolais, Santa Gertrudis, Criollo and Zebu) which were studied in the blood groups laboratory of the University of Havana (M ITAT , 1975) the following was observed (table 5) : In Cuba, allele BG!,O,TE'!,11&dquo; was only found in the Charolais breed, being the most frequent allele in this breed. The very frequent allele B B Q G '()I P ' B &dquo; is characteristic of the Charolais breed in Cuba (R IBAS & M ITAT , 1975). On the other hand, the BB !!YD' allele was observed in the Holstein, Charolais and Santa Gertrudis.
The allele with the fourth highest frequency in the Charolais breed, the B b allele, was found in all other breeds. This was expected because within the B! category, alleles may have been included which are not detectable by the reagents currently available.
The allele B P , E ', I ' I &dquo; was not found by M ITAT (1975). This was, however, according to R IBAS & M ITAT (1975), one of the most frequent alleles in the Cuban Charolais breed and in the present sample it had the fifth highest frequency. This allele did not seem to be present in other breeds of cattle in Cuba.
The B yl &dquo; allele found in the Cuban Charolais breed was also seen in Hosltein, Santa Cc!M!M and Criollo breeds and the B B , OIJ II allele which M ITAT (1975) reported to be only in Zebu cattle was found to be at a high frequency in Cuban Charolais.
The allele Bo!E'! has only been reported in the Cuban Charolais and Criollo breeds (M ITAT , 1975). This may indicate the presence of the latter breed in the formation of the Cuban Charolais.
Finally, the allele B BYP ' Y ' has only been found in the Cuban Charolais. Therefore, the Cuban Charolais appeared to be distinctly different from other breeds found in Cuba.
E. -C system Table 6 presents the forty three phenogroups in the C system. This high number confirmed the genetic variability of the breed, as already observed in the B system.
F. -S system The analyses were performed in a way similar to that used for the two previous systems, considering, however, the non-linear subtypes in this system (G ROSCLAUDE & M ILLOT , 1962;G ROSCLAUDE , 1963G ROSCLAUDE , , 1964 (table 7).
The notation U', indicates the presence of U' without U' 2' This phenogroup is characteristic of the Zebu breed (R IBAS , 1978 unpublished data) while it is not present in European breeds.
It may therefore be concluded that the Cuban Charolais showed a great variability. Furthermore, our data showed that the Cuban Charolais differs from other breeds found in Cuba. However, the presence of the allele U' l (U' without U' 2 ) may indicate that Bos indicus « blood » is present in Cuban Charolais.
Re V u pour publication en juillet 1981. | v3-fos |
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} | s2 | A survey of European primitive breeds of sheep
This paper surveys the primitive (i.e. unimproved) sheep of Europe using data (notably the fleece type) from surviving breeds in place of the more commonly-used skeletal and textile remains. Only bone evidence exists for the Neolithic sheep, and its coat is assumed to have been little different from that of the undomesticated wild ancestor. Textile remains show that Bronze Age sheep had less hairy, but still brown, fleeces like those of the Soay sheep that survives feral in the Saint Kilda islands off north west Scotland. Within this less hairy fleece type both textile remains and surviving sheep show a range of variation from relatively hairy to woolly animals. Textile remains show that white sheep appear in the Iron Age. Much wool was, however, still coloured, having either 100 p. 100 pigmented fibres (black or brown) or some pigmented fibres and others not (grey or roan). Surviving breeds of this type (often with a primitive short tail) have a similar fleece structure to the Soay but with a range of colours, white, black and grey, in addition to the brown of the Bronze Age Soay. The present paper concentrates on this type which was probably the predominant sheep in Europe until after the Middle Ages when modern, improved breeds began to emerge.
Introduction
Skeletal remains provide the most common source of evidence in archaeozoology, supplemented by remains derived from skin, e.g. R YDER (1962,1973). The present paper utilises descriptions incorporating tail length, horn, colour and fleece type data of surviving unimproved breeds, and incidentally provides a compelling argument for the preservation of such breeds (R YDER , 1976).
A synthesis of biological and historical evidence on the origin of British breeds made it relatively easy to group modern breeds into broad types, and to postulate waves of introduction (R YDER , a, 1968 a). It is less easy to discern the same patterns on the continent of Europe because the affinities are less obvious, and the greater number, and wider distribution, of breeds makes it difficult for one person to see them all (R YDER , 1968 b ;B ROOKE & R YDER , 1977, 1979. There is no conclusive evidence for wool textile manufacture in Neolithic Europe, and it is probable that the coat of domestic sheep then had outer kemps and fine underwool, showing only a gradual change from that of the wild ancestor. Textile remains from the Bronze Age, however, coupled with skeletal remains, indicate that the sheep of this period was comparable with the Soay which now remains feral only in the Saint Kilda islands, which are outliers of the Hebrides on the very edge of the continent ( fig. 1). The Soay shares with the wild ancestor : large horns in the ram, a short tail, and a moulting fleece. It also has a coloured fleece with white belly, which sets it apart from other primitive breeds. The fleece of the Soay is, however, much less hairy than the coat of the wild ancestor (see below).
It was originally suggested and b) that there was little change in British sheep until a new type was introduced about Roman times ( fig. 2). Apparently, however, the European type which forms the theme of the present paper dates back to Iron Age. Textile remains indicate either all the wool fibres naturally pigmented (black or brown), some fibres pigmented and some white (grey or roan) or all non-pigmented (white). The fleece is otherwise the same as the Soay having woolly as well as hairy types in which the hairy fibres are in fact fine kemps (R YDER , 1969 a).
Surviving breeds of this type have a range of colours, black, white and grey, in addition to the brown of the Soay, but coloured sheep with a white belly are rare. They usually retain a primitive short tail, and the skeletal dimensions are similar to those of the Soay ( fig. 5), and since they also have the same fleece structure, the main distinction from the Soay is the greater range of colour, so the type was named the (European) Short-tailed, vari-coloured by R YDER (1968 b). They seem to have been the predominant type in Europe until after the Middle Ages.
Textile remains indicate that most Roman sheep were white, and illustrations suggest that only the rams were horned. The suggestions of R YDER (1964 a) summarised in fig. 2, were that crosses between white, Roman sheep, and the brown, native Soay could have produced types that later emerged as breeds such as the Cheviot and Welsh Mountain to the north and west. These are horned in the rams only, and any pigment is brown rather than black. Such a derivation is no doubt too simplified if only because by the time the Romans arrived the Soay was no longer the dominant type. On its own in Britain, however, the Roman sheep could have given rise on the one hand to the primitive longwool and on the other to the shortwool. These types were found amoung Roman textiles (see below) but they did not become common until after the Middle Ages. On the continent the true fine wool was the more important type, emerging later in Spain as the Merino breed.
The third main type in Britain is the horned and hairy black-faced type. There are suggestions linking this with the Heath sheep of the North European Plain via a Danish introduction, but one need not postulate an exotic origin for every British type, although it is not yet clear how much evolution took place within Britain. The British representatives of the European vari-coloured typethe Orkney and Shetland breedshave been associated with Norse settlers, but if this type goes back to the Iron Age they may have introduced a similar breed and not a distinct type. Fig. 3 summarises the probable origins and relationships of modern British breeds from historical records over the last 200 years.
Changes in the fleece following domestication are shown in fig. 4. The big difference in diameter between the coarse kemps of the outer coat, and the fine, woolly under coat in the wild Mouflon (top) is very striking. Fleeces of existing sheep have been used in fig. 4, but all diameter distributions (fleece types) except that of the wild animal at the top have been found in ancient textiles. The evolutionary changes that appear to have taken place are, first, a narrowing of the coarse, bristly kemp fibres forming the outer coat of the wild sheep to produce much the less-coarse, hairy fibres (fine kemps) of the hairy Soay. The underwool became a little coarser, few, if any, domestic sheep having wool as fine as that of the wild sheep. Further narrowing of the hairy fibres, presumably as a result of selective breeding by man, changed them into wool fibres of medium diameter to give the fleece of the woolly Soay. These two fleece types occur as hairy medium and generalised medium fleeces, with the same natural pigmentation as the Soay, in Bronze Age wools.
The evolutionary term generalised is used for the latter fleece type because it formed an important link. Not only was it apparently derived from a more primitive nairy type, but it was able to give rise to several more-highly evolved fleece types as follows. A continued narrowing of the medium fibres by breeding could have resulted in a fine fleece with a symmetrical diameter distribution like that of the modern Merino ( fig. 4 bottom, right). If, one the other hand, the finer fibres had become coarser, then the medium wool diameter distribution of the modern longwool would have been obtained (top right). Thirdly, if both changes had taken place together, and the range of fibre diameter had become shortened, the distribution would have been comparable with that of the modern shortwool (right, middle). In this the mean fibre diameter has a value between that of the fine and medium values.
The true hairy (carpet wool) type of fleece seems to have arisen from the hairy medium wool by a change of most of the kemps into hairs (fig. 4, bottom, left). The origin of hairs, which are coarse and kemp-like in summer, but lack a medulla and appear like wool fibres in winter, appears to have been associated with the change from a moulting fleece to one of continuous growth (see below). The hairy, carpet fleece first appeared in the Iron Age, but was uncommon until after the Middle Ages. Most Roman wools, although white, were of either hairy medium, or generalised medium type (R YDER , a, 1981 but longwools, shortwools, and fine wools were developing at that time. A range of fleece types from hairy medium through generalised medium to true fine (rarely) has been found in the modern Orkney breed, and some Northern Short-tails have a true hairy fleece (R YDER , 1968 a).
In these histograms the fibre diameter is shown on the horizontal axis, and the number of fibres on the vertical axis. Revised version of the illustration in R YDER (1969 a and 1973), from R Y D ER (1982).
Dans ces histogrammes Ie diamètre de la fibre est en abscisses, Ie nombre de fibres In addition to allowing studies of the evolution of fleece structure, the survival of primitive breeds with a high proportion of coloured animals has allowed investigations of colour inheritance. An interpretation worked out by A DALSTEINSSON (1970) with Icelandic sheep, apparently also applies to Soay, Orkney and Shetland sheep and may even be universal (R YDER , 1980). The Soay has light and dark brown individuals, dark animals being genetically black, and most Soays have a white belly ( fig. 1). There are, however, self-colour Soays in which the belly is coloured, and dark self-coloured animals appear all black. The colour genotypes found in the Soay, Orkney and Shetland are shown in table l.
The colour pattern genes of the A series are on a different chromosome from the colour genes of the B series, and those higher in the series are dominant over those 'lower down, thus black (B) is dominant over brown (b) and the black phenotype can therefore be either homozygous (BB) or heterozygous (Bb). Brown (aabb) is the simplest (homozygous recessive) genotype ; a is the gene for self-colour and this is recessive to A « the gene for mouflon-pattern a term that A DALSTEINSSON use for black with white belly. In the Soay there are two genotypes for light mouflon-pattern, and four for dark mouflon-pattern. The all-black and all-brown genotypes of the Orkney and Shetland (and probably also of other Northern Short-tails) are the same as those in the Soay, and provide a link between these Bronze Age and Iron Age types : a polled, self-colour, light Sooy with a woolly fleece is almost indistinguishable from a brown Shetland.
White (A 11Jh ) is dominant to grey (A U ) and both are dominant to mouflonpattern (A w ) and self-colour a. There are at least nine white genotypes in the Orkney and Shetland breeds. The grey gene (A 9 ) causes a mixture of coloured and white fibres ; when the black gene (B) is present the mixture is grey, for which there are four genotypes. When there is only the brown gene (b) the mixture of brown and white fibres is named grey-brown, which the Norwegians name a brown skimlet », grey being termed « black skimlet !. It is usually the coarsest fibres that are pigmented.
The changes in fleece and other characteristics during successive periods of antiquity are summarised in table 2.
Material and methods
Most breeds described in this paper have been seen by the author and the fleece measurements given were made on wool and skin samples taken at the mid-side position (immediately behind the last rib, and half way down the body). Staple length was measured against a ruler, and fibre diameter was measured at the base of the staple on 100 fibres using a projection microscope at a magnification of 500 X. The skin was processed histologically and follicle counts were made on horizontal sections by the method in Appendix I of R YDER & S TEPHENSON (1968).
Fibre types
Although all fibres in the fleeces of sheep are collectively named wool, there are three main types : kemps which form the outer coat of wild sheep, wool, which forms the underwool, and intermediate (heterotype) hairs. Kemps are very coarse fibres with a wide internal (hollow) medulla. Kemp is usually short since the fibres do not grow long before shedding. Wool fibres are usually fine, and any medulla is relatively narrow. Hair (in sheep) is usually long and it varies over the length (hence the name heterotype). It is relatively highly evolved (see above), and so hair is uncommon in the primitive breeds of the present paper.
Criteria on which fleece type is decided Although the mean diameter, and the diameter distribution are taken into account, the upper limit of the diameter range is the deciding factor (see fig. 4). Thus a fleece with a symmetrical (statistically normal) distribution, and a maximum fibre diameter between 30 and 40 pm [one pm (micron) = 0.001 mm] would be regarded as a true fine type. The shortwool, too, has a symmetrical diameter distribution, but has a mean diameter of about 25 !t,m compared with a mean of about 20 !!,m in the fine wool. A skewed-to-fine diameter distribution (in which most of the fibres are fine) appears to be a primitive feature and if this occurred in a fleece with an upper limit above 35 !,m, it would be regarded as a fine variety of the generalised medium wool (i.e. fine, generalised medium wool).
The generalised medium wool is characterised by a skewed-to-fine distribution, and a maximum diameter about 55 pm. The true medium wool has a symmetrical diameter distribution with an upper limit of about 60 !m, whereas the hairy medium wool has a skewed-to-fine distribution with a few hairs greater than 60 !,m diameter.
The hairy type has a continuous distribution, which is at the same time skewed-to-fine, with a relatively high proportion of hairy (i.e. medullated) fibres that are often over 100 pm in diameter.
Secondary/primary follicle ratio
In wild animals outer coat hairs are grown in primary follicles, and the finer underwool in secondary follicles. Since the secondary follicles produce fine fibres, the more secondaries a domestic sheep has, the finer will be its fleece. The mean S/P follicle ratio of a breed therefore gives a measure of the relative fineness of the fleece (R YDER , 1962;R YDER & S TEPHENSON , 1968).
Breed survey
The breeds surveyed have been classified into different groups for convenience, and these are lister in table 3.
Northern Short-tail
This group of sheep has a predominantly hairy medium/generalised medium fleece type, and coloured animals are almost invariably self-coloured.
a) Romanov and Finnish Landrace
These hornless breeds are noted for their large litters. Most modern Romanov sheep are grey owing to a mixture of black hairy fibres and white wool, but E WART (1919) imported some all brown (presumably 66) sheep into Britain from Leningrad. Most Finnish sheep are white, with 10 p. 100 black and 10 p. 100 grey.
Fleece and skin measurements from R YDER (1968 b) are shown in tables 5 and 6.
In the Romanov the white wool was longer than the black hairy fibres which were kemps rather than heterotype hairs, so that the fleece type was hairy medium and not true hairy. The Finnish sheep mostly have relatively fine, lustrous wool with a curl. The uniformity of the group is shown by the grading of Finn wool as Shetland in Britain.
b) Swedish
Most Swedish sheep today belong to the native Landrace, a term which is now used to describe the white, fine-woolled variety that is akin to the Finnish Landrace. The majority of the Swedish Landrace sheep, however, belong to the Gotland, grey furskin variety that became localised on the Baltic island of the same name, and there is a remnant of the ancestral Goth sheep on the small island of Lilla Karsl6 of south-west Gotland (see fig. 6 a). The following description is based on a visit to this island by the author in june 1976 (see Anat,sTEirrssorr et al., 1978). The sheep are mainly horned (horns having been bred out of the Gotland variety) and grey, the face and legs usually being black. The fleece colour is, however, very variable ; there are light and dark grey individuals, as well as piebald animals, and many have a light-coloured belly giving them a superficial mouflonpattern. The grey colour usually derives from a mixture of black and white fibres, and some sheep have tan fibres in addition. Others had white kemp fibres that are more usual in true hairy breeds.
The rams have a height of about 72 cm and the ewes 68 cm (the sheep being characteristically long legged) and the weights are 75 kg and 45 kg respectively (EnMnrr, 1964). Older ewes usually produce twin lambs (even in the primitive strain on Lilla Karsl6) and 5 p. 100 have triplets.
An appreciable number of fleece measurements have been made in the past on· the improved Gotland and an overall diameter range of 12 '!,m to no more than 80 jim was obtained (table 4). The less improved individuals had a skewed-to-fine fibre diameter distribution, being of either hairy medium, or generalised medium type, but the more improved animals had a high proportion of true medium wools. Fleece samples had no more than 5 p. 100 medullated fibres, but skin samples had more than 30 p. 100. A consistent feature in skin samples had been the relatively high secondary/primary follicle ratio.
Samples from the primitive variety on Lilla Karsl6 comprised nine (39 p. 100) identified as true hairy, and fourteen (61 p. 100) as hairy medium. The overall diameter rrange was 14 to 190 pm, and as well as having more medullated fibres, the hairy sheep had a greater mean diameter but a smaller mode. Hairy animals also had more pigmented fibres, which is in keeping with the statement of E KMAN (1964) that black hairs tended to be coarser than white ones. The skin indicated that fewer secondaries than primaries were pigmented, and that not only was the intensity of pigmentation lower, but it was concentrated on only one side of the fibre. These unimproved animals already had the unusually high S/P ratio (table 5).
One hundred yarns in textiles from the seventeenth-centry Wasa warship wrecked in Stockholm harbour had on overall fibre diameter range of 10 to 140 [1 m compared with Il m in the Lilla Karsl6 fleeces. There was (1 p. 100) fine wool, 5 p. 100 were shortwools, 19 p. 100 were medium wools, 13 p. 100 were generalised medium wools, 51 p. 100 were of hairy medium type and there were 11 p. 100 hairy fleeces.
The hairy wools had a mean fibre diameter (average of the individual means) of 36.7 11 m compared with 41.4 11 m in the Lilla Karslb fleeces, the values for the hairy medium wools being 31.3 and 39.7 [1 m respectively. Therefore not only did the Wasa wools have fewer hairy fleeces, but those represented were relatively finer than those of the Lilla Karsl6 sheep.
The Wasa wools were comparable with a medieval group of yarns mostly from Uppsala which were supplied by D' M. Nockert along with some Swedish Viking wools for comparison. The medieval wools comprised ten hairy medium wools, and two generalised medium wools having an overall diameter range of 12 to 110 iam, and overall mean fibre diameter of 35.7 am, and an average mode of 28.2 am. I These were coarser than the Viking samples which comprised two hairy medium wools, four generalised medium wools, and two of true fine type. The overall diameter range of these was 12 to 80 pm with an overall mean diameter of 29.9 I tm, the mean mode being 25.9 itm. These findings contrast with findings from Scotland where the Viking samples were hairy, and the medieval samples relatively fine (R YDER , 1968 a). The observations of AnnLSTErrrssorr et al. (1978) suggested that both the Goth and improved Gotland sheep had two new grey genes A!9 (Gotland grey) and A 1 9 (light grey) at the Agouti locus.
c) Norwegian
The native short-tail of Norway is the Spael, in which the following colour types are recognised : mouflon-pattern (coloured with white belly), brown, black, some of which fade to grey, and othcrs to brown (as in other Northern Short-tails). Then there are « skimlet sheep with a mixture of coloured coarse fibres, and white (or paler) finer ones, so that a « black skimlet individual appears grey, as in the Romanov or the grey Orkney (see fig. 6 b), although the light grey shade in Norway is descrived as « blue !. The « badger-face has a white back and a coloured belly. The same range of colours is well represented in Iceland (see below).
There is also variation in coarseness (tables 5 and 6) ; finer fleeces tend, as in the Finn sheep, to be lustrous and to have a shallow curl, and coarse ones tend to be « tippy », i.e. to have staples with a hairy tip (R YDER , 1986 b).
d) Danish
The Northern Short-tail still remained in Denmark early in the nineteenth century, but later in that century there was a Danish Heath sheep which is said to have been a cross between the Northern Short-tail and the German Heath breed. The Danish Heath has been described as white-faced, hornless and short-tailed, yet a specimen in the Zoological Museum, Copenhagen is black, horned and long-tailed (cf. German Heath below).
e) Faroese
This group of islands was settled from Norway in the early ninth century and has a breed of Northern Short-tail. A feral variety survived on the island of Lille Dimon until the middle of the last century, and there are stuffed specimens of this in the Zoological Museum, Copenhagen ( fig. 7 a, b, c) because these are brown self-colour animals one cannot be sure whether or not they are like the Soay. There is, however, no reason to believe that they were not Northern Short-tails, and so the Soay remains unique.
Sheep were introduced from Shetland and Iceland in the eighteenth century, and the modern Faroese breed has the same range of colours as other Northern Shorttails, with relatively hairy fleeces like those of Icelandic sheep (A D n L STEirrssorr & W ARDUM , 1978). Fleece measurements are shown in table 3. The only skin sample examined had large central primary follicles with a wide latticed medulla, and smaller lateral primaries with either a non-latticed medulla, or no meddula at all. The secondary (underwool) follicles were much smaller, and the S/P follicle ratio was 4/ 1.
f) Iceland and Greenland
Most settlers of Iceland came direct from Norway about AD 900, and present-day Icelandic sheep stem from animals introduced at that time. Most sheep today are horned in both sexes, animals sometimes having four horns, but hornless sheep are common. The fleece is hairy, the coarse outer coat having a mean length of 21 cm, and the soft, fine underwool an average length of 6 itm. Despite the hairiness the mean fibre diameter is only 27 lim.
As in Shetland, Icelandic sheep were plucked in the past indicating the primitive tendency to moult. In common with the Soay, many of the hairy fibres shed at a different time from the wool, so that the wool can be obtained largely free from hair. Although in the Northern Short-tail most hairy fibres are kemps, many of those in Icelandic sheep appear to be heterotype hairs. Although heterotypes are not typical of this group of sheep, this observation supports the suggested origin of heterotype hairs from kemps (see introduction). Indeed, this occurrence may indicate a stage in the evolution towards the typical carpet type.
Present-day Icelandic sheep are mainly white, often with a tan head and feet, and varying proportions of tan fibres in the fleece, although such animals are still genetically white. From 10 p. 100 to 20 p. 100 of the sheep are coloured, black, brown, grey, brown-grey (brown skimlet) and other rare colours such as badger-face and reverse badger-face another name for coloured with white belly also called mouflonpattern a term which may cause some confusion. Each colour can be unbroken or broken (e.g. piebald) (A DALSTEINSSON , 1970). Norse settlers took a similar sheep to Greenland, and table 3 shows the measurement of an archaelogical specimen from that country.
g) The British Isles A hundred years ago the sheep of the Northern Isles of Britain formed a single population. Since then the Shetland has been selected for white, fine wool, so the Orkney with hairy and woolly fleeces now represents the ancestral type. These islands were under Norse rule from the ninth to the fifteenth centuries, and the sheep show clear Scandinavian influence in their short tail, and colour range ( fig. 6 b). But these sheep as well as those of Scandinavia, may be relics of the Iron Age (see introduction). Fleece measurements and follicle population details are shown in tables 3 and 4. The mort primitive sheep remain now only on North Ronaldsay the most northerly island of the group (R YDER , 1986 a and b). The other main feature of these sheep is the retention of a spring moult, so that in the past sheep were plucked instead of being shorn. The same word « roo ! is used in Iceland for plucking, indicating the ancestry of the custom.
Other parts of Britain with Norse settlers include the Hebrides, the Lake District and the Isle of Man. The Hebrides formerly had a sheep similar to the Orkney and Shetland, among with four-horned and piebald animals were common. Characters from the Old Hebrideare sheep probably persist in the Hebridean Blackface (R YDER , 1975). The possible relationships between the Soay, Northern Short-tail and four-horned sheep are shown in fig. 8.
Here it can be seen how selection of black, four-horned sheep could have given rise to the modern black, Saint Kilda (Hebridean) breed, and selection of brown, four-horned individuals could have led to the brown Loghtan breed of the Isle of Man. Finally, selection of piebald, four-horns could have given the Jacob breed, the origin of which has caused much speculation. Unlike the Saint Kilda and Loghtan, this does not have a short tail, and its fleece is less primitive, so other influence might be involved. But an exotic origin is unlikely when the breed could have originated in Britain.
The Herdwick of the Lake District has a hairy fleece like that of the black-faced carpet type, yet it has a white face and dark grey lambs suggesting Scandinavian influence. R YDER (1964 a) thought that the hairy fleece might have been acquired more recently, but it is now realised that such fleeces are found in Scandinavian sheep ; i again this does not have a short tail.
Skeletal evidence from prehistoric sites in Ireland indicate an animal similar to the Soay, and some four-horned skulls have been found. Textile remains from the first few centuries AD yielded six hairy medium wools in which the coarser fibres were more densely pigmented than the fine ones. Seventeenth century textiles still had this difference in pigmentation between the coarse and fine fibres and comprised 14 hairy medium wools, eight generalised medium wools, four true medium wools, and one fine wool (R YDER , 1969 b).
Descriptions and illustrations of Irish sheep do not appear until the nineteenth century, and in the west there was a horned breed known as the Kerry with a short tail and coloured as well as white sheep. Here, too, was the primitive Cladagh, and descriptions of remnants of this breed were given by NODDLE & R YDER (1974). One fleece samples was of hairy medium type with brown coarse fibres (as in the textile remains) being therefore brown skimlet. The overall diameter range was 12 to 70 [t m, with a mean of 32.8 ± 12.9 ,m and a mode of 20 u,m. The secondary/primary follicle ratio in the skin was estimated to be 4/1. The identification of the Cladagh with the Northern Short-tail of Britain (e.g. the Orkney and Shetland) is supported by the fact that its name means « shore sheep because it eats seaweed like the Orkney sheep on North Ronaldsay.
Attempts have been made recently to collect together remnants of Cladagh sheep, and a flock is kept at the Belclare Research Centre, Galway. This has mostly hornless, white-faced animals, with few coloured sheep, although at least one coloured with white belly has been recorded. Results from fleece samples (R YDER unpublished) indicate that the sheep are very variable in appearance, and therefore not pure. Three samples appeared hairy and had fibre diameter measurements supporting this identification. Six had pointed staple tips but were fine and lustrous, comprising two generalised medium wools, two medium wools and two shortwools. Three had a broad wave, two being medium wools, and one a shortwool. Three samples appeared like British Down wool, two being shortwools, and one a generalised medium wool. Only one was coloured (grey) and this was of primitive generalised medium type. There was hardly any medullation in the skin, but the proportion of medullated fibres in the fleece samples ranged from zero to 35 p. 100 with a mean of 8 p. 100. The S/P ratio was 3.7/ 1, and 25 p. 100 of both the primary and secondary follicles were inactive.
South West Europe France
The short-tailed breed of the island of Ushant (Ouessant) off north-west France was linked with the Northern Short-tail by R YDER (1968 b). The rams are horned, and the ewes polled, and there is a tendency to moult in spring. The pair with a black lamb described by R YDER (1968 b) as dark brown, are now realised to have been faded black animals. Illustrations of the island, show a predominance of white sheep, some with a tan face, but others appear to be grey, and some white sheep with a black face could be light grey animals ( fig. 7 d). More recent breed descriptions list only black and « brown » animals. According to L AUVERGNE (1976) no sheep remain on the island, and in the few flocks preserved on the mainland the colour range has become reduced to all black or all white animals.
A single fleece sample measured by R YDER (1968 b) was over 100 mm long and had a fibre diameter range of 14 to 52 !,m, with coarser fibres 56 and 58 !am in diameter, and a mean and mode of 28 and 18 8 jim respectively.
Illustrations of the native breed of the island of Corsica show it to have a hairy, carpet-type fleece, and the sheep appear to have the same colour range as the Northern Short-tail. L AUVERGNE & AnnLSTErNSSOrr (1976) found that the colour patterns were determined by a series of six alleles at the Agouti locus. Five of these : white, grey, badgerface, black with white belly and black are the same as those in Iceland. The sixth, producing a red patch around the eye has so far only been found in Corsica.
In all, six out of nine colour genotypes were the same as in Iceland from which the authors concluded that the sheep of the two islands had a common ancestry. This conclusion supports the theme of the present paper, namely, that during the Iron Age and for some time later, the sheep of Europe were of vari-coloured stock. How this conclusion is affected by the fact that Corsica was never invaded by the Celts ( DE BEER, 1965) is not clear.
Spain
There are references to coloured sheep in Spain going back to antiquity when Pliny mentioned black and brown sheep (Natural History VII, 191). In the nineteenth century Majorca in the Balearic Islands had a primitive breed named the Arta (SAL VA TO R , 1897). This was horned, and apparently had a short tail. The body was white, and the face tan, with two white stripes. One primitive sheep on Ibiza was described as black, and to have small horns and wool of medium quality. Another was said to be polled, and to have a short tail.
Apparently the only coloured breed remaining on the mainland is the Guirra kept on the coast opposite the Balearic Islands (B ROOKE & R YDER , 1977). All except 5 p. 100 of the animals which are black, are born reddish-brown and become paler with age, the final colour being cream. A DALSTEINSSON (1970) identifies this colour as « red like that in the French Sologne breed which is also born « red and goes paler with age.
Of the five fleece samples taken, one animal had wool of almost Merino fineness, ranging in diameter from 16 to 36 um with a mean of 24.6 and a mode of 27 !m, but its S/P ratio was only 5.7, lower than expected even for a Merino cross. The remaining flour fleece samples had hairy fibres (mean percentage of medullated fibre 14.25 p. 100) in addition to the bulk of the coat which ranged up to about 50 pm in diameter. The mode of each was 24 pm, but the means ranged from 27.3 to 34.8, with an overall mean of 31 [t m. The mean S/P ratio in the skin was 5.4/1. By the classification of R YDER (1969 a) these are hairy medium wools.
Heath sheep
This group of black-faced horned sheep formerly extended from the Netherlands to Poland. Some of the breeds have a short tail, and a hairy fleece, and R YDER (1964 a and 1968 b) thought from their general appearance and fleece type, that they had links with the black-faced, horned group of Britain. The grey fleece and short tail, however, probably indicate a closer association with the Northern Short-tail.
Netherlands
The three breeds of Heath sheep of Holland are the Drenthe localised in the north east, the Veluwe which occurred in belt across the middle of the country, and the Kempen breed of the south. The Drenthe is mostly horned, and has a straight nose ; the fleece is hairy and variable in colour, as is the face which can be black, tan or white. The Veluwe breed is larger and related to the German Bentheim breed. This sheep is mostly hornless, the nose is convex, and there is black around the eyes and on the legs. The fleece is less hairy. The lambs of both the Drenthe and Veluwe breeds are piebald to about the age of 6 months.
The Kempen breed is hornless and white, as are the lambs, and the hair on the face is « shiny ». Each of the three breeds has a long tail, although some descriptions referred to the Drenthe breed as having a short tail. Although the Kempen is now rare, the characters of the Drenthe and Veluwe are retained in the Schoonebeker (cf. fig. 6 c) hybrid of which one flock is kept at Ruinen. This has horned and hornless animals and varying face colours ( fig. 6 c). These are black, tan, together with patterns including speckled, black around the eyes, and badger-face. A DALSTEINSSON (1970) points out that tan-faced sheep are genetically white, the tan fibres being restricted from the fleece since the animals are homozygous for the recessive colour-restricting gene (ss), see also the genetic analysis by L AUVERGNE & B OTTEMA (1978) (cf. the rare Portland breed in Britain). The colour of the fleece is black, brown, grey, white or piebald, and the wool is generally hairy, but is more akin to that of a hairy Shetland sheep (hairy medium type) than the true hairy fleece of the Scottish Blackface. Some animals had cast parts of their fleece.
The legs and tail are long and thin, and D' A.C.v. VAN (R YDER , 1968 b) were in fact German Heath sheep ( fig. 6 d).
Fleece measurements of wool samples taken in 1974 are shown in table 6. These can be compared with a group of 17 yarns from Dutch Terpen sites dating from throughout the first millenium A.D. These comprised 3 hairy fleeces, 10 hairy medium wools, 2 generalised medium wools, 2 true medium wools, and one fine wool (R YDER , unpublished).
Germany
The Heidschnucke of Germany has a black face, horns, and a short tail. The colour is grey, and the lambs are born black, but the sheep frequently have a black belly like the badger-face genotype of the Northern Short-tail. There also appear to be some brown animals. The fleece is hairy (table 6) but there is a hornless white variety with a less coarse fleece. The hairy fibres include kemps as well as heterotype hairs. The S/P follicle ratio of 4.4 indicates a greater proportion of fine fibres compared with the Scottish Blackface (S/P = 3.7).
Poland
The Wrzosowka breed appears to have affinities on the one hand with the Heidschnucke, and on the other hand with the Romanov of Russia (see table 7). The colour is predominantly grey, but N AWARA (1976) reported 10 p. 100 black animals, with about 50 p. 100 dark grey and 40 p. 100 light grey in an experimental flock at Cracow. Of 20 skin and fleece samples received from the same source 5 were dark grey, 3 medium grey, and 12 light grey. The length of three months growth of wool was about 80 mm. The fibre diameter measurements are shown in table 5 and skin follicles details in table 6. Twelve samples were indentified as true hairy type, and eight as hairy medium wools. Another (tailed) breed of Poland is the Swiniarka, which is white faced, and horned in the rams only. It appears to have a hairy medium fleece (J ANKOWSKI , personal communication).
Alps
The Bundner breed of Switzerland shares the distinction of the Soay in Britain of being a sheep whose skeleton was compared with prehistoric remains found during the nineteenth century. It has therefore appeared frequently in the literature (e.g. Z EUNER , 1963) but with inadequate breed description. Sadly the sheep became extinct in 1960. In the Grisons Museum, Chur, in 1976 I saw the stuffed skin of the last (brown) sheep to die. I also saw the grey animal illustrated by previous authors such as Z EUNER ( fig. 9 a). The tail was of medium length, but the fleece was of hairy medium/generalised medium type like the Northern short tail, and not as hairy as that of the Heath sheep. The face and legs were black. The small horns described by others as « goat-like x probably indicate the presence of the polling gene (A DAL - STEINSSON, 1977). The Steinschaf of Austria seems to be similar to the Bundner, and both appear to be representatives of a type that was formerly more widely distributed throughout the Alpine region including Bavaria and Italy (see below). The rams are usually horned and the ewes polled, and there are black, brown, and grey animals in addition white.
The Steinschaf is seen in Italy as the Della Roccia breed, which is addition to white, has 37 p. 100 black and 3 p. 100 brown animals. The plate 51 of MASON (1967) suggests a hairy Shetland type of fleece, but the tail is long.
Italy
According to Pliny, Piedmont had a grey sheep in Roman times, which accords with the Della Roccia above. The Massa breed of Tuscany has either dark grey or brown sheep. Illustrations in breed literature indicate a primitive fleece, but the tail is of medium length.
The Carapelle of Foggia has been described as a black Merino. The survey of B ROOKE & R YDER (1977) located only one ewe remaining ( fig. 9 b). This was a horned sheep with faded black wool having a mean fibre diameter of 31 p.m and an S/P follicle ratio of 5.3. Samples taken from the local fine-woolled Gentile di Puglia for comparison had a mean fibre diameter of 25 pm and an S/P ratio of 8.9. This suggested that the carapelle has a primitive fleece type like the Northern Shorttail, since it is neither a fine wool nor a carpet type. But the tail does not appear to be short.
Balkan
Previous authors have divided Balkan sheep into the hairy Zackel type, and the finer-woolled Ruda. The survey of B ROOKE & R YDER (1977) obtained evidence that the primitive type might exist among the Zackels rather than the Ruda sheep. The Racka of Hungary ( fig. 9 c) has corkscrew horns in both sexes and there are black and white varieties, the face and legs of the latter being tan (cf. the Dutch Heath). The fleece is less hairy than the carpet type, half being hairy medium fleeces, more curly, and sometimes lustrous (R YDER , 1974 b). Fleece measurements are shown in table 5. There was no difference in fibre diameter between black and white animals, nor between the sexes. There were apparently no kemp fibres, and the coarser fibres of the black fleeces tended to be more densely pigmented than the finer ones, which is a common feature in the Northern Short-tail. Follicle population details are given in table 6. The S/P follicle ratio of 2.4 was low, being even less than the figure of 3.7. in the Scottish Blackfare.
The Dubrovnik breed of Yugoslavia is hornless and white-faced, and has a long tail ; 10 p. 100 of the sheep are coloured. The accepted view is that this breed originated from a fairly recent cross between the Merino, and the local hairy Zackel type, and some of the fleeces were relatively fine (table 5). But the S/P ratio figure of 4.7 obtained bv B ROOKE & R YDER (1977) (table 6) was lower than that expected from a Merino cross. The Merinos that are known to have been introduced are likely to have had an S/P ratio of at least as high as 10, and so the Dubrovnik would be expected to have an S/P intermediate (6.6.) between this and the Yugoslav Zackel value of 3.3.
It therefore seems more likely that the Dubrovnik breed is a relic of an ancient fine wool, which accords with historical evidence of the area in classical times and during the Middle Ages. The fine fleeces, were, however, much closer to the true fine wool, than those found elsewhere in the Balkans (see below). MASON (1969) listed the Karnobat and Shumen as the only medium wool breeds in Bulgaria along with ten Zackel breeds (table 8). The Karakachan and Karnobat were included in the present survey as the only Bulgarian breeds discovered with a coloured fleece of primitive structure. During a visit in l9 g O L AUVERGNE obtained evidence of more breeds (table 8) but it is not clear to what extent these are varieties of other breeds, or their names synonyms. More details of Bulgarian breeds are given by R YDER (1982). The Karakachan breed is associated with the nomadic Sarakatsan shepherd people, and so is found in other Balkan countries, notably Greece. Only two hairy fleece samples were obtained in Greece by B ROOKE & R YDER (1977, 1979 ; another flock wa3 seen and sampled in Bulgaria by the present author in september 1976. The samples comprised 6 hairy fleeces, and one hairy medium wool. There were black, brown, grey and white sheep, the colour being very variable, even over the body, and some of the grey animals appeared to have become so with age ( fig. 9 d). The dark grey animals were typically black on the face and legs, while some light ones had a white face or black around the eyes. According to A DALSTEINS - SON (1977) this pattern is brought about by the effect of the piebald (colour restricting) gene on recessive black, which also makes the body white. The fleece measurements and skin follicle details are given in tables 6 and 7. The hairiness of the fleece is indicated by the high mean diameter and average mode, as well as the high percentage of medullated fibres (table 6).
The Karnobat breed of Bulgaria is of less-coarse Ruda type, and has been thought to have associations with the Tsigai (Ruda) breed of Romania, which in turn is thought to be related to the Thraki breed of Greece and the Kivircilc of Turkey. My observations in Bulgaria showed it to be similar to the Drama of Greece (see below) and the Panagyruri,shte and Shumen (Bulgarian) breeds, and B ALEVSKA & P ETROV (1972) consider from skull measurements that the last two breeds and the Karnobat are of Zackel rather than Tsigai type.
The Karnobat sheep I saw were horned in the rams only, and the tail which had been short is now long, or of medium length. The fleece appeared to be of hairy medium/generalised medium type, and not hairy. The animals were brown, but different from the usual brown in that the head and legs were black. The sheep therefore appear to be genetically black, but with a tendency to fade readily to brown (as seen in the black patchse of the Jacob breed (see also L AUVERGNE and al., 1981 for the fading process). This conclusion is supported by the observation of darker patches on the body, and of a range of shade between individuals from almost black to light brown. Some sheep appeared to have faded to grey, but no true grey animals (with black and white fibres) were observed. The same colour was observed by AD AL S TEIN SSO N , DOL L ING, LAUVERGNE & RYDER in Merinos in Australia, and may be a different genotype from black and brown (R YDER , 1980). Fleece measurements and skin follicle details are shown in tables 6 and 7.
The Drama breed of the mountains in northern Greeces bordering on Bulgaria is probably a variety of the Vlachos Mountain Zackel type illustrated in Plate 110 of MASON (1967). The black sheep recorded by B ROOKE & R YDER (1977) had a relatively finer fleece than the remainder ( fig. 10 a) and were also similar to the Chalkidiki breed (see below).
As with other Greek breeds the appearance was very variable, there were horned and hornless sheep, black, white and grey fleeces, speckled faces and legs, as well as black around the eyes, and a moderately long tail. Fleece measurements are shown in table 6 and skin follicle details in table 7. I had hoped to find the remnants of an ancient fine wool in the Balkans, and was looking for a true fine wool. In the event this and the following breed provided the answer with a generalised medium fleece, which was what R YDER (1969 a) identified as the ancient fine wool. It was immediately obvious that the sheep had fleeces similar to the Shetland, and this accorded with the theme of the present paperthe primitive type surviving in southern Europe, being the same as that in the North.
The Chalkidiki breed of the peninsular of the same name in southern Macedonia has horns in the rams only, and a tail of medium length. Ten per cent of the animals were black, and there were dark and light grey, as well as white sheep. In these the face, legs, and sometimes the belly, were black, although others were white, speckled, or had black around the eyes. Somes of the animals had fine kemps as in the hairy Shetland ; fleece measurements are shown in table 6 skin follicle details are given in table 7. The similarity of the S/P ratio with that of the Drama breed suggests a relationship between the breeds.
Soviet Union
Since it is not known how many truly native breeds remain in the U.S.S.R., and since those known in the west are not well documented, the following coloured breeds recorded during a visit in 1979 will be described.
The Karachaev ( fig. 10 b) is a mainly black, horned breed with a fat tail and a carpet-wool fleece, and 20 000 are still kept privately in the Caucasus Mountains. Unlike more modern Russian breeds it is kept outside all the year. There is 115 p. 100 lambing, although twins have not been encouraged because of the custom of transhumance in which the sheep are taken to the high mountains during the summer. It is a triple purpose breed, the lambs being killed at one month so that the ewes can be milked. The curly, black lamb skins are used to make hats.
The ewes produce 1.5 kg of wool in two shearings, and weigh 35 kg, while the rams reach 55 kg. Animals seem on the march in a mixed flock ( fig. 10 b) were very variable, some apparently being grey. Others seen at a show had wool finer than carpet type, the fleece being more like that of a hairy Shetland or the Greek Chalkidiki breed. The structure and colour of the-fleece suggest a relationship with the European type of the present paper, but the possession of a fat tail implies a different broad grouping, and suggests either that this fleece structure and colour range had evolved before the fat tail developed, or that the same fleece characteristics evolved independently in different broad groups.
The Kulunda breed was seen on the steppe of the same name in the Altai region of Siberia. Although located several thousand miles away it appeared superficially similar to the Karachaev, being a black, fat-tail Not all the sheep had a fat tail, however, and there were both horned and polled animals. As with the Karachaev the fleece appeared to be of hairy medium rather than a true hairy type. Most of the animals were black, but there were a few grey and brown animals, as well as white sheep, which may have been crossbred ( fig. 10 c).
Discussion
Some of the breeds should perhaps have not been included in this survey, and there may well be other known, or more important, unknown, breeds that should have been included. But it is hoped that the present descriptions, however, inadequate, will stimulate more detailed studies of the breeds in question, as well as stimulate the search for little known breeds worthy of being put on record.
Several authors have used a diffusion model not only to study the spread of domesticates, but to estimate the rate of genetic change (e.g. L AUVERGNE , 1979). Such a model may work with plants, but has severe limitations with animals : waves of migration did not radiate outwards in regular fashion, but followed fairly narrow routes which differed in their rate of progress, and evolution was not restricted to the centre of origin.
If one accepts the evidence of POPLIN (1979) that the Mouflon sheep on Corsica are not truly wild but feral domesticates then here we have a survival of European Neolithic sheep that is little, if any, changed from the wild ancestor. Survival has however, depended more on the isolation of an island than on the distance from the centre of domestication.
The next most primitive breed, the Bronze Age Soay, is located on the very edge of the continent, but again survival was due to insular isolation. Most of the breeds of the present survey are thought to date from the Iron Age to the Middle Ages, and these are apparently scattered at random throughout Europe, although it is perhaps significant for the diffusion hypothesis that they are universal throughout northern Europe. The criteria on which these were chosen are the possession of a short tail, a primitive fleece structure, and in particular, a range of colour. It is not implied that these have undergone no selective change since the Iron Age (although the Soay appears not to have changed) since the colour range of some has been restricted, and they are clearly very variable.
Other factors casting doubt on the diffusion model are the location of the Merino, the breed with the most highly evolved fleece, in Spain a long distance from the centre of domestication in the Middle East, while the modern sheep at the centre of domestication appear primitive rather than highly evolved, although the hairy, carpet-type fleeces of these are now thought not to be primitive but relatively highly evolved (see fig. 4). It is however, difficult to define « primitive and « highly evolved » in this context, and in any case some breeds have both primitive and highly-evolved characters, the carcass in the Merino for example not being well developed.
One can speculate that the best fit of a diffusion model to the observed or likely sheep distribution in Europe took place about 1 000 B.C. Then the true fine (Merino) fleece was appearing in the Middle East, but had not yet begun to spread around the Mediterranean (R YDER , 1982). The varie-coloured type is likely to have advanced some distance into South East Europe (i.e. assuming that it had not evolved independently in at least one location in Europe itself), and further north and west the Soay is likely to have been the main type, while in outlying areas of northern Europe (and islands such as Corsica) the Neolithic « hair sheep was probably still dominant. But even the most cursory knowledge of the numerous migrations across Europe since that time, must dissuade one from expecting the present distribution to give more than the vaguest hints about movements and evolutionary changes. At the outset of the present survey, for instance, I was expecting to find remnants of the true fine wool that must have existed in South East Europe in classical times, but in the event it was apparently the more primitive vari-coloured type that has survived. Having cautioned against the over-optimistic use of a diffusion model, one must nevertheless accept that there is still considerable scope for the use as « markers » of such genes as those for colour, as suggested by L AUVERGNE (1979). Mutations observed by A DALSTEINSSON (1970) in Icelandic sheep from « mouflon-pattern » to white, from white to self-colour, and from self-colour to grey suggest one way in which the range of colour described in the present paper could have arisen ( fig. 8). Another way of obtaining white animals would have been to select for greater and greater areas of white in piebald (spotted) animals (L AUVERGNE , 1975). This would, however, have resulted in recessive-white sheep, and breeds of this type apparently do not exist. Since coloured animals in modern breeds frequently have white markings A DALSTEINSSON (1977) considers that this indicates selection for white in combination with the spotting gene.
Since neither method of selection is likely to have affected the colour genes, these are almost certainly still present in modern breeds, but are masked by the inhibiting white and spotting genes. If therefore the colour genes could be revealed by crossing with homozygous recessive brown rams, breed relationships might be elucidated (R YDER & S TEPHENSON , 1968) (see also pp. 321-322 of R YDER , 1980). Reçu pour publication en octobre 1981. | v3-fos |
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} | s2 | Toward monitoring specific DNA lesions in the gene by using pollen systems.
Specific gene systems expressed in cereal pollen could contribute uniquely to the problem of monitoring our environment for mutagens. This paper considers the development of a mutagen monitor with quantitative endpoints that reflect particular types of lesions at the DNA level, and lesions in particular components of the gene.
Introduction
Two of the best understood genes in higher organisms, alcohol dehydrogenase-1 (Adhi) and waxy (wx), happen to be expressed in higher plant pollen grains. By using specific dysfunctional alleles of these genes, revertant frequencies may be quantified by pollen staining procedures, as evidenced by several papers in this volume. One could plant maize or barley lines homoallelic for any of hundreds of wx or Adhi alleles and quantify sundry insults to the DNA in terms of revertant grains (darkly stained) per total grains (light stained). In the discussion which follows, I hope to show that one of the unique contributions pollen systems may make as a mutagen monitor is to provide readily quantifiable endpoints which reflect particular types of lesions at the DNA level, or lesions in particular components of the gene. The Ames test (1) presently provides this sort of information for some transitions, transversions, and frameshifts in coding sequence DNA of a bacterial gene. However, the "gene" in higher organisms is certainly more complicated that its microbial analog. The abundance of repetitive DNA in the genomes of most higher organisms, including man, and the fact that many specific genes in higher organisms display coding sequences which are intervened by noncoding DNA should emphasize that fundamental differences exist between "a gene" in microbes and man. Pollen systems have the potential of monitoring genetic parameters unique to higher organisms. In * Department of Genetics, University of California, Berkeley, California 94720. January 1981 order to argue this assertion further, some background on current "theory of the gene" follows.
Definition of the Gene and Its Functional Components
A "gene" is taken to be the most generous chromosomal unit of cis function. Therefore, a gene contains noncoding as well as coding sequences. Demerec's "cistron" or complementation group is one measure of the gene; Benzer's (2) use of the cis-trans test at the RII locus in T4 exemplifies this measure. Given that a gene may contain intervening sequences, cis-acting components beyond promoters, or even junk or spacer DNA, a superior means to measure the gene is by summing all chromosomal breakpoints which, by disconnecting the cis unit of function, either alter or obliterate the gene's product or alter the rate of cell/tissue/organ specificity, timing, fidelity, stability, circuitry with other genes, etc., of product expression. It might be useful to visualize the DNA of the gene as coding, regulatory component, spacer, recombinational, or junk.
It is obvious from comparisons of protein synthesis among tissues or organs of the same individual that gene expression is highly regulated both qualitatively (tissue-restricted gene expression, like 3-globin) and quantiatively. In other words, genes are programmed to be on or off (high or low; early or late) in tissue-specific ways. In theory, this could be accomplished by utilizing tissue-specific DNA components of the gene, or by evoking a regulatory gene whose product affects the expression of certain genes but not others, or a combination of both of these extremes. Variant and mutant alleles that specify regulatory-type changes in gene expression have been particulary valuable in identifying functional components of the gene.
The case for the existence of cis-acting quantitative gene components is built from phenomenological studies of mutant or variant alleles as compared to their wild-type progenitor alleles. Goldschmidt (3), in his review of the theory of the gene, argues aptly for a multicomponent gene integrated into a larger physiological system. McClintock (4) has studied numerous cases of derivatives from unstable alleles which are heritably altered in the timing or cellular pattern of expression (or mutation) during maize development. A quantitative series of DS-induced waxy alleles which differ in the ratio of branched to unbranched starch specified are exemplary (5). Studies using the R anthocyanineconditioning locus in maize have identified an abundance of allelic variation involving quantitative parameters (6). A recent study by Kermicle (,) has mapped a quantitative component of P distal to the component that specifies organ-specificity. Unfortunately, chromosomal position effects (8) and early studies on unstable alleles suffer from lack of a biochemically accessible gene product. Specific alleles of genes in Drosophila, maize, and mice differ in cis regulatory properties involving tissue/organ specificity or developmental timing. Examples are esterase-5 (9) and alcohol dehydrogenase-1 in maize (10,12), 3-glucoronidase (13), ,3-galactosidase (14), H2-antigen (15), aryl-sulfatase (16), and f3glucoronidase (17) in mice, and aldehyde oxidase (18) and alcohol dehydrogenase (19) in Drosophila melanogaster. The evidence from the above cases does not rigorously exclude the involvement of coding sequences. The behaviors of these alleles are clearly regulatory, are specified by a site in or near electrophoretically marked coding sequences and act in cis.
Chovnick and co-workers (20) have proved that a cis-acting site specifying the total amount of xanthine dehydrogenase per fly is genetically separable from the coding sequence.
One should not conclude from the above studies that all quantitative organ-specific gene regulation is a property of individual genes. For example, Abraham and Doane (21) found that the presence of a-amylase in the midgut ofDrosophila melanogaster is specified by a gene which acts on the ao-amylase structural gene in trans. Similarly, Dickinson (22) discovered three clear cases of specific negative gene regulation by soluble factors among closely related Hawaiian picture-winged Drosophila.
Taking all of these bits of information, it may be safely concluded that a gene in higher organisms is 14 more than a coding sequence and an on/off switch. Organ specific, quantitative components have been observed. As of yet, the intriguing behaviors of these variants or mutants have not been reduced to the level of nucleotide sequence and sequence arrangement.
Gene Structure in Higher Organisms
A typical gene contains a structural gene component that carries information which specifies the amino acid sequence of its polypeptide product. Until about five years ago, it was assumed that the coding sequence was transcribed in one continuous 3' -5' unit, called "the structural gene" component. Since 1977, it has become increasingly clear that many or most polypeptides in higher organisms are encoded in pieces of DNA with fragments of noncoding DNA between them (23)(24)(25)(26)(27)(28)(29)(30). The structural gene is composed of coding and intervening sequences. As was shown clearly for a yeast tRNA gene (31), the intervening sequences are transcribed into a large, primary transcript that is subsequently processed within the nucleus by two or more cut-and-splices before a translatable message is pieced together and leaves the nucleus. The great majority of a structural gene's length can be intervening sequences. There is homology for nucleotides at splice-joints among widely divergent genes; this makes it possible that one or a few processing enzymes may be involved (32). From studies of ,B-globin gene fragments expressed in SV40, it seems that a pair of splice-joints is required for the production of stable messenger RNA (33). It is not clear whether or not the regulatory step of primary transcript -mRNA processing is used as an on/off switch, and/or for quantitative gene regulation. Several ongoing studies are seeking function and evolutionary origins of intervening sequences by comparing homogenous structural genes for conserved or diverged sequences or sequence locations (34).
It should be kept in mind that the structure of a gene need not be specified entirely by its nucleotide sequence. As with any other machine, the gene has various active and inactive three-dimensional shapes which could be controlled by various chromosomal RNAs or proteins, or by the gene's shape in the sister chromatid at the previous mitosis [see T. M. Sonneborn (35) for concept of structural inheritance]. Wu, Wong, and Elgin (36) have used accessibility to chromatin digestion to show that Drosophila heat-shock genes change shape at about the same time they are induced to transcribe.
Environmental Health Perspectives The 5' end of the message is translated first. For some primary transcripts, the 5' end either remains intact or a very few nucleotides are removed to form the 5' end of the message (37,38). However, many messages have about 25% of their nucleotides in a 5' leader in front of start triplet ATG. Presumably, this later sequence is involved in translation by ribosomes (39). Similarly, the 3' end of the message is distal to the DNA stop triplet TAA: In addition to post-transcriptionally added poly(A) at the 3' terminus of many messages, there is also about a 20-nucleotide trailer which may contain an TA-rich conserved sequence (40).
About 30 nucleotides in the 3' direction of the start triplet ATG constitute an AT-rich presumptive promoter sequence which may be common to all eucaryotes (32,41,43). Although the primary structure of this "Pribnow box" is known, there is no causal evidence implying any function at all. Indeed, several intriguing stem-loop structures exist near coding sequences, but extrapolating from structure to function is not easily accomplished.
Limitations of Reversion and Forward Mutation Assays
Mutant frequencies are generally quantified by using one of two general methods. The first method measures forward mutation, where a functional gene is mutated to dysfunction. The second method measures reversion, where a dysfunctional, mutant gene is reverted back to function. Although both methods measure DNA alteration, they provide vastly different endpoints.
From the previous summary on the nature of the gene, it should be clear that a forward mutation test with a dysfunction endpoint will register any mutant lesions which results in lowering the level of expression of a gene's product below the dysfunction threshold. For example, the waxy (wx) gene in cereals specifies a starch branching enzyme; branched starch in endosperm or pollen stains blue with an 12-KI solution. If wx does not function, then relatively unbranched starch stains red with I2-KI. It is certainly possible to stain nonmutant pollen with I2-KI and count red grains. In theory, such a forward mutation method would register as mutant any DNA lesion which leads to dysfunction: substitutions, deletions, or insertions in coding sequences; and any alteration in regulatory components which rendered the gene dysfunctional in pollen. Fortunately, pollen's requirement for a complete haploid genome acts as a filter for gross chromosomal aberrations. Unfortunately, it would be naive to expect every red pollen grain to be a wx mutant. January 1981 Perhaps they are merely sick. Stadler (43) estimated that the spontaneous wx mutant frequency in maize-scored as wx seeds-was below 7 x 10-7. The frequency of red pollen in a normal plant is approximately 10-5 (44). Therefore, these red gametophytes present an inscrutable genetic endpoint. With the Adhi system in maize pollen (45) a similar result is routine: up to 0.1% of healthy, Adhi + pollen does not stain positive (blue, opaque) for ADH. However, these unstained gametophytes proved to be inviable (Cheng and Freeling, unpublished). In units of ADHi/germinatable pollen, the Adhi + -* ADHspontaneous forward mutant frequency was estimated to be below 2 x 10-7 (46).
While forward mutant assays are unbiased in that the entire, naturally occurring gene is a target, obtaining data with genetic resolution at 10-6 may not be possible. For the Adhi gene, allyl alcohol vapor treatment can select rare ADH-ungerminated gametophytes among millions ofADH + s. This could permit recovery and molecular characterization of the spectra of mutants generated by a treatment or environment (47). It should be noted that mutant spectra data are quantitative to the extent that a distribution of mutant types could be displayed, but reliable estimates of mutation rates can not be reasonably expected from mutant recovery frequencies after selection.
Revertant assays are easier to quantify reliably because the restoration of an enzyme activity should be unrelated to disease or inviability. The term "phenotypic revertant" is meant to include virtually any way a cell regains the function of a mutant gene. "True reversion" is where the change in DNA sequence which caused the mutant behavior is reversed to the original sequence. "Second-site" reversion includes all other alterations in the coding sequence which "complement" the original mutant lesion via some intrapolypeptide folding mechanism. Phenotypic reversion also includes ways to regain a gene's function without altering the gene at all: suppression by unique tRNAs (supersuppression) and other mechanisms, activation of a silent isoenzyme gene, conversion by an isoenzyme gene, and so on. In the absence of revertant recovery and characterization, or other indirect controls, phenotypic revertants are a complex endpoint.
In order to estimate the spontaneous phenotypic revertant frequency for specific genes in pollen, one should begin with several dysfunctional mutants. Using 14 independent alleles of wx in maize, Nelson (48) obtained a median revertant frequency (blue pollen/total pollen) of 7 x 10-6. I used seven Adli1-Sdeficient alleles induced with ethyl methanesulfonate by D. Schwartz to obtain a median revertant fre-quency of 5.7 x 10-' (46). These values are in good agreement and are well below ty5pical intragenic recombination frequencies (2 x 10-10-3). Even so, these five per million spontaneous revertant frequencies are much higher than expected on the basis of the below 0.5 per million estimates of spontaneous forward mutant frequencies for maize wx and Adhi. Such excessive revertant frequencies are not uncommon among eukaryotes; I have compiled the relevant data elsewhere (46). As a rule, it should be easier to wreck a machine than repair it. A solution to this apparent excessive reversion paradox is necessary in order to understand what revertants of an allele really meant at the DNA level.
When an allele generates a revertant frequency above i0-5, it is called "unstable" or "mutable." There is much evidence that mutable alleles reflect unstable integration of a piece of DNA in the gene (49,50). There is a tendency to pick unstable alleles for mutagen monitoring because fewer pollen grains need be scored in reversion assays. A wx allele in common use (44,51) has high spontaneous reversion frequency. While I can imagine the judicious use of mutable alleles as monitors, their present use is premature.
The Future of Pollen Mutagen Monitors
If specific gene reversion systems in cereal pollen are to be used, they should contribute uniquely where other monitors fail. As plants, cereals are certainly appropriate when there is some risk that crops are activating chemicals which might enter the food chain. As inexpensive, in situ monitors for volatile mutagens, plant systems excel. A third unique contribution of pollen systems is to monitor specific lesions in DNA or lesions in specific gene components. In previous sections of this paper, I have shown that the "gene" in higher organisms is composed of quantitative organ-specific functional components and is structurally complex as well. Such complicated genes seem to be confined to organisms displaying the classical embryological phenomena of determination, competence and somatic heredity. Ideally, pollen systems would monitor mutational events which could not be monitored in the Ames or any other microbial test. If lesions of a particular type or in a particular gene component prove diagnostic for one or more cancers, the monitor would be most powerful.
I suggest two specific approaches toward developing truly elegant pollen mutagen monitors.
Approach No. 1. This approach requires com-plete DNA level understanding of select mutant alleles with their normal progenitor alleles, it is possible to identify the exact change in DNA which causes the mutant behavior. This comparison could be done at the level of amino acid sequencing if the mutant site were in a coding sequence, but would require DNA-level comparisons if the site were in noncoding gene components. Particular alleles would then be chosen. Hypothetical examples include an insertion in coding sequence; a small inversion in nontranscribed "quantitative component;" a missing splice-joint; and so forth. A spectrum of revertants of these known lesions would be recovered and characterized molecularly to better understand just what a "phenotypic revertant" of a particular mutant allele means. Approach No. 2. Comparative, quantitative mutagenesis using several molecularly distinct alleles and several known mutagens and carcinogens. Do active carcinogens generate certain kinds of DNA lesion (like chromosomal breaks) more efficiently than others (like point-mutations)? I have argued elsewhere (12) that regulatory-type mutants at a gene seem to result from chromosomal breakagetype events as opposed to small changes confined to coding sequences. If certain kinds of DNA lesions preferentially cause cancers, then we need a monitor for such diagnostic lesions. I believe that pollen systems-particularly Adhi in maize, but other systems to a high-resolution, mutagen monitor with DNA-level endpoints. | v3-fos |
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} | s2 | Endocrine patterns of the post-partum cow.
Milked dairy cows generally have a shorter post-partum interval to ovarian cyclicity than suckling dairy or beef cows. In milked and suckling cows, there is a strong seasonal influence with spring-calving cows remaining anoestrous longer. Increasing the suckling intensity further delays the onset of ovarian cyclicity, probably by increasing the frequency or strength of its inhibitory influence on hypothalamic activity. Plasma FSH levels rise in most cows 5-10 days after calving and thereafter the random changes observed have little relationship to the onset of cycles. Recovery of FSH release therefore occurs earlier post partum than recovery of LH release. Hyperprolactinaemia is not a cause of reproductive failure in milked or suckling cows because there is no correlation between plasma prolactin levels and the onset of ovarian cycles. Plasma LH concentrations undergo significant changes directly related to the initiation of ovarian cycles, with low plasma levels immediately post partum, followed by an increase in basal secretion and the development of clear LH episodes. This pulsatile pattern appears earlier in dairy than in beef cows and is further delayed by suckling compared to milking. Before the first ovulation there is an increased frequency and peak height of LH episodes leading to a rise in plasma LH levels and eventually to a preovulatory-type LH surge which results in the first ovulation. These changes in the pattern of LH release appear definitive in the initiation of ovarian activity in post-partum cows.
Introduction
In contrast to the situation in many eutherian and marsupial mammals, lactation in cattle is not an absolute block to ovarian cyclicity, conception or pregnancy. However, there is clear evidence that the frequency of milking, milk yield, nutritional status, the frequency and intensity of suckling and environmental factors influence the oestrous cycle and conception (Lamming, 1978). Furthermore, the production of dairy cattle capable of higher individual milk yields has increased the economic importance of maintaining high levels of fertility (Esslemont, 1974). In view of the lack of detailed information on plasma hormone levels in lactating and non-lactating cows it is important to define gonadotrophin and ovarian steroid hormone profiles in post-partum cows and to elucidate the nature of endocrine changes associated with subfertility.
Concentration on endocrine problems is justified because improved techniques of veterinary control of cattle diseases and the application of artificial insemination have dramatically decreased cow and calf losses due to venereal infections. This paper covers two areas of study. The first section defines the incidence and nature of 156 G. E. Lamming et al.
subfertility of animals in their natural environment. During the past decade milk progesterone radioimmunoassays have been developed and widely used for this purpose as well as for detecting pregnancy in normal cows (Gadsby, Heap, Henville & Laing, 1974;Lamming & Bulman, 1976;Bulman & Lamming, 1979;Foote et al., 1979;Gunzler et al., 1979;Van de Weil, Kalis & Nisir Husain Shah, 1979). The second section gives information on changes in plasma hormone levels during early cycles after parturition, when there is peak milk yield or an intense suckling stimulus, because this is the time at which the next conception is required. Since steroid hormones and gonadotrophins are released in episodes, frequent plasma samples are required to define more precisely the patterns of hormone release.
Progesterone profiles of post-partum dairy cows
Using milk progesterone profiles to monitor ovarian cyclicity we have shown that the average dairy cow, i.e. not suckling, resumes ovarian cyclicity 24.1+ 0.6 days (N = 505) after calving (Lamming & Bulman, 1976;Bulman & Lamming, 1978). For brevity the validity criteria for all the radioimmunoassays quoted from our own work in this paper are given in Table 1. The data in Table 2 for the distribution of the post-partum interval in milked dairy cows indicate that 95% had resumed ovarian cycles within 50 days post partum. A short period of elevated progesterone (>3 ng/ml milk and <10 days' duration) precedes the first full-length oestrous cycle in Endocrine patterns of the post-partum cow 157 approximately 50% of milked cows and oestrus is frequently not observed until the end of the first complete cycle. When data collected by the herdsmen and milk progesterone profiles were compared there was a gradual increase in the proportion of cows observed in oestrus with succeeding ovulations (see . A full analysis of factors influencing the post-partum interval of dairy cows and its effects on fertility has been published elsewhere (Bulman & Lamming, 1978;Bulman & Wood, 1980). Briefly this showed a significant variation (P < 0-01) in the interval to ovarian cyclicity according to the month of calving, with animals calving in the spring (March-May in Britain) taking longer than those calving during the rest of the year. The extreme averages were 52 + 15 days in April and 20 + 1 days in October (mean + s.e.m. N = 5 and 120 cows respectively). There appeared to be a trend towards longer post-partum intervals in older animals, especially those of more than 5 lactations, but the effect just failed to achieve significance at P = 0-05. There was no correlation between yield per lactation and the post-partum interval to the start of ovarian cycles. If a 'normal' cow is defined as one in which regular ovarian cycles resume within 50 days of calving and oestrus is observed after the second full-length cycle, then about 23% of the milked dairy cows that we have studied were 'abnormal'. In addition to delayed cycles (5%), there were problems of temporary cessation of ovarian cycles (5%), prolonged luteal activity (2%) and silent oestrus (11%). This last category included cows in which two consecutive ovulations were unaccompanied by observed oestrus. In all these groups the intervals from calving to conception tended to be longer than for the normal cows, although only the cessation of ovarian cycles gave a significant difference. A further analysis of profiles of 600 post-partum milked cows has revealed a similar incidence of subfertility (Lamming, 1980).
Milk progesterone profiles in post-partum beef cows
Ovarian activity was monitored by radioimmunoassay of milk progesterone in samples collected 3 times weekly from suckling beef cows of 3 herds. Table 3 indicates the times to resumption of ovarian activity as indicated by consecutive milk progesterone levels of >3 ng/ml. Initial short cycles (>3 ng/ml for <10 days) were present in 46 (52.9%) of the cows. Because there were large differences between herds (cows in Herd 1 calving at various times of the year, those in Herd 2 from February to March and those in Herd 3 from September to October) the data were re-analysed on the basis of month of calving. Cows calving between January and June took significantly longer (P < 0.001) to resume ovarian cycles (85.8 + 2.3 days, N = 44) than did those calving between July and December (26.9 + 1.6 days, N = 43), as found for dairy cows (see above). Deficiencies of nutrient intake could be implicated but the effect was highly significant even after removal of any effect of bodyweight at calving, thus suggesting that the season of calving can influence the time of return of ovarian cyclicity and that seasonal differences in photoperiod may be important.
Pituitary gonadotrophin content and response to LH-RH
Pituitary LH content increases from low levels pre partum and immediately post partum to higher levels at 10 and 20 days post partum. Pituitary FSH content decreases from Days 1 to Endocrine patterns of the post-partum cow 159 20 (Labhsetwar, Collins, Tyler & Casida, 1964;Saiduddin et al., 1968;Wagner, Saatman & Hansel, 1969) and is accompanied by increased plasma FSH levels (Dobson, 1978). This evidence supports the concept that early post-partum follicular development may be initiated by FSH secretion, whereas the time of first ovulation is more closely related to the level of LH. These changes probably reflect an increased pituitary response to LH-RH. There is increased responsiveness to LH-RH in the milked dairy cow at 10 days post partum (Kesler, Garverick, Younquist, Elmore & Bierschwal, 1977;Fernandes, Thatcher, Wilcox & Call, 1978;Lamming, 1978;Schallenberger, Schams & Zottmeier, 1978;Foster, Lamming & Peters, 1980: see also Table 4), while the suckling beef cow requires 20 days (autumn calving) or 30 days (spring calving) to achieve full pituitary response to synthetic LH-RH (Text- fig. 2; Webb, Lamming, Haynes, Hafs & Manns, 1977). Note the significantly decreased LH response at 10 days post parturn. (From Webb et al., 1977.) Work on rats (Aiyer, Chiappa & Fink, 1974) and sheep (Crighton, Foster, Haresign & Scott, 1975;Crighton & Foster, 1977) as well as that on cows (Kittok, Britt & Convey, 1973;Zolman, Convey & Britt, 1974;Foster, 1978a, b) suggests that pituitary responses to LH-RH are influenced both by steroid hormones and LH-RH. In cows, 2 injections of LH-RH (100 14) given 90 min apart during the luteal phase of the cycle induced a much greater total LH release than did a single injection of 200 ug LH-RH (Table 4; for full details see Foster, 1978a, b). It is possible that changes in pituitary responsiveness with time post partum and differences between cows at a given time is controlled by the pattern of endogenous LH-RH release and that this situation may partly explain variations in response between cows to injections of synthetic LH-RH.
The transition from relatively low pituitary responsiveness to LH-RH during the pre-partum and immediate post-partum period to higher responsiveness between 10 and 20 days post partum appears a natural sequence of events following removal of the suppressive effects of high levels of steroid hormones during pregnancy. Our studies on plasma LH patterns provide circumstantial evidence that an increased frequency of LH-RH release with time post partum influences changes in plasma LH profiles and lack of LH-RH release is probably one major factor responsible for lack of reproductive activity in post-partum cows. Once ovarian cyclicity is established, pituitary response to injected LH-RH is influenced by ovarian hormone levels, with both LH and FSH release declining during the luteal phase (see Table 4 restored will induce ovulation in most anoestrous dairy cows (Schams, Hofer, Hoffmann, Ender & Karg, 1973;Britt, Kittok & Harrison, 1974;Bulman & Lamming, 1978;Bulman, McKibbin, Appleyard & Lamming, 1978), but in beef cows suckling 2 calves a single injection may cause ovulation and a short period of elevated plasma progesterone followed by a return to anoestrus (see Text- fig. 3, Cow 1). Two spaced injections of LH-RH given at 10-day intervals, the first designed to cause ovulation and mimic the first short progesterone rise, the second given as the plasma progesterone declined to basal levels (see Text- fig. 3, Cow 2), induced normal oestrous cycles (Webb et al., 1977). However, during a study of short-term changes in LH, FSH and progesterone concentrations, Foster, et al. (1980) measured very large LH and FSH responses to an injection of synthetic LH-RH in a 10-day-post partum milked cow which had already naturally exhibited the transient plasma progesterone rise (see Table 4; Text- fig. 4). The cow subsequently ovulated on Day 16 post partum.
Plasma FSH and LH concentrations
The availability of sensitive radioimmunoassay procedures has led to more detailed study of the endocrinology of cows post partum. The results of our studies of plasma gonadotrophin and prolactin in milked and suckling dairy cows to 30 days post partum are summarized in Table 5.
Before parturition, plasma LH and FSH concentrations were low and FSH values remained low , 1977.) previous evidence of a different mechanism of control in the cow for these two gonadotrophins (Foster et al., 1980). However, in all the ovulating cows that we have studied, we have observed a rise in the basal level of plasma LH and the development of an episodic pattern of LH release before ovarian cyclicity recommenced. Furthermore, in the ovulating animals (of which Cow 1 is typical), an LH surge of the magnitude seen at ovulation preceded the transient rise in plasma (or milk) progesterone and, after a decline of plasma progesterone concentration <1 ng/ml, a similar LH surge again occurred and was followed by ovulation and an oestrous cycle of normal length (Webb, Lamming, Haynes & Foxcroft, 1980). Schams et al. (1978) have also recorded a significant preovulatory-type peak of plasma LH before the first full cycle. Immediately before the first LH peak we have noted an increase in the frequency and peak height of LH episodes (see Text- fig. 5), indicating a possible increase in pituitary sensitivity to, and more frequent release of, endogenous LH-RH. This pattern of LH release develops as stages in a defined sequence, each pre partum to 30 days post partum. A total of 9 cows were used of which 5 suckled 4 calves each and the remaining 4 were machine-milked twice per day (n 196).
* All the pre-partum values were combined because the cows had not been allocated to a group at th s time.
-1 Significantly different, P < 0.05 (Student's t test). suckling on mean plasma LH levels and particularly on the time of appearance of the episodic pattern of LH release. In milked Friesian cows episodic patterns of plasma LH developed between Days 10 and 20 post partum while few episodes were seen in Friesian cows suckling several calves (see ). We have also observed a marked seasonal effect on LH profiles in suckling beef cows. In autumn-calving cows, which recommenced ovarian cycles at about 40 days post partum, there was a delay in the increase in mean plasma LH until about 25-30 days and a marked delay (>30 days) in the appearance of significant episodes of LH release (see Text- fig. 7). In spring-calving cows which did not cycle until at least 60 days post partum, there were surprisingly high basal levels of LH in most cows until Day 12 (2.67 + 0.4-6.82 + 0.2 ng/ml) but then a decline, perhaps related to higher circulating plasma oestradiol from developing ovarian follicles (see Text- fig. 7). Following the decline from high levels of LH at Day 12, an irregular pattern of LH release developed at about Day 20 post partum, with a more consistent typical pulsatile pattern of release by Day 40. During this period pulses occurred at approximately 2-4-h intervals and were similar in magnitude to those seen in milked cows at 18-20 days post partum (Text-fig. 7).
These results suggest that although a pulsatile pattern of LH secretion may be observed early . LH profiles of plasma samples collected at 10-min intervals from 09:00 to 17:00 h for milked and suckling Friesian cows. Cows G and D were studied before and after parturition. (n = 48) in plasma samples collected at 10-min intervals from 09:00 to 17:00 h from 3 spring (a) and 4 autumn-calving (b) suckling cows. Plasma LH profiles (c, d) are shown for representative cows (X = spring, A = autumn) at various intervals post partum. Note the high mean plasma LH levels in the spring-calving cows (a) until after Day 40. In Cow A an irregular episodic pattern developed by Day 23 with a more distinct pattern by Day 38, with 7 LH episodes in 8 h. This cow ovulated on Day 40 and was in the late luteal phase on Day 54, when there was again a lack of clear LH episodes in the profile. The 3 spring-calving cows are typical of 6 out of 8 animals sampled at this time. The high mean LH levels (see Cow X, Day 14) occurred without clear episodes of release. Note the irregular pattern on Day 20 and the 4 clear episodes above the lower basal LH level on Day 62. This animal was anovulatory for more than 100 days (see text), and the hormone pattern was typical of that in other anoestrous cows. Such animals do, however, develop the regular episodic LH pattern before the first ovulation.
in the post-partum period, especially in milked cows, this is not necessarily followed immediately by a preovulatory-type LH surge and in such animals ovulation occurs later. In milked cows a significant relationship has been measured between frequency of LH episodes and the occurrence of the first progesterone rise (Peters, Lamming & Fisher, 1981). In suckling cows there is a marked delay in the presence of these LH episodes. The identity of the immediate trigger to the first preovulatory-type LH surge is not yet known. Working on the 28-day cycle of the rhesus monkey, Knobil (1980) has postulated that the arcuate nucleus is the centre for a control system that signals, at approximately once per hour, the release of a bolus of LH-RH to which the pituitary responds by releasing pulses of FSH and LH. Monkeys with lesions in the arcuate nucleus were given infusions of 1 1.tgLH-RH/min for 6 min every hour. An increase or decrease in the dose of LH-RH or a change in the frequency of pulses resulted in a reduced pituitary response. Knobil (1980) further suggests that suckling inhibits the neural oscillator present in the arcuate nucleus. Our work with the cow confirms the importance of using small repeated pulses of LH-RH in inducing ovarian cyclicity post partum.
As indicated in Text- fig. 6, there was an increase in the basal concentration of LH several days before the first preovulatory-type LH surge which preceded the first progesterone rise, a phenomenon also noted by Chenault, Thatcher, Kalra, Abrams & Wilcox (1975). This rise may initiate the development of ovarian follicles and the resulting positive feedback due to oestrogen, because Shemesh & Hansel (1975) observed increased oestradiol production in vitro by follicles collected from cows on Day 16-17 of the cycle after stimulation with LH. A significant positive relationship between increasing LH and oestradiol before the preovulatory LH surge is known to occur in sheep (Hauger, Karsch & Foster, 1977).
Plasma prolactin concentrations and the effect of hyperprolactinaemia
The mean prolactin levels from daily samples and serial samples taken frequently over an 8-h period from the last 2 weeks of pregnancy to 90 days post partum have been determined for autumn-calving milked and autumn-calving suckling cows, and the data for the first 30 days post partum are shown in Table 5. Concentrations were low pre partum (16-0 + 2•5 ng/ml), increased around parturition (139.9 + 3.2 ng/ml) and then declined linearly up to Day 25 (r = 0.7, P < 0.01) in the suckling animals, while those in the milked group showed a gradual increase over the same period (r = 0.64, P < 0.001). Thereafter, up to Day 90, concentrations remained significantly (P < 0.01) higher in the milked cows (27.1 + 2-1 ng/ml) than in those suckling calves (10-1 + 1.0 ng/ml). We have found no correlation between prolactin levels in individual cows of either type and the time to resumption of ovarian cycles or milk yield. These results were unexpected because previous workers have reported higher prolactin concentrations in suckling beef cows than in milked cows (Hart, Bines, Balch & Cowie, 1975;Hart, Bines, Cowie & Balch, 1975). However, Webb (1977 and Webb & Lamming (1981) have shown that suckling beef cows that calved in the spring have plasma prolactin patterns similar to those of the autumn-calving suckling dairy cows cited above, with prolactin levels in agreement with those reported by Ingalls, Convey & Hafs (1973). It is possible that, if suckling beef animals are infrequently handled and venepuncture is used, stress effects may be implicated as a causal factor of the higher mean plasma prolactin levels sometimes recorded (Webb & Lamming, 1981).
The linear decline in mean plasma prolactin concentrations in suckling cows from parturition to 80 days post partum (Webb & Lamming, 1981) and the significantly higher plasma prolactin levels in milked than in suckling cows suggest that higher prolactin levels in cows cannot be implicated in explaining differences between breeds and the effects of suckling on the return of post-partum cyclicity, a view contrary to suggestions for other species (human female: Rolland, Lequin, Schellekens & de Jong, 1975;sheep: Kann, Martinet & Shirar, 1978). Our view is supported by the results of Schallenberger et al. (1978) who found no effect of the dopamine agonist, bromocriptine (Sandoz, Basle), on FSH and LH basal levels or release rates in cows, although plasma prolactin values were significantly depressed. We conclude that hyperprolactinaemia cannot be a cause of the longer post-partum interval to resumption of ovarian cycles in suckling cows. However, suckling clearly has a marked inhibitory effect on reproductive behaviour. This inhibition is possibly mediated via a neural pathway from the mammary gland.
Significance of the early progesterone increase
In the post-partum period approximately 50% of suckling beef and milked dairy cows exhibit a plasma progesterone increase of short duration before full cyclicity, and this incidence is Endocrine patterns of the post-partum cow 167 similar to that observed by Corah, Quealy, Dunn & Kaltenbach (1974) and Schams et al. (1978). Tribble (1973) thought that this progesterone probably came from luteinized follicles; we have shown that short progesterone cycles occur more frequently in animals which exhibit ovarian activity soon after parturition and believe that this requires further study especially in relation to the level of follicular development before ovulation. Schams et al. (1978) suggested that this short early cycle was caused by inadequate levels of LH or its receptor. However, our results from examination of basal plasma LH levels at this time could not demonstrate a lack of luteotrophic support to account for the lower plasma progesterone or earlier luteolysis during this first cycle. A more likely explanation would be defects of follicular development leading to a lack of steroidogenic ability of the luteal structure, which might be related to a lack of LH receptor activity. For example, corpora lutea occurring in anoestrous sheep following intramuscular injection of synthetic LH-RH did not secrete progesterone (Haresign, Foster, Haynes, Crighton & Lamming, 1975), but if PMSG was administered before the LH-RH normal progesterone release occurred (Haresign & Lamming, 1978), suggesting that follicular maturation before ovulation may be important.
The first short cycle in cows is normally followed by ovulation and a corpus luteum of normal life-span and secretory activity although oestrus is often not observed until the end of the second cycle. There is no evidence from our work, or that of Schams et al. (1978) that plasma LH concentration changed substantially during the transient progesterone rise, so it must be assumed that the second LH surge and subsequent normal luteal activity are influenced more by the stimulatory effect of removing a negative feedback influence of progesterone, rather than a possible positive or modulating effect of low levels of progesterone on the hypothalamus and/or pituitary to produce a preovulatory-type LH release, as previously suggested by Lamming (1978). However, the 'organizational' role of the progesterone produced by the first ovarian cycle on physiological and behavioural changes at the second ovulation is probably important. The fact that an LH-RH challenge given as the plasma progesterone level declines produces a massive release of LH and FSH (see Table 4) supports the view that, once the pituitary response to LH-RH is restored in the early post-partum period, inadequate pituitary sensitivity or availability of LH cannot be the reason for the lower progesterone release during the first short cycle.
C onclusions
Lactation in the cow does not provide a complete block to pregnancy, but milking and suckling can inhibit ovulation at the hypothalamic, pituitary or ovarian levels. The lack of ovarian follicular activity generally observed at the end of pregnancy is continued during early lactation.
There is good evidence of suppressed pituitary sensitivity to synthetic gonadotrophin releasing hormone (LH-RH) during the early post-partum period extending to 10 days in milked dairy cows and 20-30 days in suckling beef animals.
Plasma FSH levels in cows are low immediately post partum but generally increase within 5 days with levels fluctuating widely. There are no consistent changes associated with the first preovulatory LH surge and ovulation.
Plasma prolactin levels rise in milked cows from parturition to approximately 30 days post partum (peak lactation) and then steadily decline. In suckling cows prolactin levels decline linearly from parturition, and are usually lower than in milked cows. There was no significant relationship in individual animals between plasma prolactin values and the onset of ovarian activity and hyperprolactinaemia cannot be implicated as the causal factors in the maintenance of anoestrus.
During early lactation in the milked cow and later in the suckling cow, the low plasma LH concentrations are generally followed by increased pulsatile LH release, leading to a preovulatory-type LH surge. This probably occurs due to (i) a change in pituitary sensitivity to hypothalamic stimulation by an increased frequency of releasing factor release and/or (ii) a positive feedback effect of oestradiol from the developing follicles. The first preovulatory-type LH surge is preceded by episodes of LH release of increasing frequency and peak height and this increases basal LH levels. It can be induced by injections of synthetic LH-RH.
In approximately 50% of cows the first preovulatory-type LH surge is followed by a small transitory rise in plasma progesterone lasting for 5-10 days. This precedes the second •preovulatory LH surge, ovulation and the first full ovarian cycle. In other animals the first preovulatory-type LH surge is followed by ovulation, with or without oestrus and an oestrous cycle of normal length. The physiological effects of progesterone from the first ovarian cycle are probably important in affecting (1) a possible neural oscillator co-ordinating hypothalamic activity and particularly releasing factor release, (2) pituitary and ovarian activity, and (3) oestrous behaviour.
The mechanism responsible for the increase in frequency and peak height of LH episodes and the first LH preovulatory-type surge before the low transitory rise in plasma progesterone in post-partum cows is, as yet, undisclosed but appears to hold the key to the resumption of cyclic ovarian activity in the cow post partum. | v3-fos |
2019-04-03T13:06:09.800Z | {
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} | s2 | Nutritive value for growing pigs of pekilo protein and torula yeast grown in spent sulphite liquour
The digestibility and nutritive value of pekilo protein (Paecilomyces varioti), and torula yeast (Candida utilis) were determined for four pigs weighing 35 —5O kg. The proportion of pekilo or torula was 30 %of the barley-based diet. The crude fat was determined by the standard ether extraction method and by the HClether method. The first method indicated a low fat content and a highly negative digestibility of fat, but the second showed that thepig is able to digest and absorb a large part of the ether-insoluble fat. The energy value thereby increased 14 % for pekilo and 12 % for torula over those obtained by the conventional ether method. The same energy value was obtained for barley by both methods. The energy values (HCI-ether method) obtained for pekilo and torula were 0.96 and 0.95 F.U./kg DM (F. U. = 0.7 kg starch), or 13.41 and 12.98 MJ ME/kg DM, respectively. The protein values, corrected for nucleic acid nitrogen, were 434 and 3 58 g DCP/kg DM. The only significant difference between pekilo and torula was the DCP value (P< 0.001). The nitrogen balances in the pekilo and torula trials were very high and identical (24.4 and 24.0 g N/d), confirming the value of these feeds as protein supplements to cereal feeds for pigs.
Introduction
Two kinds of single-cell protein (SCP), the mycelium forming microfungi pekilo {Paecilomyces varioti) and torula yeast (Candida utilis), are cultivated in the sulphite spent liquor of the pulping industry in Finland.
Pekilo and torula are much alike in composition (Table 1), but pekilo is rougher in texture. Now that production had been going on for several years, the process techniques and with them the quality of products have been stabilized.
The value of pekilo and torula in animal feeding is similar. Both have been successfully used as the sole protein supplement for growing pigs (BARBER et al. 1971, ALAVIUHKOLA et al. 1975, HANSSEN 1978, from the freshly weaned stage onwards (BOBROV et al. 1978). In the feeding of broilers and calves, however, some restrictions have had to be introduced (BECK and GROPP 1974, KIISKINEN 1978, KOSSILA and KIISKINEN 1978. The amino acid composition and the protein value of pekilo and yeasts are well known, their energy values much less clear. Accordingly, the purpose of the present study was to determine the energy values of the domestic-produced pekilo protein and torula yeast for growing pigs. The digestible crude protein (DCP) values and the nitrogen balances were determined simultaneously.
Materials and methods
Pekilo and torula were products of the normal industrial production. Their compositions, and the composition of the barley used as basic feed in the trials, are shown in Table 1.
The digestibility trials were carried out with four castrated pigs weighing 35 50 kg. The proportion of pekilo or torula was 30 % of the barley-based diet. In the basic digestibility trial with barley, 16 %of skim milk powder was incorporated for protein supplement. The preliminary period was 10 days and the collection period 7 days. The details of the digestibility trials have been described in an earlier paper (SALO and ALAVIUHKOLA 1980).
Results and discussion
The crude fat was determined both as normal ether extract and as HCI-fat (boiling in 4 N HCI before extraction with ether). Digestibility coefficients and energy values were calculated on the basis of both fat determinations (Tables 1 and 2). The digestibility coefficients found lie within the wide range presented in the literature, though at the lower end of the range (ANON. 1970, BREIREM and HOME 1970, NEHRING et al. 1970, SCHULZ and OSLAGE 1976, ANON. 1979. The nutritive data for pekilo and torula agree very well with each other, better than the F.U. =0.7 kg starch data in many experiments for two different batches of yeast. Perhaps the cultivation medium has a greater effect on the nutritive quality of SCP than does the strain of microbe. And perhaps the SCP from sulphite liquor has a little lower digestibility than the SCP from some other energy sources, as some literature data suggest (NEHRING et al. 1970, ANON. 1970). In addition to protein the microbe products contain about 35% carbohydrates and 4 10 % fat. The composition of both these groups differs greatly from that of conventional feeds.
The carbohydrates resemble the hemicellulose of higher plants in solubility, but their composition is quite different. The principal units arc glucose and mannose, whereas xylose and arabinose, the essential polysaccharide components of higher plants, are wholly lacking. Hexoseamines are also typical of the microbe products. Sugar and starch, on the other hand, occur only in traces and solubilities in the amyloglucosidase and pepsin incubations are small (SCHULZ andOSLAGE 1976, SALO 1977). The digestion of yeast cell wall polysaccharides by the digestive enzymes of the calfs small intestine is very small (GAILLARD and WEERDEN 1976). Consequently, yeast and pekilo have proved to be a somewhat problematic feed for calves (SCHULZ andOSLAGE 1976, KOSSILA andKIISKINEN 1978). The division of the cell wall compounds into crude fibre and nitrogen free extracts is artificial and serves no purpose here, as can be seen from the digestion coefficients, which are almost identical for the two groups. Moreover, the crude fibre sontent of SCP depends essentially on the filtration technique (SALO 1977).
The pigs proved to digest the carbohydrates of pekilo and torula rather well with the assistance of the bacterial flora of their large intestine. Not so well, however, that the energy values approached those of the common cereal grains.
The crude fat is another group that differs from that of conventional feeds. The fat content is variable and can rise several-fold, when the SCP is hydrolysed with 4 N HCI before extraction with ether (SCHILLER et al. 1972, SALO 1977, HANSSEN 1978. The digestibility coefficients found here suggest that the alimentary canal of pig hydrolyses more fat into an ether soluble form than can be absorbed into the blood circulation, resulting in a highly negative digestion coefficient (Table 1). On the other hand, the pig does digest a good part of the ether insoluble fat, with the result that the energy values calculated by the HCI-fat method are 12-14 % higher (Table 2) and in all probability truer than the values arrived at by the conventional ether method. In respect to barley there is no difference in energy value between these two fat determination methods: the HCI -method shows a higher fat content, and lower digestibility correspondingly.
The digestible crude protein values of Table 2 are corrected for nucleic acid nitrogen. It is well known that 10-20 % of the nitrogen of yeasts is included in the nu- (SCHULZ andOSLAGE 1976, ROTH andKIRCHGESSNER 1980), and the same is true for the pekilo (SALO 1978). Further, it is known that the pig digests about 9 5 % of the nucleic acid nitrogen, but less than one third of it is retained in the body, the rest being excreted in the urine KIRCHGESSNER 1977, 1980). The DCP values of Table 2 are roughly corrected on the basis of this knowledge (15 % content and 30 % utilization). The palatability of pekilo and tornia was good at the 30 % level of the diet. The retention of nitrogen was very high, considering that the normal restricted feeding was used (Table 3). The results only strengthen the many earlier findings that pekilo and yeast are very promising protein supplements to cereal feeds for pigs. | v3-fos |
2019-04-03T13:06:12.312Z | {
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} | 0 | [] | 1981-08-01T00:00:00.000Z | 92543583 | {
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} | s2 | Genetic and management adaptation of field bean (Vicia faba L.) in Finland
The investigation of field bean adaptation in Finnish climatic conditions was carried out at the University of Helsinki in 1976—77. The main objectives were to study the effects of seeding time and population density on the quantity and quality of the yield and the vegetative features in the development of two different types of field bean varieties. Field bean yielded 4061 kg/ha in 1976. In 1977 only 2042 kg/ha was harvested due to the lack of light during the grain filling period and the presensc of plant diseases. Delayed seeding lowered yields in both years. Maximum yield was obtained with the seed rate of 240 kg/ha. Two weeks delay in the seeding speeded up flowering by two days. Temperature sum in degree days from seeding to emergence was 140— 170°C, from seeding to flowering 618—637°C and from seeding to maturity 1670—1890°C. LAI was 5.7 for early variety and 4.3 for late variety at the time ofpod setting representing very effective situation for CGR. Number and distribution of internodes, pods and seeds were primarily influenced by population density and secondly by the differences between varieties.
Introduction
The field bean was one of the most important crops for human nutrition in Europe until the early 17th century. Later it was replaced by potato and maize. In Finland field beans were cultivated since the early 16th century. Bean cultivation was concentrated in the western part of the country. Eastern type native cultivars have been maintained in Eastern Finland until today, although the rest of the Europe have long ago adopted breeded varieties (KIVI 1975).
Field bean is a long day plant. Minimum temperature requirement for germination being +l°C. The plant can tolerate short periods of -4°C growing conditions. Optimum temperature for growth lies between 23-28°C according to varieties. Field bean is considered very sensitive to the stress conditions caused by either lack of light or water ( OSVALD 1959).
As a plant, with a heavy seed weight field bean's moisture requirements for germination are great. Deep tillage, good aeration of seed bed and relatively deep sowing ensure good emergence and favourable root development. Heavy soils are recommended for the cultivation of the crop (HULTKVIST & SVENSSON 1975).
According to OSVALD (1959), a 2 000 kg/ha grain yield together with 4 000 kg/ha straw yield contain 113 kg N, 13 kg P, 54 kg K and 27 kg Ca. As other legumes also field bean's K and Ca requirements are high. In Sweden and in Denmark N-fertilization is not recommended but inocculation in the soils without earlier field bean cultivation (SJODIN et al. 1972). In Finland a relatively cool spring together with minor bacterial activity give a good reason for the use of fertilizer nitrogen. HOVINEN (1977) recommends 40-60 kg N/ha for the start fertilization.
As a crop with a relatively long growing season requirement field bean should be seeded early in the spring. However, only a noticeable delay in the seeding date (30 days) have lead to the reduced yield (HULTKVIST and SVENSSON 197 5), (CHRISTENSEN 1972). Similar results have been obtained by ROWLAND (1978) in Canada and BARRY and STOREY (1979) in Ireland.
In Sweden, BENGTSSON and BINGEFORS (1975) have studied factors relating to the rate of seeding. They have concluded that the seeding rate is dependent primarily on the variety and secondarily on the row spacing. Narrow raw spacing (12 cm) yielded more than stands with row distance 50 cm. Narrow row spacing gives requirement for a higher seed rate. Higher population density can be used with the varieties with low seed weight, although seeding rates are lower.
In Finland HOVINEN and KIVI (1975) have studied the growth behaviour of the most important Swedish varieties together with some Finnish native field bean cultivars. The variety with the highest production proved to be Primus from Svalöf, which matured in 1 30 days. Finnish native cultivar Mikko yielded 45 % less, but matured 20 days earlier.
In this investigation the primary aim was to study the variety and management questions of field bean. Study objectives were native cultivar, early maturing (110 growing days) Mikko with 1 000 seed weight = 220 g and late maturing (130 growing days) Swedish variety Aria with 1 000 seed weight = 350 g. Other objectives were seeding date, seeding rate and the effects of the studied management to the productivity, yield quality and to the vegetative features in the development of the two varieties.
Materials and methods
Field bean trials with different management and biological studies were carried out at the University farm in Helsinki in 1976-77. The research program consisted of the following type of experiments: Field observations consisted of the dates of emergence, flowering and maturing and the existence of plant diseases. Stand density plants/m 2 was determined by counting the plants in the rows 3xl m/plot. Plant height was measured once per week at three places of the plot. The scale for lodging was o-loo %. The plots were harvested with combine machine and dried to 1 3 % water content in DM.
Laboratory studies: LAI was measured twice in 1976 by using a planimeter at the time of flowering and pod setting. Together with LAI measurements was determined the number of leaves per plant. At the time of maturity the number of internodes per plant, number of internodcs per plant carrying pods, number of pods per intemode, number of pods per plant, number of seeds per pod and per plant and the distance from soil surface to the first internode carrying pods was determined . Botanical study consisted of material from 2 X 1 m length of seeding row.
Crude protein was determined by the Kjeldahl method.
Weather conditions
The growing seasons 1976 and 1977 were both cooler than average ( Table 1). Precipitation of both growing seasons was close to normal. Heavy rainfall in July of 1977 developed an extra stress factor causing Chocolate spot disease (Botrytis cinerea) to attack plants and destroy the normal development of the stand. Both the temperature sum in degree days and the cumulative radiation sum (Wh/cm 2 ) were higher in 1976 than those of 1977 ( Fig. 1.).
Results and discussion
Growing time, temperature and radiation requirements: The time period between the date of seeding and the time of emergence varied from 11 to 17 days, primarily according to the spring temperature conditions (Table 2). Weather conditions were related to the seeding date. As a consequence, in early seeding, many temperature degrees are likely to be lost due to the warming of the seed bed, as can be seen in Table 3. During the time period of the stand emergence, around 12 % of the total radiation of the growing season was used for nonphotosynthetic purposes (Table 4).
From seeding to flowering the field bean stand used a temperature sum of 616 637°C in degree days (Table 3). Plant development seemed to be bound tightly to the temperature conditions around the plant. A one week delay in the seeding time speeded up the development by one day for both varieties ( Table 2). The vegetative phase of variety Mikko was only two days shorter than that of variety Aria. The real variety differences were developing in the generative phase of plant development, and the variety Aria with a bigger seed size used more time for legume grain filling than the variety Mikko with 40-50 % less seed size (Tables 2 and 3). Since the development of the plant was primarily bound to the temperature sum, more light was lost in the early seedings than in the late ones (Table 4). (1957). Maturity observations in 1977 were disturbed by the strong appearance of Chocolate spot disease caused by Botrytis cincrea. However, important observation was that two weeks delay in the seeding date introduced only two days shorter growing time by both varieties. Results show the same trend as observed by CHRISTENSEN (1972).
Yields:
In Scandinavia and in Finland the yields of field bean have varied between 500 and 5 000 kg/ha (HOVINEN 1977). Yields in 1976 represented relatively high yield levels as results 4 240 kg/ha for Mikko and 3 880 kg/ha for Aria show. Better productivity of Mikko compaired to Aria was not earlier experienced (HOVINEN 1977). The growing season 1977 represents about 50 % of the yields observed in 1976. Mikko also showed better productivity in rather poor growing conditions in 1977 (Table 5). Statistically the seeding densities 118 plants/m 2 for Mikko and 68 plants/m 2 for Aria in 1976 and 114 plants/m 2 for both varieties in 1977 produced highest yields although yields slightly increased when seeding rates were raised up to the highest level studied. Two year results show 200 kg/ha better average yields for the earliest seeding than the seeding two weeks later, although the yield difference was not significant. In Fig. 2. reduced net yields show distinct decrease in late seeding especially at high seed rates. Aria shows the same trend as pointed by BENGTSSON & BINGEFORS (1975) and THOMPSON and TAYLOR (1977) when the optimum seeding rate was 80 plants/m 2 .
According to the results obtained (Tables 5 and 7), the temperature sum had primary effect on the plant development. On the other hand, light was primarily responsible for the yield formation. The temperature sums in degree days are almost equal in 1976 and 1977 (Table 6). Poor light conditions representing 12 %-units less total radiation in 1977 during the grain filling period resulted in a diseased stand and 50 % less yield than in 1976.
5. Field bean as a protein rich crop is valuable raw material in animal feeding. Its protein completes cereal grain protein by containing more lysin. Lack of sulphur containing amino acids restricts the use of field bean protein alone as a protein source for nonruminants. Also tannic acids and some glycosides of field bean grains are harmful in animal diet (NEHRING et al. 1972 1976 and 28.4 % in 1977). In 1977 the differences between varieties were smaller due to the less favourable growing conditions (Table 5). Delayed seeding date resulted in a lower protein content in field bean grains, as experienced by ROWLAND (1978) in Canada and CHRISTENSEN (1972) in Denmark. This trend was interfered by Chocolate spot disease in 1977 by infecting the early seedings and early variety more heavily than late seedings or late variety Aria. Seeding rate had no statistically significant effect on the raw protein content of both field bean varieties as shown also by BENGTSSON and BINGEFORS (197 5) in Sweden.
Seed weights for Mikko and Aria in 1976 were 215 and 331, in 1977 160 g an 246 g respectively. A relatively cool growing season together with plant diseases in 1977 did not allow a complete grain filling, especially when lack of light was also a limiting factor. Normal seed weights for Mikko and Aria are 220 g and 360 g, respectively. Field bean seed weight decreased as seeding date was delayed or seeding rate increased (Table 5) as shown also by CHRISTENSEN (1972).
Stand characteristics;
The stand height of variety Aria was at the end of August in 1976 100-110 cm against Mikko with an average stem length of 80 cm (Fig. 3). Stands with low seed rates developed faster than the ones with a high population density. The trend was reversed at the end of the growing season. Results arc equal to those obtained by BARRY andSTOREY (1979) or CHRISTENSEN (1972). Because of the greater height of the stand more lodging (24-41 %) could be found amongst the variety Aria, whereas the short variety Mikko stayed relatively upright. Leaf area index (LAI) was mostly influenced by variety and seed rate. Mikko had higher LAI at the beginning of pod setting; average 5.73 for Mikko against 4.28 for Aria. LAI increased when the population density of both varieties increased (Table 7). LAI 3 was reached at the time of flowering only with early seeding and with high seed rates. According to BULL (1968) at the time of LAI = 3, daily temperature has the most important effect on plant growth. When LAI is raised beyond LAI 3, leaves and developing pods have increasing competition for light which can be considered the next limiting growth factor. In this study stands reached LAI 4 at the time of pod setting. LAI 4 is known to be the most effective leaf area for crop growth rate (CGR) of field bean.
Number and distribution of internodes, pods and seeds were mostly influenced by population density based on seed rates. Dense population with high seed rate had less pod carrying internodes per plant, less pods per internode and fewer seeds per pod than the stands with a sparse population density (Table 7). Results agree with those obtained by BARRY and STOREY (1979) and THOMPSON and TAYLOR (1977). Also distinct differences between varieties could be found. The variety Aria had a greater number of internodes, more pod carrying internodes and pods per internode but less seeds per pod than the variety Mikko. Late sccdings showed a greater number of internodes and better production of pods as found by BARRY and STOREY (1979) in Ireland. The highest concentration of pods was located in the fourth and fifth internode (Fig. 4). Pods showed closely the normal distribution around those internodes.
The distance from the soil surface to the first pod carrying internode was 25.9 cm for Mikko and 29.4 cm for Aria. Pod distance varied according to the population density. Variation among Mikko variety was from 24.1 cm at seed rate 160 kg/ha to 29.8 cm at the seed rate of 400 kg/ha. Pod distances were higher than those obtained by HOVINEN (1977) but a similar trend to that obtained by BENGTSSON and BINGEFORS (1975) in Sweden.
Summary and conclusions
The investigation of field bean adaptation in Finnish climatic conditions was carried out at the University of Helsinki in 1976-77. The main objectives were to study the effects of seeding time and population density on the quantity and quality of the yield and the vegetative features in the development of two different types of field bean cultivars. The following results have been drawn: 1. Yielding ability and quality characteristics of field bean vary from year to year. Field bean still has certain primitive features such as long growing season requirements, and low drought and disease resistance. Regardless of the relatively cool summer field bean yielded in 1976 4 061 kg/ha. In 1977 the lack of light and attack of deseases resulted in an average yield of 2 042 kg/ha. Early seeding produced the highest yield. Seeding rate of 240 proved to be optimum for the maximum yield.
2. The time period from seeding to the stand emergence varied from 11 to 17 days representing temperature sums in degree days from 140 to 170°C. From seeding to flowering field bean used 46 to 50 days or from 618 to 6 3 7°C in degree days. Growing time from seeding to maturity varied from 119 to 135 days according to the variety and to the seeding time. High population density increased the growing time from 2 to 3 days. Temperature sum in degree days from seeding to maturity varied from 1670 to IB9O°C.
3. The delayed seeding date resulted lower protein content of the grain dry matter. Seeding rate had no significant effect on the protein content of both varieties studied.
The seed weight decreased when the seeding date was delayed or the seeding rate increased. 4. LAI was most influenced by variety and seed rate. LAI at the beginning of pod setting was 5.73 and 4.28 for Mikko and Aria, respectively representing the most effective situation for CGR. 5. Number and distribution of internodes, pods and seeds were mostly influenced by population density. Also distinct differences between varieties could be found. The highest concentration of pods was located in the fourth and fifth internode. Pods showed closely the normal distribution around those intcrnodcs.
The pod distance from soil surface to the first pod carrying internode varied according to the population density. | v3-fos |
2019-03-20T13:13:34.697Z | {
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} | s2 | Inhibitors of bovine parvovirus, coronavirus and rotavirus in precolostral and fetal bovine sera
Eleven, 11 and 2 of 11 precolostral sera from normal calves and 13, 14 and 9 of 14 sera from normal fetuses, 7 to 10 months of gestation, neutralized bovine parvovirus, coronavirus and rotavirus, respectively. When assayed by single radial immunodiffusion, all the sera contained IgG at 360 to 1400 mg/dl, and some of them had much smaller amounts of IgM or IgA. Most of the neutralizing activities against bovine coronavirus and rotavirus were readily inactivated by treatment with acetone or 2-mercaptoethanol. Some sera fractionated by Sephadex G-200 gel filtration or starch block electrophoresis had neutralizing activities against bovine parvovirus or coronavirus in fractions containing no detectable amounts of immunoglobulins. These observations seem to indicate the presence of substance(s), other than immunoglobulins, capable of inhibiting replication of bovine parvovirus, coronavirus or rotavirus. The chemical nature and the mode of action of the inhibitors await elucidation.
INTRODUCTION
Transplacental transfer of maternal immunoglobulins does not occur in cattle, and the newborn animal acquires maternal antibodies by ingesting colostrum (Brambell, 1970;Solomon, 1971). On the other hand, bovine fetuses develop the ability to produce antibodies upon antigenic stimulation relatively early in gestation (Solomon, 1971;Osburn, 1973;Schultz, 1973;Schultz et al., 1973). Many workers have reported the presence of antibodies against various viruses in sera from colostrum-deprived calves and fetuses (Kniazeffet al., 1967;Dunne et al., 1973;Homer et al., 1973;Hubbert et al., 1973;Schultz et al., 1973;Miura et al., 1974;Rossi and Kiesel, 1974;Kurogi et al., 1975). On the other hand, there have been reported studies suggesting the presence of virus-neutralizing substances, other than immunoglobulins, in bovine sera (McFerran, 1962 a, b;Kanamitsu et al., 1967;Doggett et al., 1968;Urasawa et al., 1968 a, b;. Recently we have reported that high percentages of normal precolostral and fetal calf sera neutralize bovine parvovirus, coronavirus and rotavirus (Sato et al., 1980). In the present study, experiments were carried out to relate these neutralizing activities to immunoglobulins in the sera, and the results obtained suggested that some precolostral and fetal calf sera might contain substances, other than immunoglobulins, capable of inhibiting the replication of these bovine viruses.
MATERIALS AND METHODS
Sera. Serum samples were obtained by jugular puncture from normal newborn calves before or after ingestion of colostrum and from some of their dams in 1973 and 1974, and by cardiac puncture from normal fetuses, 7 to 10 months of gestation, in 1979. The sera were stored at --20°C and inactivated at 56°C for 30 min before use.
Viruses. The K-71 strain of bovine parvovirus was originally isolated from an aborted fetus (Sugimura, et al., 1974) and had undergone 5 passages in primary cultures of bovine kidney (BK) cells when used in the present study. The Lincoln strain of bovine rotavirus (Mebus et al., 1971) was used at the 7th passage level in BK cell cultures. Calf diarrhea coronavirus (Mebus et al., 1973) was found in our laboratory to replicate with cytopathic effect in cultures of the BEK-1 cell line derived from bovine embryonic kidney (Inaba et al., 1976). In the present study the virus was used at the 7th BEK-1 passage.
Neutralization tests. Neutralization tests with bovine parvovirus and rotavirus were carried out in tube cultures of BK cells and those with bovine coronavirus in tube cultures of BEK-1 cells. The medium for cell growth was Eagle's minimum essential medium (MEM) containing 10% tryptose phosphate broth, 10% bovine serum and antibiotics, and the maintenance medium was MEM containing 10% tryptose phosphate broth, 0.5% sodium glutamate and 0.05% yeast extract. Cultures were prepared in 11 X 100 mm tubes by seeding 0.5-ml volumes of growth medium containing 2.5 X l0 s cells/ml and incubating at 37°C for 4 days. Serial twofold dilutions of the serum or fractionated materials were made with maintenance medium, and each dilution was mixed with an equal volume of maintenance medium containing 200 TCIDs0 per 0.1 ml of virus. The virus-serum mixtures were incubated at 37°C for 90 min, inoculated in 0.1-ml volumes into 2 tube cultures per serum dilution. The inoculated cultures were incubated at 37°C for 90 min for virus adsorption, fed with 0.5 ml of maintenance medium and incubated in a roller drum at 37°C for 8 to 9 days with parvovirus or for 6 or 7 days with coronavirus and rotavirus. The titer was expressed as the reciprocal of the highest serum dilution showing neutralization in at least one of the two tubes.
Treatment of serum with acetone. One volume of test serum was mixed with 20 volumes of chilled acetone, shaken vigorously for 5 min, and centrifuged at 500 × g for 5 min at 4 ° C. The supernatant fluid was carefully aspirated from the sediments, which were again treated with acetone in the same manner. The resulting sediments were dried under vacuum at room temperature and resuspended in sufficient PBS, pH 7.2, to make a 1:4 dilution based on the volume of serum initially introduced.
Treatment of serum with 2-ME. One volume of test serum was mixed with one volume of 0.1 M 2-ME {2-mercaptoethanol) in PBS, pH 7.2. After standing at 4°C for 24 h, the mixture was dialyzed overnight against 0.02 M iodoacetamide in PBS at 4 ° C. It was further dialyzed against PBS for 2 days at 4 ° C. A sufficient amount of PBS was added to the resulting material to make a 1:4 dilution based on the volume of serum initially introduced.
Assay and identification of bovine immunoglobulins. Quantitation of immunoglobulins was made by single radial immunodiffusion according to the method of Kniazeff et al. (1967) using rabbit anti-bovine IgG, IgM and IgA (Miles Laboratories, Elkhard, Indiana). Of each test serum, 0.005 or 0.01 ml was transferred to a well and tests were run in parallel with a standard serum having known concentrations of immunoglobulins. The plates were incubated at room temperature for 22 h, and then examined for the appearance of precipitation rings. Ring diameters were measured to determine Ig concentrations in relation to controls. The lower limit for the detection of IgG, IgM and IgA was 1.6, 4.0 and 5.0 mg/dl, respectively.
Irnmunoelectrophoresis.
A well of 3-mm diameter was punched in the center of a 80 × 120 mm agar gel plate prepared with 1% Noble agar in veronal buffer (pH 8.6, ionic strength 0.05). Test serum (0.03 ml) was placed in the well and run by conducting an electric current at a rate of 5 mA/cm for 150 rain. After completion of the electrophoresis, rabbit antiserum against bovine whole serum was poured into troughs cut on both sides of the well parallel with the electric current and allowed to react for 48 h in a moist chamber at room temperature with the serum components separated electrophoretically.
Starch block electrophoresis. The method of Miller and Bale (1954) was employed. Potato starch in veronal buffer (pH 8.6, ionic strength 0.05) was packed in a 36 × 5 × 1 cm cell, and a trough, 1.5 cm wide, was cut and filled with starch in 3 ml of the test serum and an electric current was conducted at a potential gradient of 5 V/cm for 28 h at 4°C. After completion of the electrophoresis, the starch layer was cut into 12 strips, each of which was extracted with PBS, pH 7.2. The fractions were concentrated by ultrafiltration in collodion bags and 1:2 dilutions were made with PBS based on the volume of serum initially introduced.
Sephadex G-200 gel filtration. This was carried out by the method of Flodin and Killander (1962). A 2.5 × 70 cm column of Sephadex G-200 (Pharmacia Fine Chemicals, Uppsala) was equilibrated with a solution containing 0.15 M NaC1 and 0.01 M phosphate buffer of pH 7.2, and 3 ml of test serum diluted 1:2 with PBS was applied thereupon. Gel filtration was performed using the above buffer at a flow rate of 18 ml per hour at 4°C and filtrates were collected in 3-ml amounts. The protein content of the fractions was measured as the extinction at 280 nm. The presence of IgG~, IgG2, IgA and IgM in the fractions was tested for by the slide microgel diffusion precipitin test (see above). The fractions were also tested for neutralizing titers against parvovirus, coronavirus and rotavirus.
Immunoglobulin contents
Serum samples from 11 normal precolostral calves and 14 normal fetuses, 7 to 10 months of gestation, were subjected to single radial immunodiffusion to determine immunoglobulin contents. Serum samples from 3 pairs of cows and their calves were also included in the tests. The results are shown in Table I. The serum samples from the cows and their colostrum-fed calves had immunoglobulins, IgG contents being highest, followed by IgM and then IgA. The colostrum-fed calves (C-I, C-2, C-3) showed higher Ig contents than their dams (M-l, M-2, M-3), although they had smaller or similar amounts of IgG and only trace amounts or no IgM and IgA before ingestion of colostrum (P-l, P-2, P-3). Of the 11 colostrum-deprived calves (P-l, to P-11), all had IgG, 360 to 1400 mg/dl and 7 calves, or 64%, each had much smaller amounts of IgM, trace to 42 mg/dl, or IgA, trace to 14 mg/dl. Of the 14 fetuses (F-1 to F-14) all had IgG, 360 to 1400 mg/dl, 10 fetuses, or 71%, had trace to 20 mg/dl of IgM, and 9 fetuses, or 64%, had a trace only of IgA.
The neutralizing activity shown in these sera was little affected by treatment with acetone or 2-ME, although some sera with low titers became negative after these treatments (Table II).
Neutralizing activity to bovine coronavirus
As shown in Table I, one (M-l) of the 3 cows had no detectable neutralizing activity and the colostrum-fed calves (C-l, C-2, C-3) all had positive titers. The activity of these sera was little affected by acetone or 2-ME treatment, excepting the serum (C-l) from one of the calves which had a low titer and became negative after 2-ME treatment (Table II). All the serum samples from the colostrum-deprived calves (P-1 to P-11) and the fetuses (F-1 to F-14) showed this activity, which was reduced by acetone treatment, and particularly by 2-ME treatment (Tables I, II).
Neutralizing activity to bovine rotavirus
Serum samples from the cows (M-l, M-2, M-3) and their colostrum-fed calves (C-I, C-2, C-8) had neutralizing titers ranging from 8 to 64 (Table I). The activity of these sere was not much affected by acetone or 2-ME treatment (Table If). Only 2 of the 11 colostrum-deprived calves (P-1 to P-11) were positive with low titers of 2 and 32, while 9 of the 14 fetuses (64%) had titers ranging from 2 to 128 (Table I). After acetone or 2-ME treatment all the sera became negative excepting one precolostral serum (P-3) which showed a titer reduction from 32 to 8 following acetone treatment (Table II).
Starch block electrophoresis
Precolostral calf serum P-8 was subjected to starch block electrophoresis to relate the immunoglobulins contained in the serum to the neutralizing activities for the viruses. The original serum sample contained 1400 mg/dl of IgG, but no IgM or IgA. Of the 12 fractions obtained, only the 4th and 5th fractions were found to contain IgG at 640 and 280 mg/dl, respectively. The original sample had neutralizing titers of/> 512, 16 and 2 for parvovirus, coronavirus and rotavirus, respectively. Neutralizing activity to parvovirus was shown in the 8th and 9th fractions in titers of 128 and 32, respectively, while it was not shown in the other fractions excepting the fraction of origin (the 6th) which had a low titer of 8. None of the 12 fractions showed any neutralizing activities for coronavirus or rotavirus. Immunoelectrophoresis demonstrated gamma-globulin in the 4th fraction, alpha-and beta-globulin in the 8th fraction and alpha-globulin and alubumin in the 9th fraction.
Sephadex G-200 gel filtration
The serum samples examined were those from a cow (M-3) and her calf, colostrum-fed (C-3) and colostrum-deprived (P-3). In addition 2 samples each from precolostral calves (P-2, P-8) and from fetuses (F-3, F-4) were examined. Each fraction obtained by gel filtration was tested for the presence of IgG~, IgG2, IgM and IgA and for virus neutralizing activities. The results are shown in Figs. 1 to 4.
As shown in Fig. 1, fractions of the cow serum M-3 had neutralizing activity against parvovirus and contained IgG~ and IgG2 and some early fractions also contained IgM. Sample C-3 from the colostrum-fed calf of this dam showed a similar pattern of parvovirus neutralizing activity, although the activity began to appear in somewhat earlier fractions than in the case of M-3 serum (Fig. 2). Precolostral serum P-3 of this calf contained only IgG~, but no IgG2, IgM or IgA. Neutralizing activity against parvovirus began to appear before the appearance of IgG~ and some early fractions containing IgGl showed neutralizing activity (Fig. 3). M-3, C-3 and P-3 serum samples showed neutralizing activities against coronavirus and rotavirus in fractions of the IgG elution region (Figs. 1--3). The other precolostral serum samples, P-2 and P-8, contained eluted neutralizing activities against parvovirus and coronavirus in fractions before IgG1 eluted (Figs. 3). No fraction obtained from P-2 and P-8 showed neutralizing activities against parvovirus or rotavirus. Fetal serum F-3 contained IgG~, but neutralizing activity against coronavirus was shown in fractions containing no immunoglobulin. No fraction from F-3 showed neutralizing activity against parvovirus or rotavirus (Fig. 4). On the other hand, fetal serum F-4 contained IgG1, and neutralizing activity against coronavirus was restricted to fractions containing IgG~. Neutralizing activities against parvovirus and rotavirus were not detected in any of the fractions (Fig. 4).
DISCUSSION
The present study has confirmed the previous report (Sato et al., 1980) that high percentages of normal precolostral and fetal calf sera neutralize bovine parvovirus, coronavirus or rotavirus (Table I). Furthermore, all of the 11 precolostral sera and the 14 fetal sera examined in the present study were shown to contain immunoglobulins; all of them contained IgG, 360 to 1400 mg/dl, and some contained much smaller amounts of IgM or IgA (Table I). These findings were taken to indicate that these calves and fetuses had produced neutralizing antibodies to these viruses as the result of intrauterine infection. Several reports are available on the occurrence of immunoglobulins in sera of precolostral calves and fetuses not overtly stimulated by antigens (Pierce, 1955;Kniazeff et al., 1967;Klaus et al., 1969;Schultz et al., 1971;Sawyer et al., 1973;Schultz et al., 1973).
On the other hand, most of the neutralizing activities against bovine coronavirus and rotavirus in the precolostral and fetal sera were lost by treatment with acetone or 2-mercaptoethanol, whereas the neutralizing activity against bovine parvovirus in these sera was not much affected by these treatments. Since immunoglobulins are not affected by acetone treat- ment, and IgG is resistant to 2-mercaptoethanol, these findings seem to indicate the possibility that some substance(s} other than immunoglobulins may be responsible for most of the neutralizing activities against bovine coronavirus and rotavirus, whereas the neutralizing activity against bovine parvovirus may be due to specific immunoglobulin. The results of studies on some precolostral and fetal calf sera with Sephadex G-200 gel filtration and starch block electrophoresis seem to indicate the presence of substance(s}, other than immunoglobulins, capable of inhibiting the replication of bovine parvovirus or bovine coronavirus. Thus, precolostral serum P-8, fractionated by starch block electrophoresis, had neutralizing activity against bovine parvovirus in fractions in which no immunoglobulin was detected by single radial immunodiffusion and immunoelectrophoresis. Sephadex G-200 gel filtration of P-8 sample also demonstrated neutralizing activity against bovine parvovirus in fractions con- taining no detectable amounts of immunoglobulins (Fig. 3). These findings suggest that the neutralizing activity against bovine parvovirus shown in P-8 serum may be due to some substance(s) other than immunoglobulins.
Precolostral sera, P-2 and P-3, were also studied by Sephadex G-200 gel filtration. Neutralizing activity against bovine parvovirus was detected in fractions before the elution of IgG1 and in a few early fractions containing IgG1 (Fig. 3). This finding seems also to support the view that the sera may contain some substances, other than immunoglobulins, capable of inhibiting the growth of bovine parvovirus. With P-2 serum, neutralizing activity against bovine coronavirus was also detected in fractions containing no immunoglobulin (Fig. 3), while P-3 serum had neutralizing activities against bovine coronavirus and rotavirus in fractions containing IgG~ (Fig. 3).
Sephadex G-200 gel filtration of fetal serum F-3 gave results suggesting the presence of some substance(s) other than immunoglobulins capable of inhibiting the replication of bovine coronavirus (Fig. 4). On the contrary, fetal serum F-4 showed neutralizing activity against coronavirus in fractions containing IgG~, suggesting that the activity might be due to immunoglobulin (Fig. 4).
These discussions seem to indicate that some precolostral and fetal calf sera may contain substance(s), other than immunoglobulins, capable of inhibiting the replication of bovine parvovirus, coronavirus and rotavirus.
Further studies are needed to elucidate the chemical nature and the mode of action of the inhibitors. The possible presence of nonspecific inhibitors should be kept in mind in testing sera for neutralizing antibodies to these bovine viruses. | v3-fos |
2018-08-14T23:15:53.148Z | {
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} | s2 | Identification and Differentiation of Species in Cooked Meat by Vertical Plate Gel Electrophoresis
Since quality control for adulteration in meat is very important, the vertical plate polyacrylamide gel electrophoretic technique was studied for the detection and identification of cook e d and canned species extracts. Meat samples from beef, pork, lamb and horse were heated for one hour from 65-75°C at intervals of about 5°C. Be' cause most of the research on the effect of cooking meats on the denatured serum proteins and the resultant electrophoretic patterns were done on beef, beef cooked at 70°C was selected as an initial trial for protein solubilization in order to choose an extraction and applying procedure for cooked meats. The beef was extracted with a variety of potential protein solubilizing agents such as sodium dodecyl sulfate (SOS), urea, Triton X-100 and BOI reagent in an attempt to solubilize the proteins. A single gel containing urea or SOS in conjunction with a discontinuous buffer system was studied as the electrophoresis system. Amido black lOB and Coomassie brilliant blue R-250 were compared as staining dyes. Constant power and constant voltage were compared as power supplies. Polyacrylamide gel electrofocusing was tried. Raw meat extracts were utilized as reference patterns.
' cause most of the research on the effect of cooking meats on the denatured serum proteins and the resultant electrophoretic patterns were done on beef, beef cooked at 70°C was selected as an initial trial for protein solubilization in order to choose an extraction and applying procedure for cooked meats. The beef was extracted with a variety of potential protein solubilizing agents such as sodium dodecyl sulfate (SOS), urea, Triton X-100 and BOI reagent in an attempt to solubilize the proteins. A single gel containing urea or SOS in conjunction with a discontinuous buffer system was studied as the electrophoresis system. Amido black lOB and Coomassie brilliant blue R-250 were compared as staining dyes. Constant power and constant voltage were compared as power supplies. Polyacrylamide gel electrofocusing was tried. Raw meat extracts were utilized as reference patterns. i i A system of tris-chloride buffer at pH 6.7 containing 2M urea and 10% glucose gave the best reslts as an extraction procedure. A single 7% cyanogum gel containing 4M urea in conjunction with a discontinuous buffer system was utilized as the electrophoresis system. Amido black was found to be more sensitive so it was selected as staining procedure. Also, additions of mercaptoethanol to the gel and sample improved the electrophoretic patterns. It was found that with constant power, the front solvent was a very sharp straight line and the bands were sharper than with constant voltage.
Characteristic differences were discernible between beef and lamb versus pork and horse meat heated at 65°C, but the distinction was decreased with increasing temperature.
Canned beef (corned beef) and mixture of canned pork and ham (SPAM) gave some bands but did not give proper separation of the bands. So with more study on solubility and gel electrophoretic patterns heat denatured proteins could be improved and achieved. Use of a densitometer might also improve the utility of the procedure.
Polyacrylamide gel electrofocusing did not work and that may be because the thickness of the gel which generates heat, or other causes, as discussed in Appendix C.
INTRODUCTION
With increases in world population and the limited ability of growing animals to produce meat, meat prices continue to increase, especially some of the kinds which many people prefer (i.e., beef and lamb).
Since quality control for adulteration in meat is very important, electrophoresis with polyacrylamide gel has been studied and successfully applied by Payne (1963) to raw pro-' ducts. This method, the disc polyacrylamide gel electrophoretic technique, developed by Ornstein (1964) and Davis {1964) , has proven to be successful for separation and identification of animal serum proteins. Distinct electrophoretic patterns were obtained between meat and fish species using this technique (Payne, 1963;Mancusa, 1964).
The vertical plate polyacrylamide gel electropho~etic (VPE) technique, which was developed by Raymond (1964) has also been successfully used for the detection and identification of raw meat and fish species Rand, 1972a, 1972b).
The effect of cooking beef on the denatured serum proteins and on the resultant electrophoretic patterns has been studied. Laakkonen ~ ~· (1970) found that changes in the electrophoretic patterns of bovine muscle took place during low temperature and long-time heating . Fogg and Harrison (1975) have studied the effect of two end points, 25 oc and 45*C in semitendinous beef muscle on the electrophoretic patterns of the sarcoplasmic proteins. They concluded that heating to 25°C decreased the number and intensity of the slowest migration protein components of the sarcoplasmic fraction and the effects were more pronounced at 45°C. Lee et al. (1974) 11. lOg sample + lOM urea (ultra pure urea) was prepared according to Krzynowek and Wiggin (1979).
( u )
Each mixture was transferred to a 50 ml plastic centri- Urea-polyacrylamide Gel Electrophoresis: The vertical plate polyacrylamide gel electrophoretic technique described by for the differentiation of meat speces was used with some modification.
Formulation for all reagents used in this method are shown in Table 1. The gel solution was prepared prior to each run, according to the formulation shown in Table 1. The tris, Cyanogum, urea and TEMED were dissolved in 90 ml of water and adjusted to pH 8.9 with 3N HCl, followed by filtration through Whatman #1 filter paper. The volume of the gel solution was adjusted to 150 ml, and 0.2 ml of 10% Tween 80, 0. lml mercaptoethanol and o. 13g of ammonium persulfate were added with gentle agitation on a magnetic stirrer. The gel solution was poured at room temperature into an EC Apparatus Co. EC-470 vertical electrophoresis cell which was assembled horizoinally. A slot form for 10 samples was inserted, and the gel solution allowed to solidify for 40-50 min. The cell was then connected to a circulating refrigerated water bath, and coolant at 10°C was introduced into the cooling plates . The temperature in the refrigerated waterbath was reduced to 0°C over a 1 hour period and maintained at that temperature for the balance of the run. The excess gel above the slot form was removed and the cell was placed in a vertical position. The tris-glycine electrode buffer, precooled overnight to 2°C was added to the cell and allowed to overflow into the lowest chamber. The slot form was then carefully removed, and bromophenol blue was added to the buffer in the upper chamber to mark the position of the glycine-chloride front.
fonditions for Electrophoresis 1 -Constant Voltage -Was carried out as described by .
Staining and Destaining
After completion of the run, the power was turned off and the cell was disconnected. The electrode buffer was drained off and the cell was disassembled. The gel was removed and stained for 15 minutes in the Amido black lOB stain shown in Table 1 (1) Triton X-100 with ME (TM), (2) Triton X-100 with SDS (TS), (3) Triton X-100 with urea (TU), (6) lOM urea (U) , (7) BDI reagents, (9) tris-HCl buffer containing 2M urea and 10% glucose (BGU) . · ' Figure 3. Effect of urea concentration on cooked beef meat extracts, in presence of mercaptoethanol.
• mercaptoethanol which provided a slightly better pattern.
The addition of mercaptoethanol to the running gel was also studied . Figure 4a shows e 1ectrophoretic patterns obtained from cooked meats without addition of mercaptoethanol to the running gel . Figure 4b shows The effect of Amido black 108 and Coomassie brilliant blue R-250 on the electrophoresis of meat protein as a staining procedure was compared. Cooked beef meat extracted with extraction buffer containing lM, 2M and 4M urea containing mercaptoethanol and 2M urea without mercaptoethanol were applied to a gel in duplicate. After the electrophore t ic run was accomplished, the gel was divided down the middle.
One side of it was sta i ne d i n . Amido black 108 accor di ng to (Ogita and Markert, 1979). As seen in Figure 5, Amido black was sensitive and gave sharper ' Figure 4a. Electrophoretic patterns obtained from cooked meats without addition of ME to the running gel.
I resolution -better than Coomassie blue. Wilson (1979) also found the same results. Therefore, Amido black 108 was selected as the stai.n of preference, following the procedure of coduri and Rand (1972) Samples 1,2,3 and 4 stained with Amido black lOB; 7,8,9, and 10 stained with Coomassie Blue.
'
The possibility that the increased volume resulted in dilution of the so~uble material and there may be less soluble material present in horse meat were considered as probable causes for the weaker staining of extraction containing horse meat . The increased sharpness and appearance of more bands after heating may be due to a low molecular weight fragment derived from one of the proteins initially present (Cohen, 1966).
These figures show the electrophoretic patterns obtained with VPE method using only one gel. Characteristic differences were discernible between beef and lamb versus pork and horse at 65°C, but the distinction decreased with an increase of the heat process temperature. Therefore, this study did With the vast increase in world population and the limited ability of growing animals to produce meat, meat prices are getting higher day after day, especially some of the kinds which most of t:l)e .people prefer (i.e., beef and lamb)·. Fennema (1976) predicts that the industrialists will look more and more to areas of substituted, fabricated or synthetic foods as a possible solution to world food problems. Various electrophoretic techniques such as: moving boundary electrophoresis, zone electrophore-sis, polyacrylamide gel electrophoresis and thin layer isoelectric focusing have been used for the separation and identification of proteins.
Since the quality control from any adulteration in meat is very important, electrophoresis with polyacrylamioe gel has been successfully applied by Payne (1963) as a method for separation and identification of animal serum proteins using the disc polyacrylamide gel electrophoretic technique. As developed by Ornstein (1964) and Davis (1964), the method has prov~n to be a good method for the detection and identification of mixtures of animal proteins. Distinct electrophoretic patterns were obtained between meat and fish species using this technique (Payne, 1963;Mancuso, 1964 '1964). Nakanichi and Raymond (1962) reported that VEP could be used in the routine process of blood proteins. Laakkonen et al. (1970) found this technique was useful for meat an a 1 y s i s . were the first to establish that distinct electrophoretic pattern could be obtained from the sarcoplasmic extracts of different meats --beef, lamb, pork, and horse, using vertical plate technique. Also, they applied this technique for the differentiation on fish and shellfish species {1972b).
Coduri, Bonatti and Simpson (1979) applied a vertical
Plate gel electrophoresis to the separation of pigmented and nonpigmented trout and salmon species.
In order to import meat from many countries of the world the mea _ must be cooked to 69°C to insu~e destruction of the foot and mouth disease virus if present (Heidelbaugh and Graves, 1968), a number of methods therefore have been tried to determine the cooking temperature such as extractability, coagulation test, determination .of acid phosphatase activity and direct spectroscopy of extracted meat pigments. Cohen (1966) studied the protein changes related to ham processing temperatures and found that the rate of ·heatng as well 24 as the temperature reduced the amount of extractable proteins.
In 1969 he studied the determination of acid phosphatase activity in canned hams as an indicator of temperatures attained during cooking. He found the method lacked accuracy when appli~d to hams processed to higher internal temperatures. Ooesburg and Papendrof (1969) stated that the degree of heating of some fish muscle could be calculated from the coagulation test. Helmke and Froning (1971) studied the effect of endpoint cooking temperature on the color of turkey meat. They found that the extracted pigment from turkey meat, which was cooked to 82°C, had a spectra quite different than that observed at lower temperatures.
None of these methods, however, has proved to be acceptable because they were only applicable to narrow ranges of cooking temperature or other experimental conditions.
Since cooked meat will be denatured and protein solubility will decrease, solubilizing agents such as urea and sodium dodecyl sulphate (SOS) are necessary to solubilize the protein in cooked meat. Since SOS and urea are two of the most commonly used reagents in protein chemistry, their applications in protein denaturation, solubilization, dissociation and purification are much too numerous to summarize here.
An advantage of these agents is the ability to obtain constant electrophoretic mobilities of proteins independent of isoelectric point and amino acid composition, which can be slightly modified during cooking (Lee~~., 1974). (1980) reported that when beef muscles were cooked at temperatures between 60-80°C, protein bands gradually disappeared and could not be detected above 80°C. Deschrreider and Meaux (1974) (Lee _tl ~·, 1974;Mafinen _tl ~·, 1979 andCalderoni andBazan, 1980), and it appeared to be a good procedure to try for analysis of cooked meats. Therefore SDS-polyacrylamide gel electrophoresis was performed according to Ogita and Markert (1979) with some modifications as shown in Table 2. Table 2. The proportions of 3g of the IA solution to water were varied to give different gel concentrations up to 20% acrylamide gel (Table 3), then ID solution was added.
Preparation of the stacking gel . -The stacking gel mixture was prepared by mixing the solution as given in Table 3. A 4% acrylamide stacking gel was optional for nearly all experiments.
Preparation of the sample. -Cooked beef, lamb, pork and horse meats were prepared as described by Ogita and Markert (1979)
RESULTS AND DISCUSSION
Polyacrylamide gel electrofocusing procedure as recommended by E-C Apparatus Corporation was tried with some modification. Initially the heat generation proved excessive after short time of the run at the recommended 20 watts (w).
So, the run was repeuted using lOW but heat generation proved excessive after a period of time and therefore, the power was cut to 2.5W. Even so, there was still a small heat ef-' feet on the gel. Figure 9 shows polyacrylamide gel electrofocusing of raw, cooked (beef, pork, lamb and horse meats) and canned meat (corned beef and mixture of pork and ham).
It shows no migration or bands, and that may be because of the thickness of the gel which generates heat or because the sample contained salt, which contributed to generaLed heat, or both. Also it could be a problem caused by oxygen generated at the anode. This is partially dissolved in the medium, and may oxidize the SH-groups of protein _ (Goal~~·, 1980).
An overview or the times for running, fixing, staining, and destaining of electrophoresis and isoelectric focu_ing in polyacrylamide gel is shown in Table 4. These results indicated that electrophoresis is less consuming of time and more applicable for routine work. | v3-fos |
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} | s2 | Product news
The program will provide aspiring engineers with hands-on experience by incorporating Ansys’ industry-leading electronics simulation into its curriculum of high-speed digital engineering topics from signal and power integrity to electromagnetic compatibility. The ten-course master’s program will integrate the Ansys Electronics Desktop Student software product, which students can easily download to access Ansys HFSS, Ansys Maxwell, Ansys Q3D Extractor, and Ansys Icepak. Ansys’ student products are a key component of the Ansys Academic Program, which supplies universities with affordable software for use in the classroom and in research, while providing students with free resources for selflearning. As a subset of the PMP-HSDE program, CU Boulder will offer certificate programs tailored to specific design skills and tools. The master’s and related certificate programs will be offered through the department of electrical, computer and energy engineering. The Ansys Electronics Desktop Student simulation package provides students with the opportunity to gain firsthand experience in highand low-frequency electromagnetics, electromechanical and electrothermal analyses, and more, while cultivating a greater talent pool of well-trained, career-ready electronics engineers for potential employers. Currently, engineering students across more than 3300 universities in 90 countries are developing industry-ready simulation skill sets with Ansys’ products. In Spring 2023, CU Boulder will introduce the on-campus program with the potential to expand access through an online option after a successful launch. For more information: https://www.ansys.com/
[4] Application of near IR to the analysis of food. Dr. B.G. Osborne, Flour Milling and Baking Research Association, Chorleywood.
Proc uct ews
Atlantic City revisited 1981 A review of exhibits at this year's Pittsburgh Conference The 32nd Annual Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy was held from 9-13th March at Atlantic City, NJ, USA (A report on the scientific meetings appears on page 100). In true tradition it was bigger, better attended and had more lectures presented at it than before. As a regular visitor one was left with the opinion that perhaps there is really very little new under the sun. Microprocessors were first presented several years ago and now they are mostly common place, while visual display units and graphics displays increase in numbers and sophistication. This year there was a considerable interest in computer systems and networking. The show presented the latest state of the art instrumentation, and these instruments are more and more sophisticated however, do they fulfill the true aims of the analyst? Very often it is sad to note that instrument companies have developed their systems without regard to the needs of the analyst; particularly they do not often allow facilities for filing data and interogation of the data at a future date.
It is impossible to cover all the aspects of instrumentation on show which related to automation, however the few which are discussed here typify those on show. Computer interface UTI has developed a microprocessorbased interface which links their range of mass spectrometers with virtually any computer on the market. The Spectra-Link is compatible with the IEEE 488, RS-232C and RS-449 interface standards. The unit includes software stored in read-only memory (firmware) for five operating modes, Four of these (spectrum scan mode, total pressure mode, specific.peak mode, and calibration mode) automatically perform most of the common tasks required to control the unit. The fifth, direct control mode, permits even more operator flexibility by allowing the user to write totally original programs. UII, 325 N. Mathilda Avenue, Sunnyvale, CA 94086, USA.
IR Spectrophotometers
Perkin Elmer's new product line of low cost infr.ared spectrophotometers, the Model 1300 series, was exhibited. There are three models; the 1310 has a single slit program and two scan speeds, the 1320 and 1330 have three scan speeds and two appropriate slit programs.
The series can be interfaced to the Model 3500 infrared data station providing an improvement of spectral quality and the facility to use the spectrophotometer for 'Search' applications in the identification of unknowns.
Editor's Note:. The address of the manufacturer/supplier appears in italics at the end of each item. In some cases this address will be that of a subsidiary to the manufacturing company as the address given is that from which the information has been obtained.
Surface tension measurement
Madison-Kipp demonstrated their new automatic SensaDyne 5000 surface tensiometer. Using a differential maximum bubble pressure technique the SensaDyne displays liquid surface tension valves directly in dynes/cm along with process fluid temperatures on digital displays with a resolution of 0.1 dyne/cm and 0. lC. An optional printer provides user-programmable, continuous or intermittent records of surface tension, temperature and time. The system has been designed for research and quality assurance applications where it replaces static measurement devices. It is claimed to be the first commercial instrument for dynamic surface tension measurements.
Liquid chromatograph
Spectra-Physics claim that their SP 8100 LC system is the world's first instrument to feature an intelligent autosampler capable of automatically reading sample identification bar codes on the vial and controlling the LC parameters without, operator intervention. The SP8100 is a modular compact chromatograph specifically designed for repetitive routine analysis, small batch or methods development work. The intelligent autosampler capability allows up to 80 different samples to be loaded and analysed without operator input or intervention. The integral autosampler option, which can be retro fitted at any time, automatically reads a bar code label on each standard vial and then sets up all the instrumentparameters for that sample. The operator can change or insert new samples at any time, even during repetitive batch runs, leaving the chromatograph to make all necessary adjustments and LC file selection. Spectra-Physics GmbH, Siemensstrasse 20, D-6100 Darmstadt, W. Germany.
Multi-purpose balance
The Model 5006 electronic balance from Arbor Laboratories has a capacity range of 0 5000 grams and a resolution readability to 0.01 grams. In addition to handling routine weighings this balance can be interfaced with complex computer or peripheral systems. A 15-position stability switch located on the back of the balance reduces operator errors. This option determines when the environment stabilises and then allows the balance to start a weighing cycle; in unstable environments the balance automatically inhibits input/output transfers which eliminates incorrect measurement recordings.
Solution handling system
FiAtron's SHS-200 is a microprocessor controlled system with which most repetitive analytical solution handling tasks can be readily automated. It is designed to fully utilise the many advantages of flow injection analysis. In addition it allows the injection of a programmable volume of sample and reagent into the carrier stream. This 'variable sample volume mode' can readily accommodate small as well as very large sample or reagent volumes without physically changing sample loops. Additional operational modes include stopflow, dispensing and calibration. FiAtron Systems Inc., 6651 N. Sidney Place, Milwaukee, WI 53209, USA.
Organic-free water
Millipore exhibited their new disposable cartridge for batch preparation of HPLC-grade .organic-free water. Called Organex, the cartridge contains a proprietary organic scavenger resin which is said to remove virtually all trace organics from distilled or deionised water. The Organex cartridge is designed for use in an inexpensive borosilicate glass holder which can process a one litre batch of organic-free water in five to seven minutes. The holder also accepts a 0.45 membrane disc filter to remove all residual particulate matter larger than the rated pore size.
Thermometric titrator
The Sanda Thermo-Titrator system provides an enthalpimetric method using temperature change to indicate the course of a particular reaction between analyte and reagent. Thermometric | v3-fos |
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} | s2 | Albinism in US Charolais cattle
Summary Complete albinism occurred in 7 calves in two purebred Charolais herds. One calf exhibited nystagmus, opisthotonus and solar dermatitis. All calves had photophobia, acting blind in bright daylight. Two calves were necropsied. Irides, retina, skin and hooves contained scanty amounts or no pigment. Ganglionic layer of retina had a paucity of neurons. Inheritance of this defect is likely to be recessive.
Reported here is complete albinism in purebred Charolais calves.
-Material and methods
Defective calves for this investigation were part of a long-term study of congenital defects in cattle (L EIPOLD et al., 1972, L EIPOLD , 1978. Calves were observed clinically, were humanely euthanitized and necropsied immediately. Tissues were taken from brain, spinal cord, eyes, endocrine glands, internal organs and skin. Tissues were fixed in 10 p. cent buffered, neutral formalin, processed routinely, sectioned at 6 microns and stained with hematoxylin and eosin (H & E).
Complete albinism was reported from two purebred Charolais herds. The first calf brought to us was a 2-week-old purebred bull Charolais calf, and it was presented to us because of blindness. Six such blind calves were seen over a 5 year period in this purebred Kansas Charolais herd. They were sired by the same bull and all 5 dams were half sisters. One dam had an albino calf in successive years. After removal of the bull no more cases of albinism were observed. Bull and all dams involved had normal eye color.
A 5-week-old male purebred Charolais calf from a 50 head purebred cow Charolais herd in Missouri was presented to us in lateral recumbency. It had opisthotonus, nystagmus, and appeared to be blind. The owner had not encountered any other animal with pigmentary anomalies previously. The animal was euthanatized and necropsied.
Both calves necropsied had skin which was whitish to pink and the hair was white and silky ( fig. la and lb). Areas around eyelids lacked pigment. The tongue was whitish-pink and hooves were yellowish-white. Skin over face, head and distal limbs revealed lesions of solar dermatitis and had a tendency to peel off ( fig. 1 a and 1 b).
The irides were faintly pink ( fig. 2). Whitish conjunctiva were densely injected with blood. Ocular fundi were pink and vessels of the retina were prominent. In transverse sections, the tapetum nigrum and tapetum lucidum were indistinguishable and were pigmentless.
-Histopathologic changes
None of the layers of epidermis had melanin. Hairs were devoid of pigment. Epidermis was thin. Some sections of skin had mild infiltration of polymorphonuclear leukocytes in the dermis.
Retinal pigment layers did not contain pigment and scanty amounts were present in the ciliary body and limbus ( fig. 3). Irides were completely free of pigment. A paucity of neurons was observed in the ganglionic layer of the retina.
-Discussion
Coat color in animals is dependent on the amount of pigment in epidermis, depth at which it is located, and density of the medium between pigment and light scattering surface (Fox, 1953). Mammalian coat color is almost entirely dependent on presence or absence of melanin in the skin and hair, while eye color is determined by intraocular melanin and hemoglobin (S EARLE , 1968). In mammals, melanins exist in distinct forms as eumelanin (brown or black) and pheomelanin (yellow or reddish). Thus, the genetics of coat color in mammals is largely concerned with hereditary factors affecting these pigment granules by altering their number, shape, arrangement and position or by substituting one type of melanin to another (Fox & V EVERS , 1960). Eumelanin is derived from tyrosine and coverted to dopa (3.4dihydroxyphenylalanine) by catalytic action of the enzyme tyrosinase. DOPA is then oxidized to dopa quinone, undergoes further oxidation and polymerization to become melanin (HARRIS, 1959). Genes may act on any of the biochemical steps in the synthesis of melanin within the cell, and thus result in abnormal coat color (F ITZ - PATRICK et al., 1958).
Clinical, opthalmologic and microscopic findings in the present case are consistent with complete albinism as elaborated by L AUVERGNE (1968), L EIPOLD et al. (1968), & G REENE et al. (1973). Tapetal hypoplasia and optic disc colobomas as described by G ELATT et al. (1969) were not observed in these albinotic Charolais calves.
Albinism appears to be a rare defect. In Swiss Brown cattle in Switzerland, the recessive gene was estimated to house a frequency of 0.002 (W INZENRIED & L AU -VERGNE , 1970). In a US survey of bovine birth defects involving over 70,000 births, no albinos were encountered, making the frequency of the albino gene, if recessive, to be less than 0.004 (L EIPOLD et al., 1972). G REENE et al. (1973) concluded that the type of inheritance of albinism in Beef Shorthorns appeared to be recessive. The pattern of occurrence of albinism in these two purebred Charolais herds is suggestive of a recessive mode of inheritance. However, further investigations are warranted to elucidate the exact nature of inheritance of complete albinism among Charolais cattle. | v3-fos |
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} | s2 | HIGH MORTALITY OF GREAT GRAY OWLS IN MANITOBA — WINTER 1980-81
Winter 1980-81 in Manitoba was relatively mild and dry, with little snow cover. Small mammals presumably suf¬ fered from these conditions and also from alternative thawing and freezing in late winter and early spring. In no previous winter have we tried so often to lure birds across dry grass or roads. Great Gray Owls, practically limited to a diet of small mammals, appeared in Manitoba in small numbers in scattered localities from The Pas in northwestern Manitoba to Sprague in the extrem southeast. A concentration of 10 wa reported along PR 308 from Sprague tcj Moose Lake (20 mi.) through January and February. Indications were tha voles were abundant along the roadsid
relatively mild and dry, with little snow cover. Small mammals presumably suf¬ fered from these conditions and also from alternative thawing and freezing in late winter and early spring. In no previous winter have we tried so often to lure birds across dry grass or roads. Great Gray Owls, practically limited to a diet of small mammals, appeared in Manitoba in small numbers in scattered localities from The Pas in northwestern Manitoba to Sprague in the extrem southeast. A concentration of 10 wa reported along PR 308 from Sprague tcj Moose Lake (20 mi.) through January and February. Indications were tha voles were abundant along the roadsid Figure 1. rights-of-way. On 22 February we saw 10 owls along that route (Figure 1).
Beginning in January, numerous sightings were reported along the Trans-Canada Highway (PTH 1) east of Winnipeg, especially between Hadashville and Falcon Lake (28 mi.) ( Figure 2). Accordingly, we spent most of our time on weekend sorties in that area. The total number of owls involved, based on the high number of acciden¬ tally killed owls, and ratio of banded to unbanded owls, was estimated as 100 to 150, probably the largest con¬ centration yet recorded in Manitoba.
Our largest daily counts were 16 on 8 February (Prawda to Falcon Lake, about 23 mi.), and 16 on 8 March (Mc-Munn to Falcon Lake, about 20 mi.). From one to 16 birds were reported by 20 different observers on 25 days in January; from one to 10 by seven per¬ sons on 20 days in February; from one to 12 by 18 persons on 21 days in March; and from one to nine by six per¬ sons on 10 days in April (to 19 April).
During the period from 1 January to 19 April, 50 dead Great Gray Owls were recorded (5 in January, 6 in February, 29 in March, 10 in April). All of these were victims of collisions with vehicles travelling on PTH 1 (one was found less than 2 mi. away on an adjacent road). Fifty is a minimum figure. Manitoba Highways staff, for example, admitted to having thrown three large owls onto the Hadashville garbage dump (before we contacted them) and, undoubtedly, some birds made their way into adjacent woods before succumbing to injuries. Probably predators, such as fox and coyote made off with others. Common Ravens fed voraciously on dead owls, occasionally eating most of the flesh and internal organs in a matter of minutes. Sometimes ravens carried the remains of owls some distance from the highway. It became a race between us and the ravens to retrieve birds. Whenever possible we made the rounds of the highway early in the morning, searching for live birds and the remains of others. In three instances birds were found dead less than an hour after we had made a complete circuit of the divided highway between Hadashville and Falcon Lake. We suspect that most owls were killed in the early morning, late evening or at night, but several were killed during daylight hours. Birds lying on the highway or shoulder were easy to spot, but many were in the ditch or in grassy cover some distance from the road. The telltale signs of a victimfeathers scattered along the shoulder of the road -often were overlooked until the late evening sun backlighted strewn feathers, making them conspicuous. The feathers glowed in the low light, looking like full-blown crocuses, as beautiful as disheartening. Each time we found feathers, we walked the ditch and right-of-way, looking for an owl or its remains. Sometimes only a wing, tail or stripped carcass remained, but each remnant was retrieved and recorded.
Fortunately, a number of residents of the area picked up dead birds for us, thus ensuring the retrieval of a max¬ imum number of owls. One person, a school teacher, drove this route twice daily, reporting the presence of live birds and picking up casualties. A few birds (or remains) were found after the thin snow cover disappeared and after shallow ditch water dried up. One Great Horned Owl and two Barred Owls were also found.
That birds were readily visible throughout the period is evident by records of sightings by at least 32 people. In addition, numerous residents were aware of the presence of owls and presumably a lot of travellers noticed them. Sometimes during the day they were unusually conspicuous, sitting up¬ right on the highway shoulder, perching on roadsigns, or gliding and flying across the highway. We banded 24 birds in the area (20 immatures and 4 adults; 8 males and 16 females), bring¬ ing our overall total to 338.
Road-killed Great Gray Owl.
Gordon Elde Road-killed Great Gray Owl.
Gordon Elder
Where did all these owls come from? Band returns indicate that this oc¬ currence of owls was comprised of local residents and birds from northern Minnesota. An adult female, banded by us on 7 June 1980, at a nest near Spruce Siding, was found dead 9 mi. west of Falcon Lake on 17 March about 14 mi. east of her nest site. Two banded young from nests in the Spruce Siding area, both banded 7 June 1980, were found. One was captured alive on 4 March, 3 mi. east of McMunn, 20 mi. east of its nest site; the other was found dead on 7 March, 36 mi. east of its nest site. Those are small distances, in¬ deed, suggesting that winter movement was minimal. Two banded young from our nests just across the border in Minnesota were also found. Banded 1 June 1980, one bird was captured 4 January, 9 mi. west of Falcon Lake (it was found dead nearby, 25 January); the second was captured 28 March; both were about 50 mi. north of their respective nest sites. An adult female, captured by us 5 April, at the junction of September 1981. 39 (3) PTH 1 and PR 308, had been banded as an adult by Steve Loch 10 April 1978, at Floodwood, Minnesota (pers. comm., 1981, about 223 mi. southeast, just west of Duluth. The absence of winter Hawk Owl records or band returns from a heavily banded resident and winter population of Great Gray Owls to the north at Lac du Bonnet (42-50 mi. north) suggests that there was little influx into this area of northern owls.
One of the most striking aspects of this population was the high ratio of immatures (young from summer 1980) to adults. Among birds captured or found dead throughout the province at this period, 88% of 50 dead birds were im¬ mature. These are readily identified by a number of plumage characteristics. Even a few tail feathers or a single wing were sufficient to determine whether a bird was an immature or adult.5) Of 24 birds banded on or within 4 mi. of PTH 1, 20 were immatures. We found the immature/adult ratio in striking contrast to our previous ex¬ perience. For example, in winter 1979-80, 32 of 36 owls banded by us were adults, including 10 captured on or close to PTH 1. Note that adults seemed to predominate in the notable winter in¬ cursion of Great Gray Owls into northeastern North America in 1978-79.7 Evidently, in summer 1980, Great Gray Owls had exceptionally good reproductive success in southern Manitoba and northern Minnesota. We had 13 active nests that summer (our highest number: 6 in Manitoba, 7 in Minnesota) and Steve Loch, in eastcentral Minnesota, had 17 active nests (pers. comm., 1980). A higher mortality of young birds is expected, both on the basis that there are more of them in the population (usually), and assuming that older birds may have had more ex¬ perience with highway traffic or seek prey in sites away from highways.
Great Gray Owls evidently were con¬ centrated along PTH 1 because of an abundance of voles (meadow mice) in the heavily grassed and extensive rights-of-way. In places, the grassed right-of-way extended back from the highway for as much as 100 yards. Low populations of prey species elsewhere (and competition with adults?) are presumed to have caused some of these birds to wander in search of a suitable winter hunting ground. Small mammals presumably had a tough winter in 1980-81, the sparse snow cover throughout the area making them more vulnerable to low temperatures. On the grassed rights-of-way, small mammals probably became more vulnerable as the snow melted, and as meltwater filled low-lying areas and later froze. Bog and forest habitats differed in that, generally, there was less grassy cover and more snow initially. Bogs, with an insulating layer of sphagnum moss were frozen (underneath 6 inches or so of moss) into May. Although in late April there was abundant sign that voles had been numerous earlier, we saw not a single one, and guessed that deep frost, flooding, and heavy predation (for example, Rough-legged Hawks re¬ mained in the vicinity of PTH 1 and elsewhere in southeastern Manitoba, all winter) had depleted vole populations.
Owl movements and concentrations (and nesting) are related to prey species numbers, especially for a species so limited in its prey selection as the Great Gray Owl. Probably, the availability of voles from January to mid-April was the main basis for the unusual con¬ centration of owls in this region, although recent observations by Steve Loch (pers. commun., 1981) indicate other complicating factors, as indicated by the three Great Gray Owls still in the area 12 May. Not since the winter of 1968-69, when a presumed influx was reported along PTH 1 and elsewhere in the province has this region supported so many owls.3 4 (We have since learned that Great Gray Owls are resident in this area and that "influx" may be slightly misleading.5) The availability of exten¬ sive edges of forest with good potential perches must also have contributed to the phenomenon. It seems likely, too, that some aspect of social interaction may have been involved or, at least, resulted from the unusual concentration of birds. Interactions -friendly or aggressive -were observed on numerous occasions through the period of observation.
Another factor that must be con¬ sidered as a basis for the concentration and high mortality of owls is the fact that PTH 1 consists of two double lanes, eastbound and westbound, in places separated by a wide stand of trees (up to one mi.) providing four separate edges. In general, we found more birds (and carcasses) along the south edge of the east-bound lane, though birds oc¬ curred in all areas. Owls occasionally moved from one edge to another, crossing, in effect, two highways. Moreover, traffic on PTH 1 was unusual-Robert R. Taylor Coming in to a lure.
Great Gray Owl. ly heavy. We experienced considerable difficulty conducting our search and capture activities at times owing to a continual movement of large trucks and other vehicles travelling at high speed. Often, we were attempting to capture birds, working directly from the highway shoulder, trying to ignore vehicles pass¬ ing behind us as close as 2 or 3 yards. Our efforts were complicated by a need to cfrive slowly, watching for traffic while looking for live and dead owls. There were many aggravating situations.
There has not been in Manitoba, to our knowledge, as large a concentration of Great Gray Owls or as heavy a mor¬ tality, as was experienced in late winter 1980-81. What was apparently the largest influx of Great Gray Owls on the continent occurred in winter 1978-79 in Robert R. Taylor southern Ontario and the northeastern United States.1 2 6 7 Most of these birds were far from suitable breeding habitat) and many perished from accidental collision with vehicles, shooting and starvation. In the Manitoba situation, on the contrary, most victims were in good, healthy condition and many were un¬ usually fat, right up to the end of the period. It is thus surprising that we have! not yet (10 May) found any nesting birds! in any of several man-made nests in the vicinity of PTH 1, or elsewhere. Possibly] a scarcity of prey species at a critica period may be a limiting factor.
The unusual appearance of a large number of Great Gray Owls in southeastern Manitoba along PTH 1 in late winter 1980-81, coupled with a heavy mortality, must be considered s| biological phenomenon of little known cause. Information gleaned from this occurrence of owls (twenty-one specimens which were mostly "moderately fat" to "heavy fat" prepared as scientific study skins by the senior author) somewhat softens the blow, but in our minds it will remain an astonishing event. Hopefully, it will not be repeated, for an annual mortality of this extent would pose a threat to maintenance of, at least, local populations. | v3-fos |
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} | s2 | 1981 Report of Agricultural Research, Southeast Kansas Branch Station
This annual research report is to inform area farmers of what is being attempted and accomplished at the Southeast Kansas Branch Experiment Station to serve the area. During 1980 the Station headquarters was moved from Mound Valley to Parsons, the location of one of the units of the Station since 1967. Effort at the Mound Valley location continues without reduction. The third unit of the Station is near Columbus. This report covers four areas of research emphasis: crops, forages, beef cattle, and soil and water management. The information is intended for producers, industry cooperators, and other interested persons.
This report covers four areas of research emphasis: crops, forages, beef cattle, and soil and water management. The information is intended for producers, industry cooperators, and other interested persons.
Page two is a summary chart of temperature and precipitation for 1980. Effects of weather sometimes make it impossible to interpret experimental results. This weather chart may help explain some of the reported results. The small grain variety test is to help southeastern Kansas growers select winter wheat, barley, and spring oat varieties best suited for the area. Spring oat conclusions : Lang has been the best variety over the past several years. It has good resistance to red leaf disease, which is sometimes a problem when oats are near the heading stage. 6
Effect of Seeding Rate on Yields of Selected Wheat Varieties
Semi-dwarf and soft wheat varieties dominate the wheat acreage in southeastern Kansas. The effect of seeding rates on these newer varieties has not been evaluated.
Procedure: For the past three and Trison) were seeded at 60, 90, with 70 lbs N, 50 lbs P 2 o 5 , and 50 years four varieties (Newton, Hart, Centurk, and 120 pounds per acre. Plots were fertilized lbs K 0 per acre. 2 Results: The effect of seeding rate on final yield within a given variety was rather small in most cases, more pronounced when wheat was planted late in the fall with soil conditions not ideal for germination. Under those conditions (1978), the 120-pounds-p er-acre rate gave best results.
Under optimum germinating conditions, 90 pounds per acre seemed to be more desirable. Wheat seeded at 60 pounds per acre normally resulted in good yields, although in one late-fall seeding weeds were more of a problem due to reduced population and less tillering.
Conclusions: Optimum seeding rates for semi--dwarf and soft wheat varieties do not appear to differ from rates for standard varieties. With a late planting, increasing the seeding rate helps off-set reduced tillering. (See Table 1 There were no significant differences between fall and spring N treatments. Results from applying N as anhydrous ammonia in the fall did not differ from results from applying urea in fall or spring as a broadcast application.
Conclusion: Expanded studies of optimum N rates with semi-dwarf and soft wheat varieties are planned in 1981. Results at this time are inconclusive. But several years' research has shown no significant difference between fall and spring N applications.
Wheat Response to Nitrogen Where Soybeans Were Previously Grown
Wheat often follows soybeans in many cropping rotations in southeastern Kansas. The benefit from any residual nitrogen provided by the soybeans for the next crop, is not fully known.
Procedure: In the fall of 1979, 25-bushel-per-acr e soybeans were harvested from a site, which was then planted to wheat (Newton variety). Nitrogen in the form of urea was applied before planting at 20, 40, 60 and 80 lbs of N per acre. 10 Results: Yields in 1980 were well below average due to an extremely hot, dry stmmler. Highest grain yields came from the June 20 planting for all three hybrid maturities (Table 2).
Results
Conclusions: Grain sorghum of any maturity planted in mid to late June seems to flower after the expected hot, dry period in late July and early August.
An early May planting also will mature before the hot summer period in some years, however, the early planting is more susceptible to herbicide and cold injuries. More information is needed concerning the optimum maturity range for a given planting date.
Evaluation of Grain Sorghum Herbicides
Grain sorghum. herbicides are compared to evaluate their effectiveness in controlling the problem weeds commonly found in southeast Kansas sorghwn fields.
Procedure: In 1980 we evaluated grain sorghum herbicides currently labeled and several experimental herbicides that have not been approved.
Results: Excellent crabgrass and pigweed control were achieved throughout the growing season with Bicep applied after planting before weeds or sor~hum plants emerge. Funk's seed safener apparently prevented crop injury from Bicep. However, some seedling injury was observed where Bladex or Igran was used. Results of labelled herbicides are presented in Table 3. Due to sunnner drought conditions, grain yields were not obtained.
Conclusions: Where annual grasses are a problem in grain sorghum fields, Bicep seems to give longer residual control than other grass herbicides. One disadvantage , however, is that the safening agent for Bicep has not been available for many of the commonly grown hybrids in the area. ±./ Treatments were incorporated shallow with a field cultivator prior to planting.
Evaluation of Grain Sorghum Herbicides in Reduced Tillage Systems
Reduced tillage systems with grain sorghum will not be practiced by farmers in southeastern Kansas unless weeds can be effectively controlled.
Procedure: Grain sorghum herbicides were compared in two reduced-tilla ge systems. In one system, grain sorghtUD. was planted with no tillage (no-till) where soybeans were previously grown. In the other system the land was disced once early in the spring and then in early May grain sorghum was planted with a no-till planter.
Timing of herbicide treatments also were compared~ some were applied two weeks ahead of planting; otil,ers, applied innnediately after planting.
Results: Acceptable weed control was achieved with both tillage systems in 1980 when grass pressure was light. Drought conditions during the summer resulted in poor grain yields, so yield comparisons between tillage systems were not possible.
Conclusions: Previous research with reduced tillage has shown herbicide effectiveness as erratic, so more research is needed before recommendations can be made.
Evaluation of Corn Herbicides
Although corn acreage is limited in southeastern Kansas, it is an important cash and feed crop for farmers who have the land and water necessary for growing corn. Keeping the crop clean of troublesome weeds is highly important in achieving optimum yields.
Procedure: In 1980 we evaluated currently labelled corn herbicides on two soil types in Labette county.
Sutan tank-mix combinations, incorporated with a disc, gave the best overall control.
Herbicides incorporated with field cultivator were evidently too deep and gave somewhat less effective control than similar treatments applied after planting and before plants emerged.
Results: Weed control ratings for the different treatments are presented in Table 4, but yield data were not obtained because of drought conditions. Conclusions: In many cases, proper application method and determining the optimum herbicide rate for a given soil type are essential for optimum weed control and are as important as deciding which herbicide combination to apply. When specific weed problems are encountered, be sure the weeds to be killed are listed on the label of the herbicide selected. Shallow IncorE = incorporated with a field cultivator before planting.
Evaluation of Herbicides in Established Alfalfa Stands
After alfalfa has been established a few years, winter annual weeds and crabgrass often invade. Controlling such problem weeds with herbicides would increase alfalfa's productivity in southeastern Kansas.
Procedure: Alfalfa herbicide treatments were applied to an established field in Neosho county in early December and early March to control winter annual weeds and crabgrass.
Results: Excellent control of several winter annual species was achieved with the herbicides tested (Table S).
Conclusion: Applying herbicides when alfalfa is dormant in late fall or early spring effectively controlled winter annual weeds commonly found in established alfalfa fields. South easter n Kansas is the leadin g soybea n-prod ucing area in Kansas so extens ive variet y testin g is of great benef it to produ cers.
Proced ure: In 1980, 35 soybea n varie ties of connne rcial and unive rsity origin were plante d June 23 at the Columb us field in Cherok ee county .
Resul ts: Extrem ely dry, hot condi tions during the summer and the critic al pod-f illing period severe ly depres sed soybea n yields of all matur ity groups .
Three -year averag es of the more commo nly grown variet ies are shown below. Compl ete soybea n variet y result s are compi led in Agric Expt. Statio n Report of Progre ss 393. Concl usions : For the past 3 years, late summer weath er has not been norma l in southe astern Kansa s, and yield differ ences among matur ity groups have not been large. Under more norma l condi tions, Group V maturi ng variet ies (Essex and Forres t) should perfor m better than earlie r-matu ring varie ties.
Effec t of Planti ng Date on Yields of Early, Medium , and Late Matur ity Soybea ns Soybea n variet ies of variou s matur ities are plante d in southe astern Kansas from mid-Ma y until mid-Ju ly. It is impor tant to select a variet y and planti ng date that will give maximu m yields and still fit the desire d croppi ng sequen ce.
Proced ure: In 1980 five soybea n variet ies (Willi ams, DeSoto , Crawfo rd, Essex, and Forre st) were plante d on four dates (May 29, June 11 and 23, and July 2) at the Columb us field.
Results: All varieties, regardless or maturity, yielded better when planted in late June or early July. Previous year's research gave similar results. Planting after mid-June allows many of the varieties to reach the critical podfilling stage during late August and early September when temperature and moistt:re conditions normally are more favorable. However, longer season varieties like Forrest, also have yielded well when planted in late May and early June. Planting Forrest maturity varieties later than June in extreme southeastern Kansas delays harvest until early November.
Conclusion: Planting more than one variety of late group IV and group V maturity at several different dates improves chances of avoiding the hot, dry period as soybeans reach the critical pod·-filling stage. In recent years planting soybeans in narrow rows has -increased popularity in southeastern Kansas; better weed control with herbicides and improved planting equipment were largely responsible. Narrower rows also have been advocated as a way to boost soybean yields 5 to 15%. F.owever, the yield benefit from narrower row spacings has not been fully researched with the longer-season varieties grown in southeastern Kansas.
Results: Varieties of later maturity, like Essex and Forrest, did not vary significantly in yield regardless of row spacing. The past four years' research have shown the same results. Varieties of earlier maturity, such as Crawford and Williams, yieldecl somewhat more in 7-inch than in 30-inch spacings.
Conclusions: Results to date show that longer-season varieties do not yield any higher when planted in narrower rows. However, under more favorable fall conditions, narrow row might give better response.
Effects of Cropping Sequence on Soybean Yields
Soybeans are the major cash crop for ~~ny farmers in southeastern Kansas. Typically they are grown in several cropping sequences with wheat and grain sorghum, or in doublecropping systems. More information is needed to determine how different cropping sequences influence yields and net profits. Conclusion: More studies on wheat and soybean cropping sequences are planned for 1981 to evaluate double-·cropping effects of wheat and soybeans compared with fui1 season crops, or a combination of the two with three crops in two years [wheat-doublecrop soybeans] -soybeans.
Residual Effects of Phosphorus on Soybean Yields
Many of the soils in southeastern Kansas are low in available phosphorus. When phosphorus fertilizer is applied, part of it becomes unavailable over time so it cannot be taken up by the plant-root system. The amount of such phosphorus fixation is not fully known for the clay-pan, acid soils of southeastern Kansas.
Procedure: In 1978, we initiated a study to see if heavy, first-·year applications (200 pounds P 2 o 5 per acre) would be as effective for soybeans as 100 pounds P 2 o 5 per acre applied every other year, or 50 pounds per acre every year. After 4 years, all plots will have received the same amount of P 2 o 5 • The two P sources used were diammonium orthophosphate (AOP, 18-46-0) and ammonium polyphosphate (APP, 15-62-0).
Results: Largely due to the dry topsoil during 1980, P applications did not significantly increase soybean yields. In both previous years annual P treatments of 50 lbs P 2 o 5 per acre had increased yields 2 to 5 bushels per acre on this low testing soil (10 lbs of available P).
Conclusions: Where soils are testing less than 15 pounds of available P per acre, soybean yields have increased 3 to 5 bushels per acre from phosphorus fertilizer.
Effects of Direct Phosphorus and Potassium Application on Soybean Yields
Soybeans have not responded consistently to phosphorus and potassium fertilizer applications in southeastern Kansas, especially in soils testing low to medium in available nutrients. Additional research is needed to determine under what soil conditions a fertilizer response is likely.
Procedure: For the past three years, two rates of P and K (40 and 80 lbs per acre) applied separately or in combination and two methods of application (broadcast and knifed) have been studied on several soil sites in Cherokee county.
Results: Soybean response to the different fertilizer treatments have been very small; however, abnormally dry soil conditions existed each of the past three years. With more favorable soil moisture, the root system of the soybean plant might make more efficient use of the applied fertilizer.
Conclusion: Results remain inconclusive regarding the profitability of fertilizing soybeans on soils testing in the medium fertility range.
Effects of Primary Tillage Methods on Weed Control in Soybeans
Tillage methods have changed dramatically in recent years with chisel plows, plowing discs, off-set discs, and other types of reduced tillage implements. Compared with the moldboard plow, most of the newer implements leave more crop residue, but how they affect weed control in soybeans has not been evaluated fully in southeastern Kansas.
Procedure: In 1980 soybeans were planted where grain sorghum had been grown previously. Half of the plot area was plowed, and the other half chiseled. Two incorporated herbicide treatments and three surface-applied treatments were compared on both the plow and chisel plots.
Results: There were no significant yield differences between the plow and chisel treatments, nor were there any differences in weed pressure. At one location, where lack of rainfall prevented the herbicide applied before plants emerged from being activated, the incorporated treatments gave much better weed control. However, at another location where rainfall was not a limiting factor after planting, there were no significant differences for herbicides incorporated before planting and those applied after planting before plants emerged.
Conclusion: More research data are needed on herbicide and tillage interactions before firm conclusions are reached.
20 "' f.-0 Soybean Herbicide Performan ce When producing soybeans, it is vitally important to control weeds without injuring soybean plants. Extensive research is devoted to the performan ce testing of soybean herbicide s and applicati on methods.
Procedure : In 1980 several different herbicide studies were conducted on soybeans. Details of the applicati on methods are discussed in the results section of each study.
Results: Abnormall y dry condition s during the summer reduced the effective ness of herbicide s in controlli ng weeds. A brief summary of individua l studies follows: 1) Incorpora ted and preemerge nt herbicide applicati ons for velvetlea f control.
Sencor and/or Lexone gave effective velvetlea f control when applied with the disc or field cultivato r before planting. Applicati ons made after planting before plants emerged resulted in poor control due to dry soil and inactivat ion of the herbicide . Likewise, applicati ons of Lorox, Goal, and Modown made after planting before plants emerged were ineffecti ve.
2) Incorpora ted and preemerge nt herbicide applicati ons for pigweed control.
Incorpora ted treatment s of dinitroal ine herbicide s (Treflan, Tolban, Basalin, and Prowl) in combinati on with Sencor and/or Lexone gave good pigweed control.
Air temperatu res exceeding 100 F, low relative humidity, and dry topsoil were the major factors that contribut ed to the very poor results obtained with all post-emer ge herbicide s that were applied after the soybeans and weeds had emerged. Adding crop oil did not improve broadleaf weed control. Weeds like cocklebur and velvetlea f have to be actively growing to translocate the herbicide into the plant. 4) Incorpora ted and post-emer gent treatment s for rhizome johnsongr ass control.
Incorpora ting Treflan, Tolban, or Basalin at the rate of 2 pounds active ingredien t per acre gave 80% control of johnsongr ass rhizomes and seedlings the first year applied. Several post-emer ge herbicide s applied after the soybeans had emerged and when johnsongr ass was 12 to 18 inches tall were ineffecti ve due to summer drought. 5) Herbicide evaluation with soybean~lanted no-till :In wheat stubble.
Lack of grassy and broadleaf weed pressure prevented any meaningful results. A good stand of soybeans was obtained in the wheat stubble, but nearly all soybean plants died during the i:ot. dry weather.
Conclusions: In 1980 where soil conditions were e>~tret!.1ely dry, incorporated herbicides were more effective than those applied before soybeans emerged, but in more nearly normal years, the difference was less pronounced.
Where broadleaf weeds like velvetleaf, cocklebur, and moringglory are a problem, sprays should not be applied after soybeans emerge when weeds are severely drought stressed. Weeds must be actively growing for herbicides to be effective. In addition, proper timing of post·-emergent herbicides is essential to control weeds.
Post-emergent treatments to control shizome johnsongrass need to be researched more in southeastern Kansas. Likewise, more inforn1ation is needed with soybeans planted no-till in wheat stubble before herbicide recommendations can be made. Cattle producers commonly face the question of how much supplemental protein to feed to grazing steers dry wintered on hay and pasture. With high interest rates and fluctuating cattle markets, overfeeding protein is economically prohibitive. Yet to economically maintain and i~prove winter gains~adequate supplemental protein must be provided. This study was designed to determine the level of supplemental protein needed by 500-lb steer calves wintered on mixed grass hay (fescue e.nd bromegrass) and fescue pasture.
Two pastures were assigned to each level of supplemental protein: .17, .34, .51 or .68 lb per head daily. All cattle were implanted initially with 36 mg Ralgro and were wintered on fescue pasture anrl big round bales of mixed grass hay (fescue and bromegrass) fed ad libitum in round slant-·bar feeders. The hay contained 9.1% crude protein-On a dry matter basis. Cattle on all protein levels received the same quantity supplementary energy (1.0 lb TDN/head /day), which was provided by adjusting the amount of soybean meal (51.4% crude protein on a dry matter basis) and ground milo (10.6% crude protein on a dry matter basis). Cattle were fed daily in 8-foot, round·-bottom metal bunks. Initial and final weights were taken after a 16-hour shrink from feed and water. This study was terminated March 31~ 1980.
Results: Feeding .51 lb of supplemental protein per head daily produced significantly higher average daily gains (P<::'.05) than .17 or .34 lb of supplemental protein and .68 lb per head daily resulted in no further improvement (P7.20) over the .51 lb level (Table 9 ).
Conclusion: Approximately one-half lb of supplemental protein is needed by 500-lb steer calves wintering on fescue pasture and grass hay (fescue and bromegrass). The .51-lb-per-day rate produced highest animal perfornmnce and lowest cost of gain in this study.
Effect of Alfalfa Creep on the Performance of Fall-dropped Suckling Calves
The milking ability of a beef cow and the quantity and quality of pasture or other feed available to her and her calf largely determine the ability of her calf to reach its genetic potential at weaning time. After a beef calf is 90 days old, its dam's milk usually supplies only about half the nutrients it needs for maxium growth. So abundant high quality feed usually must be provided in a creep for the calf to attain maximum growth. This is particularly important for fall-dropped calves because less high quality forage is available during winter. Grain mixtures have been used in creep rations, but high grain prices now often make that practice unprofitable, so more economical creep rations need to be developed. This study examined the performance of fall·-dropped calves creep fed alfalfa hay.
Procedure: Sixteen fall dropped Hereford and Hereford x Angus calves (10 heifers and 6 steers) were equally divided November 19, 1979, into 2 groups by weight, sex and breed. One group received high quality, long form alfalfa hay ad libitum in a creep; the other group received no creep feed. The alfalfa hay contained 17.0% crude protein on a dry matter basis. Each group of calves and their respective dams were wintered on 15-acre fescue pastures and were fed big round bales of mixed grass hay ad libitum in round slant-bar feeders. All calves were implanted with 36 mg Ralgro at the beginning of the study. All calves were weaned May 2, 1980, when they were approximately 7 months old.
Results: Daily gain by calves that received alfalfa hay was 19.4% higher (P<.05) (50 lb. more in 165 days) than gain by calves that received no creep. For each additional lb of gain the creep-fed calves required 5.35 lb of alfalfa hay. Cow weights and intakes of grass hay between the two treatments were similar.
Conclusions: Creep feeding alfalfa hay effectively and economically increased weaning weights of fall-dropped calves without adding extra condition.
Effect of Level of Milo and Monensin Supplementation on Rate of Gain of Steers Grazing Brome Pasture
With high interest rates and fluctuating cattle markets, cattle producers must decide whether or not to supplement grazing steers with grain. Cool season grasses like fescue and smooth brome produce well during spring and again in fall, but not during the summer grazing period. A 1979 study established that energy supplementation at 2 to 4 lb of rolled milo per head daily optimized efficiency of grain utilization by 525-lb Hereford steers grazing brome pasture from May 7 to September 25. Another study described below was conducted during the summer of 1980 to determine the effect of level of milo and monensin supplementation on rate of gain of steers grazing brome pasture.
Two levels of milo ( 2 or 3 lb per head daily) and 2 levels of monensin( O or 200 mg per head daily) were evaluated in the following combination: 1) 2 lb milo and 0 mg monensin; 2) 2 lb milo and 200 mg monensin; 3) 3 lb milo and 0 mg monensin; and 4) 3 lb milo and 200 mg monensin. All cattle were implanted initially with 36 mg Ralgro. Brome hay was provided ad libitum to all cattle beginning July 3 when pastures became short. Cattle were fed grain daily in 8-foot, round-bottom metal bunks. Initial and final weights were taken after the 16 hour shrink from feed and water. This study was terminiated August 19, 1980.
Results: The results are presented in Table 11. The data were analyzed by milo level and monensin level and these results are listed in Tables 12 and 13 , respectively. There was no significant difference (P >.20) in average daily gain between feeding 2 or 3 lbs of rolled milo per head daily. However~ feeding 200 ppm monensin per head daily resulted in a 20.2% increase in average daily gain (r """.01).
Conclusions: Feeding 200 mg monensin along with 2 lbs of rolled milo per head daily was the most profitable combination evaluated in this study for supplementing yearling steers summer grazed on cool season grasses.
OTHER RESE.ARCH
Two projects were completed using products not yet approved for use with beef cattle by the Food and Drug Administration. Brief summaries of these studies are listed below.
Implants for Grazing Cattle. A study was conducted to compare rate of gain in grazing steers implanted once with F.algro or an experinental long lasting removable implant with an effective life of 200 days. Both implants significantly (Pc4.05) increased average daily gain over the nor.implanted control group, but there was no significant difference (P>.20) in daily gain between the two implants in this 200-day study.
An Experimental Antibiotic and Ralgro For Finishing Cattle_. An antibiotic feed additive that is not yet approved for use with beef cattle was evaluated in combination with P.algro in a finishing study. Ralgro significantly (P < .05) improved average daily gain while the experimental antibiotic decreased feed intake and significantly (P ~ • 01) improved feed conversion with no effect on gains. The two products in combination further improved feed efficiency beyond that obtained with the experimental antibiotic alone.
FORAGE CROPS RESEARCH J. L. Moyer
Forage Agronomist
Performanc e of Alfalfa Varieties in Southeaste rn Kansas
Interest in alfalfa production has been renewed with declining alfalfa weevil. This test is to help answer the question, "What variety should I plant?" when one establishe s alfalfa.
Procedure: Twenty-fou r alfalfa varieties were seeded at 12 lb/a in spring, 1978. Benefin (Balan) at 1 1/2 lb a.i./a was applied before alfalfa emerged and 400 lb/ a of 6-24-·24 preplant fertilizer were used. Seven varieties originated at Federal and State experiment stations; the other 17 were from six seed companies.
In 1979, 200 lb/a of 6-24-24 was applied after the first cutting, and grasshoppers were sprayed with Furacan between the second and third cuttings. Another 200 lb/a of 6-24-24 were applied after the last cutting. The 1980 crop was sprayed with dimethoate in April to control aphids. Young grasshoppe rs were sprayed between cuttings with Sevin.
Results: Drought limited alfalfa production to two cuttings in 1980 (Table 14). The first cutting produced an average of 1.7 tons/a: the second, only 0.73 tons. Two varieties yielded significan tly above the average of cut 1, one in the second cutting, and two were above average in total yield. Yields in 1979 averaged 4.76 tons/a from four cuttings, ~ith no significan t difference s among varieties. Threeyear production totals ranged from 7.22 to 8.21 tons/a, and averaged 7.82 tons/a. Conclusion : Data from three full crop years should be obtained before conclusion s are drawn. So far three varieties have produced more than 8 tons/a, and seem ~~re promising than the four which have yielded less than 7.5 tons/a (Table 14). Fertility of old fescue is generally low beneath the top few inches of soil. The situation has developed as fescue removed nutrients from the root zone and fertilizer was top-dressed on the surface. Studies were begun in 1979 to see if placing fertilizer in the root zone increased yield, quality, or fertilizer uptake compared with surface-broadcasting.
Procedure: In 1979, liquid fertilizer was applied on the Terry Weidert farm in Labette county by broadcasting through flat-spray nozzles, "dribble'' on the surface from the boom, or 11 knifed 11 6 to 8 inches deep through an injection tube behind narrow shanks 15 inches apart. Rates were 50, 100, or 150 lb N/a as urea-ammonium nitrate (UAN), and 0 or 40 lb P 2 o 5 -K 2 o/a. The soil tested 12 lb/a available P and 85 lb/a exchangeable K, both in the 11 low 11 range.
The same location was used in 1980, but different rates and carriers of nitrogen fertilizer were used to compare broadcast and knifed applications.
The same rates of N and P-K were used with UAN, but anhydrous ammonia knifed into the soil replaced "dribble" treatments.
Another Weidert farm used in 1980 in Neosho county got liquid UAN, 10-34-0, and 0-0-10 at 12, 100, or 150 lb N/a, 0 or 40 lb P 2 o 5 /a, and 0 or 40 lb K 2 0/a by knifed or broadcast methods. This site was also low in available P and K.
Results: Detailed reports of these experiments are in the Kansas Fertilizer Research Reports of Progress 372 (1979( ) and 389 (1980( ). Both 1979( and 1980 experiments at the Labette county location showed significant yield and crude protein (%N x 6.25) responses to N, and to P-K fertilization. Yield and protein responses to N were even greater at the Neosho county location. Since P and K applications were separate there, we found forage yield and P content responses to added P, and to added Kin the presence of P.
Application method affected forage yield and composition (see Table 15 ).
Knifing UAN produced significantly more forage than broadcasting the same material in both 1980 experiments, and crude protein content was significantly increased by knifing in all experiments. Knifed ammonia was intermediate in both yield and crude protein between knifed and broadcast UAN in Labette county in 1980.
Conclusion: Knifing N and P fertilizer increased tall fescue hay yield by about 400 lb/a, and enhanced hay quality over surface broadcast applications. In addition, fertilizer efficiency was increased by knifing. More work is needed to predict conditions necessary for an economic crop response to knifed fertilizer ap 0 plications. :£/ Means of 12, 100, and 150 lb N/a, 0 and 40 lb P 2 o 5 /a, and 0 and 40 lb K 2 0/a rates.
Effects of Ag Lime Application on Yield, Quality, and Fertilizer Response of Established Tall Fescue and Interseeded Red Clover
Forage legumes in tall fescue can increase forage quality and reduce production costs. But many tall fescue meadows and pastures require lime to establish and produce legumes. A 6-year study funded by the Kansas Limestone Association, was started in November, 1979, to evaluate effects of lime on yield, quality, and fertilizer response by tall fescue interseeded with red clover. The 1980 drought confounded results, but mechanical renovation increased protein content of fescue and decreased yields except where lime was applied. Native meadows produce a lot of the forage in southeastern Kansas. But native hay yields are relatively low, ·and fixed costs on the land increase. Fertilizing can increase returns but it helps weeds enter a meadow.
These experiments were to learn if judicious use of fertilizer, along with burning or other weed control methods, could increase forage production and quality in a native meadow without increasing weeds. At Big Hill, on land managed by the Kansas Forestry, Fish and Game Commission, four years of treatment are being followed by two years of observing residual effects.
Procedure: Treatments, begun in 1976, consisted of burned and unburned blocks with 8 fertility levelsa control, and 30 lb N/a with 0, 10, or 30 lb/a of phosphate and/or potash applied annually through 1979. No treatments were imposed in 1980 so residual effects could be measured on forage yield, crude protein, and botanical composition of the hay, and pH, P, and K levels in the soil.
A new weed control experiment was begun at Parsons in 1980. Three fertilization treatments, control, 24-24-24 and 48-48-48, were applied to burned or unburned plots. Forage yields were taken.
Results: Yield increases were obtained at Big Hill in 1980 from some previously burned, fertilized plots. Control (no fertilizer) treatments, both burned and unburned averaged l.2l; ton/a @12% moisture, while burned plots previously receiving 30-30-30 produced 1.70 ton/a. Nitrogen-only treatments produced no carryover yield response on unburned plots, and no significant response on burned plots.
Crude protein of forage in 1980 was higher in unburned than in previously burned plots, opposite from yields. The same trend appeared in the residual fertilizer responses, that is, the lower-·yielcling treatments were higher in protein than the high-yielding plots.
Available soil P was higher in plots that had received 30 lb/a of phosphate than in any other plots. Burning made little difference in available P content. Available soil K was significantly higher on burned plots that had received 30-0-10 than in any other plots. Burned plots generally had higher available soil K than did unburned plots.
Burning increased warm-season grasses before 1980 but fertilizing caused few, if any, changes.
At Parsons burning reduced yields 30% ~ while fertilizing with 24-24-·24 increased yields 14%, and 48-48-48 increased yields 43% over the control's The summer of 1980 was characterized by one of the oost severe droughts in southeastern Kansas records. Such stress causes nitrates to accumulate in ~any summer crops, and sorghums often increase the prussic acid producing compounds in their tissues. Either compound can become toxic to livestock: and death losses were reported in southeastern Kansas from animals eating drought-stricken summer forage. Toxic response to nitrate or prussic 2cid is a complex interaction of diet and animal condition. The KSU Cooperative Extension Service considers feeds generally safe if nitrate concentrations are below 5,000 ppm and prussic acid, below 100 ppm. More than 9,000 ppm of nitrate or 200 ppm of prussic acid are considered toxic.
We sampled and tested many of our forages at Parsons and Mound Valley in 1980 to study the safety of various forages, sampling variations among fields, and seasonal effects on postharvest changes in concentrations•of nitrate and prussic acid. Usually we could not replicate samplings to accurately estimate uncontrolled variation, so our data should be interpreted cautiously.
Procedure: Samples were assayed by a commercial laboratory. Results were reported as ppm nitrate and ppm prussic acid (HCN) on a dry matter basis. Samples sent "fresh" or green were placed on ice when collected, and either frozen until shipped or shipped directly by bus in sealed plastic bags to arrive in about 16 hrs. Dried samples were either cured hay or sampled standing then dried in an oven at about 160 F for at least 24 hours.
Whole plants were cut, chopped, and sampled, or hay was randotr.1.y sampled to represent proportions of all above--ground plant parts. A 36-inch probe one inch in diameter was used to remove several cores from big round bales.
Sampling Factors
Fresh samples shipped for assay seemed to contain less nitrate and prussic acid than samples dried then shipped (see Table 17 ). Such changes may be greater than appear here because field--curing also allows some reductions, particularly in prussic acid. Table 17.
Nitrate, prussic acid (on dry basis), and moisture content of sorghum-sudangrass cut August 22 and either fresh-frozen or allowed to field-cure three days. Variation in toxic compounds among ple.nts of the same variety in the sair.e field was observed. Two five-a~re fields ~f forage sorghum of different maturities, DeKalb hybrids FS4A and FS25A , planted two weeks apart had similar 3-plant averages of more than 10,000 ppm nitrate July 25, but nitrate varied from 8,000-12,000 ppm among individual plants within each field. Plants of a hybrid grain sorghum averaged 1600 ppm nitrate, but ranged from 800 to 2700 ppm.
Much of the plant-to--plant variation could be attributed to differences in plant condition. A prematurely dead corn plant sampled July 25 had 8800 ppm nitrate, while a green plant in the same field had 300 ppm nitrate.
Seasonal Effects
Seasonal differences in a.mounts of nitrate and prussic acid of field-grown sorghums were observed. The day after the July 25 sampling a 1.07-·inch rain was received at Mound Valley, so forage sorghums wer~ re-sampled July 28. Nitrate content apparently declined slightly from 10,000 ppm July 25 to less than 8,000 ppm. But prussic acid content, high July 25 (more than 300 ppm), apparently increased to more than 500 ppm.
Sorghums generally declined in both nitrate and prussic acid contents by fall (Table 18 ). Silage sorghum at Mound Valley showed the same trend between July 28 and October 14, declining by about 2/3 in nitrate and 1/2 in prussic acid content. Hay samples cut in summer changed little in nitrate or prussic acid contents by fall (Table 19 ). Prussic acid content declined ( 50%) after hay was made into big round bales, while nitrate did not decline by October. Effect of Sorghum Type Sorghum types differed in concentrations of toxic compounds at a given time (Table 20 ), partly because the types were at different stages of maturity, especially for nitrate content. Generally, sudan seemed the safest type both July 28 and in October (Table 18 ). Of the types tested, silage-type sorghums appeared most dangerous in midsummer. Conclusions: Several general observations can be made from these data, most of which are verifia.ble from other reports. In sampling nitrate, and especially prussic acid, we found: 1) Toxin changes in samples, especially green samples, during storage and shipment were often substantial. This means one should stabilize samples other than hay before shipment, probably by at least partial drying, and then minimize storage time. One must then know moisture contents to estimate as-fed levels.
2) Plant-to-plant variation was observed, due mostly to condition of individual plants. If one samples a standing crop, plan~ in various conditions should be sampled in their approximate proportion in the field.
Levels of the two toxins were affected by several things: 1) Sudan was safest for sorghum types, forage sorghums most dangerous~ and sorghum-sudan and grain sorghum were intermediate.
2) Toxins were highest in mid-to--late summer and declined in surviving plants during fall.
3) Nitrate content was more stable after cutting and during hay storage than was prussic acid, but both declined somewhat during processing.
38 4) Plant recovery from drought tended to reduce nitrate concentration, but increase prussic acid.
OTHER FORAGE RESEARCH
The following projects are underway, but only preliminary results have been obtained.
Method and Frequency of P and K Fertilization on Alfalfa. Single and annual fertilizer applications by knifing or broadcasting were begun in August, 1979. The two cuttings taken in 1980 showed no differences between treatments.
Warm-season Grass Observation Nursery. Forty-five accessions of warm-season grasses, including 27 Old World bluestems, were established in spaced plantings in 1979, and evaluated for growth, regrowth, winter survival, and spring recovery. Several Old World bluestems rivaled the less palatable switchgrasses and Indiangrasses in production and outproduced the native bluestems.
Bermudagrass Variety Performance. Thirteen varieties were established in 1980, nine from sprigs and four from seed. Rate of spread was recorded, and several covered the plot by sunmer's end. One particularly aggressive line had to be hand-weeded from neighboring plots.
Cow-calf Performance on Tall Fescue and Red Clover-Interseede d Pastures.
Four of eight 5-acre fescue pastures were fertilized with 0-40-40, limed, and interseeded with 17 lb/a of coated 'Kenstar' red clover. The other four were fertilized as usual 50 lb Nin August). Pasture records of animals carried were kept for each pasture, but red clover seedlings died from drought. Reseeding will be performed spring, 1980.
Birdsfoot Trefoil Performance Test. Ten cultivars established in spring, 1980, grew too little for yield determination. All appeared in excellent condition for winter, and will be harvested in succeeding years.
Effects of N and P Rates and Application Methods on Winter Wheat
Purpose: Many soils in southeastern Kansas respond to P application. The rising cost of P fertilizer increases interest in getting the highest efficiency possible from applied P.
Procedure: This study was established to compare methods of applying P and P rates of 30, 60, and 90 lb/a for winter wheat. Methods of P application were dual-knifed (simultaneously injecting NH 3 and 10-34-0 eight inches deep on 15inch centers), broadcast, and banding wifh the drill. The study was established on an area that had only 4 lb/a available phosphorus. N rate was constant at 75 lbs N/ a.
Results: Wheat yield response to added P was phenomenal (Table 21 ), increasing from 6 bu/a with no P 2 o 5 /a added to nearly 52 bu/a with 90 lb P 2 o 5 /a. Broadcast P gave significantly lower yields than either dual-knifed or banded P.
Conclusions: This work shows that P fertilization effectively increases yields where soil P level is low; 30 pounds of P 2 o 5 per acre can produce an additional 32 bushels per acre of wheat on P-limiting soils. If P 2 o 5 costs $0.28 per pound and wheat is $4.00 per bushel, a return of $128 per acre can be achieved from an $8.40 per acre investment. These results also show that P fertilizer efficiency can be increased by either banding or dual-knifing. Both of these methods place the P in concentrated zones, which allows less P fixation. .5 Evaluations of Fer~ility-Tilla ge Management Systems Purpose:Intere st in reduced or no-tillage systems is increasing, so research is needed to determine if the systems are adaptable to southeastern Kansas. Research is also needed to see if fertility management needs to be changed under these systems.
Procedure: Work was continued in 1980 at two sites to compare conventional, reduced, and no-tillage systems. Several fertility management variables were included.
Results: Grain sorghum yields in 1980 were severely reduced by a season-long drought. Even with depressed yields, some trends were noted. The no-till system gave the lowest average yields, while reduced and conventional systems were equal. Fertility management was important. Knifing in N, P and Kon the conventional and reduced systems generally resulted in higher yields than broadcast applications. The surface-applied nutrients became positionally unavailable during the dry summer. Applied N efficiency and recovery were poor on the no-till systems when UAN was the N source.
Conclusions: To date this work shows that reduced tillage systems work well in southeastern Kansas, while no-tillage systems probably sacrifice yields. Fertility management is critical under reduced or no-till systems. This work will be continued.
Effects of Primary Tillage Methods on Soybean Yields
Purpose: Primary tillage methods used for soybeans in southeast Kansas vary widely. Many producers still use moldboard plows as their primary tillage tool.
Procedure: This study was established at two locations in 1980 to compare different primary tillage methods for soybean production. The moldboard plow, chisel, disk, soil saver, and no .. till were compared.
Results: 1980 results showed no significant yield effects due to tillage used. In fact, the no-till method gave the highest yields at the Parsons Field. This work will be continued in 1981.
Conclusions: More work is needed to see if primary tillage method affects soybean yields in southeastern Kansas. 42 Effects of N, P, and K Rates and Application Methods on Grain Sorghum Yields Purpose: Because grain sorghum is an important crop in southeastern Kansas this research compares fertilizer rates and methods of application.
Procedure: This study was continued in 1980 on the Parsons Field, which has 22 pounds of available P per acre and 130 pounds of available K per acre. N rates used were O, 50, 100, and 150 lbs N/A as NH 3 . P-K rates were 0 and 50 pounds of P 2 o 5 and K 2 o per acre. The P and K were applied , broadcast and knifed, as potassium tripolyphosph ate (0-26-25) liquid. When knifed. P and K were applied simultaneous ly with the NH. 3 8 inches deep on 15-inch centers.
Results; 1980 yields were severely reduced by drought, and probably limited fertilizer response. The only significant yield increase in 1980 was from applied N fertilizer.
Conclusion: Summarizing the 1979 and 1980 data, it is clear we can expect a good yield response to applied N and that 100 lb N/A is probably near the economic optimum N rate. Also, we can expect a response to P-K even on soils with as much as 22 lb of available P and 130 lb of available K shown by soil tests. Knifing P-K gave the highest yields both years, but not significantly higher than broadcast applications .
Effects of N, P, and K Rates and Application Methods on Soybean Yields
Purpose: Soybean response to fertilization in southeast Kansas has been inconsistent. Response is dictated by soil type, levels of available nutrients in the soil and by weather, so research is needed to detert'.line when and where response will be likely.
Procedure: Work was begun in 1979 and continued in 1980 to evaluate soybean response to fertilization and placement of fertilizer. P-K were knifed preplant in at rates of 30, 60, and 90 lbs/a of P 2 o 5 and K 2 o. Potassium tripolyphosph ate (0-26-25) liquid fertilizer was used to facilitate knifing in the P and K.
Results: 1980 yields were low due to the hot, dry summer, so neither fertilization or its placement significantly affected yields.
Conclusions:
Results to date have not been consistent. Poor environmenta l conditions at critical growth stages have limited yields and fertilizer response.
Effects of P Rates and Application Methods on Irrigated Corn Yields
Purpose: Southeastern Kansas now has an estimated 25,000 acres of irrigated field crops, mostly corn. Due to the soil types of this area~ research is needed to find optimum fertility rates for irrigated corn.
Procedure: This irrigated corn work was begun in 1980. P 2 o 5 rates used were 40, 80, 120, and 160 lbs P 2 o 5 /a either broadcast or knifed. Tfie knifed P was injected 8 inches deep on 15-inch centers. P was supplied as 10-34-0; N, as NH 3 to a constant rate of 240 lbs N/a. The corn was sprinkler irrigated from a pit. Scheduling was by water depletion using 12-and 24-inch tensiometers. The site was low in available P.
Results: Yields were low in 1980 because of extremely high temperatures during pollination so kernel set was poor. Even with the low yields, the applied P significantly increased yields. Knifed P produced significantly higher yields than broadcast P (Table 22 ).
Conclusions: More work is needed to determine optimum P fertilizer rates for irrigated corn in southeast Kansas. This study was done in conjunction with the P study on irrigated corn just discussed.
Procedure: K rates used on this irrigated corn were O~ 50, 100, 150, 200, and 250 lbs K 2 0/a. The site was fairly low (llO lbs K/A) in available K.
Results: 1980 yields were limited by extreme temperatures during pollination, however applied K significantly increased yields. (Table 23).
Conclusions: More work is needed before definite recommendation s can be made concerning fertility rates for irrigated corn.
Effects of P Rates and Application Methods on Irrigated Soybean Yields
Purpose: The acreage of irrigated soybeans in southeast Kansas is increasing. Research is needed to determine fertility recommendation s for irrigated soybeans.
Procedure! P rates of 40, 80, 120, and 160 lbs P 0 /a and broadcast and knifed methods of applying the P were evaluated. The ~ite was low in available P.
The beans were irrigated by sprinkler irrigation from a pit, but the shortage of water limited irrigation to about 2 inches in one application. .Ammonium Polyphosphate (10-34-0), the P source, was applied preplant. The broadcast applications were incorporated, the knifed P was injected 8 inches deep on 15-inch centers.
Results: With just 2 inches of supplemental water and an extremely hot sunnner, the yields were respectable. Response to applied P was excellent and the knifed P gave 3 more bushels per acre than broadcast P (Table 24).
Conclusions: More work is needed before definite fertility recommendations can be made concerning fertility requirements for irrigated soybeans in southeast Kansas. 45
Effects of K Rates on Irrigated Soybean Yields
This study was done in conjunction with the irrigated-soybe an P study previously discussed.
Procedure: K rates of 0, 50, 100, 150, 200, and 250 lbs K 0/a were incorporated before planting. The site was low in available K. Supplemental irrigation was limited to 1 inch for lack of irrigation water.
Results: Yields reflected the shortage of water, which probably limited yield response to K. No significant yield effects occurred.
Conclusions: More work is needed before definite fertility recommendation s can.be made concerning K fertilization of irrigated soybeans.
Irrigatio n Schedulin g for Soybeans
Purpose: Soybean irrigatio n in southeast ern Kansas usually calls for less than 4 inches of applied water. Since producers have only li~dted water to put on~ they need to know the best time to apply it.
Procedure : This study was establish ed to find out the best time during the growth of the soybeans to schedule irrigatio n. The variety used was Essex. All treatment s in 1980, except the control (not irrigated ) received 3 inches of water before soybeans' reproduct ive growth stage.
Results: 1980 results show that soybean irrigatio n significa ntly increased yields with no significa nt differenc e between watering at bloom or at top pod fill. Watering at both times produced the highest yields (Table 25 ). This work will be continued .
Conclusio ns: To date, this research indicates soybeans respond well to suppleme ntal irrigatio n at either first bloom or top pod fill growth stage. Purpose: Although we have considerable irrigated corn in southeastern Kansas, little information is available on variety performance. We need to know plant populations necessary to optimize irrigated corn production.
Procedure'. This study evaluated twelve popular corn varieties grown in the area. Plant populations evaluated were 18,000, 22,000, and 26,000 plants per acre. Corn was seeded at 30,000 seeds per acre with final populations obtained by hand-thinning. The corn was irrigated from a large pond with a centerpivot system.
Results: Results are shown in Table 26 . Yield differences in varieties tested were small but two varieties produced significantly lower yields (LSD .10). The 26,000 population gave lower yields than either other population.
Conclusions: These data should be reviewed as inconclusive because hot, dry weather reduced yields and conclusions should be based on more than one year's data. The work will be continued. | v3-fos |
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} | s2 | INTERNATIONAL CONTRIBUTIONS TO THE IMPROVEMENT AND MARKETING OF KANSAS WHEAT
Kansas is so well known for its major crop that it is identified around the world as the “Wheat State.” Wheat was a major factor in the settlement of Kansas in the 1800s, luring pioneers to the state and shaping its farms, towns, and agricultural traditions. Today, the crop supports thousands of producers; their communities; and large industries for supplies, transportation. processing, and marketing. The lives of many Kansans revolve around the life cycle of wheat. Producers plant the crop when conditions are favorable in autumn; anxiously hope that the plants will survive cold, drought, and wind during winter and spring; and then suspend most other farming operations and social activities to harvest the grain in early summer. Equipment and fertilizer dealers extend their hours of operation to provide supplies that producers need during critical periods. Elevator operators add help and stay open late to take in the grain during harvest, and railroads and truckers add equipment to move it to market. Media and markets follow the progress of the crop, its condition, and the yield and quality of the harvested grain. Millers then process more wheat for food in Kansas than in any other state. The crop that directly or indirectly provides the daily bread for so many Kansans is not native to the state or even to the US. Wheat originated centuries ago in a part of the world that gave mankind many of its most important foods, and the basic characteristics of the plant that we grow were shaped by early humans. Even today, many of the traits for developing improved varieties in Kansas come from numerous other countries.
Origin and Spread of Wheat
Wheat originated in the "Fertile Crescent" area of Iran, Iraq, and Syria. It was domesticated about 10,000 years ago by nomadic hunters/gatherers, who harvested natural stands of wild wheat as they moved from place to place. Because men were hunters, women most likely gathered the wheat and domesticated it. In turn, wheat probably fostered the domestication of mankind. Besides being highly nutritious, the grain needed no preservation and was transported easily, qualities that enabled humans to end their nomadic ways, establish villages and cities, and trade for their needs.
The women gatherers' concept of yield was probably much different than our measurement of bushels per acre. They likely were concerned with the amount that could be gathered in a given time or for a given effort. In selecting plants that could be harvested rapidly and easily, they gradually changed the wild wheats into types that we know today. Wild wheats, for instance, often have diffuse spikes (heads) with only a few small kernels, but to speed harvest, the women gatherers likely selected compact spikes that contained many large kernels like modern wheats. Another change, one that defines domestication, was the evolution from shattering types that were self-seeding to nonshattering wheats that could be harvested with a stone sickle.
Domesticated wheat spread from the Fertile Crescent to Egypt, the Mediterranean region, Central Asia, and Europe from about 4000 to 5000 BC. It became the major food staple in many of these areas as agriculture changed from nomadic gathering to cultivated farming. Many of the leavflat, and steamed breads that are consumed today were developed during this era.
By biblical times, the concept of yield had changed to the increased amount of seed harvested from the new crop. A fourfold increase was probably typical, so families had to give up one-fourth of their precious food reserve to plant the next year's crop. The inclination to use low seeding rates probably favored profuse tillering, which makes wheat highly adaptable to a wide range of conditions today. During the Middle Ages, as the population increased and land became limiting, the concept of yield as production per area became important.
Columbus introduced wheat into the West Indies during his second voyage. and the first crop was grown in the New World in 1494. Spaniards took bread wheat to Mexico during 1510 to 1520, from where it spread into the area that is now the southwestern US. Explorers later in that century took it to the East Coast, where settlers at several places grew the first wheat crops in the early 1600s.
Start of the Wheat State
The first crop of wheat in Kansas was produced by the Shawnee Methodist Mission in 1839. Production in the state grew steadily, reaching 10,000 acres in 1863; 100,000 acres in 1869; and 1,000,000 acres in 1876. Yields were low, usually 10 to 20 bushels per acre, and the first l,000,000-bushel crop was not harvested until 1866.
Settlers coming to Kansas brought small quantities of the wheat varieties that they had grown in the eastern US and Europe. These varieties usually came from areas with mild climates, however, and were adapted poorly to the state's environment. Many different types were introduced. Spring wheat, which matured late and often was injured by heat and rust diseases, predominated until 1875. The winter wheat that was grown was mostly soft grain: it was easier to mill with the equipment available at the time, but plants often lacked winter hardiness.
The situation changed slowly but steadily after the wellknown introduction of Turkey Red hard red winter wheat from Crimea to south central Kansas by German Mennonites from the Ukraine. Turkey Red was not a pureline variety, but a type with substantial genetic variability introduced from several areas of the Ukraine. Many other early names, particularly Crimean and Kharkof, are synonyms for this varie ty. The first crop of Turkey Red was planted in Marion County in 1873 and harvested in 1874. Production increased slowly because seed supplies were short, but the wide adaptation of Turkey Red, invention of the steel roller mill in 1878, and severe winter-killing of other varieties in the 1890s promoted its spread. Turkey Red occupied over 82 percent of the wheat acreage in Kansas and nearly 30 percent of the wheat acreage in the US in 1919, when the first variety survey was made. It remained the most popular variety in Kansas until 1939 and in the US until 1944. Turkey Red, without a doubt. established the wheat industry in Kansas and became the standard for judging all other varieties.
In contrast to the wheat seed, much of the technology for planting, growing, and harvesting the crop was homegrown. Conditions in the Great Plains were so unlike those that settlers had known in the eastern US or Europe that few familiar practices could be applied directly. The prairie sod had to be broken and the soil worked, first by oxen and in later years by horses and mules. Improper preparation of the land often formed poor seedbeds for planting, wasted soil moisture and nutrients, and encouraged weeds that competed with the new crop. Broadcasting the seed, the usual practice, caused uneven stands that easily winterkilled. Seeding rates used in eastern Kansas had to be reduced in western parts of the state, so that the plants didn't deplete the limited soil moisture. A further reduction was required when broadcast seeding with its inefficient seed coverage and plant establishment was replaced by the grain drill and its precise placement of seed in the soil. The optimum planting date also was debated for many decades. Planting too early exhausted soil moisture and increased losses from pests and winter-killing, and planting too late didn't give seedlings time to become established and hardened before winter set in.
Production of wheat was extremely laborious with the equipment that settlers had. Although the steel plow was available for breaking the prairie sod in the 1830s, harrows were not widespread until the 1870s, grain drills until the 1890s, reapers until 1880-90, and tractors until the 1910s. The self-propelled combine, which probably typifies wheat production more than any other machine, was introduced in the 1920s.
Chemical fertilizers and pesticides became important for wheat production after World War II. Until then, producers depended on the native fertility of the prairie soil to provide the nutrients needed for growth of wheat and on cultural practices and resistant varieties to control weeds, diseases, and insects. During the 1940s, nitrogen fertilizers became available from converted ammunition plants, and pesticides such as 2,4-D were developed by research.
Improvement of Wheat for Kansas
Many agencies and individuals have contributed to improvement of wheat in Kansas over the years. Kansas State Agricultural College (now Kansas State University, KSU) and its branch experiment stations were involved from the beginning. Today, over two-thirds of the wheat acreage in Kansas is occupied by modern varieties developed by KSU.
Development of new crops typically follows the order of introduction of varieties from other areas, reselection of adapted varieties, and hybridization (genetic crossing) of adapted varieties to germplasm with desirable traits. Improvement of wheat for Kansas has followed these steps. Kansas State University began research on wheat, mainly evaluation of introductions. in 1874 and started a selection program in 1906. The first improved variety, Kanred, a s e l e c t i o n f r o m C r i m e a n , w a s r e l e a s e d i n 1 9 1 7 . Reselection. which has yielded many other popular varieties. including Eagle and Karl 92, is possible because of the large genetic variability for many traits in adapted wheats. Hybridization to introduce new germplasm into wheat started at KSU about 1917, and the first hybridized variety, Tenmarq, was released in 1932.
Two Kansans were well-known plant explorers who introduced much of the germplasm for improvement of wheat. Mark Carleton, a USDA wheat scientist, during overseas explorations of the Ukraine and other areas in 1898-99 and 1900 collected hard red winter wheats that were used extensively in US breeding programs and hard amber spring wheats that helped start the US durum industry. S. C. Salmon, a former USDA wheat geneticist at KSU, introduced Norin 10 from Japan after World War II. This semidwarf wheat, which was the basis for the Green Revolution in many developing countries, is now in the pedigrees of varieties that occupy about 90% of the Kansas wheat acreage.
Some Variety Milestones
Certain varieties are recognized as significant advances in improvement of wheat for Kansas over the years. Most wheat producers and scientists would agree that these varieties include Turkey Red, Blackhull, Tenmarq, Triumph, Comanche, Pawnee, Wichita, Bison, Scout/Scout 66, Eagle, Newton, Karl/Karl 92, TAM 107, Jagger, and 2137. They significantly advanced yield potential; grain quality; or resistance to diseases, insects, and environment tal stresses in Kansas and are the only ones that were grown on over 20 percent of the state's wheat acreage.
The pedigrees of these significant varieties illustrate the importance of germplasm from other countries. Blackhull was selected in 1912 from a field of Turkey Red (the introduction from Russia) and released in 1917 by Earl G. Clark. a 14 year-old farm boy from Sedgwick, KS. It was grown on 34.9 percent of the Kansas wheat acreage in 1934. Tenmarq, the first hybridized variety released by KSU, was a cross between Marquis and a selection from Crimean. Marquis was a Canadian spring wheat from a cross between Hard Red Calcutta from India and Red Fife, also a Canadian spring wheat that purportedly was selected from a shipment from Poland to Scotland. Marquis greatly contributed to the excellent milling and baking qualities of Kansas wheat and made Tenmarq a popular parent for many later varieties. Tenmarq occupied 36.8 percent of the state's wheat acreage in 1944.
Triumph is the name for a group of varieties released by Joseph Danne of El Reno, OK in the 1940s and 1950s. All of their pedigrees contain Blackhull, Kanred, and Florence, a spring wheat from Australia. The Triumph varieties, which were noted for early maturity and excellent quality, accounted for 28.3 percent of the Kansas acreage in 1963.
Comanche was the first of a number of varieties released by KSU in the 1940s that contained the excellent grain properties of Tenmarq. This cross between Oro (a selection from Turkey Red) and Tenmarq was released in 1942; it had good resistance to rust and virus diseases and high yields of excellent quality grain. Pawnee, a cross between Kawvale (a soft red winter wheat from Indiana) and Tenmarq, was released by KSU in 1943. Its high yield and test weight; short, stiff straw; and resistance to several diseases made Pawnee one of the most popular varieties of its era in Kansas. It was grown on 38.7 percent of the acreage in 1951. Wichita, released by KSU in 1944. was a cross between Early Blackhull and Tenmarq. The variety matured early, which enabled it to escape many diseases, and produced grain that had high test weight and fair milling and baking properties. It was the most popular variin the state in 1955, accounting for 26.0 percent of the acreage. Bison, a cross b e t w e e n Ore-Tenmarq a n d ChiefKan (another variety developed by Earl G. Clark from Blackhull and a soft wheat) was released by KSU in 1956. It was a hardy variety that produced a good yield of grain with excellent baking properties. Bison was the leading variety in the state in 1961, occupying 27.1 percent of the acreage.
Scout-type wheats dominated production in Kansas during the late 1960s through the 1970s. These varieties included Scout and Scout 66, released by the Universit y of Nebraska in 1963 and 1967, respectively, and Eagle, released by KSU in 1971. All of them had the pedigree Nebred-Hope-Turkey x Cheyenne-Ponca. Scout 66 and Eagle were selections from Scout: Nebred and Cheyenne were selections from Turkey; Hope was a spring wheat from Marquis x Yaroslav, an emmer wheat from the USSR; and Ponca was a Kansas variety with Kawale, Tenmarq, and Marquillo (a variety from a cross between Marquis and an Italian durum wheat) in its pedigree. Production of Scout/Scout 66 reached 48.1 percent of the Kansas acreage in 1970, and that of Eagle reached 23.0 percent in 1978. The varieties combined excellent hardiness and yields with exceptional milling and baking properties.
Semidwarf wheats, which are now the most widely grown varieties in Kansas, became popular in the 1980s. Newton, a variety that contains Pitic 62, a Chris sib, Sonora, Klein Rendidor, and Scout in its pedigree, was released by KSU in 1977 and was grown on 41.1 percent of the state's wheat acreage in 1982. Pitic 62 was a semidwarf variety from Mexico; Chris was a spring wheat from North Dakota with Kanred; Marquis; and varieties from Brazil, Ethiopia, South Africa, and the Mediterranean in its pedigree; Sonora was a local variety from Mexico; and Klein Rendidor was a variety from Argentina. Newton was widely adapted, produced outstanding yields, and had excellent resistance to diseases and lodging.
Karl and Karl 92, released by KSU in 1988 and 1992, respectively, combined high yields with exceptional grain quality. Their pedigrees contain Plainsman V, Kaw, Atlas 50, Parker, and Agent. The pedigree of Plainsman V, a high-protein variety from Goertzen Seed Research of Scott City, KS, is unclear but probably consists of an unknown variety crossed with Aegilops ovata, a wild relative of wheat, and backcrossed with other varieties. Kaw and Parker are Kansas varieties; Atlas 50 is another high-protein variety derived from Frondoso, a variety from Brazil; and Agent is an Oklahoma variety that contains Agropyron elongatum, another wild relative of wheat. Karl/Karl 92 were grown on 23.6 percent of the Kansas wheat acreage in 1994. TAM 107, released by Texas A&M University in 1984, was from a cross between TAM 105 and Amigo. TAM 105 was from a cross of an unknown short wheat with Scout, and Amigo was a germplasm line from Oklahoma that contained several varieties of wheat and part of a chromosome from Argentine rye. TAM 107 was popular with growers because of its high yields and excellent resistance to stress; it occupied 20.6 percent of the state's wheat acreage in 1995.
Jagger, a cross of KS82W418 and Stephens, was released by KSU in 1994 and became the leading variety in the state in 1999, accounting for 29.2 percent of the wheat acreage. KS82W418 was a KSU experimental line from a cross between Plainsman V and KS75216 (a white wheat sib of Newton), and Stephens was an Oregon soft white winter wheat that contained the French variety Nord Desprez in its pedigree. Jagger produced high yields of excellent quality grain, was used widely for pasture, and was resistant to a large number of diseases and to aluminum toxicity.
The variety 2137 was released by KSU in 1995 from lines donated by Pioneer Hi-Bred International. It has a complex pedigree of Sturdy, TAM 101, a commercial variety, and several experimental lines from Missouri. Sturdy, a Texas variety, contains wheats from Argentina and Korea in its pedigree, and TAM 101, also a Texas variety selected from germplasm developed at KSU, contains wheats from Japan and the Mediterranean. Excellent yields, test weight, and resistance to leaf rust made 2137 popular with Kansas growers, and it was grown on 22.0 percent of the wheat acreage in 1999.
Changes in Characteristics of Kansas Wheat Varieties
Modern varieties of winter wheat in Kansas have many of the traits that were selected by early gatherers and farmers. They all also contain, on average, about 50 percent of Turkey Red hard red winter wheat in their pedigrees. However, present-day varieties have been changed considerably from these older wheats. They are more productive, shorter, and responsive to fertilizers; mature early; resist pests; and have better quality grain for bread and other products.
Recent research at KSU identified the traits that contributed most to increasing yields and quali ty of wheat in Kansas. The major changes from Turkey Red to the newest varieties were in the harvest index (the ratio of the weight of the grain to the weight of the whole plant) and the number of kernels per area of soil. Modern varieties produced only slightly more total dry matter than old varieties, but their harvest index was much higher because they were more efficient in translocating nutrients from the straw to the grain. The number of kernels per unit of soil was greater in modern varieties because more of their tillers survived to form spikes, and the spikes were longer and set more kernels. Semidwarf wheats from Japan and Korea contributed most to these characteristics of Newton, Karl/Karl 92, TAM 107, Jagger, and 2137.
Genes for the semidwarf trait in wheat originated in Japan. Norin 10, a popular source of the semidwarf trait, was a Japanese variety with Fultz-Daruma and Turkey Red in its pedigree. Fultz was a selection from Lancaster, which itself was a selection from a Mediterranean wheat. and Daruma provided the dwarfing gene.
Two other changes in the wheat plant, early maturity and resistance to lodging, also added to yields. Early maturity, which enabled plants to escape hot, dry conditions during late spring, was developed by selecting from the natural variation in wheat. Resistance to lodging, which permitted high plant densities and use of nitrogen fertilizer for increased yields, was improved gradually by selection in early varieties. The greatest advance, however, came from genes for the short, stocky stems of semidwarf varieties from Asia.
Resistance to the diseases and insects that plague wheat has come from reselection of adapted varieties, hybrtdization with introduced germplasm, and introgression of genes from related species. For example, compared with their parent varieties, Kanred (the selection from Crimean released by KSU) had greater resistance to stem and leaf rusts, and Eagle (a selection from Scout) was more resistant to stem rust and loose smut. An early variety of durum wheat from Italy provided resistance to Hessian fly that is still used in many Kansas hard red wheats. Wild relatives of wheat from the Fertile Crescent and rye from Argentina are sources of resistance to several foliar diseases and vectors of virus diseases of Kansas varieties.
The baking quality of wheat largely depends on the protein content of the grain and the ability of the protein to produce desirable loaf characteristics. Turkey Red wheat milled easily and had excellent baking properties when the grain protein was adequate. However, the protein contents of most varieties fell as the native fertility of the prairie soils declined. Increasing the grain protein content was difficult because of an inverse relationship with the high yields of new varieties, so breeders focused on improving the quality of the protein. Marquis, the spring wheat from Canada, greatly enhanced the protein quality of the varieties that had it in their pedigrees. Steady advances also were made by selection; for instance, Eagle was noted for its excellent baking quality. The most progress came in the 1980s, when the protein quality that had been improved over years of selection was combined with genetics for high grain protein content that originated from the Brazil variety, Frondoso. and the wild relative, Aegilops ovata. The results were the high protein, high quality varieties Karl and Karl 92, the only ones for which millers paid premium prices to Kansas producers.
Overseas Markets and Food Aid
International markets are as important for selling Kansas wheat as international sources are for improving the crop. Countries on all of the continents that provided germplasm for developing new varieties for Kansas also purchase much of the grain that the state's growers produce. Wheat grain and flour exports for Kansas totaled $774 million in 1998, which was 61.3 percent of the $1.262 billion farm value of the crop and provided an average of over $22,000 for each of the state's approximately 35,000 growers. Assuming an economic multiplier of 5, international sales of wheat added nearly $3.9 billion to the state's economy in 1998.
Egypt, which purchased nearly 77 million bushels, was the largest buyer of US hard red winter wheat during the 1998-99 marketing year. Mexico, Nigeria, Japan, and Israel followed with total purchases of nearly 158 million bushels. The Near East, the location of the Fertile Crescent that provided wheat to mankind, bought more US hard red winter wheat than any other region, a total of 155 million bushels.
Overseas food aid is another important use of Kansas wheat. Shipments of wheat in many cases are life savers for people who are suffering hunger because of crop failures, wars, droughts, floods, hurricanes, and other calamities. The abundant wheat crop in Kansas enables the US to provide aid to people in other countries and relieve their suffering.
Donations of food to other countries in 1999 made the US government the largest wheat exporter in the country. Wheat was the major commodity donated to most countries, accounting for nearly 62 percent of the 8.1 million tons of nonemergency food shipments. Ironically. Russia, the source of much of the germplasm for improving wheat in Kansas, was the major recipient of food under the largest US aid program, and wheat grain and flour accounted for 91 percent of the food donated.
Conclusions
Kansas owes its most important crop to international sources. • Early humans and farmers of the Middle Ages developed the plant type of wheat that is grown today. Turkey Red hard red winter wheat from Crimea established the wheat industry in Kansas. Modern varieties of wheat grown in Kansas contain germplasm from many countries.
International markets are as important for selling Kansas wheat as international sources are for improving the crop. | v3-fos |
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} | s2 | Identification of the chromosomes involved in a wheat-rye translocation using isozyme markers
SUMMARY Monosomic and disomic substitutions of Imperial rye chromosome C for 4 A of Chinese Spring wheat were analysed for two enzyme markers, alcohol dehydrogenase (ADH) and 6-phosphogluconate dehydrogenase (6-PGD). The gene(s) coding for ADH and 6-PGD were located on the short and long arms of rye chromosome C respectively. Isozyme analyses revealed that the substitute chromosome was a translocation involving alpha arm of wheat chromosome 4^1 and long arm of rye chromosome C. Pairing behaviour of the substitute chromosome with rye and wheat chromosomes in hybrids with rye addition line C and ditelosomic 4A (alpha) confirmed this.
INTRODUCTION
The homoeologous relationships of wheat and rye chromosomes is fairly well established. Homoeologies between five rye chromosomes, 1, 2, 3, 5 and 6R and the corresponding wheat chromosomes have been described earlier (Zeller & Fischbeck, 1971;Sears, 1968;Acosta, 1961;O'Mara, 1946;& Riley, 1965;respectively). In order to assess the homoeologous relationship of the group 4 chromosomes of wheat with chromosome C of Imperial rye, we attempted to substitute the Imperial rye chromosome C for 4 A of wheat and found that the substitution line obtained was highly fertile (33 % seed set) compared to nulli-4/1 (0% seed set). Since the male fertility genes were located on the alpha arm of 4 A (Sears, 1954), it was felt that the C chromosome of rye can substitute for the loss of them (Prabhakara Rao, 1975). Koller & Zeller (1976) suggested that the chromosome CR of Secale cereale L. consists of the short arm of Secale montanum Guss. chromosome 4R, its centromere region plus a segment of montanum 7 RS including the Re (Red coleoptile) locus. They have also shown that 4A/CR, 4B/CR and 4D/CR substitutions were characterized by poor fertility and reduced vigour. In order to resolve this problem we felt that a further check of the chromosome constitution of our substitution line was necessary. The karyotype analysis did not give much information about the chromosome constitution. Hence isozyme markers located on the opposite arms of chromosome C were used to infer the chromosome constitution and this was further verified by the analysis of the pairing behaviour of the substitute chromosome.
Meiotic analyses were made in Feulgen stained squashed preparations of pollen mother cells from anthers fixed in acetic alcohol (1:3).
RESULTS
Chinese Spring wheat with an added pair of Imperial rye chromosome C was crossed with monosomic 4A of Chinese Spring, and the 42 chromosome F t plants (20"W+l'4A + l'C) were selfed. No pairing was observed between the wheat (4A) and rye (C) chromosomes. The rye univalent could be easily distinguished since it was much larger than the wheat univalent. A 41 chromosome F 2 plant (20" W + l'<7(?)) with one large univalent was selected as monosomic substitution and selfed to get the disomic substitutions (20" W+1"C {?.)).
ALCOHOL DEHYDROGENASE ZYMOGRAM
The ADH zymogram of the 42 chromosome substitution plants showed three bands corresponding in mobility and relative intensities (1:4:4) with the hexaploid wheat zymogram (Fig. 16, c). The gene for the fast moving dimer (band 1) of this triplet was m (a) . 1 located on the alpha arm of chromosome 4^4 of wheat (Hart, 1970). The gene(s) coding for ADH was located on the short arm of CR of Imperial rye (Mahajan, 1975). The three band pattern obtained in the substitution plants as against the single band found in the nulli-4^4 plants (Fig. Id) suggests that the 4 A alpha arm of wheat is present, and the short arm of CR is absent. This eliminates the possibility of these plants carrying a complete rye chromosome, and the presence of 4 A alpha arm explains the high fertility observed. Fig. 2. 6-phospho gluconate dehydrogenase (6-PGD) zymogram phenotypes of (a) Chinese Spring wheat; (b) Chinese Spring wheat + Imperial Rye mixture; (c) Imperial rye; (d) Triticale (Chinese Spring-Imperial + Imperial rye mixture); (e) Triticale (CS + Imperial); (/) 4.4 (alpha)/Cii L translocation discussed in this paper; (g) CS + C: disomio addition line.
6-PHOSPHOGLUCONATE DEHYDROGENASE (6-PGD) ZYMOGRAM
The 6-PGD zymogram phenotypes of the substitution line showed three bands (Fig. 2/) corresponding to the wheat (6-PGD-3), rye (6-PGD-1) and the hybrid (6-PGD-2) dimers, suggesting that the rye gene(s) coding for this enzyme are also present. The genes coding for this enzyme have been located on the long arms of chromosomes C and F of Imperial rye (Narayana Rao & Prabhakara Rao, 1980). There is no possibility of the long arm of the chromosome F being present (since the starting material was CS + C addition) it is inferred that a part of the long arm of C chromosome of rye is present.
The above zymogram analyses indicates that the substitute chromosome carried segments of wheat chromosome 4^4 and rye chromosome C.
PAIRING BEHAVIOUR OF THE SUBSTITUTE CHROMOSOME
In a cross between the ditelosomic 4^4 (alpha) and the disomic substitution, a heteromorphic bivalent was observed (Fig. 3), confirming the presence of the 4.4 alpha arm in both the parents. In a cross of the disomic substitution with the addition line CS + C, a trivalent was observed confirming that the substitute chromosome has homology with the chromosome C of rye as well.
The above biochemical and cytological analyses reveal that the substitute chromosome available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0016672300020784 was not complete CR as reported earlier (Prabhakara Rao, 1975), but is a 4^4 (alpha)/C-R £l translocation. The cytological analyses also suggest that this chromosome has segments of 4^4 and CR large enough to form chiasmata. The substitution line originated from the F x carrying wheat (4^4) and rye (C) Fig. 3. Mataphase-/ of J*\ from a cross ditelosomic 4 A (alpha) x A A (alpha)/Cii L showing a heteromorphic bivalent (arrow).
univalents. Since wheat and rye univalents are known to misdivide at the centromere (Sears, 1952), it can be assumed that breakage and reunion has occurred at the centromere region. Alternatively, it could be the result of a crossing over between the wheat and rye chromosomes. It would be possible to infer the break points more precisely if more isozyme marker are available for these two chromosomes.
DISCUSSION
Isozyme markers are being routinely used in determining the chromosome constitution of mammalian somatic cell hybrids and addition lines (Ruddle & Nichols, 1971;Mouse News Letter, 1975). They are being used mainly to confirm the cytological data. It was suggested that the enzyme markers can be used to determine the chromosome constitution of materials not amenable to cytological analysis (Tang & Hart, 1975). In this study we have demonstrated the utility of isozyme markers in the determination of chromosome constitution of wheat rye translocations. Since this was the first attempt in plants, we have verified the results through cytological analysis. In principle, segments of alien chromosomes not detectable by pairing behaviour can be identified by the use of isozyme markers. For this to be possible a number of isozymes have to be analysed and the genes coding for these isozymes located on to the chromosomes. Presently nine out of the fourteen (homoeologous) arms of wheat and of rye are marked with at least one enzyme marker (Hart, 1979). | v3-fos |
2018-04-03T01:21:36.116Z | {
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} | s2 | Influence of Sodium Bicarbonate on Growth and Health of Young Calves1
Fifty-four Holstein and Jersey calves were assigned at 4 days of age within breed and sex to one of four treatments: control consisting of colostrum, milk replacer, and starter; buffered colostrum and replacer (.6% sodium bicarbonate) and starter (2% sodium bicarbonate); acidified colostrum (1% propionic), untreated replacer, and starter; and acidified, buffered colostrum (1% propionic, .6% sodium bicarbonate), buffered replacer (.6% sodium bicarbonate), and starter (2% sodium bicarbonate). The feeding regimen was colostrum once daily, day 4 to 14; milk replacer once daily, day 15 to 28; and calf starter ad libitum, day 4 to 84. Bull calves were fed for 42 days and heifers for 84 days. Calves fed acidified colostrum refused more feed and were less efficient from day 4 to 14 than calves fed buffered colostrum. Bulls were more sensitive to acidified colostrum than heifers. Starter intake, total dry matter intake, and average daily gains were similar for all calves during days 4 to 84. Rumen fluid from calves fed diets with sodium bicarbonate was higher in acetate and lower in propionate and lactate than that from calves fed diets without sodium bicarbonate. Sodium bicarbonate improved intake of acidified colostrum during the first 2 or 3 days of feeding but had no other effect on gain or feed intake.
INTRODUCTION
Milk and colostrum are two liquid feeds commonly fed to young calves. In current dairy Received December 4, 1981. ~Scientific Journal Series Paper No. 11934, Minnesota Agricultural Experiment Station, St. Paul 55108.
2 Department of Animal Science, Cornell University, Ithaca, NY 14853. practice, excess colostrum and nonsalable milk often are preserved for future feeding by fermentation or addition of acids. Otterby et al. (10) reported decreased acceptability of diet and depressed weight gain in calves fed colostrum acidified to pH 3.9, but responses improved when pH of colostrum was increased with sodium bicarbonate (NaHCOa). Foley et al. (7) observed that serum gammaglobulin and IgG concentrations of neonatal calves fed buffered colostrum were higher than those of calves fed fermented colostrum. In addition, Bullen et al. (3) reported that bovine colostrum with NaHCO3 had increased bacteriostatic activity. Kellaway et al. (8) and Emerick (5) reported NaHCO3 may improve performance of calves fed high concentrate diets pre-and postweaning. In contrast, Wheeler et al. (12) observed no improvement in feed intake, weight gain, rumen fluid pH, or fecal starch of calves fed pelleted diets containing 35% forage and NaHCO3 compared to calves fed rations without NaHCO3. However, feed efficiency was reduced, ratio of acetate-propionate increased, water intake increased, and more free-gas bloat occurred in animals fed NaHCO3.
The objective of this research was to study effects of NaHCO3 additions to liquid andstarter feeds on growth and health of calves.
EXPERIMENTAL PROCEDURE
During fall, 1979, and winter, 1980, colostrum was collected from several cows for six milkings immediately postpartum and frozen until approximately 115 kg were available. The frozen colostrum was thawed, pooled, mixed, and divided into four portions. One portion was used as control colostrum (diet 1). A second (diet 2) was treated with NaHCO3 (.6% wt/wt). The third (diet 3) was acidified with propionic acid (1.0% wt/wt). For diet 4 colostrum was first acidified with propionic acid (1.0% wt/wt), then buffered to approximately pH 6.0 with NaHCO3 (.6% wt/wt). After treatment, the colostrum was placed in 3.8-liter plastic con-tainers and stored at -20°C. The process of treating raw colostrum was repeated as necessary to meet feed requirements of calves on trial.
Forty-two Holstein and 12 Jersey calves were assigned randomly at birth to one of four dietary treatments (Table 1) in a randomized block design with blocks being sex and breed. All calves were fed dam's colostrum for 3 days. At day 4, calves were weighed and fed experimental colostrum once daily until day 14. Calf starter (2% NaHCO3 was included in starters of calves fed diets 2 and 4) was available ad libitum from day 4 throughout the experimental period. Prior to feeding, colostrum was thawed and mixed with warm water so that total liquid offered was 8.5% of day-4 body weight whereas actual colostrum offered was 6% of body weight. Liquid feeds were offered at 1500 h. From day 15 to 28 milk replacer was fed to maintain a solids intake equal to that offered during colostrum feeding. The milk replacer was a commercial product containing 20% or more protein from milk sources. Sodium bicarbonate was added at .6% wt/wt to reconstituted replacer for calves fed diets 2 and 4. Calves were weaned at day 28. Water was available free-choice throughout the trial. Bulls were fed for 42 days and then sold, but heifers were fed for 84 days. The calf starter contained 41.7% shelled corn, 22.9% oats, 20.9% soybean meal, 8.3% wheat bran, 4.2% dried molasses, .8% trace mineral salt, .4% ground limestone, .4% dicalcium phosphate, and .4% vitamin premix. Calves were kept in individual pens bedded with wood-shavings in an artificially lighted and ventilated calf barn. All calves were treated at birth with an oral bovine rota-coronavirus vaccine. ~ Daily observations were on health with number and type of medications administered recorded. Feces were scored (1 to 4) for scours (4 = severe diarrhea). Body weights were taken on days 4, 14, 28, 42 (all calves), and 84 (heifers only). Each portion of colostrum was analyzed for protein, fat, and total solids (1). Protein, dry matter (DM), and acid detergent fiber (1) were determined on the calf starter. Mineral contents of colostrum, milk replacer, and starter were analyzed by emmision spec-~Norden (Smith Kline), Lincoln, NE 68501.
Composition of Diets
The percent crude protein, fat, and total solids of the pooled colostrum before treatment were 5.64 -+ .69, 4.29 -+ 1.00, and 15.76 + .66 (mean +-standard deviation). Table 2 shows the mineral content of the colostrum, milk replacer, and calf starter. Sodium of the buffered colostrum, milk replacer, and starter was 1845 ppm, 2404 ppm, and 20713 ppm and within range to elicit normal calf growth and feed intake (8,10). The concentration for the starter with NaHCO3 was high and may have been a consequence of sampling or contamination. Percentages of dry matter, crude protein, and acid detergent fiber for the calf starter were 92.9 + 1.9, 15.7 + .8, and 8.19 + .51 (mean + standard deviation).
Performance of Calves
Health of calves was good throughout the course of the trial with few differences among treatments.
Illness was primarily diarrhea, and average scour scores of calves during days 4 to 28 were lower than 3, indicating few problems (Table 3). One calf fed diet 4 died of unknown causes at day 20. In contrast to the study of Wheeler et al. (12), no bloat was observed even though no forage was included in the diet.
Calves fed the acid-treated colostrum (diet 3) had higher total colostrum refusals (Table 4) from day 4 to 14 than calves fed diets 2 or 4 (P<.05). Bulls fed diet 3 refused 7.4 kg colostrum/calf compared to 2.6 kg/calf refused by bulls fed diet 4. Bulls also tended to have higher and more variable refusals and were more sensitive to acid than heifers (Table 4). We were unable to find other research reporting these differences. Almost all colostrum refusals occurred during the first 2 to 3 days of feeding. After 3 days, refusals essentially were zero unless the calf was ill. This confirms (10) indicating improved acceptability of extremely acid colostrum (ca. pH 4.0). As expected, DM intakes from liquids (Table 5) during day 4 to 14 were less (P<.05) for calves fed diet 3 than for calves fed diets 1, 2, or 4. Calves consumed all milk replacer offered unless they were ill. Thus, differences in DM intake from liquids during day 15 to 28 reflect differences in feed allocation. Table 5 shows that differences in DM intakes from liquids were compensated partially by starter DM consumption during day 4 to 14 and 15 to 28 of the feeding periods.
Starter DM intakes were similar (P = .10) among treatments. After weaning (day 29 to 84), consumption of starter with or without NaHCO3 was similar for all calves. Four-to 14-day-old calves fed diet 2 gained .29 kg/day and did not differ from gains (.11 kg/day) of calves fed diets 3 and 4 ( Table 4). Average daily gains (ADG) were similar among treatments during all feeding periods. Wheeler et al. (12) also found no differences in ADG when calves were fed rations with or without NaHCO3, but others (5, 8) have observed differences. Table 4 shows bulls fed diet 3 from day 4 to 14 did not gain because of lower feed intake, a consequence of decreased acceptability of the acidified colostrum. Heifers gained more than bulls during this period. Heifers fed diet 2 gained more (P<.05) per day aDiets consisted of colostrum and starter (4 to 14 days), milk replacer and starter (15 to 28 days), and starter only (29 to 84 days). bDiet 1 = control; 2 = .6% NaHCO~ in colostrum, 2% NaHCO 3 in starter; 3 = acidified colostrum; 4 = acidified colostrum with NaHCO 3 added (.6% wt/wt), 2% NaHCO 3 in starter. cSE for n = 13. SE for n = 14 is (SF n = 13) (13/14). d'eLeast square means in the same row with different lower case superscripts differ (P<.10). However, if one calf with persistent scours during this period was deleted, treatments did not differ at (P<.10).
From day 4 to 14, calves fed diet 2 were more than three times as efficient (Table 6) as calves fed diet 3 (.52 kg gain/kg feed vs..16 kg/kg). However, 15-to 28-day-old calves fed diet 2 were the least efficient (.39 kg gain/kg feed), whereas calves fed diet 4 were the most efficient (.59 kg gain/kg feed). There was no breed effect. Intakes and gains fluctuated from day 4 to 28 and may have caused inconsistent feed efficiencies. Differences in feed efficiencies early in life probably mean little because ADG and feed efficiencies from day 29 to 84 were similar for all treatments. Table 7 shows that rumen fluid from 84-dayold heifers fed NaHCO3 (diets 2 and 4) was higher (P<.05) in molar percent acetate and lower (P<.05) in molar percent propionate than rumen fluid from heifers fed diets 1 and 3. Contents for butyrate, isobutyrate, valerate, isovalerate, and total VFA were similar for all calves. Other researchers (2,9,12) reported similar changes in VFA profile when buffers were added to high concentrate rations. The lactic acid concentration of rumen fluid from heifers fed diets without NaHCO3 (.20/~moles/ ml) was higher (P<.07) than heifers fed diets containing NaHCO3 (.09 /zmoles/ml) and possibly related to concurrent changes in amount of propionate ( Table 7). The average pH of rumen fluid from heifers fed diets without NaHCO3 (6.43) and diets with NaHCO3 (6.50) did not differ. Two percent sodium bicarbonate in the starter did not elevate rumen pH as in (2). Differences in ration composition and maturity of calves probably accounted for the varied results between studies. Considering that NaHCO3 did not affect rumen fluid pH, feed intakes, or health from day 29 to 84, it is not surprising that calf performance was similar for all treatments ( Table 4).
fData from 43 to 84 days for heifers only. and fed throughout the entire 84 days of the experiment. | v3-fos |
2018-04-03T04:59:55.493Z | {
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} | s2 | Studies with an unclassified virus isolated from diarrheic calves
A transmissible agent (Breda agent) was isolated from a calf with diarrhea and shown to be infectious by inoculation orally into gnotobiotic and conventionally reared calves. The “Breda” agent had the morphology of a virus and possessed a hemagglutinin. Antigenic studies showed the virus to be antigenically different from bovine coronavirus, parainfluenza 3 virus, bovine rotavirus, bovine parvovirus and bovine pestivirus (BVD). Attempts to culture the virus in cell or organ cultures or in embryonated eggs, were unsuccessful. The virus was either spherical or kidney shaped, with 7–9 nm peplomers on the surface. A few particles possessed coronavirus processes of 17–20 nm, but these were arranged irregularly and were thought to be tissue debris. Three out of eight experimental calves developed severe diarrhea and the lesions in the small and large intestines were similar to those reported for coronavirus. The virus replicated in the jejunal and ileal regions of the small intestine and in the spiral colon, as judged by immunofluorescence. The virus multiplied in all experimental calves and was excreted in the feces; excretion correlating with the onset of diarrhea or a change in the appearance of the feces. There was little or no malabsorption measured by the uptake of D-xylose and the fact that infection of both the crypt and villus epithelial cells was observed, suggests that the pathogenesis may be different from rotavirus and coronavirus. Fourteen of fortyseven calves in the outbreak were infected with the virus, virus was not identified in other farm outbreaks of the disease.
INTRODUCTION
The pathogenic role of viruses in diarrhea of human beings, calves and other animals has been established. Rotaviruses have been isolated from diarrhea occurring in all animals studied, including human beings, and their pathogenesis determined experimentally in calves, pigs and mice (Mebus et al., 1971;Woode et al. 1974reviewed by McNulty, 1978 andFlewett and. Coronaviruses commonly cause diarrhea in calves (Mebus et al. 1973;Bridger et al., 1978). Although rotavirus and coronavirus are the 0378-1135/82/0000--0000/$02.75 © 1982 Elsevier Scientific Publishing Company viruses most frequently associated with diarrhea in young calves, recently the presence of calicivirus-like agents and astroviruses , and parvoviruses (Storz and Bates, 1973}, have become recognized. Many outbreaks Of the dise~e are associated with a combination of different viruses and pathogenic bacteria, and as an example of this it has been possible to identify from one calf with diarrhea at two days of age: Salmonella dublin, coronavirus, astrovirus, and three antigenically different calicivirus-like agents; most, if not all of these agents are known to be pathogenic (Woode and Bridget, 1978;Bridger and Hall, 1979). This finding is not surprising if one accepts the hypothesis that the fecal-oral route is the main method of spread of the agents. Thus the disease is complex and a continual search is being made worldwide for new viral pathogens causing scouring in animals. As a result of these studies, a new coronavirus pathogen has been identified recently in the pig (Pensaert and De Bouck, 1978).
Although 70--80% of diarrhea outbreaks may be identified as to their etiological cause, a percentage remains undiagnosed. Some of these may be due to such agents as chemical and fungal toxins but other viral agents may be present which are yet to be identified.
This paper describes the isolation of a new transmissible agent, thought to be a virus, present in an acute epizootic of calf diarrhea which could not be diagnosed etiologically as due to any of the known viral pathogens. By use of the recently described method for determining the presence of coronavirus hemagglutinin in diarrheic feces (Van Balken et al., 1978) a number of calves from this outbreak were shown to have in their feces at high titer, a hemagglutinin which was antigenically distinct from coronavirus and a particle observed by electron microscopy with a morphology different from the other enteritis inducing viruses known to infect the bovine species.
Source of virus
A beef herd in Iowa had experienced severe neonatal calf diarrhea for at least three years, in which the mortality rate was high during excessively cold weather. No etiological agent had been associated with the majority of affected calves, although an occasional isolation was made of rotavirus or coronavirus. During the period from 25 March--1 May, 1979, a detailed investigation was made in an attempt to identify the presence of a viral agent. Of the first 69 calves born, 39 developed diarrhea and 6 died. The remaining 30 calves remained normal. Diarrhea commenced at any age from 2--20 days, but usually at 3--5 days. The clinical syndrome was usually severe and similar to that seen with rotavirus or coronavirus infections. There was yellow to white semisolid or watery diarrhea, profuse in quantity, rapidly leading to severe dehydration. The first 69 calves born were all housed immediately after birth and 66 placed in direct or indirect contact with other scouring calves, as diarrhea commenced with the third calf born. This practice was thought to have contributed to the rapid spread of infection and development of disease at an early age. However, from 13 April the calves were born and reared at pasture, but this practice did not eliminate the disease. From 13--19 April, 12/30 calves born developed diarrhea and three died after 3--5 days duration of diarrhea. Between 22 April and 1 May, 29 calves were born, all of which remained clinically normal. Diarrheic samples were studied by electron microscopy for the presence of viruses. One preparation (B276) had a large number of viral-like particles which were described as atypical coronavirus. This agent was shown to be different from coronavirus by serological tests, and further studies were made in order to characterize the agent as a virus, which was named "Breda" agent after the town in Iowa from which area it was isolated.
Animal inoculation
A sample of a diarrheic feces from calf B276 (the "Breda" agent) was diluted 1:3 (v/v) in phosphate buffered saline (PBS), pH 7.2, centrifuged at 6000g and the supernatant fluid passed through a 0.45 pm membrane filter. Gnotobiotic calves (GC) were produced and maintained using a method described recently, modified by an open caesarean method (Matthews et al., 1981). Following caesarean section under deep general anesthesia, the calves were exposed briefly to the air of the operating theater, while being passed through a germicidal trap containing 10% Wescodyne (Wests Chemical Pro-duction}. The calves were inoculated orally with 5 ml of the 0.45 ~m filtrate at 0--8 days of age. Colostrum-deprived (CD) new born calves were placed in isolation and inoculated as above. Two calves (CF) were fed colostrum, one 8 h before (CF 2) and one 7 h after (CF 1) virus inoculation. Fecal samples and the body temperature were collected and recorded respectively twice daily. For pathological studies, the calves were killed either at the first sign of diarrhea or 24 h later (Calves CD 1, CD 2, and GC 1).
The above preparation of the "Breda" agent was inoculated orally (0.1 ml per rat) into two litters of new-born rats and intracerebrally (0.01 ml) into three litters of new-born mice. The rats and mice were observed for clinical signs and fecal samples were removed daily for testing for the presence of the agent.
D-xylose absorption test
The method used has been described previously . Seventeen hours after the last feed, the calves were given by mouth 25 g of D-xylose (Fisher Scientific Co.), dissolved in 500 ml of water, corrected to pH 5.5 with HC1 and sterilized by autoclaving at 120°C for 60 min. Blood samples were withdrawn into tubes containing EDTA at 1 and 2 h pi. The plasmas were separated and deproteinized: 0.4 ml of plasma was diluted in 2.8 ml of water and 0.4 ml of 10% solution (w/v) of ZnSO4-7 H20 and 0.4 ml of 0.5 N NaOH were added. One ml of the supernatant fluid was added to 5 ml of 2% (w/v) grade 1 ;Sigma Chemical Co.) in glacial acetic acid and saturated with thiourea (Fisher Scientific Co.). The tubes were held at 70°C for 10 min and then cooled and held at 20°C for 70 min in the dark. The reaction was read in a spectrophotometer (Coleman Junior) at 520 nm. Control tubes included a standard that consisted of 10 mg D-xylose in 100 ml benzoic acid, a water "blank" and an unheated mixture of each plasma sample with the bromoaniline stain. The concentration of D-xylose in blood was calculated as mg/100 ml of blood. The reduction in absorption rate when calves had diarrhea was expressed as a percentage of the normal absorption rate.
Antisera
Serum was taken from the experimentally infected gnotobiotic calf, GCO, before inoculation and 27 days later. Antisera were prepared also in guinea pigs by intramuscular inoculations of 106 HA units of "Breda" agent in 0.1 ml volumes twice a week for three weeks. None of the preinoculation sera contained antibody to the "Breda" agent.
Antiserum to bovine coronavirus, kindly supplied by Dr. C.A. Mebus, had a titer by immunofluorescence (IF) assay of > 160 to coronavirus and neutralized 3072--6144 coronavirus HA units. Rabbit antiserum to bovine coronavirus and conjugated with fluorescein was kindly supplied by Dr. Bass of Norden Laboratories. Bovine rotavirus antiserum prepared in a gnotobiotic calf was kindly supplied by Dr. A. Torres-Medina; this antiserum had a neutralizing antibody titer of 2560 with bovine rotavirus. Antiserum to the bovine parvovirus, strain HADEN, was prepared in a rabbit and had a HI titer of 1024 with the homologous virus. Antiserum to parainfluenza 3 (PI3) virus, kindly supplied by Dr. R. Van Deusen, National Animal Disease Center (NADC), had a titer of 160 with the homologous virus.
Hemagglutination and hemagglutination inhibition
A modification of the method for cornavirus identification in calf feces (Van Balken et al., 1978), was used for the detection and identification of both bovine coronavirus and the "Breda" agent in feces. Serial two-fold dilutions of the fecal samples diluted 1:3 in PBS and centrifuged at 6000g were made in 10 pl of PBS. Fetal calf serum was diluted 1:5, adsorbed with packed rat erythrocytes until all spontaneous hemagglutination was lost and 10 pl of a 1:40 dilution added to each well. Ten ~l of 1% rat erythrocytes in PBS with 0.1% bovine serum albumin were added to each well and incubated for 1.5 h at 20°C. For antigenic identification of the "Breda" agent preinoculation serum samples and antisera prepared against the agent in guinea pigs and in calf GCO were absorbed with rat erythrocytes, diluted 1:40 with PBS and used in the above test in place of the fetal calf serum. A reduction in HA titer of 64 fold or greater by antisera when compared with the titer in the presence of non-immune preinoculation serum or sera prepared against bovine coronavirus, parvovirus and PI3 virus, was taken as evidence of antigenic specificity. This was called the HAHI test. The same method was used for the identification of coronavirus, with bovine coronavirus antiserum kindly supplied by Dr. C. A. Mebus. For the hemagglutination inhibition (HI) test for determining antibody in field and experimental serum samples, two-fold dilutions of sera were made in PBS in 10 tal quantities. Ten tal of eight HA units of virus were added, the mixtures agitated and incubated for 30 min at 20°C. Ten tal of 1% rat erythrocytes were then added to each well and the test incubated for 1.5 h at 20°C. All serum samples were adsorbed with rat erythrocytes prior to testing. A HA unit was defined as the highest dilution of the sample showing hemagglutination and was expressed as the reciprocal of the dilution commencing with 1:6.
Adsorption and elution of "Breda" agent with rat erythrocytes
Rat erythrocytes were washed three times with PBS at 4°C and 0.2 ml of packed cells were mixed with 0.2 ml of "Breda" agent containing approximately 200,000 HA units/10 tal. The mixture was incubated for 30 min at 20°C and 30 min at 4°C. The cells were then washed six times in 14 ml of cold PBS (4°C). The cells were finally resuspended to 0.5 ml in PBS and incubated at 36.0 + I°C for 90 min. The cells were pelleted at 1000 rpm and the supernatant removed.
Primary calf kidney (CK cells), primary bovine thyroid cells (CTh), human rectal tumor cells (HRTla) and Madin Darby Bovine Kidney cells (MDBK)
were prepared in tubes with flying coverslips. The growth medium was Eagle minimum essential medium (MEM) with 0.25% lactalbumin hydrolysate and 10% fetal calf serum. Prior to infection of cultures with virus, cells were washed twice with the medium devoid of serum and after inoculation maintained in this medium for 7--14 days. The virus preparation was diluted tenfold in serum-free MEM and dilutions from 10-°--10 -7 inoculated into tubes. Cultures were observed for the development of cytopathic effects (CPE) and tested for the presence of hemagglutinin. They were fixed at 24 h intervals for immunofluorescence (IF) studies. The cells were then passed serially three times with 0.2% trypsin, into both established monolayer cultures or new monolayer cultures at seven days and fourteen days post infection. Each passage was examined at seven days for the presence of virus by HA, IF and CPE. In addition, a preparation containing 200,000 HA units of the "Breda" agent was treated with 500 tag of trypsin at 37°C for 30 min, prior to inocula tion into cell culture. The media for these cultures were then supplemented with 50 pig trypsin (Difco). The cultures were studied for the presence of viral HA, IF and CPE. The virus preparation used was shown to be infectious by inoculation into the experimental calves.
Egg inoculation
Ten-day-old chicken embryos were inoculated by the allantoic route with 0.1 ml dilutions of virus in "serum free" MEM. Allantoic fluid was harvested at daily intervals and the embryos examined for gross lesions seven days post infection. The allantoic fluids were tested for the presence of viral HA. Two further passes of the fluids were performed in 10~lay-old embryos. Trypsin treated virus, as above, was inoculated also into 10-day-old chicken embryos.
Organ culture
Bovine tracheal organ cultures, prepared using the methods previously described by Bridger et al. (1978}, were inoculated with ten-fold dilutions of "Breda" agent. As controls, bovine coronavirus also was cultured. Medium was removed at intervals and tested for the presence of viral HA.
Imm unofluorescence
(1) Gut ring sections, 1--4 um thick; frozen sections were fixed in acetone for 10 min and air dried. A 1:40 dilution of the gnotobiotic calf antiserum to the "Breda" agent (GCO} was applied for 1 h, the section washed and then reacted with fluorescein-conjugated rabbit antiserum to bovine immunoglobulins (Cappel Laboratories}. As controls, sections were stained with preinoculation GCO calf serum and conjugate, and conjugate alone, and the fluorescence compared. Fluorescent cells, thought to be lymphocytes, were demonstrable in the lamina propria but not in the epithelial cell region. The sections also were reacted with the rotavirus antisera by the above method, with the bovine coronavirus conjugated antiserum, by the direct immunofluorescent method, and with goat antiserum to PI3 virus, and with fluorescein conjugated rabbit anti-goat IgG sera kindly supplied by Dr. Prem Paul, NADC and Cappel Laboratories. (2) Tissue culture coverslips, inoculated with the "Breda" agent and with cell culture passes, were removed at 24 h intervals for up to seven days, washed in distilled water, air dried, fixed in acetone for 10 min and reacted with the antisera following the methods described in (1) above. HRT~s cells infected with two isolates of bovine coronavirus were fixed for immunofluorescence after 72 h.
Electron microscopy (EM) and immunoelectron microscopy (IEM)
The fecal hemagglutinin preparations were pelleted at 100,000 g for 1.5 h in a Beckman L5-65 ultracentrifuge. The pellet was resuspended in a few drops of water. One drop of the pelleted virus preparation was mixed with 15 drops of water, one drop of 1% bovine serum albumin and two drops of 4% potassium phosphotungstate, pH 6.8. After 5--10 min incubation, the mixture was sprayed onto carbon-collodion coated grids and examined with an electron microscope. For immunoelectron microscopy, the viral preparation was incubated with an equal volume of a 1: 5 dilution of antiserum in PBS at 37°C for 60 min and overnight at 4°C. The serum--virus mixture was pelleted at 100,000 g for 60 min and resuspended and stained as above. The measurement of particles was obtained by mixing tobacco mosaic virus (TMV) with the fecal virus preparations. Actual magnification obtained on electron micrographs was calculated based on the 18 X 300 nm dimensions of TMV.
Pathology
Tissues for histopathological examination were removed under pentobarbitone anesthesia. A short portion of intestine from the anterior, middle and posterior (50 cm from the ileo-caecal valve) regions of the small intestine and from the spiral colon, were opened and laid flat on cardboard before plunging into 10% buffered formalin solution. Other organs fixed with formalin included abomasum, liver, kidney, lung and bladder. Two or three blocks of each fixed tissue were dehydrated and embedded in paraffin wax; sections were cut at 5 pm and stained with hematoxylin and eosin.
Field survey for the presence of "Breda" agent and coronavirus in herds
As part of the survey into the etiology of calf scours, diarrhea samples were collected from a number of herds in which severe diarrhea had occurred, and these were tested for the presence of bovine coronavirus and the "Breda" agent by hemagglutination, the specificity of the HA being determined with the specific antisera for each virus (HAHI) and by EM studies.
Clinical signs of infection in experimental calves
The three colostrum-deprived calves (CD 1, CD 2 and CF 1) developed diarrhea at 24, 28 and 72 h post infection respectively. The feces changed in color from dark brown to orange/yellow and shortly afterwards to a brilliant yellow, profuse in quantity and watery in consistency. CD 1 was acutely depressed, shivered constantly, and for the next 18 h refused food, remained depressed, stood with difficulty and appeared dehydrated and weak when the autopsy was performed 48 h pi. Calf CD 2 at 23 h pi was depressed, shivered frequently and was unable to stand without assistance. The calf was anesthetized for post mortem examination at 28 h pi, at which time diarrhea was first observed. The third calf, CF 1, developed hyperpnea fi112 respirations/min)24 h pi, which declined to 80 respirations/min at 40 h pi and remained at that level for the remainder of the calf's life. The diarrhea was severe in this calf, yellow in color and watery in consistency and dehydration, as judged by the skin pinch test and sunken eyes, became apparent within 72 h pi. The calf refused one feed each day from 24 h pi and remained acutely depressed. At 72 h pi, a bilateral watery eye discharge developed and finally at 94 h pi the calf was unable to stand, refused feed and died at 96 h pi. The other colostrum-fed calf (CF 2) remained clinically normal throughout the experimental period, although the feces became yellow in color which correlated with appearance of viral hemagglutinin in the feces {Table I). The first gnotobiotic calf (GCO) inoculated at eight days of age, developed a mild scour 24 h pi, which was green in appearance and changed to a semi-solid with flecks of yellow 24 h later. This calf returned to normal within 24 h and remained in good health until autopsied 27 days later. Calf GC 1, which was inoculated at two days of age, developed a profuse scour 72 h pi, with yellow floccules floating in a whey-like fluid. At 96 h, the fecal motions changed to semi-solid in consistency and greenish yellow in appear-ance but at 120 h watery profuse diarrhea reoccurred. This calf retained its appetite, but when autopsied at 120 h pi it had lost weight, was dehydrated as judged by a skin pinch test and the ribs were prominent and the eyes sunken. GC 2, which was inoculated at 72 h of age, did not develop diarrhea and remained in good health although the semi-solid greenish/yellow colored feces increased in volume until seven days pi at which time the fecal samples were difficult to obtain. Calf GC 3 was inoculated at 24 h of age with the virus filtrate diluted 1:100 and at 48 h pi. The feces changed from dark green to orange-yellow in color. Nine hours later the feces were very loose, mucoid and contained yellow floccules but at 72 h pi, it became semi-solid and greenish-yellow. Anorexia was not observed (Table I).
All the new-born rats and mice remained clinically normal for three weeks following inoculation. Two litters of mice and their dams developed diarrhea at three weeks together with a control litter. However, the "Breda" agent could not be detected in the feces or intestinal contents of these animals.
D-xylose absorption studies
Studies were not performed on CD 1, CD 2, CF 2, GC 2 or GC 3. Calf CF 1 was inoculated at birth and thus no normal absorption rates were obtained, but when in severe diarrhea, it had blood levels of D-xylose at 1 and 2 h post feeding of 65 and 75 mg/100 ml, respectively. These are similar to normal levels for non-diarrheic calves. Calf GCO, with mild diarrhea, showed reductions of D-xylose absorption at 1 and 2 h, when compared with preinoculation absorption rates of 35% and 16% respectively, and calf GC 1, with severe diarrhea, reductions of 53% and 42% from preinoculation absorption rates. Studies with rotavirus showed reductions of 60--100% in the absorption rate .
Hemagglutination and hemagglutination inhibition
In attempts to determine the optimal temperature for hemagglutination, the standard preparation of B276 was titrated and incubated for 1.5 h at 4 ° C, 20°C (room temperature) and 37°C. However, no difference was observed in the titer at the three temperatures. The hemagglutinin assay was read at 1.5 h but was stable for a further 20 h at room temperature. No agglutination occurred when the viral hemagglutinin was reacted with human group O, bovine, hamster, guinea pig, chicken, turkey or goose erythrocytes.
The HA titers of feces from experimental calves before inoculation were 3.68--6.6 log2. Fecal samples obtained from naturally infected and all the experimentally infected calves with diarrhea had HA titers which peaked in different calves between 11.6 log2 and 25.6 log2. Hemagglutinin was detectable for 3--4 days ( Table I).
The guinea pig and calf antisera diluted 1:10, generally reduced the HA titer of virus to the same titer as in the preinoculation samples from the same experimental animal, or when the HA titer was high, as with B276, neutralized 15.6--16.6 log2 HA units. In contrast, antisera prepared in gnotobiotic calves against bovine coronavirus and bovine rotavirus only neutralized 1--3 log2 HA units, guinea pig antisera to bovine parvovirus neutralized 1--2 log2 HA units and goat antiserum to PI3 virus neutralized 3--4 log2 HA units, of preparation B276. Dr. McClurkin, NADC, was unable to isolate BVD virus from the B276 inoculum, and the antiserum prepared in calf GCO did not have IF antibodies to BVD.
The HI titer of serum prepared in GCO against eight HA units of B276 rose from a preinoculation titer of 2.5 to 6.6 log2, 27 days pi. In contrast, most field sera had HI titers ranging from less than 2.6 to 5.6 log2. Six to eight serum samples were obtained from each of four unrelated herds and the HI titers determined. The range of titers in Herd I were 3.6--5.6 log2, Herd II 4.6--6.6 log2, Herd III 2.6--3.6 log2, and Herd IV 3.6--4.6 log2. Herd I animals were those which included calf B276 in the "Breda" herd. The convalescent serum of this animal neutralized approximately 6--7 log: HA units of virus at a 1:10 dilution and had a titer of 3.6--4.6 log2 against eight HA units. In two adult heifers, which were inoculated orally and twice weekly subcutaneously with 17.6 log2 HA units of virus, the preinoculation HI titers were 4.6 and 5.6 log2 respectively and rose to 5.6 and 6.6 log2 respectively after 21 days.
In attempts to remove from sera the low level of "non-specific" inhibition of hemagglutination, the sera were treated with kaolin and/or heat inactivated at 56°C/30 min. Both treatments reduced the non-specific inhibitory titer two-fold but also reduced the specific HI titer by the same amount. It was considered that the HI test was an unreliable method for the determination of antibodies to the "Breda" agent in field animals, largely due to the apparently poor antigenic response, as measured by this test, to natural infection and the small difference between the HI titer of "negative" sera and the convalescent titer.
Electron microscopic studies
The agent had two distinct morphologies, approximately spherical and sausage shaped, which frequently was curved to resemble a kidney shape (Fig. 1). The spherical particle measured 89 + 7 nm × 75 + 9 nm with peplomers 7.6--9.5 nm long. The other particles measured 120 + 15 nm× 32 + 8 nm with similar peplomers. A minority of particles possessed poorly defined peplomers similar in size and shape to coronavirus (17--24 nm). An electron-dense indentation was frequently observed in the Center, or slightly off center, of many of the spherical particles. The proportion of kidney shaped to spherical particles varied considerably between samples.
There was an excellent correlation between the presence of particles observable by EM and the presence of specific hemagglutinin in feces of both experimental and naturally infected animals, with the exception of calves CF 1 and CF 2. These had low HA titers (11.6 log2) and the characteristic particles were not readily observed.
As a confirmation of the antigenic specificity of the "Breda" virus, immunoelectron microscopy was performed on bovine coronavirus in a fecal preparation and two different preparations of "Breda" agent (from B276 and CD 1), with antisera against coronavirus and "Breda" agent. Both sera coated and agglutinated their homologous viruses, and coronavirus antiserum coated but did not agglutinate the "Breda" agent particles. In contrast, the "Breda" antiserum neither coated nor agglutinated coronavirus. A mixed preparation of the two viruses, when reacted ~l~h each antiserum, also showed these effects. The "Breda" agent coated with homologous antiserum is shown in Fig. 1.
Adsorption and elution of the "Breda" agent with rat erythrocytes
Bovine coronavirus adsorbed to the rat cells and was eluted after 90 min at 36.0 + I°C. Approximately 7.0 log: HA units of coronavirus was adsorbed and 6.0 log2 HA units recovered after elution. No HA was observed in the final washing solution prior to incubation at 36.2°C. In contrast, no elution was observed by the "Breda" virus after 90 min at 36.0°C, but after 16 h at 36.0°C, 8.0 log2 HA units were released together with lysis of the red cells.
Virus excretion in the feces of experimentally infected calves
Fecal HA titers before virus inoculation were 2.6--5.6 log2 and rose after 24--48 h coinciding with the development of diarrhea or a color change of the feces. The fecal HA titers peaked at 11.6--12.6 log2 in calves GC 1, CF 1 and CF 2, at 18.6 log: in calves GCO and GC 2 and 14.6 log: with calf GC 3. The highest HA titer observed (25.6 log:) occurred in the feces obtained from the naturally infected calf B276. No correlation was observed between titer of HA in the feces and severity of infection, but the number of particles observed by EM corresponded approximately with the HA titer.
Virus excretion in the feces of experimentally infected rats and mice
The "Breda" agent was not detected in the feces of the new-born rats or mice or their dams at any time post infection and the diarrhea developing at three weeks appeared to be related to other causes.
Histopa thology
CD 1: Villi of the jejunum and ileum were atrophic and small patches of villous epithelium had desquamated from villous tips. Mixed cellular and fibrinous material was exuding into the lumen from the denuded areas. Most of the villous surface was covered by irregular squamous to cuboidal cells with basophilic cytoplasm. There were occasional synechiae between villi. Villous lamina propria was congested and contained abundant nuclear debris. Fresh hemorrhage was observed in occasional Peyer's patch lymph nodules most of which were hypocellular centrally. The surface epithelium of the colon was irregular and contained abundant cellular debris, some of which accumulated in the surface lamina propria. The abomasum was congested and had multifocal, small submucosal hemorrhages. Jejunum, cecum, rectum, liver, and kidney were not remarkable.
CD 2: The appearance of the villi and villous epithelium in the small intestinal sections was unremarkable. The colonic mucosa had many villous folds and dilated crypts. The lungs contained multifocal petechial hemorrhages and vesicular emphysema.
GC 1 : The ileum had mild villous atrophy characterized by shortened villi covered with basophilic, cuboidal epithelium, and some areas of elongated crypts. A diffuse, low-grade neutrophilia was observed in the lamina propria and submucosa. Most Peyer's patch lymph nodules were hypocellular centrally. Dilated crypts and scattered crypt abscesses were present in all sections of the small intestine. Surface epithelium of the spiral colon had areas of necrosis. The spiral colon also had dilated crypts and diffuse neutrophilia of the lamina propria. Liver, kidney, and urinary bladder were unremarkable.
There was no evidence of bacterial colonization in any of the histologic sections.
Immunofluorescent studies
Calves CD 1, CD 2 and GC 1 were examined for specific immunofluorescence (IF) in cells of the small and large intestine. Calf CD 2, showed the least tissue damage and the most extensive evidence of infection by IF. Sections of the mid gut (jejunum} and ileum showed IF positive epithelial cells, mainly in the lower 50% of the villus and extending deeply into the crypt area. Scattered individual cells of the upper part of the villus also fluoresced specifically. No IF positive cells were observed in the epithelium of the anterior small i~. Specific IF in cells was observed throughout the length of the fold of the spiral colon including the crypt cells, and most of the folds were involved. Calves CD 1 and GC 1 were examined 24 h after the commencement of diarrhea and consequently there was more villus damage. Immunofluorescent epithelial cells were randomly scattered among the villi and crypt areas of the jejunum, ileum and spiral colon. No IF was observed in these sections when reacted with bovine coronavirus or bovine rotavirus antisera (Fig. 2 a, b, c).
Coronavirus infected HRT~s cells immunofluoresced specifically with coronavirus antiserum but no fluorescence was observed at a 1:20 dilution with GCO convalescent antiserum.
Farm survey for the presence of bovine coronavirus and "Breda "" agent in feces
From the Breda herd, 47 diarrheic calves were sampled at least once and a few sampled two to four times. Fourteen were shown to be infected with the "Breda" virus by HAHI and eleven by EM, with three doubtful positives. One calf was infected with coronavirus, one with rotavirus and one possibly with all three agents.
A total of 339 fecal samples from a variety of farms other than the Breda herd were examined. Fifty-five were positive for coronavirus by HAHI, 53.~by EM, and one by EM only. None was positive for the "Breda" virus by HAHI or EM. Twelve samples had HA titers (8.6--11.6 log:) which were not related to either coronavirus or "Breda" virus antigenically and were all EM negative. With both viruses, only those samples with high titers of HA had readily observable particles with all the morphological characteristics. The range of HA titers in feces for coronavirus in field cases was 7.6--17.6 log2, and for the "Breda" agent 7.6--25.6 log2.
Attempts to isolate virus in organ cultures, cell cultures and embryonated eggs
All attempts to isolate the virus in cell cultures, intestinal and tracheal organ cultures and embryonated eggs were unsuccessful. Cell culture preparations were taken to 3--5 passes and the preparations were examined for the presence of viral specific immunofluorescence and HA. As controls for the cell culture methods, fecal bovine coronavirus was shown to replicate in trachea organ cultures, HRT cells, MDBK cells and bovine thyroid primary cell cultures, and bovine rotavirus replicated in the primary and secondary CK cells. No detectable HA developed in the allantoic fluid of all inoculated eggs, which were sampled at day 3 and 7 pi. Eggs that died were shown to be contaminated with bacteria. Pretreatment and treatment at each pass with trypsin had no apparent influence on the infectivity of the virus for these culture systems.
DISCUSSION
The first intention of the present study was to confirm the infectious nature of the particle, as virus-like particles, the so-called pleomorphic "fringed-particles", are common in feces but these (Woode, unpublished data} are not infectious. Apart from the fecal preparation of calf B276, which contained the "Breda" agent with large numbers of similar sized and shaped virus-like particles, most of the hemagglutinin positive calf samples contained fewer particles with less well-defined morphologies and were originally dismissed as non-viral in nature or considered to be "atypical" coronavirus particles. Although all early attempts to culture the agent in vitro were unsuccessful, from precedents established with other enteritis inducing viruses, we assumed that the natural host would be the most sensitive system for demonstrating infectivity (Woode, 1979}. The "Breda" agent was shown to be infectious in both gnotobiotic and conventionally derived calves as all the experimental calves inoculated orally excreted the agent for 3--4 days in the feces and one serial passage of the agent through two calves was successful. The agent can be tentatively classified as a virus on the bais of its infectious nature in calves, the size, shape, presence of the hemagglutinin and the specific immunofluorescence in the cytoplasm of infected epithelial cells.
These studies have not confirmed that the virus is pathogenic in all nonimmune calves.
It is possible that the severe clinical effect observed in 4/8 experimental calves, which is similar to the percentage suffering severe diarrhea on the farm, is controlled by individual variation in response. Similar observations have been reported for rotavirus infection in pigs (Woode and Crouch, 1978). An alternative interpretation of the data, however, is that this agent requires a microbial flora before virus infection, to demonstrate a full clinical effect, although there could be exceptions. All three colostrum-deprived calves developed severe clinical signs of disease in contrast to the gnotobiotic calves, in which only 1/4 was severely affected.
The studies have not elucidated fully the pathogenesis of the agent. The initial studies show that the pathology of the small intestine is consistent with a rotavirus (Mebus et al., 1971, Woode et al., 1974 or a coronavirus (Mebus et al., 1973, Bridget et al., 1978 infection. The immunofluorescent data of the small intestine and colon, and the pathology of the colon, shows a greater similarity with coronavirus infection rather than with rotavirus. Only with lamb rotavirus has infection of the colon been demonstrated {Snodgrass et al., 1977). The watery nature of the diarrhea which is commonly observed with gnotobiotic calves infected with coronavirus and with calf GC 1 in this study, is probably due to the colonic lesion and consequently, reduction in the ability to absorb water. However, the immunofluorescent studies suggest that the pathogenesis may differ from coronavirus in that most immunofluorescent cells were observed in the lower 50% of the small intestinal villus and throughout the crypt region, and in the colon throughout the length of the villus and crypt. This would imply that the primary site of infection is in the crypt cells and infected cells migrate up the villus before being shed as the viral cytopathic effect develops.
The minimal malabsorption effect as measured by D-xylose also suggested that the pathogenesis may differ from rotavirus and from the calicivirus-like agents . Further studies are planned to determine the pathogenesis of the agent.
The hemagglutination method proved to be the most useful method for identifying antigenically the agent in fecal samples. From these data and from data obtained from immunofluorescent studies of the agent in gut sections, and coronavirus in gut sections and tissue cultures, the "Breda" virus appears to be antigenically distinct from other bovine hemagglutinating viruses. These include bovine coronavirus (as characterized by Sharpee et al., 1976), parvovirus, and rotavirus, parainfluenza 3 virus in this study, and reovirus 3 (Woode, unpublished data). In addition, bovine pestivirus (BVD) could not be isolated from preparations containing the "Breda" virus, and antiserum to the "Breda" virus did not possess antibodies to BVD. The HI test for studying sera for the presence of antibody was considered to be unreliable and thus serological data suggestive of widespread distribution of the agent on farms should be treated with reservation. The virus may be common in the U.S. as a similar agent has been observed in one calf in South Dakota and in calves in Ohio. and antiserum to the "Breda" virus reacts specifically with the Ohio isolate (Saif, personal communication, 1981), however, it appears that these two latter viruses are different serotypes.
The failure to demonstrate "Breda" virus elution from erythrocytes, in contrast to the elution obtained with bovine coronavirus, demonstrates another dissimilarity between these two viruses.
The morphological data indicate both similarities and differences when "Breda" virus is compared with coronaviruses in general and bovine coronavirus in particular. The morphological description of bovine coronavirus as observed by the authors and others (Sharpee et al., 1976;Bridget et al., 1978) is: generally round, 110--180 nm in diameter with peplomers (17--24 nm) and a centrally located electron-dense area. An inner layer of short peplomers (10 nm) is seen on some particles. In contrast, the "Breda" agent is more pleomorphic with at least two distinct shapes: ovoid (89 X 75 nm) and sausage or kidney shaped (120 X 32 nm). The majority of particles possess peplomers (7.6--9.5 nm). Like coronaviruses, the "Breda" virus frequently shows an electron-dense region. This appeared to represent the cleft region seen in the kidney particles and may be similar to the internal component reported in avian infectious bronchitis virus, a coronavirus (Bingham and Almeida, 1977). In the authors' experience, the short peplomers are not visible on bovine coronavirus particles, when they lack the 17--24 nm peplomers. The short peplomers seen on the "Breda" agent are similar to those of many enveloped viruses from a variety of families (Dalton and Haguenau, 1973).
Definitive taxonomic classification of this agent will require characterization of its physical and chemical properties. Antiserum to the "Breda" virus is being examined for the presence of immunofiuorescent antibodies to a number of animal coronaviruses.
The histopathological lesions of villous atrophy with basophilic cuboidal to squamous epithelium were similar to alterations attributed to intestinal infections with bovine rotavirus and bovine coronaviruses (Mebus et al., 1971(Mebus et al., , 1973. The absence of such changes in calf CD 2 was presumably due to the early time of autopsy of this calf, as the intact villi demonstrated extensive virus-specific immunofiuorescence. The epithelial irregularity and necrosis seen in the colon of calves CD 1 and GC 1 indicate a role of colonic dysfunction in the pathogenesis of diarrhea produced by infection with this virus. The inability, at this stage of the research, to culture the virus by in vitro techniques is similar to the experiences of most workers with other enteritis inducing viruses. The calicivirus-like agent of the calf and the Norwalk virus of human beings (Wyatt et al., 1979) have not yet been cultured, and until the trypsin method for the culture of rotavirus (Theil et al., 1977) and the use of HRT 18 cells for coronavirus (LaPorte et al., 1979) were developed, these agents could not be cultured readily. Fortunately, modern virology methods do not require the in vitro cultivation of viruses for antigenic and nucleic acid characterization of viruses and the large quantities of virus obtained from feces permit us to use this source of virus for most studies, including the development of vaccines. | v3-fos |
2014-10-01T00:00:00.000Z | {
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} | s2 | Thermostatic regulation of blood samples in blood gas analysers: results and improved inethod applied to 11 different models
As part of a general assessment of blood gas analysers [1] the thermostatic regulation of blood samples in the machines’ sample chambers was studied with the aid of a thermal probe placed on one of the electrodes. The experiment was necessary because the analysers’ readings assume a temperature of37C; in principle the instruments should maintain this temperature to within 0" C. The influence of temperature varies with each parameter being measured (for example pH, percentage oxygen [pO2] and percentage carbon dioxide [pCO2]) and according to the nature of the solution being analysed [2]. For protein solutions, and especially for blood samples, the average variations are: for pH, 0.011 units per degree; for pCO2, 4%/C, and for pO2, 7%/C. In addition, cooling of the electrodes modifies these variations: for pO2 this may vary by as much as 10%/1C; for pCO2 the effects of temperature are in part compensatory, reducing the variations to -2% error/lC [3, 4]. Aqueous buffers are less sensitive to temperature; they are not therefore suitable for thermostatic control [5]. The measuring electrode in each instrument was modified. The sensing part of the pO2 or the pCO2 was replaced by the heat-sensitive end of a thermocouple. The aim ofthis investigation was to examine the temperature of the sample itself within the sample chamber, without disturbing the normal flushing, calibrating and measuring processes which are very elaborate in some ’automatic’ instruments. Thermocouple
Introduction
As part of a general assessment of blood gas analysers [1] the thermostatic regulation of blood samples in the machines' sample chambers was studied with the aid of a thermal probe placed on one of the electrodes. The experiment was necessary because the analysers' readings assume a temperature of 37C; in principle the instruments should maintain this temperature to within 0" C.
The influence of temperature varies with each parameter being measured (for example pH, percentage oxygen [pO2] and percentage carbon dioxide [pCO2]) and according to the nature of the solution being analysed [2]. For protein solutions, and especially for blood samples, the average variations are: for pH, 0.011 units per degree; for pCO2, 4%/C, and for pO2, 7%/C. In addition, cooling of the electrodes modifies these variations: for pO2 this may vary by as much as 10%/1C; for pCO2 the effects of temperature are in part compensatory, reducing the variations to -2% error/lC [3,4].
Aqueous buffers are less sensitive to temperature; they are not therefore suitable for thermostatic control [5].
The measuring electrode in each instrument was modified. The sensing part of the pO2 or the pCO2 was replaced by the heat-sensitive end of a thermocouple.
The aim ofthis investigation was to examine the temperature of the sample itself within the sample chamber, without disturbing the normal flushing, calibrating and measuring processes which are very elaborate in some 'automatic' instruments.
Thermocouple
The thermocouple used was a Constanant which has a diameter of mm, a very short response delay of 3 s, and a theoretical sensitivity of 4 MV/C. Thus 0.01 C can be estimated ensuring a precision 0. IC.
The thermocouple was fixed on each instrument's jacket in place of the pCO2 electrode. The thermocouple was passed through a silicon rubber plug (cut to size), and pushed firmly to the bottom of thejacket in order to keep the system watertight. A large diameter trocar (3 mm) was used to pass the thermocouple through this stopper. Once the trocar is removed, the thermocouple remains held in place by the elasticity of the stopper (see figures and 2). Each electrode assembly fitted with the thermocouple was then calibrated in a water bath with a circulating system, at three or four different temperatures ranging from 36C to 38C.
Materials and
The same high-precision thermometer (manufactured by Walter-Kessler, Z.A. Courtaboeuf, F 91403 Orsay, France), graduated to 0.02C, was used throughout the investigation. A calibration curve was drawn for each instrument (figure 3). Zero was obtained by plunging the two ends of the thermocouple into a thermos flask filled with distilled water and ice. The iced water served throughout each experiment as a fixed temperature of reference.
Recorder
The recorder used was a Kipp and Zonnen micrograph BDN5. Temperatures were recorded during the instrument calibration with gas; during three successive measurements of one blood sample previously maintained at 25C, and during the three successive measurements of the same blood sample previously maintained at / 4C. In each case the manufacturer's operating instructions were followed and the timing programmes of the instruments were taken into account. The thermocouple system indicates the temperature variations in the sample chamber when the gas is passed through, although the values obtained, due to the gas, are not necessarily exact. The continuous recordings of temperatures from within the sample chamber are illustrated in figure 4.
Results
The results of the study are shown in table 1; the instruments are listed there according to their degree of automation. The notes below relate to the table.
(a) In this instrument the galvanometer indicating temperature reverts to 37C 30s before complete thermal equilibrium of the sample. Figure 4(a)is from an instrument, without a pre-heater (speed of paper was 10 mm/min.) and ft(ture 4(b) is from an instrument with a pre-heater (speed of paper was 5mm/min.); a=rinsing, b=entry of sample, c= final temperature.
(b) The instrument confirms its results after about 45s although the thermal balance of the sample is reached after 2min. 10s at 37.12C.
(c) After rinsing with the non-thermostated solutions the temperature of the measuring chamber is slow in returning to its exact level (3 min.). In addition it was noted with this instrument that too great a speed of gas flow significantly lowered the temperature of the chamber (the calibration gases do not pass through the preheater). The flow rates advised by the manufacturer should be followed. (f) The thermal equilibrium of the sample is reached after about 2min. 30s at 36"85C. The instrument thus confirms its results before thermal equilibration is reached, hence the difference in values observed. (9) After reassembly of the instrument the test was not carried out until the temperature warning light was extinguished. The table shows temperatures of samples when the operator, or automatic instrument, confirms the result, but not necessarily the temperature of the sample at calorific balance.
Accuracy
The lowest temperature measured was 36.24C and the highest 37.16C. One company, Coming, stands out as ensuring a high quality of sample thermoregulation on the whole range of their instruments, irrespective of the level of automation. All other models show greater or lesser inexactitude, erring always by default.
Repeatability
Table demonstrates that, on the whole, the repeatability of temperature is very good for a given instrument.
Influence of sample temperature
It can equally be seen that the initial temperature of the sample does not affect its final temperature in the sample chamber. This holds good whether the instrument has a pre-heater or not. This fact means that if measuring has to be deferred (due to workloads in the laboratory or any other reason) the blood samples can be refrigerated (at +4C).
Comments
It was noticed in the course of these investigations that too high a flow rate of gas in the sample chamber during calibration cooled the chamber (from between and 1.5C)and this invalidates calibration values.
In some instruments (AVL particularly), the copious flow of cleaning fluids which are not thermostatically controlled leads to significant cooling in the sample chamber, which then takes about 5 min. to regain its correct temperature this reduces the analytical throughput of samples. For instruments which have no fixed time input the steady state of sample temperature may be reached if the response time of electrodes is slow. On the other hand, when the response time is fast, the results are read before the steady state.
When pre-heaters are provided with the instrument the blood sample enters the sample chamber at a temperature very near to the steady state.
Conclusion
Twenty instruments--11 models from five manufacturers. were tested; it was concluded that only one company offers instruments which conform technically with the documentation supplied.
The other companies' instruments fail in accuracy but good repeatability of sample thermostatization is obtained; therefore it should be possible to overcome this problem by correctly adjusting the instruments.
Call for papers
The 14th Annual Symposium on the Analytical Chemistry of Pollutants and the 3rd International Congress on Analytical Techniques in Environmental Chemistry will be held from 22-24 November 1984 at the Palacio de Congresos in Barcelona, Spain. After the two meetings several workshops on specific topics will be held--these include a workshop on ion chromatography and another on the chemistry and analysis of hydrocarbons in the environment. Those wishing to submit a paper or poster should send the organizers a 200-word abstract by 15 December 1983. Papers will be refereed and the official language is to be English.
Notes for Authors
Journal of Automatic Chemistry covers all aspects of automation and mechanization in analytical, clinical and industrial environments. The Journal publishes original research papers; short communications on innovations, techniques and instrumentation, or current research in progress; reports on recent commercial developments; and meeting reports, book reviews and information on forthcoming events. All research papers are refereed.
Manuscripts
Two copies of articles should be submitted to the Editor. All articles should be typed in double spacing with ample margins, on one side ofthe paper only. The following items should be sent: (1) a title-page including a brief and informative title, avoiding the word 'new' and its synonyms; a full list of authors with their affiliations and full addresses; (2) an abstract of about 250 words--this should succinctly describe the scope of the contribution and highlight significant findings or innovations; it should be written in a style which can easily be translated into French and German; (3) the main text with sections and subsections numbered; (4) appendices (if any); (5) references; (6) tables, each table on a separate sheet and accompanied by a caption; (7) illustrations (diagrams, drawings and photographs) numbered in a single sequence from upwards and with the author's name on the back of every illustration; captions to illustrations should be typed on a separate sheet.
Illustrations
Line diagrams are preferred to photographs. Original copies of diagrams and drawings should be supplied, and should be drawn to be suitable for reduction to the page or column width of the Journal, i.e. to 85 mm or 179mm, with special attention to lettering size. Photographs may be sent as glossy prints or as negatives.
Proofs and offprints
The principal or corresponding author will be sent galley proofs for checking and will receive 50 offprints free ofcharge. Additional offprints may be ordered on a form which accompanies the proofs. | v3-fos |
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} | s2 | Effects of transplacental exposure to chlorinated phenols.
Female rats were exposed to 0, 5, 50 or 500 ppm of 2-chlorophenol (2-CP) or pentachlorophenol (PCP). The study was designed to produce progeny which were exposed to the chlorophenolic compounds both prenatally and postnatally. Percent conception, litter size, birth weight, and number of stillbirths was determined at parturition. Hematologic parameters and body weights of the progeny were recorded at weaning age (3 weeks). Effects on reproduction were observed in both the 2-CP and PCP-exposed groups, as indicated by decreased litter sizes and increased number of stillborn. The data indicate that these chlorinated phenolic compounds may be feto- or embryotoxic at high doses. Effects on hematologic parameters were not observed. Further study involving transplacental and chronic exposures to these chlorophenolic compounds appears warranted.
2-Chlorophenol is a commercially produced chemical used as an intermediate in the production of higher chlorinated phenols. The generation of waste sources from the commercial production of 2-chlorophenol, its chemically derived products, and the inadvertent formation of 2-chlorophenol due to chlorination of organics in drinking and waste waters are potential sources of environmental contamination (1).
Pentachlorophenol (PCP) and its salts have been widely used in agriculture and industry since 1936 (10). Approximately 200 million pounds of PCP were produced worldwide in 1977 (11). Principal uses of PCP are as a pesticide and wood preservative (12), and contamination of livestock occurs by licking treated wood or drinking from vats used to treat wood (6). Industrial exposure and consumption of contaminated food and water are the principal sources of exposure to humans (13).
This study was conducted to assess the effects of two chlorophenolic compounds, 2-chlorophenol and pentachlorophenol, on reproduction and hematology. The experimental design included prenatal and postnatal exposure to the chlorophenolic compounds.
Materials and Methods
Female Sprague-Dawley rats were weaned at 21 days of age and divided into groups of 12-20 rats each. The rats were placed on dietary regimens containing 0, 5, 50 or 500 ppm 2-CP (Aldrich Chemicals, 97% pure) in the drinking water or PCP (ICN Pharmaceuticals, K & K Laboratories No. 18497, 95% pure) in the feed. The female rats were housed four per cage in stainless hanging wire racks. Feed and water were available ad libitum. The rats were bred at 90 days of age (10 weeks exposure). The dams were transferred to polyearbonate cages with stainless steel lids and hardwood shavings to litter. The study was designed to produce progeny that were exposed to the chlorophenolic contaminants both prenatally and postna-tally. Percent conception, litter size, birth weight, and number of stillbirths was determined at parturition. Body weights and hematologic parameters (white and red cell counts, hemoglobin, packed cell volume and mean corpuscular volume) were recorded at weaning age. Blood samples were analyzed using a Coulter counter, Model ZBi. The dams were terminated at weaning and liver and kidney tissues were collected for analysis of chlorophenolic content. Gas chromatography was used to analyze 2-CP and PCP in tissues by the methods of Erney (14).
Results
Parameters relating to reproductive performance that were observed include percent conception of EXON AND KOLLER the dams, litter size, birth and weaning weights, percent stillborn, survival to weaning and body weight gain of the dams (Tables 1-4). Percent conception was greater in all treatment groups as compared to controls (Tables 1 and 2). Litter size was significantly (p -0.05) decreased in groups of dams treated with high levels of 2-CP (Table 1). Litter size was also decreased in groups given 500 ppm PCP (p -0.10) ( Table 2). Percent of stillborn pups born to dams receiving either 2-CP or PCP was generally greater as compared to controls. This increase was significant (p -0.05) in the 500 ppm 2-CP group (Table 1). Body weights at weaning were generally decreased in PCP-exposed groups. No consistant effects were observed in regard to survival to weaning (Tables 3 and 4). The body weight gains of dams prior to breeding appeared to be unaffected by the treatments. bExclusive of stillborn pups. bExclusive of stillborn pups. bA pool of tissues from five rats/group. The accumulation of the chlorophenol compounds in tissues of dams was comparatively minimal (Table 5). Pentachlorophenol levels in liver and kidney tissues were considerably lower than 2-CP residues in similar tissues. Residues in kidney versus liver tissue were approximately equal. Feed samples were also analyzed for PCP content ( Table 5). The analysis correlated reasonably well with the target concentrations of 5, 50 and 500 ppm. Purity of the PCP mixture used in preparation of the PCP diet was analyzed by the Dow Chemical Company ( Table 6). This analysis incidated that the PCP formulation was only 85.5% pure as compared to the 95% purity advertised by the manufacturer. The dioxin content was also determined ( Table 6).
Hematologic parameters examined for all treatment groups included red and white blood cell counts, packed cell volume (hematocrit), hemoglobin and mean corpuscular volume (Tables 7 and 8). No significant effects of chlorinated phenols on hematologic parameters were observed.
Discussion
The chlorophenols tested, 2-CP and PCP, appeared to alter certain reproduction parameters in rats. Stimulated conception rates, reduced litter size at the highest doses (500 ppm), and increased number of stillbirths were observed in chlorophenol-treated groups. These results indicate that high levels of these compounds tend to be fetoor embryotoxic. a2-Chlorophenol was given in the drinking water either prenatally by exposing the dams from weaning through parturition (90 days) and postnatally from parturition.
bAll hematologic parameters are reported as mean (standard deviation). 2) aPentachlorophenol was given in the drinking water either prenatally by exposing the dams from weaning through parturition (90 days) and postnatally from parturition.
'All hematologic parameters are reported as mean (standard deviation).
The results are consistent with those published by others (15). The majority of commercially prepared technicalgrade PCP is a mixture of about 85-90% PCP and the remainder primarily consists oftetrachlorophenol and chlorinated phenoxyphenols (16). Small amounts of other impurities include chlorinated dibenzo-pdioxins, chlorinated dibenzofurans and chlorinated diphenyl ethers. The mixture purchased for this study was listed by the manufacturer to be 95% pure PCP. However, analysis indicated that the mixture contained actually 85.5% PCP and a variety of other phenolics and impurities. Nevertheless, exposure to this substance in the environment would be as prepared containing both the PCP and associated impurities. Once adverse effects are identified from exposure to the manufactured product, then analytical grade PCP must be evaluated to determine if the effects are produced by PCP, its impurities or from a combination of these products. | v3-fos |
2017-07-29T04:25:18.781Z | {
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} | s2 | Status and results of merging different strains of Brown cattle in Europe
In the European Braunvieh-populations since 1965 American Brown-Swiss bulls were used to increase genetic variation and milk production. While in the beginning of this period not all countries in Europe were involved, the situation now has changed : All countries are using combinations of American and European genotypes and the majority of test bulls has 50 and 75 p. 100 BS-genes. At present there are no indications of a complete replacement of European genotypes, but a combination is aimed at. While the early analysis of data showed large breed differences with negative heterosis and recombination loss, newer data showed an advantage in milk yield, fat and protein yield, while the protein content was slightly lower. The progeny of 5/8-and 3/4-BS-buils were on average as good in milk-production as the purebred American bulls, indicating that a combination of Euro-pean and American strains is successful. Rather limited information on fattening ability and carcass-quality indicate, that the crosses are taller, have a comparable or higher rate of gain with lower dress-out-p. 100. Higher internal fat leads in connection with heavier bones to a decrease in high priced cuts and to inferior carcass grading. With regard to secondary traits, the calving intervall of progeny from 100 p. 100 BS-bulls tends to be longer, but longer gestation and lower culling rates could have caused this findings. Milkability and calving ease were influenced slightly in a favourable direction. Finally the possibilities of comparing European populations according to the IDF-proposals are discussed. Links between the European populations at present are only through the use of American bulls, most of them were progeny-tested and used in Europe in planned matings. An approach should be taken, to use this material to get hints about the base differences between the European countries. Nevertheless it should be tried to organise an exchange of semen from test bulls across countries and to get an unbiased proof on these in each of the cooperating countries. We studied the heterosis effects in characteristics having fundamental influence on meat production as a result of crossbreeding Hungarian Simmental cattle with Herefords. Evaluating a considerable number of data, Fi population was found to be superior not only to the average of the two purebreed populations, but also to that of the better parent population in major maternal characteristics. As a result of crossing, the following results were found as a consequence of heterosis effects : 7-8 p. …
In the European Braunvieh-populations since 1965 American Brown-Swiss bulls were used to increase genetic variation and milk production. While in the beginning of this period not all countries in Europe were involved, the situation now has changed : All countries are using combinations of American and European genotypes and the majority of test bulls has 50 and 75 p. 100 BS-genes. At present there are no indications of a complete replacement of European genotypes, but a combination is aimed at.
While the early analysis of data showed large breed differences with negative heterosis and recombination loss, newer data showed an advantage in milk yield, fat and protein yield, while the protein content was slightly lower. The progeny of 5/8-and 3/4-BS-buils were on average as good in milk-production as the purebred American bulls, indicating that a combination of European and American strains is successful.
Rather limited information on fattening ability and carcass-quality indicate, that the crosses are taller, have a comparable or higher rate of gain with lower dress-out-p. 100. Higher internal fat leads in connection with heavier bones to a decrease in high priced cuts and to inferior carcass grading. With regard to secondary traits, the calving intervall of progeny from 100 p. 100 BS-bulls tends to be longer, but longer gestation and lower culling rates could have caused this findings. Milkability and calving ease were influenced slightly in a favourable direction.
Finally the possibilities of comparing European populations according to the IDF-proposals are discussed. Links between the European populations at present are only through the use of American bulls, most of them were progeny-tested and used in Europe in planned matings. An approach should be taken, to use this material to get hints about the base differences between the European countries. Nevertheless it should be tried to organise an exchange of semen from test bulls across countries and to get an unbiased proof on these in each of the cooperating countries.
Population genetic examination on the heterosis effects in crossbreeding Hungarian Simmental cattle with Herefords F. SZABO
University of Agricultural Sciences Keszthely, Faculty of Agricultural Sciences Keszthely, Hungary
We studied the heterosis effects in characteristics having fundamental influence on meat production as a result of crossbreeding Hungarian Simmental cattle with Herefords.
Evaluating a considerable number of data, Fi population was found to be superior not only to the average of the two purebreed populations, but also to that of the better parent population in major maternal characteristics. As a result of crossing, the following results were found as a consequence of heterosis effects : 7-8 p. 100 at the age of first calving, 6-9 p. 100 in the period between two calvings, 5-8 p. 100 at calving rate and 2-3 p. 100 in the rate of weaned calves.
Live weight, boned meat and meat production for I day of life and the percentage of meat, pectoral and abdominal tallow and tallow as a result of boning in the case of F l population gave better results than the average of Hungarian Simmental and Hereford, but fell short of Hungarian Simmental.
No heterosis effect was found in judgement scores of live animals and in killing out percentage. | v3-fos |
2016-05-17T11:56:25.055Z | {
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} | s2 | Study on milk proteins loci in some decreasing Italian cattle breeds
The genetic polymorphism of the structural loci of five of the six main lactoproteins, a-lactalbumin, (3-lactoglobulin, a, l(3and x-casein, was examined in Grey Alpine, NoricaPinzgau, Aosta Black Pied, Aosta Red Pied, Piedmont and Chiana cattle breeds. The analysis is concerned with the gene frequencies at the five loci, the frequencies of the allelic combinations controlled by the genetic unit formed by the three casein loci, the linkage disequilibrium in this cluster of loci, the genetic distance between the populations and the heterozygosity.
Casein samples were also run with the buffer of P ETERSON & K OPFLER (1966) to subtype p-casein A into A l , A 2 and A 3 .
The frequencies of the allelic combinations controlled by the genetic unit formed by a s¡ -Cn, (3-Cn and x-Cn loci were calculated by the iterative method (C EPPELINI et al., 1955) and the linkage disequilibrium in this cluster of loci was examined for loci pairs (WEIR & C OCKERHAM , 1978).
The genetic relationships between the six breeds were evaluated by two methods suggested by BOUQUET & G ROSCLAUDE (1968) and by N EI & R OYCHOUDHURY (1974).
The genetic variability was evaluated for the single locus, for the whole of the loci considered and for the casein cluster (N EI , 1978). Important observations are concerned with the rare variants which may suggest interesting relationships between breeds, even if it is necessary to keep in mind that electrophoretically identical variants may derive from different alleles and so conclusions drawn from the presence of rare variants should be supported by biochemical investigations.
The a-La A variant occurs in Piedmont and Chiana breeds, both considered of Podolic origin. Since the Chiana is supposed the oldest Italian cattle breed and was a sacrifical beast in ancient Rome (MASON, 1966), it is worth considering also the hypothesis that the presence of the a-La A variant is the result of introgression from Bos indicus, possibly brought to Rome as tribute (BAKER & M A rrwELL, 1980). The presence of this variant in Piedmont breed is in agreement with the hypothesis, supported by several studies on genetic polymorphisms and by the presence of bifid processes to the last thoracic vertebra, that the breed derives from cross-breeding of Aurochs type with zebu from Pakistan (M ALETTO , 1973). The finding in Pin!gau and Aosta Red Pied breeds of P-Lg D variant, first described in Montbeliarde , is in agreement with the classification recently proposed by BAKER & M ANWELL (1980), who consider these breeds derived from the same group, namely Upland brachyceros. A SCHAFFENBURG (1968) had already observed that the P-Lg D allele was a characteristic of cattle of the Simmental type, although its occurrence in Danish Jersey suggested that the D variant may be more widely distributed. Subsequent studies on other cattle breeds showed that this variant is prevalently present in those which are considered of the same origin ; so the j 3-Lg D allele may be a very useful marker in differentiating breeds.
The rare a si -Cn D, discovered in Flamande a) and then observed in five additional French breeds (G ROSCLAUDE , 1979), was till now described in four Italian breeds : Brown Alpine, Reggio, Rendena and Chiana (Russo & M ARIANI , 1978) ; its occurrence in Aosta Black Pied and Piedmont shows that in Italian breeds the D variant is rather spread although with low frequencies.
The very rare fi-Cn A 3 variant was only observed in Grey Alpine and Piedmont breeds.
We indicated as x-Cn! the postulated allele which determines the new variant observed in Grey Alpine (Di S TASIO & MERLIN, 1979) ; however, the hypothesis on the existence of this allele was not till now confirmed since we lack segregation data, while biochemical analysis is not yet completed.
Apart from Piedmont breed in which Osi -Cn locus reveals a statistically significant lack of homozygote genotypes CC, the other breeds are in genetic equilibrium for all loci examined. Table 3 shows the results of the analysis of ŒSI!Cnp-Cn -x-Cn cluster. The Œ SI -Cn B — p-Cn! — !-CnA haplotype, predominant in almost all the breeds of Bos taurus origin, is the most frequent in Grey Alpine, Pinzgau and Piedmont breeds ; the Œ scC n Bp-Cn A ' -x-Cn B and a . ,-Cn B -!-CnA2 -x-Cn u combinations are the predominant ones in Chiana and Aosta Red Pied respectively ; a.,-Cn B -(3-Cn Bx-Cn B haplotype is predominant in Aosta Black Pied and shows a frequency similar to that observed in Normande (G ROSCLAUDE B t al., 1966 b).
With regard to !-CnA3, it was pointed out by G ROSCLAUDE (1979) that in European breeds this allele appears associated with ΠsI -Cn c , while our investigations show it associated both with ΠSI -Cn c (in Piedmont) and with ΠSI -Cn B (in Grey Alpine). On the basis of the phylogenetic tree proposed by G ROSCLAUDE we may suppose that the (3-Cn A 3 variant observed in Grey Alpine is not the same as the one observed in other European breeds ; obviously, this hypothesis should be confirmed by biochemical investigations.
It is worth noting the presence at o tsi -Cn — p -Cn cluster of some types considered as recombinants : as i -Cn C -,(3-Cn Al and Œs I -Cn c -(3-Cn B . The first was observed in Grey Alpine, Pinzgau, Aosta Red Pied and Piedmont ; the second was observed in Grey Alpine, Aosta Black Pied, Aostct Red Pied and Piedmont. In table 3 we indicated also the a si -Cn C — j3-Cn C type for which we have no direct evidence. The existence of these recombinants do not alter the linkage disequilibrium which is significant between all the three loci, with the exception of Pinzgau, where it is significant only between a,,-Cn and (3-Cn and between ,(3-Cn and x-Cn ; in Aosta Red Pied we did not observe linkage disequilibrium between the three loci (table 4).
The existence of polymorphism of the a-lactalbumin system in Piedmont and Chiana breeds allowed us to study the relationships between the a-La and P-Lg loci in breeds of Bos taurus origin. On the basis of the genotypic distributions we found no association between the two loci, in accordance with the data reported on Malagasy zebu (G ROSCLAUDE et al., 1974) and on Somali zebu (Di S TASIO et al., 1979). However, considering the low frequency of a-La A allele, it is necessary to extend the study on a larger number of samples.
The genetic connections between the six breeds examined were evaluated by two methods, trying to make the most of the limited number of loci studied. The method described by BOUQUET & G ROSCLAUDE (1968) expressess the genetic resemblance (R) as an index ranging between 0 and 1 ; we calculated R using genotypic frequencies at P-Lg, c t .1-Cn, p-Cn and x-Cn loci instead of the allelic frequencies at the single loci. The measure of genetic distance proposed by N EI & R OYCHOUDHURY (1974) intends to estimate the number of net codon differences per locus between populations ; of the three different suggested estimates we calculated the minimum distance, D!,.
In the present work the two methods gave similar results (tables 5 and 6).
We found the highest similarity between Piedmont and Grey Alpirze, and between Piedmont and Chiana, according to the hypothesis of their common Podolic origin.
The investigated loci were utilized as marker to get a relative estimate of the genetic variability of the populations (table 7).
Grey Alpine and Piedmont breeds showed high values both at the whole of the loci and at the casein cluster, whereas the other breeds, especially Aosta Red Pied, showed a lower variability perhaps depending on their small sizes, limited breeding areas and adopted mating systems.
IV. -Discussion and conclusions
In small populations, and also in large ones divided by their reproductive structure in many sub-units, we observe an accentuated phenomenon of reduction of the effective population size. In fact, the genetic variability in a population depends upon the rate of the allelomorphic genes in the genotype, on the frequencies of which act the evolutionary process (natural selection, migration, genetic drift, etc.) and the deviations from the random mating determined by adopted mating systems. The investigations on marker loci allow us to evaluate the evolution of the populations by means of gene frequencies, whose variations may be indicative of changes occurring in the whole genotype.
We could estimate the modifications of the genetic variability in three of the breeds examined : Chiana, for which the previous study on milk proteins was completed with the analysis of the acid variants of the p-casein (B ETTINI & M ASINA , 1972), Piedmont and Aosta Red Pied, for which were calculated also the casein haplotype frequencies (V OGLINO & C ARIGNANO , 1975).
In the Chiana breed we observed that the heterozygosity values are increased for all loci, with the exception of the (3-Cn locus almost unvaried. However, from our results the Chiana compared with the other breeds shows a relatively high variability for the single loci and for the whole of the loci considered, but a low variability for the casein cluster. This situation, together with the absence of recombinant types, which are related with the genetic effective population size, shows a certain uniformity of the breed.
From available information on the pedigree of analyzed cows it was possible to identify the genotype of two bulls employed in A.I. which revealed the same genotype : a src n c -!-CnA2 -x-Cn A /a u ,-Cn B -(3-Cn^' -x-Cn B . The use in A.I. of these bulls may explain the relatively high frequencies of a9!-Cn! -P-C N A ' -x-Cn A and ag i -Cn B -!(3-CnA' -x-Cn B types in the breed. Our purpose is to identify the genotype of the other bulls in A.I. in order to have also for the males the informations on the five loci examined. With respect to Piedmont breed we calculated that the heterozygosity values both at the single casein loci and at the casein cluster are increased. This may be a result due to the preferential and large utilization in A.I. of bulls possessing alleles with low frequencies which in a short time spread in the population. This result is in agreement with the data reported by Di S TA sio et al. (1977) based on the study of some blood biochemical polymorphisms.
On the contrary, in Aosta Red Pied breed we observed a reduction of the variability in all cases. We may suppose that the limited breeding areas, the particular geographic environment and inbreeding are the main causes of this situation, although a low genetic variability may be an original characteristic of the breed.
Up to the present the problem on the critical value of the variability, beyond which the negative effects on productive and reproductive characteristics appear, is distant from a satisfactory answer, involving many and complex phenomena whose discussion is beyond the aim of the present paper. However, the presence in these breeds of recombinant types at the casein cluster suggests that their population size is up to now not much reduced.
Indeed the rather limited number of samples examined may affect the conclusions presented here, but our results will be completed with those obtained by laboratories which studied other markers (about twenty loci) in the same breeds. It is hoped that our genetic investigations will contribute to a rather comprehensive picture of these cattle populations, some of them being to-day in hard need of an immediate action to be saved.
Received for publication in January 1982. | v3-fos |
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} | s2 | Influence of diets low in protein or lysine on the intestinal flora of chicks with reference to cecal contents.
To determine the effect of a certain diet on the intestinal flora of chicks, the cecal flora of chicks fed on a low protein or low lysine diet was examined. The cecal flora of chicks fed on the low protein diet was similar to that of chicks fed on a normal protein diet, but the total count of bacteria, Eubacterium and Enterobacteriaceae in the cecal content of chicks fed on the low lysine diet containing a formulated amino acid mixture minus lysine was significantly lower than that of chicks fed on the control diet. The total count of Lactobacillus in the cecum was remarkably reduced by feeding the amino acid diet, especially the low lysine diet. Levels of most free amino acids in the cecal contents of the low protein group were significantly lower than those of the control. Lysine, leucine, phenylalanine, methionine, histidine, glycine and tyrosine of the cecal contents in the low lysine group were significantly lower than those of the control group.
To determine the effect of a certain diet on the intestinal fl ora of chicks, the cecal flora of chicks fed on a low protein or low lysine diet was examined. The cecal flora of chicks fed on the low protein diet was similar to that of chicks fed on a normal protein diet, but the total count of bacteria, Eubacterium and Enterobacteriaceae in the cecal content of chicks fed on the low lysine diet containing a formulated amino acid mixture minus lysine was significantly lower than that of chicks fed on the control diet.
The total count of Lactobacillus in the cecum was remarkably reduced by feeding the amino acid diet, especially the low lysine diet. Levels of most free amino acids in the cecal contents of the low protein group were significantly lower than those of the control. Lysine, leucine, phenyl alanine, methionine, histidine, glycine and tyrosine of the cecal contents in the low lysine group were significantly lower than those of the control group. Key Words cecal flora, low protein diet, low lysine diet, cecal contents, free amino acids It is well known that intestinal microflora of chicks has a beneficial effect on the host of sparing nitrogen, under the condition of a nitrogen-free diet (1, 2) or a low lysine diet (3). On the other hand, there are some competitive interactions between the host and its intestinal microflora under the condition of an adequate diet (4). However, little is known about the intestinal flora of chicks given a low protein diet or a low lysine diet. In this work, intestinal flora and nitrogen compounds of chicks given a low protein diet and also a low lysine diet were examined, in order to determine the relationship between the intestinal flora and nitrogen-sparing action. a Purified egg protein made by Taiyo Foods Co ., Ltd. (Yokkaichi, Japan) was used. b,c Composition is the same as that in diets used by Ishibashi (9) . Crop and cecal contents were weighed and aliquots thereof were transferred into anaerobic transport media (5) and cultured promptly. The remaining aliquots were used for the analysis of nitrogen compounds.
Bacterial procedure. Bacterial techniques used were essentially the same as those of Mitsuoka et al. (4) with some modification. Media and cultivation are summarized in Table 3. The compositions of the media have been described in previous papers (4)(5)(6)(7). Five different non-selective media and 10 different selective media were used for the isolation of bacteria. All media except modified Medium 10 and (NH4)2SO4 medium were poured into ordinary Petri dishes. Modified Medium 10 and (NH4)2SO4 medium were prepared by the "plate-in-bottle" method (4). Samples were weighed and homogenized with 10 or 100 volumes of sterile anaerobic diluent in a homogenizer for about 30 sec in a CO2 atmosphere. From appropriate dilutions, 0.05ml of sample was spread with a sterile glass rod over a half to a quarter of the whole surface of each agar medium, and incubated under the conditions given in Table 3.
After incubation, the number of colonies of each recognizable type was counted for all plates. A colony of each type was selected and identified. Organisms were subjected to Gram staining and were classified into broad groups on the basis of morphological and biochemical characteristics. In a suitable dilution for each culture plate, the number of colonies of the same bacterial group was counted and expressed as counts per g of wet material. When the count on the non-selective media was higher than that on the selective media, the former was regarded as the accurate viable count of the corresponding bacterial group. A direct microscopical count was also measured using the procedure for direct milk count. Chemical analysis. Total nitrogen and 5% trichloroacetic acid (TCA)-soluble nitrogen (non-protein nitrogen, NPN) of cecal contents were determined by the micro-Kjeldahl procedure. Ammonia nitrogen was determined by Conway's meth od (8). Free amino acids were determined using an automatic amino acid analyzer (JEOL, JLC-6AH).
Body weight gain and cecal contents
Results for body weight gain and cecal contents are shown in Tables 4 and 5.
Final body weights and body weight gains in the low protein and low lysine groups were significantly lower than those in the respective control group, but cecal contents of the deficient groups were not significantly lower. Table 7.
Cecal flora of chicks fed on a low protein diet and a normal protein diet.
a,b See Table 6 .
Effect of the low protein diet on microbial flora of chicks Microbial floras of crops and cecums of chicks fed on the low protein diet are shown in Tables 6 and 7 respectively. No significant difference was found between the flora of the low protein group and that of the controls. Figure 1 shows the counts for growth on (NH4)2SO4 medium, expressed as percentages of total bacterial counts in the cecal contents of chicks. The counts obtained from the low protein group were greater than those of the control.
Effect of the low lysine diet on microbial flora of chicks
The cecal flora of chicks fed on low lysine diet is shown in Table 8. Total counts Fig. 1. Bacterial counts grown on (NH4)2SO4 medium as % total bacterial counts in the cecal contents of chicks given the low protein diet and the normal protein diet. of bacteria, Enterobacteriaceae, and Eubacterium in cecal contents were significantly lower than those of chicks fed on the control diet. The counts for the other bacterial groups under the condition of feeding the low lysine diet seemed to be lower than those on feeding the control diet. The number of lactobacilli in the cecal contents of chicks receiving the protein diets was about 108 (Table 7), and the number of lactobacilli decreased remarkably on feeding the amino acid diets, especially the low lysine diet ( Table 8).
Effect of the low protein and the low lysine diets on nitrogenous compounds of cecal contents
Results for nitrogen compounds in cecal contents are summarized in Tables 9-12. Concentrations of total nitrogen and ammonia nitrogen in the cecal contents FLORA OF CHICKS 507 of the low protein group were not significantly lower than those of the control group. The non-protein nitrogen concentration of the low protein group was significantly lower, and most of the free amino acids except phenylalanine , arginine, proline, and tyrosine, were significantly lower than those of the control (Table 11). Total nitrogen and non-protein nitrogen in the cecal contents of chicks fed on the low lysine diet were not significantly lower than those of the control . Lysine, leucine, phenylalanine, methionine, histidine, glycine and tyrosine of cecal contents of chicks fed on the low lysine diet were significantly lower than those of the control (Table 12). Ammonia nitrogen concentration in the cecal contents of the low-lysine group was significantly lower than that of the control. DISCUSSION Intestinal flora and fecal flora of chicks have already been examined by Ochi et al. (12) and Kimura et al. (13), and it is well known that intestinal flora become established 2-4 weeks after feeding (12). In our present data , the counts of total bacteria and Enterobacteriaceae were higher and those of lactobacilli were lower , than those of the earlier data (12). The counts for the other bacterial groups in our CECAL FLORA OF CHICKS 509 present experiment were of the same level as that of the earlier data (12). These differences were thought to derive from our use of purified diets. The body weight gain and the concentrations of non-protein nitrogen and of most amino acids were significantly reduced by the intake of the low protein diet, but the cecal flora was not significantly changed. Most of the predominant bacteria (80%) isolated from the cecal contents of chicks given the low protein diet were grown in the (NH4)2SO4 medium. In contrast, only a few bacteria (8%) isolated from the cecal contents of chicks given the normal protein diet were grown in the same medium (Fig. 1). These results suggest that the predominant bacteria of the cecal contents of chicks given the low protein diet were adapted to the low nitrogen environment in the cecum, and acquired an ability to grow on the medium which contains ammonia and small amounts of amino acids and proteins.
At a low level of lysine, total bacteria, Enterobacteriaceae and Eubacterium counts were reduced. It is uncertain whether the decrease in these bacterial counts is a cause or a result of the beneficial effect of nitrogen reutilization of the host fed on the low lysine diet (3). A decrease in bacterial counts in the cecal contents of chicks was observed only at a low level of lysine in this experiment, but the concentrations of nitrogenous compounds in the low lysine group were not the lowest among the four diet groups (normal protein diet, low protein diet, normal lysine diet and low lysine diet). Therefore, the decrease in the bacterial counts could not be explained by the decrease of the nitrogenous compounds. Lactobacilli, which were reported to exist in counts of 109 per g wet weight of the cecal content of chicks (12), were reduced or not detected in the cecal contents of the amino acid diet groups, the low lysine group in particular (103.5).
Barnes (14) suggested that the cecum of chicks is a site for reabsorption of water and non-protein nitrogen, and Salter and Fulford (15) concluded that the intestinal flora has little influence on the digestion of dietary proteins but may play an important role in the degradation of endogenous proteins. Proteins in the cecum of chicks given amino acid diet were endogenous, because the test diets did not contain proteins. Proteins in the cecum of chicks given the protein diets were also thought to be endogenous because proteins in the cecal contents of the chick given the protein diets were as few as those of the lysine diets (Tables 9 and 10). These results support the suggestion of Salter and Fulford (15). Protein and amino acids in the test diets given to chicks might be digested and absorbed up to the time of the ingesta reaching the cecum, only endogenous proteins existing in the cecum.
Remarkable differences of cecal content weights existed between the low and normal protein diets and between the low and normal lysine diets but were not significant, because individual variation was great. | v3-fos |
2018-04-03T01:10:20.107Z | {
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} | s2 | Some properties of starches of opaque-2, sugary-2 opaque-2, and waxy opaque-2 mutants of two broad-based synthetic cultivars of maize.
Starches of the opaque-2, sugary-2, opaque-2, and waxy opaque-2 endosperm mutants of two broad-based synthetic cultivars (Temp HA and Temp HB) of maize adapted to the more temperate areas of the world are, in general, similar to the respective starches of inbred maize background with regard to amylose percentage and the distribution of linear alpha-D-(1 leads to 4) linked unit-chains of amylopectin, starch-granule susceptibility to amylases, and properties examined by X-ray diffractometry and photopastegraphy. Changes in starch content and morphology of starch granules by scanning electron microscopy in dissected endosperms of the two broad-based synthetic maize cultivars following germination 0, 2, 4 and 6 days after planting were also investigated.
Since the discovery of opaque-2 (o2) maize (Zea mays L.) as a high-lysine cereal (1), the urgent need for cultivars adapted to the more temperate areas of the world has been recognized at Purdue and the International Maize and Wheat Improvement Center, E 1 Batan, Mexico. Two broad-based synthetics , temperate (Temp) HA o2 and Temp HB o2 were developed at Purdue from diverse germplasm from around the world and the U. S. Cornbelt (2).
Furthermore, considerable effort has been concentrated on the development of double-mutant combinations with o2, sugary-2 opaque-2 (su2o2), waxy opaque-2 (wx o2) and brittle-2 opaque-2 (bt2o2) (3). The protein quality is at least maintained or is even superior and kernel vitreousness is improved in Temp HA su2o2 and Temp HB suzoz cultivars compared to the oz synthetics. The wx oz double mutant has a protein quality equivalent to oz and is a modified starch type. All of these improved quality maize cultivars, however, have reduced agronomic yield when compared to the more elite normal hybrids, and from this standpoint their acceptance has been limited. This paper describes the isolation and characterization of maize starch granules from kernels of Temp HA o2, Temp HB o2, Temp HA su2o2, Temp HB su2o2, and Temp HB wx o2 broad-based synthetics. Inbred lines 0h43 su2o2, 0h43 wx o2, 0h43 ae, B37 ae near isogenic conversions and the Oh43 and B37 normal counterparts were used as controls in some characterizations for comparison. The properties of the starches of these improved high-quality protein maize synthetics have not heretofore been investigated. Amylose percentage and the distribution of average chain-lengths of amylopectin were determined by the use of gel filtration after debranching starch with Pseudomonas isoamylase. Some physical and chemical properties of the starches were examined by scanning electron microscopy (SEM), X-ray diffractometry and photopastegraphy, together with evaluation of starch granule susceptibility to amylases. Changes in starch content and morphology by SEM of starch granules in dissected endosperm of maize cultivars adapted to the temperate areas following germination 0, 2, 4 and 6 days after planting were also investigated.
MATERIALS AND METHODS
Maize kernels. Mature maize (Zea mays L.) kernels of several endosperm mutants in two broad-based synthetic backgrounds were used: Temperate (Temp) HA o2, Temp HB 02, Temp HA su2o2, Temp HB su2o2 and Temp HB wx o2. In addition, inbred lines Oh43 su2o2, Oh43 wx o2, Oh43 ae, B37 ae near isogenic conversions and the Oh43 and B37 normal counterparts were used as controls in some characterizations for comparison. The materials were grown at the Purdue Agronomy farm.
Starch granules. Starch granules were prepared by a method reported previously (4).
Other methods. The methods for debranching of starches with Pseudomonas isoamylase, gel filtration of debranched starch on a column of Sephadex G-75, analytical methods for fractionated materials (5), and the methods for determi nation of starch-granule susceptibility to amylases (4) have been reported prep viously. Preparation of germinating seeds, procedures for specimen mounting and observations of starch granules in dissected maize kernels by SEM have been described earlier (6,7). Starch contents of maize kernels were determined by a method reported previously (8,9). X-ray diffractometry was recorded by a method of Hizukuri et al. (10), and the procedure for photopastegraphy has been described earlier (11).
RESULTS
Elution profiles of debranched starches by gel filtration and average chain-lengths of apices of peaks II and III Table 1 summarizes the distribution of carbohydrate in each fraction (Fr.) and average chain-lengths (CL) of apices of peaks II and III for isoamylase-debranched maize starches of several genotypes of Temp HA, Temp HB, inbred Oh43, and the normal Oh43 counterpart.
Fraction I appeared near the void volume of a Sephadex G-75 column in the normal maize which had typical amylose and amylopectin. This fraction cor responded to amylose of the original starches because the wavelength of maximum absorption (umax) of iodine-carbohydrate complexes of the fraction were in a range of longer wavelength (620-640nm). Fractions II and III originated from amylopec tin of the original starches because the degree of polymerization (CL) of their peaks was 40-45 and 15-20 glucose units, respectively. Starches of wx o2, which consist only of amylopectin, had no Fr. I but Frs. II and III. This finding also indicated Fr. I originated from amylose and Frs. II and III from amylopectin (12).
Contents of Fr. I were 23-24% in the normal and o2, 39-41% in su2o2 and 0% (12), and the chain lengths of the peaks of both Frs. II and III were similar in the normal, 02, su2o2, and wx 02 starches examined.
X-ray diffraction patterns of starches Figure 1 shows X-ray diffraction patterns of native starches of Oh43 normal, Oh43 ae, Temp HA su2o2 and Temp HB su2o2. The patterns of the native su2o2 starches are similar to the normal which corresponds to an A-type crystalline Table 2. Susceptibility to amylases of maize starch granules of several genotypes of two synthetic cultivars and inbred Oh43. a Crystalline preparation from Rhizopus niveus . b Data were expressed relative to enzyme degradation percentage of the normal Oh43 maize starch, namely % degradation of the normal Oh43 maize starch equals 100(4). structure, but were less crystalline. Not shown were the X-ray diffraction patterns of native starches obtained from o2 and wx o2. The patterns of these starches were very similar to each other and to the normal and corresponded to an A-type crystalline structure which is typical of native starches usually found in cereals.
Photopastegrams of starches
The normal and o2 starches showed two-step gelatinization curves, which are typical of normal-type starches of cereals (Fig. 2, curves a, b, d and e), while the wx o 2 starch had a one-step gelatinization curve followed by a downward slope in a range of higher temperature, which is typical of waxy-type starches of cereals (Fig. 2, curve h). The su2o2 starches are gelatinized to a lesser degree (Fig. 2, curves f,g) and show gelatinization curves which are very similar to that of the ae starches having 31% amylose and 15% intermediate fraction (Fig. 2, curve c).
Starch-granule susceptibility to amylases Table 2 shows values of susceptibility of starch granules for the broad-based synthetic maize cultivars and the normal inbred Oh43 to either pancreatin or crystalline glucoamylase of R. niveus. The susceptibility values for 02 starches were nearly identical to those of the normal. The su2o2 starches were digested 2.6-2.7 and 2 times as fast as the normal starch by pancreatin and glucoamylase, respectively. The values for wx o2 starch granules showed quite different susceptibilities . The value for the glucoamylase hydrolysis was the highest among the starches examined.
SEM of endosperm starches of Temp HB o2 and Temp HB su2o2 germinating kernels
Enzymatic erosion of starch granules was visible in the dissected endosperm of all of the maize genotypes in the Temp HA and Temp HB backgrounds following germination 4 and 6 days after planting. However, for brevity, we show photo electronmicrographs of starch granules of the Temp HB 02 and Temp HB su2o2 mutants only (Figs. 4 and 5).
Almost none of the starch granules were attacked by enzymes in Temp HB o2 endosperm 2 days after planting (Fig. 4a). Many starch granules with pores were observed in Temp HB o2 endosperm 4 days after planting (Fig. 4b). The pores on the surfaces of starch granules were bigger in Temp HB o2 endosperm 6 days after planting and sometimes the inner structure of a granule could be observed (Fig. 4c). A few granules seemed to be attacked in Temp HB su2o2 endosperm 2 days after planting (Fig. 5a). We were able to observe many starch granules attacked in Temp HB su2o2 endosperm 4 days after planting (Fig. 5b). Almost all starch granules were attacked in Temp HB su2o2 endosperm 6 days after planting and the inside step-shaped structures of starch granules were frequently observed (Figs. 5c-e). This investigation was supported in part by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan, the Agency for International Development under contract Purdue University, AID csd/2809 and AID/ta-C-1211, "Inheritance and Improvement of Protein Quality and Content in Maize" and the Lilly Foundation. | v3-fos |
2019-08-17T22:12:17.676Z | {
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} | s2 | BOOK REVIEWS
Book reviewed in this article: Digestive physiology and nutrition of ruminants: Digestive Physiology and Nutrition of Ruminants Vol 3 Practical Nutrition Veterinary virology: The Professor Margaret Sabine Virology Course. Advances in Veterinary Virology Veterinary biology and medicine of captive amphibians and reptiles: Veterinary Biology and Medicine of Captive Amphibians and Reptiles Growth and development in cattle: Patterns of Growth and Development in Cattle Laboratory data and biochemical values: Veterinary Laboratory Data. B. Rushton Laboratory data and biochemical values: Biochemical Values in Equine Medicine. D. J. Blackmore and D. Brobst Production diseases of farm animals: Proceedings 3rd International Conference on Production Diseases in Farm Animals Veterinary cancer medicine: Veterinary Cancer Medicine. Numerous authors. Eds G. H. Theilen and B. R. Madewell Diseases of cattle: Diseases of Cattle A to Z Tumours in domestic animals: Tumours in Domestic Animals. Ed Jack E. Moulton
For those seeking resources on sports coaching, there is no shortage of available possibilities. One may find topics distinguished by sport (coaching swimming, coaching basketball), by level (coaching youth athletes, coaching intercollegiate athletes), by approach (psychological, physiological), and by strategy (offensive, defensive). The literature on sports coaching is seemingly limitless yet the vast majority focus on what could be called the technical side of coaching. Convention would have us believe that effective coaching involves following a certain script or recipe. One need only attend the right coaching workshop, read the right book, or follow in the footsteps of the right mentor coach, to become a successful coach. Books on coaching often follow an approach similar to Rainer Marten's work, Successful Coaching (2004). This extremely popular work targeted for high school and club sport levels includes sections on coaching philosophy, motivation, pedagogy, physiology, and management. While the book touches on ethical issues, sports ethics certainly is not the overall focus. Furthermore, the fascination with this technical side of coaching has unwittingly prevented the consideration of coaching in normative terms.
It is this gap in the literature editors Alun Hardman and Carwyn Jones aim to address. The Ethics of Sports Coaching, another insightful work in the Ethics and Sport series published by Routledge, examines ethical issues directly related to the nature of coaching. The authors do not discount that coaching involves technical expertise and planning but that the nature of coaching, which involves the complexity of human relationships, entails significantly more. In this sense the guiding framework for this book is that 'coaching is fundamentally a moral practice ' (2011, 2). The series of original essays from a variety of scholars focus on normative concerns surrounding sports coaching, from foundational issues such as what it means to be a 'good coach' to ethical matters related to youth sports, dangerous sports, and Paralympic sports. The editors target both academics and practitioners although the work is more likely to serve as either a valuable reference tool for researchers or a text for undergraduate or graduate coursework related to sport studies or sport philosophy. The editors are also conscious of coaching education programs and, to this extent, hope the volume impacts the way these programs are designed and implemented at all levels of instruction.
Hardman and Jones write that their work accomplishes two tasks: 'First, it articulates an alternative conception of coaching as a moral enterprise. Second, it exemplifies how ethical reflection might proceed in a variety of morally-laden contexts ' (2011, 4-5). In many respects the authors point towards ways of thinking that challenge convention with regards to coaching. The general public, for example, may rarely think of coaching in moral terms (sideline histrionics and chair-throwing notwithstanding). Conversely, some individuals may view coaches as inherently selfish and at odds with any ethical framework. Others may believe that while pursuing ethical norms may be ideal, coaching involves real-world pressures and contexts that supersede 'doing the right thing'. After all, elite coaches are more likely to be concerned with winning and notoriety than displaying compassion or humility. In sum, addressing the topic of ethics in sports coaching is a daunting proposition albeit one critical for our broader profession of sports philosophy.
The book is organized in a very straight-forward manner, beginning with conceptual issues related to coaching and then progressing towards topics focused on specific coaching contexts. The work is divided into four sections preceded by an introduction which explains the book purpose and overall structure. The first section examines the nature of coaching itself, addressing normative aims in coaching as well as the role virtues play with regards to coaching conduct. In the second section, the authors focus on issues of character and behavior such as practical wisdom and coaching identity, decision-making in the context of objectivity and subjectivity, and the applicability of virtue ethics for the coaching practice. The third section moves to examining specific coaching contexts, most notably various type of athletes -this includes addressing issues related to coaching at the youth sport level, males coaching females, and coaching Paralympic sport. Finally, the fourth section takes a look at coaching in regards to broader issues that impact the coaching practice -these include performance enhancement, coaching dangerous sports, and expatriate coaching.
All of the essays demonstrate a familiarity with the broader philosophical landscape related to sports and coaching ethics. Mike McNamee situates his essay within a neo-Aristotelian framework of virtue-based ethical theory. Paul Davis examines sports coaching in the context of Nagel's notions of objectivity and subjectivity. Essays throughout the book demonstrate conceptually clear, well-reasoned arguments and point towards the ongoing discussion in the sport philosophy and mainstream philosophy literature. One of the clear strengths of this book is that the authors not only demonstrate philosophical expertise but athletic and coaching experience and prowess too. The short list of such experiences and levels of proficiency include a Paralympic champion (Anne-Mette Bredahl), elite-level rugby participant (Steve Olivier), and elite level coach (Sigmund Loland).
In terms of the overall organization and flow, chapters in general tend to build on each other and are, for the most part, related. For example, in his chapter on the The Normative Aims of Coaching, Loland refers to the Aristotelian conception of phronesis or practical wisdom. At this point the editors cross-reference the chapter written by Oyvind Standal and Liv Hemmestad titled Becoming a Good Coach: Coaching and Phronesis. Similarly, in her chapter, Coaching Ethics and Paralympic Sports, Bredahl notes the importance of coach education 'as mentioned elsewhere in this book'. The essays, while often in agreement, also prompt reflection and, at times, differing points of view. In his essay Males Coaching Female Athletes, Michael Burke posits that behavioral codes implemented by sports organizations can help prevent potential ethical problems. Conversely, McNamee cautions that codes of conduct or other approaches relying on rules or principles 'are not exhaustive of the basic facts of moral life, a picture of which is incomplete without reference to the virtues' (2011,33). On this point the editors would contend that this represents the diversity of ideas surrounding coaching ethics and encourages additional dialogue in the area. As a final note in regards to organization, an editorial postlude would provide a fitting summary and perhaps encouragement towards applying these ethical concepts to sports coaching and the continuing dialogue surrounding such issues.
One of the challenges of presenting a collection of works is maintaining a tight sense of focus on the topic at hand -in this case ethical issues related to coaching. While virtually all of the essays maintain this focus, some follow more closely than others. For example, the chapter on Rethinking Luck and Justice, while insightful in its own right, is somewhat tangential to the issues of ethics and sports coaching. In their conclusion, Richard Bailey and Martin Toms the authors offer implications for coaches -to recognize and even challenge 'the structural unfairnesses within talent development systems' (2011, 161)but the issues are not as central as some of the other essays.
In a number of ways the essays represent starting points for continued discussions related to ethical issues of sports coaching. For example, Bredahl, writing from a perspective of clinical psychology as well as a Paralympic crosscountry skiing and biathlon, examines coaching ethics as it relates to Paralympic sports. She notes the need for coaches to 'treat athletes with respect' and 'the particular difficulties associated with the athlete's impairment and their individualized experience of it ' (2011, 141). To some degree all athletes have areas of impairment -some physical, some emotional, some intellectual, some social. As Hardman and Jones rightly contend, the nature of coaching involves the very messy task of dealing with humans. Future works might focus on the extent of this messiness and how coaches might work to relate with and successfully coach athletes with a variety of ''impairments'' in this sense. Other possible topics that could serve as fodder would be how to handle issues with parents, and sport specialization at an early age. In addition, in that The Ethics of Sports Coaching examines coaching from a largely Westernized point of view, future projects could examine ethical issues surrounding sports coaching from an Eastern viewpoint.
To summarize, The Ethics of Sports Coaching makes an outstanding contribution to the sport philosophy literature. The work addresses an important aspect of sport ethics in an insightful and well-reasoned manner. The authors approach the topic from an academic perspective yet with clear experiential understanding of the complexity of sports coaching and human relationships. The book will no doubt encourage continued discussion around this significant topic. | v3-fos |
2014-10-01T00:00:00.000Z | {
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} | s2 | Use of biological assay systems to assess the relative carcinogenic hazards of disinfection by-products.
Other workers have clearly shown that most, if not all, drinking water in the U.S. contains chemicals that possess mutagenic and/or carcinogenic activity by using bacterial and in vitro methods. In the present work, increased numbers of tumors were observed with samples of organic material isolated from 5 U.S. cities administered as tumor initiators in mouse skin initiation/promotion studies. Only in one case was the result significantly different from control. In studies designed to test whether disinfection practice contributes significantly to the tumor initiating activity found in drinking water mixed results have been obtained. In one experiment, water disinfected by chlorination, ozonation or combined chlorine resulted in a significantly greater number of papillomas when compared to nondisinfected water. In two subsequent experiments, where water was obtained from the Ohio River at different times of the year, no evidence of increased initiating activity was observed with any disinfectant. Analysis of water obtained at the comparable times of the year for total organic halogen, and trihalomethane formation revealed a substantial variation in the formation of these products. Considering the problems such variability poses for estimating risks associated with disinfection by-products, a model system which makes use of commercially obtained humic acid as a substrate for chlorination was investigated using the Ames test. Humic and fulvic acids obtained from two surface waters as well as the commercially obtained humic acid were without activity in TA 1535, TA 1537, TA 1538, TA 98 or TA 100 strains of S. typhimurium. Following treatment with a 0.8 molar ratio of chlorine (based on carbon) significant mutagenic activity was observed with all humic and fulvic acid samples. Comparisons of the specific mutagenic activity of the chlorinated products suggests that the commercial material might provide a useful model for studying health hazards associated with disinfection reactions by-products.
Introduction
Development and widespread application of coupled gas chromatography and mass spectrometry has provided a much greater appreciation of the nlumber and variety of organic chemicals that can be found in most drinking waters (1). For the most part, the large numbers of chemicals which are identifiable are present at very low concentrations, usually in fractions of a microgram per liter (1). However, in total, the amounts of organic material found in drinking waters derived from surface *Toxicology and Microbiology Division, Health Effects Research Laboratory, U.S. Environmental Protection Agency, Cincinnati, Ohio 45268. sources are often in the range of several milligrams per liter, hardly a negligible amount. It is now recognized that a very small fraction of the chemicals present in drinking water lend themselves readily to analysis even with today's modern analytical tools.
As a consequence of the above considerations, efforts were begun to obtain an estimate for the general magnitude of the problem without expending the huge sums of money to test individual chemicals as they were identified. A number of research groups around the world began to examine test mixtures of organic chemicals extracted from drinking waters. The test systems employed varied from the application of the Salmonella! microsome assay (2-4) for mutagenic chemicals 216 developed by Ames et al. (5) to chronic studies conducted in rodents (6)(7)(8). In each of these cases, carcinogenic and/or mutagenic activity was detected in finished drinking water. In the U.S., Loper and co-workers (2) reported mutagenic activity oforganic chemicals concentrated from the drinking water of first five, then six U.S. cities by a technique developed by Kopfler, et al. of our laboratory (9). Following up on these initial observations it was shown that these concentrates also contained chemicals capable of transforming BALB/3T3 cells (10) and a preliminary report demonstrated the ability of certain of the samples to initiate tumors in the mouse skin (8).
The work of Bellar and Lichtenberg (11) in the U.S. and Rook (12) in Europe changed the view of drinking water problems substantially. These workers documented that the organic chemicals most commonly found in drinking water and usually at the highest concentrations were produced by chlorination. These chemicals were, of course, the trihalomethanes. From this initial observation has c6me the gradual appreciation of a problem which is a direct result of currently accepted methods for producing a microbiologically safe drinking water. Before this, chemical hazards in drinking water had been viewed simply as one of industrial and/or agricultural contamination of the source water. Now any assessment of health hazards in drinking water must consider the qualitative changes in chemical composition that arise from water treatment-in particular disinfection.
Our laboratory has been concentrating on two problems. First, efforts have been directed towards determining if chemicals obtained from drinking water can be demonstrated to be carcinogenic in vivo. Second, an attempt is being made to develop a base of information which can be applied to a wide variety of water supplies. This latter effort requires that sufficient understanding of processes which contribute to the formation and occurrence of carcinogenic chemicals in drinking water be developed so that the risks involved can be minimized. It includes consideration of the carcinogenic activity of the products of four alternative disinfectants; chlorine, chlorine dioxide, ozone and chloramine.
With regard to disinfection by-products it has been established that a substantial fraction of the organic material found in surface water sources is of natural origin. A substantial portion of this natural background is composed of humic and fulvic material that has been shown to be a major precursor of trihalomethanes following their chlorination. Recovery of the humic material from water in quantity is difficult, but humic acid obtained from peat is commercially available. The question is whether differences in the chemical properties of BULL ET AL. humic material obtained from these sources result in the formation of clearly different toxic byproducts. Therefore, we report here an initial attempt at determining whether humic acid provides a good model for the generation of biologically active by-products of disinfection for more comprehensive testing.
Sample Preparation
The five cities studied were selected as being representative of water supplies contaminated by various sources, or as having no known contamination by municipal, industrial or agricultural sources. Miami was chosen as representing ground water containing large quantities of natural organic chemicals; New Orleans, a surface water source impacted by industrial wastes; Ottumwa, a surface water contaminated by agricultural runoff; Philadelphia, a surface water impacted by municipal waste; and Seattle, a pristine surface water obtained from a protected watershed in the Cascade mountains. In all cases finished drinking water was used.
The concentrated organics were prepared for EPA by Gulf South Research Institute by the procedure described by Kopfier et al. (9). The samples are concentrated from multiple 200 liter quantities of tap water by reverse osmosis using a cellulose acetate membrane (CA) with sufficient concentrated HCI added to maintain a pH of 5.5. The reject from the CA membrane is passed through a heat exchanger to maintain the water temperature at 15°C. Part of the reject stream is diverted through a Donnan softening unit to avoid salt precipitation. The CA permeate is adjusted to pH 10 and then concentrated by reverse osmosis using a nylon membrane. The nylon permeate is discarded. Both the CA and the nylon concentrates are then adjusted to neutral pH and extracted with pentane and methylene chloride. The aqueous phases are adjusted to a pH of 2 with HCl and again extracted with methylene chloride. For purposes of this study these fractions were combined and the solvent removed to produce the reverse osmosis extract sample (ROE).
The residual aqueous concentrate remaining afteiextraction with the organic solvents is passed through a column of purified XAD-2 resin at pH 2. Following the removal of metallic oxides and other inorganic agents by eluting with 1M HCl and distilled water the organics are then eluted from the column with 95% ethanol. The ethanol is removed from the eluate by vacuum distillation and eluates from both columns are combined to produce the XAD sample.
A series of six experiments has been initiated to examine the effects of seasonal variation on the ability of different disinfectant treatments to produce carcinogenic by-products. The testing of the first three of these has progressed to a point where results can be reported. Routine analysis for general water parameters, however, are only available for the last three experiments. Consequently no parallels between biological and chemical results are as yet possible. In the first three studies, settled, coagulated and filtered Ohio river water was subjected to chlorine, chloramine, chlorine dioxide, or ozone disinfection. Disinfectant demands were determined on all samples prior to adding the disinfecting agents. The disinfectant contact times varied but were designed to result in residuals that were at a practical minimum. The doses of disinfectants amounted to 2.0-2.5 mg/l. chlorine, 1.0-3.0 mg/l. ozone, 2.0-3.0 mg/I. chlorine dioxide, and 2.0-3.0 mg/I. combined chlorine. A control consisted of filtered, but nondisinfected water. Waters and concentrates obtained in March, July and October of 1980 were assayed for various physical, chemical and microbiological parameters for quality control purposes (data not shown). All water samples were then concentrated by reverse osmosis using a cellulose acetate membrane. The May 1977 samples were concentrated 100-to 180-fold. Subsequent samples have all been concentrated to 400-fold, using freeze-drying to supplement reverse osmosis concentration where necessary.
Humic and fulvic acids were isolated from two natural lakes, Black Lake and Lake Drummond, by Christman and co-workers of the University of North Carolina as they have described previously (13). The major differences between these two lakes is the distribution of the total organic carbon between the recoverable humic and fulvic acid fractions. Black Lake contains a much higher proportion of its TOC as humic acid. Both lakes are remote from direct industrial contamination. Commercial humic acid was obtained from Fluka of Switzerland. These materials were prepared by adding 4 g of humic material to 500 ml deionized water. The pH was adjusted to 12 with NaOH and the solution was stirred overnight in the dark. The solution was then centrifuged at 16,000g for 20 min and the supernatant was brought to pH 3 by addition of HCl. The centrifugation was repeated. The supernatant was diluted to 1 liter, and the pH was adjusted to 7. The resulting total organic carbon (TOC) content was 2.5 g/l. Chlorination of the humic solution was accomplished by adding enough NaOCI (pH 12, 10 g/l.) to give a chlorine to carbon ratio of 0.8:1. After readjusting the pH to 6, the solution was diluted with sufficient deionized water to yield a TOC concentration of approximately 1 g/l. The solution was kept in a Teflon-capped bottle with no head space at room temperature for 5 days, at which time chlorine 217 consumption was essentially complete. The final pH of this solution was 2.5 and the total chlorine residual was less than 1 ppm. The sample was then maintained at 4°C until time of assay for mutagenic activity. At the time oftesting in Salmonella strains, the pH was adjusted to 7.
Analytical Methods
Measurements of trihalomethane (THM) concentrations, total organic halogen (TOX) and total organic carbon (TOC) were performed by Alan Stevens and his staff of the Drinking Water Research Division of the Municipal Environmental Research Laboratory, U.S. Environmental Protection Agency, Cincinnati or by Dr. Robert Lingg of the Health Effects Research Laboratory, U.S. Environmental Protection Agency, Cincinnati. Trihalomethane concentrations were determined by using the purge and trap method described by Bellar and Lichtenberg (14). Total organic halogen (TOX) was analyzed directly in ambient water samples by using a Dohrmann Total Organic Halogen Analyzer. In studies involving chlorination of humic and fulvic acids 0.2 ml of the sample was diluted to 50 ml before the analysis was performed. Total organic carbon (TOC) analyses were performed using the Dohrmann Total Organic Carbon Analyzer. Again ambient water samples were analyzed directly and chlorinated humic acid samples were diluted from 1.0 ml to 50 ml before analysis.
Mouse Skin Initiation/Promotion Assays SENCAR mice were obtained from Oak Ridge National Laboratories, Oak Ridge, Tenn. The SEN-CAR mice are a strain of mice selected for their sensitivity to developing skin tumors in response to 7,12-dimethylbenz(a)anthracene (DMBA) (15,16). The animals were 6 to 9 weeks of age when started on study. The dorsal hair of each mouse was shaved with electric clippers three days before the initial dose of the test compound and then shaved once weekly during application of the promoting agent to facilitate the application and the observation of tumor occurrence. All animals received distilled water and Purina Laboratory Chow ad libitum throughout the study.
Experiments with the five cities concentrates utilized total doses of 150 mg/kg (4.5 mg/mouse) of combined ROE fractions and the XAD samples applied subcutaneously in the back of the mice in six injections over a 2 week period. The vehicle used was 0.1 ml Emulphor (a polyoxyethylated vegetable oil). Two weeks after the last injection, a 218 promotion schedule was begun involving the application to the shaved back of each mouse of 1 ,ug of 12-O-tetradecanoylphorbol 13-acetate (TPA) 3 times weekly in 0.1 ml acetone. After 20 weeks of application, TPA treatment was suspended and the animals were held for observation for an additional 28 weeks.
During the course of this and the following experiments the animals were weighed weekly and observed for tumor incidence. The incidence of both papillomas and carcinomas was charted weekly. Skin lesions were included in the cumulative papilloma count when they persisted for a minimum of 3 weeks. Following completion ofthe promotion period the animals are held for observation until they reach one year of age and then are sacrificed. Major organs and all grossly observed lesions are sectioned and fixed in a 10% buffered formaldehyde solution for subsequent histopathological diagnosis (histopathological confirmation made by Dr. John Orthoefer, HERL/Cincinnati).
In each of the three studies involving aqueous concentrates doses were administered subcutaneously in the shaved dorsal area of the mice. A total dose of 1.5 ml of the aqueous concentrates (6 x 0.25 ml) was given per mouse over a 2 week period. Negative controls received the same volumes of normal saline. As positive control either DMBA in doses of 7.5 or 25 ,ug/mouse or urethane at a dose of 9 mg/mouse was administered by the same routes. Both compounds were given subcutaneously in a 10% Emulphor vehicle. Two weeks following the last initiating dose, a tumor promotion schedule was begun wherein the mice received TPA topically to the shaved dorsum three times a week for 20 weeks, at a dose of 1.0 ,ug TPA in 0.2 ml acetone per application. It should be noted that in the first experiment (May 1977) a slightly different promotion protocol (than other experiments) was used. This involved application of2.5 ,ug TPAlmouse three times weekly for 18 sacrifice (17). Assay for mutagenic reversion of the test bacteria to histidine independence were performed according to the standard plate method of Ames (5). All assays were carried out in duplicate at three sample concentrations both in the absence and presence of S9 (25 jil/plate). Positive controls were assayed concomitantly with the test samples. For assays without S9 activation, these consisted of sodium azide for TA 1535 and TA 100; 2-nitrofluorene for TA 1538 and TA 98; and 9-aminoacridine for TA 1537. For assays with S9 activation, 2-aminoanthracene was used for all strains. Specific genetic markers (histidine dependence, ampicillin resistance, UV sensitivity and crystal violet sensitivity) were confirmed for each strain before use in assays.
Five Cities Samples
The results obtained with the combined reverse osmosis extracts (ROE) from the five cities are shown in Figure 1. Although in all cases the tumor response was higher than in the control group, the sample obtained from Ottumwa stands out. It is notable that the increase in the development of tumors occurred after termination of the promotion schedule.
Results obtained with XAD fractions followed a similar course (Fig. 2). Here all samples again ended up at a higher tumor incidence than that observed in the control group. An excess in the total number of observed tumors per mouse can be used as evidence of carcinogenicity. No significant differences among proportions of animals with and without tumors for the ten test groups and control were found. This negative result might be due in part to the relativelv low response rate (10-30%, N -40 in each group). This type of data is called censured data, since both premature deaths and the end of the study precludes the full expression of the measured parameter in this case tumor incidence.
Graphical comparisons did not reveal any gross differences in censoring patterns among the groups. An additional type of evidence for carcinogenicity would be a shorter time to first tumor distribution in exposed mice compared to control mice. Since the longest response times are observations that are censored the estimates of survival time (time without tumor) are truncated at the end of the study period. As a result, the mean time to first tumor will be underestimated, since very long times are not observed. However, if tumors developing very late in the study period may be due to other factors than exposure (e.g., age), then the time to tumor related to exposure may not be underestimated. .mwwwaw 220 BULL ET AL. cVehicle was 10% Emulphor (a polyoxyethylated vegetable oil applied in six injections of 0.25 ml each). bAfter treatment indicated water was subjected to reverse osmosis with cellulose acetate to the level indicated by the concentration factor (initial volume/final volume).
The mean time to first tumor for each animal was used as the response variable in the following analysis. Comparisons of the distribution of these values for the XAD and ROE concentrates were made within each of the five city groups, using both the generalized Savage and the generalized Wilcoxon Statistic. Since no significant differences were found, the XAD and ROE groups were combined for each city to provide larger groups for the comparison of cities and control. No significant differences were found among the time to first tumor distributions of the five cities and the control using both test statistics (p > 0.20). However, all cities did have a shorter mean time to first tumor (Kaplan-Meier product limit estimates) than the control with Miami and Ottumwa being over two and three weeks shorter, respectively (Table 1). An alternative technique to analyze this type of data is the proportional hazards (or Cox regression) model. In this analysis, both concentrates from Ottumwa showed regression coefficients significantly different from zero, indicating a shorter mean time to first tumor in the Ottumwa group than in the controls. However, no other city concentrate group shows a significant difference from the control. It is notable, however, that all the ROE fractions resulted in shorter mean times to first tumor, ranging from a decrease of 1.9 weeks with the Seattle sample to a decrease of 3.8 weeks with Ottumwa. The XAD samples on the other hand, were much closer to the mean time to first tumor for the control animals with the exception of Ottumwa.
Alternate Disinfectant Reaction Products
The results of the first experiment testing aqueous concentrates from alternatively disinfected drinking waters are shown in Table 2. The overall chisquare analysis comparing the proportion of mice with tumors in the disinfectant groups (saline excluded) was highly significant (p < 0.001). It was found that most of the tumors (classified as papillomas by gross observation) were observed in groups of which 16, 20 and 28% of the animals treated were found to have tumors, respectively. When the overall chisquare was partitioned, these treatments combined were all significantly different from the combined responses observed with the nondisinfected water (0%) and chlorine dioxide treated water (0%).
Although there were small differences in the degree to which these samples were concentrated by reverse dNumber of animals when not same as tumor count. aResults in SENCAR mice after 1 year on study (40 mice/exposure group). bControl animals injected with 0.9% saline. 'Historical control incidence is 8 ± 3% based upon six experiments with a total of 190 animals.
osmosis, there was no correlation between tumor response and degree of concentration (r = 0.4, p -0.50). Consequently, we concluded that the differences were probably due to the reactions between disinfectants and trace organic material present in the water. Papillomas are nonmalignant tumors and in themselves are not life-threatening. Burns and Albert (18) have shown that although carcinomas can arise from or independently of papillomas, the number of papillomas which arise are predictive of later carcinoma development. In Table 3 it can be seen that greater development of carcinomas did occur generally in the groups having more papillomas even though the incidences do not parallel precisely. Most of the carcinomas are to be found in the chloramine, chlorine and ozone-treated water groups. However, the response level of malignant tumors was too low to be statistically significant (p = 0.36).
When the numbers of syst,nic tumors that were observed on termination of this study were examined (Table 3) it was found that eight systemic tumors were found in the chlorine dioxide group. This included four lung adenomas (in three animals), three mammary gland tumors and one stomach tumor in the 50 animals treated with this sam-ple (this includes 25 animals which did not receive topical TPA applications). Increased numbers of lung adenomas (5) were also observed with animals that received the chloramine-treated water. These tumors were not observed at all with the nondisinfected water. Only one lung adenoma and two mammary tumors were observed with the 50 animals injected with saline. The overall chi-square analysis comparing the number of animals with systemic tumors for this experiment is significant at p = 0.05 and the chlorine dioxide group is significantly different from the combined nondisinfected and saline controls at p = 0.01. Other treatments were not significant different statistically.
It was obvious that a single experiment of this type provides far too little information upon which to generalize concerning the relative safety of alternate disinfectant reaction products. Consequently, a series of five additional experiments has been initiated to examine the repeatability of the first experiment over the course of several seasons. Two of these five experiments have progressed to the point where the development of skin papillomas can be analyzed. Table 4 summarizes the results obtained from the experiment just described and these two subsequent experiments. It is clear that these latter two experiments are providing a different picture than that seen in the first experiment. A major difference is that a response rate greater than that observed in the control group is being observed in both experiments for the nondisinfected water. This might be the result of the greater concentration factor involved in these experiments compared to the first. All samples were uniformly concentrated 400-fold by a process initially involving reverse osmosis, but followed by freeze-drying. Whatever the reason, it is clear that the carcinogenic activity associated with alternatively disinfected samples did not significantly exceed that in the nondisinfected samples in these two experiments. It should be pointed out that if taken in total and against historical controls, the average tumor incidence in the chloramine group is almost double the simultaneous control incidence and more than twice the historical control response. In addition, this increased incidence has been consistent across all three experiments. Although the differences are not statistically significant when compared to the simultaneous controls, if this trend is maintained through the remaining three experiments statistical significance may well be achieved.
There has been insufficient time since the completion of the latter two studies to confirm the gross pathological findings at terminal sacrifice. Consequently, comparison ofthe systemic tumors between experimental groups is not yet possible.
Data obtained from analyses ofthe nonconcentrated water used in subsequent experiments provides some insight into the variability of these results ( Table 5). The organic chemical content of the Ohio River tends to be at a minimum during late winter and early spring and increases 60-80% with the lower flow of later summer and fall. These changes result in even more dramatic effects upon disinfection by-products. For example, total organic halogen (TOX) concentration is increased much more dramatically by chlorine, chlorine dioxide and combined chlorine in late summer than in the sample collected in March. In the case of chlorination, the trihalomethane (THM) portion of the TOX is 10-fold that observed in March. It should also be noted that under the conditions of late fall, ozone is capable of significantly increasing the TOX, whereas no significant alterations are observed in March or August.
Mutagenesis Assays
No net increase in the number of revertants was observed with Salmonella strains TA 1535, TA 1537 or TA 1538 treated with commercially available humic acid either before or after the addition of chlorine. As shown in Figure 3 the sample of humic material prior to the addition of chlorine was also without activity in strains TA 98 and TA 100 in the presence or absence of rat liver S9 fraction. However, if the sample is treated with chlorine it gives a clear cut response with both TA 98 and TA 100. A linear dose-response curve was observed in both strains. This activity is apparently destroyed by the incorporation of Aroclor 1254-induced rat liver S9 fraction.
In Table 6 the activity of the humic and fulvic acid fractions from two natural lakes (Lake Drum- . Effect of chlorination on the production of mutagenic activity in solutions prepared with commercially available humic acid. The Ames Salmonella/microsome assay and chlorination of samples were performed as described in Methods. Assays were carried out both with and without S-9 added. Each point represents the average of duplicate determinations. 223 mond and Black Lake) along with that obtained using humic acid obtained commercially from Fluka are shown. In all cases involving the humic acid fraction, the same clear dose-response relationship is seen with the chlorinated material. The unreacted material was without activity as indicated earlier for the commercial humic acid. In all cases the activity of the chlorinated material was decreased by the incorporation of rat liver S9 fraction. It is notable in Table 7 that the degree of chlorine incorporation appears to be quite similar. The TOX/TOC ratio was 0.34 with Black Lake humic, 0.34 with Lake Drummond fulvic, 0.36 with Black Lake fulvic, 0.38 with Fluka humic and 0.44 with Lake Drummond humic. It appears that the specific mutagenic activity relative to either TOC or TOX is virtually identical for TA 100 with humic and fulvic acids from all three sources. In the case of TA 98, however, the specific activities observed ranged from no measurable mutagenic activity with the chlorinated Lake Drummond fulvic acid to a maximum specific activity with the chlorinated Lake Drummond humic acid. Chlorinated fulvic acid from Black Lake gave approximately 21 to 58% of the specific mutagenic activity in TA 98 when compared to the three sources of humic acid treated with chlorine.
Discussion
The present work adds to growing evidence that chemicals with carcinogenic activity are found in Table 6. Comparison of mutagenic activities in chlorinated humic and fulvic solutions derived from a commercial source and two natural lakes. (11) aValues are mean values of duplicate place counts with range values in parentheses. Humic and fulvic samples were assayed in separate experiments with control values indicated by (A) and (B), respectively. Significant precipitation of material occurred during acidification step in sample preparation. This resulted in a TOC concentration approximately 25% of other humic acid samples (see Table 7). incidence and a reduction of the mean time to first tumor in mouse skin initiation/promotion assays demonstrates the presence oftumor initiating activity in samples obtained from at least one city.
Although not statistically significant, increases in tumor incidence and reduced mean times to tumor were observed in the other four cities studied. These data provide confirmation of the presence of chemicals with carcinogenic activity in the same samples which produced mutagenic affects in Salmonella strains TA 98 and TA 100 (2) and increased transformation in BALB/3T3 cells (10). The importance of these results lies strictly in the fact that a tumorigenic effect can be seen in an intact animal model. These data should not be understood as demonstrating that cancer hazards are any greater in Ottumwa than the other cities studied. Nor are there implications of a specific level of risk attributable to cancer causing agents in any of these waters. At least two factors militate against the use of mouse skin initiation/promotion studies for assessing relative risks. First, the system responds selectively to different carcinogen classes, giving rise to a large number of false negative results (19,20 and Bull et al.,unpublished data). Secondly, the utilization ofa promoting agent greatly enhances the tumorigenic effect of tumor initiators. In certain instances (e.g., topical ethyl carbamate application) it is virtually impossible to produce tumors in the absence of promotion.
Given the fact that a hazard of undefined proportions can be seen in drinking waters two very important questions must be addressed. The first of these is, "what is the source of this carcinogenic material?" The second is, "what is the magnitude of the risk to the consuming population?" Several authors have shown that drinking water treatment including chlorination substantially increases the level ofdirect acting mutagens (3,21-23). Organic chemicals resulting from the treatment of wood pulp with chlorine (24,25) also result in production of direct acting mutagens in Salmonella tester strains. Ozonation of municipal wastewater has been shown to increase both the direct and indirect mutagenic activity present (26). Kool et al. (22) demonstrated that chlorine dioxide also resulted in increased levels of direct acting mutagens in strain TA 98. Consequently, it is clear that material present in drinking water sources is capable of reacting with disinfectants to produce biologically active products.
Testing of aqueous concentrates of alternately disinfected water as tumor initiators in the mouse skin illustrates many of the problems that arise in hazard evaluations of complex mixtures. Eventually such evaluations must deal with the individual processes that impact the chemical composition of the mixture to which man is exposed. In the first experiment a rather straightforward conclusion that reactions of ozone, chlorine, chloramine and chlorine dioxide resulted in a clear increase in the carcinogenic activity relative to that in the source water seemed possible. Subsequent experiments, however, failed to confirm this pattern. A major difference was that the nondisinfected water appears to possess activity not apparent in the earlier experiment. If one can assume that the series of analyses of water performed in 1980 reflects gross seasonal variations in the organic content and the reactivity of this material with disinfectants, it is clear that the variability suggested by the tumorigenesis data is clearly possible. Therefore, it becomes clear that consideration must be given to the variability in the source on the interpretation of results.
A second aspect of this variability is that disinfectants are very reactive chemically. Although the measures supplied in Table 4 concentrate on one aspect of disinfectant reactions, halogen substitution, all the alternate disinfectants are strong oxidants. Given the presence of biologically active chemicals in a source water it is not unlikely that 224 BULL ET AL.
such chemicals will be destroyed by disinfectants as easily as new chemicals with biological activity might be produced. A third major source of variability is that testing is obviously being conducted at the limits of the sensitivity of this assay. This makes it predictably difficult to differentially determine the contribution of disinfectant to formation of carcinogenic products from the variations of the source contribution. Bioassays based on toxicological effects do not distinguish among the responsible chemical species. Therefore, it becomes very difficult to determine what strategy is necessary to reduce hazards or even to arrive at a circumstance which results in the lowest overall risk because of an inability to develop the data for systematic decision making.
Contributing to the lack of systematic or predictive information is the selective responsiveness of certain bioassays to certain classes of chemical carcinogens mentioned above. The mouse skin certainly suffers from this difficulty (20). Consequently, future work must take an approach which comprehensively handles the question of relative carcinogenic hazards irrespective of the chemical classes present. An approach involving the use of a matrix of testing systems to define relative carcinogenic hazards has been previously described (19).
The above described difficulties argue for a better fundamental understanding ofthe chemical reactions that give rise to mutagenic, carcinogenic and otherwise toxic chemicals during disinfection of drinking water. It has been fairly well established that the organic substrates producing frihalomethanes and other reaction products of chlorination are quite varied. Perhaps the most widespread chemicals in surface sources of drinking water are a poorly characterized group of natural substances referred to as humic and fulvic acids. It is well established that chlorine, ozone and chlorine dioxide react with this class of chemicals to reduce color or ultraviolet absorbance present in many surface sources of drinking water. In particular, reactions of these chemicals with chlorine have been studied extensively (13). It is clear that they can serve as a source of trihalomethanes (27) and it is known that many other reaction by-products are formed which are yet to be identified. It is important to realize that these reactions include oxidative processes as well as chlorine substitution reactions.
From the use of the rather gross parameter TOX, a general case can be made that reaction of chemicals in drinking water results in the formation of both trihalomethanes and non-trihalomethane TOX. Choosing conditions which maximize chlorine substitution reactions (pH < 7) we have demonstrated virtually equivalent levels of chlorine substitlution in commercially obtainable humic acids 225 and humic and fulvic material isolated from surface water sources.
In a broad sense there is also a similarity in the level and type of mutagenic activity produced by chlorination of commercially available humic acid and comparable material isolated from surface water. In no case were any of these materials mutagenic without being chlorinated. Virtually identical specific activities ofdirectly effective mutagens were observed in Salmonella strain TA 100 in all cases following treatment with chlorine. Incorporation of Aroclor 1254-induced rat liver microsomes reduces the mutagenic activity. These observations are consistent with the types of activity which are observed upon chlorination of water (21)(22)(23). Comparisons of mutagenic activity in Salmonella strain TA 98 was more variable. Fulvic acids isolated from water gave rise to nondetectable to low mutagenic activity in TA 98 following treatment with chlorine. Chlorinated humic acids derived from the same waters gave the highest specific activities in TA 98. Commercially obtained humic acid upon chlorine treatment produced the median specific mutagenic activity in TA 98.
On the basis of these results it would appear reasonable to utilize commercially available humic acids as models for studying hazards associated with disinfection by-products. Recovery ofsufficient material from surface sources to perform life time carcinogenesis bioassays would be prohibitively expensive. However, this is the only method acceptable for estimating the magnitude of risks to human populations. Consequently, the use of such a model system seems essential to provide guidance to the study of disinfectant reaction products at ambient concentrations. The results of these studies can form the basis for identifying important disinfectant by-products by providing clues to how these materials might be isolated from drinking water. Obviously a substantial effort will be necessary to compare the chemistry of disinfection products produced at the high concentrations used here and the much lower concentrations involved in drinking water disinfection. Understanding of the chemistry involved will form the basis of determining how directly experiments conducted with commercially available humic acids can be applied to estimating risks arising from disinfection by-products. Even if it is not possible to make a direct comparison at the very least the use of the commercially available material should allow the design of methods for recovering and detecting the active chemicals from actual drinking waters. This in itself will be a major contribution to arriving at estimates of risk posed by disinfection by-products.
In summary, it seems much too early to decide the relative hazards associated with alternative disinfectants. Of all the issues involved the question of disinfection reaction by-products is perhaps the most difficult. Depending upon the variety of water characteristics which exist in the real world, production of by-products might be quite diverse. At alkaline pH, considerable portions of the organic chlorine substitution appears to be in the trihalomethanes (27). At lower pH, such as those used in the present study, much more of the organic chlorine substitution is in the non-THM fraction of organically bound chlorine. It is only after a body of data has been established that has enough depth of information to allow generalizations to be made confidently can the problem of disinfection byproducts be considered solved. Therefore, the greatest need is to provide the basic core of information that can be modified systematically and logically to fit local conditions. The development of a basic experimental model and a means of field validating the results and predictions of the model under a variety of circumstances seems to be an essential step in developing a workable solution to the problem. | v3-fos |
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} | s2 | Fungus diseases of raspberry {Rubus idaeus L.) in Finland
. During 1978 1981 a total of nearly 200 raspberry cane samples were investigated originating mainly from southern and eastern Finland. The samples consisted of young and already fruited canes injuried to different degrees. Phoma sp., the conidial stage of Didymella applanata (Niessl) Sacc., causer of raspberry spur blight, was most common of the isolated fungi; the perfect stage did not develope until afer preservation in cold. The peak of pycnospore release on raspberry canes was in July 1981. Other weak wound pathogens were isolated: Fusarium avenaceum (Cda. ex Fr.) Sacc., which occurred in 22 % of the transfers, F. culmorum (W. G. Sm.) Sacc., Botrytis cinerea Pers. ex Fr. and the secondly most common Phoma species, P. exigua Desm. v. exigua Maas. The fungi mentioned above were mainly obtained from different depths of the raspberry stem tissues, the least on an average from the pith. Altemaria alternata (Fr.) Keissl. was the most common ’surface fungus’, Phialophora spp., wood rotting saprophytes, common also in the pith. Leptosphaeria coniothyrium (Fckl.) Sacc., the most strong of the pathogens, was rare in the samples. The occurrence of Cylindrocarpon destructans (Zinssm.) Scholten in root samples changed 8-70 %. The cv. Muskoka is more susceptible to spur blight than cv. Ottawa. On the other hand, the larvae of raspberry cane midge (Resseliella theobaldi (Barnes)) produced plenty of injuries in Ottawa-samples originating from Mikkeli in 1980 and thus increased also the fungal damage.
Introduction
Raspberry (Rubus idaeus L.) is cultivated in Finland up to southern Lapland, but mainly cultivation is centralized in WSW Finland and Kymenlaakso. The area of raspberry cultivation for commercial purposes, is relatively small being 100 hectares (ANON. 1982). This is partly due to the fact that foreign raspberry cultivars are not sufficiently winterhardy in Finland (SAKO et ai. 1980). Also commonly occurring virus-and fungal diseases reduce the fruit crops. The most common fungal disease is raspberry spur blight caused by Didymella applanata (Niessl) Sacc. (KOCH 1931, LABRUY-ERE & ENGELS 1963, GJAERUM 1974, MISIC et al. 1975, WILLIAMSON & HARGREAVES 1981. On the other hand, the great damages considered to be caused by this fungus, may be partly produced by other agents (SEEMULLER D. applanata, as well as an other fungus, Leptosphaeria coniothyrium (Fckl.) Sacc., which causes raspberry cane blight are both wound pathogens, although they can also penetrate uninjuried young canes (SEEMULLER 1974). In Scotland, raspberry damage caused by L. coniothyrium has noticeably increased due to mechanical harvesting . On the other hand, STALDER (1965) in Germany has established cane death caused by frost similar to that of fungal cane blight. Much attention has been paid to the feeding wounds of raspberry cane midge (Resseliella theobaldi (Barnes)) larvae as entrances to L. coniothyrium and other fungi into the vascular cane tissue (PITCHER 1952, NIJWELDT 1963, WILLIAMSON & HAR-GREAVES 1979. According to PITCHER & WEBB (1952) the damage is called midge blight.
In Finland, raspberry spur blight has been known for a long time (HÄRDH 1936). Fungiside trials have been carried out to try to find means to control the disease, but none of the fungisides have been sales licensed in Finland (BREMER 1981 The aim of this study was to discover and identify the most common fungal pathogens, which on their part have effect upon the cultivation security of raspberry.
Material and methods
The material for this study comprised of some 200 raspberry canes collected in 1978 1981 from the following localities: The South Savo Experimental Station and The Institute of Horticulture, both belonging to the Agricultural Research Centre, the Department of Plant Pathology at Viikki (N ) and 10 private raspberry plantations in southern and eastern Finland (Fig 1).
The majority of the samples, 40-60 cm long pieces from the base of the canes were collected in August-October. Of these, about two thirds originated from first year canes and one third from fruited canes. Transfers to the nutrient media were made mainly as three partial transfers: a piece from the epidermis, the periderm and the pith, all being from the same part of the stem. The pieces from the epidermis and the periderm often included cells from the outer cortex. Cells from the vascular tissue were also possible in the pieces from the periderm. Transfers were made also from buds, leaves, petioles and roots.
The nutrient media and the cultivation methods were mainly similar to those reported earlier (RUOKOLA 1981). The majority of cultivations were made from samples of the cvs. Muskoka and Ottawa. The cultures were incubated at a temperature of 20-25°C for 3-4 weeks, the fungi were identified and before the final examination the cultures were kept at a temperature of 10°C for a month.
Results and discussion
A total of 290 transfers were made to the nutrient media, comprising of about 30 % of samples from Mikkeli. The partial transfers, 2 3 pieces, from different depths of the stem tissues (cf. p. 100) and the isolates of the same fungus obtained, respectively, have been mainly counted as one. Of the transfers the Phoma fungi grew clearly best (Table 1); their portion was more than twofold compared to the occurrence of the secondly most common, Fusarium avenaceum and Phialophora fungi. As regards D. applanata, the low frequency is partly due to the fact that the perfect stage developed only in cultures which were kept in a refrigerator over the winter. When comparing the number of isolates of the principal fungal species, found from the epidermis (%/151 samples) to the number isolated from the periderm, correspondingly, the ratio changed between different fungi (Fig. 2). Nearly the same amount of the most common fungi, Phoma spp. and F. avenaceum were found from both tissues, but from the pith noticeably less. Whereas, Alternaria alternata was 3 3 times as common in the outermost cortex as in the other stem tissues investigated. Wood rotting saprophytes, The causer of spur blight is an ascomycete, which SACCARDO (1882) placed in the genus Didymella Sacc. The fungus has also an imperfect stage which, according to literature (KOCH 1931, BLAKE 1980, CORLETT 1981) is assigned to the genus Phoma Sacc.
The fruit bodies (pseudothecia) of D. applanata mature in hibernated canes of raspberry. The ascospores discharged during the summer are carried by air currents and raindrops onto the young raspberry canes. Infection often takes place through the leaves and petioles (LABRUYERE & ENGELS 1963). Because of this the lower part of infected canes is covered with violet-brown discoloured lesions, chiefly at the nodes. Towards the autumn the lesions extend to cover practically the whole basal part of the young canes. Partially the diseased cane show a splitting of the bark, air pushes beneath the epidermis, and the cane turns silver. According to GJAERUM (1974) the fungus produces the perfect stage only in the main shoot, the conidial stage also in the laterals and leaves.
As regards the several observations, carried out at Viikki during the growing season of 1981, the pseudothecia of D. applanata matured from the beginning of June until early of July (Fig. 3 D). May, 1981 at Viikki was rather warm, but rainless and June quite cool and rainy. It also rained in abundance in July and August.
Violet-brown lesions around often already dead axillary buds were observed in the basal parts of young canes, particularly cv. Muskoka at the beginning of July. Light brown pycnidia quickly developed in the lesions (Fig. 3 A, B). From the pycnidia discharging pycnospores spread the disease. The lower leaves of infected canes turned partially yellowish and angular, brown spots were seen on the leaves. At the same time spur blight symptoms also occurred in wild raspberry in the neighbourhood. The pycnidia of the cvs. Muskoka and Ottawa continued to be exhausted until the end of September, and the dark fruit bodies were already developing. D. applanata fungus penetrates into the plant through different injuries, which may be caused by pruning, wind rubbing, natural splitting of the epidermis, hoeing or night frost. The wounds caused by larvae of insects, particularly of cane midge, also promote the penetration of fungal pathogens into the plant (NIJVELDT 1963, SEEMULLER & GRUNWALD 1980. In Holland it has been established by LABRUYERE & ENGELS (1963) that out of the buds, occupied by both D. applanata as well as B. cinerea, only 50-60 % developed normally. Infected buds often freeze during the winter. Leaves wither and the fruit crop remains low even in light diseased canes. On the other hand, WILLIAMSON and LAWSON (1979) have experimentally established that the crop compensated for the laterals lost at the base of the canes by increases in the number of fertile laterals at the top and in mean berry weight. However, in cool climates the damage may turn out to be even greater.
In raspberry cane samples kept moist and at a low temperature of about 10°-+ 10°C during the winter pseudothecia of D. applanata, developed slightly. Whereas, there were plenty of perithecia of another ascomycete, particularly in the samples from Piikkiö. A similar fungus, established in the old stems of arctic bramble, was indentified to D. applanata (RUOKOLA 1981). A later examination, however, showed that the fungus was similar to an ascomycete, which LARSEN (1952) supposed to be Didymella mesnieriana (Rehm & Thiim.) Sacc., but which MUNK (cf. LARSEN 1952) identified to belong to the genus Sydowiella Petrak. In Denmark this fungus was found from the dead canes of raspberry.
Botrytis cinerea, gray mold, was only isolated noticeably from samples originating from Mikkeli during growing season 1978 (Fig. 2). According to LABRUYERE & ENGELS (1963) the fungus causes damage mainly by destroying buds (cf. p. 103). On the other hand, WILLIAMSON & HARGREAVES (1981) have established that buds of infected nodes are only weakly suppressed in spring by the metabolites of B. cinerea and D. applanata. In the present study B. cinerea caused a deep infection even in raspberry canes, cane botrytis (cf. HOCKEY 1952). The fungus produced brown lesions on the canes in which the conidiophores with conidia (Fig. 4) and later dark sclerotia were formed. Fusarium avenaceum. The fungus often occurred as mixed colonies together with other fungi; sometimes its yellow sporodochia were to be seen on the epidermis of the cane. F. avenaceum was obtained in 63 % of some strongly diseased cane samples from Järvenpää (N ). JARVIS and HARGREAVES (1972) have established that F. avenaceum was probably responsible for damage in which many lateral buds of red raspberry failed to grow and some others which had grown out, later wilted. The fungus was also occasionally isolated from the roots of diseased plants and from their soil. Out of 34 root samples examined in the present study, F. avenaceum was only isolated from two and F. sulphureum from one. F. culmorum was isolated from samples of Ottawa originating from Kesälahti (Kb) in 1979 and respectively, from samples originating from Mikkeli in 1980; it occurred in 17-20 % of the samples.
Cylindrocarpon destructans, which has been found to cause raspberry root rot (BERKELEY 1936) was rather common in the root samples of the present study. The frequency of the fungus was on an average 26,5 %, but it changed due to the origin of the samples. In the samples from Piikkiö which were only weakly diseased, C. destructans occurred in 8,3 %, and respectively, in samples rather strongly diseased originating from Sipoo (Af) in 70 %. Coniothyrium fuccelii, the conidial stage of Leptosphaeria coniothyrium was only in about 2 % of the transfers from samples. They commonly originated from strongly infected canes. The fungus was also isolated from the roots. L. coniothyrium causes dark brown lesions at the basis of the primocanes. In next spring whitened lesions begin to produce black pycnidia which, after rain exude mugilage containing black 1-celled pycnospores . This wound parasite which affects the vascular tissue of cane blocks the translocation in the sector supplying the failed or withering buds and laterals.
The comparison between the raspberry cultivars According to SÄKÖ et ai. (1980) the cv. Muskoka is more susceptible to spur blight than Ottawa. A reliable comparison between the cvs. was not possible in the present study, because samples chosen for examination were mainly diseased ones. Based on relatively concise material taken for comparison, it can only be established that pathogenic fungi were found on average the same amount from both cvs. Muskoka and Ottawa (Fig. 6). Phoma sp. was established in 80 % of Muskoka cane samples originating from Piikkiö in 1980 and in 40 % of samples of Ottawa, respectively. Whereas in the samples from Mikkeli Ottawa seemed to be more susceptible to Phoma sp. than Muskoka (Table 2). However, in 1980 plenty of injuries caused by cane Resseliella theobaldi, causer of midge blight In the years 1980 and 1981 samples from Mikkeli were obtained, in which many injuries caused by cane midge larvae were established. The larvae, pink and 3 mm long, live in the outher cortical tissues (Fig. 5) causing brown patches or stripes which extend even into the vascular cane tissue (cf. WILLIAMSON & HARGREAVES 1979). The lesions are formed mainly on the young primocanes, whose outer periderm cell wall the larvae are able to penetrate (SEEMULLER & GRUNWALD 1980). Obviously the larvae also have the capability with their entsymes to degrade the suberin of the periderm cell wall by which means they also open ways for several fungi. SEEMULLER & GRUNWALD (1980) have even established that none of the fungi occurring in raspberry canes are able to grow into the vascular tissue via an intact periderm.
During the growing season 1981 in Mikkeli cane midge had two generations (P. DALMAN, pers.comm.). Larvae and their damage were observed mainly on the cv. Ottawa, because its outer cortex easily splits, thus opening entrances for insects. In 1980 there was also clearly more fungi isolated from this cv. than from Muskoka ( Table 2). The principal fungal species found from cv. Ottawa were mainly the same as those isolated by PITCHER & WEBB (1952) and LABRUYERE & ENGELS (1963) from the larval feeding wounds. They also record F. culmorum as a frequent isolate while WILLIAMSON & HARGREAVES (1979) didn't found it at all. In the samples of Ottawa from Mikkeli in 1980 the fungus occurred in 20 % of samples.
In Finland stem gall midge ( Lasioptera rubi Heeg.) and coganberry cane fly ( Pegomyia rubivora Coquillet) also injure raspberry stands (BREMER 1981). Thus like cane midge they probably promote the entering of wound parasites to the canes. According to literature (NIJWELDT 1963, LAWSON 1981 the damages caused by cane midge and spur blight have been reduced by the removal of the first young shoots in spring and later by thinning the shoots grown too closely. In Finland, the commercial tissue culture propagation of crops in raspberry has proved to be profitable, although the originally healthy stands were already strongly infected by D. applanata two years after planting (BREMER 1980). | v3-fos |
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} | s2 | Sex linked dwarf gene (dw) in White Leghorn laying hens under normal or hot temperature
White Leghorn females from 7 sire families segregating at the sex-linked dwarf locus (Dw and dw) were studied in individual cages at moderate (13-23 °C) and high (25-34 °C) temperature from 18 to 39 weeks of age. Traits concerning body weight, feed efficiency, egg production to 39th week of age, egg quality along with plasma uric acid and glucose at 18 weeks were measured. In heated group plasma uric acid at 39 weeks, dry matter and moisture in faeces along with daily water intake were also recorded. Results are as follows :
1. Adult body weight in dwarfs was 38 p. 100 lower than that of normals in control and 32 p. 100 in heated group whereas food consumption of dwarfs was respectively 36 and 37 p. 100 less than of normals in control and in heated group.
2. Reduction of egg number from age at first egg to 39th week of age in dwarfs was 20 and 41 p. 100 of normals in control and heated group respectively. Total egg mass per 28 days in dwarfs was reduced by 34 p. 100 as compared to normals in control group and by 37 p .100 in heated group. dw/Dw egg weight ratio increased from 0.85 to 0.89 from 27th to 39th week of age. During the first 7 weeks of laying in control group p. 100 double yolk, soft and broken eggs were highly significantly lower in dwarfs ; in both groups clutch size was significantly lower, but days of laying pauses were not significantly higher.
3. Age at sexual maturity in dwarfs was only one day later than for normals (156 v. 155) in control group while in heated group and unusual delay of sexual maturity in both normal and dwarfs was observed which appears to be due to an effect other than heat (201 and 182 days for dwarfs and normals respectively). 4. Egg shell thickness was lower for dw hens in both environments, the difference being higher and highly significant in heated group. 5. A very highly significant effect of genotype was observed in rectal temperature in both control and heated group, dwarf having a lower rectal temperature than normals. 6. A very significant effect of genotype and sire family was observed in plasma uric acid measured at 39 weeks in heated group. The difference between genotypes was in the same direction (higher value for dwarfs) but comparatively less than that observed at 18 weeks. Mean level of plasma uric acid at 39 weeks was increased compared to that at 18 weeks.
7. Phenotypic correlations of egg shell traits with body weight, observed food consumption, egg mass, egg number and clutch size were all positive and mostly significant in dwarfs while they are unsignificant and generally negative in Dw genotype.
Introduction
Utilization of the sex linked dwarf gene in layer populations appears to be a subject of controversy. FRENCH & N ORDSKOG (1973) state that better feed efficiency should be searched by selection for low body weight in conventional hens whereas B ERNIER & A RSCOTT (1972) think that improvements already obtained justify the pursuit of selection of dw « mini birds !. ME R nT et al. (1974), SE LVARAJAH (1974) and H ORST & P ETERSEN (1978) suggest that dwarf hens can be at an advantage in hot climates. From this point of view, B ANERJEE et al. (1981) observed that studies are needed on the effect of the dwarf gene on feed efficiency, egg production, egg and physiological traits in layer populations reared from the beginning under hot environmental conditions. The realization of such conditions represents part of the present paper, the main purpose of it being to evaluate effects of the dw gene on production and physiological traits of laying hens in a small-sized Leghorn strain.
Material and methods
-Experimental birds and conditions
The birds used and the experimental conditions up to the age of 18 weeks have been described earlier (B ANERJEE et al., 1981). We shall recall the main points which are useful for the understanding of the present paper. Dwarf and normal White Leghorn females were obtained from 7 heterozygous sires (Dw dw) mated with dwarf females. Two hatches were obtained at a 2 weeks interval in September, 1980. The chicks were reared on deep litter till 18 weeks of age, the first hatch under fluctuating normal temperature (between 15 and 20 °C) after 4 weeks of age, the second under higher temperature (between 25 and 30 °C). The first hatch corresponded to the « control group and the second to the « heated » group. After 18 weeks of age 249 Whiie Leghorn females comprising 138 (66 Dw ; 72 dw) individ.ual.s in control group and 111 (61 Dw ; 50 dw) in heated group, were transferred to individual laying cages contained in two separate houses. The control group was maintained under normal temperature with an average daily minimum and maximum of 17.4 and 18.3 °C respectively ; the heated group was kept at hot temperature with 27.6 and 31.1 °C as average daily minimum and maximum. The experimental period lasted till 39 weeks of age for both groups. During this period the birds received 14 hours of light and 10 hours of darkness per day. Water and feed were given ad libitum to both groups. The feed contained 16 p. 100 total protein and approximately 2 600 Kcal/kg M.E. and 3.5 p. 100 calcium.
It must be added that, between the two experimental groups, additional environmental differences other than ambient temperature cannot be ruled out. Difference in day of hatch can be considered as a minor source of variation for the traits recorded here. A more important factor is the presence of mortality, mainly by Marek's disease as this stock had not been vaccinated. This mortality was considerably higher in the heated than in control group. (From 18 to 39 weeks of age, percent mortality in the control group was respectively 6.1 and 21.2 for Dw and dw females ; the corresponding figures in the heated group are 27.5 and 28.2).
-Traits measured or calculated
Only birds surviving beyond the end of test period are considered.
Traits measured or calculated during the experimental period between 18th and 39th week of age, symbols used and their sequence in the succeeding tables are listed in table 2. Data on egg shell, double yolks, soft eggs and broken eggs, blood spots in eggs, dry matter and moisture in feces, are presented in percentage. On the other hand, the data on 18 week body weight, plasma uric acid and glucose at 18 weeks have been reproduced from the previous article (B ANERJEE et al., 1981). As table 2 shows, a few variables were recorded only in control group, because their measurement was not possible or not satisfactory because of low variation in the heated group owing to delayed sexual maturity. Some other traits (plasma uric acid, water intake, water and dry matter in feces) were measured only in heated group. Concerning the trait « residual food. consumption », the procedure used for obtaining it is as follows : during several periods of 28 days each (3 for control, 2 for heated group), the per bird per period observed food consumption (0), mean body weight (W) for each period, body weight variation between the start and end of the period (A W) and total weight of eggs laid (E) were recorded. For each individual a mean value of the three or resp. two periods for the above characters was obtained to calculate the predicted feed intake (T) by a multiple regression equation : T=aWq+b4W-!cE This equation has been described by B YERLY (1941) and utililized by LESSON et al. (1973), Gous et al. (1978), Mc D ONALD (1978), B ORDAS & M ERAT (1981). The optimum value of a in the present study has been taken as 0.5 with the purpose of giving minimum error variance to the regression. For each of the control and heated groups one regression equation was established after verifying that in each group, the coefficients a, b, c were not significantly different for the Dw and dw genotypes.
The utilized equations are : The difference between observed and predicted feed intake (O-T) for each individual is taken as residual feed consumption (R).
-Statistical analysis
For each mesure, a within group two factor analysis of variance was done, sources of variation being Genotype, sire Family and Genotype X Family interaction. A three factor analysis of variance with between groups component of variation was not carried out as variation between groups caused by factors other than temperature mainly associated with Marek's disease (see above) was suspected. Within group within genotype phenotypic correlations between characters were obtained. Correlations with the two genotypes combined (on a within genotype basis) were calculated after testing correlations for homogenity between genotypes. Correlations with genotypes pooled were tested, on the other hand, for homogeneity between treatments, and conversely a pooled estimate (treatments grouped on a within treatment basis) for each genotype was carried out for correlations which revealed a between genotypes heterogeneity. .;&dquo;; ! 1 ' & d q u o ; ' Table 1 presents the average and range of maximum and minimum temperature (°C) for control and heated group during the experimental period. The means of the two genotypes and the percent of dwarf to normal genotype for each character measured under control and heated group are presented in table 3. The two factor (genotype, family) analyses of variance within group, for the same characters are given in table 4. Within genotype within group phenotypic correlations between 20 of the characters measured for control group and 22 for heated group were estimated. However, in tables 5 and 6 corresponding respectively to control and heated group, the correlation value for each genotype is given only when there is significant heterogeneity between these genotypes ; in other cases a common value (on a within genotype basis) is presented. In addition, for each genotype a pooled estimate across environments was calculated when no statistical heterogeneity between treatments appeared. The result is presented in table 7, only for correlations differing significantly on the whole for Dw and dw birds.
Discussion
We shall mainly discuss the effects associated with the dw gene, and the phenotypic correlations between traits within genotypes, separately within each experimental group. Effect of treatment per se and response of genotype to treatment will be commented with some caution, especially for traits correlated with sexual maturity, owing to the afore-mentioned higher mortality and large delay in onset of laying in heated group.
A. Means and analysis of variance a) Body weight, body weight variation food consumption and efficiency In general the reduction of the adult body weight in dwarfs has been reported to be around 30 p. 100 in females (HUTT, 1959 ;B ERNIER & A RSCOTT , 1972 ;H ORST & P ETERSEN , 1978). At a given age, the larger the adult size of the strain where dw gene is introduced, the smaller the relative reduction of body weight according to 1VI R EDDY et al. (1974). The present report seems consistent with this opinion, as the average adult body weight of the population was low and body weight reduction in dwarfs was high. The average mature and 18-week body weight reductions in dwarfs as compared to normals were 38.4 and 36.5 p. 100 in control and 32.0 and 33.0 p. 100 in heated group respectively. On the other hand, these figures suggest that body weight reduction due to the dw gene is relatively less at higher temperature which seems to be is in agreement with the finding of M ERAT et al. (1974), H ORST & P ETE R SEN (1978).
Body weight change may have been influenced by onset of egg production as it was significantly superior for the Dw genotype between 18 and 27 weeks in control group where peak of egg production took place, while in the corresponding period for heated group, where onset of sexual maturity was considerably delayed, dwarfs gained as much weight as normals. In the period from 27th to 39th, week on the contrary, dw hens have respectively in control and heated environment a lower (not significantly) body weight gain and higher body weight loss.
The reduction in observed feed consumption in dwarfs, both in control and heated group (respectively 35.7 and 36.9 p. 100) is higher than those reported by MP RAT et al. (1974), H ORST & P ETERSEN (1978) who observed a reduction between 25 and 30 p. 100 of normals. Working with birds with higher body weight than the present experiment, A RSCOTT & B ERNIER (1973) found that the percentage difference in body weight between adult dwarfs and nondwarfs was nearly proportional to the difference in feed consumption and FRENCH & N ORDSKOG (1973) observed that dwarf pullets weighing 25 p. 100 less than nondwarfs consumed 19 p. 100 less feed. In our case the observed feed consumption of dwarfs in control group was less reduced by about 3 p. 100 compared to their body weight reduction while in heated group feed consumption was more reduced by about 5 p. 100 compared to body weight reduction. This relatively higher reduction in feed consumption compared to body weight reduction in dwarfs in our heated group seems mainly to be due to the considerably lower egg production in dwarfs compared to normal in this group. In a previous experiment (ME heat cauded a relatively higher reduction in food consumption in normales compared to dwarfs. The significantly lower R (residual food consumption) at 5 p. 100 level in dwarfs in heated group (joined to the fact that in both groups R was positive in Dw and negative in dw genotype), suggests a better feed efficiency of dwarfs at higher temperature and possibly also at moderate temperature. This was not found in the report of P ROD ' HOMME & M ÊRAT (1969) in a population of larger body size. The highly significant sire family effect for residual feed consumption in control and in heated group, observed in our study, has also been reported by B ORDAS & ME . This sire family effect on R may suggest a possible improvement of this trait through sire family selection. On the other hand, it must be mentioned that r 2 (the square of the correlation coefficient between observed and estimated individual food consumption) in our study was in the order 95-96 p. 100 as also observed by N ORDSKOG (1973) in white Leghorn. In brown-egg stock MF RAT et al. (1974,1980), B ORDAS & ME RAT (1976,1981) reported this value to be around 80 p. 100. Thus it seems that in Leghorns scope of improving the residual consumption is considerably less than in other breeds.
Feed efficiency, in terms of feed consumed per unit of egg mass produced, was 2.7 for dwarfs compared to 2.8 for normal in control group while it was 2.5 in heated group for both genotypes. Thus in our case, heat appeared to improve feed efficiency to the same level in both genotypes. ME RAT et al. (1974) found a slightly better feed efficiency for dwarfs at high temperature and H ORST & PETER-SEN (1978) working with higher body weight laying hens observed. a better feed efficiency for dwarfs while in a lower body weight group, a lower feed efficiency for dwarfs compared to normals was found in a heated group. It appears that dwarf gene has a beneficial effect on feed efficiency mainly when introduced in a line with not too low body weight. b) Egg production traits Reduction in egg number from 1st egg to 39 weeks of age in dwarfs was 20.2 and 40.9 p. 100 respectively in control and heated group. Delay in age at sexual maturity in dwarfs was only one day in control while in heated group it was 19 days on average. Clutch size was significantly lower in dwarfs than in normals in both control and heated group but the reduction in control group was particularly high. Laying pause days and number of pauses were higher in dwarfs but not significantly different from Dw genotype. The consequence of this was reduced laying rate. Corresponding to this and to lower mean egg weight, egg mass per 28 days (E) in dwarf hens, is reduced by 33.8 and 37.5 p. 100 respectively in control and heated environment as compared to normals.
Previous estimates of reduction of egg production in dwarfs have not been consistent ranging from zero to 36 p. 100. A reduction in laying rate of 10 p. 100 or more has been reported with medium or light stocks (H UTT , 1959 QUISENBERRY, 1972 ;SUMMERS, 1973 ;DORAN & QUISENBERRY, 1974). The present higher reduction in control group may be mainly due to lower adult body weight of the population. This is in line with observation of a positive correlation between egg production and body weight among dw hens of small to moderate size (M!RAT, 1969). This reduction in control group is little less than observed by H ORST & P ETERSEN (1978) (22.3 p. 100) in a population of dwarf White Leghorn.
The total egg mass reduction in dwarfs is also higher than reported. in the literature (21-28 p. 100). This higher reduction may be expected because of the higher body weight reduction and consequently egg weight reduction in dwarfs. Age at sexual maturity is generally delayed by 7-10 days in dwarfs (H UTT , 1959 ;MF RAT , 1969 ;BERNIE R & ARS COTT , 1960 ;RICARD & COCHEZ, 1972). HORST & PE TERSEN (1978) reported this delay to be 9-15 days while L ECLERCQ & B LUM (1975) and G UILLAUME (1975) observed a significant opposite difference. So in the present report the delay of one day in control group is less than generally found before. Concerning clutch size, MG RAT {1972) reported a lower reduction of it in dwarfs compared to the present experiment, but body weights were higher in the former study. Small clutch size in dwarfs may be attributed to less active vitellogenesis (J AAP & M OHAMMADIAN , 1969). On the other hand, delayed ovulation of a hormonal or nutritional nature corresponding to short term calcium need cannot be ruled out as dwarfs appear to consume significantly more food than normals during the days of egg formation (B ORDAS & M ÉRAT , 1976).
As mentioned before, in the heated group some factors other than temperature, possibly associated with Marek's disease contamination, are likely to have interfered with egg production traits, mainly by delaying sexual maturity. This may explain why reduction in egg production in heated group was higher than reported by various authors. On the other hand, laying rate during the control period is not so much reduced. This comes from the fact that although clutch size is obviously less in the group at higher temperature, this is partly compensated by the lower mean number of pause days. Finally, egg mass per 28 days is not much lower in heated compared to control group, which partly corresponds to the limited reduction of body and mean egg size by the former environment.
c) Egg traits
Reduction in egg weight in dwarfs was 14.2 and 10.7 p. 100, when measured during the first and last 15 days of test period (27-39 weeks) respectively in control group. In heated group this reduction was about 1 p. 100 more during the last 15 days of the test period. A highly significant sire family effect and a significant genotype X family interaction effect was observed in egg weight measured during the first 15 days of test period, suggesting the existence of genetic modifiers of the effects of the dw gene on this trait. In comparison, the ratio of mean egg weight of dw hens compared to normals has generally been estimated around 0.90 or more. (1976) observed 8.5 to 10 p. 100 reduction in egg weight due to heat. The absence of a similar detectable effect in our data may be attributed to abnormally higher age at sexual maturity and relatively lower reduction in adult body weight in our heated group.
The reduction in albumen and yolk weight in dwarfs as compared to normals in the present experiment was the same (14 p. 100) in control group. In heated group the corresponding reduction in yolk weight was higher compared to albumen (20.5 v 11 P . 100) hence yolk/albumen ratio is significantly lower for dw hens in heated group. A highly significant genotype X family interaction was observed for yolk/ albumen ratio in heated group. However, concerning the egg components, albumen and yolk, caution is necessary in interpreting effects of genotype according to treatment, as the measurement was done at a later age in heated group. For albumen height, it was reduced significantly at 5 p. 100 level in dwarfs in control group, while in heated group a highly significant increase was observed for this genotype. Sire family interaction effect for albumen height was significant at 5 p. 100 level only in control group.
Previously MF RAT (1970 compared egg traits of dwarf and normal hens and reported no difference in yolk/albumen ratio as observed here in control group.
A reduction in .egg shell thickness was present for dwarfs, relatively higher in heated group, and the difference between genotypes was very highly significant in this group and at 5 p. 100 level in control group. While sire family effect was significant at 5 percent in both control and heated group, interaction was only significant in the latter group. There was no significant difference between genotypes for percent egg shell but in heated group dwarfs have lower and in control group higher percent egg shell compared to normals. Sire family effect was highly significant only in control group. Percent double yolks, soft and broken eggs during the first 7 weeks of laying were very highly significantly lower and percent blood spots was lower at 5 p. 100 level in dwarfs in control group. In heated group it is recalled that these traits were not measured due to the delay in sexual maturity. Percent broken eggs in dwarfs from 27th to 30th week was higher in heated and lower in control group compared to normals, though not significantly different.
In previous experiments, a lower egg density in Leghorn dwarf hens was reported by B ERNIER & A RSCOTT (1960) and in an experimental cross by G LEICHAUF (1974). While these reports correspond with the present one, ME RAT (1972) Reduction of egg shell thickness in dwarfs especially in the heated group appears at least partly due to insufficient availability of calcium from feed due to lower consumption and from body due to lower body weight in dwarfs. H ORST & P ETERSEN (1978) observed a lower shell quality and thickness of eggs in dwarfs maintained under higher temperature compared to normal temperature.
d) Anatomical and physiological traits
On the whole, rectal temperature was 0.1 °C higher in heated group. Whuc effect of genotype was highly significant in both groups, sire family effect was significant at 5 p. 100 level only in heated group. The reduced body temperature in dwarfs may be due to their lower metabolic rate caused by lower thyroid activity (M ÉRAT & G UILLAUME , 1969, and others cited by G UILLAUME , 1976).
As has been already reported (B A NERJE E et al., 1981) a highly significant increase in plasma uric acid at 18 weeks of age was observed in heated compared to normal group. A very highly significant difference between genotypes, dwarfs ranking higher than normals, appeared in both groups, although the mean difference was less in heated group. As mentioned also in the same paper, plasma glucose at 18 weeks was slightly less in dwarfs than in normals and also in heated. group compared to control group, although this was not significant. Plasma uric acid at 39 weeks of age, measured only in heated group, was also highly significantly different between genotypes, but this difference was comparatively less than at 18 weeks Sire family effect which was non significant at 18 weeks was highly significant for plasma uric acid at 39 weeks. From 18 to 39 weeks level of plasma uric acid increased but the increase in dwarfs was considerably lower than in normals. Increase of plasma uric acid with age has already been reported in the literature (BELL & FREEMANN, 1971).
Water intake in dwarfs was reduced by about 30 p. 100 in heated group in comparison with normals. As reduction in observed food consumption by dwarfs was 37 p. 100, the ratio of water to food consumption was higher in dwarfs. On the other hand, dry matter in feces was significantly lower in dwarfs at 0.1 p. 100 level while percent moisture in feces was significantly lower for this genotype at 1 p. 100 level. Genotype X family interactions for the latter two characters were also significant at 5 p. 100 level. There is an apparent discrepancy between the afore-mentioned higher ratio of water to food consumption, and the lower moisture percent in excreta for dw hens. This discrepancy remains to be explained.
B. Phenotypic correlations
In the following, at the first place correlations are compared for dwarf and for normal hens. In addition, significant correlations which are common to both genotypes will be commented. Finally, possible effect of treatment on certain correlations will be discussed, but in view of the wide difference between the two environments for some traits like age at first egg, this will concern traits not associated with the former. a) Effect of genotype at the Dw locus on phenotypic correlations Tables 5 and 6 show that within each treatment a number of correlations are significantly heterogenous between the Dw and dw genotypes. For most of these correlations it is possible to pool environments and to obtain an unique estimate for each genotype. Part of these pooled estimates are no more significantly different between Dw and dw birds. In such cases, the meaning of a significant between genotypes heterogeneity found in only one environment is not clear and may be due to sampling, so that it is safe to wait for further data before proposing any interpretation.
Conversely, some correlation estimates remain significantly different between Dw and dw hens after pooling treatments (table 7).
The interpretation of differences between the dwarf and normal genotypes for most cases is not clear. The most clear-cut differences concern the trait, egg shell thickness and to some extent also shell percentage. Among normal-sized (Dw) hens, shell thickness is slightly negatively correlated with all traits concerning body weight, egg mass and egg number. On the contrary, for dw hens, there is a clear positive correlation with the same traits. The tendency, although less marked, is the same for shell percent. The same can be said for the correlations of egg shell thickness with plasma uric acid at 18 weeks of age, showing in both control and heated group a higher value for dw hens.
It may be suggested that for those dw hens, which have a lower food consump! tion on average, calcium is a more limiting factor than for their normalsized sisters, so that dwarfs with higher food intake (and correlated traits such as body weight and egg laying) are able to produce thicker shells. This seems to be confirmed by the fact that partial correlations of shell traits with either body weight, egg mass or egg number with fixed food intake among dw hens are not significant : for instance, the corresponding values for egg shell thickness are respectively + 0.05, + 0.14 and -0.07 (N.S.).
It does not seem that such positive correlations between egg shell traits and food intake and egg production were observed previously in dwarf stocks. The present result may be due to especially small size of dw hens in our Leghorn strain.
On the contrary, in a previous publication (M ÊRAT , 1969) in a medium-sized strain, a positive correlation was observed on dwarf hens and not on their normal counterparts, between egg production and adult body size. This was confirmed in more recent years on the same stock (unpublished data). This trend seems to be present here only in the control group, not in the « heated group. However, in this last group delay in sexual maturity may have obscured this tendency. Table 7 does not include traits which were measured only in one environment. Tables 5 and 6 show significant differences between genotypes at Dw locus for a few correlations concerning these traits. In the heated group, uric acid at 39 weeks of age is negatively correlated with egg shell thickness only for dwarfs. Water in feces shows among dwarfs positive correlation with egg and yolk weight, a negative one with total pause days, the corresponding correlations being low and of opposite signs for normals. Dry matter in feces shows a positive correlation with egg weight and shell thickness only for dwarfs. Finally, in the « control environment, percentage of soft-shelled eggs shows correlations of opposite sign with egg weight for Dw and dw females. To date it is not possible to propose an interpretation of these differences. b) Significant correlations common to both genotypes and treatments A number of the observed correlations correspond to expected associations, e.g. of food intake with body weight, body weight gain, egg mass ; of egg number with egg mass and of both with age at first egg, mean clutch length and pauses ; of mean egg weight with shell thickness ; of adult body weight with 18-week body weight and body weight gain ; and the negative correlation of mean egg weight with yolk/albumen ratio, corresponding to the known fact that large eggs have a smaller proportion of yolk.
On the other hand, as the main purpose of this paper concerns effects associated with the sex-linked dwarf gene, we shall limit further the discussion of significant correlations (as common estimates to both genotypes and treatments) to those which to our knowledge have not been mentioned previously in the literature. On the whole body weight is positively correlated with clutch length and negatively with pauses. This may be due to the small size of the strain used here, including Dw birds. To a lesser extent, the trend is the same for body weight gain. Of interest are also the correlations of uric acid in plasma at 18 weeks of age : pooling genotypes and treatments, the correlation (based on 187 d.f.) is -0.21 with egg number (p < 0.01) and -0.20 with mean clutch length (p < 0.01). This fact is the more interesting as this blood parameter is measured before sexual maturity, and so is not a mere indicator of laying rate. It remains to be verified if earlier measurements of uric acid in plasma could be used as an aid for early selection for egg production.
A few significant correlations concern traits measured only for one treatment. This is the case for per cent double yolked eggs in the control group (negative correlation with yolk/albumen ratio and egg shell thickness) ; for per cent softshelled eggs (negative with egg shell thickness, as found by S ILBER & M!RAT, 1974). In the heated group, uric acid in plasma at end of laying recording is negatively correlated with body weight, weight gain, egg mass, food intake, shell percent, and rectal temperature : these correlations are largely different from those observed for plasma uric acid at 18 weeks, which is in agreement with the low correlation which appears between plasma uric acid measurements at the two ages. Also in heated group, dry matter in feces shows a positive correlation (as might be expected) with food intake, and with body weight and egg mass. Its correlation with R (« residual food intake !) is positive but low and not significant.
c) Correlations differing between treatments
A number of correlations differ between environments only by their value but not by their sign. Those differing also by their sign are generally significantly different from zero in one environment and of opposite sign but insignificant in the other environment. Among them, those concerning age at first egg are difficult to interpret, in view of the very large difference between treatments for this trait. On the other hand, the following deserve to be mentioned : percent egg shell with feed intake, egg mass, mean clutch length (positive in heated group, slightly negative in control) and with pauses and uric acid at 18 weeks (negative in heated group) ; rectal temperature with egg mass and egg number (negative in control group) and pauses (positive in control group).
Conclusion
The present study was aimed to contribute to the problem of possible utilization of the sex-linked dw gene for egg production. In this respect, it seems safe to conclude from our results that introduction of the dwarf gene for such use cannot take place into a population already of too small size such as was our Leghorn strain. This is in agreement with the positive correlation between egg laying and adult body size that we observed previously in small size dwarf strain (M ÊRAT , 1969 and more recent inpublished data) and also with the positive correlation shown in the present paper in dw birds between body size or feed intake on one hand and egg shell thickness or percent on the other hand. This too small size of the stock used here may also contribute to explain the absence of any clear advantage of dw hens for egg laying traits at higher temperature in our experiment, contrary to a slight advantage observed in previous results.
However, it may be noticed that in spite of the rather poor egg laying overall performance of dwarf hens in this strain (especially in the conditions which occurred in « heated » treatment) food efficiency of dwarfs as measured by the ratio of food consumed to egg mass produced, was not worse than for their normal fullor half sisters. This conclusion would be reinforced if the lower proportion of abnormal or cracked eggs (at least at normal temperature) is taken into account.
Finally our data may show that the main problem for possible use of dwarf egg laying stock would be to improve rate of lay, especially mean clutch length which is much lower among dwarf layers than for Dw hens. Selection on this last trait or on time interval between successive ovipositions can be suggested. It remains open to question if additional traits would be useful as an aid to such selection. The negative phenotypic correlation observed between plasma uric acid at 18 weeks of age (before the onset of laying) with clutch length and egg number may deserve further observations, to know if these correlations are present also at the genetic level and if they are found in other populations.
Received for publication in March 1982. | v3-fos |
2018-12-05T11:45:51.043Z | {
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} | s2 | Temperature-sensitive, protoplast-forming os-1 varient of Neurospora- a few tricks
Temperature-sensitive, protoplast-forming os-1 varient of Neurosporaa few tricks. Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This technical note is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol29/iss1/13 Selitrennikoff, C. P. Protoplasts of Neurospora can be formed by treating hyphae with one of a number of "cell-wall'ases". However, Temperature-sensitive, protoplast-forming resulting protoplasts often have residual cell-wall and os-1 variant of Neurospora a few tricks. will not grow and divide. The slime variant of Neurospora will, under certain conditions, grow as a population of protoplasts. Unfortunately, such cultures will not participate in crosses thus making genetic manipulation difficult. In efforts to overcome this and other difficulties, we have used a temperature-sensitive osmotic mutant (os-1 (NM233t)) in order to form protoplasts. The general method has been previously published (Selitrennikoff et al., 1981 Experimental Mycology 5: 155-161) and included here are some details. Mycelial isolates of os-1 (NM233t) are available in either mating type from the Stock Center and can be used as a male or female parent in order to construct any desired genotype. Progeny can be grown on agar slants at either 25° or 37°C. Macroconidia are inoculated (1-10 x 105 cell/ml) into 250 ml flask containing 50 ml of the following medium: 10% (w/v) Sorbose; 2% (w/v) Sucrose; 1 x Vogel's salts, 400 μg/ml Polyoxin B. Polyoxin B is an inhibitor of chitin synthetase activity and is purified from Polyoxin AL Wettable Powder (available either overthe-counter in Japan or from the Kaken Chemical Co., Ltd., 28-8 2-Chome, Honkomagome, Bunkyo-Ku, Tokyo, Japan). One hundred grams of powder are dissolved in 1 liter of water and insoluble material is removed by filtration using Whatman #1 paper. The brown filtrate is applied to a Norite column (granular form the powder packs to a very slow (100 ml/day) column). The column is washed with water and the Polyoxin eluted with 60% aqueous acetone. The dirty-brown fractions are pooled and the acetone is flushed out. The resulting brown "goo" or powder is dissolved in water and applied to a Dowex 50 (8X,H+ form) column. NH4OH The Polyoxin is eluted with 0.3N and the resulting solution freeze-dried twice (to insure complete removal of NH4OH). Typical batches result in a product 70% pure based on U.V. absorption with yields of 25.35%. Polyoxin powder can be stored desiccated at 4°C (or -20°C) for long periods of time. Cultures are grown at 37°C with shaking (140 rpm) and are filtered daily through sterile glass wool into fresh medium. After seven days a stable culture of protoplasts results. It is important that during the initial transfers that the cell density be maintained at 5 x 105 cells/ml or greater. Cultures are then filtered into flasks containing medium lacking Polyoxin and transferred daily (with filtration) to fresh medium for another seven days. These, then, are filtered to medium in which Sorbose has been replaced by 7.5% Sorbitol and growth allowed to continue for yet another week (with, of course, daily fitration). The population of protoplasts that results is stable for up to one year of growth and protoplasts can regenerate a mycelium (including macroconidia) upon a temperature shift to 25°C. However, far reasons that are not clear, the resulting mycelium is different morpholocially from the original os-1 strain. Protoplasts may be stored at -70°C in their medium for at least two years. These temperature-sensitive protoplasts are very useful for a number of studies, including hosts for exogenous DNA's, (Supported in part by NSF grants to CPS.) Department of Anatomy, University of Colorado Health Sciences Center, Denver, Colorado 80262. Stevens, J. N. and R. L. Metzenberg Preparing Neurospora DNA: some improvements. Our need to prepare DNA suitable for restriction enzyme analysis from rather large numbers of different strains led us to develop an easy method for making DNA (Metzenberg and Baisch 1981 Neurospora Newsl. 28: 20-21). Since then, we have made some small modifications that result in significantly improved yield and quality of product without increase in effort. The changes are as follows: (1) "Extraction buffer" has been modified by substituting triethylamnonium ethylenediamine tetraacetate (TEA.EDTA) for lithium EDTA, and by adding a modest concentration of NaCl. The evolution of the procedure started with the sodium salt of EDTA, which comes out of ethanol as an intractable syrup. We then switched to the lithium salt, which does not "syrup out" even at high concentrations, but may crystallize out of ethanolic solutions, especially once the lab has been seeded. The triethylamnonium salt has given no trouble of any kind except that we have come full circle and must add a small amount of sodium ion to insure precipitation of the DNA! The TEA.EDTA is made as a 500 mM stock solution by suspending 73 g (250 mmoles) of the free acid of EDTA in 350 ml of water and adding, with continuous stirring, triethylamine base to give pH 8.0 8.2. (The base should be redistilled if colored impurities are evident.) In our hands, the volume of base required is 108.5 ml, but no doubt this will vary, depending on the water content of the triethylamine. The stock solution is then diluted to 500 ml. We have stored it frozen, though this may not be necessary. The final concentrations in "extraction buffer", then, are: 50 mM; and bacterial proteinase, 250 μg/ml. TEA.EDTA, 250 mM; Triton X-100, 0.5%; NaCl, (2) Extraction of the powder for 2-3 days at 33° or 37° with gentle agitation appears to give somewhat larger yields than overnight extraction without any agitation. This seems to be especially true of scaled-up preparations. (3) All precipitations, either with ethanol or ethanolic perchlorate, are done with reagents and the preparations at room temperature. No incubation of the ethanolic suspensions, either at 4° or at room temperature,
Protoplasts of Neurospora can be formed by treating hyphae with one of a number of "cell-wall'ases". However, Temperature-sensitive, protoplast-forming resulting protoplasts often have residual cell-wall and os-1 variant of Neurospora -a few tricks. will not grow and divide. The slime variant of Neurospora will, under certain conditions, grow as a population of protoplasts. Unfortunately, such cultures will not participate in crosses thus making genetic manipulation difficult. In efforts to overcome this and other difficulties, we have used a temperature-sensitive osmotic mutant (os-1 (NM233t)) in order to form protoplasts. The general method has been previously published (Selitrennikoff et al., 1981 Experimental Mycology 5: 155-161) and included here are some details.
Mycelial isolates of os-1 (NM233t) are available in either mating type from the Stock Center and can be used as a male or female parent in order to construct any desired genotype. Progeny can be grown on agar slants at either 25° or 37°C.
Macroconidia are inoculated (1-10 x 105 cell/ml) into 250 ml flask containing 50 ml of the following medium: 10% (w/v) Sorbose; 2% (w/v) Sucrose; 1 x Vogel's salts, 400 µg/ml Polyoxin B. Polyoxin B is an inhibitor of chitin synthetase activity and is purified from Polyoxin AL Wettable Powder (available either overthe-counter in Japan or from the Kaken Chemical Co., Ltd., 28-8 2-Chome, Honkomagome, Bunkyo-Ku, Tokyo, Japan). One hundred grams of powder are dissolved in 1 liter of water and insoluble material is removed by filtration using Whatman #1 paper. The brown filtrate is applied to a Norite column (granular form the powder packs to a very slow (100 ml/day) column). The column is washed with water and the Polyoxin eluted with 60% aqueous acetone. The dirty-brown fractions are pooled and the acetone is flushed out. The resulting brown "goo" or powder is dissolved in water and applied to a Dowex 50 (8X,H+ form) column.
NH 4 OH
The Polyoxin is eluted with 0.3N and the resulting solution freeze-dried twice (to insure complete removal of NH 4 OH). Typical batches result in a product -70% pure based on U.V. absorption with yields of 25.35%. Polyoxin powder can be stored desiccated at 4°C (or -20°C) for long periods of time.
Cultures are grown at 37°C with shaking (140 rpm) and are filtered daily through sterile glass wool into fresh medium. After seven days a stable culture of protoplasts results. It is important that during the initial transfers that the cell density be maintained at 5 x 10 5 cells/ml or greater. Cultures are then filtered into flasks containing medium lacking Polyoxin and transferred daily (with filtration) to fresh medium for another seven days. These, then, are filtered to medium in which Sorbose has been replaced by 7.5% Sorbitol and growth allowed to continue for yet another week (with, of course, daily fitration). The population of protoplasts that results is stable for up to one year of growth and protoplasts can regenerate a mycelium (including macroconidia) upon a temperature shift to 25°C. However, far reasons that are not clear, the resulting mycelium is different morpholocially from the original os-1 strain. Protoplasts may be stored at -70°C in their medium for at least two years. These temperature-sensitive protoplasts are very useful for a number of studies, including hosts for exogenous DNA's, (Supported in part by NSF grants to CPS.) Department of Anatomy, University of Colorado Health Sciences Center, Denver, Colorado 80262. | v3-fos |
2018-12-21T13:15:25.559Z | {
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} | s2 | Evaluation of Soybean Varieties for Iron-deficiency Chlorosis
Iron-deficiency chlorosis is a physiological disease caused by decreased iron in the soybean plant. Iron chlorosis is common when soybeans are grown on calcareous soils found in Kansas. Iron is required for formation of chlorophyll, the green pigment found in plants. When iron is limiting, iron chlorosis symptoms may be expressed in soybean plants. Interveinal chlorosis is the most common symptom, in which the leaves become yellow, with the veins remaining green. In severe cases, the leaves become white or necrotic, with stunted plant growth. Seed yield loss is a function of the severity of yellowing that occurs to the plant.
Introduction
Iron-deficiency chlorosis is a physiological disease caused by decreased iron in the soybean plant. Iron chlorosis is common when soybeans are grown on calcareous soils found in Kansas. Iron is required for formation of chlorophyll, the green pigment found in plants. When iron is limiting, iron chlorosis symptoms may be expressed in soybean plants. Interveinal chlorosis is the most common symptom, in which the leaves become yellow, with the veins remaining green. In severe cases, the leaves become white or necrotic, with stunted plant growth. Seed yield loss is a function of the severity of yellowing that occurs to the plant.
Planting soybean varieties with resistance to iron-deficiency chlorosis represents an effective method to protect against losses to the disease ( Figure 1). Resistant varieties increase protection for the younger leaves and the growing point, decreasing death rates and improving chances for recovery. Screening soybean varieties for iron-deficiency chlorosis is critical. Soybean growers need updated information on the chlorosis response of soybean varieties to Kansas soil and growing conditions. To address this need, this research evaluated currently grown commercial soybean varieties for resistance to iron-deficiency chlorosis.
Procedures
Two hundred thirty five soybean varieties, with three checks [resistant checks A14 and A15; susceptible check KS4202RR] with known responses were planted on July 1, 2005, in a greenhouse at Kansas State University to observe their chlorosis response to soil with a high pH. The soybean varieties were grown in soil collected, 0 to 6 inches deep, from an agricultural field near Zeandale, Kansas.
Plants expressing chlorosis have been observed growing in this field in the past. The soil type for this field was Haynie sandy loam with a soil pH of 7.8. To enhance plant growth, phosphorus, zinc, and copper solutions were added to the soil. A .5 quart container was filled with approximately one pound of this amended soil, and 5 seeds of each entry were planted approximately 1 inch below the soil surface, and thinned to 2 plants after emergence ( Figure 2). After emergence, the containers were placed in large, black plastic flats for bottom watering with a sodium nitrate solution.
Iron uptake values were recorded as chlorophyll (greenness) measurements with a Minolta SPAD-502 Chlorophyll Meter. Ratings were averaged from three plots for each entry. Chlorophyll concentrations were taken when the first and second trifoliate leaves were fully expanded, 12 and 17 days after emergence, respectively. The chlorophyll concentrations were taken on the center leaflet of the fully expanded trifoliates of two plants per container.
Results
Chlorophyll concentrations differed among the 235 varieties evaluated (Table 1)
Keeping Up With Research 140
scores of 27.7 and 28.7, respectively. The score of 28.7 was the highest average reading among the entries evaluated. Germplasm lines A14 and A15 were released in 1987 for parent stock in soybean breeding and genetics programs. These checks were identified after seven cycles of selection for improved resistance to iron-deficiency chlorosis on calcareous soils in the field. In previous research, the iron-chlorosis-resistant check A15 had been shown to do well in defending against iron-deficiency chlorosis in Kansas soils. Although both A14 and A15 possess superior chlorosis resistance, they are not adapted varieties for Kansas conditions.
No commercial variety possessed resistance significantly greater than these two checks. But out of the 235 varieties tested, 34 commercial varieties possessed average chlorophyll readings that were not significantly lower than the A15 resistant check. These 34 varieties are adapted to Kansas and possess a variety of agronomic characteristics sought by soybean producers.
This study has successfully identified commercial soybean varieties that possess resistance to iron-deficiency chlorosis. Producers are encouraged to use these results with information from seed companies/dealers to select varieties to help eliminate or reduce yield losses from irondeficiency chlorosis. | v3-fos |
2016-05-12T22:15:10.714Z | {
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} | s2 | Population genetic examination on the heterosis effects in crossbreeding Hungarian Simmental cattle with Herefords
increase genetic variation and milk production. While in the beginning of this period not all countries in Europe were involved, the situation now has changed : All countries are using combinations of American and European genotypes and the majority of test bulls has 50 and 75 p. 100 BS-genes. At present there are no indications of a complete replacement of European genotypes, but a combination is aimed at.
In the European Braunvieh-populations since 1965 American Brown-Swiss bulls were used to increase genetic variation and milk production. While in the beginning of this period not all countries in Europe were involved, the situation now has changed : All countries are using combinations of American and European genotypes and the majority of test bulls has 50 and 75 p. 100 BS-genes. At present there are no indications of a complete replacement of European genotypes, but a combination is aimed at.
While the early analysis of data showed large breed differences with negative heterosis and recombination loss, newer data showed an advantage in milk yield, fat and protein yield, while the protein content was slightly lower. The progeny of 5/8-and 3/4-BS-buils were on average as good in milk-production as the purebred American bulls, indicating that a combination of European and American strains is successful.
Rather limited information on fattening ability and carcass-quality indicate, that the crosses are taller, have a comparable or higher rate of gain with lower dress-out-p. 100. Higher internal fat leads in connection with heavier bones to a decrease in high priced cuts and to inferior carcass grading. With regard to secondary traits, the calving intervall of progeny from 100 p. 100 BS-bulls tends to be longer, but longer gestation and lower culling rates could have caused this findings. Milkability and calving ease were influenced slightly in a favourable direction.
Finally the possibilities of comparing European populations according to the IDF-proposals are discussed. Links between the European populations at present are only through the use of American bulls, most of them were progeny-tested and used in Europe in planned matings. An approach should be taken, to use this material to get hints about the base differences between the European countries. Nevertheless it should be tried to organise an exchange of semen from test bulls across countries and to get an unbiased proof on these in each of the cooperating countries.
Population genetic examination on the heterosis effects in crossbreeding Hungarian Simmental cattle with Herefords F. SZABO
University of Agricultural Sciences Keszthely, Faculty of Agricultural Sciences Keszthely, Hungary
We studied the heterosis effects in characteristics having fundamental influence on meat production as a result of crossbreeding Hungarian Simmental cattle with Herefords.
Evaluating a considerable number of data, Fi population was found to be superior not only to the average of the two purebreed populations, but also to that of the better parent population in major maternal characteristics. As a result of crossing, the following results were found as a consequence of heterosis effects : 7-8 p. 100 at the age of first calving, 6-9 p. 100 in the period between two calvings, 5-8 p. 100 at calving rate and 2-3 p. 100 in the rate of weaned calves.
Live weight, boned meat and meat production for I day of life and the percentage of meat, pectoral and abdominal tallow and tallow as a result of boning in the case of F l population gave better results than the average of Hungarian Simmental and Hereford, but fell short of Hungarian Simmental.
No heterosis effect was found in judgement scores of live animals and in killing out percentage. | v3-fos |
2016-05-04T20:20:58.661Z | {
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} | s2 | Y chromosome variants in cattle Bos taurus and Bos indicus
Summary It is shown that comparison of the size of the Y chromosome between individuals may be made as reliably by computation of measurement relative to the X chromosome as by proportionation relative to the genome in the cell. By either method agreement was found with previous studies : first in placing the Y in the size range, for the cattle karyotype, between autosomes 22 and 26 ; second by finding the Y in the Charolais breed to be larger than other breeds. The value of the C-band method for recognition of the Y in Bos indicus, in particular, is reaffirmed and acridine orange is used to confirm the hypothesis of pericentric inversion as the basis of the relationship between the Y of Bos indicus and that of Bos taurus. The G-band pattern showed seven discernable regions in either species, so that the terminal part of the q arm in Bos indicus is homologous with the p arm in Bos taurus. The overall size and centromeric index of the Y are defined for each of 18 breeds of Bos taurus and 8 breeds of Bos indicus. It was found that the Y might be expected to be distinctive in certain breeds or groups : Afrikander, Augus, Claarolais, Friesian, Guern.rey, Hereford, Jersey, Murray Grey, Shorthorn and Simmental. Furthermore the Y of Simmental bulls was found to be of the same order of size as that of Charolais, although differing in centromeric index, whilst the Romagnola Y was found to be one of the smallest of Bos taurus Y chromosomes. The Y in Bos indicus was found to have short p arms in a subtelocentric conformation, in a select group of Sahiwal ; other breeds also had subtelocentric rather than terminal point centromere in the Y. The Y is established as a marker for paternal descent in defined cases.
I. -Introduction
The first description of the karyotype of cattle, using hypotonic treatment of cultured embryo cells, was by M ELANDER (1959) who confirmed earlier determinations of the diploid number 2n = 60 and noted the metacentric form of the sex chromosomes in Bos taurus. C ROSSLEY & C LARKE (1962), using blood cultures, found the Y to be a submetacentric for several unspecified breeds of British cattle.
K IEFFER & C ARTWRIGHT (1968) described the Y chromosome of Brahman and Santa Gertrudis breeds as acrocentric. The karyotype of a presumptive pure-bred Bos indicus breed was recorded in studies of Sahiwal cattle in Australia by H ALNAN (1971) and in India, together with Red Sindhi, by G UPTA , S INGH & R AY -C HAUDHURI (1974). The Red Sindhi in Australia was later found to have the same karyotype as that of the herds in India i). Karyotypes of Australian Brahman, Santa Gertrudis (King Ranch), Australian Milking Zebu (AMZ), Belmont Red and Afrikander cattle were severally studied and reported showing the Y chromosome to be acrocentric in all those of Bos indicus paternity, excepting the Afrikander with a metacentric Y and presumptive Bos taurus male descent (H ALNAN , 1971(H ALNAN , , 1972H ALNAN & FRANCIS, 1976). In those studies the selection of the acrocentric Y chromosome from the autosomes was arbitrary. G UPTA , SicvcH & R AY -C HAUDHURI (1974) used C-banding to distinguish the Y chromosome in the Sahiwal and Red Sindhi cattle. confirmed this and the earlier studies of the Banteng cattle (F ISCHER , 1969 ;H ALNAN , 1974). Thus C-banding is considered to be the method of choice for distinguishing the Y from the smaller autosomes in Bos indicus.
C RIBIU & P OPESCU (1974) and C RIBIU (1973) (1979) described the Y in a Jersey bull as metacentric rather than submetacentric. Nevertheless, there is as yet no overview for the morphology of the Y chromosome in cattle ; unlike the situation in human studies where Y polymorphisms are expected between races or individuals (Mnxirro, 1975).
In studies in this laboratory it has frequently been found that the karyotype of an individual animal or the karyotypes of all the individuals in a select close-bred group or breed were distinctive and specifically recognisable, even without G-banding. This was often seen to be contributed to in part by the individuality of the Y chromosome. This paper describes the basis for those conclusions and adds to the collected work on cattle Y chromosomes.
II. -Materials and methods
Techraiques
All the results were derived from studies of the metaphase chromosomes of peripheral blood culture as previously described (H ALNAN , 1977). Preparations were stained by orthodox Giema's stain, G-Band (H ALNAN , 1976 ii & iii) and C-Band, modified to local conditions (P OPESCU , 1973 ;GurTn et al., 1974) ; photographed and printed to a final magnification for working of 4 000 X , for measurement and karyotyping.
Orthodox staining provided the core of the measured material described. In the case of every animal at least thirty metaphase cells were analysed. Where a count was made of cells according to sex, as for example in the case of chimeras or mosaics, it was found that the definitive sex of the cell could not be determined in every cell due to either overlapping or probable breaking of the cell with concomitant chromosome loss. A standard was set for the assayable cells : being no more than five overlappings and numerical completeness. Under this criterion both sex chromosomes would be expected to be able to be visualised in 87 p. 100 of female cells and 72 p. 100 of male cells according to determinations in Bos taurus (H ALNAN , 1971).
Measurem,ent arzd statistics
Measurements were made on photographs of orthodox stained preparations using a scale attached to a magnifier, calibrated to one tenth of a millimeter and engraved on a glass face, which was placed in contact with the photograph. The gauge, known as a Barton-eye gauge, was tested for reliability and accuracy by comparison with other methods (dividers and ruler, micrometer, ruler direct) and between different operators on the same photographs. Four sets of measurements were made on a cell, each by each operator, and analysed by Spearman's coefficient of rank correlation : they gave correlations of 98.5, 97 and 97 p. 100. The standard error of regression coefficient was 3, 3.5, 4 and 2.5 p. 100 using the gauge. Analysis of variance for regression by each of the four methods of measurement gave F values above 832 for 59 degrees of freedom. The tests in earlier work have been previously described and were more extensive than those outlined here (H ALNAN , 1971). The measurements were translated into relative size for the chromosomes by computation to parts per centum of the genome or total length of all the chromosomes. Then, using these figures, the Y chromosome was expressed as a proportion of X and X + Al (C RIBIU , 1975) in both cases significant difference was based on the criteria suggested by M ATERN & S IMAK (1967).
Results
In preparations where the chromosomes were resolved by orthodox Giemsa staining in all of the Bos taasrus breeds in the study, the Afrikander and the Belmont Red (derived from Bos Taurus), the Y chromosome was identifiable as a small metacentric or submetacentric chromosome. The significant types are shown, by breed, in figs. 1 & 2.
The Y chromosome of the pure Bos indicus breeds (Sahiwal and Sindhi) and the Brahman, Santa Gertrudis and Belmont Red (derived from Zebu males) was generally described as acrocentric ( fig. 2).
The Y in Banteng (Bos sondaicus, Bibos banteng or Bali cattle) was indistinguishable from that of the Friesian breed but insuffucient numbers were measured to include in the tables. The same is so for Shorthorn, like the AIS ; South Devon, like Guernsey ; Brahman crosses and the Zebu & British X Banteng as for paternal breed of origin.
The morphology of the Y, according to the rules proposed by L EVAN et al. (1964), was found to vary between breeds as shown in figs. 1 & 2 ; tables 1 & 2. The Bos taurus Y varied in centromeric index, expressed as P or * the percentage length of the p arm relative to the sum of the lengths of the p and q arms (P or * = p/p -I-q), from metacentric to submetacentric or 49 p. 100 to 27 p. 100. The size of the Y was found to vary between breeds. Expressed as a percentage of the diploid genome or as a percentage of the X, when measurements were corrected relatively by per centum of the genome, the Y was found to be typical in size for a particular breed.
Over all animals studied it varied between 1.0 p. 100 and 1.5 p. 100 of the genome.
The most common size being close to 1.2 p. 100. length and centromeric index, for the Y in relation to the X and shows that the largest autosome varies so little as not to significantly alter the relative size of the Y if added to the X in calculation. Thus the size and proportions of the Y, in relation to the X, were used to construct a graphical figure or analogue, table 2, to show visually the relative characteristics of the Y in different breeds. There is a descending size and an increasing index suggesting a form of reciprocity such that : as the Y increases its little arm proportion seemingly decreases. The relative measurements of size per centum may be read off the top. ' The Charolais and the Simmental breeds were found to have the largest Y chromosomes, furthermore, whilst similar in size they could be distinguished by difference in centromeric index (P or * ). The Romagnola breed had one of the smaller Y chromosomes recorded.
On the criteria of size and the centromeric index together it was found that not only in Charolais and Simmental but also in other breeds the Y was characteristic. The Charolais has the largest Y on relative size at 50 p. 100 but one of the smallest indices at 30 p. 100. The Jersey is almost the reciprocal with 34 p. 100 for size and 44 p. 100 for index. It was found that the visual impression of symmetry in the index was subject to about a 5 p. 100 error, thus when a chromosome is measured an apparent median point centromere will be found to be median region. Constancy of the Y was found in crossbred stock such that the Y in Murray Greys was identical to the Y of Angus the breed of paternal origin.
In Bos indicus the Y was always acrocentric fig. 2 & table 2. That is to say the centromeric index where p arms could be discerned, was less than 20 p. 100. It was found that in the select group of Sahiwal studied the Y was consistently subtelocentric (acrocentric not telocentric) in the other breeds this subtelocentric conformation was less marked and less consistent between cells. The Y was repeatedly recognised in the first instance without resort to C-banding as shown in fig. 3 an orthodox karyotype from the Sahiwal. The positive identification of the Y chromosome in Bos indicus is facilitated by C-band staining fig. 4. The measurement of the Y was less precise after C-banding than with orthodox stain. There was no hesitation that the size of the Y for Bos indicus compared to A22 to 26 and did not differ from Bos taurus.
In preparations treated with acridine orange (B OBROW & M ADAN , 1974) the Bos taurus Y was found to have a bright fluorescent yellow segment for most of the p arm and the Bos indicus Y showed the same effect of fluorescence in the t region of the q arm figs. 5 & 6. This response in colour was also found in the centromeres of the autosomes and in Bos indicus in the terminal region of a limited number of autosomes (H ALNAN et al., 1981).
The G-banded karyotype from a Sahiwal bull is presented in fig. 6 and from a Murray Grey in fig. 7 fig. 8. Fundamentally seven discernable landmarks in the Y were found here in both species. Whilst the G-banding does not serve to identify the Y as readily as C-band staining it is in agreement with the acridine orange results showing a darker « t region for the q arm in Bos indicus, an increased pale region 21 merging with q13 of Bt : the homology noted earlier.
ttt. -juiscussion The estimation of relative size of the Y chromosome has been simplified here, both in original measurement and in calculation of relative proportion, being the application of methods developed and tested in an earlier study (H ALNAN , 1971).
The sizes of the Y chromosomes of some of the breeds studied here were in close agreement with the conclusions of other authors in particular for the Charolais (C RIBIU & P OPESCU , 1975 ;C RIBIU , 1975) Since the results here, the previous studies by this laboratory and the results of numerous other authors provided estimates of size which did not differ (G USTAVS - SON , 1969 ;H ALNAN , 1971 andP OPESCU , 1973) and since the method of relative computation used here markedly reduces the laborious measurement of all the chromosomes, it is suggested that the Y : X computation has potential for routine laboratory service.
The proposal that the Y chromosome is not one of the smallest is the restatement of the well established observations of the earlier workers in the field, it is emphasised that this is the case in the face of the occasional tendency to underestimate its size. At as true modal length of 3 microns the Y has a potential of ten resolvable segments in theory ; in practice no more than seven landmarks are likely to be defined using the G-band method.
The size and relative arm lengths of the Y chromosome are determined for 18 breeds of Bos taurus and 6 breeds of Bos indicus for the first time comparatively in one text, the first characterisations by breed being by C RIBIU (1975). This has made possible the proposal that in specific cases the Y chromosome is sufficiently distinctive to be used as a reference for the determination of disputed paternity by breed. The dependability of the estimates of relative size and proportion has been established by statistical tests : first using the measurement and computation of relativity for the whole cell or genome and then deriving the more simplified method using fewer chromosomes.
It is shown that the Y chromosome for Charolais and Simmental could not be mistaken the one for the other, neither should either of them be confused with many british breeds, for example : Angus, Friesian, Guernsey, Hereford, Jersey or any of the asiatic breeds. Furthermore, there is distinct difference in size between some breeds of the continental European cattle. Nevertheless, the size of the Y chromosome was always found to be within the range comparable to autosomes 22 to 26 so that the Charolais Y would be in the 22 zone, whereas the Jersey would be found in the 26 grouping. This presents the question of the significance of the difference in size which M ATERN & S IMAK (1967) considered to be notable where it exceeds 8 p. 100 of the average length of the two chromosomes. The difference in the relative length of the Y in Charolais and Angus was more than 21 p. 100 making the distinction, in size alone, pronounced. It is of interest that the Murray Grey, product of Angus male line and original Shorthorn cow, was found to have a Y indiscernable from the Angus (H ALNAN , acd). Further to which the likeness between the Charolais X Angus cross and the Greys calls for an independent test for which the Y difference seems to be a candidate, although not the only factor. C-banding has been used in cattle studies for the determination of the distribution of constitutive heterochromatin and for the identification of the Y chromosome (PO PESCU , 1973 ;P OPESCU & B OSCHER , 1974 ;G UPTA et al., 1974 ;. In Bos indicus C-banding is indispensable for the definite recognition of the Y, however, fluorescent staining with acridine orange also facilitates recognition, whilst G-banding provides the means of seeing alterations in finer structure. With the C-band method the Y has the dark uniform staining throughout, as previously described by the authors noted above, the similarity in the overall density of stain to the X as mentioned by P OPESCU & B OSCHER (1974) is helpful in reading fresh preparations.
Morphologically the Y of all Bos indicus breeds studied here is considered to be acrocentric, which means that the centromere will be found at a variable distance from the terminal point of the small arm ; sometimes there will be no visible chromatin beyond the centromere and at other times the Y will have distinct p arms. The criteria for the definition of terms have been proposed by L EVAN et al. (1964) so whether the anglicised expressions or greek-stem terms are adopted their rules are helpful and to be recommanded as clarifying thought.
In optimum preparations in every Sahiwal bull studied by us the Y chromosome has been visibly subtelocentric, a definition which we do not see as significantly differing from acrocentric according to POTTER and U PTON or POTTER et al. (1979). In other breeds of Bos indicus the Y was for practical purposes indistinguishable from the telocentric state. These points serve to raise the question of possible Y polymorphism in Bos indicus in contradistinction to the theme here which proposes relative stability of the Y in Bos taurus. It follows that a definition for the line of demarcation in centromere index which would place the chromosome in the asiatic cattle class is desirable. Thus, it is proposed that the line be set at the ratio p : q of 2 : 8, wherein p will usually be less than 2 and q greater than 8. There seems to be a natural mensurate gap between the 20 p. 100 and 30 p. 100 zone.
The confirmation of the concept, proposed by B ASRUR (1969), of the relationship of the Y of Bos taurus to the Y of Bos indicus being a pericentric inversion is reiterated here (H ALNAN et al., 1981) based on the use of acridine orange it adds a dimension to the interpretation of the G-band pattern in the asiatic breeds and provides an additional method of examining the Y in the European breeds. A dark band in the telomeric region of the Bos indicus Y, by G-banding, was not found in Bos taurus and appears to be the expected corollary to the bright fluorescent tip with acridine orange. The G-band pattern has otherwise been delineated by L IN et al. (1977) indicating a centromeric region and three regions in each arm in Bos taurus. The proposal for Bos indicus recognises the same number of regions leaving one on the p arm, which may not be resolved in many cases and finding five in the q arm. Since the completion of this work we note with pleasure that P INHEIRO et al. (1980) described the two types of Y in the Ibage cattle showing the same rearrangement between the acrocentric and metacentric forms.
C RIBIU & P OPESCU (1975), in some measure, presupposed the possibility that the Y chromosome might be characteristic for certain breeds by their delineation of the unusually large size of the Y in certain of the european breeds. This study derived from accumulated data over a period of eleven years has extended that primary concept to hypothesise that there is a grouping of breeds which do have similar Y chromosomes and which may have a similar origin in stem stock from which they were derived by human selection. This is of course compatible with the concept of Y polymorphism in man (M AKINO , 1975) if it be accepted that in man breeds have not been defined in relation to variation in the Y.
Received for publication in January 1982.
Acknowledgments
We wish to thank A.R.G.C., A.M.R.C., D.R.C., The Chromosome Research Foundation for financial assistance and Professor R.M. B UTTERFIELD for fostering our efforts within his department. We are deeply grateful to Stan H!LniTCx, Anthony H ORDERN and John H ALLSTR O M for their encouragement and interest. To L ANIER Australia we owe a note of special appreciation for the loan of a word processor. | v3-fos |
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} | s2 | Damping-off of sugar beet in Finland. I. Causal agents and some factors affecting the disease.
The fungus Pythium deharyanum auct. non Hesse is the main cause of damping-off on sugar beet in Finland. The fungus is found especially in diseased seedlings during the first two weeks after emergence. Later on, when the plants have one or two pairs of true leaves,Fusanum spp. can be isolated to a rather great extent. However, pathogenicity tests with three differentFusanum species have shown that this fungus is unblc to cause damping-off on sugar beet when inoculated into peat substrate. Among the fungi tried in this respect, only Pythium deharyanum and Phoma betae Frank showed clear pathogenicity. Sugar beet seedlings that outlive the disease grow slower, and their quality at harvest in the autumn is poorer than that of healthy beets.
Introduction
The damping-off of sugar beet is caused by seed borne and also by soil borne fungi. Among the seed borne damping-off pathogens Phoma betae Frank is the most important (NOLLE 1960). The degree of P. betae infection in the seed varies considerably depending on the origin of the seed. According to LEACH (1941), the European sugar beet seeds are mainly contaminated with P. betae, while in some cases seeds from the U.S.A. have proved to be clean from infection. Polygerm seeds have proved to be more heavily infested with P. betae than monogerm seeds (SCHULTZE and BOHLE 1976).
Among the soil borne fungi causing damping-off of sugar beet, species of the genus Pythium Pringsh. are the most widely distributed (BUCHHOLTZ 1938, NOLLE 1960, PESHEL 1969, KUHNEL 1978. Within the genus, the species P. deharyanum Hesse, P. ultimum Trow and P. aphanidermatum (Edson) Fizp. have often been isolated (BUCHHOLTZ 1938, HILLS and LEACH 1952, GATES and HULL 1954, TILL 1968, LINNASALMI 1970, BÖTTCHER and BEHR 1980. Species of Aphanomyces de Bary give rise to damping-off on sugar beet in Europe and America. In Europe, A. laevis de Bary (GREIS 1942) seems to be somewhat common and in the western hemisphere A. cochlioides Drechs. (BUCHHOLTZ 1938, COONS et al. 1948 Maataloustieteellinen Aikakauskirja JOURNAL OF THE SCIENTIFIC AGRICULTURAL SOCIETY OF FINLAND PAPAVIZAS and AYERS 1976). Rhizoctonia solani Kuhn has also been isolated as a damping-off pathogen of sugar beet but has not been found to be as important as for instance P. deharyanum STEWART 1927, HILLS andLEACH 1952). Another organism, Fusarium spp., has commonly been found in investigations concerning the damping-off disease of sugar beet, but the actual importance of this fungus is controversial. According to HODGES (1936), MÖLLERSTRÖM and KLINTEBERG (1964) and BÖTTCHER and BEHR (1980) Fusarium species can be considered as primary pathogens on sugar beet seedlings although contradictory opinions are also existing (GATES and HULL 1954, LINNASALMI 1970, KUHNEL 1978. Damping-off investigations from Northern countries are only available by few research-workers, among others BJÖRLING (1945), MÖLLERSTRÖM and KLINTEBERG (1964), RASMUSSEN (1967), LINNASALMI (1970) and MÖLLER-STRÖM (1974). In Finland there have been severe outbreaks of damping-off on sugar beet in certain areas during the last few years, thus there has been need for a more profound investigation into the causes.
The aim of this study is to survey the most common causal agents of damping-off on sugar beet in Finland and also to study some factors affecting the disease. The investigations have been carried out in co-operation with the Finnish Sugar Beet Research Centre.
Introductory experiments
Introductory experiments on the damping-off disease of sugar beet were started in the late autumn of 1978. Soil samples were collected from 48 sugar beet fields where damping-off occurred (Fig. 1). One half of each soil sample was sent to the Sugar Beet Research Centre, where the soil type, pH number and the content of humus, clay and phosphorus, potassium, sodium and magnesium were determined in the laboratory. The other half of each soil sample was examined, from a plant pathological point of view at the Department of Plant Pathology, University of Helsinki. The bait method was used to determine the degree of soil infection. The experiment was carried out at high (+lB h2O°C) and low (+8 -l-10 o C) temperatures in a greenhouse. Untreated Monohill sugar beet seeds were used. Under the course of the experiment, healthy and diseased seedlings were counted at emergence and at about one 1 week intervals after emergence. Seedlings with damping-off symptoms were removed from the pots and the causal agents of the disease were determined in a laboratory.
Field experiments
Disease control experiments have been carried out during 1979 l9Bl in co-operation with the Sugar Beet Research Centre. The localities of these experiments were chosen according to the degree of disease found in the soil sample investigation (Fig. 2) and only the most severely diseased fields were chosen. All trials except those at Viikki Experimental Farm and at Pohjankartano Sugar Beet Experimental Farm were on fields of sugar beet growers. The sugar beets in the experimental areas were cultivated using the same techniques and fertilization levels as the farmer.
Seedlings with visible signs of damping-off were collected from all localities 10 days and 20 days after emergence. The samples were taken to the Department of Plant Pathology where the causal agents of the disease were determined.
The differences in the quantative and qualitative development between healthy and diseased, but living plants was studied on two localities in 1979 (Helsinki and Porvoo) and on five localities in 1980 (Helsinki, Janakkala, Salo, Mietoinen and Köyliö). After singling, 120 plants with disease symptoms were marked on all localities. In the middle of July and August and at the end of September, 40 diseased plants and 40 healthy plants from the areas between the diseased ones were taken for the determination of root-and topweight. At the end of September, the sugar content and the content of amino-N, potassium and sodium were also determined at the Sugar Beet Research Centre.
The isolation and identification of damping-offfungi
The causal agents of the disease were determined by putting pieces of diseased plant parts on agar into petri dishes (0 9 cm). The samples were first carefully rinsed under running water. Thereafter they were surface sterilized for 1 min. in 1 % NaOCl and after that for some seconds in 94 % ethanol. When the samplses had dried they were put on corn meal agar with 100-300 ppm streptomycinsulphate. The dishes were kept at room temperature (+2O -+24°C) and examination of the fungi with a stereo-and a lightmicroscope was started after about two weeks.
Pathogenicity experiments
The pathogenicity of some fungi isolated from sugar beet plants in 1980 was tested in autumn of the same year. The test organisms were inoculated into light colour peat moss 1 week before the sugar beet seeds were sown. The basic fungus suspensions that were inoculated into pots with 1.5 1 of peat, were prepared by homogenicating the mycelium of 1 petri dish (0 9 cm) into 100 ml of sterile water. Dilutions were then made from this basic suspension. The seed used was untreated naked Monohill. Observations were made just after emergence and thereafter during one week intervals. The pathogenicity tests were carried out in a greenhouse with a temperature of about +lB -+2O°C.
Weather conditions
There were considerable annual variations in the weather during May and June 1979-1981. May was in 1979, and especially in 1981 warmer than normal. However, in 1980 especially at the end of the month May was very cool (Fig. 3). June was cooler than normal in 1981 with some very heavy rainfalls. During 1979 and 1980 the precipitation was somewhat normal in June and the same applies for May during all three years.
Disease symptoms
In this investigation the main importance has been attached to postemergence damping-off symptoms, although pre-emergence damping-off appeared in some cases.
Beet seedlings are usually healthy at the time of emergence, the first disease symptoms not appearing until I-l/i weeks later. Infection at this stage usually leads to complete wilting and rapid death of the plant. A watersoaked, brown to grey or black area extends up and down the hypocotyl or the upper portion of the young taproot from the point of entry of the pathogenic organism. Discoloration may also, in later stages extend up into the petioles of the cotyledons. The collapse of the hypocotyl of the seedling is followed by desiccation. Under favourable conditions the following development may take place within I-2 days (Fig. 4).
When sugar beet seedlings with I-2 pairs of true leaves are attacked by damping-off pathogens they usually survive for several days after the initial symptoms appear (Fig. 5). Often such diseased plants recover and persist through the growing season. However, they have a slower growth than healthy plants and their neck collar is weak and typically ''strangled" thus, later in the summer they easily break at the root collar due to strong winds or agricultural proceedings. In the autumn at harvesting the abnormally developed beets can easily be separated from healthy beets (Fig. 6).
Damping-off fungi
The first isolations of damping-off fungi were made from sugar beet seedlings growing in the greenhouse. In this investigation out of the damping-off mycoflora of 48 soil samples Pythium deharyanum auct. non Hesse appeared to be the most common fungi (Fig. 7). The total number of fungual isolations made was 2309 belonging to 26 species. On an average, the P. deharyanum isolates constituted 87.3 % of the total number of isolations made. Next to P. deharyanum, Fusarium spp. (2.8 %), Phoma betae Frank (2.2 %) and Chaetonium glohosum Kunze (2.2 %) were the most common fungi. P. deharyanum was isolated to a greater extent just after emergence than at later seedling stages (Fig. 7). The amount of the fungus was 95.6 % 5 days after emergence in comparison with 77.6 % 35 days after emergence. With Phoma betae and Fusarium spp. the case was in this respect the reverse. Temperature had no clear affect on the relationships between the pathogens (Fig. 7, Table 1). Damping-off occurred to a significantly higher degree at high temperatures than at low, but emergence was poorer at low than at high temperatures (Table 2). This presumably was due to a greater pre-emergence damping-off at the lower temperature. The total number of fungus isolations from sugar beet seedlings growing in the field during 1979-1981 was more than 4000 (Table 3). Here, just as in the soil sample investigation, the fungus P. deharyanum proved to be the most common fungus, especially at the earliest seedling stage (Fig. 8). The procentual number of P. deharyanum at the early seedling stage was in 1979 71.7 % and at a later seedling stage 36.4 %. The corresponding values for 1980 and 1981 were 68.5 %, 20.6 % and 72.2 %, 21.1 % respectively. Fungi belonging to the genus Fusarium occurred more commonly at later seedling stages than at early seedling stages. P. betae was isolated to a rather small extent from seedlings attacked by damping-off under field conditions. The number of P. betae isolations varied between 0.5 % and 3.3 % according to locality, collecting time of the samples and year. The occurrence of the fungus did not depend on the stage of seedling development. Alternaria tenuis Nees occurred rather commonly in 1980, Ulodadium consortiale (Thiim) Simm. in 1979 and Olpidium sp. in 1981 (Table 3), but the pathogenicity of these fungi is questionable.
Pythium debaryanum auct. non Hesse The genus Pythium was established by Pringsheim, who placed it in the family Saprolegniaceae. Since that time many taxonomic details of the genus have been studied and characteristics for new species described. The relationship with other Oomycetes has been established and the genus was put into a new family, Pythiaceae, in 1897 (HENDRIX and CAMPBELL 1973). During this century valuable supplements to the botanical position of the genus have been found by among others MATTHEWS (1931), MIDDLETON (1943) and WATERHOUSE (1967).
The fungus survives in soil by resting spores and by saprophytic growth. Pythium spp. do not compete well with other micro-organisms and they 234 Fig. 9. Pythium debaryanum. Oogonium and antheridia. x 1500. Fig. 10. The pycnospores of Phoma betae.
x 1200. grow saprophytically only under circumstances where other organisms are not present or have reduced growth due to unfavourable conditions in the soil. However, survival by resting structures is more important than survival by saprophytic growth. As the case is with many other soil fungi, the mycelium of Pythium spp. undergoes lysis in lack of suitable nutrient sources or when the sources are colonized by competiting micro-organisms. The main mechanism of survival for short and intermediate periods is by zoospores and sporangia and by oospores for long periods (HENDRIX and CAMPBELL 1973).
The Pythium species found in this investigation has, according to WATER-HOUSE (1967) been determined as P. debaryanum auct. non Hesse. The fungus grew rapidly on CMAor PDA-substrate covering the surface of the agar in petri dishes (0 9 cm) after about two days. The aerial mycelium was hyaline, unseptate, single hyphae up to 10 /xm wide. The formation of oogonia was abundant on CMA substrate, especially when adding wheat germ oil to the substrate. The oogonia were globose, terminal or intercalary and 25.3 /xm (20-32 /xm) in diameter. Oospores were 20.2 /xm (15-24 /xm) in diameter, smooth and apleurotic. There were up to 5 (1 -5) antheridia, usually 2-3 from the same or from another hypha than the oogonium. They were clubshaped, with a long stalk and 6-lo /xm in diameter (Fig. 9). Sporangia formed only in one case. They were sphaerical to oval, 20-28 /xm in diameter.
Phoma betae Frank
The first observations of P. betae as a causal agent of damping-off on sugar beet were made by FRANK (1894). The morphology of the fungus has been described by EDSON (1915) and especially by BJÖRLING (1945), who also described the sexual form of the fungus, Pleospora betae. P. betae is a seed borne fungus and the main mechanism for survival over longer periods is in the seed. The ability of the fungus to survive in the soil or in plant residues in the soil is questionable varying from a few months to two years (POOL and MC KAY 1915, BUGBEE andSOINE 1974). According to MC KAY (1952) P. betae cannot survive in soil without suitable plant residues. P. betae grew well on CMA-substrate. The mycelium of the fungus did not show as rapid a growth as that of P. debaryanum. Young hyphae were hyaline, septate, becoming brownish at older stages, 2 10 /xm in width. Different isolates of the fungus formed varying amounts of pycnidia. The pycnospores were sphaerical to rodshaped, hyaline, 3 5 jum long and about 2 /xm wide (Fig. 10).
Pathogenicity of the fungi
The first pathogenicity test with 6 fungus species isolated from sugar beet seedling in 1980 shows that only Pythium debaryanum and Phoma betae are capable of causing damping-off on sugar beet when inoculated into peat. In this experiment, a tenfold dilution of the original spore suspensions was made and this led to a more serious infection when inoculating with P. betae, but with P. debaryanum the dilution had no significant effect (Fig. 11).
The pathogenicity of 12 isolates of P. debaryanum and of 5 isolates of P. betae was tested. The results show that there were differencies between the isolates in this respect (Fig. 12). Compared with the control all but nr. 3 and 11 of the Pythium isolates were pathogenic, the most highly pathogenic being nr. 4, 5 and 12. In this experiment a ten-or hundredfold dilution of the basic spore suspension led to a rapid decrease in the disease causing ability. Out of the five isolates of P. betae examined, only one did not cause disease. Tenand hundredfold dilutions of P. betae still gave rise to damping-off, which is in contrast to the corresponding results of P. debaryanum. P. debaryanum attacked the plants faster than P. betae (Fig. 13). Two days after emergence 11 % of the plants growing in pots inoculated with P. debaryanum showed signs of damping-off, when the corresponding number in P. betae was only 1.5 %. This difference, however, was soon equalized in such a way that both treatments showed the same amount of disease at the end of the experiment. Fig. 11. The pathogenicity of some damping-off fungi to sugar beet seedlings.
Disease and soil factors
The occurrence of damping-off and the correlation of the disease to the soil type, pH-number and some soil minerals important to sugar beet were investigated in 47 soil samples. The percentage of damping-off varied between 8 and 100. The contents of phosphorus, potassium, sodium and magnesium also varied to some degree. The content of humus was on an average 9.9 % (1.7-44.3) and the pH-number 6.2. (5.1-6.9). There was no correlation between postemergence damping-off and any of the soil factors measured. However, when both pre-and postemergence damping-off were taken into regard there were weak negative correlations between the amount of damping-off and the content of potassium, sodium or magnesium (Table 4). Effect of damping-off on the quantitative and qualitative development of beet plants When comparing the quantitative development of beet plants attacked by damping-off but surviving, to that of healthy plants, it is evident that they develop at a different rate than the healthy plants (Fig. 14). In the middle of July 1979, the root weight of the diseased plants was only 47 % of the healthy ones. The corresponding value for 1980 was 50 %. In the middle of August the root weights were 63 and 77 % respectively. In September, just before harvest the infected beets had a root weight of 44 % in 1979 and in 1980 77 % of the healthy ones. The development of the top weights followed a pattern similar to that of the root weight (Fig. 14).
In September 1979 and 1980 some quality factors of the beet roots were also determined. The average results from two years investigations show that the disease only slightly influenced the content of sugar, potassium and sodium, but that the content of amino-N compounds was higher in diseased plants than in healthy plants (Table 5). However, in localities with very serious attacks opf damping-off, as in Porvoo in 1979, the disease also leads to a lower sugar content in the beet roots.
Discussion
The fungus Pythium debaryanum auct. non Hesse occurs widely in Finnish sugar beet fields, and is by far the most important causal agent of damping-off on sugar beet in Finland. Evidence of this is available from a soil sample investigation, field investigations and pathogenicity tests. These results are in agreement with several other works (among others BUC-HHOLTZ 1938, KUHNEL 1978. P. debaryanum seems to have a very quick mode of action, attacking the sugar beet seedlings mainly within two weeks after emergence, under certain conditions even before emergence. The outbreak of disease at this early stage leads almost always to the death of the seedlings. When the attack takes place later, many of the affected plants may continue to live and are invaded by secondary pathogens and saprophytic fungi. P. debaryanum being a weak competitor with these (HENDRIX and CAMPBELL 1973) is no longer commonly found in 3 5 weeks old seedlings. To get a correct picture of the causal agents of the disease one must therefore collect samples at short intervals beginning from the emergence.
Globally the fungus Phoma betae Frank is somewhat as common as Pythium spp. on beet (DUNNING 1971), but in this investigation the fungus played no important role. However, 25 years ago P. betae was as common as P. debaryanum in Finland (LINNASALMI 1970). Because P. betae is a seed borne beet pathogen, the reason for its decline as a causal agent of dampingoff must be that the seeds nowadays are quite clean from infection. Overwintering of the fungus in plant residues in the soil under Finnish conditions has not been investigated.
Another organism, Fusarium spp., was isolated quite commonly from beet samples collected at later seedling stages. According to GATES and HULL (1954) Fusarium spp. may be weak damping-off pathogens in acid soils with pH-numbers lower than 6.5. In this investigation the pathogenicity of only one isolate of F. culmorum, F. oxysporum and F. sambucinum was studied. These isolates did not cause disease when inoculated into peat. However, knowing the great variation within the genus Fusarium and among isolates within different Fusarium species, and that the peat substrate is not the most suitable one for sugar beet, it is difficult to draw any conclusions about the real pathogenicity of the fungus. On the whole, the actual role of Fusarium spp. in the damping-off complex of sugar beet needs further investigations.
On the basis of field and greenhouse observations the damping-off disease of sugar beet can in Finland be divided into two distinct types, which are very similar to those described by WARREN (1948). Firstly, a very dangerous type which rapidly results in the death of the plants. This phase begins with the germination of the seed and continues to the time, when the first true leaves develop. Secondly, a cronic type which begins after the first phase and continues for some weeks. The later stage of this type of damping-off has often been given the name ''strangles" (BOYD 1966, SCHOLLMEYER 1980 and has been said to be mainly due top abiotic factors. However, under Finnish conditions, it seems clear that the symptoms referred to as ''strangles" have their origin in typical damping-off symptoms, which are caused by different micro-organisms. In this investigation no correlation between damping-off and the pHnumber or the amount of humus and clay in the soil was found. Concerning the pH-number these results differ from many other works (BUCHHOLTZ 1938, MC KAY 1952, in which a rise of the pH-number has led to a decrease in the disease. In this investigation there were no soils with the pH-number over 7, possibly explaining the lack of response. The importance of the soil type for the occurrence of damping-off is contradictory (GATES andHULL 1954, REMY 1950). According to many works the physical qualities of the soil are more important than the soil type (GRAM 1927, COONS and STEWART 1927, LIKAIS 1948. The weak preventive effect of potassium, sodium and magnesium, that was found in this study, is in agreement with the works of YALE and VAUGHAN (1962). In some other studies especially the application of phosphorus prevents the disease (MÖLLERSTRÖM and KLINTEBERG 1964). Already in 1945 BJÖRLING suggested, that beet plants recovering from the attack of damping-off, develop into beets, which are quantitatively and qualitatively inferior to healthy beets. In the present study the correctness of this statement was justified. But it was also seen that recovering is very much dependent on the weather conditions of the year. In 1980, when the summer was warm and precipitation sufficient, the plants recovered better than in 1979.
Acknowledgements and participation. The results of this study were produced with the co-operation of the Department of Plant Pathology at the Helsinki University and the Finnish Sugar Beet Research Centre. Dr. Agr. and For. Risto Tahvonen and Lie. Agr. and For. Mauritz Vestberg from the Department of Plant Pathology have been responsible for the mycological experiments. Dr. Agr. and For. Kyösti Rainioko and Agr. Nils Nuormala have, on behalf of the Sugar Beet Research Centre, participated in the planning of the experiments and have been responsible for the practical side of establishing and conducting the field experiments.
The Finnish Foundation for Sugar Beet Cultivation and the Finnish Association of Academic Agronomists have supplied Mauritz Vestberg with a grant to help promote the study. Agricultural experts and agronomists from different sugar factories have helped establish and complete the experiments and a number of farmers gave their fields to be used for the experiments. The head of the Department of Plant Pathology Prof. Eeva Tapio has, by her encouragement and support made it possible for the completion of the experiment. We would like to express our warmest thanks to all the above. 2 | v3-fos |
2016-05-18T08:15:58.591Z | {
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} | s2 | Coat colour variants of village pigs in Papua New Guinea
Summary A preliminay description and genetic analysis of coat colour variation in Papua New Guinea village pigs is presented. Data were obtained from a series of deliberate matings and from surveys of coat coloration in two villages. A number of pigmentary variations caused by alleles at the Agouti, Extension and Brown loci were found and are described, including a possible new allele at the Brown locus (Bcausing brown spots on the basic red background colour. The segregations observed in the deliberate matings can be interpreted in terms of two alleles at the Agouti (A and a) and two at the Extension loci (E + and EP). Results from the village surveys show a high incidence of agouti phenotypes [Arelative to black [a], and a very low incidence of white designs (belt or points). These results suggest a still small genetic influence of exotic pigs in the villages, in spite of deliberate distribution, provided that the allele causing black colour (a) is present in the native Papuan stock. The allele E (spotted) giving black patched on red (or secondarily white) background may also be considered as a part of the native stock. The results are consistent with a hypothesis concerning an early arrival of pigs in Papua New Guinea relatively soon after domestication, followed by a period of isolation.
A preliminay description and genetic analysis of coat colour variation in Papua New Guinea village pigs is presented. Data were obtained from a series of deliberate matings and from surveys of coat coloration in two villages. A number of pigmentary variations caused by alleles at the Agouti, Extension and Brown loci were found and are described, including a possible new allele at the Brown locus (B k ) causing brown spots on the basic red background colour. The segregations observed in the deliberate matings can be interpreted in terms of two alleles at the Agouti (A w and a) and two at the Extension loci (E + and EP). Results from the village surveys show a high incidence of agouti phenotypes [A w ], relative to black [a], and a very low incidence of white designs (belt or points).
These results suggest a still small genetic influence of exotic pigs in the villages, in spite of deliberate distribution, provided that the allele causing black colour (a) is present in the native Papuan stock. The allele E P (spotted) giving black patched on red (or secondarily white) background may also be considered as a part of the native stock. The results are consistent with a hypothesis concerning an early arrival of pigs in Papua New Guinea relatively soon after domestication, followed by a period of isolation.
I. -Introduction
The pig is the main domesticated livestock species in Papua New Guinea, despite the introduction over the last 50 years of cattle, goats and sheep (H OLMES , 1980). Remains dating back 5 000 years suggest that the pig was introduced by man during the introduction of agriculture (B ULMER , 1966).
Disagreement exists as to the taxonomy of the native pig (Z EUNER , 1963 ;E PSTEIN , 1971) due to a lack of knowledge of its origins.
There are over one million village pigs in Papua New Guinea (D ENSLE Y et al., 1978), some of which are crossbred from the use of exotic breeds such as the Berkshire, Tamworth, Saddleback and Large White. Such crossbreeding, which has been encouraged by missions and the Department of Primary Industry (P URDY , 1971), could lead to a situation where the characteristics of the native pig become difficult to assess.
An initial study on the growth and carcass characteristics of the native pig was published by M ALYNICZ (1973 a).
The present paper is devoted to a description and genetic analysis of coat colour variation in village pigs.
II. -Resume on genetics of coloration 1. General considerations on mammalian coat colour Coat colour in mammals is due to melanins, a family of pigments whose biochemical production is controlled by a few loci known by their mutants in many species : Albino C, Agouti A, Extension E and Brown B.
The chemistry of coat colour genetics has been described recently by P ROTA & S EARLE (1978). The Albino locus (C) controls the first step in the oxydation of tyrosine, the basic raw material for melanin synthesis. The Agouti (A) and Extension (E) loci, working outside the melanocyte (A) and within the melanocyte (E) respectively, control regional and total extension of eumelanins (black or brown) and phaeomelanin (yellow/red) in the body and in individual hairs. The mutants at the B locus affect eumelanin only, changing black to brown.
Besides these main basic pigmentation loci there are several other loci where mutants may affect the coat colour in various ways.
Coat colour in the pig
The genetics of coat colour in the pig has been reviewed by S EARLE (1968), AnnLSTEiNSSOrr (1974), and more recently, by O LL mER & S ELL iER (1982). Variants at the Agouti (A) and Extension (E), as well as at loci for Dilution (D), White (I) and White belt (Be) have been detected. This species is also characterised by a variegated allele of Extension which gives black patches on a red background. This pattern can be confused with black piebaldness if the red colour background disappears. Center, Goroka, in the Eastern Highlands to examine the inheritance of coat colour in native pigs. Three breeding groups, consisting of agouti (wild), black and spotted pigs, were established. Agouti was defined according to DE F REDRICK (1971) as a pattern characterised by juvenile striping and a « grey body in the adult stage with zonated hair : black, yellow, black. Spotted was taken to include black patches on a white or red background.
2. In 1980, a small survey of village pig coat colouration was carried out at Ngasawampum village, 25 km West of Lae in the Markham Valley of the Morobe Province (67 pigs) and at Kenewenka village, 25 km North of Goroka in the Eastern Highlands Province at an altitude of about 1 800 m (40 pigs).
This survey was done in order to check the range of phenotypic variation and to make an initial evaluation of the phenotypic frequencies of pig coat colour patterns in the villages.
3. Some results from crossbreeding at the experimental pig unit of the Faculty of Agriculture, University of Papua New Guinea in Lae, which were collected in 1980, were added to the Goroka results. Figure 1 shows the geographic situations mentioned.
4. To facilitate a more exact description of the colouration of the European wild pig than that found in the literature (S EARLE , 1968 ;S NETHLAGE , 1980) and a comparison with the agouti colouration found in the Papua New Guinea pig, two examinations of wild pig populations were performed (by J.J. L AUVERGNE ) in France. The first was in Chambord Park (Loir-et-Cher) after game cropping and the second was on a private station breeding wild pigs at La Lauze (Vaucluse, S.E. of France). The definition of black and spotted pigs used in 1974 was found to be satisfactory as a basic for classification in 1980 ( fig. 2 a and 2 b). In the latter survey, a variant of spotted was seen in which the black patches are replaced by brown ones, the back-ground remaining white or red ( fig. 2 c).
For adult agouti pigs in Papua New Guinea, the main pattern may be named « white belly agouti » : the body is agouti grey (i.e. with zonated black-yellow-black hair) with a light belly and a white snout ( fig. 2 d).
Some minute variations are observed in this pattern. Sometimes the white designs are not well marked. Occasionnally black patches in the skin give the appearance of « black spotted agouti ».
A more important variation may be seen with the replacement of the light belly of the previous pattern by black, the disappearance of the white snout and the presence of some black on the underjaws, legs and mane ( fig. 2 e). This pattern is homologous in appearance with the « badger face pattern of sheep and goats (see ADALSTEINSSON, 1970 ;LAUVERGNE, 1978).
In the juvenile stage, agouti animals are characterized by a longitudinally striped coat, black and yellow ( fig. 2 f). Between animal variation and the age of attainment of the adult coat are yet to be described and defined. b) Genotypic interpretation a) Spotted. Previously what has been called « spotted » in this study was known as « black spotting » (in French « domino » ; L AUVERGNE 8c C ANOPE , 1979) and is considered to be the effect of the variegated allele at the Extension locus, as mentioned above, and not that of an allele at a locus for piebaldness.
Homologous situations are found in the guinea pig, rabbit, bovine and dog (S EARLE , 1975). The allele inducing « spotted in the breeds of the western world has been named ei or E P . fi) Brown spotted. The most likely interpretation for the brown spotted pigs seen in 1980 is an allele at the B (Brown) locus, probably recessive, transforming black eumelanin into brown. B 7is proposed for this allele (k for Kenewenka : the village where this variation was identified for the first time). y) Agouti. A preliminary interpretation is that the agouti white belly pattern is induced by the wild type allele at the Agouti locus : « white belly », symbol A w . This is the interpretation proposed by S EARLE (1968) who does not however seem to consider the possibility of variations in the wild pattern under the control of alternative genes at the Agouti locus. In France, where a phenotype very similar to the Papua New Guinea agouti has been found at La Lauze, there is another wild pattern with uniform agouti-grey except for the underjaws, mane and legs having some black. The « badger face type agouti described above may also be induced by another allele at the Agouti locus : A b (b for badger face). The early presence of the white belly agouti pattern in Papua is also suggested by a photograph from B REHM given in E PSTEIN (1971).
White designs
In the first samples in 1974 no white designs were included as they were rather scarce in the village population. In 1980, however, white belt and black with white points designs were observed. These variations may be attributed to the introduction of Saddleback and Berkshire pigs ( fig. 3 a and b).
According to O LLIVIER & S ELLIER (1982), the belt is induced by the Be allele
which is dominant over the be wild allele. No name has been proposed for the allele inducing black with white points.
B. -Analysis of segregations
Results
In the 1974 data, no distinction was made between the agouti phenotypes. The results of the Goroka and Lae studies are given in table 1.
Interpretation
The segregations in table 1 may be interpreted with a dihybridism hypothesis : -in Agouti, two alleles : A Wand a self black, a ; -in Extension, two alleles : E+ (wild) and E P (variagated or spotted), provided it is admitted that the agouti piglet of line 1 (table 1) is a mistake.
The dominance and epistacy relationships between these alleles may be interpreted as in table 2 where the phenotypes corresponding to various genotypes are given in accordance with the phenotypes observed in the matings shown in table 1.
The phenotypes of A w A w E+ E P and A w A-E P E P are probably mainly spotted, as A w a E+ E P is spotted.
Such an interpretation of experiments made with few animals and which does not take account of possible incomplete dominance must be considered with caution. However, a study of the literature on pig colouration has not revealed any studies on epistatic relationships between loci A and E (see S EARLE , 1968 andO L LW IER &S ELLIER , 1982). Such experiments need to be carried out with lines whose genotypes are known perfectly. For example the lack of black phenotypes among the progeny of matings between agouti parents could simply result from lack of heterozygotes in the parental sample or recombination sampling rather than a wrong hypothesis. The results show a high incidence of agouti phenotypes in the two villages, relative to black. There is also a low incidence of white designs.
As both villages are close to major centers of exotic pig distribution, and as most distributed pigs are black with white designs (Berkshire or Saddleback), red (Tamworth) or white (Large WhitelLandrace), the low incidence of the piebald and white as well as the absence of red phenotypes suggests a small genetic influence of exotic pigs at the village level. The black allele must be considered as present in the native Papuan stock.
This low proportion of exotic phenotypes is consistent with a high mortality recorded among exotic pigs distributed into the village environment in the early seventies. Average mortality was 30 percent within six months of arrival (M ALY -N icz, 1973 b).
In Vanuatu, Q UARTERMAIN (1981) noticed a high frequency of black and agouti phenotypes, relative to red, spotted or white phenotypes, particularly among feral pigs and in inland villages. DE F REDRICK (1971, 1977 points out the universality of agouti pigs in the Solomon Islands and that black and spotted pigs are also common. In both countries it is probable that the influence of exotic introductions has been greater than in mainland Papua New Guinea since white and red pigs are not uncommon in coastal locations or on certain islands.
V. -Final discussion and conclusion
The situation is consistent with an early arrival of pigs in Papua New Guinea (see B ULMER , 1966) relatively soon after their domestication in the Middle East (I SAAC , 1970 ;B6 K 6 NYI , 1976 : 7 000 BC), when few colour mutants (a and E P ) had yet arisen, followed by a period of isolation with the appearance of new mutants such as B k in B. In that respect the situation is similar to that of the Sheep after its domestication, also in the Middle East, as analysed by L AUVERGNE (1979 a and b). Accumulations of variants with visible effects (mainly colour genes) can be used as markers to date when a given population started to migrate. In general, the first to migrate are the most archaic with the least variants and are found at the periphery.
More precise mendelian analysis is clearly needed and could be pursued by the creation of pure and segregating lines of native pigs. Work is needed to : 1) study allelism and dominance in the various series : Agouti, Extension, Brown ; 2) analyse epistatic behaviour, especially between Agouti and Extension genotypes ; 3) study all new variants that have arisen or can be found.
The other conclusion from this study is that the allelic situation, with exotic breeds carrying alleles different to those of native populations, provides an easy way to measure the degree of genetic invasion by exotic genotypes, as seen above. Such measurement of colour gene or phenotype frequencies has already been proposed by C ASABIANCA and Mo L ENAT (1977) for the native pig of Corsica which is facing upgrading by the Large White. In Corsica the frequency of white phenotypes was used, while in Papua New Guinea the frequencies of white designs and red colouration can be used.
Received for publication in January 1982.
Acknowledgments
The manuscript has been read by the D&dquo; Ph. D REUX (E.N.S. Zoologic, Paris) et S. ADn L S T EiNSSOrr (Agricultural Research Institute, Reykjavik, Iceland) who have suggested useful improvements. Thanks are also due to the managers of Chambord Park and La Lauze breeding unit. | v3-fos |
2017-07-29T04:25:16.607Z | {
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} | 0 | [] | 1982-07-15T00:00:00.000Z | 13633785 | {
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} | s2 | Egg and yolk production traits in relation to ovum development, liver and liver moisture weight in dwarf and normal White Leghorns
Data on egg and yolk production and on ovum development were obtained from White Leghorn Dw and dw hens belonging to 4 sire families between 52 and 56 weeks of age. Ovum development was followed by the fat-soluble dye incorporation technique. At the end of this perdiod, the birds were killed and body weight, liver weight and liver water content, as well as rapidly growing ovum weight and ovum number, were recorded. We obtained the following results. 1) Average daily yolk production and weight of active ova were higher in the Dw genotype. The number of actively developing ova was higher in Dw hens, but the difference between genotypes was significant only on postmortem examination. 2) The average number of follicles undergoing rapid development at any time, estimated from eggs laid during the dye-feeding period and the number observed on postmortem examination, corresponded well in both Dw and dw genotypes, suggesting that there was no intra-peritoneal absorption of yolk in this stock. 3) For cultch sizes greater than one, the dwarfs took 3 h longer than the normals to lay their first egg. This might be due to a longer stay in the oviduct or to delayed ovulation. 4) Although the overall difference was not significant, it was noticed that the rapid development of the ova in clutches of all lengths and at any position in the clutch was shorter in dwarfs. 5) The time between the end of ovum yolk deposition and the laying of the resulting egg was significantly longer in dwarfs. 6) The weight of the liver was significantly correlated with yolk weight and the duration of rapid ovum development in both Dw and dw genotypes. The laying rate was not correlated with liver weight, while the rate of yolk production was positively but non-significantly correlated with this trail. in both Dw and dw genotypes.
I. Introduction
H UTT (1959) observed that the sex-linked dw gene reduced the rate of egg production in egg-type hens. These conclusions have been confirmed by other authors but, mainly due to body weight differences of the populations studied, there appears to be some disagreement concerning the extent of these effects.
Although ovarian follicular growth and maturation have been followed with the fat-soluble dye incorporation technique by several workers (WARREN & C ONRAD , 1939 ;L ACASSAGNE , 1957L ACASSAGNE , , 1960L ACASSAGNE , , 1962BACON & S KALA , 1968 ;Lvosm, 1978) in laying hens, the effect of the dw gene on these characters has only been reported in a broiler population by J AAP & M OHAMMADIAN (1969). The latter authors observed that dw reduced the rates of yolk deposition in the ovary but not yolk production in pullets weighing an average of 2.6 kg at 36 weeks of !age. (1968) and L EVEILLE (1969) concluded that the liver was the main site of fatty acid synthesis in chickens but G ARLICH et al. (1975) and S HIVAPRASAD & J AAP (1977) did not find any consistent association between liver weight and the rate of yolk and/or egg production in laying hens. This paper reports an attempt to determine the extent of the depressive effect of the dw gene in a low body-weight population on ovarian function and yolk production and the influence of the liver on these traits in White Leghorns.
A. Birds and experimental conditions
We used birds from the « heated group of the experiment described earlier by B ANERJEE et al. (1981). These b'ÎŒ'ds, sampled at 39 weeks of age on the basis of their egg production, included 10 Dw and 10 dw females belonging to 4 sire family groups. They were put into cages under normal temperatures (about 15 to 20 °C).
The experiment was carried out for 36 days when the birds were between 52 and 56 weeks of age. During this time no mortality was observed, and the nate of egg laying and clutch size were low. Throughout the experimental period the birds received 14 h of light and 10 h of darkness per day. Water and feed were given ad libitum ; the feed contained 16 p. 100 of total protein and approximately 2 600 Kcal/kg ME. B. Traits measured or calculated Starting on 28-9-1981, each of the 20 birds was fed daily between 9 and 9.30 h a 0.68 ml gelatin capsule containing about 15 mg of either Sudan Black B (B) or Sudan IV (R). These dyes, fed in the sequence R, B, R, B, B, R, B, R, B, R, B, B, etc., gave a non-repetitive sequence of 12-day dye deposition in the ova of the subsequently laid eggs. This sequence allowed to determine to the nearest day when the first dye was deposited in the ovum entering rapid development the period during which yolk material was actively deposited and when rapid development stopped. Starting from 29-9-1981, all the laid eggs were hard-boiled each day ; after they had been cooled under running tapwater and immediately opened, the dye sequence deposited in the ovum was determined. The same person examined the dye sequence throughout the experiment. These 12-day sequences of dye adminis!tration were continued for a 36-day period. At the end of this period, all the birds were killed, and we collected or calculated the following data : (1) egg number, rate of lay, clutch size and egg and yolk weight (of boiled eggs) during the dye-feeding period ; (2) the hour and the time between two successive ovipositions during the dyefeeding period; the hour of laying was recorded at 9, 10, 11, 13, 14 and 17 h ; (3) the duration of rapid ovum development (in days) by the fat-soluble dye incorporation technique ; (4) the time between the end of yolk deposition in the ovum and egg laying (in h). We assumed that the interval was constant between the time the dye was fed and the time of its deposition in the yolk. This time was calculated as follows : the dye was fed each day between 9 and 9.30 h. It was supposed that after .about 4 h, i.e. at 13 h, the dye had entered all the ova which were rapidly developing. Thus, in the dye sequence in each ovum laid, the last dye entering the ovum was presumed to have entered at 13.00 h the day it was fed. This time was taken as the minimal estimation of the end of rapid ovum development. The period (in h) between the end of this development and laying was studied ; (5) the rate of yolk deposition in the ovary. It was estimated in two ways : (i) by the daily rate of yolk production calculated from eggs laid during the dye-feeding period, just prior to slaughter, plus eggs in the oviduct on postmortem examination and (ii) by the total weight of rapidly developing ova, i.e. all those stained with dye. The estimate based on daily egg production assumed that all yolks ovulated were recovered as laid eggs or that the percentage of yolks lost between ovulation and oviposition was similar in normals and dwarfs ; (6) number of active or rapidly developing ova during the dye-feeding period and on postmortem examination. During the dye-feeding period, trrs number was obtained by counting the number of ova taking a certain dye of the dye sequence. These counts were made for each dye fed to a bird during the period and an average number of active ova was obtained for each bird. The number of active ova on postmortem examination was obtained by couting all the dye-stained ova in the ovary of each bird ; (7) body, liver weight and percentage of dry matter in the liver on postmortem examinat:on.
C. Statistical analysis
Mean differences between genotypes were tested by the paired t-test. Each pair consisted of a normal Dw hen and a dw hen which was a full or half-sister of the former. The phenotypic correlations were calculated for all traits within genotypes. The correlations of the two genotypic groups were combined and calculated when they were homogeneous. Table 1 presents the mean and the dw/Dw ratio (in percent )of the traits measured. Table 2 gives egg and yolk weight, time of laying, time between two ovipositions, time between the maturation of the ovum and its laying and the time required for rapid .ovum development as related to clutch length and rank in the clutch. Table 3 shows the phenotypic correlations of all traits measured with the combined genotypes.
IV. Discussion
A. Means Table 1 shows a highly significant difference for body weight and a significant one for egg and yolk weight. Egg number and clutch size were not significantly different between Dw and dw during the 52-56-week period, although there was a very highly significant difference for these traits up to 39 weeks of age in a larger sample as well as in the present sample. Average daily yolk production and weight of active ova were significantly higher in the Dw genotype. The number of active ova estimated during the dye-feeding period was not significantly lower for dwarfs, while on postmortem examination it was significantly higher in the Dw genotype.
Although not significantly different, the time required for rapid development was longer for Dw hens (8,91 vs 8.18). A similar trend was observed by J AAP & M OHAMMADIAN (1969) working on broiler dams.
The average number of follicles undergoing rapid development at any one time, estimated from eggs laid during the dye-feeding period and the number of follicles observed on postmortem examination, corresponded well (Dw : 5.67 vs 5.20 ; dw : 4.54 vs 4.60). In the Dw genotype a slightly lower number on postmortem examination was due to the fact that two birds went out of production towards the end of the experiment. J AAP & M OHAMMADIAN (1969) however observed a higher number of follicles on postmortem examination in the Dw genotype and expl.ained this discrepancy as due to intra-peritoneal absorption of yolks in the Dw broiler dams.
While the time between two ovipositions was not significantly different between the genotypes, the estimated time between the end of ovum yolk deposition and laying was significantly higher in dwarfs. However, we cannot exclude a possible difference between Dw and dw hens as to the interval between dye feeding and dye deposition in the yolk. Liver weights in Dw and dw genotypes were significantly different, representing 2.25 and 2.51 p. 100 of body weight, respectively. This is within the range of the 2 to 4 p. 100 of body weight reported by H AFEZ (1955) but lower than those reported by S HIVAPRASAD & J AAP (1977) (3.3. to 3.6). The percentage of dry matter (36.9 and 31.7 in Dw and dw genotypes, respectively), although not significantly different, suggested that the moisture count of dwarf livers was higher than that of normals ; this was in agreement with the work of S HIVAPRASAD & J A A P (1977), although those authors found a higher dry matter percentage and a much smaller difference between the Dw and dw genotypes. Table 2 shows that within each clutch size egg and yolk weights decreased in successive clutch positions in both Dw .and dw genotypes. Dwarfs had no clutch size above 4 and thus we could not compare the genotypes. As the clutch size increased from 1 to 4, the time of laying and the interval between two ovipositions appeared to decrease in the corresponding position of the clutch in both the Dw and dw genotypes. For all positions of the clutch up to clutch size 3, the time of laying in dwarfs was a little later compared to the Dw genotype.
In both the Dw and dw genotypes, the hour of laying increased with successive clutch positions. The estimated time between the end of yolk deposition in the ovum and ovum laying followed a pattern similar to those of the hour of laying and the time between two ovipositions, i.e. it decreased in each corresponding position of the clutch as clutch size increased from 1 to 4, and increased in the successive positions of the same clutch in both Dw and dw genotypes. For the first egg of clutches greater than one, this variable was 3 h longer in dwarfs. Thus, the first egg of the clutch in dwarfs may stay longer in the oviduct or require more time to ovulate.
The duration of rapid development of the ovum was shorter in dwarfs for clutches of all lengths and at all positions of the clutch. Within the genotype this duration represented a difference in ovum weight, and tended to be slightly less in the last egg having a clutch length of four. Also, rapid ovum development tended to last longer in the middle of clutches with more than three eggs. These variations observed in the length of rapid ovum development agree with the works of Lncns- SAGNE (1960) and BACON & S KALA (1968) who found that the last egg of clutches of more than one egg and the first egg of clutches of four or more eggs seemed to develop more rapidly than the eggs in the middle of these clutches.
B. Correlations
The phenotypic correlations between body, egg and yolk weights of combined genotypes were all positive and highly significant. The dependence of egg and yolk weights on body weight and of egg weight on yolk weight seemed to be closer in dwarfs. The highly significant correlation between body weight and the weight of active ova was more marked in dwarfs. The association of body weight with the number of active ova in the ovary on postmortem examination was significant. The correlation between body weight and duration of rapid ovum development was positive but non-significant.
The correlations of body weight with liver weight and the percentage of dry matter were positive and highly significant, suggesting a dependence of the liver characters on body weight, particularly in low body-weight birds.
As expected, the correlations of rate of lay with clutch size, average daily yolk production and weight and number of active ova were all positive and highly significant. Similarly, a number of correlations relating rate of lay, clutch size or yolk production traits with time parameters of ovum development or oviposition were signnficant. However, the positive association between egg or yolk weight and duration of rapid ovum development deserves to be mentioned, as no such correlatio,n appeared with daily yolk production. The same may be said for the positive correlations between the yolk weight and the number of active follicles ; these correlations were significant in the postmortem estimate of the latter trait, while small and non-significant correlations appeared between yolk weight on the one hand and rate of lay and clutch size on the other. Another correlation which is not explained a priori is the one between the duration of rapid ovum development and the estimated delay between the end of yolk deposition in the ovum and ovum laying. There appeared to be no correlation in either genotype between rate of lay and liver weight and the percentage of hepatic dry matter. The correlations of the time between two successive ovipositions with clutch size, average daily yolk production and hour of laying were significantly different in Dw and dw genotypes. They were negative in both genotypes but were very highly significant in the Dw genotype. The correlation with hour of laying was positive in the Dw genotype but negative and significant in the dw genotype. These correlations in the Dw genotype suggest that more productive, early layers take less time to form their egg, while the dw genotype seems to need more time to form an early-laid egg. A proper physiological explanat,ion for the above difference between the two genotypes is lacking at present.
The correlations of liver and percentage of hepatic dry matter wilth yolk weight were positive and highly significant, whereas the corresponding correlations w:th average daily yolk production were positive but not significant. S H I VAPRAS A D & J A A P (1977) observed that the rate of yolk production appeared to depend on liver weight and total liver lipids only in a strain carrying the dw gene.
Both liver traits were positively and significantly correlated with the duration of rapid ovum development. This association appears to reflect the positive significant correlation of liver weight with yolk weight which, in turn, was significantly correlated with the duration of rapid development. Average daily yolk production appeared to have no relation to the duration of rapid ovum development. Received 1) La production moyenne journalière de jaune et le poids des ovules en accroissement rapide étaient plus élevés dans le génotype Dw. Le nombre des ovules en développement actif était plus grand chez les poules Dw, mais la différence entre génotypes n'était significative qu'à partir de l'examen post-mortem. | v3-fos |
2018-04-03T01:45:35.423Z | {
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} | 0 | [] | 1982-01-01T00:00:00.000Z | 25508667 | {
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} | s2 | Effects of dietary levels of protein and fat on DDT (1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane) and liver lipid metabolism.
In order to examine the effect of dietary protein and fat on DDT metabolism and liver lipid concentration, rats were supplied with calory adjusted diets consisting of various amounts of protein and fat. The results suggested that dietary protein and fat changed the liver lipid concentration. They also showed that dietary protein and fat affected the residual concentration of DDT and its metabolites in the liver and adipose tissue. The change of the concentration of lipids in liver accompanied a change of the residual concentration of DDT in liver. This fact indicates that one effect of dietary protein and fat on the metabolism of DDT is attributable to the metabolic change of lipids in liver. Dietary protein accelerated the metabolism of DDT and reduced its residual concentration in liver. The results suggest that regression equations exist between the residual concentration of DDT in liver and (1) dietary factors and (2) lipid concentration in liver; In (DDT) = a x 1n (x1) + b x 1n(x2) + c x 1n(TL) + d (1) In(DDD or DDE) = a' x 1n(TL) + b' x 1n(PL) + c' (2) where x1, x2, TL, and PL are the dietary protein, dietary fat content, total lipid, and phospholipid concentration in liver, respectively. a, a', b, b', c, c', and d are constants. The concentrations of DDT and its metabolites estimated from equations (1) and (2) agrees well with the measured concentrations in liver.
Summary
In (1) Composition of experimental diets. ( fat affected the residual concentration of DDT and its metabolites in the liver and adipose tissue. Figure 1 shows the concentration change of DDT and its metabolite, DDD, in the liver and adipose tissue in rats fed on high fat diet (20%). When dietary protein content increased, the residual concentration of DDT in the liver and adipose tissue decreased. The concentration of DDD in the liver and adipose tissue increased according to the decrease of DDT in the liver and adipose tissue.
The distribution of the concentration of DDT and its metabolites was examined in all groups. Table 2 shows the skew and kuratosis of the distribution. The data show that the concentration of DDT and its metabolites follow a log. normal distribution. It is considered that the logarithmic value of the concentration of lipids in liver fits normal distribution better than the original one. Therefore, to normalize the distribution, the logarithmic value of each measurement was calculated. Figures 2 and 3 show the relation between the concentration of total lipids in the liver and dietary factors. From more than 10% dietary fat, the total lipid concentration was highest in rats fed on the diet with 20% protein. The con centration of tota! lipids in the liver increased when dietary fat increased. Table 4. Coefficients of the regression equation between DDT, DDD, DDE (ng/g) and the dietary factors, and lipids concentration in the liver. * Significant level of correlation coefficient (p<0 .05). ** Significant level of F-value (p<0.01). R2, coefficient of determination; F, F -value.
where, x1, x2, TL, and PL are the dietary protein, dietary fat content, total lipid, and phospholipid concentration in liver, respectively, a, a', b, b', c, c', and d are constants. Table 4 summarizes the partial correlation coefficients of the regression equations (1) and (2). From the equations, the concentration of DDT and its metabolites were functions of lipid concentration, dietary protein, and fat content. The estimated concentration of DDT and its metabolites from the equations agreed well with their measured concentration in liver.
DISCUSSION
In earlier experiments, it was evident that the concentration of lipids, especially of triglyceride, in the liver decreased according to the administration of DDT. The minimum dose of DDT which has an effect on the liver triglyceride content is more than 13mg/kg body weight. A dose level of DDT of under 10mg/kg body weight has no effect on the lipid concentration in the liver (21). In this experiment, the dose level of DDT is 7.2mg/kg body weight, therefore, the concentration change of lipids in the liver is due to the effect of dietary protein and fat.
On the other hand, the liver is a vital organ which metabolizes and excretes the fat-soluble chlorinated hydrocarbons such as DDT and PCB. The time pattern of DDT excretion was explained by a compartment model. In early investigations, the excretion of DDT and DDE could be adequately described by a two-compartment model (8,9).
The concentration change of DDT and its metabolites in the liver and adipose tissue in rats fed on high-fat diet is also clarified. When dietary protein increases, the residual concentration of DDT decreases in the liver and adipose tissue, whereas that of DDD increases. DDT is metabolized to DDE and DDD by the dehy drochlorinase and the hepatic microsomal drug-metabolizing enzymes which contain two independent systems; one requiring cytochrome P-450 and the other requiring FAD (22,23). It is well known that the activity of hepatic microsomal drug-metabolizing enzymes is influenced by dietary protein (24). Therefore, the metabolism of DDT is influenced by the consumption of protein.
Recently, it has been reported that poly-unsaturated fatty acids have an effect on the hepatic microsomal drug-metabolizing enzyme systems (25). Dietary fat also increases the lipid concentration in the liver because fatty acid incorporation increases.
Because they are fat-soluble chemicals, chlorinated hydrocarbons accumulated in the human body may be combined with lipids (10). Therefore, it is assumed that the metabolism of DDT may be explained by taking the metabolism of lipids in the liver into account.
The results suggest that regression equations exist between the concentration of DDT in the liver and dietary factors (protein and fat), and lipid concentration in the liver. The concentrations of DDT and its metabolites estimated from the equations (1) and (2) agree well with the measured concentrations in the liver. In the equations, it is decided that the concentration of DDT and its metabolites is connected with the lipid concentration in the liver. DDT metabolism is also modified by the dietary protein and dietary fat content.
From the results described above, the activity of the drug-metabolizing enzymes is influenced by the dietary protein and fat content. Therefore, it is necessary to investigate the relation between the fat-soluble chemicals metabolized by the hepatic drug-metabolizing enzymes and the dietary factors, and to confirm their metabolism with respect to lipids and drug metabolism in the body.
The first target organ of fat-soluble chlorinated hydrocarbons at low con centration was the liver (25), and their biological effect and toxicity changed in proportion to the residual concentration in the liver (26). Therefore, we may say that increasing the dietary consumption of protein increases the metabolism and excretion of chlorinated hydrocarbons that remain in the body. On the other hand, My thanks are due to Professor I. Wakisaka for very helpful discussions and encouragement.
I am also indebted to Dr. S. Hirosaki and Mr . M. Furukawa for their useful discussions. I wish to thank Mrs. Phyllis Ogawa for the revision of the manuscript in English. | v3-fos |
2018-04-03T06:04:44.108Z | {
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} | 0 | [] | 1982-03-01T00:00:00.000Z | 45515959 | {
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} | s2 | Phenoxy acid herbicides cause peroxisome proliferation in
Phenoxy acid herbicides cause peroxisome proliferation in Chinese hamsters. 70-73. An increase in either the size or amount of peroxisomes was obtained in the liver cells of Chinese hamsters after the animals were exposed to the phenoxy herbicides 2,4-di chlorophenoxyacetic acid (2,4-D) or 4-chloro-2-methylphenoxyacetic acid I(MCPA). At the dose level studied, 2,4-D was found to be more potent than MCPA in increasing the number of peroxisomes. A phenoxy acid derivative, clofibrate, one of the peroxi some proliferators known to possess carcinogenic properties in rodents, appeared to be still more potent in inducing peroxisome proliferation than either of the herbicides studied. Further investigations are warranted to clarify the significance of peroxisome proliferation to the toxicity of phenoxy herbicides.
Increased risk of soft tissue sarcomas has been reported among workers exposed to phenoxy acid herbicides (3,4,5,7). The mutagenic activity of phenoxy herbicides has been, however, low or nonexisting in the studies performed (11). Recently, Reddy & Azarnoff {IO) suggested a novel class of chemical carcinogens not possessing mutagenic activity in bacterial mutagenesis assays but acting via the excessive production of hydrogen peroxide (H202) and causing proliferation of peroxisomes. All the hypolipidemic peroxisome proliferators, including the widely used drug clofibrate, have also produced liver tumors in rats {6, 10). Furthermore, an overall excess of deaths from a variety of cancers, mainly gastrointestinal, has been reported for men receiving clofibrate therapy (6). Since clofibrate is closely related to phenoxy acids chemically, it was of interest to determine whether the commonly used phenoxy herbicides 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-chloro-2-Reprint requests to: Dr H Vainio, Institute of Occupational Health, Haartmaninkatu 1, SF-00290 Helsinki 29, Finland. 0355-3140/82/010070-04 methylphenoxyacetic acid (MCPA) could also act as hepatic peroxisome proliferators.
Materials and methods
The test animals used in the study were 12-to 20-week-old Chinese hamsters weighing 34-40 g. They were bred in our laboratory from an outbred colony which originates from the Shell Research Centre (Sittingbourne, Kent, UK).
The phenoxy acid compounds considered were 2,4-D and MCPA, kindly provided by the manufacturer, Kemira Oy (Finland). Both treatment solutions consisted of commercial herbicide products wearing the trade names "Vesakontuho tasku" (solution containing 2,4-D) and "Vesakontuho MCPA" (solution containing MCPA). The amounts of 2,4-D and MCPA in the herbicide solutions are 550 g/kg and 500 g/kg, respectively, and both chemicals contain only one effective compound.
A single dose of 100 mg/kg of the phenoxy acid compound dissolved in physiological saline was given daily for 9 d by gavage to three animals in each exposure group (the dose volumes ranged from 0.11 to 0.14 ml, depending on the weight of the animal). The three control animals were treated similarly with saline only. The animals were killed on the 10th day of the experiment, 24 h after the last dose.
In the positive control experiment, the same single dose of 100 mglkg of clofibrate {Klofiran®, Remeda, Finland) dissolved in olive oil was injected subcutaneously into three animals daily for 7 d (the dose volumes ranged from 0.27 to 0.35 ml). The corresponding control animals were injected with olive oil only. The animals were killed on the eighth day of the experiment, 24 h after the last dose.
Two biopsies from each animal (three controls and three animals from each exposed group) were taken from the medial lobe immediately after the killing and fixed by immersion in 2 0/0 phosphatebuffered (pH 7.4) glutaraldehyde for 2.5 h.
Minced liver samples were washed for 0.5 h in pho3phate buffer (pH 7.4). The specimens were then incubated for 90 min at 38°C in a catalase localization medium [alkaline diaminobenzidine medium (8») and washed in phosphate buffer (pH 7.4) overnight. The specimens were postfixed in 1 Ofo phosphate-buffered (pH 7.4) osmium tetroxide, dehydrated, and embedded in Epon. Ultrathin sections were stained with lead citrate and examined with a JEM-I00 CX electron microscope.
The areas of peroxisomes were determined from 12 animals {3 per group) in electron micrographs {6,600 X) projected on a drawing board (final enlargement 100,000 X). The outlines of the peroxisomes were drawn and used for later morphometric evaluation. The areas of 330 peroxisomes (between 80 and 90 in each group) were analyzed. For the calculation of the frequency of peroxisomes per unit area of cytoplasm, the peroxisomes of 40 hepatocytes, taken by random sampling, were counted {10 per group, total number of peroxisomes 5,754) in electron micrographs with an enlargement of 3,000 X.
The areas of peroxisomes and hepatocytes were examined with the aid of a semiautomatic measuring device (Hipad digitizer and ABC-80 data processing unit). The statistical difference from the control group was calculated with Cochran's approximate t-test (2).
Results
The mean size of the peroxisomes of the control rats was 1.38 flm 2 (table 1). The 2,4-D treatment increased the mean area slightly but not significantly to 1.46 flm 2 • A significant increase in size was seen after MCPA and clofibrate treatment, to 2.02 and 2.37 flffi2, respectively.
The mean frequency of peroxisomes per 100 flm 2 of cytoplasm was 17.7 in the controls (table 2). MCPA treatment increased the frequency slightly but not ... P < 0.001, NS = not significant. ... p < 0.001, NS = not significant. A slight increase in the smooth, and decrease in the rough, endoplasmic reticulum was seen after clofibrate treatment, the reticulum being normal in the other groups. Mitochondria were normal in all cases.
Discussion
Little is known about the possible mechanism by which exposure to phenoxy acid herbicide::; may be associated with increased risk of soft-tissue sarcoma and malignant lymphoma, as suggested on the basis of epidemiologic studies done in Sweden (3,4,5). Although in many cases mutagenic and carcinogenic effects are experimentally correlated (1), the phenoxy acid herbicides do not appear to have direct interaction with DNA (deoxyribonucleic acid). Phenoxy acids are not known to bind to DNA, neither have they pos-::;essed mutagenic activity in the Salmonella/microsome assay (11).
The present data show that the phenoxy herbicides 2,4-D and MCPA are able to increase either the size or amount of peroxisomes in the liver cells of Chinese hamsters. Thus the possibility exists that phenoxy acid herbicide::; could act indirectly by a mechanism similar to the one suggested recently for tumor initiation by known peroxisome proliferators. This novel class of chemical carcinogens includes, eg, clofibrate, a phenoxy acid derivative, and other related hypolipidemic drugs, as well as phthalate ester di(2ethylhexyl)phthalate (8,10). Their mechanism of action has been suggested to occur via excessive production and/or breakdown of H 2 0 2 • Thus the initiation of neoplastic transformation would be the result of continued production of DNA-damaging oxygen radicals as a result of the persistent proliferation of peroxisomes.
In summary, the data obtained suggest Received for publication: 6 October 1981 that the phenoxy herbicides 2,4-D and MCPA act as peroxisome proliferators. Taking the existing evidence into account, further 'studies should be carried out to clarify the potential significance of this peroxisome proliferation in the toxicity of phenoxy herbicides. | v3-fos |
2018-05-08T18:22:32.865Z | {
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} | 0 | [] | 1982-01-01T00:00:00.000Z | 219580374 | {
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} | s2 | Immune response to some E. coli antigens in swine
and treated as all-or-none traits in genetics analyses. A significant sire effect was found for all serum traits except albumin, with heritability estimates between 0.1 and 0.2. The disease characters showed heritability estimates between 0.04 and 0.08. Immunoglobulin and total-protein concentrations seemed to have a negative genetic correlation to diseases.
In cattle, sheep and goat, polymorphism was observed for only Three alleles in cattle, three in sheep and two alleles in goat were observed.
In pig, the earlier described polymorphism pre-albumin (Pa) was identified as a i protease inhibitor. Two Pa alleles have been reported in pig.
In dog and mink, polymorphism was observed only for Pi-1. Three Pi-1 alleles were observed in several breeds of dogs. In mink, the polymorphic post-albumin (Pa) described in literature, was identified as a l -protease inhibitor.
The a,-PI fractions in all these species are present in high concentration in plasma and thus could be visualised by general protein staining of gels after electrophoresis. The possible association of a,-PI phenotypes with respiratory and inflammatory diseases in domestic animals was also briefly discussed. Immune response to some E. coli antigens in swine The humoral immune responses to two E. coli antigens, K88 and 0149, were studied in 60 pigs after 9 sires and from 19 litters. One animal per litter was in addition kept as a control. The animals were immunized subcutaneously with a whole cell suspension of E. coli at the age of approximately 10 weeks. Ten of the animals were given a second immunization 3 weeks after the first one. Blood samples were taken immediately before immunization, 1 and 2 weeks post-injection. The total amount of specific antibodies in serum to K88 and 0149 antigen were analysed by E LISA technique. A significant increase in antibody titers was obtained after immunization, although a pronounced individual variation was seen. The animals were given a constant dose of the suspension without regard to differences in body weight. However, no effects of body weight on the immune responses were seen. The effect of sire was highly significant (p G 0.001) indicating a genetic influence on the immune response. The overall correlation between the primary and secundary response was for K88 antigen 0.76 and for 0149 antigen 0.15. Nucleolus organizer regions were determined in cattle, pig, sheep, goat, dog, horse and chicken. The mapped genes were correlated to the nucleoli formed in peripheral blood lymphocyte interphases. A positive correlation was found between the number of nucleolar organizer regions per diploid genome and the nucleolar coefficient. Peculiarities of the different species concerning nucleolar formation and association/dissociation pattern is highlighted. The use in definition of immune cells in domestic animals in order to investigate cellular heterogeneity is pointed out.
Immunoglobulin levels in the blood serum of pigs as criteria of heredity and ontogenesis E. WEHRHAHN, F. KLOBASA F. HABE Institut fiir Tierzucht u. Tierverhalten, Mariensee 3057 Neustadt 1, West Germany Milk and blood immunoglobulins were analyzed in 22 young sows and in 24 older sows (DL). In addition, the blood immunoglobulin levels of two offsprings each were monitored. Older sows had higher total Ig-serum values than young sows. Their milk IgG and IgA contents also surpassed those of young sows, which however showed higher IgM contents. The initial serum pattern of the piglets mirrored the maternal Ig-secretions in the milk (passive immunization). After weeks 2-3 piglets of the young sows showed a steeper rise in the Ig production rate than piglets from older sows. They caught-on however during the fattening period and had arrived at higher IgA-levels when slaughtered.
A number of correlations between sow/piglet Ig patterns, fattening performance and carcass compositions will be reported.
A. EALES
Moredun Research Institute, 408 Gilmerton Road, Edinburgh EH17 7JH Hypothermia, a low body temperature, is an important cause of mortality in newborn lambs in the U.K. There are two major causes of this condition : (1) Excessive heat loss | v3-fos |
2018-04-03T01:07:29.439Z | {
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} | 0 | [] | 1982-10-01T00:00:00.000Z | 25469420 | {
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} | s2 | Kinetic constants of lactase of the small intestine of growing rats fully recovered from protein depletion, in relation to dietary protein and lactose concentration.
The effect of dietary composition on the kinetic constants of intestinal lactase was studied using rats depleted of protein by feeding protein-free diet from the weanling stage to 34 days of age and subsequently allowed to recover on diets containing 11.5 or 17% of protein calories (P%) and different levels of lactose (L%; 1, 15, 27 and 37%). After four days of refeeding, rats were decapitated and lactase activity was determined at different substrate concentrations by the method of Dahlqvist using homogenate of intestinal mucosa. Maximum velocity (Vmax) and Michaelis constant (Km) were calculated according to Eisenthal and Cornish-Bowden. At both levels of P%, Vmax tended to increase with the dietary lactose concentration. With diets containing 37% lactose, at P% 11 Vmax was about 60% of that at P% 17.0 Km tended to increase with L% in groups given the 17.0% protein calorie diets, but no difference was observed between groups fed at the lower level of protein. These results can be explained on the basis of interactions between dietary protein at different concentrations and inducer substrate which results in changes in isoenzyme patterns.
maltase activities after feeding fasted rats on diets with a high carbohydrate content. Saito and Suda (2), who studied dietary effects on kinetic constants of leucine aminopeptidase bound to the intestinal brush border in normal adult rats, found higher maximum velocities (Vmax) when rats were fed on high-casein diets than when given low-casein ones. On the other hand, there are no reports extent on the effect of dietary composition on digestive enzymes of growing rats recovering from protein depletion nor on the interaction between dietary protein at different concentrations and inducer substrate.
In order to provide information on these points, young protein-depleted rats were fed on experimental diets containing lactose and protein at different concentrations. At the end of the refeeding period, kinetic constants of intestinal lactase were studied.
RESULTS
At the end of the refeeding period, irrespective of P%, animals recovered their initial body weight, except for those receiving dietary lactose at a concentration of 37%, which only recovered to a level of 94%.
Michaelis-Menten constant
Km tends to increase with increased dietary lactose concentration in groups fed on 17.0% protein calorie diets (groups B) (Fig. 1). Conversely, there was no significant influence of dietary lactose concentration on the K m of the groups refed on diets providing 11.5% of protein calories (groups A). Moreover the Km for the lowest level of lactose in group B was significantly lower than that in group A .
Maximum velocity
Vmax tends to increase along with dietary lactose concentration when P% is 17.0%, the difference being significant between 1 and 37% (p<0.01).
For a dietary protein concentration of 11.500, the same trend was observed between 15 and 37%, but the difference did not attain the expected level of significance. Anomalous behavior was observed for the lowest level of lactose where the Vmax surpassed the Vmax for all the other lactose levels.3 However , due to the large values for the standard error of this group, differences were not significant at the level of p<0.01.
When diets provided 37% of lactose, the maximum velocity at P% 11 was about 60% of that at P% 17 (Fig. 2).
DISCUSSION
The results of this work confirm for lactase that the V max of enzymes of the KINETIC CONSTANTS OF LACTASE OF THE SMALL INTESTINE 481 intestinal brush border increase with the level of inducer substrate in the diet. However, Saito and Suda (2) working with adult rats, found interrelations between maltase activity and protein concentration for levels of dietary protein above 75%. Our findings show that in the case of intestinal lactase, growing rats recovering from protein depletion are far more sensitive to changes in dietary protein concentration than in the previous case. It is interesting to point out that a small change in protein concentration from 11.5 to 17.0 was able to increase significantly (63%) the Vmax of lactase as a response to the highest level of lactose in the diet (Fig. 2). Since an influence of P% was not fo und with lower levels of lactose, this increase seems to result from some adaptive process that would take place at this higher level of inducer substrate. Other differences, with regard to Saito and Suda, were found to lie in the changes observed in Km, whereas no changes were found for those authors, in the present paper significant differences were demonstrated as a consequence of changes in dietary composition related to both protein and lactose concentration. Differences in Km seem to be more related to protein than to lactose; groups refed on diets A (P% 11.5) did not show any changes as a result of increasing lactose, whereas a close interrelationship was found between Km and lactose dietary concentration in the groups fed with diets B (P% 17.0). In these last groups a trend of increase of Km with lactose concentration was observed, so that the difference between groups with 1 and 37% lactose was significant.
On the basis of the results reported here we suggest that the changes in Km and Vmax of intestinal lactase might arise from interactions between: a) Dietary lactose as the inducer substrate, and, b) Dietary protein concentration regulating amino acid availability for "de novo" enzymatic synthesis during the recovery period.
Since there are at least 3 different lactase isoenzymes known, changes in the Michaelis-Menten constant would suggest that these interactions would result in changes in isoenzyme patterns. A similar explanation was proposed by Yoshizawa et al. (8) who found changes in Km and Vmax of chick embryonic intestinal maltase as a result of changes in the inducer sugars present in an organ culture system. | v3-fos |
2014-10-01T00:00:00.000Z | {
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} | 0 | [] | 1982-11-01T00:00:00.000Z | 12002864 | {
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} | s2 | Occurrence of arsenic in plaice (Pleuronectes platessa), nature of organo-arsenic compound present and its excretion by man.
The arsenic content in 255 samples of plaice (Pleuronectes platessa) varied between 3 and 166 mg/kg. About 65% of the samples had an arsenic content above 10 mg/kg. High (low) arsenic concentration in the fillet corresponds with a high (low) concentration in milt or roe. An excretion experiment with eight human volunteers showed that after the consumption of plaice, 69-85% of the ingested arsenic was excreted in the urine within five days. The organo-arsenic compound present in plaice was isolated by means of extraction, ion-exchange and thin-layer chromatography. Field desorption mass spectrometry of the isolate showed that arsenic was present as arsenobetaine, (CH3)3AsCH2C00-.
Introduction
In general, high levels of arsenic are found in marine products. Chapman (1) observed high arsenic concentrations (2-132 mg/kg) among marine crustaceans and shell fish, whereas Schroeder and Balassa (2) reported arsenic levels ranging from 1.5 to 8.9 mg/kg in commercially available seafoods. High concentrations (2-38 mg/kg) in crabs were measured by Leblanc and Jackson (3), and concentrations varying between 0.15 and 18.7 mg/kg were found by Egaas and Braekkan (4) in 43 samples of fish products.
Chapman (1) showed that 74% of arsenic (25 mg) present in lobster was excreted within 48 hr, whereas Coulson et al. (8) found that arsenic in shrimp was excreted completely within 4 days after consumption.
Schenk and Schreibeis (9) observed that the excretion of arsenic occurred mainly during the first 48 hr after consumption. Approximately 80% of the arsenic present in cod and plaice was excreted within 3 days (5). Munro (10), working with swine, observed that 68% of the arsenic from fish was excreted in the urine and 23% in the feces.
Of arsenic from sole given to monkeys, 57-84% was excreted in the urine and 1-16% in the feces (11).
In 1977 Crecelius (12) (13) and Tam et al. (14) that ingested inorganic arsenic will be metabolized as methylated arsenic. After the ingestion of crabmeat containing an unidentified organo-arsenic compound, the urinary concentration of the four arsenic compounds did not change significantly. However, when urine samples were digested with hot 2N NaOH, high concentrations of dimethylarsinic acid were present. The behavior and toxicological properties of arsenic depends on the chemical form in which it is present. Penrose (15) mentioned that the toxicity decreases in the following way; arsines (trivalent inorganic or organic) > inorganic arsenite > arsenoxides > inorganic arsenate > pentavalent arsenicals > arsonium compounds (four organic groups with a positive charge on arsenic) > metallic arsenic.
Several investigators have made more or less successful attempts to elucidate the structure of the organo-arsenic compound(s) in marine fish. Lunde (16) and Brooke and Evans (17) reported the separation ofinorganic arsenic (As3 + and As5 + ) from organic bound arsenic in fish by distillation from hydrochloric acid. Although the distillation conditions were rather drastic, only small amounts of inorganic arsenic (0.01-2.5 mg/kg) were present.
Edmonds and Francesconi (18) showed that digestion of methanol extracts of mussels, western rock lobster and sting ray in aqueous sodium hydroxide led, after neutralization, to a solution from which the arsines, (CH3)2AsH and (CH3)3As, were generated after reduction with sodium borohydride. The organo-arsenic compound isolated from western rock lobster by Edmonds et al. (19) has been identified as arsenobetaine, (CH3)3AM CH2COO.
Penrose et al. (20) isolated an organo-arsenic compound from witch flounder which possessed a mass spectrum that resembles tetramethylarsonium. The anion-exchange and thin-layer properties of the isolated compound, however, did not correspond with those of (CH3)4AL. The structure ofthe isolated organo-arsenic compound from witch flounder has not been further elucidated.
Kurosawa et al. (22) used HPLC and field desorption mass spectrometry for the identification of arsenobetaine in the muscle and liver of a shark (Prionace glaucus).
Recently, Edmonds and Francesconi (23) showed that the thin-layer properties of organo-arsenic isolated from school whiting are identical to those of arsenobetaine. It was therefore concluded that arsenic in school whiting is present as arsenobetaine.
LUTEN, RIEKWEL-BOOY AND RAUCHBAAR Benson and Summons (24) mentioned that arsenic isolated from kidney tissue of a giant clam is mainly present as trimethylarsonium lactate and O-glycerophosphoryl trimethylarsonium lactate.
Still more information about arsenic in marine products is necessary. This paper describes an investigation into the occurrence of arsenic in an important flatfish species, plaice (Pleuronectes platessa), and gives some results of studies on the nature of an organo-arsenic compound present therein and the excretion by man.
Material and Methods
Sampling During the period February 1979-March 1980, 255 samples of plaice (Pleuronectes platessa), caught at different fishing grounds in the North Sea, were obtained from the Netherlands Institute for Fishery Investigations, Ijmuiden, the Netherlands. The edible parts, liver, milt and roe, were homogenized separately and stored at -200C until the analysis. Table 1 presents a survey of the sampling data.
Determination of Arsenic
Total Arsenic. The homogenized samples were dry-ashed overnight at 5500C in the presence of Mg(NO3)2 and MgO. After dissolving the ash in hydrochloric acid, As5+ was reduced to As3+ with the addition of potassium iodide.
Arsine, formed by the use of zinc-hydrochloric acid as hydride generator, was absorbed in a solution of silver diethyldithiocarbamate in pyridine. The absorbance at 528 nm was then measured. Methylated Arsenic. A homogenized fish sample was submitted to a Bligh and Dyer extraction (25). After evaporation of methanol from the aqueous/ methanol phase at 40°C, the aqueous fraction was digested in hot 4N KOH for 2 hr. In the case of urine samples, the Bligh/Dyer extraction procedure was omitted. After the KOH digestion the sample could be analyzed for inorganic and methylated arsenic by reduction with sodium borohydride at pH < 1. During the reduction procedure the hydrogen and the arsines formed were collected in a 2-liter bottle filled with deionized water which was placed upside down in a water bath. The collection was terminated after 2 min and the bottle was then closed under water with a septum. In preliminary experiments it was established that under these conditions the arsenic present was recovered completely. A 1 ml gas sample was submitted to gas chromatography. Detection was performed with a
Isolation Procedure for Organo-Arsenic Found in Plaice
The isolation procedure for the organo-arsenic compound found in plaice consisted of three steps; extraction, ion-exchange and thin-layer chromatography.
About 210 of plaice (166 mg As/kg) was subjected to a lipid extraction according to the Bligh and Dyer procedure (25). Methanol was evaporated from the methanol/aqueous phase at 40°C. The aqueous extract, acidified by adding HCl until a pH 3 was reached, was then passed through a cation-exchange column (Amberlite IR-120, 20-50 mesh, H+ form). Elution of the retained organo-arsenic compound was performed with 2% NH40H. After neutralization, the eluate was applied to a mixed-bed column (Amberlite, IRA-400, 20-50 mesh, OH-form, Amberlite IRC-50, H+ form). The eluate, which contained the organo-arsenic compound, was subjected once again to the cation-exchange procedure. Fractions of the NH40H elution step containing the arsenic-compound were then collected. The overall recovery of arsenic from plaice by the described isolation procedure was about 80%.
Thin-layer chromatography of the isolate on cellulose with n-butanol/acetic acid/water (60: 15:25) resulted in three spots (Rf = 0.65, 0.58 and 0.48) after exposure to iodine vapor. Only the spot with Rf = 0.58 contained arsenic. This arsenic-containing spot was then extracted with methanol and subjected to field desorption mass spectrometry.
Excretion Experiment
Eight healthy human volunteers (6 male, 2 female) each consumed 100 g plaice (80 mg As/kg). Prior to this experiment none of the participants had eaten arsenic-rich food. During the first 12 hr of the experiment the volunteers were asked to urinate at 2 hr time intervals. After 5 days the experiment was terminated. All urine samples were analyzed for the presence of arsenic according to the procedure described above for methylated arsenic.
Results and Discussion
The average and median concentrations and the concentration ranges of arsenic in fillets of plaice are given in Table 1. A frequency distribution of arsenic over the 255 investigated samples of plaice is given in Figure 1. A large variation in the arsenic content was observed. About 65% of the samples had an arsenic content above 10 mg/kg.
The arsenic content in the fillet, liver, milt and roe of 18 samples of plaice (Table 2) show that a high (low) arsenic content in the fillet corresponds to a high (low) arsenic level in the liver and milt. In some liver samples the arsenic content was several times higher than in the corresponding fillets. It is not clear to what extent this was due to accumula- tion of arsenic in the liver. Compared with the ifilets, the arsenic content in roe is low. The excretion of arsenic in the urine of eight human volunteers after 5 days varied between 69 and 85% (average 75%) of the administered dose. As an illustration the arsenic content in the urine of two volunteers is given as a function of the time in Figure 2. The bulk of the ingested arsenic was FIGURE 2. Concentration of arsenic in urine, as (CH3)3As, as a function of time after the ingestion of plaice by two human volunteers. The urine samples were analyzed for the presence of arsenic following the procedure described in the section on methylated arsenic. excreted during the first 24 hr after consumption.
Only small amounts of inorganic arsenic (< 30 ng/ml) were present in the urine samples prior to the excretion experiment. All the arsenic excreted after consumption of plaice was present as trimethylarsine in urine after alkaline digestion and reduction with NaBH4, at low pH. Other forms of methylated arsenic as well as inorganic arsenic were not present. It was established that no volatile arsines were present if the alkaline digestion is omitted prior the reduction procedure. The necessity of an alkaline digestion prior to the reduction procedure agrees with the results of Crecelius (12). However, in this investigation trimethylarsine is formed in urine after reduction, while Crecelius reported the formation of dimethylarsine. It was demonstrated that the bulk of the arsenic in plaice (95%) was present in the aqueous fraction after the Bligh and Dyer extraction. Digestion with KOH followed by a reduction with NaBH4 led to the formation of (CH3)3As. The properties of the organo-arsenic reference compounds after the digestion procedure as well as the Rf values, given in Table 3, indicate that the isolated organo-arsenic compound found in plaice was neither arsenocholine nor tetramethylarsonium.
The mass spectra obtained by field desorption mass spectrometry of the isolated organo-arsenic compound, arsenobetaine and a mixture of the two shown in Figure 3. According to the molecular structure of arsenobetaine the protonated molecular-ion (CH3)3(AsCH2COOH (mie = 179) and two fragments (CH3)4AM (m/e = 135) and (CH3)3AA + CH2 (mie = 134) are present. The peak at mie = 134 was the base peak. The mass spectrum of the isolated organo-arsenic compound showed the presence of an ion at mie = 135 as the base peak and an ion at mie = 179 with small intensity. No peak at mie = 134 was present. The addition of an equivalent amount of arsenobetaine to the isolated compound caused a significant shift in the fragmentation pattern of pure arsenobetaine. The nominal mass mie = 135 became the base peak, while the relative intensities of mie = 134 and mie = 179 decreased considerably. Protonization of arsenobetaine, probably due to impurities present in the isolated compound, increased the presence of the tetramethylarsonium ion.
High resolution field desorption mass spectrometry (R = 104, 10% valley definition) showed that the exact mass of mie = 179 of arsenobetaine and that of the isolated organo-arsenic compound did not differ significantly from each other. It was therefore concluded that the arsenic found in plaice is present as arsenobetaine. | v3-fos |
2018-12-26T19:46:36.532Z | {
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} | s2 | Nutrient Level in Saudi Arabia Wheat Flour
The chemical composition and protein quality of four varieties of wheat flour grown in Saudi Arabia (Rice Mexi-Pak, Red Mexi-Pak, Lugemi, and Moaeah), and one type of home bread (Korsan) made from Moaeah were evaluated. Analysis performed included proximate analysis, thiamine, riboflavin, iron, amino acid composition and protein efficiency ratio (PER). Protein and iron were higher in Moaeah and Lugemi. The protein quality assessed by PER was higkest for Rice Mexi-Pak and lowest for Korsan, the cooked product. Chemical scores based on the essential amino acid content of the proteins were calculated; lysine and/or methionine was the limiting amino acid in the varieties of wheat studied.
, while in some developing countries cereals contribute up to 753 of the cal6ries and 90% of the protein (Pomeranz, 1971).
Some of these nutrients are consumed directly; those in animal feed derived from wheat are consumed indirectly.
The quantity of wheat-based nutrients available for direct Approximately 60% of the wheat consumed is now from local production (Alyamamah, 1982).
Several experiments have been conducted in past years to evaluate the nutritional value of wheat flour. determined the amino acid composition and protein quality of wheat flour. They concluded that lysine was the first limiting amino acid in all cases. Sikkaat al. (1975) analyzed the mill fractions of whole wheat flour, bran, white flour and resultant atta. The protein quality index based on protein efficiency ratio and net protein retention at the 10% protein level was found tq be highest in resultant atta, followed by bran, whole wheat flour, and white flour. The chemical score based on the essential amino acid content of egg protein and on the FAO provisional pattern indicated the limiting amino acids in different fractions. In resultant atta and bran, methionine and isoleucine were limiting, whereas in whole wheat and white flour, lysine, threonine and methionine were limited. showed that toasting affected the amino acid composition and protein of wheat flour and with increasing toasting temperature, destruction of some essential amino acid increased, and protein utilization decreased. reported the nutrient levels of internationally milled wheat flour. They concluded that the nutritional quality of large proportions of flour produced throughout the world is lower than that of wheat as a result of milling process. The ash content was determined by ignition of the sample in a muffle furnace at 600°C for two hours.
2:5 Crude fiber: The crude fiber was determined using the Association of Official Analytical Chemists method (AOAC, 1979 fed rat chow, on the fifth day assay groups were assembled in lots of 10 rats in random manner, so that the weight differential was less than 5 g. Throughout the 28-day test period, feed and water were available ad libitum.
Individual food consumption records and weight gain were · determined two times per week. Upon conclusion of 28 day feeding ~tudies, PER values were calculated for each group as follows: protein efficiency ratio (PER) = body weight gain (g) protein consumed (g) 6:1 Amino acid: Amino acid content was determined by Hebert V.
6:2 Tryptophan: The method described by 6:3 Chemical score: Calculation of chemical score was based on the essential amino acid pattern of egg as reference protein.
The method of was adopted to calculate chemical scores. The ratio of the quantity of each essential amino acid in wheat protein compared to the quantity of the respective amino acid in whole egg protein was computed and stated as a percentage. The lowest of these percentages is the chemical score. In the U.S., reported a range of 1.72-2.43 ash in wheat; Keagy et al. (1980) reported 1.843 for hard and 1.873 for soft wheats; Kent (1975) reported 1.8% ash in wheat; and reported 1.17-2.96% ash in wheat. Thus, the overall means of Arabian wheat was similar to those reported by , and Keagy et al. (1980).
Crude Fiber:
Crude fiber values had an overall mean of 2.3 except for Moaeah which was 2.62%. Riboflavin: The mean riboflavin content of all four varieties was .127 mg/100 g. The range was from .12 mg/100 g (Rice Mexi-Pak, Korsan) to .14 mg/100 g (Red Mexi-Pak). have reported a riboflavin content in ranges of .1 mg/100 g to .17 mg/100 g, and has reported a mean .14 mg/100 g.
Mienrals:
Iron: -------Ta~le 2 shows the iron content of the wheat flour; Moaeah contained higher iron than other varieties. A variation in iron content of different varieties of wheat has been reported by .
Amino acids: The results of analysis for amino acids are given in Sikka et al. (1975) and Simmonds (1968).
Threonine and tryptophan did tend to be lower in and a Casein as control. Figure II shows the average weekly gain. were within the average for wheat given by FAQ (1.17-1. 77).
The PER for Korsan was lower than Moaeah, but the difference was not statistically different. The lysine content of Korsan was lower than that of Maoeah, however that difference did not make a significant difference in the utilization of the protein by growing rats.
From the overall evidence presented, it appears obvious that the nutrient value of the old varieties In view of the wide acceptance of modern milling technology even in the developing countries, these losses are not inconsequential.
Protein and protein quality: Cereals are regarded primarily as energy food; however, they are also important sources of protein. Protein is one of the most difficult nutrients to obtain because foods high in protein are usually the most expensive food stuffs. It has been recognized that the human body requires protein in sufficient amount and of specific amino acid composition. Although the quantity of protein in wheat can vary from 7 to 22%, the average value is higher than for other cereal grains. However, other protein sources such as meat are higher in protein content.
The quality of wheat protein is poor relative to other protein sources such as meat, fish, and soybeans.
Protein quality and quantity are affected by several factors including milling procedures, cooking, the variety of wheat, the location and growing conditions. Miladi, et al (1972) shoed that toasting affected the amino acid composition and utilization of protein from unsupplemented wheat flour, and with increasing toast-ing temperature, destruction of some essential amino acid increased. Rat feeding tests showed the protein utilization decreased with increasing toasting temperature.
Protein and amino acid profiles of normal and yellowberry bread wheat have been examined (Waines et al., 1978).
The investigators concluded that values for nitrogen concentration in yellow-berry kernels of bread wheat varied from 91.6 to 72.93 of those for normal kernels. Also, values for total amino acid concentration in whole wheat flour of,yellow-berry kernels were less than those of whole wheat flour of normal kernels. Lysine and threonine were higher in yellow-berry kernels than in normal kernels. Glutamic acid was lower in yellow-berry kernels than in normal kernels. Abrol et al. (1971), reported that the enhanced protein content of wheat observed following increased fertilizer application is mainly accounted for by an increase in gluten content. Levels of glutamic acid, phenyalanine, proline, and leucine increased at high fertilizer levels, while the reverse was true for lysine, valine, threonine, isoleucine and tryosine. Haber et al. (1976) had studied the amino acid composition of hard red winter wheat and tritical. They found that the amino acid composition of tritical flour showed higher lysine levels than hard red winter wheat flour.
Vitamins and Mienrals:
Wheat contains a significant amount of vitamins, but processing changes and reduces their content. Wheat also contains a variety of minerals, usually in small quantity. | v3-fos |
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} | s2 | Calcium-binding proteins in bovine milk: calcium-binding properties and amino acid composition.
The Ca2+-binding properties and amino acid compositions of two calcium-binding proteins (mCaBP-3 and mCaBP-4) purified from bovine milk were studied. mCaBP-3 was identified as a Ca2+-bound type and mCaBP-4 as a Ca2+-free type by means of ion-exchange chromatography on a DEAE-Sephadex A-25 column. In polyacrylamide gel disc electrophoresis, both mCaBP-3 and mCaBP-4 had the same mobility of Rf = 0.73 and the addition of 5 mM CaCl2 to the electrode buffer decreased the mobility from Rf = 0.73 to Rf = 0.49. mCaBP-3 and mCaBP-4 consisted of 120 and 122 amino acid residues, respectively. The molecular weights were 13,758 and 13,967, respectively. The amino acid compositions of the two milk CaBPs very closely resembled each other. Both milk CaBPs were rich in aspartic acid, glutamic acid, leucine and lysine, but did not contain trimethylated lysine and amino sugar. An interesting feature is that each milk CaBP contained eight cysteine sulfone and three tryptophan residues per molecule. From these results, it is suggested that mCaBP-3 and mCaBP-4 are identical protein and that mCaBP-3 is formed from mCaBP-4 by means of a conformational change by binding of Ca2+. Thus, mCaBP-3 is a holoprotein and mCaBP-4 is an apoprotein. Furthermore, it is suggested that milk CaBP is different from calmodulin, troponin C and vitamin D-dependent calcium-binding protein.
feature is that each milk CaBP contained eight cysteine sulfone and three tryptophan residues per molecule. From these results, it is suggested that mCaBP-3 and mCaBP-4 are identical protein and that mCaBP-3 is formed from mCaBP-4 by means of a conformational change by binding of Cat+.
Furthermore, it is suggested that milk CaBP is different from calmodulin, troponin C and vitamin D-dependent calcium-binding protein.
Key Words calcium-binding protein, bovine milk, Cat +-binding proper ties, amino acid composition Calcium is involved in the regulation of a variety of cellular enzyme systems and in most types of cell motility (1,2). It is possible that Ca2+ might not act in its free ionic form but rather requires the presence of a binding protein. Calcium binding proteins such as calmodulin and vitamin D-dependent calcium-binding protein have been commonly identified in many tissues of many avian and mammalian species (3,4 On the other hand, calcium-binding proteins such as calmodulin (7, 8), tropo nin C (8) and vitamin D-dependent calcium-binding protein (9) elicited a large conformational change by the binding of Ca2+ to the affinity sites. Although the physicochemical properties of mCaBP-3 and mCaBP-4 very closely resembled each other, the fact that they were separated by ion-exchange column chromatography on DEAE-Sephadex A-25 seems to suggest that either mCaBP-3 or mCaBP-4 causes a Ca2+-dependent conformational change.
The current research was designed to further characterize the milk CaBPs in the absence and presence of Ca2+ and to analyze the amino acid composition. 4. Other methods. Analytical disc electrophoresis was carried out essentially by the method of Davis (11) using 7.5% polyacrylamide gel. The Ca2+-binding activity was measured by the competitive binding assay of Wasserman et al. Other chemicals were of analytical grade. RESULTS
Interrelation of mCaBP-3 and mCaBP-4 on ion-exchange column chromatog raphy
Calcium-binding proteins such as calmodulin (7, 8), troponin C(8) and vitamin D-dependent calcium-binding protein (9) are known to undergo a conformational change by binding of Ca2+ Thus, a certain calcium-binding protein can be observed as two different proteins depending on the binding of Ca2+.
When the purified mCaBP-3 was again chromatographed using on a DEAE Sephadex A-25 ion-exchange column equilibrated with a regular IKN buffer, two protein peaks were detected at positions corresponding to mCaBP-3 and mCaBP-4, respectively ( Fig. 1). However, when the IKN buffer containing 1mM CaCl2 was used instead of the regular IKN buffer, the re-chromatogram of mCaBP-3 was nearly the same as original chromatogram of mCaBP-3 (Fig. 2). Further, in the calcium-free buffer system containing 1mM EGTA, the purified mCaBP-3 was eluted at the position corresponding to mCaBP-4 and was not detected at the position of mCaBP-3 (Fig. 2). On the other hand, when purified mCaBP-4 was re chromatographed using the same ion-exchange column, the protein peak was detected at the position of mCaBP-3 in the IKN buffer system containing 1mM CaCl2 and at the position of mCaBP-4 in the Ca2+-free buffer system containing 1mM EGTA, respectively (data are not shown). These results strongly suggest that mCaBP-3 is a charge-isomer of mCaBP-4 which is induced by binding of Ca2+. Thus, they indicate that mCaBP-3 and mCaBP-4 are a holoprotein and an apoprotein, respectively. 2. Effect of Ca2+ on mobility of milk CaBP in polyacrylamide gel disc electro phoresis One of the characteristics of calmodulin is that the migration rate in polyacryl amide gel disc electrophoresis changes dramatically by the binding of Ca2+ (14). When the purified mCaBP-3 and mCaBP-4 were analyzed by 7.5% polyacrylamide gel disc electrophoresis without the additional Ca2+ to the samples and the electrode buffer, the relative mobilities of the two mCaBPs were the same, the values being 0.73 (Fig. 3). In disc electrophoresis using the buffer containing 5mM CaC12, the relative mobilities of the two CaBPs decreased from Rf=0.73 to Rf=0.49. The difference of electrophoretic mobility between mCaBP-3 and mCaBP-4 was neither observed in the absence nor the presence of Ca2+. These results indicate that the electric charge of milk CaBPs is greatly affected by the binding of Ca2+.
Amino acid compositions of mCaBP-3 and mCaBP-4
The above results suggest strongly that mCaBP-3 and mCaBP-4 are the same protein and therefore, the respective amino acid compositions were analyzed. The results are shown in Table 1. mCaBP-3 consisted of 120 amino acid residues and the calculated molecular weight was 13,758, mCaBP-4 consisted of 122 amino acid residues and the calculated molecular weight was 13,967. Both milk CaBPs were rich in aspartic acid, glutamic acid, leucine and lysine. Trimethylated lysine and amino sugar could not be detected. The amino acid compositions of mCaBP-3 and mCaBP-4 very closely resembled each other. An interesting feature was that each milk CaBP contained eight cysteine sulfone and three tryptophan residues per molecule. brated with the regular IKN buffer, the protein was detected at two positions corresponding to mCaBP-3 and mCaBP-4 . However, when Ca2+-free buffer (regular IKN buffer+EGTA) or Ca2+-containing buffer (regular IKN buffer+CaCl2) was used for the column chromatography, only one peak which corresponded to mCaBP-4 or mCaBP-3, respectively, was eluted. These facts indi cate that a portion of the Ca2+ bounds to mCaBP-3 is removed by the ion-exchange resin and that mCaBP-4 binds the Ca2+ contaminating in the regular IKN buffer at trace level (<10-6M) during the ion-exchange column chromatography. Thus, these results suggest that mCaBP-3 is identical to mCaBP-4 . The facts that the amino acid compositions of the two milk CaBPs closely resembled each other and that they crossreacted immunologically (5), strongly suggest that mCaBP-3 and mCaBP-4 are charge-isomers induced by the binding of Ca2+ . Further, it is im plied that the binding of Ca2+ to the binding sites in the milk CaBP elicits a large conformational change. This conformational change by binding of Ca2+ is partic ularly characteristic in calcium-binding proteins such as calmodulin (7 , 8), troponin C(8) and vitamin D-dependent calcium-binding protein (9) .
DISCUSSION
The electrophoretic mobilities of calmodulin (14) and vitamin D-dependent calcium-binding protein (15) are delayed by the binding of Ca2+. This change in mobilities, also, depends on a conformational change of the protein. In polyacryl amide gel disc electrophoresis, mCaBP-3 and mCaBP-4 had the same mobility of Rf=0.73, in spite of the Ca2+ bound to mCaBP-3 not previously being removed from the protein. The Ca2+ bound to mCaBP-3 must be removed by the potential difference, and mCaBP-3 transferred to the Ca2+-free form, mCaBP-4. On the other hand, the mobilities of both mCaBP-3 and mCaBP-4 were delayed from Rf=0.73 to Rf=0.49 by the addition of excess calcium to the electrode buffer. This change of mobility suggests the involvement of a large change of the electric charge dependent upon a conformational change of milk CaBP by binding of Ca2+. Calmodulin characteristically contains trimethylated lysine, but not cystein and tryptophan (3). Vitamin D-dependent calcium-binding protein also did not contain cystein and tryptophan (4). Calmodulin contains aspartic acid and glutamic acid at the level of approximately 34% of total amino acid residues present (3). Milk CaBP was rich in acidic amino acids such as aspartic acid and glutamic acid, and further contains eight cystein sulfones and three tryptophans, but not trimethylated lysine. Therefore, milk CaBP is apparently different from calmodulin, troponin C and vitamin D-dependent calcium-binding protein, though the several physi cochemical properties of milk CaBP are similar to those of other CaBPs.
Calmodulin is known to function in the intracellular system (1). One of its functions is to activate phosphodiesterase via the enzyme-calmodulin complex which is induced by the binding of Ca2+ (16). Milk CaBP can also stimulate the enzyme activity such as galactosyltransferase in the presence of Ca2+, though it is abundant in secretions such as milk. This enzyme activation may involve the same mechanism as that of phosphodiesterase by calmodulin. | v3-fos |
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} | s2 | Nitrogen Fertilization of Dryland Wheat in Southwest Kansas
This report is brought to you for free and open access by New Prairie Press. It has been accepted for inclusion in Kansas Agricultural Experiment Station Research Reports by an authorized administrator of New Prairie Press. Copyright 1982 Kansas State University Agricultural Experiment Station and Cooperative Extension Service.
Nitrogen Fertilization of Dryland Wheat in Southwest Kansas Charles A. Norwood and George M. Herron Garden City Branch Experiment Station
Fertilization of dryland winter wheat was studied for many years by agronomists at the Garden City Experiment Station. These studies were conducted in several counties and were discontinued when it was felt that adequate data were available. The results of these older studies indicated that wheat seldom responded toN on silt loam soils but did respond on sandy soils. In the mid 1970s renewed interest in nitrogen fertilization developed because of the implementation of Improved cultural practices and newer varieties of wheat. Therefore, studies were from 1977-1980 with the objective of determining if the improved practices and varieties would result in responses to N.
Studies were conducted In Lane, Stevens, Kearny, Grant, and Hodgeman counties of southwest Kansas.
The tests were on silt loam soils in all counties except Stevens, which was on loamy fine sand , and Grant in 1979, which was on sandy loam. All fields had been fallowed the previous year. Nitrate measurements were determined on soil samples taken prior to fertilization in each year. Ammonium nitrate at rates of 0, 25, and 50 lbs N per acre was topdressed in March of each year.
Results
Soil test values are presented in Table 1, along with a "yes or no" indication as to whether or not an increase in yield due to fertilization was obtained. Yield data are presented in Table 2. Generally a response to N occurred if the soil test value for nitrates in the surface two feet of soil was less than 50 pounds (about 10 parts per million}. Two locations, Kearny in 1978 and western Hodgeman in 1979, did not respond although the values were 31 and 41 pounds respectively. Conversely, there was a response in Lane County in 1980, even though the soil test value was 89.
protein contents are given in Table 3. A low protein content is frequently regarded as an indication of inadequate N fertility. However, yield levels and climatic conditions also influence protein content. High or low yield levels can dilute or concentrate protein content. This may have been the case in eastern Hodgeman County in 1979, when unusually favorable climatic conditions resulted in high yields, yet protein contents were relatively low. Both yield and protein increased due to N. In contrast, yields were relatively low in Grant County in 1977, yet protein levels were high; neither yield nor protein responded to N. Protein responses occur more readily than yield responses, however. While only 11 of the 2llocations showed a yield response, 18 locations showed a protein response.
With the exception of the site in Stevens County, a sandy location that responded each year, yield responses seemed to depend on where the sites were located in southwest Kansas. A response in Kearny County occurred only in 1979, a year in which climatic conditions were highly favorable for wheat. A response in Grant County also occurred in 1979, on a sandy loam soil where such a response is likely. Further east, in Lane County, responses occurred in. three of four years. Three of the six sites in Hodgeman County responded. Thus, yield responses appear to be more likely in the higher rainfall, eastern area of southwest Kansas. Increases in protein content, however, seemed to be independent of the location.
Conclusions
Increases in wheat yield due to nitrogen fertilizer are more likely in the eastern counties of southwest Kansas. Occasional responses may occur in the western counties, however. A response to nitrogen will notre-suit every year, regardless of the location, unless the soil is sandy and moisture adequate. Increases in protein content are likely, and occur independently of the location or soil type. Rates of N between 25 and 50 pounds will usually be adequate for maximurr grain yield.
For more than a decade wheat yields have increased because of improvements in cultural practices and varieties. The higher yields result in additional depletion of soil nitrogen reserves. Thus, increased se of N fertilizer in the future is likely. Soil tests for N dre helpful in determining fertilizer requirements. | v3-fos |
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} | s2 | Effect of feeding a raw winged bean seeds on gastrointestinal functions in rats.
The primary cause of the adverse effects of feeding of raw winged bean seeds in rats was investigated. In experiment 1, rats were fed on either a raw winged bean diet or a steamed winged bean diet for 10 days. Body weight gain of rats fed on 30% raw winged bean diet was significantly lower than that of rats fed on 30% steamed winged bean diet. The adverse effect of the feeding of 30% raw winged bean diet on growth was accompanied by disorders of the gastrointestinal tract including a significant reduction in intestinal sucrase activity, not being improved with feeding of the diet supplemented with methionine. In experiment 2, rats fasted for 2 days were refed on a 10% casein diet, a 30% raw winged bean diet or a 30% steamed winged bean diet, in which most of the carbohydrate component was sucrose, for 4 days. Although body weight gain and food consumption in rats refed on these winged bean diets were lower than those in rats refed on 10% casein diet, the effects of feeding of the raw winged bean diet on body weight gain and food consumption were extremely deleterious as compared with those of feeding of the steamed winged bean diet. Significant reductions in hydrolase activities localized in the brush border membrane of the small intestine were found in rats refed on the raw winged bean diet prior to the occurrence of apparent disorders in the gastrointestinal tract. These findings suggest that the primary cause of the adverse effects of raw winged bean seed feeding the disorders in the small intestine caused by lectin or similar substances in raw winged bean seeds.
Summary
The primary cause of the adverse effects of feeding of raw winged bean seeds in rats was investigated. In experiment 1, rats were fed on either a raw winged bean diet or a steamed winged bean diet for 10 days. Body weight gain of rats fed on 30% raw winged bean diet was significantly lower than that of rats fed on 30% steamed winged bean diet. The adverse effect of the feeding of 30% raw winged bean diet on growth was accompanied by disorders of the gastrointestinal tract including a significant reduction in intestinal sucrase activity, not being improved with feeding of the diet supplemented with methionine. In experiment 2, rats fasted for 2 days were refed on a 10% casein diet, a 30% raw winged bean diet or a 30% steamed winged bean diet, in which most of the carbohyd rate component was sucrose, for 4 days. Although body weight gain and food consumption in rats refed on these winged bean diets were lower than those in rats refed on 10% casein diet, the effects of feeding of the raw winged bean diet on body weight gain and food consumption were extremely deleterious as compared with those of feeding of the steamed winged bean diet. Significant reductions in hydrolase activities localized in the brush border membrane of the small intestine were found in rats refed on the raw winged bean diet prior to the occurrence of apparent disorders in the gastrointestinal tract. These findings suggest that the primary cause of the adverse effects of raw winged bean seed feeding is disorders in the small intestine caused by lectin or similar substances in raw winged bean seeds. Key Words winged bean, lectin, small intestine, sucrase, alkaline phos phatase, leucine aminopeptidase The winged bean (Psophocarpus tetragonolobus) seems to be a crop with nutritional potential (1,2). Protein, lipid, mineral and vitamin components of winged bean seeds were similar to those of soybean The diets prepared with the ground raw seeds were toxic to rats, and the activity of trypsin inhibitor, amylase inhibitor and lectin in the raw seeds was detected , whereas the diets prepared with the cooked seeds caused fairly good growth in rats, and activities of these substances were destroyed entirely by heat-cooking (2). On the other hand , feeding of a raw soybean diet to rats caused significant growth retardation, but the adverse effect was improved by the addition of methionine to the diet (3,4) . In a preliminary experiment in our laboratory, we found that feeding of a raw winged bean meal diet to rats produced remarkable growth retardation and a reduction in food con sumption along with significant apparent disorders of the gastrointestinal tract . And these deteriorating effects were not entirely prevented by the addition of methionine to the diet. These findings suggested that the deleterious effects of the raw winged bean meal diet were caused by some toxic factors in raw winged bean seeds with the exception of trypsin inhibitor. The present study was undertaken to investigate the primary toxic factors in raw winged bean seeds through the effects of raw winged bean seed feeding to rats on the intestinal enzyme activities localized in the brush border membrane of the small intestine (5 except for the replacement of raw meal with steamed meal. A 30% raw winged bean plus methione diet was made by the addition of L-methionine at the 0.3% level to the 30% raw winged bean diet. Rats were fed ad libitum one of these winged bean diets for 10 days, being killed in the morning on day 11. The small intestine was rapidly removed, a jejunal segment representing the second 15cm segment distal to the pylorus being excised. The segment was slit longitudinally after rinsing with cold saline, and was homogenized with cold water in a Polytron homogenizer (model PCO-2-110 Kinematica, Switzerland). The resulting homogenate was used for the determinations of protein content and sucrase activity. Experiment 2. Rats weighing approximately 100g were used. The experimen tal diets were 10% casein, 30% raw winged bean, and 30% steamed winged bean . Sucrase activity was determined by the method of Dahlqvist (8). Alkaline phosphatase activity was measured by the method of Kind and King (9). Leucine aminopeptidase activity was determined by the method of Goldbarg and Rutenburg (10), and the determination of the protein content in the small intestine was made by the method of Lowry et al. (11) using bovine serum albumin as a standard. Statistical analysis was done by the least significant difference calculated according to the method described by Snedecor and Cochran (12).
AND DISCUSSION
The results of experiment 1 are given in Figs. 1 and 2. The body weight gain of rats fed on 30% raw winged bean diet was clearly lower than that of rats fed on 30% steamed winged bean diet, and the adverse effect of feeding of 30% raw winged bean diet on growth was not improved with feeding of the diet supplemented with 0.3 of L-methionine. There were differences in food consumption similar to those in body weight gain among rats fed on these experimental diets. Intestinal sucrase activity per 15cm of the small intestine-the segmental sucrase activity-in rats fed on 30% raw winged bean diet with or without supplemented methionine was significantly decreased as compared with that in rats fed on 30% steamed winged bean diet. The significant reduction in the segmental sucrase activity was accom panied with remarkably apparent disorders of the gastrointestinal tract, diarrhea or constipation. Table 1 indicates the results of experiment 2. Body weight gain and food consumption of rats refed on 30% steamed winged bean diet were clearly higher than those of rats refed on 30% raw winged bean diet, although body weight gain and food consumption of rats refed on either 30% raw winged bean diet or 30 steamed winged bean diet were significantly lower than those of rats refed on 10 casein diet. The differences similar to those in body weight gain and food consumption among these three groups were found in intestinal enzyme activities, especially the segmental enzyme activities. The adverse effect of feeding of the diet containing raw winged bean meal on growth and food consumption observed in experiment 1 was again found during the early stage of the feeding. Although the apparent disorders in gastrointestinal functions in rats refed on the raw winged bean diet were not observed in experiment 2, their intestinal enzyme activities were significantly lower than those in rats refed on the casein diet. The significant reductions in the enzyme activities was suggested to be the primary cause of the adverse effects including the gastrointestinal disorders due to feeding of the raw winged bean diet, and to be produced by heat-labile constituents-some proteins- in raw winged bean seeds, since the adverse effects were remarkably improved in rats refed on the steamed winged bean diet.
We recently reported that the segmental sucrase activity in rats fed on a high sucrose diet was affected by the quality and quantity of dietary proteins (13)(14)(15), and that the activities were maintained at a constant level, regardless of food con sumption, if animals ate the same diet (13)(14)(15)(16)(17). And a significant reduction or inhibition of intestinal sucrase activity induced gastrointestinal disorders following nutritional disorders throughout the whole body (17)(18)(19)(20). The segmental alkaline phosphatase and leucine aminopeptidase activities, however, are known to be affected not only by quality but also the quantity of the diet consumed (13,14,17). From these findings the segmental sucrase activity has been adopted as a suitable criterion for assessing the integrity of functions in the small intestine. On the basis of the consideration mentioned above, the effect of toxic constituents in the raw winged bean seeds on gastrointestinal functions was considered to be reflected in substantial reductions in the segmental sucrase activity.
On the other hand, the toxic constituents in the raw winged bean seeds were assumed to be the lectin fractions for the following reasons. 1) The adverse effects of feeding of the raw winged bean diet were eliminated by replacing the raw winged bean meal with the steamed winged bean meal. 2) As the adverse effects were not prevented by the addition of methionine to the raw winged bean diet, the toxic constituents were suggested to be others, with the exception of trypsin inhibitor (3,4,21). 3) Since feeding of the raw winged bean diet containing sucrose as the greatest carbohydrate source induced the adverse effects, the amylase inhibitor was not considered to be a main factor of the toxic components. 4) Feeding of the diet supplemented with beans containing high lectin activities caused a disruption of the brush border membrane in the small intestine, followed by significant growth retardation (22).
To verify the hypothesis, however, the relationships between the feeding of a diet containing lectin fractions isolated from raw winged bean seeds and intestinal sucrase activity in rats are required to be extensively investigated, and attempts to fi nd confirmable evidence lending support to the hypothesis are in progress in our laboratory. | v3-fos |
2018-12-04T04:41:49.991Z | {
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} | s2 | Tillage and No-preplant Tillage Compared for Grain Sorghum and Soybean Production in North-central Kansas
Two main functions of preplant tillage are to control early-season weed growth and to prepare a seedbed. Both functions, however, can be accomplished without tillage operations if herbicides are applied to control early-season weeds and a notill planter is available to plant directly into crop residue. We evaluated grain yields and weed control for three preplant-tillage systems: (1) disk as needed to :ontrol weeds and prepare a seedbed; (2) apply a · ·1erbicide in April to control weeds and grasses until planting time, disk just before planting; and (3) use a herbicide, with no-preplant tillage, to control weeds and grass before planting, then plant with a no-till planter. This study was established on the K.S.U. North-central Experiment Field near Belleville in 1975. (See Table 1.)
Tillage and No-preplant Tillage Compared for Grain Sorghum and Soybean Production in North-central Kansas Robert J. Raney, Agronomist in Charge Oliver G. Russ, Research Agronomist D. Michael Powell, Agricultural Engineer
Two main functions of preplant tillage are to control early-season weed growth and to prepare a seedbed. Both functions, however, can be accomplished without tillage operations if herbicides are applied to control early-season weeds and a notill planter is available to plant directly into crop residue.
We evaluated grain yields and weed control for three preplant-tillage systems: (1) disk as needed to :ontrol weeds and prepare a seedbed; (2) apply a · ·1erbicide in April to control weeds and grasses until planting time, disk just before planting; and (3) The cropping sequences were: (1) continuous grain sorghum, (2) continuous soybeans, (3) grain sorghum after soybeans, and (4) soybeans after grain sorghum . In all plots, crops were planted with a no-till planter in rows 30 inches wide. Grain sorghum was seeded at 52,000 seeds/acre (seeds 4 inches apart in the row); soybeans at 105,000 seeds/ acre (seeds spaced 2 inches apart) . Grain sorghum was fertilized by broadcasting nitrogen fertilizer to apply 60 pounds of nitrogen/ acre in early spring and by banding at planting with 10-20-0 at 100 pounds/ acre. Furadan was banded at planting at 13 pounds/ acre to control insects. Also , in grain sorghum plots Ramrod/ Atrazine was broadcast at 6 pounds of product/ acre at planting to provide weed and grass control.
Soybeans were not fertilized, but the seed was inoculated. When the seeds were planted, Lasso + Sencor was broadcast at 2 qt and at . 75 lb of product/ acre to control weeds and grass. Results for grain sorghum yields and weed control for 1975-81 are given in Table 2; those for soybeans in Table 3 'Grain yields corrected to 12.5% moisture. Table 4 shows average crop yields and weedand grass-control percentages for the cropping systems. • Grain yields corrected to 12.5% moisture.
Grain sorghum and soybean annual yields for each cropping system and preplant tillage system are listed in Tables 5 and 6, respectively. The wide variation in yields probably can be attributed to the rainfall patterns and total annual rainfall. Below-average grain sorghum yields in 1975 and 1976 can be traced to below-average summer rainfall for those years. One cannot make such comparisons for 1980, when chinch bugs caused a crop failure. Near-average to above-average summer rainfall produced above-average grain yields in 1977, '78, '79, and '81. Soybean yields were below average in 1976, '78, and '80. Rainfall was below average in 1976and 1980 for both spring and summer. In 1978 rainfall was near average in the spring and 6.62 inches above average in the summer; however, that summer nearly 4 inches of rainfall came in one 24-hour period.
CONCLUSIONS:
Grain sorghum. Grain yields showed no significant differences that can be attributed to cropping or preplant-tillage systems. Yields did appear to be more affected by summer than by spring rains. Broadleaf weed control was not affected by cropping systems or by preplant-tillage systems (Tables 2 and 4). The "chemical only" preplant tillage system and the continuous grain sorghum cropping system provided inferior grass control.
Soybeans. Soybean yields were not affected by cropping or preplant-tillage systems, although continuous soybeans averaged 4 bu/acre less than did soybeans following grain sorghum (Table 4). Broadleaf weed control among the preplant tillage systems showed mechanical only to be inferior to the mechanical + chemical tillage system (Table 3).
Grass control was superior for the continuous soybean cropping system. NOTE: This study was designed to compare the effects of tillage and cropping systems on grain yields and on broadleaf and grassy weed controL No attempt was made to measure soil-erosion losses attributed to the three cropping systems.
One would think that early-spring rainfall would be better conserved by the no-preplant tillage system, but that was not reflected in our 7-year yield averages.
The economical benefit from fewer trips over the field versus the additional cost for an extra herbicide application for the no-preplant tillage system was not evaluated for this report. | v3-fos |
2014-10-01T00:00:00.000Z | {
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} | s2 | Nonvolatile organic compounds in treated waters.
Over the past decade much information has been published on the analysis of organics extracted from treated water. Certain of these organics have been shown to be by-products of the chlorination disinfection process and to possess harmful effects at high concentrations. This has resulted in increased interest in alternative disinfection processes, particularly ozonation. The data on organics had been largely obtained by using gas chromatography-mass spectrometry, which is only capable of analyzing, at best, 20% of the organics present in treated water. Research in key areas such as mutagenicity testing of water and characterization of chlorination and ozonation by-products has emphasized the need for techniques suitable for analysis of the remaining nonvolatile organics. Several methods for the isolation of nonvolatile organics have been evaluated and, of these, freeze-drying followed by methanol extraction appears the most suitable. Reverse-phase HPLC was used for separation of the methanol extract, but increased resolution for separation of the complex mixtures present is desirable. In this context, high resolution size exclusion chromatography shows promise. Characterization of separated nonvolatiles is possible by the application of state-of-the-art mass spectrometric techniques. Results obtained by these techniques have shown that the nonvolatile organic fraction of chlorinated drinking water consists of many discrete compounds. Among these, some of the chlorinated compounds are almost certainly by-products of disinfection. Studies of the by-products of ozonation of fulvic and humic acids isolated from river waters have indicated a similar proportion of nonvolatile organics. Further, ozonation can result in the release of compounds that are trapped in the macromolecules.
Introduction
This paper presents a short review of research into nonvolatile organics in water, with particular emphasis on analytical methodology. In order to limit the size of the paper, the review is necessarily selective in the citation ofresearch from the scientific literature. Results of recent work at the Water Research Centre (WRC) will be used to highlight important areas of research into nonvolatile organic compounds in treated water.
The need for methods of analysis of nonvolatile organics stems from the rapid growth of interest in organics in treated water over the past ten years *Water Research Centre, Medmenham Laboratory, P. 0 (1,2). This interest arises from concern over the possible long-term health effects from ingestion of organics in treated waters, particularly halogenated species (1). The vast majority of identifications of organic material in treated waters have been generated by the use of gas chromatography-mass spectrometry (GC-MS) techniques, which provide a relatively rapid and straightforward method of analysis. It is now generally accepted that 80-90% of the organic matter present in both raw and treated waters is not amenable to this type of analysis due to insufficient volatility. Further, there is some evidence that a similar proportion of the halogenated organic matter produced by chlorination is also not amenable to GC-MS analysis (3).
Methods for the characterization of the nonvolatile organics in treated waters are thus urgently needed to give information to complement that obtained from GC-MS. Undoubtedly, concern over possible health effects of the nonvolatile organics, which represent the bulk of the organic matter in water, is heightened by the paucity of information concerning them. The rapidly expanding application of short-term bioassay tests, such as the Ames test, to drinking waters is beginning to emphasize this need for qualitative techniques for nonvolatile compounds. Many of the mutagens detected do not seem to be identifiable by GC-MS methods (4,5). There are many diverse problems associated with the characterization of nonvolative organics in treated waters, but most of these are a result of the low levels often present, the wide range of compound types and extreme range ofmolecular weights. Currently there is no analytical method available for nonvolatiles that is comparable in sensitivity and ease of application to the GC-MS approach used for volatiles. It is unlikely that a single method will be sufficient and thus the various steps in the analysis of nonvolatiles are reviewed below.
Isolation Methods
Isolation of a specific compound from a very dilute aqueous solution of a complex mixture of components is a relatively easy process, since the extraction method can be tailored to the chemical and physical properties of that compound. However, isolation of the broadest possible fraction of a complex mixture of unknowns is a considerably more difficult problem. A large variety of methods have been examined for isolation of organics from water, and these have been recently reviewed (6). Most of these have been developed specifically for volatile compounds and are, therefore, outside the scope of this paper. Relatively few methods have been developed for nonvolatiles, but several of the methods used for volatiles have been applied, with or without modification.
Research specific to the isolation of nonvolatile organics from water has been mainly concerned with the humic substances. Methods used include adsorption, precipitation and liquid-liquid extraction, and these have been reviewed in some detail (7,8). Recent research has focused on the use of XAD resin adsorption which has been shown to provide high recoveries (>80%) of humics from raw waters (9,10). However, XAD is not recommended for the concentration of humic substances after chlorination, as these are very poorly adsorbed (3).
Other techniques which have been applied to nonvolatile organics in water are carbon-chloroform extraction (11), reverse osmosis (12) and freezedrying (13,14) which suffers form the limitation of being a batch process. Result from these methods suggest that freeze-drying and reverse osmosis provide extracts containing the widest range of compounds. With freeze-drying, the recoveries WATTS ET AL. obtained for particular compound types was found to be solvent dependent, but methanol provided the highest overall recoveries (14).
Separation Methods
Separation ofnonvolatile organics ideally requires a method that avoids derivatization, does not chemically alter the organics during separation, and has high resolving power and high sensitivity of detection for separated compounds. While no method meets all of these criteria, high performance liquid chromatography (HPLC) approaches this ideal and is the method of choice for the separation of nonvolatile organics.
A variety of fractionation procedures have been used prior to HPLC, particularly where fractions are being prepared for mutagenicity testing. Thus, thin-layer chromatography, column liquid chromatography and chemical fractionation have all been applied for preseparation of nonvolatile organics, and these approaches have been recently reviewed (5,15). Modern HPLC often involves the use of several chromategraphic methods and most of these have been used-for separation of nonvolatile organics from treated waters. Size exclusion chromatography (SEC) has shown that the molecular weights of nonvolatiles in water range from a few hundred to >15,000 (3,16,17). One drawback in the characterization of nonvolatile organics according to molecular size by use of SEC is the competition between exclusion processes and nonexclusion processes such as adsorption. This is a particular problem for an unknown mixture of compounds of widely different polarity and can invalidate a molecular weight calibration produced using a mixture of known compounds.
Ion-exchange chromatography has provided high resolution separation of nonvolatiles from chlorinated and ozonated waters prior to identification and mutagenicity testing (18,19). Reverse-phase HPLC has been applied to the separation of nonvolatiles from treated water to provide fractions for mutagenicity testing (20) and mass spectometric characterization (13,14). Finally, adsorption HPLC on silica gel G was used to separate neutral organics from a drinking water concentrate prior to mass spectrometry (21).
Although HPLC shows inherent advantages for the separation of nonvolatile organics, it suffers from two limitations. There is no universal detector for HPLC which is equivalent in sensitivity to the flame ionization detector used in GC. Several highly sensitive detectors are available, but these are also highly selective and, therefore, inappropriate to the detection of a wide range of compounds. Detection of separated components is usually achieved by ultraviolet absorption and is therefore limited to those compounds containing an ultraviolet chromophore. Secondly, since nonvolatile organic fractions isolated from water usually contain a large number of compounds of diverse chemical type, even high efficiency HPLC columns cannot provide complete separation into individual components. Thus, methods for characterization of HPLC fractions are required that are capable of handling a mixture of components, which is discussed below.
Characterization
Techniques for the characterization of nonvolatile organics can be divided into two broad categories on the basis of approximate molecular weight, each requiring different procedures.
High Molecular Weight Material (>2000). This category consists mostly of humic and fulvic acids polydisperse polymeric materials, which are not amenable to mass spectral characterization as intact molecules. General methods for their characterization have been extensively reviewed (8,22) and only recent work which centers around the production of haloforms and other halogenated organics from chlorination of humic acid fractions (23-25) will be discussed. Halogenated nonvolatile reaction products ofhumic acids have been characterized by total organohalogen measurement and size exclusion chromatography (3,26). The majority of structural studies on aquatic humic materials involve some form of degradation step to produce smaller, volatile compounds for analysis by GC-MS. Included in this approach are attempts to identify the volatile by-products from chlorination (27) and ozonation (28) of humic materials. The substituted aromatic compounds identified in degradation studies have similar structures to those obtained from soil humic material, suggesting that the two types of humics have similar compositions. Recently, attempts have been made to obtain mass spectrometric data on larger fragments of humic acid (29). This has involved field desorption mass spectrometric (FD-MS) analysis of fragments formed after chlorination or permethylation of humic material, but as yet no structural information has been reported.
Low Molecular Weight Compounds (<2000). As mentioned previously, any method used for characterization of nonvolatile organics in HPLC fractions must, in most cases, be capable of working with low levels of individual compounds in complex mixtures. Thus, most of the classical methods of characterization, e.g., nuclear magnetic resonance, infrared and ultraviolet spectrometry, are unsuitable, and necessarily some type of mass spectrometry must be employed. Conventional electron impact mass spec-89 trometry using a direct insertion probe offers a considerable extension to the types of compound that can be handled by GC-MS. However, electron impact suffers from two limitations: the compound of interest has to be volatilized prior to ionization and the molecular ion is frequently of low intensity with respect to fragment ions. The first limitation can be overcome to some extent by the use of rapid heating and "in-beam" techniques, which have been recently reviewed (30). The second limitation, however, is difficult to overcome and results in extremely complex, overlapping mass spectra in the case of mixtures. Ideally then, for mass spectrometry of mixtures of nonvolatiles, a method is required that can ionize compounds in the solid state with transfer of very little excess energy. This would provide a mass spectrum consisting of intense molecular ions with few, if any, interfering fragments ions. Several ionization methods which can achieve this are available, for example: direct chemical ionization (DCI) (30), plasma desorption (PD) (31), laser desorption (LD) (32), field desorption (FD) (33) and, most recently, fast atom bombardment (FAB) (34). All of these techniques provide intense molecular ions and only limited fragmentation for certain types of nonvolatile organics and are, to some extent, complementary. At the present time, FD-MS has received more extensive application than any of the other techniques and appears to be applicable to the widest range of compound types (35). Surprisingly, the use of FD-MS for the characterization of nonvolatile organics separated by HPLC has received only limited study. However, it has been used for analysis of herbicides and their by-products in surface waters (36,37) and organics in drinking water (14,21). While it has contributed to the identification of only few compounds so far (13), it has shown that a complex mixture of nonvolatile organics can be present in potable water but it is impossible to resolve this into individual components even by modern HPLC.
A limitation of FD-MS for the identification of nonvolatiles is the lack of structural information provided. Accurate mass measurement of molecular ions can provide empirical formulae, but a number of structures will still be possible. Recently, methods for obtaining structural data from selected ions, namely, collisional activation (CA) (38) coupled with either (B/E) linked scanning (39) or massanalyzed ion kinetic energy spectrometry (MIKES) (40,41) have become more widely available. Useful fragmentation information similar to that obtained from electron impact mass spectrometry can be obtained for any ion (of sufficient intensity) in a mass spectrum, which makes the method advantageous for the analysis of mixtures. Reports of the 90 combined use of FD-MS and CA have only recently appeared, but the technique offers great promise for the identification of nonvolatile organics (42).
Chemical Studies
The broad spectrum approach to determining the nature and behavior of nonvolatile organics in water has been supplemented by laboratory studies on specific nonvolatile organics known or believed to be present in water. Much of this type of work relates to investigations of the organic by-products formed during chemical disinfection processes such as chlorination and ozonation (3,18,27,29). Most studies have been confined, due to a lack of suitable techniques, to products identifiable by GC-MS, i.e., volatile organics. Indirect evidence from such studies, such as results from TOC and TOCl measurement, suggests that a considerable proportion of the by-products are nonvolatile in nature. However, its characterization awaits the development and application of appropriate analytical methods.
The following sections of the paper are devoted to some relevant features of WRC research. In particular, an overall analytical procedure for identification of nonvolatile organics in water, which has been developed recently, is outlined in some detail. Two additional features related to "chemical studies" are included. One involves the laboratory chlorination of uracil. 5-Chlorouracil and other halogenated compounds were detected in treated water in earlier work (13). The work reported there was carried out to test the hypothesis that such a compound is formed from uracil (a constituent of sewage effluent) during water treatment chlorination. The other aspect, very much related to the subject of nonvolatiles in water, is the identification of byproducts of aqueous ozonation of humic and fulvic acids and some preliminary findings from this work are presented.
Experimental Isolation
The procedures used for isolation of nonvolatile organics from treated water have been reported in detail elsewhere (13). Basically, these involve the freeze-drying of water samples followed by methanol extraction of the recovered freeze-dried solids. Initially, adsorption by XAD was considered as a potential isolation method and in order to provide a comparison between freeze-drying and resin extraction, the following experiments were carried out. Small samples (5 1.) of a treated water were collected; one was acidified (to pH 3 by using -4N HCI), WArTS ET AL.
one basified (to pH 10 by using -AN NaOH) and the other left at neutral pH. Each of these samples and a blank consisting of double-distilled water were passed through a resin column (XAD-2), which was subsequently eluted with methanol (-400 ml). The methanol eluates were then concentrated and examined by HPLC as detailed below.
Separation: High Performance Liquid Chromatography
The equipment used consisted of two solvent delivery systems (Waters Associates, Model 6000A), a gradient former ( RI injected). Columns (20 cm x 7 mm ID) were packed in our laboratory (13) with 5 ,um particle size Spherisorb-ODS (Phase Separations Ltd.) and generally had an efficiency of about 18,000 theoretical plates (HETP, 0.01 mm; reduced plate height, 2.2) as measured from a standard mixture of phenols (43).
A linear gradient was established from two solvent mixtures consisting of 1% methanol in 0.1% aqeous acetic acid (A) and 90% methanol in 0.1% aqueous acetic acid (B). The gradient was run over 30 min from 0% to 100% B. The flow rate was maintained at 2.0 m/min.
Size exclusion chromatography (SEC) was carried out on a TSK 3000 SW (Toyo Soda Co.) column (30 cm x 7 mm I.D.) with the use of either water or an aqueous solution ofpotassium chloride and sodium dihydrogen orthophosphate (0. 1M) as eluent (1 ml/ min). Injection and detection were performed as for reverse-phase chromatography.
Characterization
Preparative HPLC fractions were selected on the basis of a relatively high ultraviolet absorption for mass spectrometric analysis on a VG Micromass ZAB IF instrument. Electron impact was carried out at 70 eV electron voltage, 200 ,uA trap current, 8 kV accelerating voltage, source temperature of 200°C and a resolution of -4000. Samples were introduced into the source via an independently heated direct insertion probe. Field desorption mass spectra were obtained on the same instrument, also at 8 kV accelerating voltage and with a total extraction voltage of 10 kV. The FD mass spectra were obtained at a resolution of4500, using 10 ,m tungsten wires with carbon microneedles (average length -40 ,m) grown in the usual manner (44). All mass spectra were acquired and processed on a VG Datasystem computer. Accurate mass measurements were made on selected peaks using either data system acquisition or peak matching. Collisional activation (CA) studies were performed by admitting the collision gas (helium) into a collision cell (in the first field free region) to a pressure sufficient to reduce the intensity of the parent ion beam by 60%. The CA mass spectrum was then obtained by scanning the mass spectrometer at a fixed ratio of B/E and recorded oscillographically.
Chemical Studies
Chlorination of Uracil. A series of laboratory experiments was carried out on the aqueous chlorination of uracil. Distilled water (2 1.), buffered (pH 7.5) with borate, was spiked with uracil (1 and 10 mg/l.) and aqueous hypochlorite added to produce a chlorine residual (12 and 1.2 mg/I.). The solutions were stirred continuously and maintained at ambient temperature (20°C) in a water bath. The experiments were carried out in the dark, and samples were withdrawn at regular intervals for determination of pH, free and total chlorine residuals (DPD method) and 5-chlorouracil production. The latter was monitored by freeze-drying the sample (10 ml), extracting the residue with methanol (3 x 5 ml) and submitting the extract to HPLC. One sample from the experiment carried out at high chlorine residual was extracted with n-pentane (2 x 5 ml) and analyzed for haloforms by GC-MS. System blanks were carried out in an identical manner to that above, but without addition of uracil.
Ozonation of Humic and Fulvic Acids. Ozone was prepared from dry oxygen (typically -15 mg/ min at a flow rate of 180 ml/min), using a Gallenkamp GE-150 generator. The output was monitored before each experiment by determining the amount of free iodine liberated from KI solution (45). Reactions were performed in a twin-necked round-bottomed flask (500 ml) fitted with a bubbler.
Solutions of fulvic and humic acids (200 mg) extracted from river water as previously described (46) were prepared in aqueous 1M NaOH (25 ml) and diluted with double-distilled water (175 ml).
Solution pH was adjusted to -6 by addition of aqueous HCl prior to ozonation (1 hr, typically 5 mg 03/mg fulvic or humic acid). By means of a technique based upon adsorption onto XAD-2 resin (47), unozonized and ozonized solutions were extracted after acidification to pH 1 with aqueous HCI. After concentration under reduced pressure ( -2 ml), the aqueous eluate was examined by HPLC. 91 The XAD-2 resin column was sequentially eluted with ether (25 ml) and methanol (30 ml). The ether fraction was concentrated (to -1 ml) by evaporation prior to GC and GC-MS analysis, and the methanol fraction was concentrated (to -2 ml) under reduced pressure prior to HPLC analysis.
The XAD-2 resin was regenerated using a modified procedure to reduce the amount of organic contaminants released from the resin (48). Blank experiments were carried out for all of the ozonations and resulted in chromatograms which were essentially devoid of peaks.
GC was performed on a chromatograph equipped with flame ionization detector (Carlo Erba Fractovap 2150), Grob splitless injector, and a wall-coated OV-1 Pyrex capillary column (25 m). GC-MS was carried out in the electron impact mode (Finnigan 4000) with computerized data acquisition and processing (INCOS 2300). A wall-coated silicone-fluid fused silica capillary column (20 m) was used with a Grob splitless injector.
HPLC equipment used was similar to that outlined earlier in the experimental section consisting of two-solvent delivery systems (Waters Associates, Model 6000 A), a gradient programmer (Water Associates, Model 660), and an ultraviolet absorption detector (LDC Spectromonitor II) operating at 240 nm. A reverse-phase column (25 cm x 4.6 mm I.D.) of C18-bonded silica (DuPont Zorbax ODS) was used with a linear gradient of water/methanol (0% to 100%) containing an ion-pairing reagent (tetrabutyl ammonium bromide: 0.01M).
Results and Discussion
Isolation As mentioned above, our studies with model compounds have indicated that freeze-drying and methanol extraction is a very efficient method for isolation of a wide range of nonvolatile organics from drinking water. However, it does suffer from two limitations: (1) only a fixed sample size can be processed in one batch and (2) a proportion of the inorganics in the sample is dissolved in the methanol extract. These problems led us to explore the possibility of using XAD-2 resin as a method for obtaining a nonvolatile organic concentrate that was relatively low in inorganics from a large volume of water sample. Figure 1 compares the reverse phase HPLC chromatograms derived from a drinking water sample at neutral pH using the freeze-drying/methanol extraction and XAD-2 resin/methanol extraction techniques. The XAD-2 extract exhibits a more pronounced "hump" of unresolved compounds and fewer discrete peaks than the freeze-dried extract.
Furthermore, a larger proportion of the XAD-2 extract (eight times) had to be used to obtain a similar ultraviolet response on the chromatograms. The XAD-2 extracts obtained at acidic and basic pH gave chromatograms similar to the neutral pH extract but with fewer discrete peaks. From this comparison of XAD-2 extraction with freeze-drying, it is possible to make the following point with respect to ultraviolet-absorbing compounds present in treated water. XAD-2 resin is less efficient at concentrating nonvolatile organics than freeze-drying and results in an HPLC chromatogram with fewer discrete peaks.
It is probable that this lower efficiency of XAD-2 is to some extent sample-dependent, since the water sample used to compare the different extraction methods was a chlorinated drinking water. It has been suggested (3) that the adsorption efficiency of XAD-2 resin for humics is significantly lower for water that has been chlorinated, as compared to the source water.
Freeze-drying and methanol extraction appears to offer the best overall recoveries for a wide range of organics. The major problem with this method remains the significant quantity of inorganics which is dissolved by the methanol. These elute as broad peaks on reverse-phase HPLC and SEC and can interfere with mass spectrometric characterization, which is discussed below. We have evaluated several methods for desalting methanol extracts (14), but none of these have been successful in reducing the level of inorganics without adversely affecting the organics. It is possible that the problem will be alleviated by the use of SEC as a further HPLC separation step.
Separation
Reverse-phase HPLC is the most suitable method for the separation of complex mixtures of nonvolatile organics isolated from treated water. HPLC chromatograms of treated water extracts consist of a large early eluting peak and many discrete peaks superimposed on a "hump", presumably consisting ofunresolved ultraviolet-absorbing compounds (e.g., Fig. la). Using preparative reverse-phase HPLC, it is possible to separate an organic extract into a large number of fractions for characterization by mass spectrometry. Unfortunately, it is not practicable to examine all of these using the newer methods of MS characterization and it is essential to develop criteria for selection of particular HPLC fractions. Previously, we have selected fractions according to either relatively high ultraviolet absorption and/or relatively high total organohalogen (TOX) values (14). However, recent MS evidence (see below) has indicated that the use of TOX levels as a criterion is severely handicapped by the presence of inorganic chlorides. Preparative reverse-phase HPLC fractions that are selected on the basis of high ultraviolet absorption are always found to consist of mixtures of compounds by subsequent MS analysis and only rarely consist of one major compound. This complexity has encouraged us to seek a further separation step in an attempt to reduce the complexity of the mixtures prior to MS characterization. Further separation according to molecular size was an attractive possibility and a high performance size exclusion column was evaluated for this purpose.
The retention volumes and molecular weights of a series of proteins, used to provide a molecular weight calibration, and some model compounds are shown in Table 1. These were obtained on an SEC column using both water (very low ionic strength) and 0. 1M KCl/NaH2PO4 (high ionic strength) as the mobile phase. With water as the mobile phase, an elution order based on molecular size was not obtained. One of the proteins, albumin, was irreversibly adsorbed on the column and the largest protein, ferritin, eluted after a smaller one, catalase. Further evidence of the occurrence of ionic effects was provided by the small retention volumes of four of the model compounds and the exceptionally high retention volume for the smallest model compound, anisole. Changing the mobile phase to one of higher ionic strength resulted in the expected elution order for the proteins and model compounds based purely on molecular size. The inherently low peak capacity of SEC columns is also .0 DECREASING MOLECULAR WEIGHT 15 10 5 RETENTION VOLUME (ml) FIGURE 2. Analysis of a reverse-phase HPLC fraction from a dring water extract using size exclusion chromatography. demonstrated in that a retention volume of only -5 ml is available for separation of compounds with a molecular weight spread of -400 to 500,000. Thus, these columns are appropriate for a crude separation of nonvolatile organics, and are best applied to fractions obtained from reverse-phase HPLC rather than to a total drinking water extract. In order to give some insight into the resolving power of SEC columns for nonvolatile organics of drinking water, a broad reverse-phase HPLC fraction was chromatographed on a SEC column. The chromatogram (Fig. 2) shows two major peaks with some fine structure superimposed suggesting that the extract contains nonvolatile organics of two broad molecular size ranges. It is not possible to determine the molecular size range from this chromatogram, since this is dependent on the nature of the compounds used for calibration of the column which cannot be extrapolated to a complex mixture of unknown composition.
Characterization
The results obtained using electron impact (EI) and field desorption (FD) mass spectrometry both show that reverse-phase HPLC fractions of drinking water extracts contain mixtures of compounds with a wide range of molecular weights in agreement with other reports (21). An example of a FD-MS of a reverse-phase HPLC fraction of a treated water extract is shown in Figure 3. This represents one ofthe more complex fractions obtained but illustrates the wide range of compounds present, since the majority of ions can be considered to be molecular ions under the FD-MS conditions used. Identification of these compounds presents many problems and the identifications reported to date (13) generally constitute cases where HPLC fractions have contained only one or two major compounds. Halogenated compounds are present, as illustrated by the FD-MS of another HPLC fraction of a treated water extract (Fig. 4). This shows 94 WATTS ET AL. several ions with isotope ratios indicative of compounds which contain chlorine and/or bromine. The methodology we are applying to attempt identification of the nonvolatile organics is shown schematically (Fig. 5). First, low resolution FD-MS is obtained on a fraction to provide nominal mass data for the organics present and information on the emitter current at which they desorb. Then accurate mass data and hence empirical formulae are obtained on each FD-MS peak of interest by using suitable reference compounds. Either a calibration table is created by the Datasystem which then automatically performs the accurate mass determination or the peak is manually matched against a reference ion and its accurate mass calculated. Structural information is then obtained for each compound by the use of collisional activation and B/E linked scanning. This technique produces the equivalent of a normal electron impact mass spectrum for any molecular ion of sufficient intensity generated by FD-MS.
An example of a FD-CA mass spectrum obtained by linked scanning is shown in Figure 6. The compound used to produce this spectrum was tetrabutylammonium chloride and the FD-CA spectrum was obtained on the tetrabutyl ammonium ion, m/z 242. The structure of this ion is clearly indicated by the elimination of methane (m/z 226), ethane (m/z 212), propane (m/z 198) and butane (m/z 184). The formation of the tributylammonium ion (m/z 185) and similar eliminations from this (e.g., m/z 169, elimination of methane) provide further structural information. The combination of this data with the empirical formula information would allow relatively easy identification of this compound.
Application of this approach is still in its early stages, but it is possible to comment on some of the problems that have arisen. FD-MS is a relatively sensitive technique, requiring only a few nanograms of compound, but produces low intensity fluctuating ion beams as compared to EI-MS. In order to carry 95 out collisional activation and linked scanning on a FD ion beam, a minimum of 0.5,u/g of each compound must be deposited on the emitter. Not only does this mean that there is a relatively high (for mass spectrometry) minimum sample requirement, but also if there is a very complex mixture, it may only be possible to examine one or two of the compounds present before exhausting all of the sample. The presence of large amounts of inorganics in methanol extracts of treated water and in some derived HPLC fractions can interfere with FD-MS in several ways; (1) they limit the absolute amount of a fraction that can be deposited on a FD emitter, (2) they can give rise to high molecular weight cluster ions for polar molecules (49) and (3) they can yield, by formation of cluster ions, FD-MS spectra with ions at similar molecular weights to compounds of interest, which can complicate interpretation of the mass spectrum (33). However, it is likely that the problems due to inorganic constituents will be overcome by either improved HPLC resolution and/or FD-MS handling techniques. Even ifthese problems cannot be solved, FD-MS with collisional activation and linked scanning is by far the most promising technique available for the identification of nonvolatile organics isolated from water samples.
Chemical Studies
Chlorination. The results obtained from laboratory chlorination of uracil are shown in Table 2. A negligible drop in free chlorine residual of the system blanks was observed during the experiments. GC-MS analysis of a low chlorine residual experiment showed negligible production of haloforms. Several points emerge from study of these results, albeit from a limited number of experiments: (1) the experiment using a high chlorine residual demonstrates that formation of 5-chlorouracil from chlorine and uracil is rapid; (2) 5-chlorouracil is appar-FD-CA mass spectrum of tetrabutylammonium ently not the ultimate product of the chlorination reaction; and (3) although the ultimate product(s) were not identified, they are not haloforms, at least under the conditions used.
The method used to monitor the course of the reaction did not indicate the appearance of any products other than 5-chlorouracil, hence the end product(s) either does not absorb in the ultraviolet region at 254 or 280 nm or is sufficiently volatile to be lost during freeze-drying or is not extracted into methanol.
Ozonation. The compound types identified by GC-MS of the XAD-2/ether extracts before and after ozonation of humic and fulvic acids are shown in Table 3. Identification is limited to the functional groups that can be recognized from the mass spectral fragmentations, and does not exclude the presence of further oxygenated functionalities in each compound. Full details of the structural assignments will be published at a later date. A typical GC-MS chromatogram of an ether extract of ozonized fulvic acid is shown (Fig. 7) to indicate the complexity of the mixture of volatile organic products.
These results show that the volatile products due to ozonation of humic acid are mainly aromatic, WATTS ET AL. while those of fulvic acid are chiefly aliphatic. Such findings are in agreement with previously published results (8). Ozonation of humic acid does not seem to liberate any volatile compounds resulting from fragmentation of the polymer skeleton, although changes are apparent among some of the components detectable after XAD-2 resin extraction of unozonized material. With fulvic acid, ozonation leads to the appearance of certain aromatic acids and normal, branched and possibly cyclic alkanes. While the aromatic acids found in the humic acid extract only after ozonation may result from oxidative cleavage of the macromolecular structure, the alkanes found in the fulvic acid extract only after ozonation were presumably trapped within the polymeric matrix. However, the release of alkanes and DDT and related compounds which are extracted by XAD from humic acid prior to ozonation may be due to a function of the macromolecular structure, which is known to be more "open" at the low pH involved in the XAD extraction procedure (22). Estimation of the proportion of volatile organics extracted before and after ozonation suggests that there is little difference for humic acid (<1%) and only a slight increase (-8% before and -12% Possibly ethers and ketones (aliphatic and mixed aliphatic-aromatic) Aliphatic acids (n-C, 18) DDT and derived compounds Alkanes (normal, branched, -C2o30 and possibly cyclic)b n-Alkanes (n-C23 32) a Present only in unozonized ether extracts. b Present only in ozonized ether extracts. e Probably also includes hydroxy acids, more predominant in ozonized sample. after) for fulvic acid. Thus, despite the severe conditions of ozonation (-10:1 w/w ozone to humic acid), only a relatively small amount of the humic and fulvic acids was converted to volatile organic compounds.
Ozonation of both humic and fulvic acid resulted in the disappearance of most of the color of the starting solutions, which could suggest that a major structural change occurred. Since no major change was detected in the volatile components, it seems that such changes should be detectable in the nonvolatile components. The reverse-phase ion pair HPLC chromatograms ofthe XAD-2/methanol extract both before and after ozonation (Fig. 8) of humic acid show similar features, i.e., three major peaks. A method is, therefore, required to provide information on the structural changes undergone by the polymeric matrix of the humic and fulvic acids. One appropriate method is FD-MS (see above), which will be applied to the nonvolatile ozonation products in the near future.
The results of TOC measurements of the aqueous solutions which are applied to the XAD column and the aqueous eluate indicate that the majority of the organic matter is not adsorbed onto the resin col- SCAN TIME umn. These water-soluble organics are also nonvolatile in nature and exhibit similar chromatographic behavior to those in the methanol extract of the XAD column. Investigations of these water-soluble organics will be reported at a later date. 10 15 TIME (min)
Conclusions
Nonvolatile organic compounds account for most of the organic matter present in both raw and treated water. Many of the industrial, agricultural and pharmaceutical compounds in widespread use are nonvolatile and undoubtedly some proportion of them find their way into the aquatic environment. Studies of the organic by-products of chlorination and ozonation of water have shown that a significant proportion of these are nonvolatile. The increasing application of short-term bioassay tests to drinking water has shown that nonvolatile organics could be responsible for some of the mutagenic response.
Until recently there have been no really suitable techniques available for the isolation, separation and characterization of nonvolatile organics from water. Consequently, few data are available on the types and amounts of nonvolatile organics present in drinking water.
Recently there have been significant analytical developments, particularly in mass spectrometry, of direct relevance to nonvolatile organics. New techniques have emerged for the characterization of nonvolatile organics and the approach using freeze-drying, HPLC separation and FD-CA mass spectrometry appears to offer great promise in this respect. Without doubt, the identification of nonvolatile organics in water would benefit considerably from a more widespread application of new techniques such as these.
Much of the work discussed in this paper was funded by the Department of the Environment. Permission from the Department of the Environment and the Water Research Centre to publish this work is gratefully acknowledged.
Much of the work discussed in this paper was funded by the Department of the Environment. Permission from the Department of the Environment and the Water Research Centre to publish this work is gratefully acknowledged. | v3-fos |
2018-12-11T14:53:50.970Z | {
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} | s2 | AN INVESTIGATION OF THE POTENTIAL OF SOY CELLULOSE AS A TABLET DISINTEGRANT
Scanning Electron Micrographs of soy cellulose (Emcosoy) particles were studied to further the understanding of the morphology of the substance and thereby to increase the comprehension of how the excipient functions in tablet systems. Bulk swelling and water uptake were studied for Emcosoy, CLD II, corn Starch, and Ac-Di-Sol. Emcosoy had maximum swelling about 56%; followed by CLD II 49% and Ac-Di-Sol 9%. No swelling was observed in Corn Starch. An ideal disintegrant should perform uniformly in pH range of gastrointestinal tract in order to disintegrate the tablet. When Emcosoy and Ac-Di-Sol were tested against various pH values, there was no significant difference in swelling of these disintegrants. Since alcohol and sodium lauryl sulfate are often used as dissolution media, the effect of different concentrations were also observed on Emcosoy and Ac-Di-Sol. There was no significant effect. Another important preformulation test reported in this thesis is a comparative study of the viscosity of different concentrations of various disintegrants. When a tablet disintegrant reacts with water a gel may be formed. If the gel is too viscous, it impedes the water penetration in the tablet. Generally it can be said that the cellulose group of disintegrants (Ac-Di-Sol, CLD II and Emcosoy) had a viscous gel compared to starch group of disintegrants (Corn Starch, Explotab and Sta-Rx 1500). If the viscosity of the gel of starch group of disintegrants has to be arranged indescending order then Explotab is followed by Sta-Rx 1500 which is followed by corn starch. If the viscosity of the gel of
Another important preformulation test reported in this thesis is a comparative study of the viscosity of different concentrations of various disintegrants. When a tablet disintegrant reacts with water a gel may be formed. If the gel is too viscous, it impedes the water penetration in the tablet. Generally it can be said that the cellulose group of disintegrants (Ac-Di-Sol, CLD II and Emcosoy) had a viscous gel compared to starch group of disintegrants (Corn Starch, Explotab and Sta-Rx 1500). If the viscosity of the gel of starch group of disintegrants has to be arranged indescending order then Explotab is followed by Sta-Rx 1500 which is followed by corn starch. If the viscosity of the gel of ii cellulose group of disintegrants had to be arranged in descending order then CLD II is followed by Emcosoy which is followed by Ac-Di-Sol.
Plastic deformation and elasticity of various disintegrants were studied using a Universal Instron Testing Machine. High plastic deformation of the disintegrant particles, may be advantageous since the deformed particles are " " energy-rich and that energy is released when the particles are exposed to water. The energy-rich particles probably swell more rapidly in water, unlike undeformed grains, which require more energy of swelling in order to well. Ac-Di-Sol particles have good plastic deformation compared to Emcosoy and Explotab particles. The three major mechanisms of ( disintegrant action are : (i) swelling (ii) capillary action (wicking) and (iii) deformation. Emcosoy particles swell considerably and they have good wicking effect, but they have poor plastic deformation.
Following the performulation study, Emcosoy (soy cellulose) was compared with other disintegrants in about 45 formulations.
About 100 dissolution profiles were obtained on various vitamin formulations using either Emcompress or Endex as the tablet matrix.
Emcosoy is as effective a disintegrant as Ac-Di-Sol and very much more effective than Corn Starch. Grateful acknowledgment is made of the invaluable cooperation and support of Edward Mendell Company,and special thanks to Dr. Joseph L.
Kanig for his advice during the study.
Appreciation is also acknowledged of the invaluable cooperation of Dr. Joan M. Lausier in solving many problems with the tablet press. Grateful acknowledgment is also made of the invaluable cooperation of Dr. Chong M. Lee
I. INTRODUCTION
There are two classes of drugs administered orally in tablet dosage form. These are: (1) insoluble drugs intended to exert a local effect in the gastrointestinal tract (such as many antacids and absorbents), and (2) soluble drugs intended to exert a systemic drug effect following their dissolution in the gut and subsequent absorption.
In the case of drug products intended to exert a s ystemic effect, the design of a dosage form which rapidl y disintegrates and dissolves may or may not be critical, depending on whether the drug is absorbed in the upper gastrointestinal tract or more generally throughout the intestinal tract. Because dosage forms must be designed to disintegrate or dissolve to release the drug in an available form at or above t h e region of absorption in the gut, the design must also be based on the solubility properties of the drug at or above its absorption site.
The present study covers the bioavailability consideration of the tablet as a dosage form, the disintegration process in general, and the information about a few widely accepted new disintegrants. The mechanism of disintegrant action will be discussed with the help of numerous references on the subject. Unfortunately, Proctor's concern was not shared by other scientists.
The need to quantify tablet disintegration by official standards was not recognized until just prior to World War II, and specifications for tablet disintegration did not appear in the USP until the fourteenth revision which was published in 1950. During the course of subsequent disintegration testing experiments, it was theorized and eventually proven that drug dissolution must follow tablet disintegration if bioavailability is to occur (6).
The topic of bioavailability has now become of interest to government; the FDA (Food and Drug Administration) has already issued specifie regulations on bioavailability.
When a drug in a dosage form is administered to a patient, the first steps in the sequence whereby the drug reaches the site of action are commonly disintegration and dissolution. The importance of these processes is year or dollar value of drug dispensed (7). The process of disintegration, dissolution, and absorption can be outlined as follows (8): Tablet or Capsule Disintegration) (1) Granules or Aggregates Deaggregation) (2) Dissolution (3) Drug in Solution (in vitro or in vivo)
Drug in Blood, Other
Fluids and Tissues Absorption (in vivo) Fine Particles (3) Before drugs can effectively pass through the gastrointestinal wall, they must be in solution. Drugs which are only sparingly soluble in the gastrointestinal content at or above their absorption site can have, as the controlling process affecting their absorption , the rate of drug solution in these fluids. In this type of s y stem, the drug goes into solution at a slow rate, absorption occurs almost immediately and is ( s not, therefore, the rate-limiting step. In one study, Nelson (9) correlated the blood level concentration of various theophylline salts with their dissolution rates.
Drugswhich exert a systemic effect must dissolve as a prerequisite to effective drug absorption. The various processes of tablet making, including the aggregation of drug into granular particles, the use of binders, and the compaction of the system into a dense tablet, are all factors which militate against a rapid drug dissolution and absorption in the gastrointestinal tract. In considering, in a general manne~ the availability of drugs from various classes of dosage forms, drugs administered in solution will usually produce the most available drug product, provided that the drug does not precipj_tate in the stcraach or is not deactivated there. The second most available form of a therapeutic agent would be drug dispersed in a fine suspension, followed by micronized drug in capsule form, followed by uncoated tablets, with coated tablets being the least bioavailable drug product in general. In formulating and designing drug products, as well as in considering methods of manufacture, the fact that the tablet dosage form is one of the least bioavailable forms (all other factors being equal) should be kept in mind (10).
Many factors can affect drug dissolution rates from tablets; hence, possibly drug bioavailability , including the crystal size of the drug, tablet disintegration mechanisms and rates, the method of granulation, ( 6 type and amount of granulating agent employed, type, amount and method of incorporation of disintegrants and lubricants, and other formulations and processing factors. Levy,et al (11), showed the effect of granule size upon the dissolution rate of salicylic acid. Salicylic acid of two mesh ranges containing 300 mg of aspirin and 60 mg of starch, were compressed at 715 kg cm-2 . The 60 to 80 mesh granules had better vitro bioavailability than 40 to 60 mesh granules.
Lachman, ~al (12), studied the effect of crystal size and granule size on a delayed-action matrix using tripelennamine hydrochloride. He notes that while granule and crystal size both affected release rate, in this instance the crystal size played a greater role than granule size in dissolution rate.
Paul, et al (13) showed that with nitrofurantoin there was an optimal average crystal size of about 150 mesh, which resulted in adequate drug excretion (hence, absorption and efficacy), but minimized emesis. This exemplifies a situation in which too rapid drug dissolution in the stomach may produce nausea and emesis; an intermediate release rate reduced this effect while achieving adequate bioavailability.
Numerous accounts of the effect of particle size on dissolution rate of steroids have been reported. In one study, Campagna,~al (14),showed that, in spite of good disintegration, therapeutic inefficacy of prednisone tablets could occur.
( 7 As discussed above, bioavailability of the drug depends on many factors.
The ensuing discussion will focus on the disintegration process, disintegrants available, and the mechanism action of disintegrants.
Disintegration of a Tablet and Disintegrants
Complete tablet disintegr?tion is defined by NF XIII (15) as: II that state in which any residue of the tablet, except fragments of insoluble coating, remaining on the screen is a soft mass having no palpably firm core." This often makes tablet disintegration a necessary first step to achieve rapid availability of the active ingredient(s). The importance of tablet disintegration was recognized as early as 1879, when a patent recommended that pills be perforated to admit gastric juice for better disintegration (16).
Reasons for Measuring Disintegration Times and Rates of Dissolution
(i) For research purposes to elucidate the mechanism involved in the processes and to determine the relative importance of the various variablesinvolved in the process of disintegration, deaggregation, and dissolution.
(ii) For developmental purposes to guide the pharmaceutical formulator in the preparation of optimum dosage forms of drugs for clinical trial.
8
(iii) For control purposes to ensure that a given pharmaceutical ( product is essentially uniform from lot to lot. (iv) For predictive purposes so that one may estimate rate(s) of absorption in man from measurement of disintegration time and/or rates of dissolution in vitro. Such predieting requires careful correlation of in vitro and in vivo results.
Our discussion of disintegration will be confined to uncoated tablets designed to release all the active ingredient(s) rapidly.
The disintegration of a tablet depends on compression force, tablet hardness,propertiesof fillers and active ingredients, properties of binders,propertiesand concentration of disintegrant and the properties of lubricants. All these factors play an important role in tablet disintegration. If we keep other factors identical (compressive force, fillers, active ingredient binders, and lubricant) in two formulations but select two different disintegrants at the same concentration for these two formulations, the in vitro bioavailability will entirely depend on the quality of the disintegrants. The drug will be released quickly from a tablet which disintegrates faster because it has better disintegrants. Thus, the disintegrants play a role in the disintegration process. The ensuing discussion will focus on different disintegrants.
( 9 Disintegrant is a term applied to substance added to a tablet granulation for the purpose of causing the compressed tablet to break apart when placed into an aqueous environment. The disintegrant in a tablet formula may be considered as a dispersing agent for the dry compacted tablet mass in the gastric milieu. Ideally, it should cause the tablet to disrupt not only into the granulated form which it was compressed, but also into the powder particles from which the granulation was prepared. The function of the disintegrant is, in effect, to counteract the action of the tablet bindersand the physical forces of compression necessary to form the tablet. The stronger the effect of the binder, the more efficient must be the disrupting effect of the disintegrant in order to release the active ingredient in the gastrointestinal tract (17).
There are two methods used for incorporating disintegrating agents into tablets. These methods are called external addition and internal addition. The most common method is the external addition method in which the disintegrant is added to the sized granulation with mixing just prior to compression. In the internal addition method, the disintegrant is mixed with other powders before wetting the powder mixture with the granulating solution. Thus, the disintegrant is incorporated within the granule. When this method is used, part of the disintegrant is added internally and part by external addition. Many experts believe that use of the two-step method usually produces better and more complete disintegration than the usual method of adding the dis-( ( 10 integrant to the granulation surface only.
Six basic categories of disintegrants have been described: starches, clays, celluloses, algins, gums, and miscellaneous. Many disintegrants have also been shown to possess binder or adhesive properties.
Since disintegration is the opposite operation to granulation (agglomeration) and the subsequent formation of strong compacts, one must carefully weigh these two phenomena when designing a tablet (18).
Formulators have tried a number of materials as tablet disintegrants with varying degrees of success. Lowenthal tabulated all the disintegrants with their pertinent references in a review article of "Disintegration of Tablet," in Journal £f Pharmaceutical Sciences (19).
In the past six to seven years, exciting developments have occurred in the area of tablet disintegrants. A nurr,ber of new disintegrants have been marketed in this period. Attention will be focused on these ''super disintegrants~ as they are called, because of their remarkable quality as disintegrants.
(i) Direct-Compression Starch: One of the most significant modifications of starch for (iv) Cross-Linked Polyvinylpyrolidone: Cross-linked polyvinylpyrolidone (PVP) is a homopolymer of N-vinyl-d-pyrolidene and has been marketed as Polyplasdone-XL (GAF Corp., New York, New York). Because of its high molecular weight and cross-linked structure, it is insoluble in water but is still very hydrophilic. The particles of PVP are porous in nature. As water is absorbed into the porous structure of the agglomerates, the lattice structure of polymer expands, causing high stress on surrounding tablet components.
The porous nature of the particles provides intraparticulate wicking of water. Like cross-linked carboxymethyl cellulose, cross-linked PVP has the ability to shrink to its original particle size when dried and then expand again when rewetted.
Both cross-linked carboxymethyl cellulose and cross-linked PVP would thus appear to be effective disintegrants in tablets made by wet granulation, as well as in those produced by direct (21); potato, corn and wheat (22); and potato, wheat, and rice (23).
Tablets made with low pressure have high porosity, and hence, too much space. When starch swells, no pressure is exerted; therefore, disintegration is slow. Medium pressure allows just enough space so that when the starch swells, it exerts pressure on the granules to cause disintegration. High pressure, producing low porosity, decreases the ability of fluid to enter; so, disintegration is again slow (24,25).
Starch swelling was claimed to be dependent upon amylase and ( 14 arnylopectin content; the amylopectin expands, and the amylase gives osmotic pressure (26) .
Borzenou and Nesmiyan (27) Many substances swell to a greater degree than the starches, but are poorer disintegrants. Amylase does not swell but has been stated to cause good disintegration (29). Although starch grains swell in water according to some views which I do not share, the rate and extent of swelling is still debated (30)(31)(32).
Whenever swelling of disintegrant particles take place with great force, it overcomes the adhesiveness of other ingredients in a tablet and causes the tablet to fall to powder.
The rate of penetration of fluids into a tablet is proportional to mean pore diameter or porosity (51-52); corn and starches increase penetration of fluids into tablets. Permeability of tablet decreases as pressure increases. The effect of starch on porosity may be due to its poor ability to bond and compress (52). In 1955, Curlin reported that although aspirin tablets containing starch disintegrated in 15 seconds, starch grains I \ were not swollen; nevertheless, a drop of dye solution placed on the tablet penetrated rapidly. He suggested that the disintegrating action of starch was due to capillary action, rather than to swelling (53).
Wicking is due to capillarity of fibers. Stiff fibers of uniform structure and resistance to collapse are required for good wicking. The fibers should have zero contact angle and should not swell (54). This would appear to rule out any wicking effect due to starch or cellulose fibers.
( 16 The existence of pores or capillaries is not the complete answer to the mechanism of action of disintegrants, because semipolar and nonpolar fluids penetr~te into tablets (55), yet do not cause the tablets to break. Also, tablets do disintegrate with minimum porosity (56).
(iii) Deformation: Plastic deformation of starch grains under high pressure has been reported by a number of investigators (57). Starch grains are generally thought to be elastic; therefore, any grains that are deformed under pressure tend to return to their original shape and size when the pressure is removed. However, it has been suggested that compression may cause more permanent deformation that the deformed starch grains are energy rich and that this energy is released when the grains are exposed to water (58). The energy-rich starch grains swell rapidly in water, unlike undeformed grains, which require more heat in order to swell.
The various mechanism of disintegration action discussed under a separate heading, interrelationships, probably occur in almost all tablet formulations.
Objective and Justification for the Present Study:
As discussed earlier, tablet disintegration has been increasingly viewed as an important factor in formulating pharmaceutical systems.
Mechanisms by which tablet disintegrants function have been investigated.
The current concern about bioavailability of drug products has made formulators very selective in the use of disintegrants. The United States Pharmacopoeia is presently considering a radical extension of dissolution test requirements to most conventional tablets and capsules, and thus a number of formulators are now re-evaluating their formulations to see whether it is now appropriate to alter the identity or quantity of disintegrant (59) .
The pharmaceutical industry has been using corn starch and guar gum extensively as tablet disintegrants for many years. Both dis-I integrants are natural source materials, but their function as a tablet disintegrant are not satisfactory. There is, however, a demand from certain segments of the pharmaceutical industry, particularly those concerned with vitamin formulations, which require a powerful disintegrant of natural origin.
Soy cellulose (Emcosoy), is an all-natural source material, derived from defatted soy beans by a special process. It contains no sugar or starch, and it has been given GRAS status (Generally Regarded M any formulators are not aware of the potential of this material because of the lack of authentic and extensive study . It is hoped that the present study will provide a reliable evaluation of soy cellulose (Emcosoy) as an effective substitute for corn starch and guar gum. There are several tests which can be used to evaluate disintegrants before using them in actual formulations. No one test is perfect; each has its advantages and disadvantages. The behavior of disintegrants alone could be vastly different compared to their behavior in compressed tablets. Some of these tests may, however, be useful as the basis of a raw material specification designed to control lot-to-lot variation.
1. Scanning Electron Micrographs: The role of particle morphology(size, shape and composition) in the production of tablets has long been discussed. This theoretical effort has found an a ·lly in scanning electron microscopy (SEM), which has made it possible to obtain direct photographs of tableting excipients and finished compacts. This instrument provides scanning electron micrographs of a range of disintegrating agents, lubricants, and glidants in an attempt to further the understanding of the morphology of those substances and thereby to increase the comprehension of how the excipients function in tablet systems.
An adhesive was placed on a metal stub, which was coated with gold or cadmium.
b. A few disintegrant particles were sprinkled on the stub.
Bulk Swelling and Water Uptake:
One relatively simple test used by many groups involved in the evaluation of tablet disintegrants is the quantification of the interaction of a bulk powder bed, composed of pure disintegrant, with water. There are several types of apparatus which can be used for this purpose. The one used in this study consistedof a calibrated glass reservoir (containing water or other approp~iate fluid), .connected at its base by a U-tube to a second glass reservoir which contained the disintegrant powder (1) where s 0 is the disintegrant level in the tube at time zero, and St is the disintegrant level in the tube at any time t.
where v 0 is the water level in the calibrated reservoir at time zero, and Vt is the water level in the calibrated reservoir at time t.
Percentage of swelling and water uptake were plotted versus time. This test is helpful in determining the ease and extent of ' bulk powder interaction with water ( 61 ).
The disintegrants used for this test were Emcosoy, Ac-Di-Sol, corn starch, and CLD II. The solvents were distilled water, ethanol/water mixtures, and sodium lauryl sulfate solutions.
Air pressure was used to remove air bubbles from the lower funnel shape portion of the reservoir. The apparatus was left undisturbed for half an hour, so the water levels in both reservoirs remained constant. The readings were taken initially at short intervals, but later at an interval of twenty minutes. Although the mechanism of disintegration is very complicated and has not been completely determined (40), the penetration of liquid into a tablet is the first step in the process of tablet disintegration.
In this test the water penetration in various disintegrants was compared.
p Where 6H is the heat wetting. If systems are not disturbed during the wetting process, 6H is independent of temperature. In this experiment, pressure p is considered constant; solving above equation.
y Cose = 6H CT (8) is obtained, where C is the positive constant; combining all abovementioned equations: k y e-/RT
2A
Since generally 6H/CT is coming between one and two, taking logarithm of equation, approximating log (6H/CT -1) to 6H/CT + C" We can obtain value of k from slope of each straight line and then Emcosoy was wetted with excess of water in a petri dish (diameter 22 cm). Then the first batch was dried at room temperature (21 C) , ; second and third batches were dried inside the oven at 40C and and 60C respectively. The cake-like mass that formed on drying was reduced to fine particles by using a Fitzpatrick Comminuting Machine. The sieve, #00, was placed at collecting end of machine.
The particles received through the "OO" sieve passed through the 100 mesh (U.S. Standard) screen.
The bulk swelling was determined by using the D-tube-type apparatus for all the batches. The batch which was deemed most promising in all respects was -compressed as formulation IV.
Tablets from· both batches were made at identical press settings,using original Emcosoy for comparison purposes. Tab (13) o is the portion of the solution which is subjected to stress. % is the torque reading.
Spindle: Big to small (1 to 7) Twenty, fifty, and a hundred rpm were used. The dial reading was taken when the pointer had completed one full return.
The easiest method of calculating viscosity is to use the Brook- ( 63). These grains returned to their original shape when the tablets were exposed to moisture.
We can classify various disintegrant particles into two categories: those permanently deformed by the compression (in other words, these particles are nonelastic particles); and those much less permanently deformed by the compression (elastic particles).
In the past, the scanning electron microscope was used for gaining The chart paper had ten big divisions, and each big division was divided into ten small divisions; so the total number of small divisions was one hundred. A mobile recorder pen moved along the base line of the paper, with the movement of piston. Whenthe piston applied pressure on the disintegrant particles, the pen moved on the calibrated chart paper; so by counting the small divisions, one can determine the pressure applied by the piston.
The piston can move down and up once or more times; that· makes it possible to apply single compression or cyclic compression.
The data was interpreted as follows: e. Disintegration: Tablet disintegration was tested by using the U.S.P. apparatus wit~ discs as described in the National Formulary XIV (68). f. Dissolution: Drug dissolution was measured using a U.S.P.
apparatus, and according to monographs in U.S.P. XX.
Plots of these readings were made to depict the dissolution process. The computer program appears in Appendix (20). This mechanisn of tablet disintegration is so effective that Emcosoy can be used as a tablet disintegrant in concentrations as low as .5%. All the disintegrants had equal initial volume in the calibrated tube, since the volume difference was used in calculating percentage swelling.
Bulk Swelling and Water
For one disintegrant,different initial weight would give different percentages of swelling as found out in Ac-Di-Sol, Table IV, and graph on page 54. Logarithm of percentage swelling vs. time would give almost a straight line (Fig. 7 ). For different initial weight, the amount of water that would travel in a powder bed would vary (Fig. 8 ) .
The amount of water travel would reduce as the weight of the disintegrant increased.
The percentage of swelling decreased with increased initial weight of the disintegrant because the particles in the bottom portion of the tube came in contact with water of the reservoir first; they tried to swell, but their swelling was reduced by the weight of particles on the top. As the weight of the disintegrant was increasing, the bulk of particles on top was increasing; at the same time the amount of particles which were in contact with water remained the same. So, the swelling was reduced with increases in weight of the disintegrant.
Average bulk swelling of Emcosoy, Ac-Di-Sol, CLD, and corn starch were compared. Time required to reach the swelling and amount of water travelled in various disintegrantswere compared ( reduced the swelling of Emcosoy significantly.
It was observed that very dilute alcohol solutions and Sodium
Lauryl Sulfate solution reduced the surface tension of water, and they also acted as a wetting agent and enhanced the absorption of water by Ac-Di-Sol; but as their concentration increased, they ceased to act as wetting agents and there was relatively small proportions of water available for disintegrant to absorb. Therefore, swelling was reduced or, in some cases, swelling did not occur.
Sn
Where L is the penetrating length at time t, r is the average radius of void space, 8 the contact angle between liquid and powder surface, g the acceleration constant of gravity, and y, n, and d are the surface tension, viscosity, and specific gravity of liquids, respectively. The penetration rate also depends on the viscosity of gel formed by the interaction of water and disingegrant. If the gel formed is too viscous, it would impede the further penetration of water in a disintegrant bed. All these factors will be discussed in great detail separately.
In addition to water, water-alcohol mixtures of different strength, The value of energy of wetting obtained by this method according to some critics is an apparent value, since they believe that the approach is too simplified to give any real values.
Wetting and Drying of Disintegrant:
Emcosoy batches were prepared under different conditions. Bulk swelling was determined for these batches. Results are tabulated in Table XX.
As it can be seen from the Emcosoy table that was dried at room temperature and at 40chad a bulk swelling less than the untreated (original) ·Emcosoy. One reason could be that these samples were dried at low temperatures, and therefore significant amountsof surface moisture was left in these samples; that could have resulted in low water uptake and less swelling. When the cake is baked, the surface area of the cake increases enormously, and the same thing can happen in the disintegrant particles.
The increase in surface area could lead to increased water uptake and increased swelling. Emcosoy dried at 60c had an improved flow and high density compared to the original Emcosoy. The color change was from yellowish white to brown. No swelling was observed in the first two minutes in the original Emcosoy, but there was 30% to 35% swelling in the first two minutes in Emcosoy dried at 60 c. Wicking effect was very much improved in the Emcosoy dried at 60 c --the time that was taken by the water to travel through the entire powder bed was 60 minutes, compared to 90 minutes for the original Emcosoy.
There was little offending odor from Emcosoy dried at 40 c and 60 c, and visible mold growth in Emcosoy which was dried at room temperature.
Published literature on the subject indicates that molds require a water activity number of 0.75, and bacteria requires 0.88. Below these values, microbial growth cannot be supported. It has been estimated that Emcosoy with a 10% moisture content = water activity number 0.5; and with 20% moisture content, there exists a water activity of 0.88. Since original Emcosoy usually contains less than 10% moisture, it theoretically cannot promote microbial growth. However, in a ( wet granulation process, this argument might not betrue all the time. Prolonged storage or improper storage may lead to higher moisture content and consequently makes this disintegrant vulnerable to microbial growth. c As discussed above, that Emcosoy dried at 60 had a better physical property than the original Emcosoy; they both were compared in actual tablet formulation. Original Emcosoy was identified as "A:' and the processed Emcosoy was identified as "B." In order to get unbiased evaluations of these disintegrants, the tablets were prepared and tested by my colleague. The results and other details are shown in a tabular form (Table XXI) .
As it can be seen from the table and contrary to expectation, processed disintegrant tabletshad higher disintegrant time; in other words, the tablets made using it took a long time to disintegrate, compared to tablets made using original Emcosoy. The reason could be poor deformation of processed disintegrant. The three major mechanisms of disintegration are: (1) swelling, (2) porosity and capillary action (wicking), and (3) deformation. As mentioned above, processed disintegrant is better than original Emcosoy in the first two categories.
There is no data to compare the third one. When the tablet is compressed, the compression may cause more permanent deformation in original Emcosoy than the processed Emcosoy. When the tablet is put into a disintegrating medium, the energy-rich Emcosoy grains swell rapidly, unlike processed Emcosoy grains, which require more heat in order to swell. The overall effect leads to an early disintegration of the ( 6 ). They compared two percent aqueous dispersions of various starches, cellulose products, and of cross-linked PVP in water-diluted hydrochloric acid, (1:100) were shaken periodically, and were allowed to stand overnight.
They found that all the disintegrantshad different swelling; in some of the disintegrants, the swelling was enhanced or supressed in acidic medium.
It is expected of ideal disintegrants that its swelling should remain unchanged or preferably increased in acidic medium, since most of the tablet disintegrates in acidic environments of the stomach. In practice, Explotab is much more superior to corn starch; the reason is that in addition to viscosity, there are other important factors, such as rate and extent of swelling, wicking effect, and deformation play crucial roles in disintegration mechanism.
Explotab, Emcosoy, Ac-Di-Sol, and corn starch were compared in actual tablet formulations. The viscosity of the gel of Explotab is greater than that of Emcosoy. The viscosity of Emcosoy is greater than that of Ac-Di-Sol. Vitamin formulations studied clearly indicated that Explotab is inferior to Ac-Di-Sol and Emcosoy. Viscosity study provides the answer. Explotab swells much more compared to Ac-Di-Sol and Emcosoy, and we therefore expect it to be better disintegrant compared to Ac-Di-Sol and Emcosoy. However, that is not true, because Explotab forms very viscous gel, and it will be shown later that it has poor deformation.
The disintegrant solutions were subjected to different shear rate, and the viscosity were determined. The rheological profiles of the various disintegrants were shown in Fig, 15 to 18 . In theory, there were all three kinds of profiles, as shown in Figure 14. In Newtonian liquids the viscosity remains unchanged with the increase in shear rate. High hydration capacity indicates that the disintegrant requires less amounts of water to wet itself thoroughly. It can be theorized that a disintegrant with high hydration capacity would relatively have smaller water uptake in order to achieve disintegration of the tablet, compared to a disintegrant with low hydration capacity. It can be said from the data that as the viscosity of the gel of disintegrant increases, the maximum hydration capacity decreases.
Sieve Analy sis:
Nogami, et al, found that there should be the critical amount of starch necessary for the ~isintegration depending upon the particle size or specific area of ingredients. The smaller the particle size of aspirin, the more amount of starch was required for the tablet disinte- gration. The tablet of the smallest aspirin (11.9 in mean particle size) did not disintegrate by the addition of 20% of starch. Thus, the particle size of the ingredients play an important part in tablet disintegration (71).
Rudnic, et al, studied the different particle size grades of crosslinked polyvinylpolypyrrolidone (Polyplasdone,G.A.F. Corporation) in direct compression tablet for~_ulati o ns showed tMt i1'l.<!!i'\'~a.ses in mean particle size enhanced powder flow, disintegration and dissolution, although hardness and friability were slightly better for tablets made from the finer grades ( 72). Therefore, particle size of the ingredients, as well as disintegrants, are equally imp ortant.
There is nothing like optimum particle size of disintegrant or matrix. Particle size varies for various disintegrants and tablet matrices . However, the par ticle size should be such that disintegrant can be mixed and distributed uniformly with the rest of the ingredients.
The particle size of Emcosoy was determined using sieving. Table XXV shows the size-weight distribution of Emcosoy as measured by U.S. Standard sieves.
Plastic Deformation and Elasticity of Various Disintegrants:
Modulus of elasticity at various attempted deformations and compressive force needed to achieve that deformation were tabulated in Table X1.1!I.The percentage of plastic deformation and the percentage re- From the elasticity data, the disintegrants were arranged in the decending order of their elasticity on page 96. The order remained unchanged for CLD II, polyplasdone-XL, and Emcosoy at 10%, 20%, and 40% attempted deformation.
Force required to achieve various percentage of deformations for various disintegrants was tabulated on page 98. The force needed to attain 20% deformation in CLD II was little more than double of the force needed to attain 10% deformation in the same disintegrant.
Similar observation was made in polyplasdone-XL, and to some extent in Emcosoy. However, when the deformation was doubled from 20% to 40%, the force needed to achieve that deformation was almost five to six times than the force needed to achieve 20% of deformation. From these results, it was concluded that it was easy to deform particles initially; but as the percentages of deformation increased, the task became very difficult and greater force was needed to achieve those deformations. It was also observed that unusually high force was needed to achieve even 10% deformation in Explotab. It might be due to high degree of cross-linking among starch glycolate molecules. Cross-linked polymers of starch are more elastic, compared to their cellulose counterparts. At 20%, attempted deformation starch polymers required much more compressive force compared to cellulose polymers.
( 95 The percentages of plastic deformation and the percentages of recovery at various attempted deformations for different disintegrants were tabulated on page 93. As it could be seen from the table, at 10% attempted deformation Ac-Di-Sol had more plastic deformation than Sta-Rx 1500 and CLD II, but at 20% attempted deformation order was reversed. It was clear from the data that the initial deformation might be more in Ac-Di-Sol compared to Sta-Rx 1500 and CLD II, but the additional deformation --the deformation after some initial range --was less compared to Sta-Rx 1500 and CLD II, which indicated that Ac-Di-Sol particles deformed initially easily but later they resisted more deformations.
The recovery was least at 10% in Ac-Di-Sol, but at 20% the recovery was more than those of Emcosoy, CLD II , and Sta-Rx 1500.
The disintegrants were arranged in decending order according to the plastic deformation (at 10% atteIT>ted deformation) on page 97 .
The deformed particles are energy rich, and that energy is released when the grains are exposed to water. The energy-rich particles swell more rapidly in water, unlike undeformed grains, which require more heat in order to swell. These data provide an answer that why Ac-Di-Sol containing formulations had better disintegration and dissolution Tables XXVIII through XXXII show physical properties of the tablet formulae I, II, and III. High weight variation, thickness and high hardness variation were observed in multivitamin formulation (Formula I). Capping was observed in a formulation having Emcompress as a matrix. Emdex matrix formulation ~ave friability less than .90%. Disintegration time was less than 20 minutes in most of the formulation Fcrn.i.larior:: Fyridc-15ne' P.c 1 37.
Dissolution profile of pyridoxine formulation containing 0. 5 Dissolution profile for Pyridoxine formulation containing 3% of Corn starch.
Emdex as a matrix.
Dis5clution Frofi1e~. Emcosoy was tested for three major mechanisms of disintegration: (i) swelling, (ii) porosity and capillary action (wicking), and (iii) deformation. Swelling and wicking were adequate in the comparison of other satisfactory disintegrants, but Emcosoy had a poor plastic deformation. The viscosity of the gel produced by Emcosoy on wetting is less than the gel produced by CLD II, but higher than that by Ac-Di-Sol. All these factors were considered separately; but in actual disintegration of a tablet, inter-relationships always take place.
Proper storage precautions are advised for this disintegrant because it has a tendency to promote microbial growth in the presence of high moisture at room temperature as observed during the wetting and drying test.
In this project, a total of 45 tablet formulations were studied, and about 100 dissolution profiles were obtained on various vitamin formulations using either Emcompress or Emdex as the tablet matrix.
The results obtained clearly indicate that: 1. Emcosoy is superior to corn starch as a tablet disintegrant at two or three percent levels; 2. Emcosoy competes favorably with other disintegrants, such as Explotab and Ac-Di-Sol, at half, one, and two percent levels The author would have liked to change the particle size of Emcosoy to determine its effect on disintegration and other properties of the tablet.
Emcosoy has one great advantage over other popular disintegrants in that it is of natural origin. If we want to retain this advantage, there is very little we can do to change its property. It would be of great advantage to reduce the proteinaceous fraction of Emcosoy composition (at present, about 17.5%) by some changes in processing procedure. The author anticipates that such changes would improve the plastic deformation of the Emcosoy particles; and consequently, the disintegration property. Low protein fraction would also help in protecting disintegrant at higher moisture level from lumping, as well as from microbial degradation. . , 15 20 Concentration /lg/ml. | v3-fos |
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} | s2 | The Origin of the Intermediates Detected in the Folding of Swine Pepsinogen*
Unfolding of swine pepsinogen to a cross-linked ran- dom coil, as measured by spectrophotometric titration of tyrosine residues after pH jumps to above pH 11, is shown to be followed by slow reversible reactions. Absorbance studies show further small changes in the ionization of tyrosine residues. Double pH jump exper- iments indicate that the unfolded protein changes from an initial form that can refold rapidly to a mixture of forms which refold slowly. Both of these reactions in the unfolded protein are distinct from irreversible denaturation and show thermodynamic features which resemble cis-trans isomerization of proline residues. The inactive folded form of the zymogen, which occurs above pH 8 and was previously identified as an inter- mediate in unfolding by urea (McPhie, P. (1980) J. Biol. Chem. 255, 4048-4052) unfolds much more rapidly above pH 11 than molecules originating at neutral pH. The initial product of this rapid unfolding is found to refold slowly. These slowly refolding forms show complex behavior on returning to neutral pH. Absorbance changes dis-close the rapid formation of partially folded forms of the protein, which must undergo further slow reactions to reach the native state. These results are interpreted to show that native pepsinogen is in rapid equilibrium with the initial unfolded form. Slowly folding forms are produced by proline isomerization after unfolding. The partially folded forms detected during refolding do not lie on the most direct route of folding, but are trapped by proline isomers, which differ from those found in the native protein.
Unfolding of swine pepsinogen to a cross-linked random coil, as measured by spectrophotometric titration of tyrosine residues after pH jumps to above pH 11, is shown to be followed by slow reversible reactions. Absorbance studies show further small changes in the ionization of tyrosine residues. Double pH jump experiments indicate that the unfolded protein changes from an initial form that can refold rapidly to a mixture of forms which refold slowly. Both of these reactions in the unfolded protein are distinct from irreversible denaturation and show thermodynamic features which resemble cis-trans isomerization of proline residues. The inactive folded form of the zymogen, which occurs above pH 8 and was previously identified as an intermediate in unfolding by urea (McPhie, P. (1980) J. Biol.
Chem. 255, 4048-4052) unfolds much more rapidly above pH 11 than molecules originating at neutral pH. The initial product of this rapid unfolding is found to refold slowly.
These slowly refolding forms show complex behavior on returning to neutral pH. Absorbance changes disclose the rapid formation of partially folded forms of the protein, which must undergo further slow reactions to reach the native state. These results are interpreted to show that native pepsinogen is in rapid equilibrium with the initial unfolded form. Slowly folding forms are produced by proline isomerization after unfolding. The partially folded forms detected during refolding do not lie on the most direct route of folding, but are trapped by proline isomers, which differ from those found in the native protein.
Above pH 8, swine pepsinogen is changed to a form which cannot activate itself to pepsin in acid solution and is precipitated by high concentrations of salt (1). However, the molecule is not denatured since the large changes in optical rotation or viscosity characteristic of protein unfolding reactions are only seen above pH 9 (2, 3). Thus, the two overlapping structural transitions in the molecule at alkaline pH involving three forms of the protein are inactivation, with minor alterations in physical properties which changes the native zymogen (N) to a folded inactive form (I), and unfolding to a crosslinked random coil form (U). Kinetic studies showed that the inactivation reaction is slow and that its product unfolds much more rapidly above pH 10 or in high concentrations of urea than molecules originating at neutrality. Furthermore, the rate of the slowest step in the refolding of pepsinogen from * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. the random coil form was shown to be equal to the rate of reversal of the alkaline inactivation (4,5). This suggested that the alkali-inactivated form is a stable intermediate in the unfolding and refolding of pepsinogen between the native and the unfolded random coil forms of the protein.
Determination of the enthalpies of activation of the rates of unfolding and refolding, in the presence of urea, indicated that the slow step involves the isomerization of proline residues in the protein (5). This was a puzzling conclusion, since this reaction is postulated to occur after unfolding, rather than as the initial step in the reaction (6, 7). Consequently further studies were undertaken on the nature and origin of this inactive form of pepsinogen (I) in the hope of producing a more detailed model of the folding mechanism. Its behavior was compared with that of the native protein (N) after pH jumps to unfolding and refolding conditions, using two probes, absorbance and potential pepsin activity.
Materials
Swine pepsinogen (Lot PG 305 608) and bovine hemoglobin (Lot HB 39H 795) were from Worthington. All other reagents were analytical grade or equivalent. Stock solutions of pepsinogen were made up in distilled water at concentrations of 2 to 4 mg/ml and pH values between 6 and 6.5. To study the properties of the inactive form of pepsinogen (I), solutions were adjusted to pH 10 by the addition of small volumes of 0.1 N sodium hydroxide with stirring and equilibrated for 15 min (5). Previous results on the pH dependence of the alkaline transitions indicated that 9 0 % of the protein would be present as the inactive form (I) under these conditions of temperature and ionic strength (4, 5).
Methods
Kinetics of Unfolding-Initial experiments on unfolding in the pH range of 10 to 12 were made in an Aminco-Morrow stopped flow apparatus, as described elsewhere (4). Equal volumes of pepsinogen solutions and 0.1 M sodium bicarbonate buffers were mixed, and unfolding was followed by the large transmission change at 295 nm caused by the ionization of tyrosine residues in the protein. Later experiments were performed in a Cary 15 spectrophotometer using the 0-0.1 absorbance scale. Equal volumes of pepsinogen solutions and bicarbonate buffer were mixed in the cuvette (mixing time of 10 s), or small volumes of 1 N sodium hydroxide solution were added to pepsinogen solutions using an Add-A-Mixer from Precision Cells Inc. (mixing time of 3 s). Absorbance changes were followed at 295 nm.
Kinetics of refolding-Aliquots of 1 N sodium hydroxide (5 p1) were added to 500 pl of pepsinogen solution in a cuvette and blended on a Vortex mixer. After a carefully measured interval, 500 pl of 0.1 M sodium phosphate buffer, pH 7, were added to the cuvette, and the time course of the resulting absorbance increase at 287 nm was measured. The mixing time of these experiments was 10 s. The procedure was repeated, varying the delay time between the additions of sodium hydroxide and of phosphate buffer.
Reappearance of Potential Pepsin Activity during Refolding-The experimental procedure was identical with that described above. At intervals after the addition of phosphate buffer, 50-pl samples were removed from the cuvette and assayed for potential proteolytic activity in a 25-mg/ml solution of hemoglobin, pH 1.8, as described previously (5). All other techniques were described before (4, 5). Unless stated otherwise, all experiments were performed at 25 "C.
RESULTS
Absorbance Changes on Unfolding-The results obtained using the stopped flow apparatus were in good agreement with those published previously (4). Mixing a neutral solution of pepsinogen with sodium bicarbonate buffers was found to produce large increases in absorbance at 295 n m , resulting from ionization of tyrosine residues in the protein. The total change in absorbance increased sigmoidally with the final pH in the range of 10 to 12. At all pH values, half of the change was too fast to measure, and the other half followed an exponential time course whose rate constant was found to increase with the final pH in this range and was a marked function of temperature. At pH 11.5, the rate constant was found to increase with temperature in the range of 15-50 "C. The enthalpy of activation was 30 f 1 kcal mol".
Experiments performed in the spectrophotometer showed a previously undetected phenomenon. Above pH 11, most of the absorbance changes accompanying unfolding occurred within the mixing time (10 s). However, approximately 2% of the increases were found to occur very slowly ( Fig. 1) and to follow an exponential time course, k = 0.004 s-'. The observation was reproducible and easily measured on the expanded scale of the Cary 15 instrument. The rate constant of this small change was independent of pH in the range of 11 to 12 and independent of ionic strength. The relative size of the slow change was found to increase if the pH jumps were made by the addition of small aliquots of 1 N sodium hydroxide, rather than of bicarbonate buffer. At pH 11.5, the rate constant of the slow change increased with temperature. The enthalpy of activation for this reaction was found to be 24 & 1 kcal mol" over the range of 15-35 "C. The wavelength dispersion of the slow change showed that it arose from the partial ionization of one or more tyrosine residues.
When solutions of pepsinogen which had been equilibrated at pH 10 for 15 min to produce the inactive form (I) were subjected to pH jumps in the stopped flow apparatus, absorbance changes seemed to be completed within the mixing timr (approximately 5 ms). Experiments in the spectrophotometer showed that the final 2% of this absorbance change also occurred slowly. The characteristics of this change were identical with those determined in pH jumps starting from the native protein (N).
Variation of Potential Pepsin Activity-Other investigators have often seen slow changes in the absorbance spectra of proteins at high pH and have ascribed them to irreversible denaturation (e.g. Ref. 8). Consequently, it seemed important to determine the effect of extended exposure to unfolding conditions on the potential proteolytic activity of the protein.
Samples of a neutral solution of pepsinogen (500 p1) were jumped to pH 11.5 by the addition of 5 pl of 1 N sodium hydroxide. After a carefully timed interval, an equal volume of 0.1 M sodium phosphate buffer, pH 7, was added to give a fiial pH of 7.3. The intermediate exposure to neutral pH allowed reversibly unfolded protein to regain the native conformation. Aliquots of the neutralized solution were assayed at various times after the pH drop for their potential pepsin activity as a measure of the recovery of native protein (N). Irreversible denaturation could not be detected in this way after 5 min at pH 11.5 although slight losses of potential activity occurred after longer periods. However, incubation at pH 11.5 for 5 min had marked effects on the kinetics of the reappearance of potential pepsin activity (Fig. 2). Thus, pepsinogen exposed for 10 s at pH 11.5 was found to be 83% in the native form (N) only 10 s after returning to neutrality. Exposure for 5 min at pH 11.5 produced protein which showed only 14% of its potential activity 10 s after the pH drop. In all cases the remainder of the activity was regained slowly.
To study the behavior of the inactive form of pepsinogen (I), solutions of the protein were equilibrated at pH 10 for 15 min and then jumped to pH 11.5 by the addition of 4 pl of 1 N sodium hydroxide to 500 p1 of solution. In this case, extended exposure to pH 11.5 had a less marked effect on the kinetics of reappearance of activity (Fig. 3). Exposure of the I form to pH 11.5 for 10 s produced 11% of the potential activity within 10 s of the pH drop to pH 7.3, whereas a 5-min incubation at pH 11.5 gave 9% potential activity 10 s after the pH drop. The remainder of the activity reappeared slowly, at a rate which decreased with increasing delay time at pH 11.5. Again, unfolding was found to be fully reversible for incubations up to 5 min at pH 11.5.
Absorbance Changes during Refolding-Previous studies on the kinetics of absorbance changes during refolding were all performed after the protein was maintained at high pH for one convenient time period, i.e. 30 s. Absorbance changes on unfolding were thought to be complete,iand irreversible denaturation was not detected (4). The present observations indicate the importance of studying the effect of varying the delay time at pH 11.5. Marked changes were found both in the size of the absorbance changes resolved in these experiments and in their time courses (Fig. 4). The size of the resolved change in molar absorptivity, estimated for each delay time and obtained by extrapolation of these curves to zero time of refolding was found to increase from Ae0 = +450 after 10 s at pH 11.5 to = + B O O after 5 min. Refolding was complete in all cases, as judged by recovery of potential pepsin activity. At early times the absorbance change followed an exponential time course, with rate constant k I = 0.02 s-', but with increasing delay time after 30 s a slower reaction with rate constant kp = 0.004 sK1 became increasingly evident. The total resolved change in molar absorptivity was found to vary in parallel with the rapidly recovered fraction of the potential Double jump experiments, measuring the reappearance of potential pepsin activity at pH 7.3, after jumping to pH 11.5 from pH 10. Solutions of pepsinogen were incubated at pH 10 for 15 min, then jumped to pH 11.5, and held there for a variable delay time. The pH was dropped to 7.3, and aliquots of the solution were assayed for potential pepsin activity at pH 1.8, as a function of time after the pH drop. Delay times at pH 11.5 were: V, 10 s and 0, 5 min. Solutions of pepsinogen were incubated at pH 10 for 15 min and then jumped to p H 11.5. They were held at this pH for a variable delay time and then mixed with an equal volume of 0.1 M phosphate buffer, pH 7.0. Refolding was measured as a function of time after the pH drop by absorbance changes at 287 nm. The delay times at pH 11.5 were: 0, 30 s and 0,5 min. Inset, the variation of the total resolvable change in molar absorptivity, A E~ (0) with the delay time at high pH.
pepsin activity, estimated from Fig. 2, and to follow apparent first order kinetics with the delay time, with a rate constant, k,, of 0.025 s" (Fig. 5). Similar effects were seen at 30 and 35 "C, except that the rate constants were found to be functions of temperature. Over this limited range of temperature the enthalpies of activation for the three reactions were found to be AH! = 2 & 1 kcal mol", AH: = 12 & 1 kcal mol", and AH:,, = 26 & 2 kcal mol".
Similar experiments were performed to study the behavior of unfolded pepsinogen molecules (U) which originated from the inactive form (I). Protein solutions were incubated at pH 10 for 15 min and were then jumped to pH 11.5. After a carefully measured interval under these conditions, the solution was neutralized and the absorbance changes accompanying refolding were measured (Fig. 6). The time course of the reaction was biphasic, again with rate constants kl = 0.02 s-l and k2 = 0.004 s-'. The estimated size of the total resolved change in molecular absorptivity was a weak function of the delay time at pH 11.5, increasing from AEO = +1450 after 10 s to +2000 after 5 min. Most of the change resulted from an increase in the contribution of the slower phase to the total change.
DISCUSSION
These studies show that the alkaline unfolding of swine pepsinogen is much more complex than was previously reported (3,4). They indicate that the rapid unfolding previously detected above pH 10 by the ionization of tyrosine residues in the protein is followed by several other reactions. A further structural transition is indicated by a small slow change in absorbance whose rate is independent of pH or ionic strength but increases markedly with temperature. Refolding studies demonstrate that this is not due to irreversible denaturation of the protein, since no loss in the zymogen's potential proteolytic activity could be detected after 5 min at pH 11.5,25 "C, when this shw absorbance change was almost 70% complete (Fig. 1). However, the mechanism of refolding is found to change over the same time period. After 10 s at pH 11.5 over 80% of the zymogen is in a form which can be activated to pepsin immediately after returning to neutrality. Five minutes later, only 14% of the molecules show this property; in each instance, the remainder of the protein became activatable through a slow reaction (Fig. 2). At this pH and temperature, unfolding from the native form (N) is complete in 3 s (4). Similar changes in refolding properties could be measured by absorbance changes (Fig. 4), which measure burial of aromatic side chains in the protein by folding. After short delay times at pH 11.5, only a small slow change in absorbance could be detected although this increased in size with the delay time. The time course of the absorbance changes on refolding also became more complex with increasing delay times at pH 11.5.
Similar observations with other proteins have been interpreted as indicating a transition in the unfolded molecule from a fast refolding form to one or more slowly refolding forms (9). There is very strong evidence that this transition results from cis-trans isomerizations of proline residues in the protein, away from the configurations found in the folded form. With increasing time under unfolding conditions, the rate of refolding becomes limited by reversal of these slow reactions at "essential" prolines, which must have the correct configuration for attainment of the native structure. Fig. 5 shows that this slow transition in unfolded pepsinogen can be approximated by a f i t order curve whose rate constant, 0.025 SKI, and enthalpy of activation, 26 kcal mol", fall in the range expected for this kind of reaction. Since pepsinogen contains 18 prolines (lo), a number of such transitions might be anticipated. It seems likely that the double jump procedure detects primarily the fastest isomerization reaction(s) in the unfolded protein, The increased complexity of refolding kinetics after longer periods at pH 11.5 suggests the presence of slower reactions. The slow absorbance change shown in Fig. 1 is also similar to that expected for proline isomerization; its rate is independent of pH but the reaction is highly temperature dependent, AHt = 24 kcal mol". At 25 "C, pH 11.5, it occurs six times more slowly than the reaction shown in Fig. 5. Garel (1 1,12) has shown that proline isomerization reactions can be detected both in a hexapeptide and an unfolded protein by absorbance changes at adjacent nitrotyrosine residues. The sequence of pepsinogen contains two Tyr-Pro bonds (10). It is conceivable that the slow absorbance change is associated with the isomerization of one or other of these bonds.
After 5 min at pH 11.5, 95% of the zymogen is present in slowly folding forms. Yet the kinetically resolved change in molar absorptivity under refolding conditions (+MOO) is much smaller than that expected from equilibrium unfolding measurements (+9OOO) (5). It has been argued elsewhere that this indicates the rapid formation of one or more partially folded intermediates during folding (4, 5). The data in Fig. 5 show that these intermediates must arise from the slowly folding forms of the protein.
(As yet, folding after very short times at pH 11.5 is too fast for study of mechanism.) To accommodate these observations, one can write a general model for unfolding-refolding reactions.
=I,
where N is the native protein, Uo the initial unfolded form, and U1, UZ, etc. are all slowly folding unfolded forms produced presumably by subsequent isomerization of one, two, or more essential proline residues. 11, Iz, etc. are partially folded intermediates produced from U1, Uz, etc. These intermediates must undergo proline isomerizations before they can fold fully to N. How can the alkaline-inactivated (I) form of pepsinogen be accommodated into Scheme 2? In agreement with previous results (3-5), the I form unfolds much more quickly than the native protein. However, the present data show that the initial product of the reaction is changed. Immediately after unfolding, all of the protein is in a slowly refoldable form as judged by two criteria (Figs. 3 and 6). Extended exposure to pH 11.5 makes the refolding reaction both slower and more complex. The simplest way to explain these facts is to identify the inactive form (I) with a subpopulation of 11, in which one proline is in the wrong configuration. Proline isomerization will destabilize the protein structure and, unlike a conformational change, its effects will persist after unfolding. Rapid unfolding would produce U1, which refolds to N slowly and can further isomerize to UZ, UB, etc.; the latter set of forms would refold even more slowly.
Evidence for structural changes from such an unfolded form is seen in the continued presence of the small and slow absorbance change after rapid unfolding. The small increases in size of the resolved refolding absorbance change with longer delay times at pH 11.5 in the double jump experiments can be taken to indicate ensuing changes in the structure of the intermediates produced by pH drops (Fig. 6). The data also show that the refolding mechanisms of the unfolded forms produced from native (N) and inactive (I) forms of pepsinogen become more alike with increasing exposure to pH 11.5. This result would be anticipated from Scheme 2, as the unfolded forms undergo further isomerization to the same equilibrium distribution. Scheme 2 implies that N and I1 are reversibly connected. The refolding kinetics demonstrate the formation of N from I1 showing that proline isomerization can occur during folding (the reason for the low A H t values of these reactions has been discussed by Nall et al. (13)), but it is generally thought that this reaction is initiated after unfolding. Since the inactivation and unfolding equilibria overlap at high pH, the possibility must be entertained that N is converted to I], through transiently formed Uo and U1. The possibility of such "unfolding" transitions between very similar forms of a protein was predicted some years ago by Lumry and Biltonen (14). However, Reeke et al. (15) have suggested that the conformational change accompanying the release of metal ions from concanavalin A includes the cis-trans isomerization of a peptide bond. Thus, such reactions may occur in folded proteins.
Earlier studies on unfolding of pepsinogen by urea did not show two phenomena predicted by Scheme 2, which were detected in these studies. All of the absorbance changes on unfolding followed a simple exponential time course. There was no sign of fast and slow phases (3). Double jump experiments failed to reveal a hidden transition after unfolding from fast to slow folding forms. Conversely, refolding experiments showed complex kinetics indicating the presence of intermediates. The temperature dependence of the rates of unfolding and refolding indicated that conversion between the inter-mediates and the native protein involved proline isomerization (5). Similar results have been obtained with other proteins such as penicillinase (16) and the a-subunit of tryptophan synthetase (17, 18) and have also been interpreted to show stable obligatory intermediates in unfolding and refolding. The connection between these studies and the present ones can be made through the theoretical studies of Creighton (19). The unfolding of a protein containing n trans prolines in its native state can be idealized as: Here, k, and k, are the rate constants of unfolding and refolding, and kc and kt are those for cis-trans isomerization, assuming all prolines to be identical. The statistical factors indicate the possibilities for isomerization between the different species, which are as in Scheme 2. Because of the large number of unfolded forms, mass action will weight the equilibrium toward unfolding by a factor of (1 + k,/kt)". Studies on short peptides have shown that equilibrium constants for cis-trans isomerization of proline residues fall in the range kc/ kt = 0.2 to 0.6 (6). Consequently, for a protein like pepsinogen, with 18 prolines (lo), unfolding could be favored by a factor of (1.2)18 to (1.6)'*, i.e. 27 to 4722. Thus, proline isomerization can drive unfolding to completion even when k,/k, is very small. The reaction will occur without the large transient population of UO that would be necessary to see biphasic kinetics of unfolding and for successful double jump experiments (6). Furthermore, the product of unfolding w i l l be a mixture of U1...U,, all with incorrect proline residues. The intermediates detected during refolding will be trapped by these prolines and will not lie on the most direct route of folding. For large proline-rich proteins information on this direct route may only be obtained through double jump experiments which first unfold the proteins under the most extreme conditions, such that k, >> k,, thereby avoiding this trap. | v3-fos |
2014-10-01T00:00:00.000Z | {
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} | s2 | Mycoplasmas and bovine respiratory disease: studies related to pathogenicity and the immune response--a selective review.
Three species of mycoplasma have been established as being of importance as causes of pneumonia in housed calves, based on pathogenicity studies and frequency of association with the disease. These three species are Mycoplasma bovis, M. dispar, and Ureaplasma diversum. M. bovis is the most pathogenic of these species but the disease outbreaks with which it is associated are sporadic. M. dispar is regularly isolated from pneumonic calves but is also found causing mild superficial and asymptomatic infections of the respiratory mucosa. The bovine ureaplasmas are serologically complex. They are distinct from ureaplasmas isolated from other non-ruminants by PAGE analysis, G + C content of DNA, and serology. A second species within the genus ureaplasma has been proposed to accommodate the bovine ureaplasmas, U. diversum. Control of mycoplasma respiratory infections of cattle based on immunization might be possible. Calves have been immunized against M. bovis and immunity has been related to antibody in the lung. M. dispar appears less immunogenic in calves than M. bovis and this may contribute to its pathogenicity.
THE COMPLEX NATURE OF BOVINE RESPIRATORY DISEASE
A variety of viruses, typical bacteria, and mycoplasmas are associated with pneumonia of housed calves, and these agents probably interact to produce more severe lesions than would be produced by any single entity. Several of the common respiratory viruses of calves (e.g., parainfluenza type 3, infectious bovine rhinotracheitis, and respiratory syncytial virus) usually cause an acute transient infection, although a carrier state sometimes follows. Decreased resistance of the lung to bacterial infection is often a consequence of viral infection; the best studied example of this is Sendai virus in mice [1]. A similar effect is apparent in calves [2,3]. Viral infections can also predispose to subsequent mycoplasma infections. However, several mycoplasmas are primary pathogens and their possibly chronic existence may be exacerbated by a viral infection. Thus the timing of viral-mycoplasmal interactions is likely to be less critical than that of viral-bacterial interactions in the production of respiratory disease [4].
Brennen et al. [5] reported increased severity of disease following the inoculation of bacteria and mycoplasmas together into the respiratory tract, compared to the disease observed when either was inoculated alone. This could simply be an additive effect of introducing two agents at the same time. Alternatively, Schewen and Wilkie [6] reported Pasteurella haemolytica had a toxic effect for bovine alveolar macrophages and this type of effect could aid mycoplasma multiplication. Mycoplasmas do not appear to predispose to secondary bacterial infection in the same way as described for viruses such as Sendai and parainfluenza type 3 [4,7]. The authors presume this is because they do not affect the same host cells.
THE RELATIVE IMPORTANCE OF THE MYCOPLASMA SPECIES ISOLATED FROM THE BOVINE RESPIRATORY TRACT A large number of species of mycoplasma have been isolated from the respiratory tract of calves. These isolations have been from both non-diseased and diseased tissues [8]. Not all of these species are proven pathogens for cattle. Furthermore, only a few of the proven pathogens are frequently isolated from housed calves with pneumonia. The three most important species, based on studies of occurrence and pathogenicity, are Mycoplasma bovis, M. dispar, and Ureaplasma sp. The occurrence and pathogenicity of each of these three species is different and each needs to be studied individually.
ISOLATION AND PATHOGENICITY OF M. DISPAR
Isolation of this glucose-fermenting species from calves is not possible on conventional mycoplasma media and a modified medium similar to that developed for M. hyopneumoniae was originally used to isolate M. dispar. Failure to grow on conventional media has probably led to a considerable underestimate of the prevalence of M. dispar in the calf population [8,9,10]. The membrane of M. dispar is surrounded by a layer of material that stains with ruthenium red. This layer is more extensive than that seen in several bovine mycoplasma species, apart from M. mycoides subsp. mycoides [11]. It has been proposed that this "capsular" material plays a role in the virulence of the organism. Attachment to bovine erythrocytes and to the epithelium of bovine tracheal organ cultures is via a trypsin-sensitive attachment site. Thomas and Howard [12] reported ciliastasis following the inoculation of M. dispar into bovine tracheal organ cultures, and this fact is likely to play an important role in the pathogenicity of this species.
Gourlay et al. [13] reported isolations from the lungs of 30 percent of calves that died with severe pneumonia or were killed in extremis and 60 percent of calves with subclinical pneumonia. Subsequent to these findings in England, isolations have been made in other parts of the world [8,9]. M. dispar was isolated from nasopharyngeal swabs taken from 93 percent of apparently healthy Ayrshire calves obtained from farms in southern England [14]. Later studies on animals from the same source have indicated that many of them have subclinical pneumonia, usually involving 2-10 percent of the lung surface, and M. dispar was isolated from lungs of 97 percent of 33 animals examined. Higher levels of colonization were apparent in pneumonic, compared to non-pneumonic, animals. Thus of 32 lungs examined, 14 had lesions involving c 2 percent of the surface and the number of M. dispar isolated from these was c 104 CCU in 12/14 cases. Of the remaining 18 lungs, which had lesions involving > 3 percent of the surface, > 101 ccu M. dispar were isolated from 17/18. The principal histopathological lesion in these animals was an alveolitis with pronounced lymphoid accumulations rarely being present. M. dispar has also been isolated from animals free from macroscopic pneumonia, more frequently from the bronchus than from the lung, with peak colonization at three months [15].
Pneumonia was apparent in gnotobiotic calves following the introduction of M.
dispar into the respiratory tract and the lesions extended over 0 to 17 percent of the lung surface. The predominant histopathological lesion in these young gnotobiotic calves involved a cellular infiltration, and thickening, of the alveolar walls [16,17]. M. dispar has also been shown to cause mastitis in cattle following its introduction via the teat canal into the mammary gland. Infection seems to be limited to the inoculated quarter and can continue for several months. Associated with the infection is a persistent and markedly raised cell count in the milk. Both virulent and avirulent strains, as judged by ability to induce mastitis, are present in the respiratory tract of cattle [18]. In conclusion, this pathogenic species is clearly a very common inhabitant of the bovine respiratory tract, being typically found in the bronchi and lungs of calves.
SEROLOGICAL RESPONSE TO INFECTION WITH M. DISPAR Attempts have been made to measure antibody to M. dispar in the sera and lung washings from several groups of naturally infected cattle in order to determine whether infection or disease associated with M. dispar could be diagnosed by serological tests.
The first group of sera examined were from five groups of eight calves selected from each of five groups of approximately one hundred animals reared on a beef unit in southern England [19]. Sera taken from these animals at about monthly intervals over a period of up to 200 days were examined for IgG antibody to M. dispar by enzyme-linked immunosorbent assay (ELISA) [20]. A small increase in the mean antibody titer of two of the five groups was observed, but the mean titer was always < 103 units of antibody. It has been proposed that this represents the serological response in colonized animals in the absence of severe disease involving M. dispar.
A second group of sera examined consisted of paired samples, the first taken at the onset of outbreaks of respiratory disease and the second about thirty days later [21]. Fourfold, or greater, rises in IgG antibody to M. dispar were noted in association with disease in several of these animals. Furthermore, sera from several animals contained > 103 units of antibody. Rises in antibody titers were also detected by single radial hemolysis (SRH) [20,21]. Thus, antibody rises to M. dispar can be demonstrated in association with some outbreaks of respiratory disease. A third group of sera examined were from Ayrshire calves. Most of these animals had pneumonic lesions and were colonized with M. dispar, as noted above. Serum antibody was not detected. However, antibody to M. dispar was found in lung washings by ELISA. Of nine lung washings examined, two had IgGl antibody to M. dispar, one had IgG2 antibody, and seven had IgA antibody demonstrable. Six gnotobiotic calves inoculated previously with respiratory syncytial virus were used as controls. None had antibody to M. dispar detectable in lung washings. In this case M. dispar appears to have generated only a local immune response and infection was presumably limited to the surface of the lung.
Other evidence for M. dispar producing infections that do not progress beyond the mucosal barrier come from an examination of experimental mastitis. When sera from infected animals is examined by SRH, which detects bovine IgM and IgGl, serum antibody can be demonstrated in some cows. Others appear not to have produced a detectable response, even though they had developed severe mastitis.
In conclusion the progression of M. dispar infection in cattle might be as follows. Calves become colonized in the nasopharynx and then in the lung. Possibly a subclinical pneumonia with a local IgA response is produced, but no serum antibody response. In association with other agents, M. dispar may be involved in more severe clinical pneumonia. In this case a serum antibody response is produced. Conversely, it can be argued that the presence of serum antibody to M. dispar indicates more than colonization of the respiratory tract, probably lung lesions and possibly spread beyond the mucosal surface to which it is usually limited.
M. BOVIS, ISOLATION, PATHOGENICITY, AND SEROLOGICAL RESPONSE
This mycoplasma has been isolated from pneumonic calves as well as other diseased cattle, notably those with mastitis, arthritis, and urogenital infections [9]. Although not as ubiquitous as M. dispar, isolation rates within a given herd, in association with pneumonia, can be very high. A serum antibody response detectable by a variety of techniques, including indirect hemagglutination, single radial hemolysis, complement fixation, and ELISA, almost invariably follows infection. This is in contrast to M. dispar and probably reflects the more invasive nature of M. bovis.
Pneumonia develops following inoculation of the respiratory tract of gnotobiotic calves. Severe clinical mastitis, sometimes with spread to other quarters, follows intramammary inoculation. Arthritis can be produced by injecting calves intravenously. Thus, this species is more pathogenic than M. dispar but less prevalent. ISOLATION AND PATHOGENICITY OF BOVINE UREAPLASMAS Ureaplasmas were first isolated from cattle in 1967. Originally, isolations were from the urogenital tract [22]. Subsequently, ureaplasmas were isolated from pneumonic calf lungs [13]. The diseases from which ureaplasmas have been isolated include: vulvitis, infertility, pneumonia, and keratoconjunctivitis. Ureaplasmas are also commonly isolated from apparently healthy urogenital tracts [9,23,24].
These mycoplasmas have been shown to produce mastitis in cattle, pneumonia in gnotobiotic calves, vulvitis, and ocular infections [16,25,26,27]. Both virulent and avirulent strains exist. Since ureaplasmas have been shown to be pathogenic, a possible role in any disease from which they are isolated should be considered.
ANTIGENIC STRUCTURE OF BOVINE UREAPLASMAS The genus Ureaplasma was proposed in 1974 by Shepard et al. [28], some 20 years after the original description of the organisms. A single species, U. urealyticum, accommodates the human isolates. Originally eight serotypes were proposed, but this serotyping scheme has subsequently been expanded [29].
The bovine ureaplasmas are serologically complex. Initial studies using antisera prepared in rabbits indicated there were three distinct groups, two of which were clusters of serologically similar, but not identical, strains [30]. Similar conclusions were reached by Ogata et al. [31]. Eight strains were selected from these studies as representing the range of antigens synthesized by the bovine isolates. Antisera against these eight strains were used to serotype fresh isolates from various countries. Most (72/77) isolates reacted with at least one of the sera, but three more strains had to be added to the original eight to complete the range of antigens expressed [32].
Subsequently one strain from each of the three antigenic clusters was selected and antiserum against it prepared in calves. All of the representative strains previously selected from our studies, as well as those of the Japanese workers [31], were found to react with at least one of the bovine antisera [33]. Thus, bovine antisera were less discriminating than rabbit antisera. It should be possible to identify all bovine ureaplasmas by using just three antisera; 96/96 strains examined were identified as being of cluster A (represented by strain A417), B (represented by strain D48), or C (represented by strain T44).
The serological relationship of antigens possessed by bovine ureaplasmas is represented in Fig. 1. The smaller clefts represent differences distinguished by rabbit antisera and the larger ones by bovine antisera and polyacrylamide gel electrophoresis (PAGE). This illustrates the relationship of the 11 selected strains and possibly not the entire spread of antigens present in nature. Thus, within the groups A, B, or C there may be a continuous spectrum of antigens with no "clefts," but with strains at distant parts of the spectrum being distinct. The clefts between clusters A, B, and C may be genuine, and strains may not exist to fill them. However, some strains have been isolated that react with antigens of clusters A and C and, if sufficient fresh isolates are examined, there may be a continuous spectrum of antigens evident for the whole of the bovine group.
For the future, it should be possible to identify bovine isolates using just three antisera and to use three antigen preparations for serodiagnosis. Finally, immunity between strains of the same serological cluster might occur. However, strains from different clusters (A and B) do not cross-protect [34].
RELATIONSHIP OF BOVINE UREAPLASMAS TO U. UREAL YTICUM Bovine ureaplasmas are distinct from U. urealyticum strains when compared by growth inhibition, metabolism inhibition, and immunofluorescence using antisera prepared in rabbits and cattle [31,32,33]. However, it appears that there is at least one cytoplasmic antigen common to both species [35,36].
The range of guanine plus cytosine (G + C) contents of DNA from U. urealyticum strains (27.0-27.8) and bovine isolates (29.0-30.2) was found not to overlap. This indicates the organisms comprise two distinct populations [37,38,39]. Analysis of the polypeptides by PAGE, in both one and two dimensions [40,41,42], indicated the bovine strains were distinct from the human. Furthermore there were clusters of strains by PAGE that were coincident with the serological clusters (see Based on these findings, it has been proposed that a second species should be recognized within the genus Ureaplasma with the bovine isolate A417 named the type strain of this species -U. diversum. Within this species there are three clusters of strains, Groups A, B, and C, represented by three strains A417, D48, and T44.
COMPARISON OF BOVINE UREAPLASMAS WITH THOSE FROM OTHER ANIMAL SPECIES
In Fig. 2 a diagram has been constructed representing the relationship between ureaplasmas from various animal species. The strains have been arranged according to G + C values with serological and PAGE studies used to further position groups of organisms.
Within the species U. diversum (cattle isolates) are the three clusters A, B, and C which are recognized by serology and PAGE. The sheep and goat isolates have similar but slightly higher G + C values. Two major clusters of these strains exist based on studies with rabbit or bovine antisera and PAGE of polypeptides [43,44].
Whether the two groups of sheep and goat strains are further groups of U. diversum, or another species, will be apparent from future studies.
U. urealyticum is distinct from the ruminant animal group. The several serotypes of this species comprise two clusters based on PAGE, and DNA homology [45]. Mouches et al. [42] showed that chimpanzee and Talapoin monkey strains were similar to U. urealyticum strains by two-dimensional PAGE. The dog and cat strains have similar G + C values to U. urealyticum, but the canine isolates are serologically distinct and comprise four clusters [46]. Kotani et al. [47] found that the feline strains contain two more serological clusters. The strains from a squirrel monkey and marmoset are distinct from U. urealyticum and U. diversum by PAGE and G + C and should not be regarded as being of either species. This diagram is certainly not complete; uncharacterized ureaplasmas from other animal species are not included and it reflects only the information available now. It would seem certain that a number of further species will be proposed in the future as more tests and comparisons are made.
IMMUNIZATION STUDIES WITH M. BOVIS AND M. DISPAR A series of experiments have been done to determine whether it was possible to immunize against respiratory infections with M. bovis and M. dispar by injecting killed organisms [48,49,50]. The first series of experiments was stimulated by the work of Pierce and Gowans [51] who found that systemic (intraperitoneal) priming, followed by local boosting, was an effective way of producing a local immune response.
Animals were injected with the killed M. bovis intramuscularly (im) twice, intratracheally (it) twice, im + it or subcutaneously (sc) three times. In summary, only animals injected im + it or sc x 3 showed increased resistance to M. bovis respiratory infection. Immunity was related to the antibody, IgG, in lung washings but not in the serum. This is consistent with the general consensus that IgG is a major component of bovine secretions [52] and of the lung rather than the upper respiratory tract in general [53].
With M. dispar im + it injections of antigen did not produce detectable protection from colonization. Calves injected 3 x sc with antigen and oil adjuvant had less mycoplasmas in their lungs (p < 0.05) than control calves. However, this difference was not as great as observed with M. bovis and a relatively low immunogenicity appeared to be a feature of M. dispar.
Further studies with M. dispar and M. bovis [54] have indicated that young calves respond poorly to injectons of M. dispar antigen compared to older animals. With M. bovis, the age effect is less marked. The poor immunogenicity of M. dispar in young calves might have affected the histopathological lesion observed following its inoculation into the respiratory tract of gnotobiotic calves. For M. dispar this was found to be primarily an alveolitis, but with M. bovis lymphoid accumulations were observed. Thus, failure of young calves to produce a proliferative response is related to absence of, or poor, immune response following infection compared to M. bovis.
No evidence for destruction of M. dispar antigens by formalin or increased response with increased dose was found. One possible explanation for the poor response of young calves is that the lymphocyte clones that recognize and react with the surface antigens of M. dispar develop late in the ontogeny of the calves' immune system. This has been described for certain other antigens [55]. Further immunological studies will provide an insight into the mechanisms of immunity to mycoplasmas in the calf respiratory tract that should enable effective vaccination procedures to be employed. | v3-fos |
2018-04-03T00:38:57.978Z | {
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} | s2 | Protein digestibility of weaning foods prepared from rice-minced meat and rice-mungbean combination in infants using a short term nitrogen balance method.
Three diets based on rice-dehulled mungbean, rice-minced meat and rice-mungbean with hull were tested with infants 11 to 20 months of age using a short term nitrogen balance technique. The results indicate that with isocaloric and isonitrogenous intake, all the subjects given either of the three diets were in positive nitrogen balance. The protein quality, in terms of nitrogen absorption and true digestibility, of rice-meat diet was superior to that of rice-bean diets. Among the rice-bean diets, it was noted that rice-mungbean with hull had a lower digestibility as compared to rice-dehulled mungbean diet. The poor digestibility of rice-mungbean with hull diet is the first limiting factor in its utilization by infants. Dehulling of mungbean before cooking is recommended for preparing weaning food for infant feeding.
children of low socio-economic group in both the rural and urban population .
It has been reported that 56% of the children in Pakistan suffer from various degrees of malnutrition, with over 7% and about 10% suffering from severe and moderately severe malnutrition respectively. Of every 10 children born , one dies before his first birthday and 25 in every 100 die before reaching their fifth year of life. Some 80% of the children under 5 years suffer from diarrhea and respiratory infection. Acute dehydration caused by diarrhea kills 30% of malnourished as against 2% of well nourished babies (8). Malnutrition is prevalent due to inadequate food intake, illiteracy, poverty, lack of clean drinking water , infectious diseases, improper weaning and feeding practices.
The best time to introduce solid food is 4-6 months, but in Pakistan 80% of the mothers breast feed their children up to 2 years, and introduce solid food at the age of 11/2-2 years. This late introduction of weaning food to the infants has been one of the major factors contributing to PEM and high incidence of infant mortality (7) .
Since the high cost of weaning food available in the market is also one reason for withholding solid food till the age of 2 years, it was considered appropriate to investigate the formulation of home prepared weaning food for infants from locally available plant and animal sources taking into consideration the normal diet pattern, habitual weaning food, price structure and acceptability of the products.
Cereal like wheat is the staple diet in Pakistan. Rice is commonly available. However, these cereals are deficient in lysine. As more than 80% of the children are dependent on cereal as a major source of protein and energy at the weaning age, it would give rise to nutritional deficiencies and adverse health (9). It has been recognized that legumes are comparatively rich in lysine and therefore a combination of cereal and legume can provide an ideal source of dietary protein , and their increased consumption is widely recommended for infants and children .
Formulation and nutritional quality of home prepared weaning food have been described previously (10). Many workers have also reported the nutritional quality of infant foods based on cereal and legume combination (11)(12)(13)(14)(15)(16) .
The digestibility of weaning food plays an important role in the overall acceptability and quality of the product.
People mostly in the rural communities withhold legumes from the diet of the weaned infants, presumably due to the poor digestibility of the bean , which may be another factor contributing to protein deficiency in this age group .
The poor digestibility of common beans and other food legumes in comparison with animal protein has also been recognized by various investigators (9 , 17), but not many works have been done with human subjects .
In the present paper, protein digestibility of 3 infant food mixtures prepared from rice-mungbean dehulled, rice-meat and rice-mungbean with hull , using a short term nitrogen balance method in human infants, has been reported and discussed . Subjects. Six male children 1-2 years of age were selected with the help of medical staff from Rajavithee orphanage. The subjects were initially treated for any bacterial and parasitic infection with proper medication, and were rehabilitated and fed milk-egg and other foods for at least 6-8weeks. When the subjects were normal weight for height, healthy and without any symptom of infection the study was undertaken. The objective and design of the study were approved by the committee on clinical investigation of the Faculty of Medicine, Ramathibodi Hospital Bangkok, before the beginning of the study. The initial characteristics of the subjects are given in Table 1.
Diets. Rice (Oryza sativa), mungbean (Vigna radiata), meat and vegetable oil were purchased from the local market. Three diets were prepared. The formulation and methods of preparation of the diets are given in appendix 1. The approximate Table 1. Initial characteristics of subjects studied for metabolic evaluation of weaning foods. compositions of the weaning foods are presented in Table 2 , while the nutrient compositions per 100 kcal are shown in Table 3 .
The experimental diets were based on rice-dehulled mungbean-oil for diet I , rice-minced meat-oil for diet II and rice-mungbean with hull and oil for diet III . The diet I and diet III were given to infants to compare the effect of dehulling on digestibility of rice-bean combination.
The ingredients in these diets were chosen considering availability cost and acceptability.
The protein, fat and energy contents per 100g of the cooked food mixtures are shown in Table 4.
Level of protein and energy intake. The protein intake was 1 .7g/kg/day, while the energy intake was 110kcal/kg/day (5). The levels of protein and energy intake were kept constant throughout the entire study .
Vitamin intake. The vitamin supplement was given daily during the metabolic period. The composition of multivitamin syrup is shown in Table 5. Each subject was orally given 0.6ml of this preparation daily . Methods. i) Chemical analysis. The protein and fat contents of the weaning food mixtures were determined by the methods described in A. O. A. C. (18), while the energy density was measured by a bomb calorimeter.
ii) Metabolic evaluation. Experimental design: The subjects were studied for 3 consecutive metabolic periods with 3day resting period in between. Each metabolic period lasted for 7days, with 4day adaptation and 3day balance periods. The experimental design for the metabolic evaluation is given in Fig. 1, during the balance period the subjects were placed in the metabolic bed as designed by Fomon (19) for complete urine and fecal collection.
Nitrogen balance studies. In the balance period daily urine samples were collected in bottles to which 5ml of toluene was previously added as preservative and analyzed for nitrogen. Feces were collected and pooled for 3 days. Carmine dye, 50mg was used as a stool marker and was given at the beginning and end of each balance period. Pooled feces were mixed and diluted with water to a fixed volume. An aliquot was analyzed for total nitrogen. Anthropometric measurements were done at the beginning and at the end of one week experimental period except for body weight which was measured daily in the morning before breakfast. The nitrogen balance, apparent digestibility or nitrogen absorption as percent of intake and true digestibility were calculated from the nitrogen balance data as follows (20). The values of obligatory N excretion were obtained from the data recom mended by FAO/WHO/UNU expert committee (21). They recommended obli gatory N losses in urine, feces and skin as 41, 21 and 10mg N/kg/day respectively.
Nitrogen balance
The results on the mean nitrogen balance data of the infants given the three weaning foods are presented in Table 6. It was noted that at a similar level of protein and energy intake of 1.7g/kg/day and 110kcal/kg/day, all the subjects given either of 3 diets were in positive nitrogen balance. Subjects given diet II prepared from rice-minced meat showed a significantly higher mean nitrogen balance of 102.3mg/kg/day than diets I and III prepared from rice dehulled mungbean and rice mungbean with hull, which were found to have a mean nitrogen balance of 68.6 and 74.6mg/kg/day respectively. No significant differences in the mean of nitrogen balance between diets I and II and diets I and III were observed, while the difference was statistically significant between diets II and III. The mean nitrogen retention as percentages of intake was lower, 25.9 and 29.0, for diets I and. III made from rice mungbean combination as compared to 38.7 observed for diet II, but there were no statistical differences in the mean nitrogen retention of the three diets. Graham et al. (17) have also reported lower nitrogen retention of beans as compared to methionine enriched beans fed to infants.
A significantly higher positive nitroge balance in infants given diet II and a positive nitrogen balance with diets I and III indicate that while diet II is superior in overall protein adequacy, diets I and II are also adequate for supporting body nitrogen balance.
Protein digestibility
The protein digestibility of the three experimental diets is shown in Table 7. The results indicate that the mean of apparent digestibility or nitrogen absorption as percentages of intake was 78.2 for diet II and 66.6 and 64.4 for diets I and III respectively. There were no significant differences in the mean nitrogen absorption of diets I and II and diets I and III, while significant difference was observed in diets II and III. Calloway and Kretsch (21) reported protein digestibility of 69% for subjects fed a diet based on cereal and bean combination. Their results are ODS 503 somewhat similar to the apparent digestibility values of 66.6% and 64.4% noted in the present work, though there were differences in the protein intake and subject age. True digestibility was calculated using an endogenous fecal nitrogen value of 21mg N/kg/day (22). As expected the values for all the preparation increased. Diet II, a rice-meat combination, showed a higher mean true digestibility of 86.1% as compared to the mean true digestibility of 74.6% and 72.7% observed for diets I and III, a rice-bean combination. A statistically significant difference in the true digestibility was found in diets II and III, but there were no significant differences in diets I and II and diets I and III. Mean fecal nitrogen was correspondingly low 57.5mg/kg/day in subjects fed diet II, while it was higher 88.7mg/kg/day and 90.1mg/kg/day in subjects given diets I and III respectively. These results agreed well with those of Naverrete and Bressani (14) who found higher fecal output of nitrogen in the bean fed subjects as compared to subjects fed ground meat. The same authors reported that the apparent and true digestibilities of beans were lower than those of meat, which are similar to the observation recorded in this study. The large fecal output of nitrogen in subjects given rice-bean diet and those reported elsewhere has been considered to be due to the poor digestibility of beans.
The present results showed that diet II prepared from rice-meat had a higher protein digestibility than diets I and III prepared from rice-bean combination. In the case of the two rice-bean diets, it was found that rice-dehulled mungbean, diet I has a higher nitrogen absorption and true digestibility than rice-mungbean with hull of diet III. It has been reported that the level of fat as a source of energy intake has no significant effect on the protein utilization provided that the body received adequate intakes of protein and energy (15). The dietary intakes in the present studies were isocaloric and isonitrogenous, therefore it seems that the improved digestibility with diet II may be due to a better amino acid pattern and the availability of amino acids for digestion and absorption with rice-meat protein, while in rice-bean diets I and III, the presence of hull which is mostly fibre may be a reason for the low digestibility of diet III. These observations are similar to those of Graham et al. (17) and Navarrete and Bressani (14).
Body weight changes
The results of the body weight changes in individual subjects given the 3 experimental diets are shown in Table 8. It was noted that all the subjects gained weight during the experimental period in which diets I and II were given. The body weight increased significantly with the first two diets as compared to diet III. The highest mean weight gain of 66g/day was noted for diet II followed by an increase of 50g/day with diet I, though there were no statistically significant differences in the mean weight gain of the subjects with diet I and diet II. For diet III, four subjects lost and two gained weight. The energy intake was isocaloric being 110 kcal/kg/day and isoproteinic 1.7g/kg/day, which is the recommended level of energy and protein requirements of this age group (22). In our study it was noted that even with the recommended level of energy and protein intakes, subjects lost weight during the experimental period in which diet III was given. The reason for the net loss in body weight may be due to the presence of hull, which is mostly crude fi bre in diet III. A high fibre diet may decrease the availability of energy from the diet, moreover a short term balance period of 7days may not be fully indicative of the dietary effect on body weight (23). A long term balance study is required to be made with infants comparing the three diets and possibly increased the number of subjects studied, because there were considerable variations in the subjects in their response to the diets. Nevertheless, it is clear from the results of this metabolic evaluation that protein quality and digestibility of rice-meat diet are superior to those of rice-bean diets. The rice-mungbean diets also show a fairly good protein quality but it is necessary to process the legume more extensively and it is recommended to remove the hull before cooking for preparing weaning food for infant feeding. Method. Clean and wash rice, add minced meat and rice in a pot with 2-3 cups of water and boil, add oil and cook till soft and tender, blend and serve.
Diet III.
Method. Soak mungbean in water for overnight, the hull was not removed from mungbean. Clean and wash rice. Put rice and mungbean with hull in a pot with 2-3 cups of water, boil, add oil and cook till soft, blend and serve rice mungbean with hull diet. | v3-fos |
2018-04-03T05:05:43.953Z | {
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} | 0 | [] | 1983-08-01T00:00:00.000Z | 24702310 | {
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} | s2 | Effect of partial hydrolyzates of casein and soybean protein on serum lipoproteins and fecal neutral steroids.
Effect of partial hydrolyzate of casein and soybean protein on serum lipoproteins and fecal neutral steroids in cholesterol-fed rats was studied. In rats fed partial hydrolyzate of casein, the levels of plasma and liver cholesterol, liver triglyceride and the ratio of serum beta/alpha lipoprotein had a tendency to decrease compared with those in rats fed intact protein. On the other hand, no difference was observed between soybean protein and partial hydrolyzate of soybean protein diet groups. The excretion of neutral steroids to feces and the amount of fecal coprostanol were increased in rats fed soybean protein and partial hydrolyzate of soybean protein.
According to the development of the studies on lipoprotein metabolism, the role of apoprotein has been noticed. The effect of dietary protein on plasma cholesterol level has also been reported in the recent years (1)(2)(3). We observed that in the cholesterol-fed rats, high casein and soybean protein diet decreased plasma and liver cholesterol concentrations and increased high density lipoproteins (4). The difference of amino acid composition was demonstrated (5-8) but the presence of another unknown factor is considerable.
Casein is composed of three subunits and has 75,000 to 375 ,000 molecular weight. On the other hand, soybean protein is composed of several subunits and has 200,000 to 600,000 molecular weight (9). This examination was intended to in vestigate whether the structure and molecular size of the protein is responsible for the difference in lipoprotein metabolism. and Y. NOZAKI METHODS Male Sprague-Dawley rats weighing initially 100g were controlled with commercial diet (type MF; Oriental Yeast Co., Ltd., Tokyo) for one week. At the start of the experiment, they were divided into four groups and were given experimental diets as shown in Table 1 for three weeks. Diets and water were provided ad libitum. enzyme and then lyophylized. The protein concentration of both products was 85%. The percent of hydrolysis measured by the method of ultraviolet absorption (10) was about 60% (Fig. 2). The molecular weight of each hydrolyzate was less than 65,000 on SDS-polyacrylamide gel electrophoresis (11) as shown in Fig. 3. Feces were collected for the last three days and dried to analyze the neutral steroids.
At the end of the breeding period, rats were fasted for 14-16h. Blood was obtained by cardiac puncture to prepare serum and EDTA-plasma. Liver lipids were extracted by the method of Folch et al, (12). Plasma and liver cholesterol concentration was assayed by the method of Zak-Henly (13) and triglyceride concentration was measured by a kit (TG-test Wako, Wako Pure Chemical Industries, Ltd., Osaka). Polyacrylamide gel electrophoresis of serum lipoprotein was done by the method of Maruyaa et al. (14). Neutral steroids were measured by the method of Miettinen et al. (15) using gas-liquid chromatography (model GC 6A, column model GC-9BM, Shimadzu Seisakusho, Kyoto).
RESULTS
The body weight gain, food intake, diet efficiency and liver weight are same at 2.0. Table 5 shows the excretion of neutral steroids to feces. The weights of feces in rats fed SB and PSB had tendency to a greater increase than in rats fed C and PC . Total neutral steroid excretion was higher in rats fed SB and PSB compared with rats fed C and PC. About 90% of the neutral steroids was cholesterol in rats fed C and PC, while coprostanol occupied 54 and 52% in rats fed SB and PSB respectively. The ratio of steroid excretion/cholesterol intake was higher in rats fed SB and PSB than rats fed PC. DISCUSSION The activity of Pronase E is the highest of all other protein digestive enzymes because of having non-specificity to the substrate. On the enzymatic hydrolysis of proteins with Pronase E in vitro, soybean protein takes ten times as long as casein (Fig. 1). This indicates that the digestibility of dietary soybean protein also seems to be lower than that of casein in vivo, though Pronase E is not a digestive enzyme of human or rat.
In our previous experiment using Wistar rats, the 40% casein diet showed the | v3-fos |
2018-04-03T00:41:57.434Z | {
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} | 0 | [] | 1983-06-01T00:00:00.000Z | 26216271 | {
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} | s2 | Separation and partial purification of wax and fatty alcohol from Okinawan sugar cane rind lipids.
A composition analysis of Okinawan sugar cane rind materials was conducted, and it was found that the main components were lipids, crude fiber and water indicating about 27%, 19.6% and 14.6%, respectively. The lipids were effectively extracted from rind materials by benzene. Wax (18%) and fatty alcohol (8%) were found to be the main components, totaling up to 96% of the lipids. Separation and partial purification of wax and fatty alcohol were attempted by means of silica gel column chromatography, thin-layer chromatography (TLC), infrared (IR) absorption spectra and gas-liquid chromatography-mass spectrometry (GC-MS). The major components of the wax were considered to be two compounds with the molecular weights of 408 (C-26) and 436 (C-28), and those of fatty alcohol were di-ol with molecular weights of 440 (C-29) and 468 (C-31). Both wax and fatty alcohol were purified by rechromatography on a silica gel column and the samples obtained seemed to be satisfactory for use in experimental rats' diets.
In the previous paper (1) we reported that sugar cane rind materials showed effects on weight control and on lowering serum cholesterol levels of rats. It is widely known that the plants, generally leaves and stems, are covered with wax or some other lipids for protective purposes.
Yomo et al. (2) reported that the protecting elements of sugar cane rind such as wax and fatty alcohol were found on the surface of sugar cane, and were also mixed in the cane juice when sugar cane is pressed. In the processing of sugar cane to manufacture refined sugar, some portion of the wax and fatty alcohol remains up to the stage of raw (black) sugar.
H. SHO, I. CHINEN, and K. UCHIHARA According to the literature (3), Avequin first separated sugar cane wax in 1841 from the whitish materials attached to the surface of sugar cane. Industrial recovery of sugar cane wax had first been demonstrated in South Africa during the period of the First World War by means of solvent extraction. However, the lipid analysis of sugar cane rind materials for the purpose of finding the element(s) which serve a lowering effect on serum and (or) liver cholesterol in rats had not been attempted before this experiment.
The purpose of the present study was to analyze the main components of sugar cane rind materials and to investigate in detail the separation and partial purifi cation of wax and fatty alcohol from sugar cane rind materials as well as to determine the molecular weight and the number of carbon atoms in the molecule.
RESULTS
Water and crude fiber contents The water content of sugar cane rind materials collected in January was slightly lower than that of the August sample, but the crude fiber content was just the opposite. The average contents of water and crude fiber were 14.6% and 19.6%, respectively.
Extraction of lipids
The total lipids, fatty alcohol and wax contents of sugar cane rind materials extracted by different solvents are shown in Table 1. As Paturau (6) demonstrated for the extraction of sugar cane wax, benzene was the most effective among the three solvents in each lipid showing about 27% for total lipids, 18% for wax and 8% for fatty alcohol. The solvent used by Folch et al. (13), most generally used for the extraction of lipid from animal tissues (chloroform:methanol mixture), was the next most effective and n-hexane was not adequate for effective extraction.
Separation and preparation of wax and fatty alcohol
In order to separate wax and fatty alcohol from lipids extracts, silica gel column chromatography was applied and the fractions obtained were qualitatively tested on TLC. For developing, n-hexane:benzene (1:1, v/v) was used as the solvent. As shown in Fig. 1, wax was eluted with benzene (fractions 1-4), fatty alcohol with chloroform (fractions 7-9) and other lipids with methanol (fractions 10-13). Therefore, the fractions containing wax (1-4) were combined as the total wax fraction, F-1, and concentrated for rechromatography on a silica gel column. The same procedure was done for fatty alcohol fractions (7-9), fraction F-2. As shown in Fig. 2, separated wax (F-1) and fatty alcohol (F-2) obtained by rechromatography on a silica gel column seemed to be purified enough to feed the rats for the experiment. Then, confirmation of those two fractions, F-1 and F-2, was alcohol spot also was clearly identified at a higher Rf value than that of palmitic acid. Confirmation of the samples was, furthermore, carried out by means of IR absorption spectra to ascertain the existence of the hydroxyl radical (-OH) and ester linkage. As shown in Figs. 4-6, -OH was observed on the fatty alcohol IR spectrum at 3,200-3,500 kayser and the ester linkage on wax at 1,740 kayser.
Determination of the molecular weight and the number of carbon atoms The GC chromatograms of wax and fatty alcohol are shown in Figs. 7 and 8. In regard to the wax, two major peaks and four minor peaks were observed at retention times of 117s, 81s, 50s, 159s, 203s and 20s in decreasing order . For the fatty alcohol, the major peak was observed at a retention time of 106s and the considered to be C29H58(OH)2 with a molecular weight of 440, and the second, the major component, to be C31H62(OH)2 with a molecular weight of 468.
Wax and fatty alcohol content of sugar cane rind As shown in Table 2, the ratio of wax and fatty alcohol contained in sugar cane rind differed in direct correlation of the degree of cane-growth. Those collected from the middle to the upper part (green part) of the sugar cane contained mainly fatty alcohol whereas the lower part (mature part) were covered mostly with wax. Therefore, the sample collected in January (mature cane) contained twice as much wax as the sample collected in August (premature cane), as shown in Table 3.
DISCUSSION
For the extraction of lipids from sugar cane rind materials, benzene, which Paturau (6) previously used for extraction of sugar cane wax, was a more effective solvent than the chloroform-methanol mixture; the generally applied solvent. Therefore, the main components of the sugar cane rind lipids are now considered to be wax and fatty alcohol. The total lipid content of the January sample was about 27% of the total rind materials, and the wax and fatty alcohol contents were about 18% and 8%, respectively, composing about 96% of the total lipid extracts.
In order to separate fatty alcohol and wax from lipid extracts, column chromatography was applied as Carroll and Serdarerich (7) and Rouser et al. (8) demonstrated, and samples were eluted with n-hexane, benzene, chloroform and methanol successively. Judging from the Rf values of each fraction obtained by TLC analysis, wax was separated by benzene, fatty alcohol by chloroform and other lipids by methanol. Those results agreed with the Rf values reported by Hashimoto et al. (14) and Malins and Mangold (15).
Partial purification of fatty alcohol and wax was effectively carried out by rechromatography of each obtained fraction on a silica gel column. | v3-fos |
2014-10-01T00:00:00.000Z | {
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} | s2 | Association of egg traits and feathering mutants in domestic fowl
A search was made for association of egg numbers and egg quality traits with genes for frizzling (n, muffs and beard (Mb), and their normal alleles in domestic fowl. Hens from the fifth, sixth, and seventh generations of a segregating population were each studied for two months late in their first production year. Egg numbers, weight, shell thickness, albumen quality, and incidence of blood spots and meat spots were compared. Hens with muffs and beard produced eggs with thin shells (P < .01) compared to hens with the wild-type allele. There were no major effects of feathering loci on other egg characteristics examined.
I. Introduction
Associations between qualitative and quantitative traits are usually explained by pleiotropy, linkage, and chance effects. Of the three, association due to pleiotropic effects is of most potential value in commercial production. But distinction between pleiotropy and other causes of association is not always easy. Chance effects should not persist if observations are made over several generations of a population or on several different populations. Linkage effects would be expected to dissipate over several generations, and associations of the same degree or kind would not be anticipated in unrelated stocks. Pleiotropy would be indicated when associations continue through many generations and where the same associations occur in diverse populations.
During reproduction of a stock segregating for frizzle, muffs and beard, and the normal alleles of these two mutants, casual observation indicated that frizzle hens were producing larger eggs than their non-frizzle flockmates. Because of the potential value of this association. the present study was undertaken to seek verification and to identify its cause.
A concise account of the two feathering mutants, frizzling (F) and muffs and beard (Mb), has been given by H UTT (1949). Both genes are incompletely dominant autosomal. Frizzling is associated with modifications of metabolism and size of body' organs, probably in compensation for poor insulation by the feather coat. No mention has been found in the literature of association between frizzling and egg quality traits. H UTT (1964) reported that sexual maturity was delayed in Mb pullets, and M ERAT (1962) observed a reduction in wattle size in the presence of Mb. In a large study of association between qualitative and quantitative traits, LowE et al. (1965) found that birds with muffs and beard had lower adult body weight, and egg weight at 32 weeks of age was reduced by 1.1 grams ; they found no association of muffs and beard with age at maturity, egg production, albumen quality, shell thickness, egg shape index, or egg weight at 55 weeks of age.
II. Materials and methods
The population under study originated from a bantam male, exhibiting frizzled feathers and muffs and beard, mated to normal Brown Leghorn females. Their progeny were mated inter se to establish a segregating closed flock. In each generation, flock mating combinations were planned to ensure a 1:1:1:1 ratio of genotypes resulting from presence of the two feathering mutants and their normal alleles ( Table 1). Birds of the fifth, sixth and seventh generations were used in the present study.
In the first year, six hens of each of four genotypes (Table 1) were studied for 52 days beginning when they were twelve months of age. They were individually caged. Egg numbers were recorded daily and all collectable eggs were measured to determine weight, specific gravity, Haugh units, and incidence of blood spots and meat spots. The study was repeated a year later on the next (sixth) generation beginning with twelve hens per genotype ; there was some mortality during the 60-day trial and data are reported only for the survivors. An identical procedure was followed in a 60-day trial with the seventh generation. Data were analyzed by analysis of variance (STEEL and T ORRIE , 1960).
III. Results and discussion
Egg production data, expressed as total eggs laid per hen during the trial, are shown in Table 1 and analysis in Table 2. Production was measured during May and June of each year when environmental temperatures were rapidly increasing and when the birds were nearing the end of their laying cycle. There were no significant differences among genotypes nor was any one genotype consistently superior to the others. There were highly significant differences among generations reflecting uncontrolled environmental effects. There were also highly significant interactions between generations and each of the feathering loci under study ; these are of doubtful biological importance and probably reflect chance events.
This study had been prompted by the observation that frizzle hens of Generation 5 were laying larger eggs than their wild-type flockmates. Formal measurement indicated that this was indeed the situation (Table 1). However, similar effects were not evident in data for Generations 6 and 7, and statistical analysis (Table 2) did not reveal any significant differences. LowE et al. (1965) had found no relation between Mb and egg weight at 55 weeks of age, which is consistent with the present findings. They had, however, found that egg weight at 32 weeks of age was reduced by 1.1 grams. It is tempting to explain this by the delay in sexual maturity in Mb pullets reported by H UTT (1964) but a significant delay is not evident in the analyses of LowE et al. (1965). Observations at a comparable age were not made in the present study. It would appear valid to conclude that weight of eggs from adult birds is not affected pleiotropically by the frizzling or muffs and beard loci.
Egg shell thickness was evaluated by measuring specific gravity of intact eggs. Hens carrying the muffs and beard gene produced eggs with thin shells (P < .01) ( Table 2). It would be of interest to obtain further data since the observed differences (Table 1) are very small, and to examine hens which were homozygous MbMb to determine whether a dosage effect exists. LowE et al. (1965) had found no association between the muffs and beard trait and egg specific gravity.
Albumen quality, as measured by Haugh units, was not affected by alleles at the two feathering loci under study. However, statistical analysis revealed a significant interaction between the two loci ( Table 2). Interpretation of this interaction is not certain, but it may be an artifact of the very large generation effects (P < .01) that were encountered. LowE et al. (1965) found no association between albumen quality and the muffs and beard locus.
Inclusion bodies present technical difficulties in measurement. In this study they were recorded as present or absent, and within a generation all observations were made by the same person. There were generation differences in blood spot incidence, which may reflect differences among observers, but otherwise relationships were negligible.
In a population segregating for many generations, such as the one under study, it would be expected that linkage associations established in the first cross would diminish with succeeding generations. There is no indication that linkage explains the associations observed here. Although of low magnitude, the association of the muffs and beard locus with shell thickness was consistent across generations which is indicative of pleiotropy. Further data are needed to verify a pleiotropic relationship. Since the present data were derived from a comparison of mutant heterozygote with homozygous wild-type, it would be very helpful to include mutant homozygotes in future studies to determine whether a dosage effect exists. | v3-fos |
2018-04-03T04:52:36.405Z | {
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} | s2 | Nitrogen requirement of amino acid mixture with maintenance energy in young men.
The effect of energy on nitrogen balance was examined in young men given amino acid mixture. The minimum amino acid nitrogen requirement for nitrogen equilibrium was determined together with the egg protein requirement. In experiment 1, the nitrogen sparing effect of energy was evaluated in four male students receiving diet containing an amino acid mixture and a constant nitrogen intake of 3.5 g N/day, which was equivalent to the nitrogen requirement with excess energy intake determined by Rose and Wixom (1). When the dietary energy supply was 45 kcal/kg, which is approximately the maintenance level, the mean nitrogen balance was negative, being -23.9 +/- 9.3 mg N/kg. However, with an excess energy intake of 55 kcal/kg, the nitrogen balance improved significantly, being -6.1 +/- 7.7 mg N/kg. In experiment 2, the nitrogen requirement of egg-pattern amino acid mixture for apparently zero balance was evaluated at maintenance energy intake in 28 Japanese young men and was compared with that of egg protein. After receiving standard diet, the subjects were given a semi-purified experimental diet containing egg-pattern amino acid mixture at nitrogen intake levels of 60, 75, 100, and 130 mg N/kg for two weeks. Then all groups except the 60 mg N/kg group were given isonitrogenous egg protein diet for another week. Energy intake was kept constant at approximately the maintenance level of 44.4 +/- 1.4 kcal/kg throughout the experiment. Nitrogen balance was not significantly different in groups given egg-pattern amino acid mixture and intact egg protein in each nitrogen intake level. From regression analysis, the nitrogen requirement for nitrogen equilibrium of the amino acid mixture was calculated to be 110.1 +/- 50.2 mg N/kg, which was not significantly different from the value of 88.4 +/- 40.6 mg N/kg of egg protein. It was concluded that the total amino acid requirement estimated by Rose and Wixom (1) was too low because they gave excess energy, and that there was no difference between the nitrogen requirement of egg protein and that of the corresponding amino acid mixture.
Summary
The Studies on amino acid requirements have been carried out mainly on require ments of individual essential amino acids and there have been few studies on the total amino acid nitrogen requirement except for those of Rose and Wixom (1). Rose and Wixom (1) estimated the amino acid nitrogen requirement to be 3.5g N/ day (50mg N/kg for 70kg body weight) with an excess energy intake of 55kcal/kg . But this is considerably lower than the value of 90mg N/kg of egg protein obtained in our laboratory with an approximate maintenance energy intake of 45kcal/kg (2) . It was necessary to investigate whether the difference between the total amino acid requirement determined by Rose and Wixom (1) and that of egg protein determined by us (2) depended on the difference in energy intake or in the nitrogen source.
There have been some studies on nutritional differences between intact proteins and corresponding amino acid mixtures. Rose et al. (3) reported that the nutritive value of amino acid mixture with the pattern of casein was inferior to that of intact casein and that with the mixture excess energy above the maintenance intake was required to obtain a similar nitrogen balance to that with intact protein. But Anderson et al. (4) reported that the nitrogen balance with an amino acid mixture simulating casein was not significantly different from that with intact casein. Thus no definite conclusion has been reached on whether the nutritive value of an amino acid mixture is different from that of intact protein. The protein sparing effect of energy has been known for a long time (5, 6). Inoue et al. (2) and Kishi et al . (7) found that the egg protein nitrogen requirement was reduced by 1.9mg N/kcal on increasing the energy intake from a maintenance level of 45kcal/kg to an excess level of 57kcal/kg. Furthermore, Inoue et al. (8) showed an interrelation of nitrogen intake, energy intake, and nitrogen balance by multiple regression analysis. But there are no reports of quantitative examinations of the nitrogen sparing effect of energy with amino acid mixture diet. In this work, therefore, we first examined the effect of energy intake on nitrogen balance with an amino acid mixture containing 3.5 g N/ day as total nitrogen, which is equivalent to the total amino acid nitrogen requirement determined by Rose and Wixom (1). Then in experiment 2 we examined the amino acid nitrogen requirement under maintenance energy conditions with an egg-pattern amino acid mixture and compared results with that for egg protein to determine whether there was any nutritional difference between the two nitrogen sources.
MATERIALS AND METHODS
Thirty two male Japanese students of 19 to 30 years old in this university, participated in this study. During the experiments they lived in a metabolic ward under our clinical supervision and were weighed daily before breakfast after voiding urine. During experiments, they continued normal daily routines, but refrained from any hard physical activities. Table 1-1 and 1-2 summarizes the characteristics of these subjects and their energy and nitrogen intakes. Subjects were given standard diet for five to seven days before experiments. Standard diet was prepared from conventional foodstuffs containing about 1.25g of protein per kg (200mg N/kg) and about 45kcal/kg of approximate maintenance energy. These nitrogen and energy intakes were enough to meet the allowance levels for Japanese men (9), and the subjects maintained constant weight during the period on standard diet as described later. After the standard period, the subjects were given a semi-purified experimental diet. Experiment 1. We investigated whether energy had a nitrogen sparing effect with amino acid mixture diet as with protein diet. During the experiment, four subjects were given semi-purified experimental diet containing amino acid mixture as the sole nitrogen source. The energy intake was 45kcal/kg, or approximately the maintenance energy level, for ten days and 55kcal/kg, or an excess energy level, for ten days, with a three-day recovery period between these periods. During the recovery period, the subjects ate conventional Japanese diet ad libitum. The 45kcal/ kg diet and 55kcal/kg diet were given according to a simple switchback schedule; namely, two of four subjects were fed 45kcal/kg diet for the first ten day period and then 55kcal/kg diet for another ten days and the other two subjects were given the two diets in the reverse order. For comparison of the requirement with that reported by Rose et al. (1,10), the same total amino acid nitrogen and essential amino acid nitrogen levels as the requirements reported by them were supplied throughout the two dietary periods; that is, 3.5g N/day of total nitrogen and 1.46g N/day of essential amino acid nitrogen. The compositions of essential and nonessential amino acids in the mixture simulated those in egg protein, as shown in Table 2, but cystine and tyrosine were replaced by isonitrogenous amounts of methionine and phenyl- alanine, respectively. The composition of the experimental diet is given in Table 3 . Energy was supplied mainly as cornstarch, sucrose, dextrin, corn oil and shortening.
Dietary fat supplied about 30% of the total energy intake. Vitamin and mineral mixtures given in the experimental periods fully satisfied the allowances of Japanese men (2). In addition, 5g of agar was given as roughage.
The mean urinary nitrogen excretions in the last four days of each experimental a Intake is given for a 64kg subject (code No . 305) receiving 100mg N/kg diet. b The content of essential amino acid nitrogen with the egg pattern is 43% of the total nitrogen. In experiment 1, the essential amino acid nitrogen supplied was equivalent to the requirement estimated by Rose et al. (10) and was 41.8% of the nitrogen intake of 3.5g/ day. c In some cases, arginine and histidine were used as chloride forms.
period were used to estimate nitrogen balance. Apparent nitrogen balances were calculated from the nitrogen intake and fecal and urinary excretions. Subject 4 did not participate in the study with 45kcal/kg diet because he had a cold. Experiment 2. In this experiment, the apparent nitrogen requirement of egg pattern amino acid mixture was evaluated under approximate maintenance energy conditions and was compared with that of egg protein. After a period on standard diet, subjects were fed a semi-purified experimental diet containing amino acid mixture simulating whole egg protein as a sole nitrogen source for two weeks. The nitrogen intakes during the period on amino acid mixture were 60mg N/kg for five subjects, 75mg N/kg for ten, 100mg N/kg for eight, and 130mg N/kg for five subjects. For comparison of the nutritional value of egg-pattern amino acid mixture with that of egg protein, all groups except that receiving 60mg N/kg were given egg protein diet for one week after amino acid mixture. Energy intake throughout the experimental period was kept constant at approximately the maintenance intake of period on amino acid mixture and egg protein, respectively, are given in Table 5. Urinary total nitrogen excretion decreased rapidly to fairly steady levels within a week on changing to the amino acid diet from standard diet, as shown in Fig. 2.
There was no significant difference between the mean urinary nitrogen excretions in the two periods, as shown in Table 5.
DISCUSSION
Rose and Wixom (1) estimated the nitrogen requirement of an amino acid mixture for apparent nitrogen equilibrium as 3.5g N/day (50mg N/kg as 70kg body weight) with a large excess energy intake of 55kcal/kg body weight. This value for amino acid nitrogen requirement is lower than the egg protein requirement with a maintenance energy intake estimated by us and others. For instance, Calloway and NITROGEN REQUIREMENT OF AMINO ACID MIXTURE 181 Table 9.
Concentrations of plasma free amino acids of men fed amino acid diet and egg protein diet. Margen (18) reported that the true minimum egg protein requirement was 4 .8g N/ day (68mg N/kg as 70.1kg body weight), which is more than the endogenous nitrogen output of 3.5g N/day determined in that study. Young et al. (19) also evaluated the nitrogen requirement of whole egg protein as 60-94mg N/kg body weight. In addition, Inoue et al. (2) reported that apparent nitrogen equilibrium was established at about 90mg N/kg of egg protein nitrogen with maintenance energy intake. All these values are considerably higher than the amino acid nitrogen requirement reported by Rose and Wixom (1). We thought that this difference might be due to the difference in energy intakes, not to the difference in nitrogen sources.
The effect of energy intake on protein requirement was studied quantitatively by Inoue et al. (2) and Kishi et al. (7), who found that the nitrogen requirement of egg protein decreased with increase in energy intake; that is, it was 124mg N/kg at 40.2kcal/kg, 90mg N/kg at 45.0kcal/kg, 82mg N/kg at 48.2 kcal/kg, and 67 mg N/kg at 57.0kcal/kg of energy intake. Thus the amount of egg protein nitrogen spared by energy was calculated as 1.9mg N/kcal on changing the energy intake from an approximate maintenance level of 45kcal/kg to an excess energy level of 57 kcal/kg. For comparison of results with intact egg protein, we investigated the effect of energy intake on nitrogen balance of an amino acid mixture in experiment 1. Results showed that at 3.5g N/day of amino acid mixture the nitrogen balance was almost zero with the excess energy intake of 55kcal/kg used by Rose and Wixom (1), but was clearly negative with approximately the maintenance energy intake of 45kcal/kg. Results also showed that the nitrogen balance improved by 1.7 mg N/kg with increase from maintenance to excess energy intake . This indicates that the nitrogen sparing effect of energy is similar with the amino acid mixture to that with protein. Thus it appears that the amino acid requirement estimated by Rose et al, is too low because of the nitrogen sparing effect of excess energy.
There has been much controversy over differences in nutritional value of intact proteins and mixtures of their constituent amino acids. Some investigators consider that amino acid mixtures have lower nutritional value. For instance, Rose et al. (3) reported that nitrogen utilization of an amino acid mixture with the pattern of casein was inferior to that of intact casein and subjects failed to achieve nitrogen equilibrium below 45kcal/kg intake even when they received 10g/day of nitrogen from a mixture of the amino acids and urea. Thus these workers always carried out investigations on essential amino acid requirements with an excess energy intake of 55kcal/kg and evaluated the total amino acid nitrogen requirement as 3.5g N/day. | v3-fos |
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} | s2 | Studies on viability and infectivity of Anisakis simplex stage III larvae in fresh salted and spiced Baltic herring
The studies involved periodical teste of salted and spiced Baltic herring for the presence of live Anisakis simplex larvae through indicing their spontaneous movements when kept in a liver extract fresh bovine blood medium in a thermostat set at 37 ° C for 4 days. The infectivity of the larvae was studies by in vitro observing their development in a medium such as above. A period of time necessery to kill all the Anisakis larvae present in salted an spiced herring was determined.
INTRODUCTION
It is very important from the standpoint of food hygiene to determine the variability of A,nisakis .simplex stage HI . larvae, yery commonjn mai;ioe .fishes. The la�ae are known Anisakis larvae present, and fresh Baltic herring processed directly after capture and not frozen. The latter can be very strongly invaded depending on the season and fish size (J. Grabda, 1974a).
It is the Baltic herring that were the object of the pro cent study. So far, no work has been done in Poland on salted and spiced Baltic herring. It was only the North Sea herring (Lubieniecki, 1970) and herring from such fishing grounds off the British Isles as the Irish Sea, English Channel, and Hebrides (J. Grabda, 1974c) that were examined. Lubieniecki (1970) studied the herring processed according to the Fisheries Central Board Standard issued in 1969, requiring 7-10% salt content in fish flesh and less than 20°Be brine.
The Standard required the uptake of 9.5 kg salt, 0.405 kg sugar, 0.614 kg spices, and 0.225 kg preservatives per 90 kg fish. Fresh deep-sea herring salted on board were processed.
Lubieniecki recorded several live larvae at l 5-19°Be brine. He found that in most cases the amount of salt required by the Standard produced a 22-23°Be brine, sufficient to kill nematode larvae over 7 days, on the third or fourth day after salting. However, be made a provision that the herring flesh salt content depended not only on the actual brine salt concentration, but also on the gonad maturity stage, flesh fat content, and salting temperature; all those factors could possibly contribute to elongate the Anisakis larvae viability.
119
Subsequent studies by J. Grabda (1974b,c,d) were carried out during a 3-yr period of 1971-1973 on deep-sea herring processed on board according to the above mentioned Standard. Each year live larvae were being found at 10-19°Be brine. The shorter herring maturation period and the lower brine salt content, the more larvae were found live.
Therefore an increase in the fish flesh salt content at the expense of certain flavour-enhancing properties was postulated, 20°Be brine and 20% salt in fish flesh being assumed as the minimum. The maturation period enabling a proper absorption of salt by the flesh, leading to death of all larvae, should be sufficiently long, preferably 2 months before marketing the fish.
It was as late as in 1973 that the Fisheries Central Board implemented the recommended increase in brine salt content; in 1976 a new standard was worked out according to which the salted and spiced Baltic herring are prepared at present.
Both Lubieniecki and Grabda studies the larvae viability only, using the method by Khalil (2°h heating at 37°C), while the studies described below were aimed at finding for how long the Aniso.kis simplex larvae remained viable and whether they retained their infecti.vity or if salt and spices weakened them so as to render them harmless to man (Cishi et al., 1974).
Large Baltic herring (25-30 cm Lt.) were caught in the Pomeranian Bay from December through March, i.e., when the Anisakis infested western herring migrated here to spawn(J. Grabda, 1974a).
After checking the degree of infestation by opening the fish abdominal cavity, the fishes were headed and placed in jars with salt-spices mL°"ture added. The jars were kept in refrigerator at about +5°C.
In this way 6 batches of herring caught on various dates were prepared in the laboratory. In each batch, the Aniso.kis larvae viability was checked 1, 2, 3, and 4 weeks after salting.
Each time the brine salinity (°Be) was measured with the Baume salinometer (J .Long Ltd.London, N.D.:A.2825) at 15°C, while the fish flesh salt content was checked by AgN0 3 titration according to the Polish Standard No. PN-74/A-86739 ,,Fish and fish products. Salt content determination". larvae viability tests The larvae viability was tested in 4 days after removing them from a fish. After l week in the brine some larvae showed spontaneous, movements usually on the first day of testing. The remaining larvae, seemingly dead, transferred to the culturing medium consisting of bovine liver extract enriched with fresh bovine blood (J. Grab da, 1976) and placed in a thermostat for 24 h at 37°C. Some larvae became ,,revived" after 24 hours. The remaining ones were left for further 24 hours in the medium. In this way their viability was checked 3 and 4 days after removal from fish. Some larvae were observed to revive after 72 hours.
The viability after 3,3, and 4 weeks from salting was tested in the some way. Studies on larvae infectivity The infectivity of the larvae was studied by in vitro cultures (J. Grabda, 1976), assuming that the larval development proceeding in the medium is an evidence of their capability to invade and develop in a live poikilotherm organism, man included, under natural conditions.
The larvae moving spontaneously on the first day and those showing movements on the second and subsequent days were kept separately. The observations were carried out until sexually mature nematodes were obtained, or until the death of the last larva. Usually, 20 larvae of each series were cultured.
RESULTS
The results of viability tests in the Anisakis simplex larvae removed from salted and spiced herring and their capability of further development are summarised in tables illustrating 4 series of experiments (Tables 1-4). After 1 week in salted herring Live, spontaneously moving larvae were being found as early as on the first day of the examination, and also after 24 hours. Generally, almost all (up to 98.2%) of the larvae in the herring tested were mobile. The brine salinity was 17-l9°Be, the viscera salt content ranging within 5.6-8.2%.
The _third moult was observed to start normally on the fourth to sixth day of the cultures; after this moult, in two cases only deaths of 3 and 7 larvae (15-35% of the cultures larvae) were recorded. In other experiments, all the larvae survived. The nematodes underwent the fourth moult, too, to metamorphose into stage 4 proceeding sexual maturity. Saome larvae (5-45%) reached maturity and produced eggs. The nematodes survived for 25 to 67 days. After 2 weeks in salted herring. The brine salinity amounted to l 7-l 8°Be, while the flesh salt content was 9.36-12.9%. No motile larvae were found on the first day. After 24 hours 12.6-84.6% of the larvae found were ,,revived". In one case, motile larvaeseemingly dead initially -were revealed on the third and fourth day. Before the third moult and during it, 11.3-68% of the cultured larvae dies off, only a few reaching maturity in two instances when 3.9-5% survived. The survival time was 14-52 days. After 3 weeks in salte4 herring. The brine salinity was l 7.5-19°Be, the salt content in flesh ranged within 11.6-14.04%. Notile larvae were found in two cultures, One of them (l8°Be brine, 11.9% salt in flesh) contained 13 (18.3%) motile larvae of which 8 died before the third moult and only 1 survived 26 days. The third moult took place at a normal time. This larva then, in our opinion, was fully capable of surviving in the human intestine and of penetrating to the intestinal mucosa, thus inducing pethological changes. The fourth moult was not observed. The other culture (18°Be brine, 11.6% flesh salt content) was found to contain 2 larvae only which performed very weak spontaneous movements and<lied on the second day. After 4 week in. salt herring. The brine salinity ranged within 18 .5-20 .5°Be, 12. 2-14 .6% being the flesh salt content range. No motile larvae were found in the cultures.
The experiments show the Anisakis larvae to retain their full viability during fish maturation in the first week after salting; thus the larvae capability of a further development and thus of invading is retained as well (obviously, the natural mortality of larvae, affecting also the cultures of fresh larvae not exposed to salt (J. Grabda, 1976) should be taken into account). This is the period when, as should be supposed, the larvae are most harmful to man.
After 2 weeks the danger is considerably smaller, while after 3 weeks from salting any posibility of the larvae invading humans is virtusally non-existent. The larval mortality rate is very high, single individuals only proceeding through the third moult and none reaching sexual maturity owing to their considerable weakening. Nevertheless, it is only after 4 weeks from the salting date that the salted and spiced Baltic herring can be regarded as entirely safe, parasitologically, for human consumption and should be allowed time to reach their full flavour properties.
DISCUSSION
When performing the experimented described above, it was seemed necessary to find appropriate criteria of larval viability. Van Thiel et al. (1960) regarded the larvae as dead when no visible movements could be observed under x20 magnification for a week after transferring the larvae to 50%-diluted sea water at 10°C. Khail (1969) differentiated between spontaneous larval movements and those induced by mechanical stimuli; he regarded non-motile larvae as dead. The author placed the larvae, removed from fish, at the Tyrode fluid at room temperature for an hour and then transferred them for 2 hours to a pepsin-HCl solution at 36°C in thermostat and observed them under a stereomicroscope.
In view of the present results, the assumption that those larvae remaining motionless after 2 hours in a thermostat are dead dose not suffice to detect live larvae in salted and spiced herring and should not be applied when checking larval viability. The examination should continue for 4 days as motile larvae removed 3 weeks after salting were observed as late as after 72 hours, the larvae shoeing at first no trace of any movement.
It is not necessary to use a pepsin-HCl digesting medium; a liver extract-blood medium and a storage at 37°C, i.e., the conditions corresponding to those under which the parasite's life cycle proceds should be applied instead.
Another issue of importance for the Anisakis invasiology is the infectivity of those larvae considered live. Pathogenic properties of the larvae are determined not only by their viability but also by their ability to penetrate the host's intestinal mucosa. Several authors carried out in vivo studies by artifical infested experimental anjmals (rabbits, guinea pigs, rate, pigleta). On the other hand, Ruitenberg (1970) used in vitro laboratory techniques in addition to his studies in vivo. He was using 188 mm high 23 mm diameter vials, filled with agar-agar to 3/4 of their volume. In a half of the vials, the medium had a amooth surface; in the other half, the medium surface was roughened by scrubbing with forceps. A 1 cm thick layer of herring blood was poured on to the agar-agar and 5 Anisakili larvae placed there. The vials were kept in a thermostat at 37°C; the larvae penetrating into the medium were counted after 1, 2, and 3 days. Most larvae were fiund to have penetrated after 1 night (18 hours), more larvae penetrating the rough-surface agar-agar medium.
Although possessing many advantages, Ruitenberg's technique is not sufficient to check the infectivity of larvae of differing viability. Therefore, during the present work, the larval life cycle was followed in an in vitro culture kept in a blood-enriched liver extract at 37°C, with corresponds most closely to the conditions created by the internal milieu of a poildlitherin, the definits host for Anisakis simplex.
Having penetrated their definite hosts, the larvae become very active as early as during the first 24 hours and penetrate the intestinal mucosa, thus inducing pathological changes in a patient (Van Thiel et al., 1960;Ruitenberg, 1970;and others). It is then sufficient to continue the culture until the 3rd moult occurs, i.e., for about 5 days.
In the present work, only the effects of salt content in fish flesh and in brine on the Anisakis larvae viability and infectivity were studies, no consideration being given to effects of spice essential cils and preservatives using in the salted and spiced herring production, the effects undoubtedly existing as shown by Japanese authors (Oishi et al., 1974). They found all the spice essential cils, particularly . the nutmeg and nutmeg flower ones as well as those of coriander, dill, cummin, clove, allspice, and cinnamon to affect the Anisakis larvae. Of the synthetic preservatives, particularly strong, deterimental for Anisakis, effects were obtained when using sorbic, benzoic, dehydroacetic, and salicylic acids. Effects of salts of those acids are, however, much weaker.
Effects of those substances are certainly additive with respect to salt and provide an additional factor detrimental to the Anisakis larvae.
Vability and infectivity of Anisakis 127 CONCLUSIONS l. The viability criterion for Anisakis simplex larvae occurring in fresh salted and spiced Baltic herring was a spontaneous movement of the apparently dead larvae removed from herring, placed in a cattle blood-enriched liver extract and kept for 4 days in a thermostat. The ,,revival" of those apparently dead larvae can occur on the 1st, 2nd, 3rd, and even 4th day. 2. The infectivity of the larvae removed from.salted and spiced herring can be checked in an in vitro culture (fresh cattle blood-enriched liver extract as a medium, 37°C) instead of an artificial infested of laboratory animals or Ruitenberg's agar-agar technique. 3. It is sufficient to keep the culture until the third moult, i.e., for 5 days. 4. A period of 4 weeks from salting is sufficient to kill all the Anisakis simplex larvae occurring in salted and spiced Baltic herring produced in Poland according to the Mafo jest badan nad zywotnosci11 larw w sledziach mafo solnych, tzw. korzennych, ostatnio bardzo preferowanych w przetworstwie rybnym. | v3-fos |
2018-04-03T04:08:33.017Z | {
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} | s2 | Intestinal changes associated with rotavirus and enterotoxigenic Escherichia coli infection in calves
Newborn calves inoculated with rotavirus, enterotoxigenic Escherichia coli (ETEC) serotype 020:K′ × 106′:K99:HNM, either alone or in combination, became depressed, anorectic, diarrhoeic and dehydrated. ETEC did not adhere to the intestine although there was extensive proliferation in the lumen. Only slight mucosal changes were induced by ETEC and the activity of membrane bound lactase remained normal. More severe mucosal damage and a decrease in lactase activity were found in newborn calves inoculated with either rotavirus or rotavirus and ETEC in combination. The most severe clinical illness was found in calves inoculated with both rotavirus and ETEC. Calves inoculated at 1 week of age with either rotavirus or ETEC remained clinically normal. Rotavirus infection produced slight mucosal changes and a reduction of lactase activity. In contrast, colostrum-fed or suckling calves up to 2 weeks old inoculated with both rotavirus and ETEC became clinically affected, showed severe mucosal damage and decreased lactase activity. There was no bacterial adhesion to the intestinal mucosa as observed by immunofluorescent labelling and light microscopy.
INTRODUCTION
Microbiological investigations of spontaneous cases of diarrhoea in calves have shown that infection with several enteropathogens, occurring in different combinations, is more common than infection with a single agent (Acres et al., 1975;Morin et al., 1976;Moon et al., 1978).
Dual infection with rotavirus and enterotoxigenic E. coli (ETEC) has been studied in newborn gnotobiotic calves (Gouet et al., 1978). These authors showed that calves infected with both agents suffered more severe diarrhoea than similar calves infected with either agent alone; they described the interaction as being synergistic.
In a previous communication (Tzipori et al., 1981a) it was established that co-infection of older calves with ETEC and rotavirus precipitates diarrhoea in circumstances where each agent alone did not. In this study we have examined the nature of this dual infection and its effect on the intestinal mucosa.
Virology
The bovine rotavirus C6 used in this experiment was isolated from a field outbreak of calf diarrhoea as described previously (Tzipori et al., 1981a). Fifteen ml aliquots of faecal filtrates (20%, v/v) which contained between 10 ~ and l0 s particles per ml, as assessed by electron microscopy (EM), were used as standard inoculum for each calf. The presence of virus in the faeces of infected calves was also determined by EM.
Bacteriology
The strain of ETEC used in this experiment (020:K'× 106' :K99:HNM) was originally isolated from a calf with diarrhoea and shown to produce stable toxin and labile toxin (Tzipori et al., 1981a). A 4-ml portion of tryptose soya broth containing 106 to 108 organisms per ml was used as the standard ETEC oral inoculum per calf. ETEC organisms isolated from faeces were identified serologically and tested for K99 by the slide agglutination test (Moon et al., 1976). Faecal or intestinal swabs collected at necropsy were plated on Mc-Conkey and sheep blood agar, and ten randomly selected colonies were tested for the presence of the K99 antigen.
Serology
The complement-fixation test using the SAll rotavirus as antigen was performed as described previously (Tzipori and Makin, 1978}.
Experimental animals and procedure
Seven specific pathogen-free (SPF) calves, derived by caesarian section and maintained in plastic isolators, were inoculated with either rotavirus (2) or ETEC (4), with one calf remaining as an uninoculated control. Fourteen calves were inoculated simultaneously with rotavirus and ETEC, which included: six colostrum-fed (CF) and two SPF calves aged up to 4 days, and five suckling (SK) and one SPF calf aged between 4 and 14 days (Table I). Five SK calves aged between 7 and 10 days were maintained as controls.
Before and after inoculation the calves were closely observed for clinical diarrhoea and faecal samples were tested daily for the presence of ETEC and rotavirus. Serum samples were collected from calves before and at the end of each experiment. Clinical illness was assessed on the basis of three criteria: (a) anorexia; (b) change of colour of faeces from orange to white-grey; and (c) increased frequency of discharge and fluid contents of faeces.
Necropsy
Under terminal anaesthesia representative pieces of intestine were taken from five equally divided sites along the small intestine (numbered 1 to 5), and from the spiral colon and caecum. From each sample, portions were taken into 10% formol saline for histology, and duplicate samples were frozen and stored at -80°C for immunofluorescence (IF) and enzyme tests. Swabs were taken from all sites for bacterial culture.
Immunofluorescence
Cryostat sections of intestines of calves infected with ETEC or rotavirus or both were tested with hyperimmune rabbit sera raised against ETEC or rotao virus. Fluorescein-conjugated sheep anti-rabbit antiserum was used as an indicator in the indirect IF test.
Enzymology
Small portions of the small intestine were assayed for lactase activity as de-scribed previously (Halpin and Caple, 1976). Lactase activity was expressed as International Units (I.U.) per g wet tissue with one I.U. equalling one/~mole lactase hydrolysed per min at 37°C.
Inoculation of calves with a single agent
Two SPF calves inoculated within a few hours of birth with ETEC, and one with rotavirus, developed clinical signs of depression, anorexia and diarrhoea. They were necropsied within 6 h after the onset of diarrhoea. The uninoculated control SPF calf was also killed at this time.
Examination of the intestine by IF showed extensive intracytoplasmic fluorescence associated with rotavirus infection. Histological examination revealed that the villi were shortened in the jejunum and ileum and were coated with immature cells. There was little or no cellular reaction except for a few macrophages in the lamina propria of the ileum. No changes were observed in the large bowel. In calves inoculated with ETEC there was no evidence of bacterial adhesion to the mucosa at any of the sites examined. Extensive luminal proliferation, however, was evident from bacterial growth, often in pure culture, throughout the intestine. Histological changes were mild; the villi in the jejunum appeared oedematous and were coated with cuboidal cells, the ileum was congested and the lamina propria of the jejunum, ileum and caecum were heavily infiltrated with neutrophils.
Two SPF calves inoculated with ETEC and one with rotavirus at the age of 7 days remained clinically healthy. They were necropsied 24 h after the onset of excretion of ETEC (2 days after inoculation) or rotavirus (4 days after inoculation) in the faeces. ETEC proliferated in the lumen of the small and large bowel but not on the mucosal surface as observed by IF. Infection of enterocytes with rotavirus was evident throughout the small intestine.
There were no histological changes associated with ETEC and only slight villous atrophy and oedema of the duodenum and jejunum were observed in calves infected with rotavirus.
Dual inoculation of calves aged up to 14 days
Six CF and two SPF calves which were inoculated within 4 days of birth developed depression, anorexia and diarrhoea. Four CF calves that were kept for 5 or 6 days after inoculation became severely dehydrated and were killed in extremis. Table I provides details of age, clinical response, age at necropsy and preinoculation complement-fixing antibody against rotavirus. There was no difference in the length of the incubation period between the two SPF calves and the two youngest CF calves.
IF studies showed that calves killed within 24 h of the onset of clinical signs had extensive intracytoplasmic fluorescence of small intestinal entero-cytes. It was patchy and focal in calves killed 3 or 4 days after the onset of diarrhoea. There was no evidence of adhesion of ETEC to the mucosal surface of the small intestine. In five of the six CF calves there was some, often intense, fluorescence due to ETEC associated with the mucosa of the large bowel which could not be removed by vigorous washings. There was evidence of luminal proliferation of ETEC (40 to 100% of luminal E. coli) at most intestinal sites examined from the eight calves.
Mucosal damage in these calves was moderate to severe, and was more severe in calves killed in extremis. Villi in the jejunum and ileum were blunt and abbreviated, with some erosion at the tips and mostly lined with cuboidal cells whose shape was probably exaggerated by oedema. Lacteals and lamina propria were distended and contained macrophages, mononuclear cells and cytolytic debris. There was a degree of fusion of villi in the jejunum of two of the calves that were necropsied in extremis. The submucosa, submucosal capillaries and the lamina propria of the entire intestine were infiltrated with neutrophils. In the ileum there was evidence of extramedullary haemopoesis. The duodenum, with the exception of one calf, and the large bowel of all eight calves showed only slight morphological changes.
Five SK and one SPF calves were inoculated with ETEC and rotavirus. The age of the calves, the clinical response and the levels of preinoculation complement-fixing antibody against rotavirus are summarised in Table I. The calves developed moderate to severe diarrhoea. There was evidence of anorexia in three of the SK calves and the SPF calf. The SK calves which were killed 2 and 3 days after the onset of diarrhoea became dehydrated and had rough coats. However, none of the calves was severely ill at necropsy. There was no difference in the incubation period between calves with high or low antibody against rotavirus. Shedding of ETEC and rotavirus (Table I) continued until necropsy.
Extensive intracytoplasmic fluorescence of enterocytes was observed in the SPF calf which was killed shortly after the onset of diarrhoea; it was less intense and focal in calves killed 2 to 3 days later. Fluorescence due to ETEC was only evident in the large bowel of four of the five SK calves. Extensive luminal proliferation of ETEC was detected in the small and large intestine (60--100% of luminal E. coli) in the six calves at necropsy.
The morphological changes observed in the mucosa of these calves were similar to those observed for the CF group.
Examination of Figs. 1 and 2 reveals that: (a) the level of enzyme activity of the ETEC-inoculated calf was similar to that of the control; (b) the activity was lower in the duodenum and jejunum in calves inoculated with rotavirus alone, or rotavirus and ETEC and necropsied shortly after the onset of diarrhoea; and (c) the activity was severely reduced in dually infected calves which were necropsied 3 to 4 days after onset of diarrhoea. Figure 3 shows that the level of enzyme activity of the 7-day-old control calves was lower than the day-old control calf and that the dual inoculation of suckling calves caused a marked reduction in activity. Figure 4 confirms that reduced activity was associated primarily with rotavirus infection.
DISCUSSION
These experiments confirm previous observations that co-infection of calves with ETEC and rotavirus can induce diarrhoea in circumstances where one agent acting alone does not (Tzipori et al., 1981a). Similar observations were made in foals (Tzipori et al., 1982a, b) inoculated with rotavirus and the same ETEC, while dual infection failed to precipitate a disease in lambs older than 3 days (Tzipori et al., 1981b). It was surprising that the ETEC serotype used in these experiments, although possessing K99, failed to adhere to the mucosal lining of the small intestine of newborn calves. This observation was confirmed by IF studies and examination of histological sections of the bowel and is in contrast to other reports of field and experimental colibacillosis in calves (Pearson et al., 1978(Pearson et al., , 1979Bellamy and Acres, 1979;Moon et al., 1978). The ETEC serotype used in these experiments did, however, induce diarrhoea in newborn calves, probably by proliferating in the lumen of the intestine. It indicates that colonization of the brush borders of enterocytes, although an important virulence attribute (Orscov et al., 1975), was not a prerequisite for induction of diarrhoea in newborn calves.
The significance of adhesion of this serotype to the mucosa of the large bowel is not clear. Layers of bacteria, presumed to be E. coli, were also observed by Moon et al. (1979) in the colon of calves with naturally occurring diarrhoea. In our experiment the ETEC induced neither mucosal damage nor influenced the level of lactase activity. The effect of ETEC on mucosal integrity has been described as variable (Barnum et al., 1967;Moon, 1974). Much more severe clinical illness and mucosal changes were reported for 0101 (Pearson et al., 1979) and 09 (Bellamy and Acres, 1979) serotypes. These differences may be due to either the low dose used in our experiments or directly related to the failure of 020 to colonize the mucosa. Smith and Huggins (1978) have shown that in vivo the K antigen in a strain with a 99 plasmid and, to a lesser extent, the 0 antigen, were important in determining whether or not a strain would colonize the small intestine. The expression of K99 in vitro has been reported to depend on the 0 antigen (De Graaf et al., 1980). They showed that 020 produced less K99 than did, for instance, 0101. It is feasible then that in calves, different serotypes may induce colibacillosis of varying severity depending on the K and/or 0 antigens. Differences in clinical response between serological 0 types have been observed in piglets, where 064 induced a more severe disease than did 020, although both types possessed the K88 antigen and produced stable toxin demonstrable by the infant mouse test (Tzipori et al., 1982).
Dual infections precipitated a more severe clinical illness and in older calves than did rotavirus alone. Mucosal changes were also more marked in animals infected with both agents than those infected with rotavirus alone. We consider that the damage in the mucosa of these calves resulted from infection with rotavirus and was exacerbated by the presence of ETEC. Villous atrophy and destruction of mature enterocytes, resulting in maldigestion and malabsorption have been suggested as the mechanism of action of rotavirus, while hypersecretion is said to result from the action of enterotoxin liberated by ETEC (Moon, 1978). Therefore, simultaneous infection of the gut with these two agents would suggest an additive effect. However, because ETEC failed to adhere to the mucosa it was difficult to assess their role in terms of the degree of colonization in the dually infected calves. Presence of undigested lactose in the lumen of the small and large bowel may have provided a more favourable environment for selective bacterial proliferation in the lumen. | v3-fos |
2020-06-04T09:14:46.061Z | {
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} | s2 | NATIVE CACTI OF SASKATCHEWAN
The Cacti are unique plants that long have caught the popular fancy. The common name “cactus”, is derived from the Greek word “kaktos” meaning “a prickly plant”, making it an ap¬ propriate designation for plants of the family Cactaceae (= Opuntiaceae). Most North Americans think of cacti as warm-desert plants of the American Southwest and Mexico, where they are Herbarium, University of Saskatchewan,
Introduction
This article attempts (1) to briefly in¬ troduce the cacti as a novel and in¬ teresting plant group, and (2) to present a taxonomic treatment of Saskat¬ chewan's native cacti, with descriptions, habitat information, and distribution maps of verified records in the province.
The Cacti are unique plants that long have caught the popular fancy. The common name "cactus", is derived from the Greek word "kaktos" meaning "a prickly plant", making it an ap¬ propriate designation for plants of the family Cactaceae (= Opuntiaceae). Most North Americans think of cacti as warm-desert plants of the American Southwest and Mexico, where they are Herbarium, University of Saskatchewan, indeed most common and diverse in numbers of species, and they often are surprised to find that there are native cactus species growing as far north as Saskatoon and North Battleford, Sask¬ atchewan, and the Peace River country of Alberta and British Columbia.
Not all cacti are strictly desert plants, although most are indeed xerophytes (i.e. drought-resistant plants that are characteristic of relatively dry sites). In the Americas, members of the cactus family may be found in a variety of local habitats, and geographically ranging from Patagonia in southern South America, northward to our Canadian Prairies. Various species are even native Prickly Pear Cactus.
Bob Godwin to the tropical rain-forests, where they often are epiphytic (i.e. growing on other plants). Some well known examples of tropical cacti that are commonly grown as household ornamentals, are the Christmas and Easter Cacti (Zygocactus and Schlumbergia spp., and their hybrids). Authorities differ considerably as to the number of "good genera" into which to group the members of the cac¬ tus family (conservatively about 30-50, although some recognize up to 120), as well as the total number of included species best recognized (usually es¬ timated as between 1500 and 2000).
An amazing variety of plant bodyforms have evolved among cacti,e.g., globular, cylindrical, columnar, flattened, jointed, branched or not, and single-stemmed or clustered from the base. Characteristically, in all succulent xerophytes, the main adaptation of the body-form to reduce water loss has been development of a decreased sur¬ face to volume ratio. Among cacti, this has involved the development of very fleshy stems and the reduction or com¬ plete loss of leaves. Only members of the more tropical, less xerophytic, and mostly shrubby, subfamily Pereskioideae, have "normal" leaves that persist. The species of subfamily Opuntioideae, including the pricklypear cacti, have small awl-shaped juvenile leaves that soon fall off. Leaves are entirely lacking in the large sub¬ family Cereoideae, which includes the pincushion and barrel cacti.
With leaves absent, or reduced and short-lived, photosynthesis (food manufacturing) occurs instead in green cells of the stem outer layers. Much of the inner volume of succulent cactus stems is composed of storage cells con¬ taining mucilaginous materials with strong water-retaining capability. The tough waxy stem surfaces greatly retard water loss by evaporation. The remarkable efficiency of this latter feature is demonstrated by the great dif¬ ficulty plant collectors have in properly drying intact cactus specimens when using normal plant-pressing techni¬ ques. Instead, to obtain satisfactory dried specimens, cacti need special handling by first slicing the stems and fruits to expose the inner tissues, or kill¬ ing them with alcohol or some other preservative before press-drying.
Spine-clusters called "areoles" are unique structures of cacti. Although other kinds of succulent plants often bear spines, these are not clustered in areoles. Cactus areoles are borne spir¬ ally arranged on the stems, positioned directly above the "leaves" or (in sub¬ family Cereoideae) the potential location of the leaves. The spine (etc.) structures in areoles thus represent parts developed from the stem axillary buds. In some cacti (including our native Pur¬ ple Ball Cactus), the areoles are borne on small cone-shaped or globular pro¬ tuberances. In some others, they may occur on spiralling or nearly vertical stem ridges. Often distinguishable within cactus areoles, are long central spines and somewhat shorter radial (or marginal) spines. Besides the obvious, long, firm spines, the areoles of many cacti (e.g. prickly pears and chollas) may also bear numerous, small, softflexible, barbed bristles called "glochids", that, although incon¬ spicuous, are probably even more troublesome to humans contacting these cacti than the former. Frequently, wooly hairs also occur in the areoles.
The flowers of cacti are solitary, usually large and conspicuous, and often strikingly beautiful. The flowering periods tend to be rather short. Cactus flowers are botanically anomalous in combining such purportedly primitive characteristics as numerous, spirally arranged, separate stamens and perianth parts (i.e. sepals and petals), together with such evolutionarily ad¬ vanced features as inferior and multicarpellate ovaries with parietal placentation (i.e. attachment of ovules in several to many rows along the sides of a single ovary chamber). Cactus flowers are radially symmetrical and bisexual (i.e. with stamens and pistils both present).
The perianth is not sharply differen¬ tiated into sepals and petals. Rather the outer perianth-parts, which are more greenish and sepal-like, intergrade with the inner perianth-parts, which are variously coloured and petal-like. The stamens are very numerous and spirally arranged, sometimes showing some basal fusion of the long filaments into groups. The pistils are composed of several to many fused carpels, with as many stigmas as carpels. The inferior ovary is 1-chambered with the numerous ovules borne in several to many vertical rows along the sides. The perianth-parts and stamens arise from above the (inferior) ovary (= epigynous) and, in subfamily Cereoideae, from the rim of a cylindrical fioral tube ( = hypanthium) that extends above the ovary. Such an extended floral tube is present in our Saskatchewan native Purple Ball Cactus (see figure 1), but is absent or very short in our Prickly Pear species (see figure 2). The cactus ovary matures into a berry-type fruit, ranging from quite fleshy to rather dry and leathery. Areoles often occur even on the outer surfaces of ovaries (later fruits) and sometimes on the floral tubes ex¬ tending above the ovaries. These may be identified by means of the diagnostic characteristics given in the following key. (In using the key, choose between the first pair of alternative leads, "la" vs. "lb"; then if your choice is "lb", choose between the subsequent pair of leads, "2a" vs. "2b").
Identification Key to the Native Species of Cacti in Saskatchewan la. Stem globular or pincushion-like, solitary or tufted only from base, un¬ branched above, not jointed into pad¬ like segments, covered with coneshaped protuberances (tubercles), each tipped by an areole (spine-cluster); leaves entirely lacking; spines more than 12 per areole; areoles with dense wooly hairs, but lacking glochids (small, sharp, soft-flexible, barbed bristles); flowers borne between the tubercles; perianth-parts narrowly lance-shaped, less than 1 cm wide; the outer greenish perianth-parts ciliate-fringed; inner perianth-parts purplish-red; floral tube (hypanthium) distinct and elongated, extending somewhat above ovary; berry-fruit juicy-fleshy, non-spiny, wedged between the tubercles.
1. Mamillaria vivipara. lb. Stems often branched above base, conspicuously jointed into a series of more or less flattened pad-like segments separated by muchconstricted joints; stem surfaces lacking cone-shaped tubercles; awl-shaped juvenile leaves present on very young stem pads but soon falling off; spines fewer than 12 per areole; areoles with tufts of glochids at base of longer spines; flowers borne from areoles on edges of young upper stem-segments (pads); perianth-parts, except outer¬ most series, ovate to triangular or fan¬ shaped, over 1 cm wide; the outer green perianth-parts not ciliate-fringed; inner perianth-parts yellow, often tinged with green, tending more orange or pinkishred with age and toward flower-centers; floral-tube above ovary absent or very short; berry-fruits relatively dry, with spiny areoles and often awl-shaped juvenile leaves on surfaces.(see 2a vs. 2b).
2a. Pads (stem-segments) mostly less than 5 cm long, less than 2.5 cm broad, not strongly flattened, at least half as thick as broad; green to often somewhat reddish, the terminal ones quite readily detaching; areoles crowded on pads, mostly less than 8 (-10) mm apart, with dense, distinctly white, wooly hairs; longer spines seldom more than 5 per areole, the largest spines mostly 2 cm long or shorter; the small young spines distinctly barbed (under 20x magnification); glochids relatively few; pad surfaces between areoles usually becoming strongly wrinkled on dried specimens; inner perianth-parts usually pale-yellow when fresh; stigmas 2 mm long or less.2. Opuntia fragilis.
2b. Mature pads mostly over 5 cm long and over 3 cm broad, strongly flattened, over (2-) 4 times as broad as thick, not readily separable, usually bright-green; areoles more distant on pads, (8-) IQ-13 mm* apart, with wooly hairs more sparse and whitish to mostly rustytinged; spines often more than 5 per areole and over 2 cm long; the short younger spines scarcely if at all barbed; barbed glochids much more abundant; pads less strongly wrinkling upon drying; inner perianth-parts usually bright-yellow when fresh; stigmas over 2 mm long . 3 .Opuntia polyacantha.
* ranges given in measurements denote nor¬ mal variation in size; numbers in parenthesis represent extremes that have been found. (-20) radial spines, all straight and rigid, reddish-brown to whitish, 1-2 cm long, the radial ones somewhat shorter than the long central spines. Dense, white, wooly hairs are usually present in the areoles, but glochids are lacking. Leaves are entirely absent, even on young cacti. The conspicuous flowers are 3-4 cm long, 1.5-3 (-5) cm wide, borne between tubercles (actually just above the groove of a tubercle) on upper part of stem. A distinct funnelshaped floral-tube (= hypanthium) is present, extending somewhat above the inferior ovary. The perianth-parts are numerous, narrowly lance-shaped, 15-30 mm long, and 2-5 (-8) mm wide; the outer ones are green, sepal-like, and ciliate-fringed; the inner ones are petal¬ like, bright purpiish-red, varying to sometimes pale purplish-pink or salmon-pink. Stamens are numerous, the filaments pinkish-red or yellowish, and anthers yellow, forming the yellow flower-centers. Pistils are compound, of 6-15 fused carpels; stigmas 1.5-3 mm long, equal in number to carpels. The berry-fruit is quite juicy, globular to ovoid, 10-15 mm long, tightly wedged between stem tubercles, smooth, lacking spiny areoles, pale-green becoming brownish with age, sweet and edible. Seeds are 1.2-1.5 mm long, 1.5-2 mm broad, with surfaces light-brown and reticulately pitted, and the hilum (seed-stalk scar) appearing lateral. (See figure 1).
These plants grow on dry, usually quite sandy, exposed hillsides and ridges in the grassland region of southern Saskatchewan, from the International Boundary, north to Saskatoon (see Map 1). The short-lived flowers appear in early summer (June), usually opening only in the mornings; the berry-fruits ripen by fall.
The plants are low-growing, prostrate-spreading to more or less decumbent (i.e. terminal pads turned upward), often branching (two pads arising from the top of one below), sometimes forming dense mats to 5 dm wide and 0.5-2 dm high. The stems are conspicuously jointed, with the segments (pads) obovate to elliptical or ovate in outline, (1.5-) 2-3 (-5) cm long, 1-2.5 (-3.5) cm broad, somewhat flattened to nearly circular in cross-section, 1-2 cm thick, at least one half as thick as broad. The terminal pads are readily detached, facilitating dispersal by animals. Areoles are mostly distanced less than 8 (-10) mm apart on stem pads, each armed with (2-) 3-7, strongly divergent, yellowish to brownish, straight spines, 1-2 (-3) cm long; the smaller young spines are barbed. Areoles also commonly bear dense, coarse, white-wooly hairs, and only a few yellowish to whitish-grey glochids. Stems, upon drying, tend to strongly contract between areoles; thus dried herbarium specimens usually appear much wrinkled. The juvenile leaves are subulate (awl-shaped), 2.5-3.5 mm long, and soon fall off.
The flowers are conspicuous, 2.5-5 cm long and broad. The perianth-parts (exluding outer bract-like series) are mostly triangular or ovate, 2-3.5 cm long, and 1.5-2.5 (-3.5) cm wide. The outer perianth-parts are sepal-like and greenish, edged with yellow; the inner petal-like perianth-segments are paleyellow, tending to pale-pinkish or orange with age especially toward the flower centers. Stamens are numerous, with the filaments yellowish to often reddish. Pistils are compound, with 10 carpels and 10 oblong stigmas, mostly 1.5-2 mm long. The fruit is a dry, ovoid, spiny berry, 1-2 cm long, about 1 cm in diameter, greenish when young, becoming tannish when mature. Seeds are numerous, 5-7 mm long, yellowish, and irregularly shaped. (See figure 2).
Brittle Prickly-pear grow on warm, dry, sandy, exposed hillsides, in the general grassland region of southern Saskatchewan, from the International Boundary, north to the Saskatoon and Battleford areas (see Map 2). They often are locally associated with, but almost always less frequent than, plants of the Plains Prickly-pear. The flowers bloom in June, but many plants do not seem to produce flowers, at least in a given year. The fruits mature over the summer to ripen in fall, although many appear not to develop after flowering. The plants are low-spreading, more or less prostrate to 2 dm high, of several to many jointed segments (pads), often forming large, prostrate mats or clumps. The larger stem-pads are broadly obovate or nearly circular in outline, 5-13 cm long, 4-10 cm broad, to about 1 cm thick, strongly flattened, at least 4 times broader than thick, not readily disjointing, bright-green to bluish-green. The areoles are about 1 cm (8-13 mm) apart on mature stempads, with longer spines (3-) 5-10 per areole, straight, creamish-white or reddish-brown, 1-5 cm long and un¬ flattened, about 0.5 mm in diameter at base; the smaller young spines are scarcely, if at all, barbed. Areoles also bear clusters of tawny, barbed, bristle¬ like glochids, and whitish to brownishtawny wooly hairs. The flowers are large and showy, 4-8 cm broad, and 2.5-6 cm long, waxyappearing, borne from areoles along the margins of young upper pads. The perianth-parts are ovate to triangular, 2.5-5 cm long and 1.5-3 cm broad. The outer perianth-parts are greenish and sepal-like; the inner perianth-parts are petal-like and pale-yellow, tending toward pinkish or reddish-orange with age at bases and flower centers. Stamens are numerous, the filaments yellowish to often pinkish-red. The pistils are compound with 10-15 carpels; the stigmas are lanceolate to oblong, 2.5-4 mm long, as many as the carpels. The fruit is a dryish, quite leathery berry, globular to oblong or more or less obovoid, 2-3 cm long, tan to brown, with areolar glochids and spines. Seeds are numerous, 5-6 mm long, irregularly shaped, yellowish to whitish. (See figure 3).
Plains Prickly-pear grow on warm, dry, usually quite sandy or gravelly, exposed and often denuded plains, hillslopes and ridge-summits, in the grassland region of southern Saskat¬ chewan, north to the Saskatoon and Battleford areas (see Map 3). The flowers appear in June, and fruits mature by fall. This is our most common cactus, locally frequent even as far north as Saskatoon. In some sand-hill areas, its frequency appears increased by overgrazing. Boivin's statement that this species is more southern in the Prairie Provinces than the Brittle Prickly-pear seems incorrect for Saskatchewan, since even at the northernmost known stations where they coexist, the Plains Prickly-pear is more abundant. Presettlement Indians, white pioneers, and even modern-day people have used the berry-fruits of cacti as a food source. The edible part is the fleshy pulp between the firm outer coat and seeds. The sweet juicy berries of our Purple Ball Cactus are edible. But the unusually dry, leathery fruits of the two prickly-pear species that are native to Saskatchewan make them less edible. The younger fleshy stem-pads of prickly-pear cacti have also been used for food after peeling off the outer coat along with the areolar spines (or else burning off the spines and glochids) and then roasting, boiling, or frying the internal parts. Prickly-pear cacti have also been used as an emergency food source for cattle, after burning off the surface spines and glochids.
Saskatchewan Distribution
Maps 1, 2 and 3 show the known documented records in Saskatchewan of the three native cactus species. The dots on the provincial maps are based entirely on personally verified herbarium specimens filed in Saskatchewan and Ottawa herbaria. Thus, the full distributional picture for these cacti in the province is likely quite incomplete. Information about other locality records to help fill in the Saskatchewan distributions would be welcomed by the author. Better distributional information seems especially needed for eastern Saskatchewan.
Brittle Prickly-pear might even be looked for in the southern fringes of the boreal forest in dry rocky clearings, as it has been reported from such habitats in eastern Manitoba.23 | v3-fos |
2019-03-19T13:11:58.992Z | {
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} | s2 | A karyotypic and anatomical study of an unidentified liliaceous plant
An unidentified sterile liliaceous plant and three possible relatives were studied karyotypically and anatomically. All these plants have a somatic chromosome number of 2n = 14. The possibility of the sterile plant having arisen as a result of a mutation appears unlikely, when compared with the possibility of hybrid origin. Chromosome morphology rules out Bulbine latifolia (L.f.) R. & S. and Aloe arborescens Mill, as possible parents. The sterile plant and Aloe marlothii Berger have similar karyotypes and. therefore. A. marlothii may be one of the parents. A close relationship between the sterile plant and the genus Aloe is further confirmed by their similar epidermal structure.
M ATERIALS A N D METHODS
Root tip material of Aloe arborescens (Hardy 6045), A. marlothii (Henderson 352), Bulbine latifolia {Mauve 4995) and the supposed hybrid {Hardy 6055) were collected in the Pretoria National Botanical Garden and pretreated in monobromonaphthalene at room temperature. After four hours, the root tips were washed in running water and fixed in Carnoy fixative (Darlington & La Cour, 1976) for 24-36 hours. After hydrolysis for 10 minutes at 60° C in IN HC1, the material was stained in leucobasic fuchsin (modified formula after Coleman, 1938;Darlington & La Cour, 1976) for 2 hours. The darkly stained parts were then squashed in aceto-orcein (La Cour, 1941). Slides were made permanent by placing them in a fridge for ±10 minutes, removing the coverslip with a needle, dehydrating through an alcohol series and mounting in Euparal. Ten cells per plant were studied.
Leaf segments were boiled for 10 minutes in concentrated H N 0 3 to prepare epidermal peels. The epidermis was separated from the rest of the leaf during this process and was removed from the debris, stained in Methylene Blue, dehydrated in alcohol and mounted in Euparal.
Voucher specimens of the material studied are lodged in the National Herbarium, Pretoria.
(a) Bulbine latifolia
A somatic chromosome number of 2n = 14 for this species, as reported by Snoad (Darlington & Wylie, 1955) and Jones & Smith (1967), was confirmed. The haploid karyotype ( Fig. 1) consists of two large metacentric to submetacentric, two large subtelocentric and three small subtelocentric chromosomes. An idiogram ( Fig. la) has been compiled from the data given by Jones & Smith (1967). The results of the present study are shown in Fig. lb.
The two idiograms illustrate small differences in the karyotypes. The present study revealed the second chromosome pair to be submetacentric, whereas the published data show a metacentric chromosome pair. The fourth chromosome pair was found to be of similar length to the third pair, but with the centromeric index slightly lower. Although Jones & Smith also found the fourth pair to be more subtelocentric than the third, the two pairs could also be distinguished by a marked variation in length. The third difference observed in this study lies in the presence of satellites on the long arms of the fifth chromosome pair. No satellites were reported by Jones & Smith (1967).
These karyotype differences between the pub lished and observed data indicate the existence of chromosomal variability in B. latifolia. A cytotaxonomic study of this species should prove valuable.
(b) Aloe arborescens
A somatic chromosome number of 2n = 14 was observed and confirms reports in the literature (Taylor, 1925: Ferguson. 1927Resende, 1937;Muller. 1941;Snoad. 1951;Sharma & Mallick, 1966). The haploid idiogram (Fig. 2) illustrates four long subtelocentric chromosomes and three short subtelocentric ones. Satellites are present on the distal part of the long arms of the fourth chromosome pair. Apart from the SAT-region on the fourth chromosome pair, no secondary constric tions, as described by Sharma & Mallick (1966), were observed.
(c) Aloe marlothii A somatic chromosome number of 2n = 14 was found and agrees with reported observations (Resende, 1937;Riley, 1959). The haploid idiogram (Fig. 3) shows four long and three short subtelocen- (Fig. 4). The third and fourth chromosome pairs have satellites on the distal parts of the long arms. These satellite-bearing chromosomes have slightly larger arm ratios than the other long chromosomes (Fig. 4). The karyotype suggests that the chromosomes might be arranged in seven homoeologous pairs with no, or very small differences being visible between the chromosomes within a pair.
Morphology and epidermal structure. The small distichous-leaved plant produces suckers. The mother plant is c. 60 mm tall with c. 9 pairs of smooth, linear-acuminate, succulent, unequal lea ves, the longest c. 60 mm long and c. 10 mm broad with a few small teeth at the apex. The morphology of the different plants can be seen in Figs 5 -8 .
The epidermis of Bulbine latifolia consists of elongated quadrangular cells (Fig. 9). Both the Aloe species, and the sterile plant, have hexagonal epidermal cells. Epidermal cells of A. arborescens (Fig. 10) are smaller than the cells of A. marlothii (Fig. 11) and the sterile plant (Fig. 12).
DISCUSSIONS A N D CONCLUSIONS
The sterile plant might represent an undescribed species or may have originated by hybridization or by mutation. If it is a hybrid, the parents must have occurred in the vicinity of the hybrid and the hybrid will have one genome from each parent. Since all possible parental species in close proximity to the sterile plant have been investigated, the similarity between genomes might have an indication of relationships. A comparison of the haploid idiogram of the possible hybrid with that of Bulbine latifolia reveals large differences. The presumed hybrid has no chromosomes that are nearly metacentric as in B. latifolia and the short-satellited chromosomes of B. latifolia are absent in the hybrid. If B. latifolia was one of the parental species, the resulting hybrid must have received at least two large metacentric chromosomes and a short chromosome with satellites from this source. This observation indicates that B. latifolia could not have been one of the parents. Further evidence for this is illustrated by the epidermal structure, where B. latifolia has a totally different epidermal structure from the hybrid which has a typical Aloe pattern (Figs 9 -1 2 ).
The epidermal structure of the hybrid corres ponds closely with the patterns found in Aloe arborescens and A. marlothii (Figs 10-12). A closer relationship between the hybrid and the Aloes studied is further confirmed by the similarity in their karyotypes. However, as the hybrid has two satellited chromosome pairs and A. arborescens only one, A. arborescens is an unlikely parent. The karyotypes of A. marlothii and the hybrid agree in almost all respects. The only observed differences were in the arm ratios of the first two chromosome pairs. These small differences might result from a statistically inadequate sample, or from the fact that the first two pairs are of almost similar length and the first and second pair might be interchanged in the presumed hybrid.
If the sterile plant is the product of mutation, the parents must have a karyotype similar to A. marlothii. The chances of a mutation transforming a fertile plant, a few metres tall, to a 6 cm sterile plant and hard thorny leaves to smooth fleshy leaves at the same time seems very unlikely. The suggestion of hybridization seems more probable.
The restricted growth habit and absence of flowers in the sterile plant suggest hybridization rather than a new species. The similarity between the sterile plant and A. marlothii with regard to karyotype and epiderm al structure make it reason able to suggest that A. marlothii might be one of the parents. Although no other possible parent was found in the vicinity of the supposed hybrid plant, a hybrid origin for the sterile plant is still possible. The second parent might have died by the time the hybrid was discovered. It is also possible that the hybrid was introduced into the area where it was found.
The results of this study indicate, therefore, that the sterile plant is a hybrid between A. marlothii and another species of Aloe. | v3-fos |
2018-04-03T04:56:42.384Z | {
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} | s2 | The attempted enrichment of beer with thiamine alkyl disulphides.
The Wernicke-Korsakoff syndrome, commonplace in Australia, might be prevented by the enrichment of alcoholic beverages with thiamine. The use of the well absorbed thiamine alkyl disulphides for the enrichment of the most relevant Australian beverage, namely beer, is examined. A liquid chromatographic method is described whereby thiamine tetrahydrofurfuryl disulphide and thiamine propyl disulphide can be detected in beer in concentrations down to 125 ng/ml. It is concluded that the thiamine alkyl disulphides offer no special advantage because their disulphide bonds are reduced by substances in beer, yielding free thiamine.
In Australia the Wernicke-Korsakoff (W-K) syndrome, a condition seen almost entirely in alcoholics after they have become acutely thiamine deficient, is so common that it does not, as elsewhere in the world, constitute a rarity (1,2). For example, in the largest mental hospital in the Australian State of Queensland, Price and Theodoros (1) found 170 cases of the Korsakoff syndrome out of a total inpatient population of approximately 1,100. The W-K syndrome comprises an acute encephalopathy (Wernicke's encephalopathy) followed in the majority of cases by Korsakoff's psychosis characterized by a variably severe but often permanent impairment of short-term memory.
Price and Theodoros supported Centerwall and Criqui's assertion (3), based on a U. S. study, that it would be cost-beneficial to supplement alcoholic beverages with thiamine in order to prevent the W-K syndrome occurring. They saw it as necessary to determine what beverages were being consumed in the critical period prior to the onset of Wernicke's encephalopathy, arguing that only the supplementation of these beverages could be justified. In Queensland, where their study was carried out, the beverage most frequently consumed during the critical period was beer, this beverage being all or part of what had been consumed in over half the cases where reliable information was available. The acute thiamine depletion leading to the W-K syndrome related to the absence of significant amounts of thiamine from alcoholic beverages plus the fact that patients virtually stopped eating during the critical period.
Malabsorption of thiamine may occur in alcoholics (4,5) and a deficiency of the folic acid necessary for the transport of water-soluble thiamines such as thiamine hydrochloride may also occur (6,7). Furthermore, the absorption of these vitamins may be interfered with even in non-alcoholic subjects if they are taken with alcohol (4,8). Hence there would appear to be merit in supplementing alcoholic beverages not with water-soluble derivatives of thiamine, but with one of the much more readily absorbed lipid-soluble alkyl disulphides (9) or related homologues (10).
Price and Theodoros (1) demonstrated the stability of thiamine hydrochloride in beer where its presence can be recognised by utilising the thiochrome reaction; this reaction provides the most frequently used method for measuring water-soluble thiamines in foodstuffs. The question arises whether the thiamine alkyl disulphides and their homologues are similarly stable in beer. However, the thiamine alkyl disulphides studied here, thiamine tetrahydrofurfuryl disulphide hydrochloride (TTFD HCl) and thiamine propyl disulphide (TPD) do not give a thiochrome reaction. Hence it became necessary to develop an alternative method for analysing these substances in which the presence of the thiamine alkyl disulphide would not be masked by interfering substances contained within beer, a highly complex chemical mixture.
RESULTS
The blank beer sample chromatogram contained a small peak which co chromatographed with TTFD thus making direct measurement of TTFD in fortified beer impossible. A correction was made by subtracting the height of this peak from the combination peak seen in fortified beer. The chromatogram of a beer sample which has been fortified with TTFD HCl and TPD is shown in Fig. 1. Three other brands of beer which were fortified and extracted yielded very similar chromatograms to that depicted in Fig. 1. The retention times were 8.4min for TTFD, 15min for TPD and 20.1min for the internal standard, nitrazepam. The detector responded to quantities of TTFD and TPD down to 50ng. Table 1 shows that when TTFD HCl or TPD, ranging in concentration from 125 to 1,000ng per ml (2,000ng per ml for TPD) were added to beer just prior to assay, there was a linear relationship between concentration and the peak height ratios of each compound to the internal standard. | v3-fos |
2018-04-03T04:33:33.547Z | {
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} | 0 | [] | 1983-04-01T00:00:00.000Z | 21045382 | {
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"Medicine"
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} | s2 | Efficiency of utilization of soy protein isolate in Japanese young men.
The nutritional quality of soy protein isolate (SPI) was evaluated in young men by the nitrogen balance method using fish protein as a standard. Twenty-one male university students were given SPI (Supro 620, Ralston Purina Co., St. Louis, Mo., U.S.A.), fish protein (cod fish) or a 50:50 mixture of the two proteins as the sole source of protein. SPI and fish were prepared as paste products (kamaboko). Four experimental periods were used in which each subject received 0.35, 0.45, 0.55 and 0.65 g protein/kg/day, respectively, given in random order. Each period consisted of one day on protein-free diet and ten days on the experimental diet, with an interval of three days on a free-choice (ad libitum) diet between periods. Energy intake was constant for each individual to maintain their body weight (44.6 +/- 2.4 kcal/kg/day). The linear regression equations obtained between nitrogen intake (X: mg N/kg/day) and the apparent nitrogen balance (Y: mg N/kg/day) were as follows: SPI, Y = 0.298X-35.2; fish protein, Y = 0.365X-31.8; mixed protein, Y = 0.423X-38.3. The nitrogen requirement for maintenance of nitrogen equilibrium determined from the regression equation was 118.1 +/- 15.4 mg N/kg/day for SPI, 87.1 +/- 17.2 mg N/kg/day for fish protein and 90.5 +/- 17.1 mg N/kg/day for mixed protein. The NPUs calculated at the respective maintenance nitrogen intakes were 38.9, 52.8 and 50.8 for SPI, fish protein and mixed protein, respectively. There was no significant difference between the nutritive values of mixed protein and fish protein. The nutritive value of SPI relative to fish protein was estimated as 82%, 74% and 74% by the slope-ratio method, and values for the nitrogen requirement and NPU, respectively.
Summary
The In Japan, soybean is an important traditional food item together with rice and fish, and serves as a good source of protein. Soybean is eaten in various forms such as miso (fermented soybean paste), soy sauce, tofu (soybean curd) and natto (fermented soybean product) as well as simply as cooked soybeans. In 1977, protein intake from soybean was estimated to be 7.6g/person/day, corresponding to be about 10% of the total daily protein intake of Japanese. In contrast, the primary use of soybeans in the United States is for oil and the residual meal after extraction of oil is used as animal feed or fertilizer.
With advances in food technology, new soybean products have been in troduced, such as soybean flours, concentrates and isolates. In recent years, the consumption of soybean products in a variety of food industries has increased rapidly for economic reasons and because of the multiple functional properties of these products such as for gelation, increase in viscosity, emulsification, dough formation, aeration, and improvement of texture (1). Soy protein isolates are used in processed meat, fish paste products (kamaboko, chikuwa, fish sausages), bakery products and dairy-type products. They are also used as protein supplements in infant formulas and hospital diets.
Besides having wide applications for use in processed foods because of their unique functional properties, soy protein isolates like other vegetable proteins have advantages over animal proteins with respect to lipid metabolism (2). Moreover their nutritional value is important, because protein malnutrition is a world-wide problem and a plentiful supply of cheap high quality protein foods is urgently required to meet the needs of the rapidly growing world population. Although soy protein has a low content of sulfur-containing amino acids, it is considered to be a good quality vegetable protein.
Thus in view of the growing use of soybeans in human foods and the importance of soy protein in protein nutrition, we studied the nutritional value of soy protein isolate and its minimum requirement for apparent nitrogen equilibrium compared with those of fish protein. We also examined the improvement of the nitrogen balance achieved by mixing an equal amount of fish protein with soy protein isolate. Anthropometric measurements as well as hematological exam inations and urinalyses were conducted in an attempt to detect possible changes in body composition and metabolic status of the subjects.
MATERIALS AND METHODS
Three series of experiments using soy protein isolate, fish protein and a mixture of the two proteins as protein source were conducted on 21 male university students. The characteristics of the subjects are shown in Table 1. The subjects were all healthy and lived in a metabolic ward of our laboratory throughout the study. During the feeding period, they continued their daily routine activities and their physical activities were roughly measured with a pedometer. Subjects C and D could not participate in one of four experimental periods with fish protein because of febrile upper respiratory infections.
The experimental design is shown in Fig, 1. After a four-day adjustment period during which subjects were given 1.25g/kg of protein and maintenance energy from conventional foods, each subject received four low levels of protein successively. Subjects were randomly assigned to each low protein level according to the Latin Feces in the last eight days at each protein level were pooled. The average daily fecal nitrogen loss in each period was used in calculating the nitrogen balance and digestibility. Square Design to avoid an effect of the previous protein intake on the results. Each period consisted of one day on protein-free diet and ten days on low-protein diet, and was followed by a three-day interval on free-choice (ad libitum) diet (Fig. 1). The protein sources were soy protein isolate (Supro 620; Ralston Purina Co., St. Louis, Mo., U. S. A.) ( Table 2) for five subjects, fish protein (cod fish as a standard) for eight subjects and a mixture of equal amounts of soy protein and fish protein for eight subjects, of whom five (Subjects A, B, C, D and E) also at each protein level was used for calculating the nitrogen balance. Feces in the last eight days of each test period were pooled. True digestibility was calculated at each level of protein intake using the value of 12.4mg N/kg for metabolic fecal nitrogen (3). The nitrogen balance response to nitrogen intake was observed for each individual and the regression equation relating nitrogen balance to nitrogen intake was calculated from pooled data together with the 97.5% confidence intervals. The minimum nitrogen requirement was estimated from the regression line. Net protein utilization (NPU) at nitrogen equilibrium was also calculated. The nutritional value of soy protein isolate was estimated from the slope of the nitrogen balance response curve and compared with that for fish protein.
Body weight was measured daily at 7:00 a. m. with minimal clothing after voiding urine and before breakfast. Total nitrogen, creatinine (4), urea (5), am monia (5), uric acid (6), sodium and potassium in urine samples were measured. Blood was drawn from an antecubital vein at 6:30 a. m. after an overnight fast of about 10hr on the last days of the adjustment period and of each period on low protein diet. Routine hematological analyses (erythrocyte and leucocyte counts, hemoglobin and hematocrit) were carried out, and total protein (7), albumin (8), urea (5), SCOT (9), SGPT (9), glucose (10), triglyceride (11), total cholesterol (12), sodium and potassium in plasma were also measured. Anthropometric measure ments to detect possible physical changes, such as measurements of the circumfer ence of the chest, waist and mid-upper arm and skin-fold thicknesses of the subscapular, suprailiac, triceps and biceps areas were conducted at the ends of the adjustment period and each period on low protein diet.
RESULTS
Date on nitrogen balance with the three proteins are summarized in Table 5. Fecal nitrogen did not increase appreciably with increase in soy protein or fish protein intake and therefore the two proteins were almost completely digested. Nitrogen balance for soy protein was significantly lower than that for fish protein (p<0.05) at the same protein intakes of 0.45, 0.55 and 0.65g protein/kg/day. There were no significant differences between the nitrogen balances of fish protein and mixed protein at any level of nitrogen intake tested.
Individual nitrogen balance data were plotted against nitrogen intake in Figs. 2, 3 and 4. From these results, regression equations relating nitrogen balance to nitrogen intake for individual subjects were calculated and are given in Table 6. Nitrogen balance in subject K given fish protein was inversely related to nitrogen intake and so these data were omitted from further analyses. The slopes of regression lines showed wide individual ranges of 0.210-0.396 for soy protein, 0.158-0.461 for fish protein and 0.171-0.620 for mixed protein. The regression lines obtained from pooled nitrogen balance data are shown in Fig. 5. The slope for soy protein (0.298) was smaller than those for fish protein (0.365) and mixed protein (0.423), but the differences were not statistically significant. The mean nitrogen intake for apparent nitrogen equilibrium (intercept of the lines for nitrogen intake with nitrogen equilibrium) determined from individual regression lines was 120.5mg N/kg (range 101-138) for soy protein, which was significantly higher than 87.7mg N/kg (range 66-104) for fish protein (Table 6). There was no significant difference between the maintenance nitrogen intakes of fish protein and mixed protein. The coefficient of variation of the maintenance nitrogen intake was 13.3% for soy protein, 16.5% for fish protein and 22.9% for mixed protein. The maintenance nitrogen levels obtained from pooled data, that is, 118.1 mg N/kg for soy protein, 87.1mg N/kg for fish protein and 90.5mg N/kg for mixed protein, were similar to those obtained from individual data ( (17) found on the basis of nitrogen balance data that although the nutritional value of textured soy protein was inferior to that of beef at 4g nitrogen intake, those of the two proteins were similar at 8g nitrogen intake per day. Zezulka and Galloway (18) reported that the average nitrogen balances obtained with soy protein isolate were -1 .21, -0.08, +0.26 and +0.79g nitrogen per day at 3.0, 4.5, 6.0 and 7.5g soy nitrogen intake per day, respectively, with a total nitrogen intake of 9g/day adjusted by adding non-specific nitrogen in the form of a mixture of glycine and alanine in isonitrogenous amounts. The maintenance nitrogen intake obtained from their nitrogen balance data is considerably lower than that obtained in this study, possibly because of the difference in the total nitrogen intake. Young et al. (15) observed a similar value to ours for maintenance nitrogen intake of soy protein isolate (judging by inspection of their figure for nitrogen balance (15); Fig. 5, p. 114). They also calculated the relative nitrogen requirement of soy protein isolate as 80% of the egg protein requirement, which is similar to our value of about 75 described above.
The lower quality of soy protein relative to fish or egg protein may be due to its lower content of sulfur amino acids. Kies and Fox (17) and Zezulka and Galloway (18) observed an improvement of the nitrogen balance by adding methionine to soy protein at low nitrogen intake. Young et al. (15) observed a similar effect when 0.6% or 1.1% methionine was added to soy protein at 0.51g protein/kg/day. But they observed a negative effect when 1.6% methionine was added to 0.51g soy protein/kg/day, although a positive effect of 1.6% methionine was still observed at 0.8g soy protein/kg/day. In the present study, we examined nitrogen utilization with soy-fish combination instead of methionine supplemen tation. Soy protein in combination with an isonitrogenous amount of fish protein had essentially the same nutritive value as fish protein alone judging from data on nitrogen balance, the slope of the regression line, the maintenance nitrogen intake and the NPU. Kies and Fox (17) and Young et al. (15) investigated the protein quality of combinations of soy and beef in ratios of 4:0, 3:1, 2:2, 1:3 and 0:4. Kies and Fox (17) obtained a linear increase in nitrogen balance with increase in the ratio of beef to soy at 4.8g nitrogen per day per person, but Young et al. (15) observed no differences in the nitrogen balance with the same combinations of beef and soy protein at 0.6g protein/kg body weight/day. Thus, the nutritional quality of soy protein may depend on the level of protein intake as well as the individual soy protein product.
It is concluded from the present study and the above discussion that soy protein isolate has a nutritive value of about 80% of that of fish or egg protein and is a good source of protein for human consumption. However, long-term studies like those of Young et al. (15) should be carried out on a wide variety of subjects to detect possible adverse effects of this protein source. Some blood analyses and anthropometric measurements were carried out by Prof. K. Oikawa and Prof. K. Fujiwara, School of Medicine, The University of Tsukuba. Paste products (kamaboko) of Supro 620 and cod fish were supplied by Ralston Purina Co., St.
Louis, Mo., U. S. A. and Fuji Oil Co., Osaka, Japan. This work was supported in part by a grant (No. 357049) from the Ministry of Education, Science and Culture of Japan. | v3-fos |
2019-03-31T13:43:09.407Z | {
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} | 0 | [] | 1983-11-06T00:00:00.000Z | 87641203 | {
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} | s2 | A preliminary survey of primitive crops cultivated in the northern Transvaal of South Africa
The different tribal economies of South Africa rely extensively on a number of primitive crop taxa which are cultivated as a primary food source. The most important of these include Sorghum bicolor, Pennisetum americanum, Citrullus lanatus, Lagenaria siceraria, Vigna unguiculata, Voandzeia subterranea and Hibiscus esculentus. Morphological variation within these and a number of less important crops is discussed. The frequency with which each crop is grown and preference ratings allotted to them by individual tribal families are compared between the three major ethnic regions of the northern Transvaal. Factors which determine preferences are also discussed and suggestions made relating to germ plasm conservation.
INTRODUCTION
Little is known about the exact nature and diversity of primitive crop taxa cultivated in South Africa, despite that many of these crops, for example. Sorghum bicolor, Pennisetum america num, Vigna unguiculata and Citrullus lanatus, exhibit a considerable range in variation and are extensively cultivated by the various ethnic groups throughout the country.
The majority of these crops are considered to have originated and evolved in North and West Africa (Harris, 1976), with South Africa represent ing merely the southern most extreme of their distibution. This may account, at least in part, for many workers (De Wet, 1972;Doku & Karikari, 1971;Stemler et al., 1977 andWestphal, 1974), investigating the origin, evolution and taxonomy of these crops, having focused their attention upon the northern half of the continent. Where reference is made in the literature to primitive crops grown in this country (Brunken et al., 1977;De Wet & Huckabay 1967;De Wet et al., 1976;Harlan & Stemler, 1976;and Fursa, 1972), it is largely inadequate, based on limited information and usually forms part of more detailed investigations centred around the region of origin of these crops.
In order to expand our knowledge of primitive crops grown in South Africa and bring it into line with that from elsewhere in Africa, a detailed study of the morphological variation and distribution of these crops is being undertaken by the present authors, together with extensive germ plasm collection. Included in this paper is some of the information so far collected from the Transvaal, which is presented essentially as an introduction to a much wider study covering the whole country. STUDY AREA AND SAMPLING METHODS Information presented in this paper was gathered during a pilot study of the ethnic regions of Lebowa. Gazankulu and Venda in the northern Transvaal of South Africa (Fig. 1), and represents part of a more extensive survey to be undertaken throughout the Republic of South Africa.
A total of 119 families from 73 villages was interviewed in the three regions. These include: Lebowa -17 families from 13 villages, Gazankulu -67 families from 31 villages, and Venda -35 families from 29 villages. With the help of interpreters, a questionnaire was completed for each family. Herbarium specimens and seed were collected to support the information gathered for the questionnaire.
It is impossible to provide definite conclusions as to the nature and variability of the crops cultivated, because only a limited number of families were sampled in each village. Additional information requires to be gathered for a more complete assessment of the situation. The extent to which each crop varied morpholo gically differed between the taxa. Phaseolus radiatus and Hibiscus esculentus showed little variation, whereas the remainder showed extreme variation not only at the regional level but also within a single village and even within individual cultivated fields. Variation within the major primitive crops is discussed below, as well as that observed in Arachis hypogaea and Cucurbita sp.
Sorghum bicolor (Sorghum).
All five cultivated races of S. bicolor recognized by De Wet (1978) were recorded. Race Caffra is the most common, followed by races Guinea and Bicolor. Caudatum sorghums were present in Venda, with a single collection from Lebowa, and race Durra was recorded twice from a single village in Venda.
Morphological variation was considerable in all the races except race Durra, with Caffra sorghums exhibiting the greatest amount of variation. Varia tion within each race was expressed largely in the size and degree of compaction of the inflorescence, the number of grains per inflorescence, the colour of the glumes, and the size and colour of the grains. Seven Intermediate races were also observed (Intermediate races according to Harlan & De Wet, 1972). Morphological variation in South African sorghums is discussed in greater detail by Arnold (1982).
Pennisetum americanum (Bulrush!Pearl millet)
This crop is cultivated in differing amounts throughout the study area. Weedy forms (subsp. stenostachyum), distinguished by their disarticulat ing spikelets, were often found growing alongside cultivated forms (subsp. americanum). The amount of variation observed within this crop differed markedly between the different regions. In Lebowa variation of the inflorescence was minimal, whereas in Venda it was often difficult to find two inflorescences from a single field that looked suitably alike.
Throughout the study area inflorescence length ranged between 10 and 55 cm, and included club as well as candle-shaped forms. Involucral bristles (3-10 mm in length) varied from § spikelet length in some individuals to three times spikelet length in others. Grain size also varied (2,1-4,1 mm by 1,3-2,5 mm) as did grain colour which ranged from cream-yellow to light or dark grey.
Vigna unguiculata (Cowpea)
Cowpeas are commonly cultivated in all three regions. Two basic forms were collected: a soft, purple-podded form characterized by purple leaves; and a hard, cream-podded form with green leaves. The beans were found to be extremely variable, particularly in colour, but also in size and shape. Colour forms included cream, beige, orange, red, mauve, purple and black and were either plaincoloured or speckled. A high proportion of diminutive, angular beans were also observed.
Voandzeia subterranea (Juga bean/African or Bambarra groundnut)
Juga beans exhibited similar although less variation than cowpeas. This was manifested mainly in the colour of the beans, which ranged from cream (often with black around the hilum) to beige, orange-brown, red, purple and black. Speckled forms of most of these colours also exist.
Citrullus lanatus (Watermelon)
Two major types are grown. The first includes the sweet melons which have a relatively spongy, sweet, cream-white or pink flesh, and are eaten raw immediately they are harvested. The second type includes the cooking melons, with an insipid tasting, slightly firmer flesh, ranging in colour from cream-white to pale yellow or salmon-orange.
Melon size ranged from 10-35 cm in length (diameter in spherical forms). Skin colour patterning is highly variable, being plain light or dark green, or cream-green coloured (cooking type) or a combina tion of these colours, either mottled, speckled, or arranged in stripes (sweet and cooking types). Intermediate patterns also exist. The seeds showed a similar range in variation, ranging in length from 0,9-1,4 cm and in colour from cream to green, light or dark brown, red and black. Speckled forms of the green, brown and red colours also exist.
Lagenaria siceraria (Gourd!Calabash)
As in the case of the watermelons, calabashes are grown throughout the study area. Variation is limited to the fruits and seeds. Two basic groups are recognized by the different ethnic tribes, based on utilization. In the first group, the calabashes are characterized by their suitability for use as household utensils (water or medicine containers, bowls, scoops for water, beer or meal, etc). Calabashes that cannot be used as utensils comprise the second group cultivated specifically for eating.
A number of basic fruit shapes can be recognized in both the utensil and eating forms, but at this stage these have not been formally categorized. Interme diate shapes have also been collected. The colour of the skin remains relatively uniform, varying between light and dark green and changing with age to cream-beige. Skin texture is either smooth or rough (covered in small ± 1 cm high rounded or angled growths). The rough forms are always eaten irrespective of their shape.
The seeds comprise two major types, those with wing-like margins and those without. A single intermediate form with the margins partly developed has been observed. Seeds range in size from 1,3-2,5 cm in length and 0,9-1,4 cm in width and vary in colour to include cream-yellow, brown and olivebrown.
Hibiscus esculentus (Okra)
Together with Sesamum indicum, this crop is grown almost exclusively in Gazankulu (by 60% of the families interviewed) and in immediately adjoining areas of Lebowa and Venda. This suggests a later introduction into South Africa than other primitive crops, possibly from Mozambique. Two forms are recognized by the families who grow this crop: the first includes plants with short, fat fruits 7,8-10,0 cm long and 3,6-3,8 cm broad, whereas those of the second form are noticeably long and thin, 11,4-13,8 cm long and 2,1-2,2 cm broad. A range of intermediates exists between these two extremes.
Phaseolus radiatus (Mung bean)
Although grown in Gazankulu and Venda, mung beans are important only in Lebowa where they are grown by 53% of the families interviewed. Morphological variation in the three regions is minimal. Throughout the study area this crop was erroneously referred to as China pea by the villagers. According to Westphal (1974), China pea is one of the many common names for Vigna Unguiculata.
Arachis hypogaea (Peanuts/Groundnuts)
This species is not native to Africa and is probably a recent introduction into tribal agriculture in South Africa. Peanuts are grown in varying amounts throughout the study area. Three distinct forms are cultivated differing in the size and colour of the nuts. These include a long red form (1,7-2,3 cm by 0, 8-1,1 cm), a small dark red form (0,9-1,4 cm by 0,6-0,9 cm) and an intermediate pink form (1,1-1,7 by 0,7-1,0 cm).
Cucurbita sp. cf. C. pepo (Pumpkin!Squash)
Like Arachis hypogaea, this crop is not native to Africa. It is considered to be a relatively recent introduction into tribal agriculture, possibly a derivative of one or more of the modern pumpkin or Hubbard squash forms grown throughout the country. Pumpkins are an extremely important crop, not only because they are grown throughout the study area, but also because selection has produced forms that are extremely hardy and reasonably well adapted to survive under low rainfall, dryland agricultural conditions. It is impossible at this stage to give a realistic assessment of the variation exhibited by this crop.
Minor crops
Crops of minor importance that are also grown include: Saccharum sp. (castor-oil bean). The variation exhibited by these crops was either negligable or not sufficiently well recorded to be discussed in this paper.
RELATIVE FREQUENCIES AND PREFERENCE RA TINGS
The frequencies with which the major corps are grown within each ethnic region are shown in Figs 2a, 3a & 4a. These data indicate what percentage of the families interviewed grew each crop. It is important to note that frequency represents merely whether the crop was or was not cultivated by individual families and not the extent (area under cultivation) to which it was grown.
At this stage of the survey, frequency is considered to be a better indicator than abundance on which to base germ plasm conservation priorities.
In Lebowa 65% of the families interviewed grew seven of the ten major crops available, with at least two crops from each of the differenct crop groups (cereals, cucurbits and pulses), including the three extra-African taxa, being represented. In Gazankulu 74% of the families grew all the major crops in varying amounts, whereas in Venda 66% of the families grew eight of the ten most important crops.
This tendency to grow as many of the major crops as possible irrespective of whether climatic condi tions favour their growth may be ascribed to a need to safe-guard against periods of climatic extremes. The drier parts of the study area are particularly prone to periods of drought which seriously affect the cultivation of maize, sorghum (in some areas), pumpkins and peanuts. When these crops are favoured in such areas the more drought resisiant taxa are also planted, but to a lesser extent, thus ensuring at least some food during bad years. Another reason for planting a large variety of crops is that besides providing a varied diet, they also provide a variety of foods with differing tastes. Also significant is that many crops have a particular desirable quality absent in other similar crops, for example, although maize is preferred in Gazankulu for meal, limited amounts of sorghum and Pennise tum americanum are also grown for making beer. Similarly, although Lagenaria siceraria is a relatively low priority crop throughout the study area, it is still cultivated, being the only crop from which household utensils are made.
Preference indicates the relative importance attached to each crop by different ethnic tribes and is seen also as a potential indicator of possible trends towards or away from specific crops. The data presented in Figs 2b, 3b & 4b represents the percentage of families that allotted a 1st preference rating to the various crops. As some families gave a priority one rating to more than one crop, the percentage totals for the different crop groups (cereals, cucurbits and pulses) generally exceeded 100%. Preferences did not necessarily reflect the frequency with which the different crops were grown. Where preferences contrasted strongly with frequencies, this was due largely to the tendency for the majority of families to grow as many of the major crops as possible. The reasons for this have already been discussed.
Cereals
In Lebowa (Fig. 2) Pennisetum americanum is less frequently grown (47% of the families) than sorghum and maize (100% respectively). This is also shown by the preference ratings. Although P. americanum is more suited to the drier parts of this region, sorghum is more frequently grown as it is considered much more palatable. In Gazankulu (Fig. 3), maize constitutes a serious threat to sorghum and P. americanum cultivation. It is grown in large quantities throughout the region and is preferred by the vast majority of families. The cultivation of cereals in Venda (Fig. 4) is closely related to the climate of the region and is also closely correlated with the preference rating for each of these three crops.
Cucurbits
In Lebowa (Fig. 2) the frequency of these crops follows the trend of preference ratings alloted to them In Gazanki'lu ( Fig. 3) pumpkins are highly preferred to watermelons and gourds despite all three of these crops being grown by 80% of the families. In Venda (Fig. 4) the three cucurbits were grown with similar frequency. The reason for the low preference rating for Lagenaria siceraria throughout the study area is due, at least in part, to the increasing supply of metal and plastic utensils from local stores.
Pulses
Vigna unguiculata, grown by 88% of the families in Lebowa, is the most important pulse crop in this region (Fig. 2). Here, unlike Gazankulu and Venda, Phaseolus radiatus is also commonly cultivated (53% of the families). This latter crop, although occasion ally eaten, is grown mainly as a cash crop. The frequency of pulses grown in Gazankulu (Fig. 3) follows closely the preference ratings allocated to these crops. A similar situation was also found in Venda (Fig. 4), with the exception of Voandzeia subterranea which is both less preferred and less grown than in Gazankulu and Lebowa, being considered a less versatile crop than Vigna unguiculata and lower yielding than Arachis hypo gaea.
FACTORS DETERMINING PREFERENCES
Whether a crop is cultivated or not and the extent to which it is cultivated, is determined largely by individual preferences (see Figs 2b,3b & 4b). Preferences, in turn, are determined by a number of factors, working individually or more commonly in combination.
Diversity o f Use
In Gazankulu maize is preferred to sorghum and pearl millet because, besides providing meal for porridge, it can also be eaten as a green vegetable during the growing season. Throughout the entire study area, and particularly in Venda, Vigna unguiculata is preferred to other pulses because, not only can the beans be eaten green, or dried and stored for later consumption, but the leaves can be eaten as spinach. Similarly, pumpkins not only have edible fruits, but the leaves can be prepared as a vegetable and the male flowers dried and stored for later consumption.
Long-term storage
Crops which can be stored are preferred to those which cannot. Cooking forms of Citrullus lanatus and pumpkins are therefore generally preferred to edible forms of Lagenaria siceraria.
Resistance to insect attack
The small seeds of pearl millet are less susceptible to insect attack during storage than most other cereals. This crop is therefore favoured more than sorghum and maize in southern Lobowa, west Venda and north-west Gazankulu.
Palatability
In the low rainfall areas of Lebowa Pennisetum americanum is considered highly unpalatable and is replaced by sorghum.
Relative yields
Preferences favour high yielding crops. Yield is generally closely related to annual rainfall and to soil type. In Venda (Fig. 5.1) pearl millet, although grown over a wide range of rainfall, is preferred to sorghum and maize in areas with an annual rainfall between 400 and 460 mm. As the annual rainfall increases from 460 to 520 mm, sorghum yields increase and surpass those of pearl millet. In these areas sorghum is preferred. Where the annual rainfall exceeds 520 mm, maize is generally higher yielding than sorghum or pearl millet and is therefore preferred to both these crops.
Although similar, the situation in Lebowa (Fig. 5.2) is complicated because Pennisetum americanum is considered unpalatable in areas of low rainfall (400 -480 mm). It is consequently replaced by the relatively lower yielding sorghum in these areas.
In Gazankulu (Fig. 5.3) the annual rainfall is between 520 mm and 760 mm for the villages sampled. Within this range, pearl millet and sorghum yield less than maize and are therefore not preferred. Where preferences were recorded for these two crops, they were shared equally with maize.
Soil type
It was generally observed for the cereals that the range in rainfall under which the respective crops grew best was increased or reduced depending on the nature of the soils. Well drained, sandy soils extend the upper rainfall limits at which sorghum and pearl millet are preferred, while reducing preference limits for maize. On the other hand, soils that tend to retain moisture extend the lower preference limits of sorghum and maize.
Commercial value
In Lebowa Vigna unguiculata, and in particularly the large speckled blue-grey seeded form which can be sold to the local milling company in Pietersburg for cash, is preferred to most other pulses. Similarly Phaseolus radiatus is grown almost exclusively as a cash crop in this region, and is therefore more frequently grown and preferred than in Gazankulu or Venda.
Susceptibility to bird damage
In many parts of the study area birds are a major problem, destroying large fields of sorghum and in particular Pennisetum americanum. In north-east Lebowa, south Venda and north-east and south Gazankulu the problem is so great that P. americanum is largely excluded from cultivation.
Nutritional value
In parts of Lebowa where sorghum is preferred it is believed that this crop prevents infantile-pellagra (caused by a protein deficiency), while maize does not. Consequently, sorghum is preferred to maize.
GERM PLASM CONSERVATION
One of the primary aims underlying this survey, besides being to assess the nature and variation of primitive South African crops, is the collection and preservation of germ plasm and the assignment of conservation priorities to these crops.
Most of the major primitive crops show a wide range in morphological variation and accordingly constitute a valuable source of germ plasm. Efforts to collect and preserve this germ plasm have been minimal in the past, largely due to a lack of realization as to its potential value. As a result manpower and funding allocated to this work has been limited. This fact is reflected in the mere 201 collections of primitive crops housed in the national seed bank (Department Agriculture and Fisheries, 1980 Poor collecting is not the only problem facing the conservation of primitive crop germ plasm in South Africa. In many parts of the study area there is an increasing tendency to move away from low yielding primative crops, and to replace these with better developed higher yielding modern crop types. This is particularly noticeable with cereals, where maize cultivation is being strongly promoted by all local agricultural authorities. This trend is also seen in the increasing importance of Cucurbita sp. cf pepo and Arachis hypogaea as agricultural crops. These changes are particularly, although not exclusively, prevalent in areas where irrigation has been made available. In these situations, the cultivation of vegetables (onions, tomatoes, chillies, cabbages and various bean types, etc), as well as fruit crops (pawpaws, oranges, mangoes, bananas, avocados and guavas) is also promoted, all of which influence the extent to which primitive crops are cultivated. Such changes in attitude do not necessarily mean that the cultivation of primitive crops is being discontinued. In most situations it is merely the reduced area cultivated (up to 90%) that causes a serious loss in germ plasm.
In assessing the situation in South Africa, it is apparent that a concerted effort is required to collect germ plasm of all primitive crops, and that this should include as much as possible of the variation expressed by these crops. In addition, the need to prescribe special conservation priority ratings to certain crops within the ethnic regions studied has emerged from this survey. Special consideration should be given to all minor crops through the study area as well as to Pennisetum americanum in the low rainfall areas of Lebowa, to Sorghum bicolor and Pennisetum americanum in Gazankulu and to Voandzeia subterranea in Venda. | v3-fos |
2019-03-19T13:08:15.419Z | {
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} | s2 | Effects of Septoria nodorum Berk. on yield and yield components of spring wheat.
Data from two experiments was analysed in order to determine the effects of Septoria nodorum Berk, on the yield of spring wheat. In the first experiment the cultivar Hankkija’s Taava was artificially inoculated with low spore concentration suspensions of S. nodorum. The resulting disease reduced grain yield by 10 %, 1000-grain weight by 14 %, and hectolitre weight by 5.7 %. An examination of the ears from the main stems revealed that the pathogen induced a reduction in all yield components but especially in grain number/ear and grain weight. In the second experiment a total of 28 cultivars or lines were studied and the correlation between grain yield/ear and disease severity was found to be negative but low. No consistent trend among the correlations was seen and some susceptible cultivars suffered only slightly from the disease while other fairly resistant cultivars showed great losses. The results are discussed in relation to compensatory mechanisms and potential disease tolerance in wheat.
Introduction
Septoria nodorum Berk., the cause of glume blotch disease of wheat, is a major wheat pathogen in many parts of the world (SHIPTON et al. 1971). Numerous reports have shown that the glume blotch disease has rapidly increased in importance in recent years causing severe reductions in wheat yields (EYAL 1981). The reason for this trend seems to be partly explained by the fact that local wheat cultivars have been replaced with short, highyielding susceptible varieties over large areas and these varieties facilitate outbreaks of the disease (SAARI andWILCOXSON 1974, RAJ ARAM andDUBIN 1977). Simultaneously there have been changes in cultivation practices such as the use of minimum tillage which promotes the epidemiological build-up of inoculum; wheat stubble being an important source of primary inoculum for disease development (HARROWER 1974, JENKYN andKING 1977). Moreover, the reduced use of crop rotation and the increased use of fertilizers may have also contributed to the occurrence of Septoria epidemics in many intensive wheat growing areas (EYAL 1981).
S. nodorum attacks wheat at all growth stages, and can infect all aerial parts of the plant (BAKER 1978). The relationship between symptom expression and yield reductions is not consistent since the pathogen is capable of causing considerable yield reduction even at a moderate level of infection (BRÖNNIMANN 1968, SHIPTON 1968, OBST 1977. The effect of early infection by this pathogen on final yield reduction may be more important than has previously been expected. Early infection may disturb tillering and influence the primary development of the ear thus reducing the potential number of sites for assimilate deposition and consequently reducing yield (SCHAREN and TAYLOR 1968).
Considerable empirical evidence indicates that major yield losses occur when infection takes place at later development stages (BRÖNNIMANN 1968, SPIERZ 1973, WAFFORD and WHITBREAD 1978. This has been explained by the observed fact that the greatest increase in dry weight of wheat grain occurs when photosynthetic assimilates are translocated to the ear after its emergence (WOOLHOUSE 1981, LUPTON 1982. Numerous experiments show that S. nodorum not only causes a heavy reduction in the rate of photosynthesis but also reduces the duration of the green-leaf-area period (SCHAREN and KRUPINSKY 1969, SPIERZ 1973, WAFFORD and WHITBREAD 1976. Hence the infection causes a decreased supply in the amount of assimilates that can be translocated to the grain, and may thus reduce 1000grain weight (SPIERZ 1973).
Precise estimates of national crop losses due to glume blotch disease are poorly known, but in England and Wales national surveys have revealed yield losses of up to 8 % in years when infection was severe (KING 1977), and the economic significance of the disease has been shown to be important (DOODSON 1981). In Finland no estimates of the economic importance of 5. nodorum are available, but there is every reason to assume that during the last few rainy years it has caused significant yield and quality reductions (KAR-JALAINEN and LAITINEN 1982).
The present paper reports on the effect of infection by 5. nodorum on the yield and yield components of spring wheat.
Materials and Methods
The data reported in this study were based on two trials carried out at the experimental farm of the Hankkija Plant Breeding Institute. The objective of the first trial was to study the effects of S. nodorum on yield and yield components using Hankkija's spring wheat cultivar Taava.
Normal plant breeding plots (8 m 2) with four replications arranged in a randomized block design were used. Standard fertilization and herbicide treatments were applied.
The first experiment consisted of the following treatments: uninoculated control, plots inoculated with S. nodorum, and plots sprayed with three kinds of fungicides. The following fungicide treatments were performed: spraying before anthesis with 2 kg/ha Benlate, with 2 kg/ha Maneb, and with a mixture of Benlate 0.25 kg/ha + Maneb 2.4 kg/ha respectively, and spraying at the postfloral stage with Benlate 1 kg/ha.
Three inoculations with S. nodorum were carried out starting before flag leaf emergence. The final treatment was made after anthesis. The inoculum consisted of about 10 4 spores/ml. The preparation of inoculum and the culturing techniques of the fungus have been previously described in detail (KARJALAINEN et ai. 1983). After inoculation all plots were irrigated to keep them wet, thus encouraging a successful disease build-up. The assessment of the disease on the different plots was made on the flag leaf and ear of 40 randomly labelled stems by estimating the percentage area covered by S. nodorum lesions. The assessment was made two weeks after the last inoculation.
The second experiment consisted of a variety test carried out with small plots to screen spring wheat cultivars for S. nodorum resistance. The details of the experiments have been previously described (KARJALAINEN et ai. 1983). The purpose of this study was to examine the relationship between disease severity and yield loss (g/ear yield). Before harvesting the labelled tillers were cut and the yield components were counted. Alter harvesting the 1000-grain weight was determined. The percentages of diseased area (leaf area values of the wheat leaves) were transformed using the arc-sin transformation. Variance analysis for comparing the yield between different treatments was calculated. Regression analysis, correlation analysis, and Path-coefficient analysis were also calculated according to LI (1975) in order to define the main effects caused by 5. nodorum on yield and yield components.
Efects of inoculation on yield and yield components
Artificial inoculation with low concentration of Septoria nodorum reduced grain yield by 10 % and 1000-grain weight by 14 % (Table 1). Inoculation also induced reduction in hectolitre weight (5.7 %). All fungicide treatments caused statistically significant yield increases: 21-30 % relative to the untreated control (Table 1). The fungicide treatments increased 1000grain weight and hectolitre weight.
The results of the data recorded of the single tillers are presented in Table 2. Inoculation reduced all yield components; the loss in ear yield was 38.5 %, in grain number/ear 17.8 %, in 1000-grain weight 28.4 %, and in spikelet number/ear 4.4 %. The fungicide treatment increased all the yield components recorded (Table 2). Relationship between disease severity and yield loss In order to test which yield components were most affected by disease stress a correlation analysis was computed. The results of the data based on single tillers obtained from the yield loss trial are presented in Table 3. The relationship between yield (g/ear) and ear severity is negatively significant (r=0.36, p<0.05), and the correlation between yield and flag leaf severity is negative but weak. The ear yield seems to be most strongly correlated with grain number/ear (r=0.71, p<0.001), and 1000-grain weight (r=0.57, p<0.01). The yield component and disease severity data was subject to pathanalysis in order to partition the correlation coefficients into direct and indirect effect. The path-analysis clearly reveals that grain number/ear and 1000 grain weight had major direct effects on the yield/ear on a single tiller basis (Fig. 1). The path-diagram not only indicates that disease directly reduces grain yield but also indirectly reduces grain number and grain weight. It also shows that grain number and grain weight had the largest direct contribution to grain yield. The results of the data recorded from 29 cultivars are presented in Table 4. The correlation between ear yield and disease severity is negative, but the coefficients are low. A detailed demonstration of the relationship between disease severity and ear yield on different cultivars is shown in Figures 2,3 Figure 4 demonstrates the combined disease rating (flag leaf value + ear value) in relation to yield loss calculated from the difference between noninoculated and inoculated plots. The greatest losses were detected in Ulla, Ruso, Tapio, Tähti, Kadett, Drabant, Allen, Maris Butler and CI 13406. The A path-diagram was also constructed from the data on cultivar trials (Fig. 5). The results confirm the previous findings ( Fig. 1) since again grain number and grain weight had major direct effects on the total yield/ear and the effects on total yield/ear due to disease were of the same magnitude. However, only half of the total variation is accounted for by these variables. Fig. 4. Combined disease rating (flag leaf value + ear value) in relation to yield loss calculated as the difference between non-inoculated and inoculated plots. The data is based of 28 cultivars and lines of spring wheat.
Discussion
The present experiments support the prevailing idea that Septoria nodorum can cause significant yield reduction even with a moderate level of infection (OBST 1977). Theresults reported here show a yield reduction of 10 % in relation to untreated control plots using a moderately susceptible cultivar, Hja Taava (see KARJALAINEN et al. 1983). The yield component which was most affected was 1000-grain weight, thus confirming the results of several experiments (BRÖNNIMANN 1968, SPIERZ 1973, NELSON et al. 1976). The hypotheses that S. nodorum causes a reduction in photosynthesis and shortens the green-leaf-area period are in accord with the results of this experiment. We found that in inoculated plots the maturation time was shorter than in the untreated control plots and further evidence can be seen from the fact that in the plots that received fungicide treatments the maturation time was 2-3 days longer than it was in the inoculated plots. This latter observation confirms the results of SPIERZ (1973) who demonstrated that Maneb and Benomyl, or their mixtures, can delay the spread of S. nodorum in the wheat crop with the result that the flag leaf remains green longer and the grain-filling period is lengthened.
Physiological studies have indicated that Septoria infection may enhance translocation because it is known to accelerate the grain maturation rate and the onset of foliar senescence (SCHAREN et al. 1975). WAFFORD and WHITE-BREAD (1976) have found that despite large lesions on the leaves and a reduction in total plant growth, S. nodorum infection appears to alter the export of assimilates from a leaf to only a limited extent, and it also seems to have only a small effect on the pattern of assimilate distribution. The results of SCHAREN et al. (1975) support this assumption since they found that the export of metabolites from Septoria-miected flag leaves did not vary greatly between control plants and inoculated plants.
The fungicide treatments caused significant yield increases in relation to untreated and inoculated plots. However, conclusions from this can only be applied to the field situation with great caution since a number of factors can affect the results of this kind of experiment. First of all, the fungicides used in this experiment only partially prevented S. nodorum infection since disease was observed on treated plots, too. The fungicides may also control other foliar diseases and this may lead to an erroneous interpretation of the yield reduction by S. nodorum (JAMES 1974, JAMES andGAUNT 1979). Furthermore fungicides may cause harmful side-effects on the plant and thus lower the yield or fungicides may have some curative properties and this may cause yield increases without preventing the occurrence of disease (FEHRMANN et al. 1978, JAMES and TENG 1979, COOKE et al. 1981. Recent findings of SMEDEGAARD-PETERSEN (1982) have also revealed that inhibition of the saprophytic fungal flora on cereal leaves with fungicides increases yield since saprophytic fungi deprive the host of energy.
The conclusion that can be drawn from the above arguments is that a comparison between inoculated and untreated control plots may be a rather reliable way to evaluate the effects of Septoria on wheat yield and this type of comparison may have applications to the field situation. The present results, where a yield loss of 10 % was seen in the inoculated plots compared to the control plots, seem to be fairly realistic, but we emphasize that this experiment resulted in fairly low yield levels even in the control, plots because of unsuitable weather conditions.
The yield component analysis showed that both 1000-grain weight and grain number/ear were heavily affected by the infection. These results agree with several experiments which have indicated that inoculation at a late developmental stage causes the greatest reduction in grain weight (JONES and ODEBUNMI 1971, WILLIAMS and JONES 1972, JONES and ROWLING 1976. These results may be explained by the observed fact that late infection mainly affects grain weight since this yield component develops later compared to other yield components which develop during early stages (TENG and GAUNT 1980). Heavy infection seems to alter all yield components and hence reductions in the total yield of single tillers are evident. However, this experiment detected that in some tillers reduction in 1000-grain weight was compensated by more grains being filled per ear. This observation agrees with those of WAFFORD and WHITBREAD (1978) and JONES and ROWLING (1976) and their conclusion that compensatory mechanisms might be too weak after heavy infection to prevent large yield losses is also confirmed by our results ( Table 2).
The present results suggest that path-analysis can be a powerful tool for analyzing the relationship between disease severity ratings and yield component factors. Constructing a path-diagram can be a clear and simple way to visualize disease effects as it partitions direct factors from indirect factors as they affect yield. For example, the present data reveals some interesting points regarding the different yield components. We found that grain number was the most important factor in determining the final yield/ear although several studies done on healthy wheat (WOOLFIOUSE 1981) show that kernel weight is the biggest contributor to the final yield/ear. Our experiments seem to confirm the idea that when wheat is attacked by S. nodorum it compensates for lower kernel weight by the development of more kernels.
The hypothesis that S. nodorum causes a reduction in yield which is not correlated with symptom expression (OBST 1977) was evaluated in this study. The results support this idea since we found that the correlation between disease severity and yield loss were low and our comparison between different cultivars with regard to yield loss in relation to disease severity shows many inconsistencies (Fig. 3). Some cultivars such as Pilot, Kadett, Drabant and Maris Butler, which have been found to be rather resistant to S. nodorum (KARJALAINEN et ai. 1983), were seen in this study to suffer great yield losses. On the other hand some cultivars such as Hja 21600 and Hja 22159, which had been found to be fairly susceptible to S. nodorum (KAR-JALAINEN et ai. 1983), were seen in this experiment to suffer only a small yield reduction.
These inconsistencies may possibly be explained by the phenomena of tolerance (BRÖNNIMANN 1968(BRÖNNIMANN , 1982 however they may also be explained by experimental error since reliable yield comparisons require a large number of replications and careful disease assessment (SCOTT andBENEDIKZ 1977, GAUNT 1981). More experiments are needed to test the above assumption that some susceptible cultivars have tolerance to S. nodorum. | v3-fos |
2019-04-01T13:12:17.284Z | {
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} | s2 | EFFECTS OF QUANTITY AND QUALITY OF CARP SPERM ON EGG FERTILISATION SUCCESS
During the artificial spawning, the carp ( Cyprinus carpio L.) eggs were fertilised with sperm added in various volumetric and quantitative proportions and assessed in terms of spermatozoa motility. The spermatozoa/egg ratio was based on spermatozoa densities in each sample. The analysis of the fertilisation success served to verify conditions crucial to the artificial fertilisation of carp. The results obtained show the percentage of fertilised eggs to increase with increasing number of spermatozoa per 1 egg, provided the spermatozoa are performing the progressive movement.
INTRODUCTION
Reproduction of fish under artificial conditions makes it possible to test the quality of eggs (Brzuska and Bieniarz, 1977) and of sperm Ginzburg, 1968;Tomasik, 1973;Winnicki and Tomasik, 1976) prior to fertilisation. So far, sperm quality vs. net result of fertilisation was tested in trout (Goryczko and Iomasik, 1975), while the results of carp egg.fertilisation were conside, n"d,011,ly,in te�m� o. f th� sperm to eggs ratio, the sperm(its texture, amount of water) being evaluated macroscopically; in some cases, the spermatozoa motility was assessed (Dyk and Lucky, 1954;Kossmann, 1973;Stein, 1975). Matlak (1970) reported positive results obtained when using 3-4 cm 3 of sperm per 250-300 g of roe. Wolny (unpubl.) considers a proportion of 200 cm 3 of roe to 13-20 drops of sperm obtained from at least 3 males and mixed to be satisfactory for carp.
It seems necessary to find an objective criterion for the carp sperm quality assessment by means of accurate microscopic methods.
The aim of the present study was to work out such an assessment method and apply it to define how the amount and quality of sperm influences the fertilisation success.
MATERIALS AND METHODS
Artificial spawning of carp was conducted in 1981 at the Laboratory of Fish Biology and Aquatic Environment hatchery, Zator. The available materials were used; the roe was obtained from 21 females of the following strains: Hungarian (8), Yugoslav (10), and Starzewo (3), while the sperm was collected from 42 males of the following strains: Hungarian (22), Yugoslav (6), Zator (12), and Starzewo (2).
Before spawning, the females were injected with the carp hypophysis homogenate: 0.5 mg/kg body weight and 2.0 mg/kg body weight at the first and second injection, respectively, the second one being administered 10 h after the first.
Some females yielded oocytes for the vital sexual maturity determination prior to the injections ( as in Brzuska and Bieniarz, 1977). The males were not injected with the hypophysis hormone.
Prior to fertilisation the sperm was evaluated in terms of spermatozoa viability and density. The viability was measured as the duration of various movement phases (in 0.01 min.) and percentage of spermatozoa performing various movements: progressive, circling, and oscillatory. The evaluation involved estimating the percentage of sperma tozoa at a given phase of movement and resulted from observing a few fields of view at about 200X magnification. The following point score scale was used: a) 100% of spermatozoa in progressive movement 3 points 100% of spermatozoa in circling movement 80% of spermatozoa in progressive movement 80% of spermatozoa in circling movement b) duration of progressive movement: The spermatQzQa deri�ity wa_ s.measured in the :BiiJ."ker_chamber. Additionally,, the total number of spermatozoa in sperm unit volume (1 cm 3 ) and in the sperm dose used was calculated. The latter served to calculate the spermatozoa/egg ratio. The fertilising capability of a given sperm sample was tested: experiment I involved 0.01-2.0 cm 3 of sperm and 30 cm 3 of roe (Table l); 1.0-7.0 cm 3 of sperm and 50-350 cm 3 of roe were used in Experiment II, while in Experiment III 90-350 cm 3 of roe were fertilised with 2.5-5 .5 cm 3 of sperm. It was assumed that 1000 cm 3 of roe contained about 800,000 eggs. Following its fertilisation in the Woynarovich solution I ( 40 g NaCl and 30 g urea in 101 water), the roe was subsequently rendered nonviscous in solutions I andII (16 g tannin in l O 1 water) (Woynarovich, 1962(Woynarovich, , 1964. Those experiments involving small roe samples were conducted in small ( about 0.6 1) Weiss jars; the remaining runs were carried out in a large incubation battery (71 jars).
RESULTS
Experiment I. The roe was collected from 2 Hungarian females. As stated in Table I, 5% of sperm collected from the Hungarian male W-2 showed progressive movements, the remaining males yielding sperm of a very good motility: 100% of spermatozoa in progressive and 80-100% in circling movements. The volumetric sperm/ roe ratio in 400 50 no. of spermatozoa in sperm dose: 21.3X10 9 -50Xl0 9 51 X 10 9 -100Xl0 9 ----mean % f ertilisation ;�; � � �!i:3 � � � ��'\" � 't-��;� .;.;. ;,;. sperm/roe volumetric ratio 20.0X 10 ( -30.0X 10 6 mm-3 3l.OX10 6 -40.0Xl0 6 mm-3 41.0XI0°-50.0X10 6 mm-3 10 3 spermatozoa per I egg Experiment! ranged from 1:15 to 1:3000. The spermatozoa density ranged from 17.0 x 10 6 / mm 3 to 32.0 x 10 6 / mm 3 , which -when converted to the number of spermatozoa per dose -gave a range of 0.17 x 10 9 to 170.5 x 10 9 (Table 1). A 30 cm 3 roe sample was calculated to contain about 24 x 10 3 eggs, the controls containing 72 x 10 3 and 280 x 10 3 eggs. The per cent fertilisation ranged from 0.0 to 97 .5% /c.; W-150 o W-30 control sample. Table 1 shows the poorest results to have been obtained from the W-2 and Z-11 males, 4.0-24.4% and 20,8% fertilisation, respectively, the highest per cent fertilisation (11.6-53.9%) being obtained for the female W-150 roe and male W-25 sperm. Better fertilisation results were obtained when using sperm of a very good and good motility. Experiment II. Fig. 1 illustrates the relationship between the fertilisation success and sperm/roe volumetric ratio, while Fig. 2 shows the per cent fertilisation as related to the number of spermatozoa / number of eggs ratio in the same experiment. The fertilisation success ranged from 6.3% to 97.5% and increased with increasing volume of sperm per roe unit volume (Fig. 1). Additionally, Fig. 1 shows the range of total numbers of spermatozoa in various sperm doses (21.3 x 10 9 -216.15 x 10 9 ). The highest per cent fertilisation was obtained in the roe samples mixed with sperm doses containing 100 x 10 9 -200 x 10 9 spermatozoa. Fig. 2 reveals a growing trend in the per cent fertilisation at a higher spermatozoa number per 1 egg. Additionally, Fig. 2 presents the spermatozoa densities (20.0 x 10 3 -50.0 x 10 3 / mm 3 ) in the samples. Experiment III. This experiment includes following the effects of spermatozoa motility in sperm portions used. The range of sperm / roe volumetric ratio was 1: 36 to 1: l 00 (Fig. 3). Based on the total number of spermatozoa (17 .5 x 10 9 -41.0 x 10 9 / cm 3 ), numbers of spermatozoa in various doses were calculated to amount from 57.0 x 10 9 to 196.9 x 10 9 . The per cent fertilisation in this experiment was found to range from 20.8% to 85 .2%. As shown in Fig. 3, the motility of spermatozoa bears a considerable effect on egg fertilisation. The maximum motility score obtained was 7. The sCGre was in a reverse proportion to the duration of phase I (the progressive movement) and in direct proportion to the number of spermatozoa performing this type of movement. A certain increase in the per cent fertilisation was found at a higher score, even at a higher i.e., less f avourable sperm/roe volumetric ratio ( e.g., 1: 100, 1 :83).
DISCUSSION
The principal objective of the three experiments performed was to compare the volumes and densities of sperm used with corresponding volumes of roe and to look for a possible relationship between the fertilisation success and the number of sperma tozoa/number of eggs ratio.
Kiselev (195 7) related the success of fertilisation to the sperm volume: the percentage of fertilised eggs increased from 0.5 to 44.4% when LO cm 3 of sperm was used for 5 X 10 3 eggs (about 6 cm 3 of roe). The results contained in table 1 fail to reveal any unequivocal relationship between the sperm volume used and per cent fertilisation. However, a tendency for an increasing fertilisation success with increasing sperm volume per roe unit volume is supported by the results of Experiment II (Fig. 1). The results of all three experiments allow to suppose that the optimal sperm/roe volumetric ratio is within the range of 1:100-1:50 in spite of a positive result (53.9% fertilisation) being also possible at 1: 300 ratio (Table 1 ).
On the other hand, a relationship between the per cent fertilisation and number of spermatozoa per 1 egg is evident. The number of spermatozoa in a sperm dose is determined by the spermatozoa density (Wolny, 1974); e.g., 2 cm 3 of sperm obtained from the male W-2 contained 64 x 10 9 spermatozoa, i.e., almost twice as many as in 2 cm 3 of sperm of the male J-37B (34 x 10 9 ). Furthermore, the higher the number of spermatozoa in a sperm dose, the higher fertilisation success as shown in Table I comparing the data for all the males.
The males used in Experiment II showed a range of total spermatozoa number in their sperm, which resulted in a range of spermatozoa number in doses used for fertilisation (21.3 x 10 9 -216.15 x 10 9 ); a mean per cent fertilisation (Fig. 1) exceeds 50% when the spermatozoa number in a dose is 100 x 10 9 -200 x 10 9 . Fig. 2 confirms the fact of increasing fertilisation success with increasing spermatozoa number/egg number ratio. On the other hand, the spermatozoa densities in Experiment II shown in Fig. 2 do not seem to be an unequivocal measure with which to assess the sperm quality unless they are compared to number of eggs. So far, effects of sperm concentration on fertilisation success have been assessed. Moczarski (1976) found a statistically significant correlation between per cent fertilisation of eggs from a single female and spermatozoa density. This single fact of the correlation occurred under the following conditions: amount of roe to be fertilised: 3 cm 3 ; amount of sperm used: 0.4 cm 3 ; spermatozoa density: 31 x 10 3 -160 x 10 3 /mm 3 . According to Mussielus (1951), the higher sperm concen tration, the better viability of the offspring and the higher mean body weight of fry. That author reports results of an experiment showing a mean of 60.13% fertilisation of roe fertilised with sperm of 26.9 x 10 6 spermatozoa/mm 3 , while a mean of 37.94% fertilisation was obtained when using sperm containing 18.5 x 10 6 spermatozoa/mm 3 .
It seems that the number of spermatozoa per a defined number of eggs can be an indicator of sperm utility provided the spermatozoa viability is considered as well. For example, the success of fertilisation was relatively high with the sperm from males W-25 and· W-:30 (Table 1) with only 50% of the spermatozoa performing the oscillatory movement and 100% moving progressively. The remaining males produced a lower per cent fertilisation, which might :have resulted from a differential spermatozoa motility; e.g., the male W-2 yielded sperm with only 50% of spermatozoa in the progressive movement, the fertilisation success being, however, also the result of a high number of spermatozoa in a dose. Experiment HI supports to some extent the results of Experiment I in terms of the spermatozoa motility effect on fertilisation success. The relationship is not unambiguous, but as can be seen in Fig. 3, samples of sperm with high scores ( 6 and 7) gave higher per cent fertilisation in most cases than those with lower scores.
When analysing the results of all three experiments one can conclude that in order to obtain a satisfactory fertilisation success there should be 300,000 spermatozoa per 1 egg provided 100% of them perform the progressive movement lasting from 0.3 to 0.4 min., the movement being probably very important in fertilisation (Zuromska, 1981).
A control sample incubated in a large battery Weiss jars gave much better results (95% fertilisation) than eggs incubated in a small battery, in spite of a smaller number of spermatozoa per l egg than in many other cases in Experiment I (Table 1 ). This difference may have been brought about by lower water temperature in the small battery; the water was not heated, while the temperature is an important factor in fertilisation (Matlak, 1969). Additionally, the roe quality may have influenced the results: the female W-150 roe showed usually a higher per cent fertilisation than the roe obtained from the female W-136. | v3-fos |
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} | s2 | Cultural Ecology of Old Cultivated Plants in the Carpathian Area
In the Carpathian area there are some plants cultivated up to this day which were earlier of major importance in Europe' economic life and had actually appeared already at the dawn of tillage in the Near East and in Iran (einkom emmer, panic-millet etc.). By now, their cultivation is limited to the marginal areas and the highlands; they can be found on sites difficult of access, in poor soil, on burned clearings of mountainous regions. They are usually grown on minor plots, because the households need only a small crop. It is only the oldest, conservative -minded generation, the poorer social strata, which cultivates them. Agricultural authors of the 1819Lh centru-ies still mention these plants, but agricultural statistics include them only rarely if at all. Our scope 9fknowledge on their growing is rather limited.
There is a particular relationship between these old cultivated plants and man, i.e. the community. Not only the plants themselves are old, but also are the primitive cultivation methods as well as their methods of consumption (unlike the cultivation of wheat, barley and rye). The people are not interested in the improvement of these plants, and in fact , they deny their cultivation to the autJ10ri.ties; on the other hand, the authorities do not pay attention either to the cultivation of einkorn, emmer, foxtail -millet, panic -millet etc: Primitive implements are used for their cultivation, hulling and giinding. As a result, an a1·chaic cultural ecosystem is being formed by man, the plants and the implements. Relying upon ancient traditions and experiences, man integrates these cultivated plants into his culture. Some of them are not used any more for human food, but only for the forage of animals or other purposes.
Helbaek was still of the opinion that Triticum monococcum ,,is the progeny of the small-grained wild wheat, Triticum aegilopoides ' (Helbaek 19.59 :36 ). Einkorn is an ancient cultivated plant in the Carpathian area also and was grown there already in the N eolilhic and the Bronze Age . However, the decline and fall of the Roman Empire also involved a regression in its cultivation. As shown by archaeo-Prof. Dr. Bela Gunda,PoBox 36,Egyetemi Neprajzi Intezet, Debrec en 10, Ungarn logical finds, it was cultivated in the Bronze Age both on the Great Hungarian Plain (Poroszl6, along the River Tisza) and in different parts of Transdanubia (finds from the Late Bronze Age are known, for instance, from Sopron). In the 4-Sth centuries the einkom was an important cultivated plant in the southern part of what is to-day Slovakia (the main sites are Nitra and Rusovce ). In th e 6-?1h centuries remains of einkorn can be found less frequently in the Carpathian area (Hartyanyi-Novaki 1975 :65;Hajnalova 1976 :228-254;Beranova 1980 :316-318). The next find of Triticum monococcum is known from 1240 in Slovakia (Banov,Beranova 197 5: 17). Then a long time passes without any evidence of its cultivation. As a matter of fact, the raising of einkorn was latent for several centuries and became gradually limited to the highlands. In the 18 th century it was still cultivated in the eastern part of the Great Hungarian Plain (Bihar), while numerous data are known from Transylvania, including the period between the middle of the 15 th century and the present time. In fact, the Hungarians, Saxons and Rumanians of Transylvania are still cultivating this plant or have eventually discontinued it in the last decade only. There are still fine sowing of einkorn to be seen in the Bihar Mountains, the Mezoseg region ( Cimpia Transilvaniei), between the rivers Maras (Mure~) and Aranyos (Arie~) in the MunFi Metalici (Hungarian Erdelyi Erchegyseg;Nyarady 1941Nyarady -1942Borza 1945: 7-9; Gunda 1966:306-309;Pentek-Szabo 1981:259-277). No data are available from the past two or three centuries about Triticum monococcum having been cultivated by Slovaks and Ukrainians in the Northern Carpathians.
Nevertheless it seems that its cultivation did not cease long ago here either. Triticum monococcum was probably often mistaken for Triticum spelta. When earlier authors (Fenyes 1843 : 549, Vol. II) are mentioning alakor from the Trencsen (Trencfn) region, for instance, the denomination applies not to the latter, but to the former cultivated plant. In the Gomor region belonging already to the Northern Carpathians the Hungarians call the earless stem of spelt alatka; deriving probably from the earlier cultivation of einkom (Hungarian alakor).
According to records of 1817, the Transylvanian peasants cultivated in years of scarcity einkorn together with the oat and potato. In the lean year 1817 the peasantry lived on bread made of einkom and oats, but einkorn was more expensive than oats: a bushel of the latter costed 2 gulden, the former about 3 gulden (Teleki 1862: 316-317).
In Transylvania Triticum monococcum is sown in spring and autumn. In the highlands the newly broken grassplots are first sown with einkom. Although of a rather modest nutritive value, einkorn grows also on weedy soils and is frostresistant. In Transylvania it is actually cultivated at a height of 900 m. above sea level. It is harvested with the sickle. Its flour is used by Hungarians and Rumanians for baking bread, but is mixed as far as possible with the flour of wheat, barley or maize. In the Kalotaszeg, Mezoseg and Torda-Aranyos regions (Transylvania)" the Hungarians are baking the einkorn flour into an unleavened flat bread on a hot slab . The flour is kneaded with whey and salt-water, while a flat stone is smeared with oil, fat or butter and used for baking the ca. 3 cm. thick flat bread. The bread is dipped in boiled whey or a soup when being eaten. Einkom bread can be conserved for a long time. Rumanian women, in fact, bake it as a gift to the shepherds. In recent times einkom has been used only as forage for pigs, homed cattle, sheep and fowl. Thus, an ancient human food has become the fodder of animals, as happened for other cultivated plants, such as barley and maize. The straw of einkom is used for weaving hats. In the Hungarian village of Szek (Transylvania) there are only a few peasants who still cultivate einkom on tiny plot of 3 -4 square metres in order to obtain the straw for weaving hats. The soil is broken up only with the spade. The einkom is cut with the sickle, and the grains are rubbed out of the ear with the hand. These small einkom-plots are in shady places to prevent the straw from growing too long, since long straw is not suitable for hat-making. The straw of einkorn is used also as litter, for pigsingeing, roofing and the tying up of vine-branches and maize-stalks. The cultivation of einkom fluctuates conspicuously. In the Szekely villages (Szekely: Hungarians of Eastern Transylvania) there are, in fact some years with a rather considerable crop, followed by other years, when hardly any peasants cultivate Triticum monococcum. Where it had still been cultivated 10 -30 years ago, we find it today only as a weed in the oat-and barley-sowings (Bertsch 1949 :31;Gunda 1966 :306-309;Pentek-Szabo 1981 :266-274).
The name of Triticum monococcumis alakorin Hungarian and alacin Rumanian. The two names are clearly related. The terminology probably comes from the language of an unknown people, with Hungarian acting as intermediary language for the Rumanian term (Peiitek-Szab6 1981 :264; a more careful opinion about the derivation of the word is expressed by Bakos 1982 :204, 414).
It should be noted, however, that an emmer find of the 9 th century is known from beyond the Carpathians near Brno (Slapanice, Bera.nova 1980 :318), in a Moravian settlement. Tri'ticurn dicoccumis also mentioned from J3ohemia (Bylan y u KutneHory) from the second half of the 13 th century (Beranova.1975: 17, Tabl . 2).
For many centuries the cultivation of emmer remained unnoticed by botanists and ethnologists on remote ,fields. In the last decades, however, this cultivated plant has been found in villages of Moravia and Slovakia. In these regions Fr.
Two or three of the four subspecies a re being raised in one and the same village (Klihn 1970 :589 -59 0). As far as I ean see, Triticum dicoccum-just like Ttiticu111 monococcurnremains within the single peasant farms; the peasants do not exchange the grains, nor sell them on the market, insisting on sowing the very grains grown on their holding. As a consequence, these cultivated plants remain for all practical purposes stuck on a farm and become so to say ,,a family -plant''. The neighbours do not even know about each-other cu ltiv ating such ancient plants. I was able to observe the same thing in the ZemplenMountains (Hungar y) in regtu·d to the cultivation of Setaria italica. According to Fr. Kilhn, Triticum di. caecum was mistaken in earlier agricultural and botanical literature for spelt ( Triticum spelta}. So far, there is no unequivocal evidence of spelt being cultivated in Czechoslovakia (Tempir 1963 : 97;Kuhn 1970 : 58 7 -594). The question will be treated further below. Here it shou ld be noted, however , that the typical area of cultivation of emmer includes the White Ca1pathians 1 the javornik Mountains, the Zvolen region (Horny Tlsovnik, Stara Huta, Doln aStre hova), and to a certain extent Eastern Slovakia as well. All the terms published by M. Markus from Western and Central Slovakia ( sobotist, sky rjz, spalda, turcianskj tenkel, oravskj okris etc.) are the different denominations of Triticum dicoccum,just as the Central and Eastern Slovakian terms okrm, autar, autir, volter, voter stand for emmer (Markus 1975 :34-35; I must admit, however, that the Slovakian paper by M. Markus is not always clear). According to F. Kiihn the terms tengel (Stary Hrozenkov), tenkel (Sobotiste), rjz (Sobotiste), spalda (Velke Karlovice, Stary Hrozenkov) etc. also signify emmer (Kiihn 1970 :590;cf. Kiihn-Hammer-Hanelt 1976: 294-295). In the J avornik Mountains the words tenkel, spalda, gengel are also used as denominations of Hordeum distichum var. nudum (Tempfr 1963 : 93-97). This barley species is sown in the highlands instead of wheat. Still cultivated in the past century in the' highlands and on the flat-land of North Eastern Hungary (Zemplen Mountains, Retkoz, Bodrogkoz etc.) the plant known as v6tir ( autir, outirj is definitely not Triticum spelta, but Triticum dicoccum The latter is cultivated also by the Hungarian and Rumanian population of Transylvania, but only in the hill-and highlands (Kalotaszeg,Koloz.svar and surroundings}.Ernmer is also known to be a cultivated planl of the Houtzuls (Ukranian ethnic group) in the North Eastern Carpathians (Bukovina), where it is called pseneca (Borza 1945:19;Nyarady 1941Nyarady -1942Szabo-Pentek 1976:46). The cultivation of Triticum dicoccum in the Carpathian area is unknown to K. Moszyiiski, who says that -among the Slav peoples -only the Serbo-Croats and the Russians along the middle course of the Volga and the lower course of the Kama as well as the non-Slavic population of these regions are raising this plant; furthennore, its cultivation can also be observed on Ukrainian areas (Moszynski 1929 :215). According to archaeological finds discovered in Poland, emmer was cultivated in that country in the 10-13 th centuries (Hensel 1965: 42). I find it rather remarkable that M. Markus quotes from the South Eastern part of Poland the name samopsa which is actually one of the names of Triticum dicoccum among the Slovaks (Markus 197 5 : 24,38). In the Polish language, however, samopsza means Triticum monococcum (Moszyiiski 1929: 214). In earlier Polish dictionaries different interpretations of samopsza are to be found (e.g. spelt).
Emmer is sown in burned clearings difficult of access, where dung cannot be carted out. Brushwood and branches are burnt and the ashy soil is hoed (Fig. 1 B). Thus the ashes will be mixed with the earth. The plough will be used only rarely for tilling such clearings. Towards the end of February or the beginning of March, but sometimes even as late as April, the glumaceous grains are sown with the hand. The sown soil is smoothed by means of a bush-harrow drawn by a man or a woman. Sown in April, the emmer ripens by the end of August or the beginning of September. In the case of crop rotation (i.e., if the burned clearing is not a fresh one), the emmer will be sown after rye. Emmer is not choosy as far as the soil is concerned; and it can stand cold as well as heat. Harvesters must take care to prevent the rachis of the ear from breaking into pieces. This is why the emmer is cut by the women early in the morning or at night with a serrated sickle or a small scythe. The half-rip ears are scorched , the gi·ains rubbed out between the palms and will thus be eaten, in an ahnost prehistoric way . 1 The em.mer is threshed with the flail, often in the barn during winter, or less frequently by treading out with a horse. The grain was dried on the oven. Before eating it, the glume had to be cleaned off. For this purpose it was hulled between two flat stones, in a mortar, a hand-mill or a foot -pestle. A mush can be prepared out of emmer with milk and water. The mush is used as the last course of a wedding menu and is also eaten on Christmas Eve. At pig-killing it is put into the white sausage. In the Hant and N 6gnid regions emmer is mainly grown for obtaining a tasty sausage. Its flour is used for baking an unleavened flat bread in hot ashes or on a flat stone. In the case of bread-baking its flour is sometimes mixed with barley-and oat-meal. Among the Slovaks (Tur6c, Z6lyom) one of the names of the bread made of emmer flour is bochnik (Old Bohemian bochnec, Polish bochen), deriving from the Old High German fochen, fochan;:,a, fochan;:,e 'bread baked under ashes' (Machek 1957: 37;Heyne 1901 : 268). Emmer is also used as fodder for horses, pigs and fowls. Pigs fed with emmer are supposed to have a more tasty bacon. In the Gomor region the Slovaks make baskets and hats of emmer straw; at the beginning of this century they sold them on the markets of towns in Northern Hungary (Tokaj, Nyiregyhaza, Miskolc, V ac, Losonc). On the way to the market they occasionally exchanged their products for maize, lard or apples.
The emmer mush was cooked in North Eastern Hungary as well; emmer-bread and flat cake are mentioned in the Bodrogkoz and Retkoz regions; pigs were fed with emmer (Ecsedi 1935 :276;Markus 1939 :390-391;Kiss 1961 :25, 67, 157, 250,346,354,369). The cultivation of emmer (Hungarian voter) is mentioned in church documents of 1802 (Balassa 1964: 70), which permits us to conclude that it was consumed also by middle-class families.
III.
The history of spelt ( Triticum spelta) is rather mysterious. It is unknown in the wild state and missing from the archaeological finds of the ancient crop-growing areas of Asia Minor and Iran, but is cultivated on the north-western table-land of Iran and in the Armenian Caucasus. pelt is known to have been cultivated in Armenia already about 3000 B. C. 2 In Germany and Switzerland it occurs rarely in Neolithic times, but rather frequently in the Bronze Age. Several hypotheses have been set up on the origin of cultivated spelt. According to some authors this wheat species is the result of mutation or of the spontaneous crossing of wild emmer and Triticum compactum ('club wheat', a variety of bread wheat; La Baume 1961 :30; Bdojan 1972 :302;Bertsch 1949 :39). Of course, there are also other opinions on the subject. "This species" -writes H. Helbaek -"has the semitoughness and structural habit of einkorn and emmer, except that its internode adheres to the spikelet by its lower end and not, as in the two others, by the upper end. Genelics have demonstrated that a similar form may be produced by crossing mmer and Aegilops p." (Helbaek 1 59 :36 h;cf. McFadden -Sears 1946: 81). However, bread wheat ( Triticum aestfoum) was certainly cultivated already in the area of Asia Minor and Iran as well as in the European area when the first rudimentary speltsowings appeared around the human settlements. A. De Candolle is probably right in supporting the hypothesis that spelt originated from bread wheat or some other wheat species in not too early prehistoric times (De Candolle 1894: 385). If Triticum aestivum or Triticum compactum played any genetic role in the evolution of spelt, then we must evidently regard spelt as a recessive deteriorated species as compared to bread wheat: the kernels are held by the glumes like a vice, the ear easily disintegrates into spikelets consisting of internodes, and the glumes and kernels are covered by their pales.
In mentioning the Serbo-Croatian (pir) and Slovenian (pira) names of spelt, K.
Moszy:6.ski affirms that all Slav peoples cultivated spelt, but stopped its cultivation on a considerable part of their region of settlements transmitting its name to the wheat-grass (Agropyron repens, Moszy:6.ski 1929 :218). In the Slovak language, for instance, pyr means wheat-grass. Spelt is also cultivated in the Velebit Mountains by the Croatians ( Degen 1936-1938. The "Alamani" spelt is a rather significant grain crop in Russia, particularly in the 2 R cently G.N . Lisilsina hns wrilten the following:" ... the findings of Triticum spelta in Transcaucasia are chronolo gically earlier than th e European ones and in most recent time its remains were among the palaoo -ethnobotanical finding from th e layers of the region along the middle course of the W alga (Maurizio 192 7 : 26 7;Engelbrecht 1916 :24-25).
Recently Triticum spelta has been found in Northern Moldavia (the middle phase of Tripolie-culture,J anushevich 1978 : 61, 63), Slovakia (Devin-Bratislava, Hallstatt-period, Hajnalova 1978:88-92). Remains of the Triticumspelta (with emmer and einkorn) from settlements of the Neolithic times are abundant in Poland (Mogila, Zawarza, Lasek, etc., . In Hungary we know sporadic spelt (?) remains from the Neolithic-Copper Age (Zengovarkony) and the Bronze Age (Poroszl6), but no finds from the Roman, the Migration or later periods (Hartyanyi-Novaki 1975: 7, 29), although spelt was definitely known to the Romans, spelt-mush having been a food of their soldiers (Markus 1975 :25). Slovak, Hungarian and Rumanian archaeological finds do not include spelt either. The fact that spelt is often missing from archaeological finds may be explained by a regres_ sion taking place in the growing of that cultivat ed plant. In Hungarian d0cum ent s spelt is mentioned at the end of the 15 th century (1498), when the peasants actually paid their taxes in spelt. At the beginning of the 15 th century, the German bmgesses of Buda sold the spel t on the market. Particulars in documents of the 15 th century as well as the Hungarian denomination of Triticum spelta (Hungarian tiinkiily, from High German Dinke~ leaded Zs. Batky to conclude that this wheat species was imported to Hungary by German immigrants from the Rhineland, through the Danube trade (Batky 1918 :27-29). This would be a secondary appearance of spelt in the Carpathian area. The cultivation of spelt was probably for several centuries what may be called a latent branch of agricultural activity within the Carpathian Basin, actually driven back to marginal areas. The earlier Hungarian name of the plant has vanished, and tb'nkiily (see above) is oflater date. Although F. Ktihn definitely denies the raising of Triticum spelta in Czechoslovakia (Ktihn 1970 :594), earlier Hungarian statistics (Batky 1918:27-28) as well as etymo_logical results (the Slovak tenkel is from the Hungarian tb'nkiily, Machek 1954 : 286) permit us to conclude that Triticum spelta, which secondarily came undoubtedly from Hungarian regions to the Northern Carpathians, was still cultivated at the end of the last century in the southern part of Central Slovakia. In some towns of Central Slova kia (Nova Ban.a Pukanec) spell seems also to have been sold (Marku s 1975 :26), while the da ta from the 18 th century published by Slovak historians from the Z6lyom, Nyitra and Hant regions (Horvath 1962 : 3 7) certainly apply to the cultivation of Triticum spelta. The documents mention white and red spelt. Early Hungarian agricultural works also allude to white, blue, black and red spelt (Pethe 1805 : 449).
In the middle of the last century Triticum spelta was frequently cultivated in the hilly and mountainous regions of the Carpathian area ( surroundings of Pozsony, Kassa). On the Great Hungarian Plain, however, not even its name was known. It was raised in Western Hungary, in Transylvania (Szatmar, Kalotaszeg, Szekely land) and in the Banat region ( Galg6czy 1855: 259). According to my own observations spelt was still found among the field crops of the Zemplen Mountains and Boldva Valley (N orthem Hungary) in the years between 1930 and 1940 ( Gunda 1937 :52;Ikvai 1967: 72).
The Rumanians are known to have cultivated spelt already in the 17 th century ( Claudian 1939: 77). According to A. Borza this plant is raised by the Rumanians rather frequently in the Eastern Carpathians (MuntiiApuseni, Maros and Aranyos valleys, Mezoseg, Szilagy etc.) but on small plots only (Borza 1943: 10;Prodan 1931 :283). In a recent Rumanian ethnographical work Triticum spelta is named alacul ro §U ( red Triticum monococcum), without mentioning where it is cultivated and how it is consumed (Butura 1978 : 152). In his monograph on Rumanian plant cultivation T. Pamfile does not mention the cultivation of Triticum spelta (Pamfile 1913). Unfortunately the Rumanian works do not always elucidate the species of glume wheats meant by the different dialect terminologies ( alac, caplagea, griu-gol, tenchiu, siicarii alba, orz muchei, griu moale etc.). In fact, Triticum monococcum, Tr. dicoccum and Tr. spelta are often designated by one and the same name. The opinion of A. Borza according to which Triticum spelta must be Roman remains in the Muntii Apuseni, is devoid of foundation (Borza 1943: 13). Triticum spelta was actually cultivated in the Carpathian area already before the Roman period. It is in any case remarkable that a Rumanian terminology of Triticum spelta is of Hungarian origin (Rum. tednc, tentiu from Hung. tb'nkoly, Tamas 1966: 776).
The Transylvanian Saxons cultivated Triticum spelta 50-60 years ago mainly in the Bistritz region (Nosnerland) in a two-course rotation system (E. Domer 1910 :220;Krauss 1943 :568). The Saxons had brought spelt with them from the Middle-Rhine and the Mosel regions already in the 12 th century, but apparently without maintaining its former name (Dinkel, Spelt). The Saxon Ualenk is from Rumanian alac (Krauss l 43 : 568). Spelt was introduced into the Banat region (Yugoslavia, Ru mania) towards the end of the 18 th century by Swabian colonists (Bertsch 1949: 46). In GaJicia and Bukowina its cultivation is also due to German settlers ( 18-19 th century; Borza 1945 : 17).
Of course, all these opinions are more or less of a theoretical nature. Into the Banat region -where spelt was cultivated at the beginning of this century by both Germans and Rumanians -it could have been introduced from Transylvania as well,just as into Galicia and Bukowina.
Unfortunately we have no detailed ethnographical observations on the cultivation and consumption of spelt. In the Zemplen Mountains and the Boldva valley (Northern Hungary) it was sown by the Hungarians in the barren soil of small plots (1930)(1931)(1932)(1933)(1934)(1935)(1936)(1937)(1938)(1939)(1940) and was harvested at daybreak with a sickle to prevent the ear-axis from going brittle. The grains were threshed out by means of a thick stick or a flail and were hulled in a hand-mill or a mortar. The glume was also rubbed off with the hand. The cleaned grains were ground to coarse meal in a hand-mill and used for cooking a mush. Poor people often made an unleavened flat cake or bread with salt-water or milk. Spelt was also given to the pigs as fodder.
IV.
L. Kunz has described in details the cultivation of the bushy rye ( S ecale cereale L. var. multicaule Ktzg .) , a biennial rye sort, in Mora via Q a vornik Mountains). This plant ( German Staudenroggen) is mentioned in earlier agricultural literature as Saint] ohn's rye, Russian rye and forest rye. In ethnographical literature, however, its cultivation is mentioned, but most infrequently. According to I. Manninen it was raised by the Estonians, but only few people liked it. Bushy rye actually requires as rich soil, and its cultivation may easily lead to disappointment (Manninen 1933 : 29).
In the J avomik Mountains it is raised as follows: The peasants lease a little forest, bum it out and sow the bushy rye in the soil manured with the ashes , Gunda 1957: 129-131) while the seed is harrowed with a twigbrush or is trodden into the soil by sheep and oxen driven onto the plot. Early in the autumn the bushy rye is gathered for fodder or is grazed, while the ripe ears will be harvested by the women with sickles at the end of August or the beginning of September of the following year. The sheaves are carried home on sunny days by the men either on their shoulders or in a cart or on a sledge. (In the Carpathians the sledge is used not only in winter, but also in summer on steep, grassy slopes, Gunda 1977 : 121-138). The ears are grasped and hurled against the wall of the barn in order to obtain the grains. In pine-clearings the bushy rye yields a 7-8-fold crop and even more on beech-clearings. Mixed with chaff, its straw is an excellent fodder for horses. Prior to World War One, the grains with high gluten contents were coarsely ground on a hand-mill and so used for making mush or bread. The meal was mixed with that of common rye. After the harvest of the bushy rye the clearing would be reforested. Occasionally the bushy rye is sown together with other plants (trefoil, oats, wheat, buckwheat), which, of course, would be harvested in the first year already. Bushy rye is sown only once in the same soil. Afterwards, the peasants make new burned clearings, i.e. they move together with the tillage. It may also happen that the burned <;:learing is sown in the first year with potatoes or foxtail millet, in the second year with bushy rye to be gathered for fodder, in the third year the ripe ears are harvested and in the fourth year the clearing will be reforested (Kunz 1969:379-385;Stastny 1971 : 97 -98). A black, sour bread was baked out of bush y rye (Stika 1980: 40). Bushy rye was cultivat ed in a similar way in the northern and eastern part of Slovakia (Orava-,Bystrica-,Snina-,Humenne-region,- Urbancova 1965 :27-28;Podolak 1972 a: 150, 152, 160;Kiihn-Hammer-Hanelt 1976 :293;Hammer 1978 : 265;Kuhn-Hammer 1979 : 171). In Orava and Trencfn the plots tilled with the hoe cover the area of the so called Vlach settlement, wheresheepbreedingwas the main occupation, while tillage was only of secondary importance. 3 The steep slopes of the hillsides could not be ploughed, but social factors also contributed to the survival of hoe-culture, as shown by the following instructive example: In the villag e of Huty near Lipto vsky Mikulas in North ern Slov akia a glass-works was found ed in the 16 th century. The men worked there , and when the glass-works failed, they earned their living as itinerant glaziers, hawking also linen and linseed oil. In spring and summer they were absent, and so tillage was the job of the women who worked more easily with the hoe than with the plough. In teams of 10-15 the women dug up the soil and with a wooden hammer broke the clods (Fig. 3). They sowed the grain and harvested with the sickle. The women carried the sheaves on their back into the barn. In winter the men threshed. Wealthy women hired men as farmhands from the poor village of Orava. This labour organisation was gradually abolished after 1920 only (Urbancova 1960 :259-267;Podolak 1972 b: 17;Fenyes 1951: 127, Vol. II). In Orava the bushy rye was sown together with barley ( 1/ 4 rye, 3/ 4 barley). Of course, barley ripened already in the first year (Fenyes 1843: 74, Vol. II). Today the Slovaks use the milling stone (Fig. 4) mainly for crushing salt, barley and oats, but formerly it was also used for grinding the grains of bushy rye ( Gunda 1961 : 46).
Relying upon an earlier publication ( 1804), Slovak ethnographers are of the opinion that bushy rye was introduced from Moravia at the end of the 18 th century into the region of Orava, Northern Trencin (Kysuca) (Csapolovics 1821:362-363; Urbancova 1975:767), where its cultivation was discontinued, as in Moravia, only after World War Two. This however, is obviously an error.
Bushy rye was actually cultivated extensively in the western part of the Carpathian area towards the end of the 18 th and the beginning of the 19 th century. Thus, it was used as food plant in County Sopron (village of Ivan) and as fodder for cows in Csakvar ( County Fejer) in the Esterhazy-domain famous for its high-level farming (Gaal 1978 :293, 546). In 1821 J. Nagyvathy writes clearly that bushy rye owes its Bushy rye is also raised in the Polish Carpathians. On the burned clearing it is sown on St.John's Day (June 26) immediately into the ashes, then it is hoed in and harrowed. In the second year it is harvested for its grain. The plots sown with bushy rye are rather small and of irregular shape. The Poles often plant potatoes in the burned clearing and sow it only in the next year with bushy rye. Its meal is used for baking bread and for making dumplings. It is also used as fodder for horses. It was also cultivated on the Polish Flatland, but was supplanted already in the 19 th century by improved ryes (Lewicka 1972:136-140· Hanelt -Schultze-Motel 1979. The cultivation of bushy rye can be followed as far as to the Don region, the former province of Stavropol (Maurizio 1927 :203).
V.
The foxtail millet Setaria italica is an ancient cultivated plant in both the Old and the New World. The presumed progenitor of Setaria italica is Setaria viridis, a weed ubiquitous from] apan to England and now widespread in North America and elsewhere (Harlan 1977 :380). The examination of its cultivation in the New World is beyond the limits of the present paper (cf. Rominger 1962;Callen 1967 :535-538) . In the O1d World it was known already in the Neolithic Yang-Shao times (China) about 6000 years ago, but was raised at the same time in Europe as well. For this reason, it is difficult to find out the original centre of its domestication (Harlan 1977: 380;Ping-ti Ho 1977 : 438). Probably it was domesticated more than once. In archaeological finds of the Carpathian area the S ertaria italica occurs only rarely. We know it from the Neolithic Age (Lengyel, County Somogy, Hungary) and from the Roman Barbaricum (Arka, County Zempl en, Hungary). In Transdanubia (Poganyzentpeter, County omogy) it appears also in the Middle Ages (Hartyanyi -Novaki 1975 :64). In the Northern Carpathians (Ockov Devin in Slovakia} it is known from the 3 rd century B. C. (Tempfr 1969 :26;Hajnalova 1975 :240-241).
In the Slavic languages Setaria italica is known by the name bar, her. The terminology was known by tl1e Slavs already when they were living together and they actually cu ltivated foxtail millet. According to K. Moszyii.ski it is rais d in Poland, Polesia, White Russia, in the Ukraine, by the Balkan Slavs and also in Hungary ( Moszyiiski 1929 :22;Obrebski 1931 :B 27;Kwasniewski 1960 :203-222). In the Velebit Mountains the Croatians still raise it in some places and bake bread of it, which crunches under the teeth when eaten (Degen 1936: 345. Vol. I).
In Minor Poland foxtail millet is used for making a mush with water or milk. Christmas Eve the mush is eaten with dried apples and pears. In Volhynia it is put into pastry or stuffed cabbages (Kwasniewski 1960: 214-216). In scanty years the Rumanians also used Setaria italica as a food plant (in the 17 th century and certainly in the following period too). It was cultivated with the hoe on clearings in the forest (Neamtu 1975 :219).
K. Moszynski is correct: foxtail millet is indeed a well-known food-plant in Hungary. In 1825 a Hungarian agricultural periodical wrote as follows: "Now here in the world is it cultivated as abundantly as in Transdanubia", where it was still sown to decades ago. It was mainly the food-plant of the Hungarian and Slovene population of the counties Vas and Zala. Setaria italica was sown in the clearing at the end of April and the beginning of May, the trees having been burned already in the previous autumn. Eventually the clearing was used for 1-2 years as pasture. The soil was tilled with the plough or the hoe. Foxtail millet is always sown in small, remote plots. It is sown thinly and is weeded by hand or with a digging-stick. It is cut off with the sickle, and the standing sheaves are left to dry on the stubble for 1-2 weeks, before being hurled against a chair or a bench to obtain the grain. The straw serves as fodder. The grains are hulled in a mortar or a wooden hand-mill. They are put into soup, or a milky mush is made of them. If there is no milk in the house, the grains are boiled in water, and some lard with garlic is put on the top of the mush. Mixed with milk, foxtail millet is also used for an unleavened flat-bread. Dumplings can be made of its meal mixed with wheat flour (Takacs 1968 :333-352;Pavel 1927: 135). In Transdanubia (Hungary) the cultivation of foxtail millet evidently correlates with its raising in Styria and Slovenia, where its cultivation and consumption are similar to those in Western Transdanubia (Gamerith 1956:97-112;Novak 1947:41;Novak 1960:44). In Hungarian foxtail millet is named mohar, muhar, a word of Serbo-Croatian origin, but in earlier times another term (kales 'millet') was also used.
According to L. Takacs the raising of Setaria italica is evidence for the Mediterranean-Alpine contacts of Hungarian agriculture (Takacs 1968 :351), but this seems to be rather doubtful, since the cultivation of foxtail millet is wide-spread in the northern regions of the Carpathian area as well. In the Hungarian villages of the Zemplen Mountains (Telkibanya, Panyok, Nagyb6zsva) Setaria italica was still sown as a food-and fodder-plant some 40-50 years ago. The green sowing was weeded out by a digging~stick (Fig. 1 E). The grains were hulled in a handmill and eaten as mush. In those times the mush made of foxtail millet was also known in the Nyirseg region (Eastern Hungary). Moravians cultivate the plant in the North-Western Carpathians (Javornik-Mountains), where the grains are sown in burned clearings. The women often loosen the soil only with a hoe (Fig. 1 C). Foxtail millet is rarely included in the crop rotation. In proper soil it is sown after bushy rye. Setaria italica is sown two or three years in the same soil, then a new clearing is made. Harvested with the sickle, foxtail millet is bound in small sheaves which are dried on the oven and then threshed with a thick-stick. The grains are hulled in a wooden hand-mill (Fig. 1 A) and then in a mortar. They are ground as coarse-meal and eaten as mush (Kunz 1961: 369 c_378· Stika 1980. In the last decades Setaria italicawas wide-spread among the Slovaks and the Carpathian Ukrainians in Eastern Slovakia, where it was raised for 3 -4 years on burned clearings, afterwards new clearings were made for its cultivation (Urbancova 1965:27-28;Podolak 1972a:164;Podolak 1972b:17;Kuhn-Hammer 1979 :167-170).
In the above mentioned Hungarian villages of the Zemplen Mountains the redomestication of S etaria italica was still known at the beginning of this century . . The grains of foxtail millet "escaped' from cultivation and growing on the edge of tillage, on the roadside and on the bank of ditches, were gathered and sown. Such plants yield a rich crop even on a poor soil and are highly resistant to frost, heat and drought. A similar redomestication was also practised with panic-millet (Tiszaigar, Tiszacsege, County Hajdu).
VI.
Like foxtail millet, panic-millet (Panicmn miliaceum) was probably also cultivated at first in China some 7000 years ago. Its original cultivation area includes the loess highland of the Shensi, Shansi and Honan territories. Panic-millet remains found in Greece (Argissa, about 7500 B.C.) are about of the same age (Renfrew 1969: 168;Hadan 1977 :380;Ping-ti Ho 1977 :438 · 439). Panic-millet was probably also domesticated in more than one place, but a very long time ago.
In Neolithic times it was raised in many parts of Europe. It was a mosL important planl of the Tripolie civilization (Ukraine) which flourished from 5800 to 3900 B. C. (Harlan 1977: 380; Bertsch 1 49 : 0 -92). In the Carpathian area (Hungary, Slovakia) its cultivation is also known from the Neolithic times and the Eady Bronze Age (Tempfr 1969 :37;Hartyanyi-Novaki 1973 :62; Hajnalova l975 :227-254). Significant panic-millet remains were discovered in Hung¢an archaeological finds of the 10 th -13 th century (Kardoskut, Tiszaorveny, Gyor), which proves that it played an important role in the nourishment of the conquering Hungarians. The Hungarian name of panic-millet ( kales) is of U grian origin. Since the plant also grows wild and escape form culture, and thus also occurs on uncultivated land, the Hungarians may have obtained it aL:eady in thefr earliest economic pe1-iod by means of gathering.
Up to the latest decades panic-millet was cultivated in every part of the Hungarian territory and is still raised in some places. Hungarian pea ants usually sow it on fallow land, tilled grass-plots, stubb les and flooded fields. If the young wheat -crop is destroyed by fro t, flood or drought, it wiU be replaced by panic -millet which ripens within 80-120 days. Minor plots of panic-millet are broken up only with the spade or the hoe before sowing. In the Great Hungarian Plain the grains were often strewn on the soil and then ploughed in. An ancient method of sowing still exists on the shores of the river Tisza. After the spring floods have passed, the muddy soil is sown with panic-millet without previous ploughing. By the time the soil surface becomes dry, the panic-millet also grow (Bellon 1981 : 238). In the eastern part of the Great Hungarian Plain ( Great Sarret) the soil was rooted by pigs and then sown with panic-millet (Takacs 1982 : 156-157). It also happened that the grain was sown in the soil trampled down by a herd of cattle. Panic-millet is rarely reaped with the scythe or the sickle; the women rather break the ears off by hand or cut them off with a knife (Fig. ID). When this is done, the straw is reaped. Occasionally the plant was uprooted (Bellon 1981 : 244). The ears were threshed or trodden out by a horse only in the case of a large quantity. Usually the grain was beaten out with a stick or a washing beater, or the ears, laid out on a canvas, were trodden out by women and children (Bellon 1981 : 246). The White Russians and Poles also tread out the grain with their feet. In Switzerland panic-millet is trodden out in dance -steps (Zelenin 1927: 48;Moszyiiski 1926: 150;Moszytiski 1929: 199;Habetlandt 1926: 354;Riitimeyer 1924: 211). The memory of crop-treading is conserved by the "treading-dance" in Pinzgau (Austria, Wolfram 1936 : 1-15). The Estonians and Latvians tread out the rye with their feet (Manninen 1933 : 106-107).
Before being prepared for eating, panic-millet was hulled in a mortar, a footpestle (Fig. 5) or a hand-mill. Up to recent times it was an important food of the Hungarians. Already in 1805 it was written that the agricultural workers in the Great Hungarian Plain ate millet-mush everyday -from Sunday evening to Saturday evening (Pethe 1805 :458). The mush is prepared by the Hungarians with water, milk, fat, mutton etc. In County Szolnok there are as many as thirteen different kind of millet-mush made. The millet can be put into sausages and soups, and is also used for baking bread and an unleavened flat cake.Not only the women, but also the men ( especially the herdsmen) are proficient in preparing millet-mush. In the last century the payment in kind of Hungarian herdsmen included panic-millet. With the decrease of millet production, millet-mush was replaced among the Hungarians by a mush-like meal made of dry noodles and pastas, with a variable cooking technique and terminology. Even today milletmush is a ritual meal: it is offered to the bride and the bridgeroom before their wedding, and to the retiring farm -hand on the last day of his service. Since the end of the 18 th century, the cultivation of maize and potatoes contributed to the regression of millet raising among the Hungarians, Rumanians and Slovaks.
Besides the Hungarians it is also characteristic of Southern Slav peoples to raise millet and to eat millet-mush. According to]. Csaplovics the millet-bread of the Croatians looked like a mass of swan-shot kneaded together with bird-lime ( Csaplovics 1819 : 114-115. Vol. I). Cultivated but rarely by the White Russians, panic-millet is wide-spread among the Ukrainians, the Poles and in southern Slovak regions (Moszyiiski 1929 :211;Obrebski 1931 :B 27). There exists a common Slav word (proso) for designating millet (Vasmer 1953. It is also rather significant in the nourishment of the Rumanians, whose milletbread is often mentioned (Claudian 1939: 778, 104;Butura 1978: 155-156;Neamtu 1975 :218). Its earlier importance is demonstrated by the role it plays in Rumanian folk healing and witchcraft. Among the Hungarians it is also an important plant of enchantment. The witch can be kept from the animals by strewing millet grains around the stable. If somebody is pursued by witches, sprinldes panic-millet behind. While the witches gather the millet, the pursued can run away or hide. There are actually several variants of such enchantments (Bellon 1981 :253).
VII.
Buckwheat is the only bread-and mush-plant which belongs not to the cereals but to the Polygonaceae. Two of its species are cultivated in the Carpathian area: Fagopyrum esculentum and Fagopyrum tataricum. Both were at first raised in the mountainous regions of Western and Central China together with the adjacent lowlands (Vavilov 1951 :21). However, we have no exact knowledge of the beginnings of their cultivation. It is also suggested that the original home of the buckwh eat reache s from T urkestan to M anc huria and that the pla nt w as introduced into China and J apan from Ma nch uria in the 5 th century B. C. (Bertsc h 1949 : 232). Since buckwheat also grows wild and thus occurs as weed, I think it was included in cultivated plants independently at several times and places. This hypothesis is supported by the fact, that the Siberian Tatars partly cultivate Tatar buckwheat (Fagopyrum tataricum) within the range of their primitive agriculture and partly gather it as weeds (Hehn 1911 :514 Ru ellin s me ntions it by the name of lllrcicum frumentum. On German territory its cul tivat ion cul mina ted in the 17 -l 8u 1 century (Bertsch 1949: 232 -233). In botanical literature it is also mentioned as Tatar, Turkish, Negro, Greek crop or millet. In Russian it is named greca, grecucha, leading M. V asmer to the conclusion, that buckwheat came to the Russians via the Greeks (Vasmer 1953. It is certainly possible that Greek merchants played a role in the propagation of buckwheat, but it cannot be ignored, that in Polish, Russian and 1949: 232). However, the route Black Sea-Venice-Alps-Amsterdam-Germany is obviously most uncertain and sinuous, and must be regarded as the secondary route of naturalization of Fagopyrum esculentum, if the above-mentioned find of the Bronze Age (Brandenburg) is taken into consideration. Tatar buckwheat probably reached the Poles through the Southern Ukraine already in the 12-13lh centuries ( or even earlier). This is suggested by buckwheat finds discover d in grain-pits (11-12 th centuries) which have been opened up near Charkow (Hensel 1965 :50). The Poles often sow the two Fagopyrum species together, because Tatar buckwheat is less sensitive to frost.
The latter grows also as a weed and would oppress the common buckwheat on the field (Galicia). In the sandy region ofTuchola ( German Tuche4 the Poles cultivate Fagopyrum tataricum , gather and consume wild buckwheat as well. It is remarkable, that the Kashubs (a northern Polish ethnic group) peddled buckwheat -groats in the 19 th century as far as Krakow (Mamizio 1927: 136,213).
Le us now glance at the terminology of buckwheat. North German Tadder, Tarre (Middle -High German Tattelkorn, Tatterkorn) is an adoption of the Polish tatarka (Kluge 1934: 83;Vasmer 195.3-1958: 81. Vol. III). Polish greczka is Ukrainian, Russian loan-word (cf. Berneker 1924 :359), just like Polish tatarka. Bavarian and Tyrolian Blende, Plent can be a reduced form of Italian polenta (Kluge 1934: 83). Rumanian ethnographers also mention frequently the cultivation and consumption of buckwheat. In Bucovina and Moldavia it is used for baking bread (Pamfile 1913: 196;Neamµi 1975 :219). The Rumanian sources do not permit us to establish exactly which of the two species is typically cultivated in peasant farms, but in the first place it can only be the Tatar buckwheat, he Rumanian terms for which -hreycd, tdtarcacan only be of Ukrainian 01igi.n, in spite of any different opinion (Cioranescu 1958(Cioranescu -1961828). Towards the middle of the last century the raising of buckwheat was still widespread among the Slovaks (mainly in the South), whereas it was not cultivated at all in the Great Hungarian Plain.
Remains of Fagopyrum sp. from the 10-13Lh century have been broug~t to the surface in Hungruy next to Lake Balaton (Hartyanyi -Novaki 1975 : 43;Arendas 1982 : 5). It is possible, that buckwheat was brought already before this into the Carpathian basin by the peoples coming from the Sou them Ukrainian steppe, but its cultivation was driven into the background. As proved by linguistic evidence, buckwheat was introduced later into Hungai.ian erdtory from several directions in more than one wave. One of its Hungarian names (hajdina, first record 1495) is known in Transdanubia.Itis a loan -word of Croatian origin. Today jt is still cultivated in the western part ofTransdanubia, but in the past century its cultivation still reached eastwards to the Danube line. Its cultivation in Transdanubia correlates with that in the Croatian, ]ovene and Styrian regions. Th e term haricska 'buckwheat' is known in Transylvania since 1614; it is a loan-word of Rumanian origin, but may have been adopted in the northern part of Transylvania from the Ukrainian as well. Pohanka 'buckwheat' (first record 1494) is a word of Slovak origin, current in the northern Hungarian dialects. The original meaning of pohdnka is 'pagan crop' (Hungarian pogany gabona). Among North Eastern Hungarian ethnical groups the name of Fagopyrum tataricum is tatarka 'Tatar buckwheat' (first record 1533), an Ukrainian loan-word. At the beginning of the last century, in Transylvania, buckwheat was also known by the name kruppa (first record 1683), a Rumanian loan-word (Bakos 1982 :237). The etymology of the Hungarian words hajdina, pohanka, haricska, tatarka and kruppa 'Fagopyrum sp.' also demonstrates the complicated history of this food-plant. Taking into account the archaeological finds as well, it must have penetrated the Carpathian area on several occasions and from different directions. The above-mentioned loan-words have obviously supplanted some ancient Hungarian tenninology, since Fagopyrum remains from the 10-13 th centuries have been discovered in Transdanubia. Fagopytum tataricum was ( or still is) cultivated by the Hungarians in the North East, in the northern part of Transylvania, by the Slovaks and the Carpathian Ukrainians in the North Eastern Carpathians. The latter are known to deforest some plots early in the spring (Zemplen region). By the month of] une the trees and bushes dry up and are then set on fire. The remaining ashes are used for manuring the soil, which is sown with buckwheat (Fagopyrum esculentum and F. tataricum) or foxtail millet. In recent times, however, the burned clearings have been sown with potatoes, oats, rye or eventually with panic-millet. The plot is used for 3 -4 years, then it is abandoned and a new clearing is burned ( Gunda 1969: 106-107;Podolak 1972 a :164;Hammer 1978 :264-265). Under Ukrainian influence, the Rumanian peasants also use mainly Fagopyrum tataricum as foodplant. As shown by historic evidence, it was most important food-plant of the 17 th century in the North Eastern Carpathians (Makkai 1954: 124,126,142, 143 etc.).
In the western part of the Carpathian area (Transdanubia) the species Fagopyrum esculentum is known as food-plant. In my opinion Tatar buckwheat is a later cultivated plant in the Carpathian area than the other species. In any case the history of Fagopyrum must still be examined in detail.
Within the Carpathian area the largest quantity of buckwheat is cultivated today in the western part of Transdanubia by Hungarians, Croats and Slovenes. This cultivation area is closely connected with the Styrian and Slovene buckwheat region of the Eastern Alps.
In Western Transdanubia, County Somogy, County Baranya (Ormansag region) buckwheat is raised and consumed in the same way as described in the agricultural literature of the beginning of the last century. At the end of] une or in July it is sown in the ploughed up stubble after the winter crop has been harvested. It was also sown in burned clearings. A negative selection can now be observed: the sound grains are consumed, while those of poor quality are sown.
By the beginning of October the buckwheat is ripe and is then reaped with the sickle. In County Baranya the few plants growing on the plots were also uprooted.
The sheaves are kept standing for a few days thoroughly dry. They are threshed with a flail. Before being cooked, the grains are slightly boiled to have the seedcoat broken, and are then dried in the oven. After drying, the grains are moistened and hulled in a mortar, a foot-pestle (Fig. 5) or a wooden hand-mill. They are also put into a wooden vessel and trodden with naked feet until they are hulled. Buckwheat is used for making soup or mush, it is put into sausages or mixed with milk and thus used for baking a flat cake or bread. A typical buckwheat meal in Transdanubia is called sterc (from Austrian, Bavarian Ster;::,); it is made of coarse meal, put in a pan with hot water and roasted, while being constantly stirred, and (Bodei 1937: 445-450;Kardos 1943 : 58-60). In the Ormans ag region hulled buckwh eat is mixed with hulled barley .and eaten as mush. In Transylvania it was mentioned already at the end of the l th century, that the Hungarians ate a thick buckwheat-mush with milk and curd. The stiff mush was eventually cut into thin slices and sprinkled with honeyed water and poppy seeds (K. Matyus 1787: 153. Vol. II;Teleki 1796: 94). Unfortunately we have no detailed informations on the buckwheat meals of the Rumanians, the Ukrainians and the Slovaks. In Moravia buckwheat is used for making bread or mush (Stika 1980: 40, 62, 63).
On field-work in Rumanian villages of the Szolnok-Doboka and Maramaros regions (Transylvania) I have observed, that Tatar buckwheat was sown in burned clearings in a two-course rotation system (buckwheat-potato or maizefallow plot). In 1940-50 it was cultivated only in small plots tilled with the hoe and left to lie fallow for several years. The authorities registered such plots as barren ground. The grains were slightly moistened and then hulled in a wooden Fig. 7 Roumanian young man husking Tatar buckwheat with foot-pestle. Rohi, Country of Szolnok-Doboka, Transylvania. Photo B. Gunda mortar (Fig. 6) or a foot-pestle (Fig. 7). Tatar buckwheat was used for making a mush. In the northern part of the Szolnok-Doboka region the Hungarians also cultivated this plant in the same years. It was sown at full moon after May 15 in a clearing burned out 50-60 years before. These plots were sited in the mountains about 8 -10 km from the village. The soil was tilled with the hoe. The buckwheat was reaped by the women with a sickle, and the sheaves were threshed with a stick on the plot itself. The straw was set aflame and the ashes were thrown over the plot. After the buckwheat the plots were sometimes sown with maize and oats. The Hungarians used the clearing for 2-3 years. In the first year they sowed buckwheat, in the second maize or potato and in the third oats, but it also happened that only Tatar buckwheat was cultivated in the plot for two or three years. After this the plot was used as sheep-run for 5 -6 years. In this century no new clearings were burned out.
VIII. CONCLUSIONS
In the Carpathian area the cultivation of einkorn, emmer, spelt, bushy rye, foxtail millet, buckwheat survived as recently as the middle of the present century, even nowadays can be found. With their cultivation and consumption thes e plants repres ent a characte ristic margin al cul ture . In preh ist oric times, in anti qui ty an d in th e Middl e Ages, an d even in th e 18 -19 th centuri es some species were also raised in the lowlands (for instance, einkorn in County Bihar, emmer in the Bodrogkoz and Retkoz regions). However, their cultivation became gradually limited to the mountainous territories (e.g. bushy rye to theJ avornik Mountains, Orava, emmer to Slovakia, einkorn to Transylvania). In Transdanubia buckwheat also retired towards the Bakony Mountains and Styria. Within the Carpathian area the mountainous districts are regions of refuge not only for the implements and customs, but also for the cultivated plants.
By the 20 th century only panic-millet remained as a typical plant of the lowlands; it was sown in a most primitive way in grass-plots or after the flood in the muddy soil ( the grains were strewn without ploughing on the muddy ground or trodden into the soil by the animals, e.g. along the river Tisza, in the Great-Sarret region).
The sowing of buckwheat was still marked by a negative selection (Transdanubia), as could be observed at the beginning of plant cultivation in the New World, for instance in the case of maize ( Gunda 1968 b: 11).
Einkorn, emmer, spelt, foxtail millet, buckwheat and bushy rye are usually cultivated on small plots far away from the villages. The plots are cleared by means of fire with no rotation of crops, or just a most primitive one. It is not the crops but the fields, that are rotated. The cleared plots are scarcely manured or n Jt at all. The ashes of the burned vegetation serve as manure. Animal work is quite insignificant. The plough is seldom used; in thejavomik Mountains it is most primitive. The soil is tilled by means of spade and hoe; the women play a major role in both the soil tillage and the harvest. Eventually the soil is tilled jointly by the women, as demonstrated by the cultivation of bushy rye in the J avomik Mountains, in Orava and by that of emmer in Slovakia. The ripe plants are often pulled up by the women, or the ears are broken off or cut off with a reaping knife (seethe cultivation of panic-millet in the Great Hungarian Plain). In the Zemplen Mountains the women cut off Triticum spelta with the sickle immediately under the ear, because the stalk can be more easily cut through at the top than at the root, where it is stronger; furthermore, the harvester has to bend and thus the work is more strenuous. Therefore a high stubble was left and then set aflame. This problem of reaping was already pointed out by A. Steensberg (Steensberg 1943: 129). Often a simple stick was used for threshing. The grains are also rubbed out of the ear with the hands ( einkom, Transylvania) or trodden out (panic-millet, Great Hungarian Plain). Often the ears are grasped and hurled against the wall of the barn in order to obtain the grains (bushy rye, J avomik Mountains).
The grains are hulled with ancient implements (wooden hand-mill, foot-pestle, mortar) and used for making mush or baking an unleavened flat bread. These plants have actually conserved an ancient period of mush and unleavened bread representative also of Europe's prehistoric forms of nourishment. In the Cai.pathian ai.·ea these cultivated plants were gradually supplanted from the beginning of the 19 th century by maize and potato. In the Great Hungarian Plain panic millet-mush was replaced by different dry noodles made of wheat-meal which are cooked in a mush -like way and have often different names in single villages ( o'!Lon, slambuc , galabwgyi, tyeraszka, rk;sami , tarlionya , etc.). This proves that they are new in the rural food-culture. Some ancient implements used for processing the buckwheat, emmer, panicmillet or foxtail millet were adopted by recent plant cultures. Thus the footpestle is used by the Hungarians for crushing paprika or for hulling pumpkin seeds (Wildhaber 1948: 192), and by the Rumanians also for hulling pumpkin seeds.
The peasants are unwilling to cultivate einkom, emmer, spelt, bushy rye, buckwheat and foxtail millet according to the principles of modem agriculture. In fact, they are most conservative as far as these plants are concerned . They try to obtain improved grain for bread-wheat and common rye and interchange the seed-grain among themselves. The wheat is sown with the sowing -machine and threshed with the thresher. Wheat-culture has reached a high stage of development on the peasant farm. On the other hand, einkom and the others are stepchildren of agriculture, vegetating on burned clearings and barren plots. In Kalotaszeg (Transylvania) the peasant working in the co-operative farm often still raises einkom on his small plot in the forest. Not even the neighbours would know about the cultivation of einkom, emmer, bushy rye etc. Thus, the emmer and all these other plants must be regarded increasingly as family plants. The small plots are not filed by the authorities who do not even know of these plants. The crops are not put on the market, except some rare cases when panic-millet is offered on Hungarian markets and alakor appears on Transylvanian markets.
Statistically the production of the above mentioned plants can be quantified only approximately or not at all. Hungarian statistical data are available from the beginning of this century on panic-mille t, buckwheat and spelt, but are not reliabl e. 4 It is a major difficult y that we are unabl e to find out from docum ents, statistical works and vernacular publications, exactly which of these cultivated plants is meant. When ancient sources mention, for instance, the Hungarian alakor or Rumanian alac, these terms can mean Triticum monococcum just as well as Triticum dicoccum or Triticum spelta. The botanical definition of Rumanian dialect plant names is especially difficult. The Latin spelta as written in documents cannot always be identified either with Triticum spelta.
In the Carpathian area the cultivation of these plants -apart from panicmillet -is mainly characteristic of slash -and-bum or swidden agriculture . This kind of tillage and plant cultivation is well known from the northern and eastern parts of Europe (Steensberg 1955 : 65 -130;Linnard 1970: 192 -197) and from the extra-European continents (Conklin 1961 :27-61;Conklin 1965;Vayda 1969). Slash-and-bum agriculture has innumerable local types within the Carpathian area. The types depend on the socio-economic and geographical conditions and mainly on the present or past prorietorship of the forests. Today the peasants are not allowed any more to avail themselves of such a form of deforestation . Different types are created on burned clearings by the application of various crop rotations, leading finally to the end of the slash-and-bum system. Of course, it must also be said, that the slash-and-bum system within the Carpathian area has not yet been examined in detail. 5 But it might be sufficiently clear by what has been said so far, that the components of the geographical and social environment and their interrelationship with the cultivated plants, human labour, the working implements and the utilization of crops represent a specific ecological complex within culture as a whole. In the geographical and social respect this cultural ecological complex is sited in the marginal areas. There is an archaic and local slash-and-bum culture surviving in the many-sided, up-to-date agrarian cultures, together with the archaic cultivation of panic-millet in the lowlands.
It should be pointed out emphatically, that in the slash-and-bum system practised in the Carpathian area the wild fruit-trees of the forests ( crab-, pear-, camel-, sorb-trees etc.) are spared. They are neither felled nor burned, but their fruits are gathered and utilized. On tillage areas, grazings or hayfields we can often see a solitary crab-tree or a wild pear-tree, reminding us of former slash-andburn tillage. If these trees grow old and begin to decay, the hollow will be occupied by a swarm of bees, the honey of which is still gathered by the beehunters. The hollow trunk is often cut out with a saw and taken home together with the swarm of bees ( Gunda 1968 a: 1-62).
The traits of slash-and-bum tillage in the Carpathian area lead us to remote regions in time and space. These traits include in the first place the cultivated plants. Hoe-culture can be followed not only as far as Africa, but back to the Neolithic Age as well. The use of clod-breaking hammers and clubs is evidently very ancient. In Egypt, for instance, they were used before the harrow. Such implements are known from East India and Tibet to France. In Switzerland (Maraviglia) pictographs from the Bronze Age represent men with clod-breaking hammers (Rtitimeyer 1924: 288;Leser 1928: 480). They were also known in the Carpathian area in the lowlands and the hilly regions (Takacs 1963 : 419-425), but have survived only in the slash-and-bum tillage of the highlands (e.g. in Orava, Nagy 1891: 129-130). The pulling of the cultivated plants and the cutting of the ears with a knife are characteristic of the beginnings of plant cultivation (Steensberg 1943-122-126;Bohrer 1972: 145-156). The treading of the grains from the ears or their rubbing with the hand as well as the eating of unripe grains obviously also belong to these traits ( cf. the consumption of emmer by the Slovaks). The consumption of unripe, roasted grains proves, that these cultivated plants are ancient not only in the botanical sense, but also in the cultural ecolo-5 In his book L. Takacs deals mainly with the clearings of Transdanubia, but without paying -allenlion to the correlation exi.sting between einkorn, em mer , spelt bushy rye etc. and th e slashand -burn system (Taka 1980). ll is worth y of note lhat sla!Jh-and -burn agricullur was recently l"ilm d inStyria (H. PrUhwald,Mitt eleu.ropa-Steiermark ,BrundwirtschaJt I -Ill .Film Cl 73,UI -llT der BHWK. Bundesstaatliche Hauptstelle fiir Wissenschaftliche Kinematographie, Wien 1982). gical respect. Already J.G.D . Clark has called our attention to the fact, that the consumption of half ripe, scorched grains has been known since the Neolithic Age ( Clark 1952 : 112). The Poles dry the half ripe rye in the oven, grind it in a handmill and then eat it. In Wtirttemberg the dried grains of spelt (German Griinkern), together with half ripe grains of einkom, emmer and spelt were still used at the beginning of this century for cooling a soup (Maurizio 1929 :141). The Shors (Altay Mountains) scorched the ears on an open fire before threshing them (Levin-Potopov 1964 :452).
One of the implements for removing the hull of emmer, panic-millet and buckwheat is the foot-pestle. It is as wide-spread all over] apan, China, the Altay Mountains, Afghanistan, the Caucasus, Eastern and Central Europe, the Alps, France and Porlugal as panic -millet itself (Vavilov -Bukinich 1929: 205;Meynen 1927: 95-100;Leser 1929: 468-469, 484;Dias 1954: 437-451;Gamerith 1965: 107). J. Dias pointed out the correlation between the cultivation of the panic-millet and the use of the foot-pestle (Dias 1954: 445 -446). I should like to complete his opinion with the observation, that the use of the foot-pestle correlates in the Carpathian area not only with the cultivation of panic-millet, but also with that of emmer, buckwheat and foxtail millet. This applies also to the wooden hand-mill, the use of which extends from the Eastern Alps over Carpathian Europe to the W olga region, N orthem Russia and the Caucasus. According to I. Manninen such a husking and grinding wooden hand-mill is used by the V epsians, the Z yryans, the V otyaks, the Cheremisses, the V oguls and is not unknown by the Turkish-Tatar peoples (Chuvash, Bashkir, Kirghiz, in Siberia the Tatars of the Minussinsk region) either (Manninen 1932: 80-81;Rudenko 1955: 120;Gamerith 1956: 100;Kunz 1961 :376). The wooden hand-mill and the foot-pestle are index fossils of ancient cultures just as much as einkom and the other above mentioned plants.
The author hopes to have called attention here to several problems of the cultivation and consumption of some old cultivated plants. In our days, when European ethnology tries to find its way, experiments with structuralism and semiotics, attempts to amalgamate with sociology, knocks at the door of citizens, collects political slogans daubed on fences and obscenities smeared on the wall of lavatories, -we should not forget the still extant survivals of ancient European folk-culture, the investigation of which is one of our most urgent tasks. Still living today and functioning on the social level as well, these relics are certainly not going to be brought to light a thousand years from now by the spade of the archaeologist. | v3-fos |
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} | s2 | Soil compaction by the tractor in spring and its effect on soil porosity
The effect on soil porosity of tractor compaction of soil in the spring was studied by taking cylindrical core soil samples. The profile samples showed that the tractor most seriously compacts the soil below the harrowed layer at the depth of 10-25 cm. Soil was compacted most severely when tillage and drilling were performed under wet conditions about one week before normal sowing time. The subsoil at the depth of 35-40 cm was compacted only under very wet conditions. The grain yield of wheat was significantly reduced when the volume of large pores was reduced to about 10 % or less. Porosity measurements showed that the severely compacted soil almost completely recovered from one spring to the next. Theoretical calculations suggested that compaction by normal traffic does not cause a shortage of oxygen at least in the inter-crumb pores of soil if the soil surface structure is not dispersed and encrusted. The decrease in crop growth by compaction is primarily due to mechanical impedance, which slows down development of the root system.
Introduction
The compacting of soil by traffic on the field in spring increases the bulk density and reduces both the pore space and the air content of soil. Primarily, soil compaction causes a reduction in the volume of large pores (o>3O /xm), The volume of mediumsize and small pores is much less influenced by such traffic (e.g. ERIKSSON et al. 1974). The greater mechanical impedance to plant roots and lower hydraulic conductivity of saturated soil in compacted fields are due to the reduction of large pores. The compacting of soil also slows down the diffusion of gases. The aim of this study was to clarify the effects of tractor traffic in the spring on porosity conditions in soil. The study also includes observations on how soon soil severely compacted during three successive years can recover if only moderately compacted in the fourth spring. The effect of traffic on the mechanical impedance and aeration of soil was also examined. Experimental A detailed description of the field experiments was presented earlier by ELONEN (1979ELONEN ( , 1980. The factors affecting the compacting of soil in spring were: 1. Tillage and sowing time Tl = Tillage and sowing about a week before the normal time and under wet conditions T 2 = Tillage and sowing at the normal time, when soil moisture conditions were favourable 2. Compacting of soil CO = No traffic by the tractor, only spring tillage and sowing Cl = Twice with a double-wheeled tractor over the whole surface area of soil at tillage and sowing C 2 = Normal compaction. Once with a single-wheeled tractor over the whole surface area at tillage, plus sowing C 3 = Severe compaction. Before tillage twice with a single-wheeled tractor 4-normal compaction. The fields were harrowed to the depth of about 8 cm. Spring wheat or barley (Laukaa field) was sown to a depth of about 5 cm.
The characteristics of the experimental soils are shown in Table 1. Fields were treated in the same way during three successive years 1975successive years -77. In 1978 the recovery of compacted soil was studied as follows: previous Tl-plots were harrowed and drilled at the normal time using a double-wheeled tractor (Cl-treatment), while previous T2-plots were compacted as in earlier years but the tillage and sowing took place about a week before the normal time.
Soil samples for porosity measurements were taken with metal cylinders of height 4.9 cm, inside diameter 7.2 cm, and volume 200 m 3. The samples were collected during the two weeks following the later sowing time, when the moisture content of soil deeper than 10 cm was still near the field capacity, or a few percentage points lower by volume. An exception was the Laukaa field where the soil tended to dry quickly after sowing. The samples were taken as follows: 1976. Mietoinen 1: on CO-, C 2 and C3-plots, from successive layers of 5 cm from the surface to the depth of 40 cm. Other fields: on CO-, Cl-, Cand C3-plots, only from the layer of 10-15 cm.
The number of replications in the field trials was 4. There was a special sampling area at both ends of the plots. The number of replications in sampling was at least 8. Samples were moistened in the laboratory by placing the cylinders on a fine sand bed, where the water potential had a value of -5 cm. Thereafter the cylinders were transferred onto ceramic plates and the water potential regulated to -lOO cm. When equilibrium was attained, the weights of the moist soils were recorded and the soils were dried in an oven. Soil density was determined by the pyknometer method (BLAKE 1965). The proportions of solid substance, water and air space in soil were then calculated. In 1978 some profiles in Espoo 1 from a soil sown with seed earlier than usual were studied more closely. Then porosity conditions were determined by equilibrating the samples of these profiles to the potentials of -2.5, -5, -lO, -5O and -lOO cm. The pore diameter corresponding to the water potential was calculated using the formula (e.g. CZERATZKI 1958): The results for the profile samples are shown in Figures 1, 2 and 5. The average water contents just before spring tillage at the depth of 5-20 cm in ploughed fields are presented in Table 2. Table 2 also presents the water contents at the water potential of -0.1 bars corresponding to the moisture content at the field capacity.
The profile samples taken in 1976 from Mietoinen 1 indicate that the traffic most severely compacted the soil below the harrowed layer of the soil, at the depth of 10-15 cm. The proportion of large pores by volume was particularly reduced. The soil was compacted most severely when the harrowing and drilling were performed under wet conditions about one week before the usual time. The large pore space in the layer from 0 to 10 cm is due to spring tillage. The effect of compaction on porosity was weak in subsoil: at the depth of 35-40 cm there was no clear difference in the pore space between the treatments. The profile samples of 1977 from Espoo 1 confirm the above results, as do the results of Mietoinen 1 in 1978 ( Figure 5). The profiles of Espoo 1 in 1978 show, however, that severe compaction under wet conditions in the spring can strongly reduce the volume of large pores in subsoil, too. According to Figures 1, 2 and 5 the minimum volume of large pores after strong compaction is found at the depth of 10-25 cm but there is also a decrease at the deeper layers. Table 3 confirms the results in Figures 1, 2 and 5. Compaction reduced the total space and especially the volume of the large pores in the layer of 10-15 cm. The changes in soil structure were greater during spring tillage in The average water contents at the depth of 5-20 cm just before spring tillage. Volume-%. Early sowing = Tl, normal sowing = T 2. Measurements by ELONEN (1979). The water contents at -0.1 bars in CO-plots are also shown. 1976 1977 1978 Water content at the Tl T 2 Tl T 2 T 1 potential of-0. (Table 2) but, rather, rainy wheather caused further moistening. As Table 3 shows, in 1977 compacting of the soil in these fields at the normal time had as harmful an effect on the volume of the large pores as the traffic at the earlier sowing time.
The results of the Laukaa field differ from those of the other fields (Table 3). Even the strongest compacting of the soil did not reduce the total pore space or the volume of large pores. Nor did the time of sowing influence the results of this field. Table 2 shows that the soil at the time of the first sowing was already drier than the moisture content at the potential of -O.l bars. On account of its high silt content (Table 1), the Laukaa field could not be tilled until the soil moisture content reached the field capacity. Table 3 shows that the percentage of large pores by volume in the Laukaa field was not much over 10 % even in the soil that had not been compacted. In the other fields the corresponding percentage was about 20 %.
Evidently the water content of soil was not the only factor determining the effect of compaction (Tables 2 and 3 and Figure 3). Especially in Espoo 1 and Espoo 2 there was no clear correlation between water content of the soil and degree of compaction. Although the soil was drier at the two sowing times in 1976 than in other springs, the reduction in the volume of the large pores by traffic was greatest in that year. Table 3 shows that the compaction of the soil was about the same whether tractors were double-wheeled or single-wheeled. On average, the volume of large pores in the Cl-plots was one percentage point higher than in the Cplots, when tillage was done a week before the normal time.
b. Porosity and yield
Yield results showed that in most cases only severe compaction (C3) caused a significant lowering of grain yield (ELONEN 1979). There was no great difference in the porosity between the Cl-and C2-treatments and therefore no great difference in yield between these treatments. In the Tl- plots, driving with a single-wheeled tractor compacted soil on average slightly more than driving with a double-wheeled tractor. Accordingly, the ClTl-treatment gave a 100-kg higher yield than the C2TI-treatment. The porosity measurements of the Laukaa field accorded with the yields of that field: traffic on the field affected neither the porosity nor the yield level. In the heaviest clay soils, Espoo 1, Mietoinen 1 and Anjala, the moisture content below the harrowed layer remained near the field capacity until the crop sprouts began strong transpiration, whereas the lighter soils lost much of the water content before the emergence of the sprouts. Apparently the measured porosities of Espoo 1, Mietoinen 1 and Anjala described the conditions in the soil for a longer period during the early summer than did the results from the other fields. Figure 4 shows how the yields of the former fields depended on the volumes of the large pores. The relative yield of the Cl-treatment is indicated by 100. Figure 4 also shows that traffic will strongly decrease grain yield when large pores in the layer at 10-15 cm are decreased to about 10 % by volume or less. c. Improvement in the strucrute of compacted soil In 1978 all Tl-plots were harrowed and drilled using double-wheeled vehicles at the normal planting time. The after-effect of severe compaction (C3) in comparison with the treatment without compaction (CO) is shown in Figure 5. In soil of Espoo 1 the after-effect was slight and in Mietoinen 1 no after-effect was observable. Table 4 confirms these results: the soils had almost completely recovered from severe compaction by following year, 1978. The yield results were in accordance with porosity measurements and there were no significant differences between the aftereffect plots (ELONEN 1979). The effect of soil compaction on the proportions of large pores of various sizes is presented in Figure 6. A detailed analysis of large pores was performed on some CO-and C3-profiles from Espoo 1. Figure 6 shows a sharp rise in the volume of air-filled pores when the interval of the water potential lies between 0 and -2.5 cm. These largest pores are apparently the air spaces in the cracks crossing the soil sample in various directions. This Fig. 5. Large pores in the fields Espoo 1 and Mietoinen 1 in 1978 space was strongly decreased by soil compaction. The increase for the interal between -2.5 and -100 cm was not so sharp. However, it should be noted that differences in porosity are not due only to tractor traffic but also to the natural compaction of soil before the spring tillage.
An approximate value of the pore density was calculated from the graphs presented in Figure 6 The pore densities calculated according to equation (6) are presented in Figures 8 and 9. Figure 8 shows that in compacted soil the density of pores > 300 /x m in diameter may be near zero. The density of pores larger than 60 /urn or larger than 30 /x m is also dependent on soil compaction. e. Calculation of oxygen adequacy in compacted soil An approximate idea of the effect of the compacting of soil on aeration can be estimated in the following way: As the profile samples showed, the harrowed layer has a great air volume and the diffusion of gases is hindered only slightly if the surface of the soil is not encrusted. The harrowed layer dries rapidly and the respiration in this layer is approximately 0. Assume that the owygen consumption at the depth of 5-25 cm is 10 1/m 2 day (MONTEITH et al. 1964, BROWN et al. 1965. In the subsoil the use of oxygen is slight (RICHTER and GROSSGEBAUER 1978). If the concentration of oxygen in the air space of soil changes slowly, the streaming of oxygen in soil is nearly in a steady state. Hence (van BAVEL 1951): where q = the diffusion of oxygen downward mg/cm 2 s C = the concentration of oxygen in soil air mg/cm 3 z the depth cm a = the use of oxygen by soil mg/cm 3 s D = the macro-diffusion coefficient of oxygen in soil cm 2 /s 10 1 02 /m 2 day in the layer of 5-25 cm = 7.7x10 2 mg/cm 3 s Boundary conditions are: 5 cm: C = Co = 0.2095 cmVcm 3 = 0.2786 mg/cm 3 25 cm: dC/dz = 0 (q = 0) Thus equation (8) (see e.g, RICHTER and GRLSSBEBAUER 1978) By substituting the values of a, D, L, C 0 and z (= 20 cm) into equation (9) we obtain C at the depth of 25 cm. The computed concentration of 0 2 is 0.23 mg/cm 3 or 0.17 cmVcm 3 . The calculation shows that if the surface of a soil is not encrusted normal compaction probably does not cause a shortage of oxygen at least in the inter-crumb pores of the soil during the growing season, when the soil most of the time is drier than at the field capacity.
Discussion
As mentioned above, the fields Espoo 2, Mietoinen 2 and Laukaa tended to dry rapidly after the second drilling (T2) if precipitation was poor. The cracking of soil soon followed, which should help the roots to grow downward, but on the other hand soil shrinkage between cracks makes the soil harder for the roots to penetrate than the pore measurements showed.
The heaviest clay soils Espoo 1, Mietoinen 1 and Anjala dried more slowly and began to loose moisture rapidly only when sprouts started vigorous transpiraton. At this time the heaviest clay soils cracked and the porosity between the cracks was lower than that obtained by the porosity measurements. However, the porosity measurements described the conditions in these fields at an early stage of the growth cycle which was apparently important for the development of the crop.
The compacting of the soil by spring traffic primarily reduced the volume of the large pores in soil. Thus the hydraulic conductivity for saturated soil was reduced in the compacted soil. The streaming velocity of water in a thin tube is proportional to the square of the radius of the tube. Hence small changes in the volume of large pores mean great changes in conductivity, as RASMUSSEN (1976) has shown for example. In hard rains water may remain on the soil surface because the infiltration rate is reduced by soil compaction. Consequently, compaction causes a dispersion of soil surface structure and thus reduces soil aeration and crop growth.
The aeration of soil depends on the rapidity of the diffusion of gases. Contrary to hydraulic conductivity, the diffusion coefficient of oxygen is determined by the total air porosity of soil. The calculation presented in this paper suggests that compaction by normal traffic does not cause a shortage of oxygen in the inter-crumb pores of soil if the soil surface structure is not dispersed and encrusted.
The reduction of growth on compacted field seems to be primarily due to mechanical impedance to crop roots. If the pore walls are not at all elastic the roots cannot penetrate pores smaller in diameter than the extending zone of the roots (WIERSUM 1957). Even a slight hinderance can greatly restrict root elongation. RUSSELL and GOSS (1974) have shown that a pressure of 0.2 bars against the roots of barley can reduce root extension to about half that of the control. Because the diameter of the laterals of the root system of crops is about 300 /am (WIERSUM 1957, FINNEY andKNIGHT 1973), the density of pores greater in diameter than 300 /xm greatly influences the development of crop roots. In the harrowed surface layers the maximum density of crop roots can be about 10-20 cm/cm 3 , but in the deeper layers the density is less than 5 cm/cm 3 (BARLEY 1970, WELBANK et al. 1974. These values are comparable to the porosity densities of pores > 300 /xm in diameter. The pore densities were calculated from the desorption section of the water retention curve. In this way the large pores, that were blocked and had a poor continuity, were not taken into consideration in the calculations. A significant finding of this study was that the severely compacted soil had nearly recovered by the following spring. The factors responsible could be the deep frost in Finland (KIVISAARI 1979) and the successive wetting and drying of soil. In winter 1977-78 the maximum depths reached by frost in the vicinity of the experimental fields were as follows: Espoo 64 cm, Mietoinen 94 cm, Laukaa 21 cm and Anjala 57 cm. | v3-fos |
2019-03-30T13:11:54.357Z | {
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} | s2 | Effect of storage (in small packages) on volatile oil and piperine content of ground black pepper
The composition of the essential oil of Sri Lanka black pepper and changes in composition on grinding and storage in various packaging materials are reported.
Introduction
Pepper (Piper nigrum L.) is one of the moreimportant spices grown in Sri Lanka. Sri Lanka pepper is reported to have special characteristics6 (high volatile oil and piperine) which gives it a special position in world trade. Traditionally, pepper has been exported as whole black pepper, however with increasing competition in the world market the need for product diversification has arisen. Among the new forms of pepper for the export market are: ground pepper (black and white), canned and pickled green pepper, buff and white pepper (containing pericarp) and dried green ~e p p e r :~ The original systematic study of the volatile constituents of pepper by Jennings 8,9,11.12.13. 15 and co-workers was carried out using Sri Lanka pepper. However, these studies were directed mainly towards the confirmation of the presence and identification of the constituents of the volatile oil. Although one study '6 gave a quantitative picture of the oil of Sri Lanka pepper, the report is nearly 20 years old. Indian varieties, on the other hand, have been extensively studied and the subject has been given much prominence in a recent review. 6 Thus there is a case for investigating the composition of volatile.constituents of Sri Lanka pepper based on gas-liquid chromatography notwithstanding the fact that the subject was considered in part in a paper on quality control!s 'Past studies 1.2.1.4 on the packaging of ground black pepper have revealed that polyethylene alone does not fully meet the requirements of a ground pepper and in order to retain volatiles, select laminates must be used. Chinenova el aL4 reported reduced losses in lacquered cello~hane, while Balsubrahmanyam elal-" found that low density polyethylene (LDPE) and high density polyethylene (HDPE) were poor barriers for volatile oil while ground pepper in a polyester / polyethylene lined carton lost the least oil. Balsubrahmanyam and Kumar2 reported a 5 month-shelf life using a paper / aluminium foil / polyethylene laminate as well as a cellophane / LDPE double pouch. Balsubrahmanyamet a1 'also reported that high temperature storage in LDPE and H D P E resulted in 'sticky' packages and oil deposition on the surface of the packaging material.
Despite this knowledge, ground pepper continues to be marketed in Sri Lanka in single pouch LDPE.
Our study on packaging of this material appeared important for two reasons:- T o our knowledge the effect of storage in various packaging material on essential oil composition has thus far not been reported.
(ii) It was of interest to determine in more detail the shelf-life of'ground pepper in LDPE as this was the packaging material currently in use in this country.
In this paper we report the following: (v) The effect of storage (in these packages) on piperine (the pungent principle of pepper). Chwi ,pad, 5 mm min-1.
Moisture content
Moisture content was determined by the Dean and Stark (toluene) method.5
Oil content
Oil content was determined using Clavenger light oil arm after hydro-distillation for 4h. For the determination of oil content grinding was preceded by freezing. This procedure prevented oil loss.
Oil composition
Oil composition was determined using a Varian 2440 gas chromatograph. Operating and other conditions are given in a footnote of Table 1. Identifications were made using retention data and peak enrichment techniques only. This has been considered adequate as all the components tentatively identified have been previously reported in pepper oil.
Piperine content
Pepper was extracted with CH2CI, and pepper oleoresin produced as described previously? 9' Piperine was estimated by the two methods detailed previously: (i) tlc-uv densitometric method7 and (ii) the tlc-uv spectrophotometric method."
Oil composition of Sri Lanka pepper oil
Oil composition of an export sample'and a sample collected frdm an estate is shown in Table 1. As reported previously"16' monoterpene hydrocarbons were present in relatively large proportions. A significant peak appeared immediately prior to P-caryophyllene (r.r.t = 3.62) conspicuous in Sri Lanka pepper and sometimes occurring in very large proportions. The peak, not reported previously, was unidentified. Beyond r.r.t. = 5.6 were 15-20 minor components whose total contribution was < 8%. No effort was made to identify these components. Table 1 also shows the effect of grinding where the levels of monoterpene hydrocarbons declined to the greatest extent. It appears that some caryophyllene o i a chromatographically similar sesquiterpene was formed during grinding.
Prelimiizary observations
Moisture content was initially 8%. After the various stages of storage, moisture content rose to 9-1496. Mouldiness was not visually observed.
Effect on oil corztent
Oil content declined markedly under all LDPE and the HDPEstorage conditions but remained constant in the aluminium foil laminate and in the tinned can. Some of the results are expressed in Figure 1. (50p m, 100 y m and 175p.m LDPE, 15pm HDPE and A1 foil laminate).
Effect on oil conlposition
Here selected known components were combined under the following categories: (1) nlonoterpene hydrocarbons. (ii) sesquiterpene hydrocarbons and (iii) oxygenates.
In all cases a greater loss of monoterpene hydrocarbons was observed. Sesquiterpene hydrocarbons were also lost, but at a less rapid rate. On a percentage basis the smallest losses appeared to be eqhibited by the oxygenates . Figures 2,3 and 4 describe the rate of loss of volatile oil fractions from LDPE (25p.m) LDPE (175.pm) and HDPE (1 5 p m). Losses of monoterpene hydrocarbons were also shown in the A1 foil laminate and tinned cans after six months of storage; as total oil content did not decline in these cases, the loss of this fraction is probably due to its conversion into other volatiles. Table 2 describes the changes in composition of oil after different treatments and illustrates that the degree of less of constituents is preferential.
Fffcct o)t i)i/~erirre content
Both methods of assay of piperine used in this study gave very closely concordant results.
The only significant decline in piperine content was in the 25pm LDPE ( Figure 5). The figure also shows the loss of volatile oils from the same package. Volatile oil and piperine losses closely parallel one another. Only identified constituents, present in significant amounts, areshown. Constituents areexpressed as a%of total oil. The piperine levels of the 50 p m LDPE packaged pepper showed a small decline (5%), while no significant decline in piperine content was shown in the case of the other packaging materials.
On washing the .outer surface of 25 p m LDPE with ether, two major components were isolated: (i) piperine-separated by tlc and its identity verified by its uv spectrum and (ii) caryophyllene oxide (tentatively identified by glc and tlc techniques).
Discussion
The composition of Sri Lanka pepper oil does not differ significantly from that reported by the original study of Jennings and Wrolstad.16 However special attention must be drawn to the additional peak at r.r.t = 3.62 which appears to be characteristic of and unique to Sri Lanka pepper oils. This peak has not been identified.
Results show that milling and packaging conditions markedly affect oil content as well as composition. The absence of volatile constituents in the samples from the open market illustrates two facts viz. : (i) little attention is being paid to the method of packaging of ground pepper and (ii) the absence of volatiles does not appear to be an important consideration in local consumer tastes.
However, this will not be true for products destined for the export market. Here, cold or otherwise controlled milling will be required to minimize heat generation and packaging materials will need to be chosen according to the requirements of shelflife. This does not necessarily rule out the use of LDPE as preferential loss of monoterpene hydrocarbons and thereby a n increase in concentration of oxygenates among the volatiles could result in a product more preferred organoleptically.
The study, confirms that both LDPE and HDPE are poor barriers for essential oils and that the thickness of LDPE has a significant effect on not only the rate ofloss of oil but also the composition of the residual volatiles.
The loss of piperine from the 25 micron LDPE is interesting and it is theorised that this compound migrates through the polyethylene facilitated by the movement of oil through the barrier. The detection of caryophyllene oxide on the outer surface of the pouch is not surprising and probably arises by the oxidation of B-caryophyllene; while the less volatile caryophyllene oxide is retained on the outer surface ofthe pouch to some extent, the other hydrocarbons are volatalised. | v3-fos |
2014-10-01T00:00:00.000Z | {
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} | s2 | Use of pattern-recognition display techniques to visualize the data contained in complex data-bases. A case study
Automation and computer acquisition of data in analytical chemistry has meant that enormous amounts of data can be obtained. The analytical chemist is faced with the problem of bringing some order into these data, so that he can understand the relationships between the variables being measured and between those variables and the object of his research. This is particularly difficult when more than two variables are being measured. Indeed, when only one or two variables are measured the analyst usually makes graphs to explain what happens. When more tha two variables are measured, it is impossible to represent the data in two dimensions. The purpose ofthis article is to show that, in such a case, pattern-recognition methods can be used to visualize the data and therefore to gain insight into them. These methods can be called display methods. Instead of giving a lot of theory, this is demonstrated with a practical example: the authentification of the geographical origin ofwine. The classical methods for the chemical analysis of wines enable the major constituents, such as ethanol content, dry matter, acidity and mineral content, to be controlled. These parameters can be useful for the detection of adulterations, but do not help with the problem of the identification of wines according to their origin. Until now, such an identification has only been possible with sensorial evaluation by experienced tasters. As the characterization of wines according to origin is important, several investigators have attempted to solve the problem by multivariate characterization of wines. Wines are analysed for a number ofconstituents and the set ofobservations made on each wine sample constitutes a pattern. If the constituents that were analysed were appropriately chosen, wines from different origins would have different patterns--so these patterns may be used for classification according to origin. Motet et al. [1] and Scarponi et al. [2 and 3-] analysed samples of three groups of Venetian DOC wines for several inorganic parameters. On the basis of the patterns obtained, and with the use of multivariate statistical techniques, the three groups of wines could be reasonably well identified. Kwan et al. [-4] also proved that the characterization of wines on the basis of inorganic contents can be useful for assignment according to origin. Schreier et al. [5] made use of the concentrations of aroma constituents derived from the grapes: the six groups of German white wines that were involved in their study could be discriminated using multivariate techniques. When the same samples were characterized by their content ofyeast metabolites, the discrimination decreased considerably. It has been suggested that amino-acid patterns may be useful for the detection of adulterations of food products (orange-juice
Introduction
Automation and computer acquisition of data in analytical chemistry has meant that enormous amounts of data can be obtained. The analytical chemist is faced with the problem of bringing some order into these data, so that he can understand the relationships between the variables being measured and between those variables and the object of his research. This is particularly difficult when more than two variables are being measured. Indeed, when only one or two variables are measured the analyst usually makes graphs to explain what happens. When more tha two variables are measured, it is impossible to represent the data in two dimensions. The purpose of this article is to show that, in such a case, pattern-recognition methods can be used to visualize the data and therefore to gain insight into them. These methods can be called display methods. Instead of giving a lot of theory, this is demonstrated with a practical example: the authentification of the geographical origin of wine.
The classical methods for the chemical analysis of wines enable the major constituents, such as ethanol content, dry matter, acidity and mineral content, to be controlled. These parameters can be useful for the detection of adulterations, but do not help with the problem of the identification of wines according to their origin. Until now, such an identification has only been possible with sensorial evaluation by experienced tasters.
As the characterization of wines according to origin is important, several investigators have attempted to solve the problem by multivariate characterization of wines. Wines are analysed for a number ofconstituents and the set ofobservations made on each wine sample constitutes a pattern. If the constituents that were analysed were appropriately chosen, wines from different origins would have different patterns--so these patterns may be used for classification according to origin.
Motet et al. [-1] and Scarponi et al. [2 and 3-] analysed samples of three groups of Venetian DOC wines for several inorganic parameters. On the basis of the patterns obtained, and with the use of multivariate statistical techniques, the three groups of wines could be reasonably well identified. Kwan et al. [-4] also proved that the characterization of wines on the basis of inorganic contents can be useful for assignment according to origin. Schreier et al. [5] made use of the concentrations of aroma constituents derived from the grapes: the six groups of German white wines that were involved in their study could be discriminated using multivariate techniques. When the same samples were characterized by their content ofyeast metabolites, the discrimination decreased considerably.
It has been suggested that amino-acid patterns may be useful for the detection of adulterations of food products (orange-juice Professor Dr Massart is corresponding author. for example) [6, 7 and 8], and in order to determine the geographical origin of foodstuffs. Gilbert et al.
[_9] made use of amino-acid patterns to prove that groups ofhoney samples from different countries could be distinguished from each other.
As amino-acids are important factors in the vinification process, and as most of the amino-acids in wines are related to the grape varieties, Ooghe and De Waele [ 10] suggested that the differentiation of wines according to origin is possible on the basis of such patterns. Therefore, French wines were analysed for 20 amino-acids. The interpretation ofthe resulting data-set is not possible with the use of univariate statistics or by visual interpretation. The evaluation of the results by comparing the values for each of the parameters separately takes no account of the relationship between these different parameters. This can cause an important loss of information, as becomes obvious from the following example. In figure the concentration of the proline content is plotted against the concentration of the glycine content in, respectively, 13 C6tes du Rhone wines and 14 Beaujolais wines. From the range of these two parameters in both groups it is clear that univariate consideration of the two parameters separately does not permit differentiation of both groups of wines. However, when the two parameters are plotted against each other, a good discrimination can be made between the two groups. Obviously, more information is obtained from bivariate interpretation of the results. If the concentration of a third amino-acid is taken into consideration, the data-set can be visualized by representing each individual wine sample in a three-dimensional space, each dimension corresponding to the concentration of one acid. This three-dimensional representation might then improve the differentiation between the groups. However, only a minor part of the information included in the data-set is used. As 20 parameters are available for each wine, each of the samples can be thought to be situated in a 20dimensional space. Visual representation ofthe data is no longer possiblemmore sophisticated techniques that utilize the information in an optimal way and that can give a visual representation ofthe results are necessary. Multivariate statistical techniques or pattern-recognition techniques can be used for this purpose.
Among the different pattern-recognition techniques, a distinction can be made between three groups--pure display methods, supervised methods and clustering techniques: (1) The aim of the pure display methods is to represent multivariate data collected for a group of samples in a two-dimensional space without significant loss of information.
(2) Supervised methods are used when the data-set consists of samples that can be divided a priori into several groups, which is the case in this example. They aim to develop mathematical decision rules that can be used for the classification of new samples.
(3) Clustering techniques are employed when the samples of the data-set cannot be a priori divided into groups, or when one ignores the a priori existence of groups. The fundamental purpose of these techniques is to find or to define groups in the data-set.
The first step in the analysis of a multivariate data-set should always be the display, as in this way an indication can be obtained of what can be expected from further multivariate investigations. In this article it is the intention to introduce and to illustrate these methods with results obtained from a specific data-set. Besides the typical display methods, some supervised and most clustering techniques also permit visualization of the data. So the application of such techniques on the same data-set is also explained. Beaujolais wines (1-14) and 13 Cbte du Rhone wines (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27). The full lines are the axes of the plot of the unscaled data; the dotted lines are the plot of the scaled data. Where [--] [-]=range of the parameters in the Beaujolais wines; and Q) (C) range of the parameters in the Cbte du Rhone wines. Computer programs The computer package 'ARTHUR' [11] was used to obtain principal component plots and non-linear maps of the data-set, the 'BMDP' package [12] was used for the application of LDA. From the 'CLUSTAN' package [13] the routines 'Hierarchy', 'Plink' and 'Tree' were used for the application of the clustering technique and for the representation of the corresponding dendrogram.
Pre-treatment of the data Before the application of the multivariate techniques, the data were transformed in order to make the range between the different variables comparable. A z transformation, or autoscaling, was applied: each variable is given variance one and mean zero. This means that from each variable the average of that variable over the entire data-set is subtracted and the result is divided by the standard deviation of that variable over the dataset. It will be demonstrated that scaling can have a great influence on the display obtained.
Data-set
195 French wines were analysed for their amino-acid pattern. An appropriate sample of wine was boiled for 20h in a nitrogen atmosphere with an excess of 6N HC1. After filtration and concentration in vacuum an adequate amount of the concentrate was injected into an amino-acid analyser (a Technicon NC-2P). The concentration ofeach of the 20 identifiable aminoacids was determined by the use of an external standard that contained each of the amino-acids in an amount comparable to that of the samples. Uj--alj X qt-azj X + q-anj X (1) The coefficients ai. (the loadings) give an indication of the importance of the original variable in the direction of PCj.
Instead of thinking of the objects as situated in the hyperspace with the position of each object given by the values of the N original parameters, the objects can be thought now to be situated in a hyperspace the dimensions of which are given by the latent variables. This coincides with the rotation of the original space. The position of the samples in this rotated sphere are then given by the scores of each object on each PC. These scores are calculated by filling in the values of the parameters Xi in equation (1).
As the first few PCs explain the greatest part of the variance within the data, the samples can be represented in a reduced space defined by these first few PCs only. The percentage of the variance explained by each PC can be calculated and gives an idea of how much of the information in the data-set is represented in the display. A PC analysis was performed on the scaled data-set plotted in figure 1. As in this data-set each sample is characterized by two parameters, two PCs can be computed. The vector V1 in figure gives the direction ofthe first PC. The second PC will be orthogonal to the first. The PCs are given by the following equations: Instead of representing the samples in the original pattern space, as was done in figure 1, they can also be represented in a rotated space--the co-ordinates of which coincide with the direction of the PCs (see figure 2). The position of, for instance, sample 1, which has in the plot ofthe scaled data the co-ordinates(-0" 158, 1-997) is computed in the following way: =0"7071 (-0"158)-0"7071 1"997 1"524 U2,1 =09"7071 (-0"158)+0"7071 1"997= 1"300.
In order to represent the data in a reduced space, i.e. in a onedimensional space in this example (on one axis), the data points can be projected on the first PC: figure 3 shows such a display.
As the fraction of the total variance explained by the first PC amounts to 57.8%, the display obtained in the reduced space gives an idea ofthe situation ofthe samples in the original space. The loadings ofboth parameters on the first PC are equal, which means that the variance represented in the direction ofthis PC is defined by both variables to the same extent. In fact, when PCA is performed on a two-dimensional data-set with autoscaled variables, the absolute value of the loadings on both PCs will always be equal to each other. In the case that more than two variables are included in the data-set, the loadings cannot be predicted a priori; the loadings of the variables on the important PC give an indication of the relations between the parameters themselves. Indeed, when two variables are strongly correlated the variance of these two variables over all the objects will vary in a similar way. In the case that a great part of the variance of one of these variables is explained, by for instance, PC 1, resulting in a high loading on this PC, a significant part of the variance within the other variable will also be explained by the same PC, so that the loading of this second variable will also be high on this same PC. Variables that have high loadings on the first few PCs will be strongly related to each other.
If PCA is performed on the unscaled data, the first PC explains 97.5% ofthe variance and the second 2.5%. This first PC is given by the equation" =0"9997 X-0"0251 X 2.
Clearly the direction of this PC is given almost solely by proline--the loading ofthis variable is much greater than that of glycine. Consequently, the information given by this PC (see figure 4) is almost the same as that given by proline itself. This is to be expected as the variance ofproline in the samples (0.8906) is much greater than that of glycine (0.1442).
If the only purpose of displaying the data is to obtain a visualization of the data-set without interest in the relations between the parameters used to characterize the samples, nonlinear display methods can be used as well. These methods produce displays in which the reduced dimensions are nonlinear combinations of the original variables. The displays one obtains are called maps. Non-linear mapping (NLM) is one of the most frequently used methods: it represents the data in a reduced dimension according to the criterion that the distances between the samples in the reduced space approach the distances between the samples in the original space. Kowalski and Bender [14] give the following comparison in order to explain the method. Suppose that each of the samples in the original hyperspace is connected with every other sample by tensionless springs. The total energy of the springs is zero when considered in the original space. Pressing the samples together in order to obtain a mapping into, for instance, two dimesions gives rise to a tension on each of the springs. NLM aims to give a representation of the data so that the sum of the tension on all the springs is minimal. Bordeaux wines, particularly, form a compact cluster in the plot. The differences between the two subgroups in the Bourgogne wines is less explicit, but a certain degree ofdissimilarity can still be seen. Table 2 gives the loadings of the amino-acids on the two first PCs. The loadings on PC have the same magnitude for all the parameters (except for proline but this has practically no importance in the first direction). This means that this PC represents mainly the difference in the concentration of the amino-acids in the samples. As the Bordeaux and the C6tes du Rhone wines all have a low score on this component, it can be concluded that these groups of wines are characterized by a lower total amino-acid content as compared to the Bourgogne wines. The direction of the second PC is largely due to the proline content. The discrimination that occurs in this direction between Beaujolais wines and the other groups is mainly due to the greater concentration of proline in Beaujolais. As mentioned above, the loadings on the most important PCs may give an indication of the relationship between the parameters themselves. In the example, only the first PC is really important. The amino-acids Asp-ac, Thr, Set, Glu-ac, Gly, Ala, Val, Met, Ile, Leu, Phe and Lys have almost the same weight on this PC; consequently, these parameters must be correlated to a high degree. This conclusion was confirmed by the application of the routine Pearson available in the SPSS package [ 15]. The correlation between each of the amino-acids mentioned ranges from 0"68 to 0"95. There is no significant correlation between proline and one of the other amino-acids.
The second display method applied was NLM. As the method is time-consuming, the NLM routine of the ARTHUR package was used to obtain maps for two groups of wine at a time. Figure 6 is the map of the Bordeaux and the non-Beaujolais Bourgogne wines; figure 7 is the map of the Bourgogne wines. Again, it can be concluded that a differentiation of wines must be possible on the basis of the parameters used in the study. In order to compare the representation obtained with NLM and PCA, PCA was applied to the same combinations of two groups of wines. The pictures obtained with NLM are similar to the PC plots. As the optimization procedure to obtain an NLM map is rather tedious, and no supplementary information is obtained about the parameters themselves (i.e. it cannot be easily concluded which parameters are responsible for the differentiation between the groups), PCA is the preferred display method. 140 Table 2. Loadings of the parameters on the two first principal components. Supervised methods that can be used for display As mentioned above, supervised pattern-recognition techniques are methods that deal with the problem of the classification of 11 Fi.qure 7. Non-linear map of 110 Bourgogne wines. Where 1 --Beaujolais wines; and 2 non-Beaujolais Bourgogne wines.
samples or objects into a group. The general procedure carried out in a supervised situation is as follows. First, with a data-set consisting of objects with known classification, a classification or decision rule is developed that separates the learning classes in an optimal way--these decision rules can be used for the classification of new samples. The computation of the decision rules corresponds with the division of the original hyperspace in as many regions as there are learning classes in the training set, each region corresponding to one class. New objects are classified according to their position with respect to the boundaries between the classes. The data-set collected on the wines can obviously be investigated with supervised techniques as the samples can a priori be divided into several groups, i.e. into groups of wines from the same region. Some supervised techniques, such as linear discriminant analysis (LDA), also give a display. In LDA, the classification functions are developed in a reduced pattern space; as in PCA, each of the directions of this space is obtained by a linear combination of the original variables. These new variables are called discriminant functions or canonical variates. The criterion used to define the discriminant function is the maximization of the ratio of the betweengroup variance to the within-group variance. The resulting discriminant functions are oriented in the direction which provides a maximum differentation between the classes. The dimension of the reduced pattern space is one less than the number of training classes. In the case of two groups, one discriminant function is obtained. Each of the discriminant functions is given by the equation: DFi=alj X -{" +a, v X,,. ( The position of an object, k, in the reduced space is given by its score on each discriminant function: dsik=a Xlk + +a, v X,,. ( The display obtained with this technique visualizes the optimal discrimination between the classes. The vector V' in figure gives the direction of the discriminant function obtained when LDA was applied to the data-set represented in the figure. Figure 8 gives the display of the data in the reduced onedimensional space.
Application to the wine data With LDA, a display of the data-set is obtained when the samples are plotted in the space defined by the discriminant functions. When a discrimination is attempted between three groups, two discriminant functions are calculated and the optimum discrimination can be represented in a twodimensional plot. If the differentiation between, for instance, four groups is investigated, the reduced space is threedimensional and so visualization becomes more difficult.
Therefore LDA was applied to only three groups at a time. The variables were introduced in the discriminant function one at a time. The selection criterion for the introduction of a new parameter is based on the discriminating power of that variable, i.e. the degree to which a newly introduced variable increases the discrimination between the groups. Variables are included in the discriminant function until the further inclusion of one of the remaining parameters does not significantly improve the discrimination. The coefficients of the canonical variates obtained in this application are given in table 3. Only the amino-acids Asp-ac, Thr, Pro, Cys, Ileu, Gaba, His, Orn, Eth, and Arg were included. It is this combination of aminoacids that gives the best discrimination between the three groups of wines under investigation.
Clustering techniques
The aim of multivariate techniques belonging to this group is mainly to find groups of similar samples in the data-set and to display these in a so-called dendrogram. The first step in clustering is the determination of the similarity between the different objects to be clustered. One ofthe possible criteria is the distance between the samples in the original hyperspace.
Consider, for instance, four objects (A, B, C, D) each measured for three different variables (X, Y, Z): The second step in the clustering procedure is the search for similar groups of objects. Many clustering methods exist, differing in the criteria used to decide which belong to the same group. One of the possible procedures is the following. In the first instance, all the objects are considered separately. Then the two most similar objects (A and B) are to form a cluster A*. The similarity between this cluster and the remaining objects is now represented by one value, for instance the mean similarity between the objects of the cluster and the other objects: Figure 10. Representation of the situation offour objects (each measured for three parameters) in the pattern space.
Again, the two most similar objects or clusters, A* and C, are joined and represented as one object: A dendrogram visualizes the relations between the different objects ofthe data-set: the total height ofthe links are a measure of the distances between the objects. The algorithm used to join the clusters is called the average linkage method, and the method used for clustering the data of figure and the wine data is known as Ward's method, or the error sums of squares method. The clustering obtained with this method is generally comparable to that obtained with the average linkage algorithm. More information on the different clustering algorithms can be found in a recent book by Massart and Kaufman [16]. Figure 11 is the dendrogram of the scaled data offigure 1. As the objects can be divided into two classes, the highest link in this dendrogram can be cut to obtain two clusters and to see whether each of these contain mainly objects of a same class. Applying this criterion, a cluster is obtained which consists only ofCgte du Rhone wines, while the second contains all the Beaujolais wines and three C6te Rhone wines. Obviously, the dendrogram visualizes the degree of discrimination between the groups of wines.
The application ofWard's method to the wine data results in the separation of the objects into two very distinct groups (see figure 12): group A is compact and consists mainly of Bordeaux wines; group B, which is more heterogeneous, can be divided into three subgroups: B1 and B3 consist of Bourgogne wines and B2 joins wines from Bordeaux and from Bourgogne. The Cgte du Rhone wines appear in all of the clusters.
Conclusion
The object of this paper is to show that display and related pattern-recognition techniques permit the investigation of multivariate data-sets. Wine analysis is considered only as an example and certain aspects are treated more fully by Ooghe and De Waele [10]. Clearly, the applications are not confined to wine or food analysis. Applications in, for example, archeometry [17], meteoritics [18], microbiology [19], medical diagnosis [20], investigations of structure activity relations [21], and environmental problems [22] have also been reported. Pattern recognition is a natural addition to automated analysis: eventually there will be instruments which can analyse samples, determine the pattern to which a sample corresponds, and suggest its geographical origin. Figure 11. Dendrogram of the scaled objects in .figure 1 (obtained by using the Ward's method). | v3-fos |
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} | s2 | Farmers Read Wheat Newspaper Most in Winter
Many farm magazines tailor more of their editorial and advertising linage to appear in late fall and winter when farmers aren't busy with field work. Creative Commons License This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 4.0 License. This research brief is available in Journal of Applied Communications: https://newprairiepress.org/jac/vol66/iss1/11 Farmers Read Wheat Newspaper Most in Winter Many farm magazines tailor more of their editorial and advertising linage to appear in late fall and winter when farmers aren't busy with field work. It's a lesson that we shouldn't forget. You wouldn't read much if you were working 18-hour days in the field, would you? In early November 1982, we did a telephone readership study of MINNESOTA WHEAT, the monthly publication of the Minnesota Wheat Research and Promotion Council. MINNESOTA WHEAT is a monthly tabloid newspaper that varies from four to eight pages. It is mailed to over 34,000 Minnesota farmers who contribute to the Minnesota Wheat Research and Promotion Council through a one cent per bushel check-off program. Funds are used for market development, promotion and research. The council helps fund wheat research at the University of Minnesota's Agricultural Experiment Station. For the past two years we've prepared some special articles for MINNESOTA WHEAT's annual progress edition in October. We did stories based on various research projects the Wheat Council helps fund. Our question was how well farmers read the articles. Published stories give benchmark figures on readership for state and national farm magazines, but I've never seen any for state commodity publications like
Many farm magazines tailor more of their editorial and advertising linage to appear in late fall and winter when farmers aren't busy with field work.
It's a lesson that we shouldn't forget. You wouldn't read much if you were working 18-hour days in the field, would you?
In early November 1982, we did a telephone readership study of MINNESOTA WHEAT, the monthly publication of the Minnesota Wheat Research and Promotion Council. MINNESOTA WHEAT is a monthly tabloid newspaper that varies from four to eight pages. It is mailed to over 34,000 Minnesota farmers who contribute to the Minnesota Wheat Research and Promotion Council through a one cent per bushel check-off program. Funds are used for market development, promotion and research. The council helps fund wheat research at the University of Minnesota's Agricultural Experiment Station.
For the past two years we've prepared some special articles for MINNESOTA WHEAT's annual progress edition in October. We did stories based on various research projects the Wheat Council helps fund. Our question was how well farmers read the articles. Published stories give benchmark figures on readership for state and national farm magazines, but I've never seen any for state commodity publications like MINNESOTA WHEAT.
In addition to questions on readership about specific articles dealing with research, I worked with the council to develop questions regarding farmer preference for frequency and length of the publication, when it was read most, and whether farmers would object to advertising in the publication (the publication did not print advertising at that time).
The Wheat Council provided us with an "almost random" sample of 130 names from their total mailing (34,000). We originaJly aimed at a sample of 100. That was done by drawing every 329th name on the list. However, telephone numbers could not be uncovered for about 15, so we drew another 30 names by taking every 1,000th name. That gave us 130 names-110 with telephone numbers.
After an initial screening question to find out if respondents received the October issue of MINNESOTA WHEAT, we had three categories of questions on the survey: -When farmers read the publication the most. We had three time periods: November through March, April through June, and July through October. Response categories were: read most, (defined as having read 75 percent or more of the publication); read some (50 percent or more); glanced at; and did not read.
-Readership for specific articles, both the research stories generated by us and stories on council activities and market development produced or solicited by staff people at MINNESOTA WHEAT.
-General questions about the publication. Staft and board members of the Wheat Council were already can· sidering making some changes in the publication before we did the study. One question the Wheat Council had was whether farmers would object to advertising In the paper. They also wondered if farmers would prefer a one·page newsletter, as opposed to the present format.
Our overall response rate was 76 percent (we reached 84 of the 110 on the list via telephone). But only 36 percent (30 respondents) said they'd received the October issue. Another 27 percent (23 respondents) answered "I don 't know." That probably meant most of them had received the publication but hadn 't yet uncovered it due to the busy harvest season.
We couldn't ask questions about readership of the Oc· tober issue if they hadn't received it. But we did ask the 23 people who "didn't know" If they'd received the October issue what months they read it the most. We also asked the questions about advertising and publication format, so we had 53 respondents for those portions of the questionnaire.
Respondents gave an overwhelming margin to November through March as the months when they read the publica· tion the most. Of the 53 respondents, an even 75 percent said they either read most (49 percent) or read some (26 per· cent) articles during these months. Another 17 percent answered glanced at; 4 percent said do not read (4 percent, no response).
From April through June, 9 percent said read most; 34 per· cent read some ; 42 percent said glanced at; 8 percent said do not read. Another 8 percent said do not read , and there was 8 percent no response .
For the July through October period, figures were 8 per· cent read most; 21 percent read some, 47 percent glanced at; 15 percent did not read and 9 percent no response.
55
Here are answers to other questions asked of the 53 respondents: - . Highest readership score was for an article about overseas marketing activities entitled "U.S. Wheat Around the World." Of the 30 respondents, 13 percent said they read most and another 13 percent said they read some . Another 17 percent said they glanced at it, while 53 percent said they didn 'I read it (3 percent no response).
That isn't particularly high readership for a specialized farm publication. Did not read figures ranged from a low of 53 percent to a high of 73 percent for the publication ' s editorial . | v3-fos |
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} | s2 | Problems of calf rearing in connection with their mortality and optimal growth: A review
Protection of calves against enteric and respiratory disorders is dependent on the passive immunity that the calf has received, its innate resistance to infection, the burden of infection in the environment and the nutrition of the calf. Superimposed on these, are the effects of management and physical environment. The plane of nutrition required for dairy heifers during rearing depends on the age at first calving, and for meat animals depends on the carcass weight and fat deposition required at a particular slaughter date in relation to time of birth.
Introduction
For comparative purposes, it is important that the time span to which mortality rates refer, should be specified. Thus, calf mortality can best be subdivided into: (a) abortions (still births at < 270 days of gestation); (b) perinatal mortality (still births at > 270 days of gestation and mortality during the first 24 h of life); (c) neonatal mortality (calves born alive that die between 24 h and 28 days); (d) older calf mortality (calves born alive that die between 29 and 84 days, or between 29 and 182 days). Under good management conditions in the developed countries, mortality rates in these classifications are approximately as follows: (a) Abortions: 2--2.5% (b) Perinatal mortality : 3.5--5.0% Of these deaths, 85% are due to anoxia and the results of prolonged parturition (Greene, 1978), mostly caused by inappropriate mating, particularly of heifers. The problem arises mainly from the effect of the sire and dam on birth weight and musculature, but partly to the size of the pelvic opening of the dam (Menissier, 1975). Cows with difficulties at successive calvings have smaller pelvic openings (Dufour et al., 1981} and a strong correlation has been found for dystokia in Holsteins at successive parities (Thompson et al., 1981). Bull calves and younger ages at calving increase the risk. The dimensions that were most closely related to calving difficulties were the circumference of the head and nose. The role of the hormonal status of the dam and the effect of the prepartum nutrition of the dam is largely unknown, although the incidence of dystokia appears to be greater with overfat animals. Rarely, septicaemia may be the cause of death in calves within the first 24 h of life.
(c) Neonatal mortality: 3% In this period, septicaemia and enteric disorders caused by particular serotypes of Escherichia coll. with or without viral involvement, or by salmonella, particularly Salmonella typhimurium, are the main cause of death. The susceptibility of the calf to these conditions is dependent on the passive immunity received from colostrum, the calf's innate resistance to disease, the burden of "infection" in its environment and the quality of the diet given after the colostrum-feeding period. Superimposed on these are the effects of management and the physical environment.
(d) Older calf mortality: 1% (29--84 days or 2% (29--182 days) This period is dominated by the occurrence of respiratory infections, salmonellosis, particularly S. dublin, and bloat arising from the failure of oesophageal groove function in veal calves, or from the use of dry diets containing insufficient long roughage. Internal parasites, causing coccidiosis and helminthiasis are also important, particularly in the period 84--182 days, but these conditions are not considered in this review.
The microorganisms
Septicaemia arising from E. coli is associated with the increased virulence and tissue invasion of strains, which produce colicine V (Smith and Huggins, 1976), whereas enterotoxaemic strains that are characteristic of calves are associated with K99 antigen (Orskov et al., 1975), which allows adherence to the mucosa of the small intestine, and with a heat-stable enterotoxin. Adherence occurs initially in the ileum and gradually progresses towards the duodenum causing stunting of the villi (Bellamy and Acres, 1979;Pearson and Logan, 1979), changes in myoelectrical activity (Bums et al., 1978), increased secretion of water and electrolytes into the lumen (Isaacson et al., 1978) and severe diarrhoea and dehydration. However, various viruses, such as rotavirus, coronavirus and epithelial cell syncytial virus may become established first and act synergistically with E. coll. Vaccination of pregnant cows with a vaccine containing K99 antigen may give protection to the calf by inhibiting adherence (Contrepois et al., 1978).
Interference with the balance of E. coil and lactobacilli in the small intestine can arise from changes in the composition of the abomasal outflow, or from the use of antibiotics (Weijers and Van de Kamer, 1965). Moreover a build-up of "infection" in a calfhouse may occur when a large number of newborn calves are introduced successively into a building (Roy et al., 1955). The rate of build-up, which is characterized by an increasing incidence of diarrhoea and mortality as time progresses, is associated with the development and dominance of enteropathogenic strains of E. coli (Wood, 1955) and is related to (a) the immune status of the calf, (b) the postcolostral diet and (c) probably the air space in terms of cubic capacity per calf and ventilation rate in the calf house. Salmonella typhimurium has increased in importance relative to S. dublin in both the U.K. and France, and the incidence of antibiotic resistance is high (Martel and Fleury, 1979;Threlfall et al., 1979). The commonest lesion is necrotic enteritis in the ileum and in the large intestine (Wray and Sojka, 1978). The apparently lower incidence of S. typhimurium in the older ruminant calf, may be associated with the finding that bovine rumen fluid that contains high concentrations of volatile fatty acids has an inhibitory effect on S. typhimurium in vitro (Chambers and Lysons, 1979). Mice rather than rats appear to be the most important rodent vector (Hunter et al., 1976), and a lower incidence of salmonellosis has been reported in individually-rather than group-penned calves {Linton et al., 1974).
In respiratory disorders, the relative importance of bacteria, viruses, chlamydia and mycoplasma has not been elucidated, but viruses appear to be established first and exacerbate the effects of secondary bacterial invasion.
Passive immunity
The immunological status of the calf is determined by the quantity of each class of immunoglobulin (Ig) ingested and the time after birth that ingestion occurs. As little as 14 g Ig given within 12 h of birth will protect the majority of calves against a septicaemia (Aschaffenburg et al., 1951), but 300--400 g Ig given within 36--48 h of birth {equivalent to 1.7 kg/ feed for four feeds from the first two milkings after parturition) are required to ensure protection against a heavy challenge of infection with enteropathogens. The Ig requirement for protection against salmonellosis appears to be non,specific and about twice that required for protection against E. coli {Fisher et al., 1976). With respiratory infections, high serum Ig levels in the first 2 weeks of life have been associated with reduced susceptibility at 2.5 months (Williams et al., 1975).
To prevent bacterial adherence, colostrum must be fed prior to the establishment of the microflora (Logan et al., 1977). Bacteria that are established in the small intestine before the first colostrum feed can be absorbed by pinocytosis in the same manner as for Ig (Corley et al., 1977). Moreover, the presence and multiplication of bacteria may reduce absorp-tion by accelerating cell migration along the villi and reduce "closure time" i.e., the length of time from birth that intact Ig can be transferred into the blood (James et al., 1976;James and Polan, 1978;James et al., 1981). Microorganisms multiplying within the digestive tract may also degrade Ig (James et al., 1976). This finding and also the build-up of "infection" in calf houses may explain why serum Ig levels are lowest and incidence of diarrhoea highest in the spring (Barber, 1979;Vajda and Slanina, 1980;Williams et al., 1980;Boyd and Hogg, 1981) although colostral Ig levels are not affected.
The later that calves are fed after birth, the lower will be their Ig absorption and Ig concentrations in their blood (Stott et al., 1979b). "Closure times" to absorption of intact Ig were reported in 1973 to be 16 h for IgM, 22 h for IgA and 27 h for IgG, and it was suggested then that if colostrum feeding was delayed, a calf might be deficient in IgM (Penhale et al., 1973), which is the Ig of most importance in protection from a septicaemia (Bywater and Logan, 1974). However, recent work indicates that closure occurs at about 24 h for all Ig classes if a calf is not fed. Feeding colostrum shortly after birth promotes earlier closure, and a delay in feeding colostrum, will delay closure up to a maximum of 32 h if calves are first fed at 24 h. Even so, 50% of calves whose first feed was delayed to 24 h were unable to absorb Ig (Stott et al., 1979a).
Attempts to increase time of closure have been unsuccessful, although calves in the presence of their dams, rather than in isolation, appear to absorb more Ig (Selman et al., 1971a;Stott et al., 1976;Fallon, 1978), but very low temperature may reduce absorption (Olson et al., 1980). However, the effect of "stress" in reducing Ig absorption has not been proven (Stott, 1980).
Innate resistance
In theory, calves should ingest more colostrum suckling their dams than is likely to be offered from a bucket, but in practice 32% failed to suckle within 6 h of birth in a study in the U.K. (Edwards, 1982), whilst in the U.S.A., 42% failed to suckle within 12--26 h of birth (Brignole and Stott, 1980). Delays in suckling due to abnormal behaviour of the dam were found to be restricted to heifers. Offspring of older animals with pendulous udders and those that had difficult calvings were likely to fail to suckle. Differences in time of first suckling occurred between the offspring of different sires and low temperatures reduced the calf's desire to suckle.
Extreme dairy breeds, such as the Jersey, Guernsey and Ayrshire, seem to be more susceptible than Friesians to E. coli and salmonella infection (Osborne et al., 1978;Wray and Sojka, 1978;Muller and Eliinger, 1981). Friesian and Jersey calves have been shown to have a higher efficiency of absorption of Ig than have Red Danish calves (Kruse, 1970) and Friesian × Ayrshire calves were more efficient than Ayrshires in absorbing Ig from Ayrshire colostrum (Seiman et al., 1971b) and Holsteins were more efficient than Ayrshires in absorbing total Ig from Holstein colostrum (Baumwart et al., 1977). Breed differences in efficiency of digestion of post-colostral diets (Roy et al., 1970) may be due to differences between breeds in the amounts of digestive enzymes (Ternouth et al., 1976). Associated with the marked decrease in susceptibility to enteric infections as calves grow older is an increase in gastric acid production and in digestibility of protein (Ternouth et al., 1976).
Comparative breed resistance to respiratory infections differs from that of enteric infections, Ayrshire and Hereford × Friesian calves being more resistant to respiratory infections than Friesian and Jersey calves (Roy, 1971).
Nutrition
The composition of the diet fed after the colostrum feeding period may affect the incidence of enteric disease. The severity of the effect will depend on the immune status of the calf in relation to the burden of infection in the environment (Roy, 1975). "Severely" heat-treated milk and non-milk proteins, which do not coagulate in the abomasum may result in reduced gastric acid secretion (an important protective barrier to the multiplication of E. coli) (Tagari and Roy, 1969), reduced gastric enzyme secretion (Williams et al., 1976), reduced proteolysis with increased escape of undigested protein into the duodenum (Tagari and Roy, 1969) and reduced pancreatic enzyme secretion (Ternouth et al., 1974(Ternouth et al., , 1975. In the U.S.A., but not in Europe, milk powders are classified so that "mildly" heat-treated powder can be selected for use in milk substitute diets. To be satisfactory for the neonatal calf, skim milk used in milk substitutes should contain at least 0.17 of the N as non-casein N. The value for raw milk is 0.25 and for UHT sterilised milk (135°C for 1--3 s) is 0.11 (Roy, 1980b).
It appears that whey protein, in the form of whey protein concentrate, which normally passes rapidly out of the abomasum can be well tolerated by the calf. The production of whey protein concentrate has been possible because of new developments in process technology, e.g., reverse osmosis and ultrafiltration.
Diarrhoea in suckled calves has been associated with changes in the composition of whole milk, as a result of dams grazing particular pastures (Shanks, 1950), or being sufficiently calcium-deficient to prevent the coagulation of their milk in the calf's abomasum (Johnston and Maclachlan, 1977).
Gastric stasis, followed by diarrhoea, may occur from allergy to the proteins, glycinin and fl~onglycinin in soyabean flours (Kilshaw and Sissons, 1979). However, only a proportion of calves appear to become sensitised (Kilshaw and Slade, 1980). The oligosaccharides of soya flour may also be detrimental to the calf. Both problems can be overcome by the extraction of soya flour with hot aqueous ethanol.
Diets containing a low fat concentration (10 g kg -1 ,dry matter) cause increased incidence of diarrhoea in comparison with diets containing a high-fat concentration (Roy et al., 1970). Although gastric proteolysis is not affected, pancreatic enzyme activity is reduced by low-fat diets (Ternouth et al., 1974(Ternouth et al., , 1975. Diarrhoea arising from low-fat diets may be caused by fermentation in the large intestine, similar to that produced by diets containing too much soluble carbohydrate. The maximum amount of hexose equivalent (glucose (g) + lactose (g) × 1.05) that should be included in a high-fat diet is about 12 g kg -1 live weight (Roy, 1980b).
Ruminal bloat may be associated with cold-milk feeding or the inclusion of some non-milk proteins in the diet, and appears to be due to failure of oesphageal groove function and resultant passage of the diet into the rumen. Fermentation of carbohydrates, e.g., supplementary glucose or those present in vegetable protein sources may cause abomasal bloat. The claim that the use of acidified milks reduced the incidence of diarrhoea has not been proven.
Weaning at a young age, i.e., 5 weeks rather than at a later age, and the physical environment, e.g., a low relative humidity at a high environmental temperature and a high relative humidity at a low environmental temperature, may all increase the susceptibility of calves to respiratory infections Roy, 1973). In contrast a very high dry matter concentration in a milk substitute to achieve very high growth rates (1.57 kg day -1) and a high carcass fat concentration may increase the susceptibility to respiratory infections and the risks of dehydration (Stobo et al., 1979).
PROBLEMS IN RELATION TO GROWTH
These are concerned with the selection of the most economic plane of nutrition required for a particular form of production. Except for veal production, calves should be weaned on to dry food at the youngest age possible to achieve a low mortality and the desired weight gain. For the dairy heifer, the optimum plane of nutrition will be dependent on the age at puberty that is required for a particular calving age. For the meat-producing animal, the optimum plane of nutrition will depend on the intended date of slaughter in relation to the date of birth, the desired weight of carcass, and the desired degree of fat deposition.
The relative economic efficiency of size of breed, in relation to feed availability and protein metabolism (Roy, 1980a), for milk or meat production has yet to be established.
Dairy heifers
Prediction in the calf stage of subsequent milk yield, composition, fertility and longevity has not been achieved. Very high growth rates during the rearing of heifers, particularly arising from the use of high-cereal diets, can be detrimental to subsequent lactation. It is not known to what extent this detrimental effect is associated with changes in metabolic hormone concentrations in the blood, particularly of growth hormone and insulin, with changes in rumen fermentation patterns leading to a high proportion of propionic acid, and with increased fat deposition in the udder. Nor is it known whether the detrimental effect occurs during any particular period during rearing, i.e., before puberty, between puberty and conception or during gestation, or whether very high growth rates during all of these periods may have an adverse effect.
The effect of environmental factors, such as light, on feed conversion efficiency, growth (Peters et al., 1978) and age at puberty (Roy et al., 1980) is still largely unknown.
Meat animals
Cattle seem unable to compensate for low growth rates during the first 3 months of life (< 10 g kg -~ body weight per day), and thus cannot catch up in weight during the first year of life (Wardrop, 1966). Although after scaling for mature weight (Taylor, 1980), little differences occur between beef breeds in carcass composition at the same stage of maturity, it is not known whether there are discrete classes of muscle:bone ratio for dairy and beef cattle.
Large gaps in knowledge exist in the factors controlling food intake in growing cattle. For instance, greater dry matter intakes may be achieved by pelleting concentrates with roughages rather than by giving them separately (Thomas and Hinks, 1982).
CONCLUSIONS
From this brief review is should be clear that problems of calf mortality and ill-health dominate the field of calf rearing. However, the establishment of optimum growth curves during different periods of life for particular forms of milk and meat production using specific breeds in particular farming systems and economic and environmental conditions is clearly going to be an area of increasing importance in the future. | v3-fos |
2019-04-04T13:12:21.398Z | {
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} | s2 | Cytochemical identification of callose deposits around plasmodesmaia of virus-infected phloem cells
s of The XIV Congress o f the Italian Society of Electron Microscopy 163 using either KOH or SnCI 2 solution which are known as callose solvents in unembedded plant tissues (Currier, Am. J. Bot. 44, 1957, 478). Sections(60-80 nm thick), cut from either Epon-Araldite or Spurr's resin embedded BYDV-infected plants, were transferred with a Deer's loop (J. Microscopy 130, 1983, 115) to small drops of KOH at different concentrations or to SnCI 2 satured solution in HCI IN for 5 to 120 minutes. They were then picked up with a grid, immediately washed in distilled water, stained with U and Pb and examined under the electron microscope. Results indicate that KOH 1 N treatment for at least 15 minutes removes the deposits from plasmodesmata, both in Epon-Araldite and Spurr's embedded samples, although the high pH of the solution tends to make sections opaque, diminishing their general contrast. However, lower KOH concentrations are not effective also after longer exposure periods. SnCI 2 treatments do not give reproducible results as they sometimes seem to be effective in removing the deposits and at other times they are not. In any case, when deposits are removed from plasmodesmata, either using KOH or SnCI2, also callose on sieve plates is removed thus confirming the identity between the two substances. X-RAY MICROANALYSIS AND ULTRASTRUCTURE OF MAIZE LEAVES NATURALLY INFECTED WITH BARLEY YELLOW DWARF VIRUS M. A. Favali, R. Osler*, C. Lorenzoni* and E. Refatti* Institute of Botany and Plant Physiology, University of Padova; *Institute of Plant Protection, University of Udine Barley Yellow Dwarf Virus (BYDW), an isometric virus of graminaceous plants, is a typical member of the luteovirus group, in being phloem-restricted, and aphid-borne but non-sap-transmissible. BYDW has been proven to be a cosmopolitan pathogen existing in several strains that vary in insect vector specificity. Three cultivars of maize plants (W646, FRI8 and B84) resulted positive after the serological test by enzymelinked immunosorbent assay (ELISA) , a useful direct quantitative assay for monitoring yields of BYDW, were used in our studies. General symptoms, such as yellowing, redding and malformations of the leaves were observed. The ultrastructural observations on the infected tissues gave us the possibility to identify the virus particles in the phloem cells and to analyze the alterations of cell organelles caused by the virus infection. Moreover, we report the data obtained by SEM X-ray microanalysis, performed on healthy and infected areas following standard and new methods of specimen preparation. Particularly interesting are the low levels of K + detected in reddish portions of infected leaves. The results of X-ray microanalysis are discussed with the ones obtained after TEM observations. POLYEDRALVIRUSLIKE PARTICLES IN MYOCYTES AND NEUROGLIA OF PERIPHERAL NERVES IN OCTUPUS VULGARIS L. AFFECTED BY RUNGGER'S DISEASE
Viral particles
were detected in 30.1% of examined specimens. In 22% of cases Rotavirus were described 3, and in one case, tubular forms were evidenced ~. In 3.5% of cases Adenovirus were observed, in two cases surrounded by adenovirus-associated virus particles 5 Parvovirus 6 25-30 nm particles were observed in 2.5% of patients. Three cases of Parvovirus-Rotavirus associations could be described too.
In one specimen large clusters of 15-20 nm particles which can ~robably be classified as Parvovirus I were evidenced.
In 0.16% of cases typical Astrovirus could be described 8.
Well (J. Microscopy 130, 1983, 115) to small drops of KOH at different concentrations or to SnCI 2 satured solution in HCI IN for 5 to 120 minutes. They were then picked up with a grid, immediately washed in distilled water, stained with U and Pb and examined under the electron microscope.
Results indicate that KOH 1 N treatment for at least 15 minutes removes the deposits from plasmodesmata, both in Epon-Araldite and Spurr's embedded samples, although the high pH of the solution tends to make sections opaque, diminishing their general contrast. However, lower KOH concentrations are not effective also after longer exposure periods.
SnCI 2 treatments do not give reproducible results as they sometimes seem to be effective in removing the deposits and at other times they are not. In any case, when deposits are removed from plasmodesmata, either using KOH or SnCI2, also callose on sieve plates is removed thus confirming the identity between the two substances. Barley Yellow Dwarf Virus (BYDW), an isometric virus of graminaceous plants, is a typical member of the luteovirus group, in being phloem-restricted, and aphid-borne but non-sap-transmissible. BYDW has been proven to be a cosmopolitan pathogen existing in several strains that vary in insect vector specificity.
Three cultivars of maize plants (W646, FRI8 and B84) resulted positive after the serological test by enzymelinked immunosorbent assay (ELISA) , a useful direct quantitative assay for monitoring yields of BYDW, were used in our studies.
General symptoms, such as yellowing, redding and malformations of the leaves were observed. The ultrastructural observations on the infected tissues gave us the possibility to identify the virus particles in the phloem cells and to analyze the alterations of cell organelles caused by the virus infection.
Moreover, we report the data obtained by SEM X-ray microanalysis, performed on healthy and infected areas following standard and new methods of specimen preparation.
Particularly interesting are the low levels of K + detected in reddish portions of infected leaves. The results of X-ray microanalysis are discussed with the ones obtained after TEM observations. The present work contributes knowledge to the area of viral tumors in Invertebrates.
POLYEDRALVIRUSLIKE
Rungger I described a seasonal epidemic between young Octupus Vul@aris L. found at summer time in the Gulf of Naples when anthropic industrial and oil marine pollutions were at their peak, and treatment was not yet initiated.
The morb consists of edema and nodular tumors in the tentacles and mantle and leads to apathy of the animal and self-multilation and appears to be fatal.
Tumoral foei appeared to consist histologically of degenerated myocytes and connective tissue and to contain hexagonal particles measuring 140 x i00 nm approximately.
According to Rungger's data, 8.4% of young animals (about i00 gg in body weight) were affected but the efforts to obtain experimental reinfection were not conclusive.
In the course of the present study, realized in 1973, three animals were found affected by mantellar microfoci alone and not yet by tentacular tumors; the microfoci were all localized on or nearby the nerves irradiating from the stellate ganglion on the ventral side of the mantle.
Within the nodules myocytes appeared necrotic and virus-like | v3-fos |
2019-03-19T13:08:58.626Z | {
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} | s2 | Inheritance of leaf resistance to Septoria nodorum Berk , in two crosses of spring wheat
The F 2 progeny of two spring wheat crosses involving susceptible and moderately resistant parent cultivars were tested in the field for their reaction to infection by Septoria nodorum Berk. The variation in symptom expression was continuous, providing no support for the existence of individually acting major genes. The broad sense heritability values for the crosses were moderate at 0.47 and 0.34 with over halfof the variation being of environmental origin. The results are discussed in relation to developing a resistance breeding strategy for wheat to S. nodorum.
Introduction
Septoria nodorum Berk., the cause of the glume blotch disease of wheat, has become a major wheat pathogen in Europe, in several parts of Asia, and in South and North America (SAARI and WILCOXSON 1974).
Resistance is the most feasible method for the control of the diseases in the Septoria complex in terms of economic advantage and long term protection (DOODSON 1981). Genetic progress in breeding for disease resistance depends on several factors, such as the availability of resistant germplasm, appropriate screening techniques, and the nature of the host resistance. Available experimental analyses indicate that the resistance of wheat to S. nodorum is under polygenic control and involves several genes (LAUBSCHER et al. 1966, NELSON 1980, MULLANEY et al. 1982, SCOTT et al. 1982. However, there is some evidence that resistance at the seedling stage is conferred by dominant gene(s). KLEIJER et al. (1977) have shown that the cultivar Atlas 66 carries a single dominant gene located on the wheat chromosome 1 B and this gene is responsible for resistance to S. nodorum at the seedling stage. Recently, SCEIAREN and EYAL (1983) have provided further evidence for this phenomenon as they have found that the resistance in highly resistant cultivars may be governed by major R-genes. Hitherto there is no evidence for the existence of single genes which alone could give an easily detectable level of resistance at the mature plant stage (SCHAREN and EYAL 1980). BRÖNNIMANN (1975) has shown tolerance, measured as the loss in 1000 kernel weight, to be polygenically inherited and controlled by additive gene action. Further, KLEIJER et al. (1980) have demonstrated, using the chromosome substitution lines of Thatcher and Timstein, that the cultivar Thatcher carries 12 chromosomes which influence the control of tolerance, nine having a large effect and three having a minor effect. However, only seven chromosomes of Timstein showed a positive effect, suggesting that the interaction effects between chromosomes are important.
Progress in improving quantitatively inherited traits is dependent on the proportion of variation due to genotype and that due to environment. The present preliminary study describes the results of an evaluation of the heritability of leaf resistance to S. nodorum on two crosses of spring wheat.
Materials and methods
The data reported in this study are based on the trials carried out at the experimental farm of the University of Helsinki. Two F 2 populations, derived from the crosses Tähti X TGS 334/74/8 and Tähti X Maris Butler, were investigated. TGS 334/74/8 and Maris Butler (obtained from the Cambridge Plant Breeding Institute) are moderately resistant varieties, while the Finnish cultivar Tähti is a susceptible one. The crosses were made during the winter of 1980 in glasshouses. The F t generation was grown in the glasshouse, and it produced, by self-fertilization, material for the F 2 populations.
Small plots were space-planted with two replications arranged in a randomized block design. Standard fertilization and herbicide treatments were applied. An artifical inoculation technique was used in which inoculum was applied onto the plants by spraying each plant separately and as evenly as possible during or soon after ear emergence. Inoculum consisted of a mixed aqueous suspension of about 10 6 conidia ml 1 obtained from several isolates of S. nodorum. The preparation of inoculum and the culturing techniques of the fungus have been previously described in detail (KARJALAINEN et ai. 1983). After inoculation the plants were kept damp by daily irrigation. The assessments of disease severity were made on the flag leaves by estimating the percentage area covered by S. nodorum lesions. The percent values were transformed using the arc-sin transformation. The assessments were carried out twice, 8-12 days after the first inoculation.
The objective of this study was to estimate the proportion of heritable variation of the total variation of disease severity using two F 2 progenies.
Heritability in the broad sense was estimated from the present data, although it is of limited value in predicting breeding success (CAHANER and HILLEL 1980). However, in this case the broad sense heritability estimates were calculated in order to arrive at an upper limit for the narrow sense heritability, and from this the additive component of the total variation can be deduced. Heritability in the broad sense was calculated using the following equation that has been cited by several authors (e.g. GRIFFITHS and LAWES 1978 In utilizing this variance method the environmental variation was approximated from genetically uniform parent populations. The standard error of the heritability was calculated assuming that the sample variances of the parents and the F 2 progenies follow the Gamma distribution. Furthermore, the standard error of the heritability was derived assuming that all sample variances are independent.
Results and discussion
The heritability figures calculated for the F 2 crosses of spring wheat are presented in Table 1. The results indicate that in general the H 2 b. values are moderate, and that over half of the variation in the level of disease severity of the F 2 generation was of environmental origin. In the present experiment the cross Tähti X Maris Butler showed a somewhat higher heritability than that of Tähti X TGS 334/74/8, but the standard error of heritability was lower in the first cross. In this experiment the inoculum was applied as evenly as possible to avoid any variation due to the uneven spread of inoculum onto the plants.
From the present experiment it may be concluded that in these particular crosses involving moderately resistant and susceptible parents, the additive genetic variance is rather low and that environmental variation accounts for a major part of the total variation of symptom expression. Ffence breeding progress depends on the use of efficient screening methods in order to detect the slight differences caused by several minor genes. The variation in disease symptom expression was continuous and there was no evidence of major gene effects. The evidence obtained from experiments described in the literature have revealed fairly high heritabilities in host resistance to S. nodorum. For example, ROSIELLE and BROWN (1980) indicate heritabilities of 63 % and 52 % on leaf and ear resistance, respectively. AASTVEIT (1982) has observed heritabilities as high as .89, but his experiment was carried out using natural infection. BRÖNNIMANN (1975) has analysed heritabilities on the basis of five parent diallel crosses measuring the inheritance of tolerance. The results showed heritabilities of .66 and .65 for the F] and F 2 generations, respectively. Contrary to these experiments, which have led to high heritability estimates, SCOTT et al. (1982) have provided evidence that in most of their winter wheat crosses the heritabilities were low and the standard errors were high.
The observed diversity in the heritability estimates may be due to several reasons. For example, only a few authors have used the same method of calculating the heritability estimates. Furthermore, standard errors for the obtained H 2 estimates have been presented only in some experiments, though in many cases they would provide valuable aid in evaluating the reliability of the results. A great deal of the variation in presented heritabilities may be due to the different procedures and different environmental conditions of the experiments. Inoculation procedures spraying techniques, spore densities etc. -which affect the reliable ranking of cultivars have also varied amongst studies (SCHAREN and EYAL 1980). Moreover, evidence is now accumulating that this host-pathogen interaction is sensitive to environmental changes. For example, FRIED and BRÖNNIMANN (1982) have observed that environmental conditions after inoculation may mask the genotypic differences between cultivars. The experiment conducted by MULLANEY et al. (1982) substantiate this, since the authors have found that wheat crosses exhibit a strong genotype-environment interaction in relation to disease expression.
It may thus be concluded that it is not possible to make comparisons between different H 2 estimates. Each experiment has its own value, applicable only to a specific population and to the specific conditions under which the experiment has been carried out. This is already widely accepted in interpreting the usefulness of heritability values in a general sense (DUDLEY and MOLL 1969). Recent studies on the genetic control of wheat resistance to S. nodorum throw some light on practical resistance breeding. For example, MULLANEY et al. (1982) have shown that leaf resistance is polygenically determined and can be explained by mainly additive gene effects. A diallel analysis by NELSON (1980) and NELSON and GATES (1982) supports the theory of additive effects in the inheritance of host reaction to S. nodorum. NEL-SON(I9BO) also found that general combining ability effects were highly significant, but specific combining ability effects were observed as well, indicating non-additive gene action for some specific crosses. It has been recently suggested that cytoplasmic inheritance might be involved in the resistance to S. no (/or«m(NELSON 1980). However, this assumption remains to be verified.
For the practical resistance breeder it is important that the resistance to S. nodorum is mainly controlled by additively acting genes, as this allows him to accumulate desired genes into populations. Experimental selection trials provide encouraging results, for example, SCHAREN and KRUPINSKY (1978) have obtained positive transgressions in several of their wheat crosses indicating that additively acting minor genes can be accumulated into breeding lines and populations. Thus, since there are good opportunities for developing the selection techniques and the resistance material presently available, it seems that the prospects for breeding S. nodorum resistant wheats are promising. The best evidence for this optimism is the fact that cultivars with a satisfactory degree of resistance have been produced, and the cultivation of these varieties has given good economic return in terms of cost-benefit analysis of crop protection costs (DOODSON 1981). | v3-fos |
2018-04-03T01:07:17.304Z | {
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} | s2 | Effect of excess L-lysine on rat growth and on plasma and tissue concentrations of copper, iron and zinc.
A 28-day feeding study was conducted to test the effect of excess dietary lysine on rat growth and the concentration of copper, iron and zinc in plasma and liver. Young male Sprague-Dawley rats were fed a 10% protein casein diet with or without excess lysine. There were no significant differences in body weight gain, food intake or plasma proteins among the dietary treatment groups. Supplementation of the basal diet with 2.1% L-lysine caused a 53% reduction in hepatic copper and a significant reduction in hepatic iron. The addition of 0.7% or 2.1% lysine to the basal diet caused significant reductions in levels of plasma copper. The 2.1% level of lysine tended to lower the concentration of zinc in plasma. The data suggest that lysine may interfere with the availability of selected minerals by reducing tissue utilization or promoting excretion, or both.
The existence of a nutritional interaction between amino acids and minerals has been shown (1,2). Several reports have suggested that amino acids influence the absorption and excretion of minerals (3)(4)(5)(6)(7). Histidine, cysteine and lysine enhanced iron uptake and absorption (S). Increased concentrations of cysteine and histidine in serum and urine were found to be associated with increased urinary zinc excretion (8). Tyrala et al. (9) reported that urinary copper losses in infants receiving free amino acid solutions were positively correlated with the excretion of glycine, methionine, histidine and lysine. Giroux and Prakash (10) studied the influence of various salts and chelates of zinc given by gavage on serum zinc levels of rats. They found that zinc sulfate-liquid mixtures of the amino acids lysine, cysteine, glycine and histidine produced higher levels of serum zinc than an equivalent dosage (10mg/kg) of zinc sulfate alone. On the other hand, one report has indicated that an excessive amount of histidine had an adverse effect on the absorption of selected minerals (6).
The formation of amino acid-mineral complexes has been proposed as one of the reasons for the amino acid-induced changes in mineral uptake, absorption and excretion (4,6,7). This postulate suggests that the possible consequences of ex 709 G. V. MITCHELL and M. Y. JENKINS cessive supplementation of diets with some amino acids may be alterations in mineral storage in the body. The present study was undertaken to examine the effect of excess lysine supplementation on rat growth and the iron, copper and zinc content of plasma and liver. This study was partly motivated by the increased human consumption of concentrated lysine supplements and concern for potential adverse effects on health due to moderately excessive levels of dietary lysine. protein diet can result in adverse effects ranging from moderate growth and food intake depression to the development of pathologic lesions and low survival rates (13). Excess lysine has a more moderate effect on growth. Levels of lysine in excess of 3% of the diet are generally required to produce depression of growth and food intake (14,15). The results presented here indicate that a lysine level as high as 2.1% of the diet died not affect food intake or growth. The kidney weights of the rats fed 2.1% lysine tended (p=0 .10) to be higher than those of the other groups. Relative kidney weights of the rats fed 2 .1% lysine were significantly higher (p<0.05) than those of the other two treatment groups (Table 2). No significant differences were found in liver and spleen weights or rel ative weights of these organs among the treatments. The effect of lysine on kidney weight in this study was consistent with results obtained in studies in which the kidneys were enlarged by feeding excess amounts of other essential amino acids (13) . Oral or intravenous administration of amino acids or high protein diets significantly increased renal flow and glomerular filtration rates in dogs (16) . Brenner et al. (17) suggested that the mechanism responsible for these increases was also responsible for the sustained hyperfiltration and accompanying renal hypertrophy , and that some circulating hormone or intermediate effector might be responsible for the increased renal perfusion and filtration induced by protein-rich (amino acid) meals .
METHODS
The effect of lysine on the concentration of plasma protein is shown in Table 3 . Excess lysine did not cause any significant differences in total protein , albumin, globulin or the albumin-globulin ratio when compared with rats fed on the basal diet. Alterations in the individual globulin fractions may have occurred without a significant change in the total fraction. However , we do not have data on the individual fractions.
Hepatic and plasma concentrations of copper , zinc and iron are shown in Table 4. The data clearly indicate that excess lysine affects copper storage . The con centration of hepatic copper in rats fed 2.1% lysine was significantly lower (p<0 .05) than in rats fed the basal diet or the basal diet plus 0 .7% lysine. Hepatic copper was reduced by 53% in rats fed 2.1% lysine compared to rats fed the basal diet .
Fasting animals absorb copper from copper sulfate considerably faster than from copper complexes (18). However, when food is given with the copper preparations, the opposite effect occurs. High concentrations of copper-binding substances in the intestinal tract may lead to the formation of macromolecular compounds with the soluble copper present in the food, and the rate of copper diffusion is thereby considerably reduced (7). Kirchgessner and Grassmann (7) also reported that, when copper-amino acid complexes were fed to rats receiving a ration low in copper, storage of copper in the liver increased compared with a copper sulfate supplement. The copper content of the liver diminished with increased polymerization of the amino acid. A comparison of these studies with the results presented here is difficult since general experimental conditions were different and preformed copper-amino acid chelates were used in the earlier study. The plasma levels of copper in rats fed 0.7% and 2.1% lysine were also significantly lower than those in rats fed the basal diet. In normal mammals, about 90% of the plasma copper exists as ceruloplasmin, and there is a highly significant correlation between ceruloplasmin levels and plasma copper levels (19). The significant reductions in plasma copper in the present study suggest the possibility of reduced levels of ceruloplasmin.
The biochemical basis for the reduced plasma and hepatic concentrations of copper in the rats fed the highest level of lysine is unclear. Our results suggest that, at least in part, excess lysine interferes with the availability of copper by reducing and M. Y. JENKINS The authors thank Benjamin Glasco for his technical assistance. | v3-fos |
2014-10-01T00:00:00.000Z | {
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} | s2 | Substrate utilization in defined media.
Substrate utilization in defined media for two flower spiroplasmas (S. floricola and FS SR-3) and honeybee spiroplasma (HBS AS-576) was investigated. Glucose, fructose, and mannose were utilized by all three spiroplasmas. In addition, HBS (AS-576) could ferment trehalose; FS (SR-3), sucrose; and S. floricola, trehalose, sucrose, and raffinose. The three spiroplasmas varied greatly in growth requirements for amino acids. Only S. floricola utilized arginine. HBS (AS-576) required at least one purine and one pyrimidine base for growth, while both flower spiroplasmas grew with only one base in the medium. Oleic acid, cholesterol, and BSA were essential to all three spiroplasmas. Palmitic acid, which was non-essential, promoted growth significantly.
INTRODUCTION
Although more than 60 isolates of spiroplasmas have been successfully cultivated, including some pathogenic to plants, insects, and vertebrate animals, and some not yet shown to be associated with any disease, the precise nutritional requirements for these isolates is still unclear [1,2,3].
Previous studies [4-12; Chang CJ, Chen TA: Phytopathology 69:1024, 1979; Lee IM: Ph.D. thesis, University of California, Riverside, 1977; Lee IM, Davis RE: Phytopathlogy News 12:215, 1978; Malloy KM, Chen TA: Phytopathology 70: 465,1980] using undefined media attempted to show the comparative capabilities of spiroplasmas to utilize various nutrients. The results were frequently conflicting. For example, Saglio et al. [11] showed that Spiroplasma citri was unable to metabolize arginine, whereas Townsend [12] reported that with a limited supply of a fermentable carbohydrate S. citri was able to metabolize arginine. Igwegbe et al. [8] showed that S. citri (Morocco isolate) could ferment mannose, whereas Davis' result [4] showed otherwise. Thus data on substrate utilization and nutrient requirement of spiroplasmas based on cultivation in a complex medium are very difficult to interpret.
We report herein the precise nutritional requirements for carbohydrates, amino acids, nitrogenous bases, ribonucleosides, fatty acids, and cholesterol of two flower spiroplasmas (S. floricola and FS SR-3) and honeybee spiroplasma (HBS AS-576) cultured in a chemically defined medium [13]. Substrate utilization in undefined and defined media is discussed.
Spiroplasma Growth
Cell growth and yields were determined as previously reported [13]. At least 10 passages in a given test medium were made before results were considered positive. Growth yields were measured at two, four, and three days after inoculation for S. floricola, FS (SR-3), and HBS (AS-576), respectively, unless stated otherwise.
Carbohydrate Utilization
Each of the following carbohydrates was tested as the sole carbohydrate source at a concentration of 0.8 percent in CC-494 [13]: D-glucose, D-fructose, D-galactose, D-mannose, D-mannitol, D-sorbitol, a-lactose, maltose, sucrose, D-trehalose, D-raffinose, and starch. All stock solutions were 20 percent, except for lactose and starch which were 10 percent and were filter-sterilized (millipore filter 0.45 /,1m).
Ribonucleoside Requirement
The same ten combinations as used in the free nitrogenous base study were replaced with corresponding ribonucleosides. The concentration of each nucleoside was the same as that used in the original medium CC-494 [13]. Deoxyribonucleosides (including 5-methy-deoxycytidine) and UTP, which were constituents of CC-494, were excluded. Fatty Acids, Cholesterol, and BSA Based on the lipid composition in medium CC-494 [13], the following four groups were tested: (1) without cholesterol, (2) without Tween 40 and palmitic acid, (3) without Tween 80 and oleic acid, and (4) without bovine serum albumin. The concentration of each ingredient remained the same as that in medium CC-494 [13].
RESULTS Carbohydrate Utilization
The three spiroplasma strains showed differences in metabolizing various carbohydrates. All could ferment glucose, fructose, and mannose. In addition, HBS (AS-576) utilized trehalose, S. floricola utilized sucrose, trehalose, and raffinose, and FS (SR-3) utilized sucrose. None could utilize galactose, mannitol, sorbitol, lactose, maltose, or starch. Glucose was the best carbohydrate source for all the spiroplasmas. There were significant differences in yield among the spiroplasmas growing in media supplemented with other carbohydrates (Table 1). Amino Acid Metabolism Both S. floricola and FS (SR-3) grew in any of the ten amino acid combinations, whereas HBS (AS-576) grew only in four: (1) 20 amino acids, (2) glucogenic amino acids, (3) nonessential amino acids, and (4) uncharged polar R group amino acids.
The medium supplemented with 20 amino acids supported the highest growth for all three spiroplasmas. Generally, deletion of amino acids from the medium resulted in lower yield. For example, HBS (AS-576) reached 2.46 x 109 cells/ml with 20 Arginine Metabolism Based on cell growth and pH change of the culture medium it was determined that S. floricola, among the three strains tested, could obtain energy through an arginine dihydrolase pathway.
Growth of S. floricola in media containing either glucose or arginine reached its peak in two and five days, respectively. S. floricola grew much more slowly in the arginine medium; its yield was reduced almost fivefold. Nitrogenous Base Requirement The requirement for nitrogenous bases differed between the two flower spiroplasmas and honeybee spiroplasma. HBS (AS-576) required at least one free pyrimidine base and one free purine base. On the other hand, any pyrimidine or purine base could support the growth of S. floricola and FS (SR-3).
Growth of the three spiroplasmas varied significantly with different combinations of nitrogenous bases. The highest yield was obtained when all five nitrogenous bases were added. Growth in the medium containing guanine and cytosine was significantly greater than that containing adenine, thymine, and uracil.
Ribonucleoside Requirement
Similar results were obtained when five ribonucleosides were used to replace the corresponding five nitrogenous bases. HBS (AS-576) required at least one ribonucleoside from a pyrimidine base and one from a purine base, whereas S. floricola and FS (SR-3) grew in a medium supplemented with any single pyrimidine or purine ribonucleoside.
The medium supplemented with all five ribonucleosides supported the highest growth for the three spiroplasmas. The addition of guanosine and cytidine resulted in higher yields than that of adenosine, thymidine, and uridine. Other combinations resulted in significantly different and reduced yields for the two flower spiroplasmas. Fatty Acids, Cholesterol, and BSA Requirement All three spiroplasmas have the same requirements for fatty acids, cholesterol, and BSA. Oleic acid, cholesterol, and BSA were found to be essential. Although palmitic acid was not required, it promoted significant growth of the three spiroplasmas. DISCUSSION
Carbohydrate Utilization
This study indicated that the spiroplasmas of distinct serogroups show differences in the utilization of carbohydrates. Of the twelve carbohydrates tested, S. floricola could utilize six whereas HBS (AS-576) and FS (SR-3) could each utilize four. A possible explanation is that S. floricola has more enzymes for carbohydrate utilization than the other two spiroplasmas.
Considering that trehalose is the major disaccharide found in the hemolymph of most of the insect habitats of HBS, it is not surprising that this spiroplasma could cleave trehalose into two glucose residues. Therefore, it is quite possible that S. floricola, at some time in its life cycle, may also reside in the hemolymph of an insect.
Both S. floricola and FS (SR-3) were originally isolated from the surfaces of flowers. It has been suspected that the two spiroplasmas multiply in the nectaries of flowers, but no direct evidence of this has ever been reported. The ability of S. floricola and FS (SR-3) to ferment sucrose is consistent with this.
Our results contradict earlier reports on carbohydrates utilization studies. For example, HBS (AS-576) was reported to use glucose, fructose, maltose, trehalose, and starch [Malloy KM, Chen TA: Phytopathology 70:465, 1980] but not mannose [4]. Since in our defined medium the presence or absence of a particular sugar can be precisely controlled, different results of carbohydrate utilization obtained from undefined and chemically defined conditions should be expected. Recently, Malloy and Chen [Phytopathology 71:892, 1981] showed that di-, oligo-, and polysaccharides are converted to glucose when incubated with horse serum or serum fraction in the absence of spiroplasma. Therefore, the validity of carbohydrate utilization as a criterion for mycoplasma taxonomy when using undefined media is ques-
tionable. Amino Acid and Arginine Metabolism
It is possible that free amino acids are present in the complex components of undefined culture media because the three spiroplasmas required all 20 amino acids for maximum growth in our defined medium. Cell yields decreased with reduced amino acids supplements. This is not surprising since the amino acids are important metabolic building blocks for spiroplasmas which carry a minimum genome size. However, it was unexpected that both S. floricola and FS (SR-3) grew in a medium supplemented with only two negatively charged polar R group amino acids (aspartic and glutamic acids) or with any of the ten amino acid combinations used in this study. This suggests that they are able to transaminate amino acids readily. On the other hand, HBS (AS-576) required at least seven uncharged polar R group amino acids in the medium, indicating a lesser transamination ability. Positive results have been reported for HBS (AS-576) and S. floricola [4] for arginine metabolism. In our defined medium, however, only S. floricola utilized arginine but growth was much less than that obtained with glucose as the energy source. Since ATP production from arginine catabolism is less than that through glycolysis, the arginine dihydrolase pathway may function as a minor energy-generating system for S. floricola.
Like carbohydrate utilization, studies on arginine metabolism obtained from defined and undefined conditions produced conflicting results. Previous studies [4,7,12] indicated that minute amounts of glucose were required for arginine metabolism of S. citri. In our defined medium, however, S. floricola required a period of adjustment before adapting to the arginine medium while HBS (AS-576) and FS (SR-3) did not survive through the first passage.
Nitrogenous Base and Ribonucleoside Requirement
Our studies indicated that spiroplasmas, like other mycoplasmas, lack the orotic acid pathway for pyrimidine synthesis and the enzymatic pathway for de novo synthesis of purine bases [14].
HBS (AS-576) could only interconvert bases within the same group (either purine and pyrimidine) whereas S. floricola and FS (SR-3) could convert not only within groups but also between groups. HBS (AS-576) required the same base precursors as Mycoplasma mycoides subsp. mycoides [15] and Acholeplasma laidlawii strain B [16]. S. floricola and FS (SR-3) require fewer base precursors for nucleic acid synthesis than any of the above organisms. Since the biosynthesis of purine and pyrimidine bases are different, it is very difficult to explain the growth of flower spiroplasmas in a medium in which only single bases or ribonucleosides were supplemented.
Deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxycytidine, and 5-methyl-deoxycytidine), ribose, and deoxyribose are constituents of medium CC-494 [13]. In both nitrogenous base and ribonucleoside utilization studies they were therefore deleted from the medium. Thus our results have clearly shown that spiroplasmas are able to synthesize ribose and deoxyribose from other fermentable carbohydrates and make deoxynucleotides from either nitrogenous bases or ribonucleosides. Fatty Acids, Cholesterol, and BSA The requirement for sterols by spiroplasmas and mycoplasmas has been recognized universally. Cholesterol is supplied from the serum in the culture media. Beside sterols, a number of glycerides, free fatty acids, phospholipids, and proteins also play important roles in spiroplasma nutrition. Only in the development of chemically defined media [13] have we realized the precise importance of these active chemical components from the serum.
In our medium CC-494, horse serum was replaced by a combination of palmitic acid, oleic acid, cholesterol, and BSA. CC-494 supported excellent growth of S. floricola, FS (SR-3), and HBS (AS-576). We found that oleic acid, cholesterol, and BSA were required by the three spiroplasmas. Palmitic acid was not essential but promoted significantly greater growth. This confirms the previous suggestion that spiroplasmas are incapable of de novo biosynthesis of long-chain fatty acids and cholesterol for their cell membranes from acetyl-CoA [14].
Using defined medium, we are convinced that spiroplasmas do require cholesterol. Such requirement is a taxonomic criterion for the family spiroplasmataceae.
Apparently spiroplasmas need BSA in the medium to act as a carrier and detoxifier for the free fatty acids [17]. Whether BSA also functions directly as a nutrient, e.g., as a nitrogen source, requires further investigation. | v3-fos |
2014-10-01T00:00:00.000Z | {
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} | s2 | Monitoring breast milk contamination to detect hazards from waste disposal.
Human milk is a repository for certain classes of long-lived, fat-soluble environmental contaminant chemicals. Some members of this class, such as the chlorinated pesticides and the chlorinated biphenyls, can be expected to be present at chemical waste disposal sites. Analysis of samples of breast milk obtained from women near such a site could provide documentation that exposure has taken place. However, background contamination is present and must be dealt with by the collection of comparison samples. Sample collection can be difficult because of the low level of chemicals being sought, and thus the possibility of sample contamination. The diagnostic and public health consequences of contaminated breast milk are not clear at this time, and thus chemical analysis of milk should be carried out in a research setting. Despite these difficulties, breast milk monitoring has been a successful tool in certain investigations of the spread of environmental chemicals.
Introduction
Uncontrolled and unquantified exposure of the public to hazardous substances is a consequence of the large amounts made, used, and discarded. The disposal process in particular presents opportunities for exposure during handling and transport, and from chemicals present in poorly operated storage or disposal facilities. Knowing what to do following an accidental exposure requires information on the extent and degree of exposure, any illness that may be attributable to the exposure, and who, if anyone, needs clinical study in greater detail. The chemical analysis of human milk yields data that may be useful during initial investigation or subsequent followup.
Some of the chemicals involved may be suspected of being able to contaminate milk either because they are known to have done so in the past or because they share physical or chemical properties with those that have done so. When this is the case, analysis of milk may seem appropriate for two general reasons. One is that breast milk is an easily *Epidemiology Branch, NIEHS, 101, A3-02, P. 0 collected fluid whose degree of contamination can serve as an index of exposure. The other is that milk is of interest in its own right because of its role as food for children. Analysis of milk has been proposed often enough that an investigation of its usefulness seems called for.
What follows is an outline of some scientific and practical aspects of breast milk analysis, and an examination of some of the assumptions that must be made from a public health and scientific point of view. The issues to be discussed are: (1) the decision that milk analysis is appropriate, (2) dealing with the existence of background contaminant levels, (3) some practical problems in the collection of samples and (4) clinical and public health implications of the data obtained.
Decision to Analyze Milk
Chemicals like DDT and the polychlorinated biphenyls (PCBs), as well as many other highboiling halogenated polycyclic hydrocarbons, have properties that favor their appearance in breast milk, even when exposures have been to low, unnoticed amounts. The chemicals cannot be excreted or metabolized once they are absorbed and are stored in the body's fat. We expect that the concentration of these chemicals in body tissues is directly related to the amount of fat in the tissue. While these chemicals could be measured in fatty tissue (about 65% fat), it is not easily accessible. Since breast milk is about 3% fat, sensitive analytic methods can detect residue levels of these chemicals there. Blood and urine, which are easily accessible, have much lower amounts of fat. Thus, an analytic method that works satisfactorily at the range of concentrations found in milk may be inapplicable at the lower concentrations of blood and urine. Background levels of these kinds of compounds tend to be in a range where, with current technology, milk levels can be determined with success, but blood and urine levels may be too low to detect or very hard to quantify. Since accidental exposures will increase levels above background, analysis of milk in such a situation may provide quantitative data when analyses of other body fluids do not. If preliminary data indicate that exposures have occurred at high enough levels, analysis of blood or urine should be considered since samples will be available from more people. Some statistical aspects of the blood versus fat choice have been treated elsewhere (1).
Whether analysis of breast milk is appropriate depends not only on the kinds of chemicals involved in a particular incident and the levels at which they occur; it also depends on the purpose of the study. There are at least four purposes for which analysis has been proposed or used. One is detection of exposure or documentation that it has taken place. A second is epidemiologic study, in which levels of chemicals are to be the index of exposure to the mother or child. A third is diagnostic use, in which some illness in the mother or child may be attributed to the chemical, depending on the level found. A fourth is advisory use, in which the mother is aided in her decision about breast feeding by knowing what level she has.
Documentation of Exposure
For detection of exposure or documentation that it has taken place, the usefulness of breast milk analysis will depend on whether there are sufficient lactating women located appropriately, whether the match between level of exposure and analytic sensitivity works out well, and whether other factors, such as age, are important for the study. For example, the Michigan Health Department used breast milk analysis to estimate the statewide distribution of contamination with polybrominated biphenyls (PBBs) (2). Since the sampling frame, the state population, was very large, the availability of adequate numbers of nursing mothers was assured. It was known from previously collected paired blood and fat samples that blood values could be undetectable when moderate amounts of PBBs were demonstrable in fat (1). Analysis of milks collected from a statewide probability sample showed that about 90% of lactating women (and inferentially of the whole population) from the lower peninsula had detectable levels. Presumably, a serum survey would have given a falsely low estimate. Thus, the added sensitivity afforded the analytic chemist by the amount of fat in milk was useful for this problem. On the other hand, in Triana, AL, a community with exceptional exposure to DDT (3), all 499 sampled residents had detectable serum levels, and a striking increase oflevel with age was noted. Because of the small population and the high chronic exposures, serum analyses were adequate and breast milk analysis would not have been very informative. Only a few milk samples would be expected and, of course, none would have come from older women.
Epidemiologic Study
The use of breast milk for epidemiologic study of the women who supply the samples is generally done only when some aspect of lactation or the determinants of the levels themselves (e.g., diet, race, age) are under study. Whether hypotheses about illnesses in children exposed via contaminated breast milk may be tested depends mostly on the number of such children available for study. Evaluation of subtle decrements in growth and development, for example, requires large numbers. However, in most exposure situations, children who are in utero or breast feeding at the time of exposure should be evaluated as thoroughly as possible. Such children may be particularly likely to display toxicity because of their developmental vulnerability. In the extreme case, one affected child can be informative. For example, Bagnell (4) noted cholestatic jaundice in a breast-fed 6-week-old whose mother lunched daily at the family dry-cleaning shop. Trichlorethylene, which was present in the shop, was present in her milk; other causes were ruled out, and the jaundice resolved with cessation of breast feeding.
For other nonlactation studies, breast milk analysis is not likely to prove useful. Lactating women come from a fairly narrow age range, they are generally quite healthy, and in many other ways they fail to represent any broader population. Besides, in any given study, women who find the process of giving samples tolerable are a further subset of lactating women. For example, we are doing a study in North Carolina of the effects of PCBs and DDT in breast milk on the health of breast-fed children; the women who volunteered for this study are a select group. For instance, only 5.5% of the volunteers are black (in a state which is 21.5% black), 54% have 16 or more years of education, and 81% are employed. Because of the very unrepresentative nature of such groups, the choice among biologic fluids for analysis in most nonlactation studies will be blood, fat or urine; breast milk analysis may be a useful "add-on" but will generally be secondary to the main thrust.
Diagnostic or Advisory Use
The diagnostic or advisory use of breast milk analysis is controversial at this time and should not be undertaken outside a research setting. For many chemicals, there is some evidence that laboratories vary substantially within themselves and among each other on the values obtained for a given sample. There is no nationwide quality assurance program as there is for, say, blood lead testing. A more serious objection is that there is neither general agreement nor available data on what level, if any, constitutes a hazard for any of these chemicals. Thus, data should be collected only when they are to be evaluated in a formal way, preferably by formal hypothesis testing. Analysis of milk outside this context does not provide the mother or her physician with any useful information, despite the formidable persuasive powers of an actual number, computer generated. Even in a research setting, these data can be problematic; this point is discussed below.
Dealing with Background Levels
There is now a substantial literature, dating back to 1951 (5), showing that it is unusual to find uncontaminated milk anywhere in the world. The data include a series of studies reviewed in 1980 (6), 1975 data from EPA on over 1000 United States women (7), 1977-78 data from Michigan (8), and our North Carolina data. The chemicals usually reported include DDT and its metabolites, PCBs, dieldrin, chlordane and heptachlor. Lindane (BHC, benzene hexachloride) is occasionally reported (9); PBBs have been reported from Michigan (2). Mirex has been sought but not reliably identified (7). There are no obvious secular trends in these data; there is substantial geographic variation in the United States, with the southeast tending to be higher than the northwest (7). The widespread prevalence of such contamination has direct implications when the testing of milk is proposed. When exposure to a point source is to be evaluated, overlap of the suspect chemicals with background is to be expected. Thus, control samples must be analyzed simultaneously. When an entire geographic area has been affected (a common situation in waste dump incidents), finding suitable controls is not easy.
Although each individual chemical will be somewhat different, the magnitude of the background problem can be illustrated by considering PCBs as an example. When planning to analyze, a convenient but arbitrary rule of thumb is to try to detect levels of twice background. Our data show a median level of 1.9 ppm milk fat; recent data from Michigan (8) show a median level of 1.4 ppm. The average adult female is about 60 kg and about 20% fat; thus in steady state, she has 12 kg of fat containing 17-23 mg PCBs. She can double her body burden, and thus double the level in her milk, by exposure to air, water, soil, food, etc., contaminated by PCBs. PCBs have quite a low vapor pressure, so the notion that 20 mg can be absorbed from the air in the short term is unlikely. Foodstuffs not produced in the area are likely to have quite low levels or be uncontaminated; however, locally raised produce or livestock can be important, as in Michigan (10). Water contamination is typically in the low parts per billion range (11) because of the low water solubility of the compounds, and thus it would contribute to body burden at a microgram/liter rate. Again, this is relatively unimportant in the short run. However, if the suspect site does pollute local water and fish are taken and consumed, substantial contributions can be made. Fish living in water chronically polluted by PCBs will bioaccumulate the chemicals and levels can reach 5 ppm or more (12). A woman consuming quite moderate amounts of such fish could absorb 20 mg easily. Another likely source of contamination is direct contact with the chemical itself or with heavily contaminated dirt from the site. Soil at an uncontrolled site might reach 50-500 ppm PCBs or more. PCBs, like the cyclodiene pesticides and many solvents, can be dermally absorbed; besides, even adults engage in some hand-to-mouth activity, and so small amounts might be ingested. There would be 20 mg in about 40 g of material contaminated at 500 ppm; over a few months, clothes, shoes, toys, or tires would be able to transport this amount, in addition to whatever contribution blowing dust might make. A consequence of these kinds of exposure routes is that a simple decrease in levels with distance from the site should not be expected. Exposure may well depend more on traffic patterns, the presence of children and their habits of play, the number of people living in a household, and their food preferences, rather than directly on distance from a given source.
Under certain circumstances, it may be possible to distinguish source exposure from background by "fingerprinting." The PCBs are a mixture ofvariously chlorinated biphenyls. The commercial mixtures, once sold in the United States as Aroclors, had numbers representing the percent chlorine by weight and thus, indirectly, the presence of the higher chlorinated congeners. "Background" PCB chromatograms usually look like something between Aroclors 1254 and 1260; this reflects the differing abilities of the congeners to bioaccumulate. There is some selection in the body for the higher congeners. When the exposure is to a relatively pure commercial grade of PCBs, such as to Aroclor 1260, chromatograms from exposed persons may differ from background both in the amount of chemical present and in the different relative amounts of the congeners.
Collection of Samples
To the analytic chemist who regularly works with residue levels of pesticides, the problem of sample contamination is obvious. Fat-soluble chemicals like DDT and PCBs are in fact ubiquitous, and the amounts being sought are small. Exogenously deposited contaminants from glassware, plasticware, fingers, foils and stoppers seem to be much easier to extract from in and around a sample than are endogenously deposited contaminants. Figure 1A shows a gas chromatogram from a collection jar in which pentane was shaken against the dull side of an aluminum foil cap (13). The initial spike is the pentane, and the rest is silent. When the procedure is repeated with the shiny side towards the solvent, the multiple peaks shown in Figure 1B are recorded; they come off at about where endogenous PCBs or other residues are expected. Sample collection and handling posed several problems for us in our field work. For example, efficient collection of the 30 ml or so of milk that we require for analysis meant the use of a breast pump for many women. Hand expression is relatively less efficient and tedious. The pump we chose, as well as many other commercial ones, uses a plastic nipple shield and tubing to avoid loss of the white blood cells in the milk; these are thought to aid in the immune function of the child, and they tend to stick to glass surfaces. We found that the plastic was an unacceptably high source of (presumably) adsorbed contaminants. Finally, we had hand-blown nipple shields and custom tubing made. Because of problems like these that arise from unexpected sources, we recommend that any collection procedure used be documented contaminant-free during actual field operations.
Public Health Implications
Breast milk is usually collected from women who plan to feed their children with it, although sometimes milk can be collected from milk banks and the like. Such women will have a stake in the results of any chemical analysis performed, and the question will arise as to whether the child should continue to breast feed. There is no body of experimental or observational data available from which to counsel mothers in this situation. In the setting of a protective clinical study such as we are doing in North Carolina, we explain that the analytic data are generated for research purposes only, that we will continue to examine the child, and that no illnesses occurring in breast-fed children have as yet been attributed to population levels of PCBs or DDT. In a situation where such rapport will not be developed, very careful thought should be given in advance as to exactly what mothers will be told the levels mean. If private physicians are to be involved in the interpretation of numbers, they must be warned of this in advance. The simple availability of a physician to the project is not sufficient, in our experience, to deal adequately with the concerns that are generated. If these are not dealt with, the potential for jeopardizing subject cooperation with whatever inves-90 ,\ tigation is underway is very high. The use of advisory levels or action levels borrowed from the food regulatory activity of the Food and Drug Administration or the advisory activity of the World Health Organization is problematic, since, for some chemicals, 30-50% of human milk samples will be expected to exceed such levels on the basis of background contamination alone (6). The recommendation not to breast feed implies that a benefit will be achieved by stopping that is greater than that usually attributed to breast feeding. In terms of morbidity and mortality decrements in industrialized countries, this benefit due to breast feeding may be regarded as slight, but it appears to be real (4) and must be taken into account when recommendations are contemplated.
Summary
Breast milk is a readily collectible and convenient source of human fat, which in turn is a repository for a variety of chemicals to which exposure may occur from contact with hazardous waste. Moderately sensitive and specific methods for breast milk testing exist at a number of laboratories. In appropriate circumstances, analysis of breast milk can give information on the extent to which contamination has spread, and to a lesser degree on the quantity experienced by individuals; however, each incident must be evaluated to decide whether testing of milk will be informative. Background contamination will always be a problem, and data for comparison must be simultaneously available except in extraordinary circumstances. Careful collection procedures must be used when testing for chemicals present at the low levels usually resulting from waste dump contamination. Finally, careful thought must be given to the impact of milk testing on lactating women. | v3-fos |
2016-05-04T20:20:58.661Z | {
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} | s2 | On the estimation of genetic parameters via variance components
Summary Variance components have been estimated by three methods using two different but overlapping data sets from a dairy cattle breeding scheme. The methods were H method III, MINQUE and a new method proposed by H in 1980. Two different statistical models of grouping sires were considered. For all methods, the exact variances of the estimators were calculated for given true variance components and assuming normality of the data. As a byproduct, the large sample variances of REML were obtained. A short discussion of the interpretation of the two estimated variance components is given for the two statistical models taking selection into account. A concise description is given of the three estimation methods employed. For a relatively simple model, it is shown that they use different weighting factors for combining means and squares.
I. Introduction
This investigation arose from a larger project with the aim of obtaining estimates of genetic parameters for the Swiss Braunvieh population. In this population a heavy amount of crossing with US-Brown-Swiss is practised. Thus, the variance components were estimated separately for three data sets: i) offspring of pure Braunvieh sires, born 1971-1972; ii) offspring of pure Braunvieh sires, born 1973-1975; iii) and offspring of F, bulls, born 1972-1975. The methods used were Maximum Likelihood (ML), Restricted Maximum
Likelihood (REML), Minimum Norm Quadratic Unbiased Estimation (MINQUE) and
Henderson's method III (H III), (H ARTLEY & R AO , 1967; P A TT ERSON & T HOMPSON , 1971;R AO , 1970R AO , , 1972H ENDERSON , 1953). For MINQUE and H III the exact variances of the estimators (for given true variance components) were calculated and the large sample variances of REML were obtained as a byproduct. The main results of this study are given elsewhere (H AGGER et al., 1982).
In this paper we concentrate on the smallest data set, dealing only with the F, bulls born between 1972 and 1975. With this data set we estimated variance (and covariance) components for milk yield, percent fat (fat %) and percent protein (prot %) using two overlapping data sets, two different statistical models and three estimation procedures, namely MINQUE, H III and a new method proposed by HE NDER SO N (1980) which in the present paper is called Henderson's method IV (H IV). For all methods used, the estimates as well as their exact variances (for given true variance components and assuming normality) were obtained. Some results on REML were again obtained as a byproduct.
Because the data set is fairly typical for many situations in Central Europe, the main objective was to determine the relative efficiency of the methods, e.g. is it really worthwhile changing from H III to MINQUE? The main criterion for judging this question was the precision achievable (variance of the estimators) by these three unbiased methods. In practice, however, the ease of computing the estimates is also of great importance, whereas the ease of calculating the variances of the estimators is rather unimportant. For practical use a rough estimate of this variance should be sufficient, since we only want to decide whether the estimate should either be ignored (variance very large), or should be used as obtained (variance rather small) or should be combined with other estimates from the literature. In the last case the reciprocals of the variances should be used as weighting factors, but even for this purpose rough estimates should be sufficient.
A. Data set
The data consisted of first lactation records collected from 1978 to 1981. Two overlapping data sets were used. Data set 1 included all daughter records from F, bulls having more than 7 daughters whereas data set 2 included all daughter records from F, I bulls having more than 19 daughters. All The difference between the two models lies in the definition of the sire groups.
In model I sires born in the same year were assembled in one group, giving 4 groups altogether.
In model II groups were formed by grandsires, i.e. paternal half sibs were assembled in one group, giving 17 groups for data set 1 and 15 groups for data set 2.
The following assumptions were made: For calculating the variances of the estimators, it was assumed that e and u were independently normally distributed. The vectors of fixed effects are of no interest in our analysis (they are, apart from the definition of sire groups, mere nuisance factors). In the two models the sire effect Ujk has different meanings. In model II it is the deviation of the transmitting ability from the true paternal half sib mean, whereas in model I it is the deviation of the transmitting ability from the true average transmitting ability of all bulls born in the same year.
In model II the assumption of independently distributed sire effects Var(u)=Ia) should be correct (apart from small maternal relationships), whereas with model I certain existing relationships (paternal halfsibs) are ignored. With model I this results in an underestimation of the sire variance. However, in addition to the last mentioned facts, the interpretation of the parameters depends not only on the model but also on the history of the population (B ULMER , 1971;D EMPFLE , 1975) as outlined.
If we symbolize the additive genetic variance and the phenotypic variance of the (conceptual) random mating base population by cr! and crP(Q! =crP-crA), we have for In the base population we have K = K, = K ji = 1. After one generation of truncation selection, where selection is characterized by intensity i, truncation point x and precision p, and where the paths are indicated by BB, BC, CB, CC (BC-Bull to Cow, etc.) we get: After repeated cycles of selection the K-values decrease further and reach an asymptotic value, but even in the extreme case (p 2 i(i-x) -1 ! we have K> ! ; 3 K, ! !; 2 2 3 2 !&dquo;&dquo;3' To give an example: a simple well organised selection scheme for milk yield is assumed with h 2 =0.25 in the base population and with selection operating only on first lactation. 70 % of the cows are bred to produce replacement heifers and 0.2 % are bred to produce bulls. The great majority of cows is either sired by selected sires or by test sires. 100 bulls are tested each year on 100 daughter records and the best 5 bulls are then used. For this example Table 2 shows the evolution of K values. These values are only approximate, since it is assumed that even after repeated cycles of selection the breeding values are still normally and independently distributed and that selection is done by truncation and not by the more realistic censoring.
C. Methods of estimation
Three statistical methods were used, MINQUE, H III and H IV. For MINQUE we have to calculate (notation as given in last section): Properties of the estimators are: V is proportional to ZZ'+ kl, where is any positive operational value used in the computation. A should be as close as possible to the true ratio of ff! 2/ cru 2.
For H III we have to calculate: The formulae for Var(a2) are similar to the ones given for MINQUE.
In order to describe H IV, the following observation is of importance: HENDERSON (1972) pointed out that there is a connection between BLUP and MINQUE via the Mixed Model Equations (MME), which is useful for both understanding and computation.
Writing the MME for the model used, we In H IV we make use of Eq.(l) and absorb all fixed effects, which leads to : Then the coefficient matrix is replaced by a matrix with diagonal elements identical to those of Z'FZ + XI and with off-diagonal elements equal to zero. This is symbolized by -The solution for u is easy to compute and is used to calculate the following quadratic form: -This quadratic form is set equal to its expected value. A second quadratic form for estimating Q e is needed and it is suggested that « any logical estimator of Q e, for example the within smallest subclass mean squares» (HENDERSON, 1980) should be utilized. The latter is undoubtedly very easy to compute but there may be other simple estimators which are more efficient.
A solution for u can also be obtained directly if Eq.(1) is modified in the following way:
D. Computational aspects
For data sets like the one described in Table 1, or larger ones, the computational aspects become very dominant. For all three procedures Eq.(I) was the starting point where, during reading in the sorted data, the region x herdclass x year x season effects were absorbed and other necessary quantities were calculated. Then for MINQUE and H IV an operational was added to the diagonal elements and u was estimated. Using the following notation it is well known that T can be calculated from the absorbed set of equations.
For MINQUE the expected values of e'e and u'u are calculated and the variances and covariances of e'e and u'u are given by: Having computed e'e and u'u with a given operational value of A, then the true variances can be calculated with these formulae for a range of true X values. A similar approach was taken for H III and H IV where well known formulae were used.
E. Comparison and discussion of the methods Before reporting the numerical results, a general discussion of the methods is useful. For discussion the most simple setting is used because otherwise the formulae are too complex to give much insight.
Using (GLS estimate regarding u i as random) of R for q, and (n;/(ni+!»)2 as weighting factor. If is zero (implying no variation within sires) the square of each sire is equally weighted, regardless of n i , which is completely in agreement with intuition. If is very large, each square has a weight proportional to the square of n ; . Thus, depending on k the weights of the squares can vary from being proportional to 1 up to n2. For a given distribution of n, there should be a ! where the weights of MINQUE are in similar proportion but not identical to n ; , the weights used in H III. For the same model a discussion of the weightings of the squares (using always w!) being in agreement with the above mentioned results, but using the F-value of the Analysis of Variance instead of X, was presented by R OBERTSON (1962).
It should be further noted that, if jju were known, then the weights used in MINQUE for q, are proportional to the reciprocals of the variance of the squares, and therefore well known weighting factors are used to combine these squares.
With H IV the LS estimate of )J L is used (as in H I1I), whereas the weights are similar but not identical to those of MINQUE.
With regard to H IV several comments can be made: i) Methods that have a high efficiency relative to MINQUE and that are easier to compute are very desirable and urgently needed.
ii) Using the obvious estimator for Q e (the within smallest subclass mean squares) quite a lot of available information may not be utilized. Consider the simple model in sire evaluation If there is a total of n daughter records from n u sires which are distributed over n,, herds, then, with H III nn u -n,, + 1 degrees of freedom (df) are used to estimate u 2 A similar number of df is used by MINQUE. For the obvious estimator only n-c df are used (c-number of filled subclasses). In the extreme case of a completely balanced block design we have (n h -1)(n,; -1) df for H III and zero for the obvious estimator, since there is only one observation in each smallest subclass. In a typical dairy sire evaluation scheme there may be few half-sibs in a herd x year x season, which would lead to a drastic reduction in df. Even in our example using region x herdclass x year x season we had 16777 df ( 15150 df) in data set 1 (data set 2) for H III and only 7395 df (6808 df) for the obvious estimator, resulting in the error-variance of ae being more than 2.2 times larger than with H III. As already mentioned, other estimators for Q e than the « obvious one could be used, like the H III estimator or the MINQUE estimator (e.g. with -> ! ). However, as can be seen from fig. 1, the MINQUE estimator for À -+ œ (sometimes referred to as MINQUE (0)) can be very inefficient; whereas the H III estimator always has a high efficiency. Choosing a different estimator than the obvious one, it should still be easy to compute, since this is the only justification for changing from MINQUE to H IV.
iii) In a progeny testing situation, where 0 contains only fixed herd effects (herdxyear x season) and u the transmitting abilities, the solutions of u are the Contemporary Comparison (CC) estimates as was pointed out by P OWELL & FREEMAN (1974). In sire evaluation there were good reasons to move away from CC and use more sophisticated methods. The question is whether the disadvantages of the CC method are carried over to H IV. One major disadvantage of the CC method lies in the fact that the competition, a sire has in a certain herd is not taken into account. It is implicitly assumed that the mean of competing sires is the same in all herds. However, if we have several subpopulations the effects of the subpopulations (the group effects) are accounted for in H IV. In the context of estimating variance components we must always have a random sample of sires and the daughters of these sires should be distributed randomly over the herds. In this case we would expect that the disadvantages of the CC method would not be of great importance in the estimation of variance components. In order to investigate if there could be more bias with H IV than with MINQUE or H III, the following example was considered: there is a number of herds available, which are considered as fixed, thus no further assumptions about them need to be made. A random sample of sires is drawn out of a well defined population. Given that bulls were mated randomly over herds, without any assortative mating and without any preferential treatment of the daughters, we would have good conditions for estimating variance components unbiased. However, what happens if after drawing a random sample of bulls, we get some information on them and order these bulls according to this information (consider the trait type score at the age of one year, where we could have a random sample of male calves, conduct a performance test and then use all bulls in a progeny testing scheme for the same trait, allowing farmers the choice of bulls). If we relabel the bulls according to the ordering (1 labelling the bull with the highest order) we no longer have E(u)=0 0 and Var (u) = I O EfI but we have instead E(u)=pJ.1.oITu and Var(u)=(1-p2)IIT!+p2VolT! where p is the correlation between the true sire value and the information on which the ordering is based. J .1.0 is the vector of expected values for order-statistics from the unit normal distribution and V o is likewise the variance-covariance matrix of the vector of order-statistics. The values for > o and V o are given e.g. by SARHAN & GR E EN BER G (1962, p. 193) and the formulae for E(u) and Var(u) are standard results for associate variables (D AV iD, 1970, p. 41). Now in the dairy industry, it is not unlikely that some farmers use only the « very best testbulls » whereas others use average or even below average bulls. This may even apply to a trait like milk yield.
With all three methods considered, we compute quadratic forms, and in the standard case set these equal to the expected values derived under the assumption of E(u)=0, Var(u)=Icr!. In the example it is possible to derive the expectation under the condition of ordering and nonrandom use of the sires and thus the bias can be calculated. Some results are given in Table 3. From the few cases investigated out of the large number of conceivable ones it seems that with larger daughter number the bias of H IV is somewhat larger than with MINQUE and that H III is more robust against this departure from the usual assumptions. It is well known (S EARLE , 1968) that H III gives unbiased estimates of the variance components if there are nonzero covariances between the factors of the model. However, the case investigated here, is different, because there is essentially a correlation between the sires of the same herd. Knowing the value of )ne sire utilised in a herd enables one to make informative predictions about the other ;ires used in the same herd. In the standard application of H III the expectation is aken under the assumption of Var (u) = IIT! which does not apply for this example.
However, from this limited inference, these results cannot be used as a strong argument against H IV in comparison to MINQUE.
III. Results and Discussion
A. Influence of the models on heritability estimates Whereas with H III only one result is obtained, with MINQUE and H IV a multitude of results are obtained depending on the values of used. The heritability estimates for a = 15 for milk yield and = 9 for fat % and protein % are reported (Table 4). The variance of these estimators from Model II is indicated in the last section in connection with the figures 7 and 8. The variance from Model I is somewhat smaller. The h 2 were estimated under the assumption that K = K ¡ = K II = 1. The resulting estimates for < J ' Ã (milk yield, MINQUE, data set 2) are 117751 kg z for model I and 138232 kg 2 for model II leading to an estimate of K,/K&dquo; of 0.85 which is well within the expected range. Now the question is which h 2 to use in practical situations e.g. for estimating sires. This depends again on the model used. If we have a model like model I (sires grouped by year, no relationship matrix) then from a bayesian point of view the applicable 'h 2 is that from model I, since it parameterizes best the a priori distribution of the transmitting ability of test bulls. If, on the contrary, we use the full numerator relationship matrix relative to the base population, the parameters of the base population should be used and thus, the estimates from model II are more appropriate. However, in theory they still underestimate the parameters of the base population since K and K jj not being unity is not accounted for in the estimation. In practice, however, it may be very difficult to determine those coefficients with any reasonable precision.
B. Efficiency of the methods
The comparison of the efficiency of the estimators is shown in the figures 1-9. There the following attitude is taken: each version of MINQUE or H IV with a given operational value of (symbolised as !) has to be regarded as a procedure in itself, since in practice only one such procedure will be utilized, where of course the true state of nature, that means the true X, is unknown. Quite often, however, we can put reasonable lower and upper bounds on it. For milk yield e.g., we are rather sure that under our condition the following is true: 0. I < h! < _4.
In addition, with paternal half sibs we have the relation B=(4—h!)/h!. Instead of and !, we can therefore use h 2 and h 2 , a parameter more familiar to geneticists.
Thus the choice of f¡2 is often not difficult and the procedure has also to be judged only in this range. All results are given relative to the best possible procedure (in the sense of minimum variance) having the properties of unbiasedness and translationinvariance and utilizing all data. For each true h 2 there exists an optimal procedure, but it is unknown to the user. The minimum variance utilised in the comparison is identical to the large sample variance of REML.
For the comparison shown in the figures the inefficiency is defined as follows: If the variance of procedure A is x times as large as the variance of the best procedure it can be roughly interpreted as follows: in order to reach the same precision with procedure A as with the best procedure the design (with the given unbalancedness and average daughter number) has to be x times as large. Sometimes, however, the higher precision may not be very crucial e.g. for the estimate of since with any procedure (e.g. H IV) we may get a reasonably good estimate.
C. Efficiency for estimating a;
In the figure 1 the inefficiencies of the procedures with respect to the best procedure are shown. As expected the efficiency of the estimator used for H IV is low since it utilises much fewer df. The H III estimator is only slightly inferior to the best estimator whereas the MINQUE estimator with h 2 much smaller than h 2 is very inefficient. There it can even occur (h 2 =1, h!=0.01), that using the reduced data set the estimate is more precise than using the full data set.
D. Efficiency for estimating Q u
In the figures 2, 3 and 4 the inefficiencies of the procedures for estimating OE ) with respect to the best procedure are shown. The (0)) this was also shown by Q UASS & Bor.G l a. NO (1979). If h 2 is large but a small value of h 2 is used, H IV decreases somewhat faster in efficiency than MINQUE and if h 2 is small and h 2 large, the efficiency of MINQUE decreases faster. The reason for this behaviour is not obvious to us and it is unclear if this is just peculiar to the present design.
iv) Comparing the figure 2 and the figure 4 for the optimal method, it can be seen that reducing the data set has quite different effects depending on h 2 . If h 2 is very low e.g. h 2 =0.01, the inefficiency is small (I.OS) wherease with h 2 =1.0 the inefficiency is large (1.88).
v) If a procedure other than the optimal one is used, reducing the data set can improve the estimate. This is true for all three methods considered.
It is at first sight surprising that an estimate can be improved by ignoring data i.e. ignoring information. For the Analysis of Variance method in the one way classification (then identical to H III) this was also pointed out by R OBERTSON (1962) and by S WIGER et al. (196!). A look at the formulae in section II.E. explains that paradox. The h 2 are applied to calculate the weights used to combine the means and to combine the squares. If the weights are far off the optimal values then it can easily happen that the estimator combinin all squares is less precise than the estimator combining only a subset of the squares. If we have two estimates of w, §i and J.L 2 with is less precise than § i With optimal weights that will never happen. With H III the weights are completely given by the method and they are in no case optimal (except all n ; are equal) but in the present data they are never very extreme. It should be observed, however, that in this data set MINQUE with h! = .05 is always better than H III (strictly speaking the superiority was determined for h 2 =.01, . 025, .05, .129, .15, .20, .25, .40, .60, 1.0), and the MINQUE with h z =.25 is inferior only with very small h 2 but is considerably better than H III over the remaining range.
A look at the formulae in section ILE. also explains the observation noted under iv). With a low h 2 , bulls having few daughters do not contribute much information. In the optimal method they are weighted not very heavily, whereas with X = 0 each bull, regardless of daughter number gets equal weight (for q ; ). With progeny testing in a random mating population it is always true that X --3, (h2,,;; 1) thus for the breeding scheme considered, the weights would differ, but not much for h 2 -+ 1. In this case reducing the data set implies ignoring a lot of valuable information.
Another observation, which is given in Table 5, indicates that with H IV the smallest variance is not achieved if h z = h 2 . For example if h 2 = .25 then h 2 = .40 gives a slightly smaller variance than h z = .25. From our calculations it is not possible to give empirically the best value of h 2 for our data set. This observation agrees with one made by HENDERSO N ( 19RO).
E. Efficiency for estimating h 2
In the figures 5 to 9 the variance of h 2 is shown. These variances were computed using the usual Taylor Series approximation (KENDALL & STUART 1969, p. 232). The main conclusion from these figures is the relatively high efficiency of H IV compared to MINQUE in spite of the low efficiency of the estimator used for Q e. In the case investigated this does not have a large effect, since the variance of h 2 is dominated by the variance of §fl. For the data set given, the lowest possible s.e. for h z are 0.006, 0.012, 0.033, 0.045 and 0.077 for h 2 =0.01, 0.05, 0.25, 0.40 and 1.0 respectively. A further observation can be made by comparing the figure 2 and the figure 6.
Though MINQUE with fi2=0.05 was always superior to H III for estimating Q!, this is not true for estimating h'. The reason is found from the figure 1, where it can be seen that for estimating cr, 2 H III has always a very high efficiency, whereas MINQUE can be quite inefficient for a large value of I h2 -h 2 /. Since for estimating h 2 both Q e and 5fl are needed, the lower variance of !72 from MINQUE is more than compensated for by the larger variance for Q e in case of h2 = 0.05 and h 2 -1.
IV. Conclusion
From the results presented and from the more theoretical considerations we conclude that in data sets and models like the ones investigated (which we believe are very common) the judicious use of MINQUE can improve the estimates of genetic parameters quite considerably compared to the H II1 estimates. The H IV estimators are, as expected, not as good as the MINQUE estimators, but they showed nevertheless a very high efficiency for estimating ar2 and h 2 . One suspected weakness of the H IV estimator against violation of the model assumptions which it inherited from the CC method does not seem to be of great importance according to our limited study. Thus if MINQUE is impossible or very difficult to compute, H IV seems to be a useful alternative. | v3-fos |
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} | s2 | Domestication of fynbos Proteaceae as a floricultural crop
Domestication of a South African group of Proteaceae, the proteas, began with their cultivation as exotics in Europe. A growing local interest in their cultivation climaxed in the publication of a popular handbook in 1958. Commercial interest in cultivation and seed sources was stimulated and led to a botanical and horticultural survey of useful species throughout their distribution range in the fynbos. Information pamphlets on cultivation requirements and seed were eventually supplied to the public as an official service. Up to 1970 cut-flowers were harvested in limited quantities, mainly from the western Cape folded mountains, and sold on the European markets. During the last decade, the export trade in fresh Proteaceae flowers has become a significant factor in the national economy. However, the original system of harvesting from the natural habitat has caused serious marketing problems, for instance, poor cut-flower quality and an erratic supply of many species. Increased exploitation has also led to unprecedented disruptive pressure on the fynbos biome system, particularly on the Proteaceae-component. It is clear that the scientific cultivation of the protea family as a floricultural crop is necessary' for its sustained growth as an economic factor, as well as for its natural conservation. The present paper gives an overview of the developments that led to the rise of the fynbos Proteaceae as a commercially cultivated crop in South Africa.
INTRODUCTION
The domestication of plants begins with cultiva tion. This is, in due course, followed by improve ment of techniques and plant material (Jacobs, 1970). In modern society these functions are usually assigned to scientific research. The domestication of fynbos Proteaceae for horticultural use has been profoundly influenced by research contributions in their country of origin. South Africa. CULTIVATION The fynbos Proteaceae were first cultivated in Europe, An interest in the exotic plants from the Cape led to the cultivation of a number of species, under highly artificial conditions, during the late 18th and early 19th centuries (Rourke, 1980). However, the 'art' was soon lost, due to the lack of systematic knowledge of the requirements of these 'strange' plants. For the next 150 years, the only significant cultivation of the family, popularly known as proteas, was at the Cape; of special interest were the attempts, since 1913, by the National Botanic Gardens at Kirstenbosch (Vogts, 1962) and since about 1940, by two or three pioneering growers (Rourke, 1980). Their success was based partly on hit-or-miss methods and partly on study and observation of the natural habitat. However, all fynbos proteas cultivated with signifi cant success were grown in the winter rainfall area, that is, in the region where they grow wild (Vogts, 1982). RESEARCH
First phase
From approximately 1960, this limited scientific approach changed. The cultivation requirements of proteas were identified, classified and described in a popular handbook (Vogts, 1958). The information was drawn from the results of scientific experimen tation, research and observation over a decade, of the behaviour of proteas cultivated outside their distribution range. This publication heralded the rapid establishment of protea cultivation and the intensification and expansion of research (Vogts, 1960;Letty, 1962;Rourke, 1980). The analysis of the basic cultural requirements of 52 species and 8 genera showed remarkable similarities within the family. Notwithstanding deviations in common needs, such as an alkaline soil required by some species instead of acid, it was generally accepted that all fynbos proteas could be considered for further investigation (Vogts, 1958).
The cultivation potential of proteas focused attention on their economic potential (Vogts, 1982). World-wide interest in the new and exotic in the cut-flower trade was indeed an impetus to bring these wild plants under human control.
At the same time, a breakthrough was achieved when South African proteas came on the European flower markets (Middelmann, 1981b). Most of these flowers were harvested directly from plants in the habitat. Hopes for the future of cultivation were, however, high and a 'South African Indigenous Flower Growers ' Association' was formed in 1965(Anonymous, 1965.
Conservation
The first serious problem which confronted the new phase of horticultural exploitation of proteas, was the protection of the natural sources of material. The exciting new interest in growing proteas for gain resulted in great numbers of prospective commercial growers harvesting plant material in the wild without the necessary know-how. Irreparable harm to rare plants, waste of seed collected at the wrong stage, and waste due to incorrect cultural methods soon led to a disastrous state of affairs. This was eventually brought under control, not so much by law enforcement, as by expert guidance (Vogts et al., 1972) and by providing reproductive material (McCann, 1977;Anonymous, 1980). These official conservation actions were based on an awareness of the need to keep the natural resources intact (Vogts, 1982).
Reconnaissance survey o f available resources: species, variants and ecotypes
It was realized by the authorities that, in order to cope with the growing interest in protea cultivation, an overall survey of the distribution area of the 300-odd species of Cape Proteaceae had to be undertaken (Nel, 1965). Localities in the south western and southern Cape ranging from Clanwilliam to Grahamstown, were subsequently investi gated systematically in different seasons from 1962from to 1972from (Vogts, 1972. All accessible and apparently suitable species were recorded as well as their habitats. Investigation of the latter included climatic data, elevation, aspect, soil analyses and associated plants (Vogts, 1972).
The most important contribution of this extensive survey project was the discovery of the variation of sub-populations within species. These were disting uishable from recognizable ecotypes with modified forms due to environmental conditions. These sub-populations were named commercial 'variants' (Vogts, 1980). A study of the variants of several species (up to the fourth generation) showed that many characteristics including flowering time of different populations of a species remain stable (Vogts, 1971;Vogts, 1980;Vogts, 1977c). It can be accepted that the discontinuous topography of the distribution area has contributed to bringing these variants to the threshold of speciation (Vogts, 1971). However, variants of some species may lose their distinguishing features in cultivation and revert back to the average, their characters obviously dependent on changeable environmental conditions. An exam ple is the brilliant red of Leucadendron salignum from the Cold Bokkeveld (Vogts, 1980).
Evaluation and selection for further research
Over 150 species in their wild state proved to have some economic potential, 86 to a very high degree (Vogts, 1972). For practical reasons, only a few could be considered for intensive research. Those with the highest score of combined favourable characteristics were chosen.
Economic and horticultural potential was asses sed on a number of points, of which the following were the most important (Vogts, 1980): Attractive and arresting appearance, colour, shape and size of flowerhead; foliage attrac tive but not dominating, Flowerhead not hidden or pendulous, Erect growth providing good flowerstems, Good cultivation potential and availability of propagation material, Stability cf characters, Desired flowering times, Postharvest quality, No obnoxious odour.
Finally, about 14 species and a number of variants of the genera Protea, Leucospermum, Leucadendron and Serruria were selected for commercial cultiva tion. Examples are the red, summer-flowering variant of Protea repens (Vogts, 1980) and ten variants of Protea cynaroides, chosen to allow year-round production of flowers of this species (Vogts, 1977c).
Second phase
The above-mentioned pioneering research and publicity were prerequisites to the cultivation of proteas on a commercial level in South Africa. They also led to limited attempts at cultivation and research in other countries, for example in New Zealand (Brits, 1978a;Thomas, 1974). These activities, however, lacked a strong financial incentive, such as that engendered by the South African export trade (Brits, 1978a).
The next stage in the domestication of proteas, spanning the last decade, has been characterized by rapid developments. These were, firstly the ex ponential growth of the protea cut-flower export trade by an annual average of 20%, to a value of about R3 million in 1979/80. This growth created an industry of national economic significance (Strydom, 1980). Secondly, a concomitant and vigorous programme of basic and applied production research developed. In this period, commercial protea activity was also initiated in at least five other countries (Brits, 1978a;Middelmann, 1981a).
In South Africa, research continues to preceed the cultivation process in a curious way. An estimated 80% of all exported proteas are still being produced by the method of reaping directly from the habitat (veld-picking). This opportunistic practice is at variance with the principles of a sophisticated cut-flower marketing system and it may, in the long run, become counterproductive (Wood, 1980). The continuing disruption of the fynbos ecology, through selective commercial exploitation, also has serious ethical implications. The practice clearly conflicts with the growing concern in South Africa for conservation.
The considerable output of production research during the nineteen seventies has, therefore, been partly in anticipation of, and partly aimed at stimulating, cultivation (Strydom, 1978).
It is expected that the demands of increasing production costs, quality standards and competition will force ever-growing numbers of producers to resort to the cultivation of their product (Weiss, 1980;Strydom, 1980). It is believed that cultivated proteas may eventually replace the veld-picked product of the Western Cape to a large extent, if not completely.
The second research phase may be defined in terms of the problems which had to be overcome, in order to achieve the development of the protea family as a modern horticultural crop.
Seed dormancy in Leucospermum
Seed propagation of the genus Leucospermum, especially L. cordifolium, has always been difficult due to the severe dormancy exhibited by the seed (Brown & Van Staden, 1973;Brits, 1980d). Basic studies of the promotive effects of atmospheric oxygen in raising embryonic cytokinin levels (Van Staden & Brown, 1977), led to the development of a technique to break seed dormancy, consisting of imbibition in hydrogen peroxiue (Brits & Van Niekerk, 1976).
Vegetative propagation
Until as recently as 1974, all proteaceous plants were commercially propagated by seed. Seed propagation had all the inherent disadvantages of a heterozygous species. Techniques have now been developed to root selected protea clones on a large scale yielding vigorous, true-to-type plants with apparently normal root systems (Admiraal, 1966;Rousseau, 1966;Rousseau, 1967a;Jacobs & Steenkamp, 1975;Vogts, Rousseau & Blommaert, 1976). Although species of Protea are often difficult to root, members of the other commercial genera are generally easy to root, with indole butyric acid, under mistbed conditions (Jacobs & Steenkamp. 1976).
The dependency of many Proteaceae on low soil pH-values, their sensitivity to higher levels of soil solutes and the poor rooting ability of some clones prompted grafting research (Brits, 1978c). Species and clones suitable as rootstocks were identified and screened (Rousseau, 1967b; Z. Welgemoed, 1979b). In the genus Leucospermum, for example, the arborescent species L. conocarpodendron subsp. conocarpodendron and L. patersonii, proved to be the most suitable, as well as several vigorous hybrids which were tested as clonal rootstocks. A mass budding technique resulting in 95% success was developed, as well as an effective technique to graft onto the unrooted cuttings of a clonal rootstock, which are then rooted simultaneously with the process of graft wound healing (Z. Welgemoed, 1979b). These developments have placed the grafting of protea clones on a practical level comparable to the rooting of cuttings as a means of mass propagation.
Nutrition
The extreme sensitivity of the family to relatively moderate levels of phosphorus (Thomas, 1974;Claassens & Fólscher, 1978;Jones et al., 1978) and the inability of most species to negotiate soil solutions in the higher pH-ranges (Vogts, 1958;Vogts, 1962) necessitated nutrition research. Some highly specific requirements have been identified, for example a preference of the family for nitrogen in the NH4+-form and low levels of available potassium and phosphorus; proteas require nitrogen in relatively moderate amounts (Van Staden, 1968;Hanekom et al., 1973;Claassens & Fólscher, 1978;Claassens, 1980).
Manipulation o f growth habit
A study of the effects of ethephon and other growth regulators has revealed that it promoted lateral bud break of plants during vegetative flush. Tests on seedlings and rooted cuttings showed that early branching of both could be significantly increased by the application of ethephon as well as other feathering agents (Brits, 1977;Brits, 1980b). The same effect, though more costly, can be obtained by manual pruning (Vogts et al., 1976a). This method is particularly useful in correcting the undesirable sprawling growth habit of many plants grown from terminal cuttings (Brits, 1980b).
Manipulation o f flowering time in L. cordifolium
The main demand for imported cut-flowers on European markets falls within the South African midsummer. A basic problem of local L. cordifolium cut-flower production is the natural spring-flowering characteristic of the species (Brits, 1977).
The inflorescence of Leucospermum species is not borne terminally on a shoot, but in the axil of a leaf close to the shoot apex. Apart from this inflores cence referred to as the primary inflorescence, a number of axillary buds develop, situated immedi ately proximally to the primary inflorescence. These buds, referred to as secondary inflorescence buds, do not normally develop beyond the stage where they are 5 mm in diameter, due to correlative inhibition by the primary inflorescence. However, on removal of the primary inflorescence manually or chemically with ethephon sprays, one or sometimes more than one of the secondary inflorescence buds develop, giving rise to a secondary inflores cence which flowers later (Brits, 1977;Jacobs & Honeyborne, 1978). It has been shown that removal of the primary inflorescence of Leucospermum cv. 'Golden Star' on October 7 shifted the peak flowering period from October to December (Jacobs & Honeyborne, 1978). Secondary inflorescence buds may, however, abscise if the primary inflores cence is removed too late (Brits, 1977;Jacobs & Honeyborne, 1978). Flower initiation in Leucosper mum has also been studied (Jacobs & Honeyborne, 1979;Jacobs & Minnaar, 1980a;Jacobs, 1980b).
Blackening of Protea leaves
The leaf colour of Protea cut-flowers air-shipped from South Africa to Europe, often undergoes a degenerative blackening during transit (Anony mous, 1965). This is unsightly and extremely damaging from the marketing point of view. Oxidation of flavonoids has been identified as one cause of the blackening in Protea leaves (Whitehead, 1979;De Swardt & Pretorius, 1980). High temper atures, low light intensity, desiccation and packing flowers with free water on the leaves all enhance blackening of the leaves (Jacobs & Minnaar, 1977a;Jacobs & Minnaar, 1977b;Jacobs & Minnaar, 1980b). Techniques of proper precooling have been developed as an efficient method of controlling leaf-blackening (Vogts et al., 1976c;Jacobs 1980a;Jacobs, 1981).
Breeding
The breeding of superior protea types for horticulture was attempted on a limited scale before 1973 (Horn, 1962a;Horn, 1962b;Brits, 1980e).
In 1973 a mass selection programme of Pro teaceae cutflower types was launched involving the scrutiny of most natural and cultivated resources of commercial Proteaceae in the Western Cape Province. Individual selections were indexed, estab lished as vegetative clones and evaluated. The primary breeding goals established were cut-flower quality, variety, yield, ease of rooting and lateness of flowering. An interspecific hybridization program me was launched using the established selections of these proteas as the main contributors. A genetic investigation of interspecies crossing compatability was initiated (Brits, 1980b). Special techniques such as the polyploidization of proteas have been attempted ( Van der Merwe, 1981).
Registration o f protea cultivars
Prior to 1973, commercial resources were undescribed and were traded collectively under their old specific names, e.g. 'Leucospermum nutans . In 1973 a national cultivar registration programme for proteas was initiated (Brits, 1980c). In addition, authority was obtained from the ISHS (International Society for Horticultural Science) for South Africa to act as the International Registrar of all protea cultivars falling within the South African genera (Anonymous, 1975).
A first group of nine Leucospermum-selections and hybrids were registered as cut-flower cultivars and released, five in 1979 (Z. Welgemoed, 1979a;A. Welgemoed, 1979) and four in 1981(Welgemoed, 1981Anonymous 1981). Protea repens: Kouga variant, which was improved horticulturally by selection for deep red colour and early summer flowering time, was also released in 1981 (Welge moed, 1981;Anonymous, 1981).
Diseases and pests o f Proteaceae
Several studies have shown the Proteaceae to be exceptionally susceptible to the root-rot fungus Phytophthora cinnamomi Rands (Van Wyk, 1973a;Van Wyk, 1973b;Von Broembsen, 1981). A survey was conducted to determine the extent of Phytoph thora cinnamomi infestation in protea nurseries and cut-flower plantations in the Western Cape (Von Broembsen, 1981). A sanitary programme was initiated to control the spread of the disease in nurseries, plantations and in the natural habitat. This included the publication and distribution of information pamphlets to producers (Brits & Von Broembsen, 1978). These measures form the basis of a long-term project to control Phytophthora cinnamomi on proteas in the Western Cape.
The commercial insect pests of proteas constitute a major problem field (Myburgh et al., 1973). The problem is illustrated by the vast number of pests, estimated at more than 200 species (Gess, 1968) and probably more than the total number of pests of all introduced horticultural crops in the Western Cape. This is because proteas are cultivated within the boundaries of their natural habitat. A wide range of destructive insects has been identified (Myburgh et al., 1973;Myburgh et al., 1974;Myburgh & Rust, 1975) and efficient control measures for some have been developed (Myburgh et al., 1976;Myburgh, 1978;Starke, 1979).
Communication between research and industry
The scattered distribution pattern of protea production areas in South Africa (Vogts, 1980) presents a considerable problem of communication. A protea information pamphlet series was initiated (Vogts, 1980;Vogts, et al., 1976b;Jacobs & Steenkamp, 1975;Vogts et al., 1976a;Vogts et al., 1976c;Myburgh et al., 1976;Brits & Von Broembsen, 1978;Jacobs & Steenkamp, 1976;Vogts, 1977a;Vogts 1977b;Vogts, 1977c;Starke, 1979;Vogts, 1979;De Swardt & Pretorius, 1980;Claassens, 1980). In addition, a mailing list of cut-flower producers was compiled on a national basis (Brits, 1978b). New information is regularly distributed free-of-charge to producers in South Africa as an extension service to the Industry. CONCLUSION The above is a summary of important problems and research contributions relating to protea culture in South Africa. The solution of these problems represents a significant advance in the domestication of the fynbos Proteaceae as a commercial flower crop. | v3-fos |
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} | s2 | Extraction of leaf protein from green crops. Chemical composition and nutritive value of products of fractionation
. Leaf protein was extracted from different green crops in 11 pilot plant esperiments. Of the crops, 4 were grass, 6 clover and one pea. The extraction of juice was on average 55 % of the fresh weight of the green crop and the values for dry matter (DM) and crude protein (CP) were 22.6 and 24.1 %. Clover gave better recoveries of protein than grass. In the leaf protein concentrate (LPC) obtained from the juice, the separation ratios for DM, CP and TP (true protein) were, respectively, 23.7 %, 48.0 and 80.7 %. Heating to 85°C gave more efficient recoveries of LPC than the combination of heating and acid precipitation. The average DM content of the pressed pulp was 30.4 %, the corresponding value for the whole crop being 16.5 %. Measured on a DM basis, the CP content of the pressed pulp was only 0.4 % units lower than in untreated forage, but the crude fibre content was 7.3 % units higher. In vitro organic matter digestibility and the pepsin-HCI solubility of crude protein were on average 5.1 and 5.5 % units lower in the pulp. The average DM of the plant juice was 6.5 % and contained 21.9 % ash, 21.5 % CP, 10.7 % TP and 29.9 % soluble sugars. Clover and pea had much higher values for CP and TP than grass. In the LPC preparations, CP and TP averaged 43.6 % and 38.5 % of DM. Heat treatment gave higher protein content than precipitation of LPC by combined heating and acidification.The in vitro digestibility and protein solubility of LPC were high, on average 85.6 % and 80.2 %. LPC had fairly high contents of lysine and methionine, 4.1 % and 1.6 g/16 g N. There were only small differences in the amino acid composition between grass and clover and between crops harvested at different growth stages. Green crop fractionation is a potential means of improving grassland production and utilization. Promising results have been obtained with plant juice and LPC fed to monogastric animals and pressed pulp residues in diets for ruminants. The economic aspects offractionation remain to be evaluated.
Introduction
Green crops can be separated by mechanical methods into two fractions, protein-rich plant juice for monogastric animals and fibrous pressed pulp for ruminants. Further processing of the green juice gives leaf protein concentrate (LPC) and deproteinized juice (WILKINS 1977, PIRIE 1978. In Finnish conditions pasture swards, both grass and legumes, have remarkably high dry matter (DM) and protein yields right up to northern Finland. By making grass silage at an early stage of growth, protein requirements of ruminants can be satistied. The supplementary protein for non-ruminants and highly productive ruminants is mostly imported. The use of leaf protein from grass and legumes could be a way to increase the country's self-sufficiency in respect of protein. The concentration of protein in grass and legumes that have been fertilized properly and harvested at an immature growth stage is greater than that required by most ruminants. The losses during ensiling of fresh grass cut at an early growth stage are considerable (NORRGAARD PEDERSEN et al. 1980); ETTALA and KOSSILA (1980) found that the total weight losses averaged 31.9 % and those for DM and CP 21.2. % and 19.5 %. The effluent losses are most important when the silage crops have a low DM content. When peas and horse beans were ensiled, the effluent losses amounted to over 30 % of the weight of the crop and the DM and CP losses in the effluent were 15 % of the values of the original crop (SYRJÄLÄ et ai. 1980, SYRJÄLÄ-QVIST et ai. 1982. By fractionating the crop, effluent losses could be avoided and grassland production and utilization could be increased. The objective of the investigation reported here was to perform leaf protein extraction on various pasture crops in Finnish conditions, to examine the chemical composition of the products of fractionation, and to asses their nutritive value and suitability for animal feeding. The results of a study of the preservation of plant juice and wet leaf protein concentrate are published in another report (NÄSI 1983).
Materials and methods
Eleven different crops were fractionated during summers 1979 and 1980: four grass crops, six clover and one pea. Table 1 shows the cutting dates, crop production, and yields of dry matter (DM) and crude protein (CP) per hectare. The leys of grass mix (timothy 40 %, meadow fescue 40 % and red clover 20 %) and pure stands of red clover were second or third years' growth. The crops were first cuts, except grass 2, which was a second cut. The fertilizer application per ha on the grass swards was N 190 kg, P 15 kg and K 30 kg and on the clover N 16 kg, P 50 kg and K 95 kg; for the pea crop it was N 70 kg, P 35 kg and K 35 kg. The grass leys and peas were harvested with a chopper and the clover was cut with an experimental harvester.
The green crops were pulped with a laboratory cutter to rupture the plant cells, after which juice was expressed hydraulically (HAF press 0.75 kW, pressure 200 kN). The leaf protein was coagulated by heating the juice to 85°C with steam injection, or precipitated by combined heating and acidification with 0.5 % v/w cone. HCI. The precipitated leaf protein coagulum was separated by cloth filtration.
Samples for analysis were taken from the green crop before and immediately after pressing. These and samples of the juice and LPC were stored in the deep-freeze until analysed. Samples were vacuum-dried at +5O°C. The DM determinations were made at 103°C. The feed analyses were made by standard methods, water-soluble carbohydrate was determined by the
Results and discussion
The extraction ratios of the plant juice and its components for the different crops are shown in Table 2. On average, 55 % of the fresh weight of the green crop was expressed as juice and the extraction ratios for DM, and crude protein (CP) were 22.6 % and 24.1 %, respectively. The values were higher for clover than for grass; the CP extraction ratio was twice as high as in grass and the true protein (TP) ratio three times as high. The extraction ratio of water-soluble carbohydrates was very high, on average 75.9 %.
The extraction of juice and its components depends mainly on the crop species, stage of maturity at harvest and crop moisture content, but it is also affected by the mechanical treatment of the crop prior to pressing and the types of press used (HOUSEMAN and JONES 1978). The extraction of protein requires efficient maceration of the crop to rupture the cells before pressing. OSTROWSKI (1976) reports that the protein recover from grass ranges between 5 and 30 %, but it is possible to achieve protein recovery of between 40 and 50 %.
The leaf protein curd averaged 12.1 % of the weight of the plant juice. The average separation ratios for DM, CP and TP were 23.7 %, 48.0 % and 80.7 % ( Table 3). Precipitation of LPC components was more efficient with clover juice than with grass. Protein (CP and TP) separation ratios were higher when protein was coagulated by heating than when it was precipitated by combined heating and acidification. The true protein recoveries were in some cases over one hundred per cent which indicates that some changes in the protein fraction had been caused by the heating treatment. Cloth filtration was not efficient enough; when the composition of the deproteinized juice (DPJ) was examined, 3.7 % of the DM was found to be true protein ( Table 7). The chemical composition and in vitro digestibility of the forage and pulped pressed forage are compared in Table 4. The pulp which remains after juice has been expressed from the green crop contains almost all the fibre of the original crop and a proportion of the crude protein, soluble carbohydrates and mineral matter. The average dry matter content increased in processing from 16.5 % to 30.4 %. The crude protein content, calculated on a dry matter basis, decreased by only 0.4 % units, but crude fibre increased by 7.3 % units. Pepsin-HCI-soluble protein was 5.5 % units lower and in vitro organic matter digestibility 5.1 % unitserlower in the pressed pulp than in the crop prior to processing.
The enegy content in the original crop averaged 14.54 MJ ME/kg DM and in the pressed crop 13.70 MJ ME/kg DM, calculated according to the equation presented by TERRY et. al. (1974). The corresponding NE values were 1.18 kg DM/FU and 1.25 kg DM/FU (1 FU = 0.7 kg starch). In the fractionation of green crops, large quantities of the more digestible nutrients are removed, leaving pulp containing larger relative amounts of cell wall material, and according to the chemical analysis the pressed pulp should have a lower nutritive value than the whole crop. But when the juice extraction is moderate as in the present experiment, where the juice DM averaged 22.6 % of the DM in the whole crop, the nutritive value does not decrease too much. When the crop is cut at an early growth stage, the protein and energy values of pressed ensiled pulp are sufficient to meet the requirements of lactating cows and beef cattle.
In several experiments pulp residues have been demonstrated to be similar in nutritive value to the whole crop in terms of digestibility of OM and DM and conversion of DM to liveweight gain (MAQUIRE and BROOKS 1973, VARTHA et al. 1973, JONES et al. 1974, HOUSEMAN et al. 1975, CONNELL and FOXELL 1976. In those experiments the pulp residues were fed to animals in fresh, ensiled and artificially dried form. GREENHALGH and REID (1975) suggested that some modifications occur in pulping and pressing which lead to improvement of pressed forage utilization.
The pulp residues obtained from grass or lucerne have been reported to ensile easily with relatively small effluent losses (JONES et al. 1974), although some workers (RAYMOND andHARRIS 1937, VARTHA et al. 1973) have reported difficulties in the ensiling process, due to the low sugar content of the pulp. The palatability of ensiled pressed crops has been noted to be relatively good (JONES et al. 1974, HOUSEMAN et al. 1975. Pressed lucerne silage fed to dairy cows had the characteristics of the conventional wilted whole crop (CONNELL and FOXELL 1976). Attention has been drawn to the substantial reduction in field dry matter losses through the avoidance of field wilting. Table 5 shows the composition of the juice extracted from grass, red clover and pea in 1979 and 1980, giving the mean values and ranges. Expressed as percentages of DM, the levels of crude protein, ash and watersoluble carbohydrates are relatively high. The composition varied fairly widely between the different growth stages. The clover juices had higher means than the grass juices for DM, CP ja TP, but lower values for ash and water-soluble carbohydrates. The pea juice contained considerably more CP in DM than the other juices. The ratio of true protein to crude protein in the juices averaged 37.2 % for grass, 59.6 % for clover and 46.6 % for pea. The protein content of grass juice was low compared with the values reported from the literature (HOUSEMAN and CONNELL 1976, CHEESEMAN 1977, HOUSEMAN and JONES 1978. This suggests that the cells were ruptured less frequently during maceration, because the protein extracted from juice originates from intracellular fluid (PIRIE 1978). The amount of protein extracted also depends on the DM content of the forage (JONES and HOUSEMAN 1975) and the pressure applied (KOHLHEB 1978). In more mature grasses the high ratio of fibre to protein lowers the protein extractability (JONES and HOUSEMAN 1975).
Grass and lucerne juice has been fed to growing pigs in a number of trials (JONES and HOUSEMAN 1975, BRAUDE et al. 1977, BARBER et al. 1981, and its nutritive value has veen shown to be high. In pigs of 40 to 60 kg nitrogen retention was equally good when juice was substituted for fish meal as a supplement for barley (JONES and HOUSEMAN 1975). Similarly, partial to total replacement of fish meal or soybean meal with fresh or preserved juice from grass or lucerne did not affect performance and green crop juice supplied a substantial amount of protein . In other trials, performance was similar when lucerne juice replaced 3.5 % fish meal for pigs of 54 to 90 kg, but was poorer when it replaced 7 % fishmeal in diets for smaller pigs (BARBER et al. 1979). BRAUDE et al. (1977) also reported poorer performance when fish meal was replaced completely by lucerne juice. The drop in performance has been attributed to sub-clinical effects of excessive mineral levels in the lucerne juice (BARBER et al. 1981). In the present study the potassium content of grass and clover was 8 g/kg juice. Clover juice had twice as much calcium as grass juice but only half as much phosphorus (Table 9).
The composition and nutritive value of the leaf protein, concentrates precipitated from plant juice by heating or by combined heating and acidification are presented in Table 6. This fraction contains the insoluble cell constituents, such as chloroplasts, together with heat-denatured cytoplasmic protein. It is therefore enriched in protein and poor in soluble material compared with the forage from which it is derived. The dry matter content of LPC was rather low, on average 12.7 %, when it was separated with fourfold cheesecloth. The crude protein content of the leaf protein samples was high, on average 43.6 % of DM, and the true protein content was also high, 38.5 %. In LPC of clover the contents of CP and TP were 3 % units higher than in grass LPC. Coagulation by heating gave about 2 % units higher CP and TP contents than precipitation heating and acidification. In some samples the crude fibre content was rather high, 8-9 % of DM, due to contamination of the plant juice during processing. According to the in vitro digestibilities, the nutritive value of the LPC products was high. The pepsin-HCI-solubility of the crude protein of LPC averaged 85.6 %. Heat coagulation gave better solubilities than the combination of heating and acidification (89.7 % vs. 77.9 %). Clover had slightly higher values than grass. In vitro organic matter digestibility averaged 80.2 %. Digestion in vitro was 6.2 % units higher for clover LPC than grass LPC, but did not differ between the two precipitation methods.
The amino acid composition of the LPC samples is presented in Table 8. The mean lysine content was 4.1 g/16 g N and it decreased a little during the growing season. The methionine content averaged 1.6 g/16 g N and threonine 3.8 g. There were only small differences between grass and clover. The amino acid composition of leaf protein has been found to be remarkably independent of the age and species of the crop from which the LPC is derived (GERLOFF et al. 1965, BYERS 1971. In feeding monogastric animals, the true protein and amino acid content is important. GERLOFF et al. (1965) and HOVE et al. (1974) reported that the limiting amino acid in LPC prepared from several species of crops was methionine, and that the other essential amino acids were present in amounts The biological value and true digestibility of LPC obtained from various green crops were found to be very high when it was prepared under optimal conditions. The drying method and temperature were found to be crucial for the nutritive value (HOUSEMAN and CONNELL 1976, MORRIS 1977, HOUSE-MAN and JONES 1978, PIRIE 1978). High quality leaf protein is a valuable feed for pigs and poultry. Enriched with methionine, it can be used as the sole protein supplement in cereal diets. LPC has replaced fish meal in rations for growing pigs without adverse effects on performance, at least with pigs over 55 kg (DUCKWORTH et al. 1961, CARR andPEARSON 1976) and given good results as a substitute for soybean meal (CHEEKE 1975). In diets for laying hens LPC has value as a source of pigment (YOSHIDA and HOSHII 1981); its xanthophyll content is high. LPC levels of 20 % in layers'diet (MORRIS 1977) and up to 54 % in broiler diets (KUZMICKY and KOHLER 1977) have been used without adverse effects. Growth-depressing substances, such as saponins have been recognised in extracted juice and LPC, but these are partly removed in the deproteinized juice during the preparation of LPC.
Conclusions
Mechanical fractionation of green crops provides a means of extracting larger quantities of protein for utilization by nonruminants, leaving pulp suitable for ruminant livestock. Mechanical extraction of leaf protein is technically and probably commercially feasible and many systems are being developed for recovery of protein from forages and other leafy materials (WILKINS 1977, PIRIE 1978. At the industrial and commercial level, efforts are being directed to producing leaf protein concentrate and drying pulp residues for green meal. On the farms, systems of green fractionation can be operated to provide plant juice for feeding pigs and processed residues for ruminants. Recent research has indicated the technical potential of green crop fractionation for improving grassland production and utilization. The nutritive values of grass juice, pressed pulp residues and leaf protein concentrate are promising. Further experimentation is necessary to identify the optimal methods of mechanical processing and to evaluate the economic aspects of fractionation. | v3-fos |
2014-10-01T00:00:00.000Z | {
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} | s2 | Growth curves of highly inbred lines of fowl and their F1 hybrids
HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. Growth curves of highly inbred lines of fowl and their F1 hybrids Helena Knížetová, B. Kníže, J. Hyánek, R. Šiler, Ludmila Hyánková, J. Plachy, Milena Vilhelmová
I. Introduction
The course of the growth of most mammals and birds, expressed as a weight change in time, is described by an asymmetric, sigmoid-shaped curve with an inflection point where the autoacceleration phase passes into the autoretardation phase. There have been frequent attempts to describe the growth of individuals in a population or in lines by means of some mathematical function. The importance of the derivation and construction of curves is that the information contained at a number of points, given by coordinates of weight and time, can be summarized in several generalizing parameters. The irregular fluctuation of weight caused by random environmental effects is eliminated when the functions are expressed graphically. Another advantage to growth functions is the prediction of animal growth rate, the determination of changes in the shape of the curves in the course of selection, and the application of derived parameters to selection trials. Growth curves can provide information for the estimation of feed requirements. The flexibility of the growth model, i.e. the ability to comprehend different shapes of the curve, is essential in choosing the function.
The three growth functions which have been applied most extensively to animal species are logistic, Gompertz and Bertalanffy curves. R ICHARDS (1959) has shown that each of these three functions is a special case in a general family of growth curves which differ primarily in the proportion of final weight at which the inflection point occurs.
A basic requirement for a mathematically derived growth curve is a measure of the goodness of fit to actual data. The authors consider this essential since it is difficult to construct a curve that agrees completely with the actual course of growth. Most of the functions are sensitive to the frequency and regularity of data on both weight and age. As a rule, a monotonic increase in weight is assumed throughout the period studied. The usual tests for goodness of fit involving residual variance are not appropriate to longitudinal data because of correlated errors among repeated observations over time . Despite this, some authors use these tests, assuming that the source of correlated errors influences residual variances in different cases in a similar way (EisEN et al., 1969 ;T!MOrr & ErsErr, 1969 ;BROWN et al., 1976).
Another approach to fitting a growth function is to compare observed and predicted body weights at important points of the curve and to evaluate the inter-individual variability and correlations of the analogical parameters estimated by different mathematical functions .
The aim of the present study was to characterize the growth of four highly inbred lines of fowl and their F, hybrids in terms of the Richards function and to analyze the differences between estimated growth curve values and observed weights. The genetic aspects of growth and live weight (variability, effects of additivity, dominance, sex linkage, maternal effect, etc.) have been analyzed in two of these lines (C, I) and their hybrids in other studies (C LOUGH & CocK, 1957 ;COCK & M ORTON , 1963 ; M ORT ON, 1973).
I1. Material and methods
Growth was studied in chickens of four highly inbred lines (F! > 99.9 p. 100) C, I (Iowa), W and M (Minor) which have been characterized in detail by F ESTING (1979). Lines C, I and W were derived from White Leghorns by sib mating at the Northern Poultry Breeding Station, Reasehealt, Cheshire, starting in 1932. Line C was developed from a pair of WL purchased from a commercial breeder. Line I was obtained from a group of three males and five females inbred WL imported from Iowa State University in 1937. Line W originated as a British commercial line WL ; a colour variant with barring pattern, that appeared in 1941, was subsequently fixed. Finally, line M was established in 1956 at the Czechoslovak Academy of Sciences from Black Minor and maintained by brother X sister mating. Of the F l hybrids, groups I X C, I X W, I X M, W X C, M X C and M X W (sire line X mother line) are represented in the present trial. With regard to demand on the same hatching, the numbers of birds in inbred lines and hybrids were low (C -55, I -23, W -26 and M -25). In total, the F hybrids were represented by 79 cocks and 77 hens.
All the chicks were reared in litter floor pens in an environmentally controlled room. The diet contained 19.3 p. 100 protein and 11.82 MJ (2 823 kcal) ME/kg. Both feed and water were provided ad libitum. Until the age of 10 weeks the chickens were weighed at 7-day intervals and, in the subsequent period, at intervals of 2 weeks (up to 32 weeks in hybrids and 36 weeks in lines).
The records up to the end of the studied period were not used in some cases to calculate the growth curve parameters of pullets and we only used data up to the first decline of live weight because applying mathematical growth functions supposes a monotonic live weight increase. Since laying began in most groups before we had finished weighing the birds, usually a decrease of live weight is seen.
The changes in the weight of each individual during postnatal growth were expressed by the four-parameter Richards function (R ICHARDS , 1959) : The parameters, estimated using the generalized least-squares method, are the following : If n = 1, the function is logistic (y * /A = 0.5). Cases in which n -0 (y * /A = 0.368) correspond to the Gompertz function and those where n = —0,33 (y * /A = 0.296) correspond to the Bertalanffy function.
Weight (y * ) and age (t * ) at the inflection point were calculated from the parameters of the curve. The inflection point represents mathematically the time at which the second derivative of the growth curve changes from positive to negative : Further derived parameters included the average absolute growth rate v (g/day) and maximal absolute growth rate v * (g/day at which the inflection point was reached) : The coefficient of determination R 2 was calculated for each individual growth curve : The coefficient of determination and the percentage deviations of the observed values of weight at individual points of the curve characterized the accuracy of curve fit to the observed course of growth. Animals with an R 2 < 0,99 were eliminated from the overall analysis. The parameters of the curves of the inbred lines and hybrid combinations were evaluated by analysis of variance ; the differences were verified by the t-test.
The F l hybrids were compared with the parent lines and deviations from midparent values were tested ; the significance was evaluated using W EBER ' S (1972) formula : In the many cases where the hybrids exceeded the parent line with the higher parameter values, i.e. F 1 > max. (P I , P z ) the differences were verified by the t-test.
III. Results and discussion
A. The course of growth Changes in the weight of chickens in individual lines and in hybrid combinations during postnatal development are illustrated by figure 1. The limited number of individuals in each group does not allow a reliable evaluation of differences in the variability of lines and hybrids. Nevertheless, the variability of weight expressed by the coefficients of variability (tab]. 1 ) seemed to increase to the age of 6-10 weeks and then markedly decrease. The expected trend to lower variability in the weight of hybrids compared with parent lines seemed to be manifested in older age classes. Differences in body weight between the sexes steadily increased throughout the course of growth. At maturity, the cocks of individual groups reached 120 to 140 p. 100 of the weight of the hens.
A comparison of F i hybrids of both sexes with the parent lines showed a marked heterosis effect (F i > max. P i , P !) in weight at 2 weeks of age. This might be explained as a better adaptation of the hybrids in the period after hatching. However, as seen from the percentage deviations from mid-parent values (tabl. 2), the relative positions of different hybrids became apparent with further development, particularly in terms of dependence on the cross combination.
B. Fit of tlae growth function
The difficulties of fitting the generalized Richards function arise mainly from the high correlation (r1, = + 0.90) between the constant k and the shape parameter of the curve (T IMON & E ISEN , 1969 ; RuTr.EncE et al., 1972). In our trials the goodness of fit of the growth curves was measured by using the coefficient of determination (R 2 ), by evaluation of the differences between the estimated asymptote (A) and the highest weight (A') observed in the period studied, and by comparing the percentage deviations of observed weight from the theoretically determined values at different points of the curve.
With respect to the determination coefficient of individual growth curves (R 2 > 0.99), we eliminated a higher number of individuals in inbred lines (6.9 p. 100) than in F, hybrids (1.3 p. 100) from the overall analysis. K IDWELL et al. (1969) also report that, in applying the Gompertz function to mice, more inbred animals (3.9 p. 100) than hybrids (0.2 p. 100) had to be eliminated. One explanation of these results might be the higher sensitivity of inbred birds to randomly changing environmental conditions and also a higher fluctuation of gains during growth (including temporary stagnation or a decrease of live weight).
An important criterion of the accuracy of the estimated curve parameters is a comparison of the asymptote (A) with the highest observed weight (A'). The data in table 3 show that percentage deviations from the estimated asymptote were usually higher in hybrid combinations than in inbred lines. A comparison of the different groups indicates that the growth curve of cockerels of inbred lines corresponds to the type 3 function (n < 0) in only 5 p. 100 of the cases, whereas it corresponds in hybrid combinations in 44 p. 100 of the individuals. A similar situation was found in pullets (18 p. 100 vs. 49 p. 100), but the case of the Iowa line was special. When weight data were used up to the age of 30 weeks (practically up to the termination of growth) to calculate parameters of the curve, the asymptote was overestimated by 10.4 p. 100. When all the data were used (i.e. up to 36 weeks ; the Iowa line is designated by I'), the estimate of A was improved (the difference between A and A' was reduced to 3.7 p. 100) but the determination coefficient was much lower (0.9860 vs. 0.9957). This means that the theoretical curve did not fit the observed course of growth ( fig. 2).
The main reason for the overestimation of asymptotic weight is the overall character of growth in the studied period as expressed by shape parameter n. This parameter, which estimates the position of the inflection point of the curve, also reflects the grade of sigmoid curving. In growth curves with less sigmoid curving, i.e. with lower values of n (n < 0), the estimated value of the asymptote is higher. In spite of that, A did not correspond to the highest observed live weight ; in the case of a given estimated n, the theoretical growth curve gave the best fit for the observed course of growth. The next factor influencing the estimation of A are irregularities in weight when the animals neared maturity. The lines were considerably different in the age at sexual maturity. The pullets of the hybrid groups started laying eggs substantially earlier (tabl. 4). The observed weight of cocks in some hybrid combinations fluctuated in the final phase of growth due to their social behaviour. In addition to this, we noted that the well-known difficulty in estimating final weight is to determine the proportion of physiologically unessential fat (L ILJEDAHL , 1970 and others).
A comparison of the differences between estimated and observed weight in chicks of inbred lines and hybrid combinations during the studied period showed that the greatest deviations were recorded in the initial stage of postnatal growth, i.e. up to about 6 weeks of age (fig. 3). The differences are comparatively low (.! 1.5 p. 100) in the region of the inflection point and this trend was the same up to the end of the period studied. In the post-inflection part of the curve, the average deviations of the curve did not usually exceed ± 4 p. 100. In this connection, it should be noted that deviations of observed weights ranging between -7.1 and + 5.2 p. 100 were also found in mice after the goodness of fit of the Gompertz function had been analyzed (LAIRD & HOWARD, 1967).
In evaluating of percentage deviations of observed weights from fitted values it should be emphasized that conspicuous differences existed between inbred lines and hybrid combinations (P < 0.01, P < 0.05) particularly in the initial phases of growth. In 2-week old hybrids of both sexes the theoretical values were significantly underestimated, whereas at 6 weeks of age they were overestimated. A reverse trend was found in inbred chickens. The pronounced positive deviation of the observed weights of 2-week old hybrids seemed to be related to heterosis effect during this period (tabl. 2). The negative deviation of inbred chickens could be attributed to their low ability to adapt to the conditions of postnatal life. The opposite trend (shown by deviations at the age of 6 weeks) may reflect the compensation of differences arising after hatching.
C. Parameters of the growth curves of inbred lines and hybrid combinations
The high degree of inbreeding (F . > 99.9 p. 100) suggests that the intraline variability in growth curve parameters could be ascribed mainly to the action of environmental factors. A greater genetically determinated variability within the F 1 hybrid group could not be expected either. On the other hand, interline differences were mainly of genetic origin, although eventually they might be a manifestation of the genotype X environment interaction.
The growth curves of inbred and hybrid groups are shown in figures 4-7 and the parameters of the curves are summarized in tables 5-8. The shape parameter (n) and the y * /A ratio show a wide range of values for individual curves (n =-0.3 to 0.8 and y * /A = 0.279 to 0.480). The average values of different groups are within the range n = -0.100 to 0.271 and y * /A = 0.347 to 0.410, demonstrating that the growth of chickens can generally be expressed by the Gompertz function (y * /A = 0.368). This function has been used to analyze turkey growth (BuFFirrcTOrr et al., 1973).
A comparison of the course of growth and the shape of curves (parameters n, y * /A) in cockerels of inbred strains reveals a similar trend in the Iowa and C lines (tabi. 5). Higher average shape parameter values and, therefore, higher values of the y * /A ratio, characterize the curves of the W and M lines. However, it should be emphasized that the W and M lines differed significantly (P < 0.01) as to the time needed to reach the inflection point (t * = 77,4 vs. 101,0 days) and, therefore, as to growth rate parameters (k, v, v * ). The post-inflection growth phase was first entered by the C line, followed by Iowa and W, with a marked delay in the Minor cockerels. The order of lines in reaching the inflection point (y * ) was the same in regard to weight as the order of final weight (A) : I < C < W < M. It should be noted in this connection that the correlation rp = 0.14, r G = 0.39 between y * and A has also been demonstrated in mice (T IMON & E ISEN , 1969). Coefficients of correlation (rp) ranging from 0.57 to 0.89 were obtained in ,our laboratory in unselected fowl populations (White Leghorn, New Hampshire, Orpington). The pullets of inbred lines showed a pattern of intraand inter-line differences similar to those of cockerels (tabl. 6). Higher y !=/A ratio values were recorded in the W and M strains ; minor pullets were also slower to reach the inflection point (t * ). The highest growth rate parameters (v, v&dquo;), recorded in the W line, were associated with a comparatively late transition to the post-inflection phase.
The course of the F l hybrid growth is shown in figures 6 and 7 and the curve parameters are presented in tables 7 and 8. Due to the limited number of chickens, we could not evaluate differences in group variability. Nevertheless, it is obvious that in inbred lines and in hybrids of both sexes, the k parameter showed the highest coefficients of variability (C.V. = 6.6 to 25.2 p. 100). A comparatively lower intra-group variability was noted in the y * (C.V. = 1.7 to 9.1 p. 100) and y&dquo;/A (C.V. = 0.0 to 9.2 p. 100) parameters.
Data on the analysis of variance of curve parameters are included in table 9.
Taking the characteristics of the parental material (F! > 99.9 p. 100) as a basis, it follows that the genetic differences between lines, and eventually between hybrid combinations, are an important source of the total variance. The highest inter-group variance was recorded for age and weight at inflection point (t !', y * ), higher values being obtained between inbred lines than between F, hybrids. The component determined by genotype was also comparatively high for average and maximal absolute growth rates (v, v!'). The situation is similar for the variance of the asymptote of inbred lines. The low value in F 1 hybrids, particularly in pullets, can probably be ascribed to an inaccurate estimation of A, since the genetic variance for maximal live weight (A') remained high (8 74.3 p. 100, y ? 54.2 p. 100). The inter-group variance for the y * / A ratio was comparatively low (22.6 to 31.3 p. 100).
A comparison of hybrids with parental lines gave estimates of heterosis in individual growth curve parameters (tabl. 10 and 11). In our experiment comparing different inbred and hybrid groups, age at the inflection point of the curve occurred within a wide range (from 63.3 to 101.1 days) on an average, the hybrids showed some shifting towards an earlier inflection : cockerels -9.4 p. 100, pullets -7.6 p. 100 (tabl. 10). Some hybrids were significantly more precocious (t'k) than the earlier parental line (tabl. 11). This manifestation of heterosis depended on particular parental lines and hybrid combinations ; a more detailed analysis of these effects would require a representation of all groups of reciprocal crosses. LAIRD & H OWARD (1967) found a shift to an earlier age at the inflection point in hybrids of inbred lines of mice.
In the growth analysis of chickens of commercial populations of egg and meat types, the parameter values for age at the inflection point were shifted to an earlier period. For instance, when the power function y = at') was applied, it was found that growth rate (b) began to decrease at the age of 7-8 weeks (R OBERTS , 1964). In another analysis applying the logistic function, the maximal absolute growth rate of broiler chickens was estimated to occur at the age of 35.5 to 48.3 days at a live weight of 763 to 1 261 g (L ILJEDAHL , 1970), although the author admitted that the weights were underestimated with respect to the growth capacity of the meat-type broiler population. This assessment agrees with the results of Wi L SOrr (1977) who applied the Gompertz function to the growth of meat-type chickens (Ross 1). The inflection point in cockerels was estimated to occur at 55 days of age at a live weight of about 2.0 kgs. ! A comparison of hybrid weight at the inflection point reveals positive deviations from the mid-parent value (tabi. 10). In some cases the hybrids significantly exceeded the parent line with higher parameters (tabl. 11 The higher values of the asymptote for the majority of hybrid combinations, compared with pure lines, are a distortion due, to some extent, to inaccurate estimation. Nevertheless, the significant differences where F 1 exceeded the max. ( p l , P 2 ) for the observed final weights in groups of males I X C, C X M, M X C and females I X C, M X W, suggest that manifestations of the heterosis effect are maintained up to maturity in some hybrid combinations. Taking into account the higher parameter values in hybrids, it is not surprising to find heterosis in average and maximal absolute growth rate (v, v'k).
D. Sex differences
As expected, the cockerels showed significantly higher estimates (tabl. 5-8) for parameters immediately related to live weight and growth rate (y * , A, v, v * ). Cockerels reached the inflection point (t * ) later but the differences were small and in most cases statistically insignificant. A shift in the age parameter at the inflection point towards higher values in the male sex was also reported by L ILJEDAHL (1970). The values of the remaining curve parameters (y * /A, k) were somewhat higher in cockerels but all these differences were insignificant. | v3-fos |
2018-12-15T06:15:09.972Z | {
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} | s2 | Effects of various feeding, breeding and management practices on milk production
This report is brought to you for free and open access by New Prairie Press. It has been accepted for inclusion in Kansas Agricultural Experiment Station Research Reports by an authorized administrator of New Prairie Press. Copyright 1984 Kansas State University Agricultural Experiment Station and Cooperative Extension Service.
; • The March 1984 DHI :sum mary of f{ansas Holstein her'ds shows some interesting correlations of various feeding, breeding, and management factors to production ( Table 1). The Rolling Herd Average (RBA) is an excellent evaluation of the efficiency of dairy herds since RHA and incomc-over-feed-cost are closely related. Although income-over-feed-cost is not profi t, it pr'ovides the income for paying the other costs of producing milk. The goal of every dairy producer should be to increase the RflA in order to improve profitability.· The following observations can be made from Table 1: Feeding Higher producing herds are fed mor·? grain and more dry matter than those with lower RH A's. The rations of higher producing Ilerds are composed up of about 12% more grain than those of lower herds. but hte higller RHA herds produce milk more efficiently, as shown by tile milk/lb of grain or milk/lb of dry matter fed. Total feed cost increases with RII A but feed cost/cwt milk decreases, which results in more incorne-over-feed-cost for higher RH A herds.
Management
All of the management factors contributing to tile RIIA cannot be evaluated by the DHI report. However, high RHA herds have a higher percent of days in milk and fewer dry days than lower producing herds.
More days in milk are accomplished with shorter dry periods, raising more replacments, and then culling first lactation heifers while they are still lactating.
Reproductive management does not seem to be much different in relation to RII A, except the number of days from calving to first service tends to be less in high RHA herds. Apparently, high production does not have a negative effect on reproduction. Most herds could shorten the calving interval by reducing the days between calving and first service.
f)
An obvious management characteristic of high producing herds is the high summit milk yield. Apparently, feeding and management programs during the dry period and early lactation are such that fresh cows peak higher in their lactation curves than those in lower producing herds. A 60-day dry period is recommended. For high summit milk yields, starting about 2 wk before calving, dry~ows should be fed the same forages as the lactating cows with about 15 lb of grain. Following calving, fresh cows should be on a high level of grain within a few days. The average age of cows in high Rfl A herds is lower than in low herds. Therefore, it cannot be concluded that high RH'\ herds have more mature cows. Also, the high RHA herds' first calf heifers are slightly youngel'. Bl'eeding Sire selection is closely related to the R[l A. All age groupS are sho~n t~be sired by higher PD$ bulls as the RH A increases. In addition, the serVice sires currently being used have a higher PO$ value and higher percentile rank in the high [{HA herds. High RHA herds use a largel' percent of proven bulls than lower RH A herds. The goal should be 80/80, which means 80'!6 of the COWS bred to at least 80+ percentile rank bulls. The other 20% of the cows should be bred to several young Al sires to help prove the next generation of bulls.
CSound breeding decisions cannot be made without identification. Table 1 shows that the percent of cows identified by sire and dam increases with the RHA.
• Figure 1 depicts the stage of lactation profile of the groups of herds summarized in Table 1. It is obvious that the hiaher RH A herds maintain the • b productIOn advantage through all stages of lactation. Thus, in order to obtain high total lactation yields, cows must staI't their lactations at a high level. | v3-fos |
2019-04-03T13:06:02.052Z | {
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} | s2 | Evaluation of barley distillers dried grains with soluble, and condensed distillers solubles in the diet of growing pigs
Two digestibility and nitrogen balance trials were conducted to evaluate the feed value of barley-derived distillers dried grains with solubles (BDDGS) and semisolid distillers solubles from barley and wheat (BDS and WDS) in rations for growing pigs. The average crude protein, lysine, crude fibre and neutral detergent fibre contents of BDDGS, BDS and WDS were, respectively: 30.6, 20.6, 30.7 %; 1.7, 1.7, 2.6 g/16 g N; 16.1, 2.0, 3.1 %\ 69.4, 2.3, 7,1 %. Available lysine was greatly reduced in all samples. The distillery by-products composed 33 % of diet DM in the barley based rations. The control diet was a mixture of barley and skim milk powder. The organic matter and crude protein digestibilities of BDDGS, BDS and WDS were 40.5, 80.4, 85.0 % and 52.4, 46.9, 77.5 %, respectively. Their FU values and DCP values were 0.50, 0.94, 0.94/kg DM and 319, 102, 253 g/FU. on the WDS diet, nitrogen retention, as g/d, was similar to that on the control diet, but on the diets with barley distillery products it was reduced due to the lower amount of protein absorbed, especially the lower lysine intake. Barley distillers by-products proved to have low feed values for pigs in this study, but the value for WDS was quite reasonable. The new integrated starchethanol process can be expected to yield more suitable fractions for use in pig rations.
Introduction
Distillers by-products can be fractioned by screening or by centrifuging into solubles and grains, and these fractions can be dried together or separately to produce DDGS or DDG. The soluble fraction can be evaporated to a semisolid (Pieper 1981). Distillers dried grains with solubles (DDGS) and whole stillage from wheat have been investigated in rations for growing pigs and it has been found that up to 5°/o and 10 % of the pig feed can be replaced with DDGS and wetstillage, respectively (Alaviuhkola 1978, Suomi 1980. Newman and Gras (1983) have reported that 5 % barley dried distillers grains (BDDG) can be used to replace barley and soybean meal in diets for growing pigs. Higher levels tended to decrease the feed conversion efficiency, although the rate of gain was not impaired by up to 10 % BDDG.
A new ethanol plant using barley as raw material will start operation in Finland in Index words: Barley distillers grains with solubles, ethanol fermentation by-products, protein sources, pig feeds. 221 JOURNAL OF AGRICULTURAL SCIENCE IN FINLAND 1987. The process will be integrated ethanolstarch production, with an annual yield of 61700 tn DM grain fractions for feed purposes (Lehmussaari 1984). The increased supply of distillers products expected in the next few years has encouraged reevaluation of these products, especially barley residues, for which little information is available regarding utilization in pig diets.
The present study was designed to evaluate three distillery products as feed for growing pigs by detailed analyses of chemical composition, and measurements of nutrient digestibilities and utilization of protein.
Materials and methods
Two digestibility and nitrogen balance, trials were carried out, with four castrated Landrace pigs in each trial, during the growth period from 73 to 109 kg. Barley distillers grains with solubles (BDDGS), and condensed wheat distillers solubles (WDS) composed 33 % of the dry matter of the diet which was based on barley meal enriched with minerals and vitamins. The control ration was a barley, skim milk powder mixture (85 % + 15 %). In the second trial semisolid barley distillers solubles (BDS) composed 33 % and 67 % of the dry matter of the diet, which was based on barley. This trial had to be abandoned during the second period, due to the severe diarrhoea of the pigs receiving the larger amount of BDS. Each test period consisted of a 9-day adjustment and a 6-day collection period. The daily feed ration was 2.7 kg, on average 85 g/kg W 0 The pigs were kept in metabolic cages, which allowed separate collection of faeces and urine. These were collected quantitatively twice daily and representative samples were frozen and stored until analysed. The pigs were weighed before and after the collection period.
Chemical analyses of the feeds and faeces were performed according to official procedures. Acid detergent fibre (ADF), neutral detergent fibre (NDF) and acid detergent lignin (ADL) were determined according to Coering and Van Soest (1970). Amino acids were determined with a Technicon TSM autoanalyzer after hydrolysis of 6 hours in 6 N HCI. The availability of lysine was measured according to Carpenter (1960). The mineral composition was analysed with an atomabsorption spectrophotometer and phosphorus was determined by the method of Tayssky and Shorr (1953).
The digestibilities of the nutrients in the distillery products were calculated by the difference method, using measured values for barley and table values for skim milk powder. The feed values were calculated according to Salo et al. (1982) and Andersen and Just (1979).
Results and discussion
BDDGS and WDS had a crude protein content around 30 % of DM, but in BDS the value was 20 % (Table 1). During yeast fermentation of grain, nearly all starch is removed, which concentrates the protein and other components. The crude fibre content of BDDGS was 16 %, which is in good accordance with the results of Newman and Gras (1983). Barley contains 10-12 % hulls (Salo and Kotilainen 1970) and in DDG this percentage is increased, which is undesirable from the nutritional point of view since hulls consist of strawlike material. Barley also contains a fibre fraction primarily derived from endospermal cell walls, /3-glucan, which is also poorly digested (Cambell et al. 1984). In BDDGS the contents of total cell wall constituents (NDF), 69.4 %, and lignocellulosic material (ADF), 33.8 %, were high and exceeded the values (65.2 % and 28.2 %) given by Newman and Gras (1983). Condensed distillers solubles (BDS and WDS) had low values for ADF and NDF, which was due to the water solubility of these materials.
In BDDGS and BDS, especially the latter, most of the essential amino acids were lower (Table 2). In both the products the lysine content was only 1.7 g/16 g N, whereas in barley it was twice as high. The available lysine was reduced to the minimal value of 0.35-0.5 g. Methionine and threonine were also decreased. WDS had a sligtly better amino acid profile than the barley distillers products. Distillers products undergo heat and other processing treatments and these cause deamination and affect reactions with sugars, thus decreasing the biological value of the protein. Alaviuhkola (1978) and Salo (1978) found reduced lysine concentrations in wheat DDGS and decreased availability, too. Satterlee et al. (1976) and Newman and Gras (1983), how- with the original grains. The sodium, potassium and phosphorus contents were higher in condensed distillery solubles than in barley (Table 1). Sodium is added in fermentation to adjust the pH (Lehmussaari 1983). The high sodium content in BDS was probably the reason for diarrhoea in the pigs receiving the larger amount of solubles in the second trial.
On the diet containing WDS, nitrogen retention was almost the same as on the control diet but on the diets containing barley distillers products, nitrogen retention was decreased. The differences are mostly due to the differences in DCP intakes, which were 191, 363, 293 and 343 g/d on the BDS, WDS, BDDG and control diets, respectively.
The lysine intakes were 12.2, 14.2, 12.0 and 19.0 g/d, respectively. The availability of lysine was also much reduced in both barley distillery products. However, the results of Newman and Gras (1983) indicated that apparent nitrogen retention was not affected when BDDG replaced soybean, composing up to 20 % of the diet. Thong et al. (1978) measured nitrogen retention with gilts and found no differences between DDGS and soybean used as protein supplements.
The digestibility coefficients indicated that WDS was similar to barley, though the crude protein was 5 %-units less digestible. BDS had slightly lower values for organic matter and NFE digestibility, but crude protein digestibility was only half that of barley. Barley BDGS was digested poorly by the pigs (Table 3). The values were lower than those measured with ruminants (Näsi 1984). The organic matter digestibilities of distillers byproducts are reported as 54-63°/o for wheat (Salo 1978), 80°7o for maize and 74 % for potato (Roth and Kirchgessner 1975); the crude protein digestibilities were 63-67 %, 53 % and 55 %, respectively. Newman and Gras (1983) did not find any differences in nitrogen digestibility between diets when up to 20 % of the soybean was replaced by BDDG.
When feed values were calculated from the chemical composition and digestibility values, both condensed solubles were found to have a FU value of 0.94/kg DM, but the value for BDDGS was low, 0.50/kg DM. The DCP value for BDS was low, about the same as in grain, but WDS and BDDGS had higher values, 253-319 g/FU.
When barley distillery by-products are used in pig diets, the hull content has to be decreased to obtain an adequate energy value, and thermal processing treatments should be estricted as far as possible, to avoid impairing protein availability. The new integrated starch-ethanol process yields fractions which are more suitable for use in pig feeding. | v3-fos |
2017-08-25T14:41:08.263Z | {
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} | s2 | The Influence of Hardarian Gland Removal and Fur Lipid Removal on Heat Loss and Water Flux to and from the Skin of Muskrats, Ondotra zibethicus
The Hardarian gland (an apocrine organ located behind the eye) was removed from one group of muskrats. Another group of muskrats was anesthetized and shampooed. The fur of shampooed and Hardarianectomized muskrats did not appear to repel water as effectively as sham control animals. Both shampooed and Hardarianectomized muskrats had higher rates of heat loss while submerged in a 4 C water bath than sham control animals. The magnitude of heat loss was less for the Hardarianectomized muskrats. Hardarianectomized and shampooed muskrats had elevated rates of evaporative water loss with an estimated cutaneous water loss of 52% and 66% of total evaporative water loss for Hardarianectomized and shampooed muskrats compared with 41 % for control animals. Coating the fur of muskrats with mineral oil tended to retard evaporative water loss. It appears that the Hardarian gland is an effective source of lipids which may be spread onto the fur from the nasal duct by both self and communal grooming. This lipid coat appears to help retard heat loss as well as water flux to and from the skin.
INTRODUCTION
The muskrat, Ondotra zibethicus, is a semiaquatic mammal which is broadly distributed from the tropics to the arctic and consequently endures extreme thermal environments (MacArthur and Aleksiuk 1979). For example, during winter they forage in ice-covered water (Johansen 1962;Miller and Irving 1967), and on hot summer days their activity is primarily restricted to the lodge (Lay 1945) in which temperatures often exceed the upper critical temperature of the muskrat (Hart 1962;MacArthur and Aleksiuk 1979). The importance of lipids in enhancing the quality of fur as a protective barrier in cold as well as hot environments has not been fully explored in any mammalian species. Lipids in the fur may be instrumental in retaining the vital air layer on which muskrats de-pend for a considerable amount of their insulation (Johansen 1962). In addition, during heat stress, lipids may retard evaporative water loss through the skin (Quay 1965;Nicolaides 1974). In view of the importance of lipids in maintenance of a nonwettable fur as a means of providing buoyancy (Johansen 1962), cold resistance, and possibly heat tolerance, an additional source of lipids to augment the sebaceous gland secretions would be advantageous to the muskrat.
One source of lipids demonstrated to have thermoregulatory importance is secretions of the Hardarian gland (Thiessen and Kittrell 1980). These glands are large, lobular tubuloalveolar apocrine organs situated within the orbital cavity directly behind the eye (Graffin 1942). The Hardarian glands in many rodents are rich in porphyrins (Kennedy 1970) and lipids (Cohn 1955;Hais, Strych, and Chemlar 1968) which are secreted into the anterior corner of the eye as well as the external nares (Davis 1929;Venable and Grafflin 1970). Thiessen and Kittrell (1980) have demonstrated that the gerbil (Meriones unguiculatus) spreads these lipids over the entire body coat during autogrooming in a way analogous to the use of preen glands of certain birds, thereby providing insu-lation against wetness and cold. The Hardarian gland, therefore, may serve an important adaptive function for the muskrat by enhancing fur lipid content and potentially increasing the barrier against water penetration to and from the animal.
It is the purpose of this study to determine the effect of total lipid removal and Hardarian gland removal on: (1) water penetration into the fur and resultant heat loss while the animal is submerged in cold water, (2) thermal conductance of the fur in cold air, and (3) cutaneous evaporative water loss in a hot air environment. In addition, this study will investigate the effect of oiling the fur on the rate of cutaneous water loss.
MATERIAL AND METHODS
Muskrats were live trapped during June and July 1982 in southeastern Wyoming (42000'N 105020'W) at approximately 2,200-m elevation. Muskrats were housed either individually in 1 x 1-m metal cages, with ample fresh water for drinking and swimming, or in groups of three in 1 x 3m stainless steel cages with continuously circulating water for both drinking and swimming. All animals were maintained on a natural photoperiod and provided fresh vegetables and fruit ad lib. along with occasional fish and frogs. Only adult muskrats of similar body mass (909 g + 44 SD) were used in the following studies.
SURGICAL MANIPULATION
Hardarianectomy was performed on six muskrats under ketamine anesthesia (1.5 mg/kg) injected intramuscularly. A 0.5-cm incision was made in the nictitating membrane at the anterior corner of the eye with a no. 20 scalpel. The superior lobe of the gland was grasped with two serrated forceps and carefully pulled out through the incision, to maintain the gland intact during its removal. After the entire gland was exposed, its anteromedial attachment in the orbital cavity was cut. The procedure was then repeated on the other side. Sham operations were performed on seven muskrats by making a cut in the nictitating membrane and inserting the forceps to touch the Hardarian gland but not remove it.
AND SHAMPOOING ON HEAT LOSS
The body temperatures of animals in all three groups (control, Hardarianectomized, and shampooed) were monitored prior to, during, and after forced submergence in cold water.
Body temperatures of sham control animals were measured with a thermocouple inserted 4 cm into the rectum and were continuously recorded on a Sargent-Welch model SRG-2 recorder. Each animal was placed in a 17 x 17 x 30-cm wire cage for 10 min in order to obtain deep core temperature. The cage containing the muskrat was then lowered into a circulating water bath maintained at 4 C (+ 0.2) so that only the nose and mouth of the muskrat protruded from the water. Body temperature was monitored for the 10 min each muskrat was submerged and for an additional 10 min after emergence.
Six muskrats were lightly anesthetized at 0400 MST, shampooed twice with a mild baby shampoo, rinsed, blow dried, and allowed to recover 5-6 h prior to submergence as previously described. Six Hardarianectomized muskrats were also monitored during presubmergence, submergence, and postemergence in a manner similar to the other two groups and at approximately the same time of day. An analysis of variance followed by a Student-Neuman-Keuls test for interaction was performed on values representing the total temperature drop by muskrats caused by cold exposure (Sokal and Rohlf 1969). An F-statistic comparing the sum square error was calculated to determine whether regression lines representing the rate of body temperature change were different among the three groups (Netter and Wasserman 1974).
THERMAL CONDUCTANCE
Six control and six Hardarianectomized muskrats were placed into a 14 x 14 x 25-cm hardware cloth cage which slid into an airtight temperature control chamber. Ambient air temperature inside the chamber was recorded simultaneously with body temperature. Rate of air flowing into the chamber at 10 C (+0.2) was determined with a hot wire anemometer. The excur-rent air was channeled to a Beckman F-3 paramagnetic oxygen analyzer to determine oxygen content. Oxygen consumption rates for muskrats were determined from formula 10 of Depocas and Hart (1957). Thermal conductance was then approximated from the formula C -M/(T,, -Ta), where M = oxygen consumption, Tb = body temperature, and T,,ambient temperature. A pooled t-test was used to test for significant differences between the means of these two groups.
AND SHAMPOONG ON EVAPORATIVE WATER LOSS
Six individuals from each group were placed into a hardware cloth cage suspended over a tray containing mineral oil and were maintained at 36 C (+0.2) in a temperature control chamber. Air previously scrubbed of water was pumped into the chamber. The excurrent air was monitored for water content by an EG&G Dew Point Hygrometer and then for oxygen content as previously described. Both evaporative water loss and oxygen consumption were measured on muskrats in the chamber for 30 min. In order to establish cutaneous evaporative water loss, certain estimations had to be made. Because the percent oxygen extraction in mammals is roughly constant (inspired air is 21% 02 and expired air is 16% 0,), the minute volume of the muskrat was estimated at 20 times its oxygen consumption. The temperature of the expired-air was measured directly with a copper-constantan thermocouple and Thermometrics V-1B micro-voltmeter to be about 33 C. From the estimated volume of expired air, the amount of water lost was calculated, assuming that this air was saturated with moisture. Cutaneous evaporation was, therefore, obtained by subtracting the calculated respiratory evaporation from the total measured evaporation. An analysis of variance followed by a Student-Neuman-Keuls test for interaction was performed on evaporative water loss and oxygen consumption of these three groups. Five muskrats were individually placed into the temperature control chamber at 36 C for 30 min to determine their baseline water loss rates. Each animal was then removed from the chamber and allowed to rest for 4 h. At the end of this time, muskrats were firmly held while mineral oil was rubbed into their fur. After an additional 1 h of rest, muskrats were returned to the 36 C chamber to provide a second 30-min reading of evaporative water loss. A paired t-test was performed on these data to determine whether oiling significantly changed an individual's evaporative water loss.
RESULTS
The adult muskrat has large J-shaped Hardarian glands weighing an average of 0.79 g and measuring about 29 mm in length and 10 mm in width ( fig. 1).
The fur of muskrats, 2 wk after surgical removal of these glands, had a dull appearance and lacked the uniform fluffiness typical of the sham control muskrats. When dipped in and out of the water, fur of the shampooed and Hardarianectomized muskrats became wet down to the skin and compressed into a dense mat. On the other hand, fur of the control muskrats remained dry at the skin and appeared light and erect after emergence from the water (fig. 2).
The body temperature of sham control muskrats that were submerged in a 4 C water bath for 10 min dropped 4 C over 20 min, and they exhibited no ill effects as a result of the cold exposure. However, shampooed muskrats under similar test conditions had significantly higher rates of heat loss (P < .005) than controls; the average body temperature decrease after 20 min was 14.3 C. Hardarianectomized muskrats also had a significantly greater heat loss than the control group (P < .05), but body temperature decreased (7.4 C) less than in the shampooed group. Both Hardarianectomized and shampooed muskrats had a temporary impairment of locomotor ability after the 10-min forced submergence in cold water. The control and Hardarianectomized muskrats did not demonstrate a decline in body temperature until after 6 min of submergence, while the shampooed group became hypothermic only after 4 min in cold water. The regression lines representing the body tempera- ture drop during the interval between 6 and 20 min postsubmergence were significantly different for Hardarianectomized (P < .05) and shampooed (P < .005) groups compared with the control group ( fig. 3). The slope of the body temperature curve of the Hardarianectomized group, however, was not as great (P < . 10) as that of the shampooed group ( fig. 3). Oxygen consumption of control (0.862 cm3/g-h) and Hardarianectomized (0.851 cm3/g-h) muskrats was not significantly different at an air temperature of 6 C. In addition, thermal conductances of control (0.034 cm,.g '-h ' C ') and Hardarianectomized (0.035 cm, .g--'.h ' C ') muskrats were also not significantly different at this ambient temperature.
Shampooing significantly increased the extent of evaporative water loss by muskrats in a hot environment (P < .05). Hardarianectomized muskrats had evaporative water loss rates which were also greater (P < .10) than sham control muskrats ( fig. 4). In addition, when muskrats were covered with mineral oil, their evaporative water loss rates in a hot environment were decreased (P < .10) (fig. 5). DISCUSSION However, in an attempt to generalize this function to other rodents, they found that lipid contents of Hardarianectomized Syrian golden hamsters, Sprague Dawley rats, and DBA/2J mice were not significantly different from those of sham-operated groups, suggesting the lack of a thermoregulatory function in these species. The present study suggests that the Hardarian gland of the semiaquatic species, Ondotra zibethicus, does serve a thermoregulatory function for retarding water penetration into and away from the fur. Johansen (1962) demonstrated that the muskrat's nonwettable fur retains an insulating layer of air at the body surface which allows the muskrat to remain warm when submerged in near-freezing water. Hart (1962) and Sherer and Wunder (1979), however, presented data suggesting that muskrats become hypothermic from 3 to 5 C when exposed to cold water for prolonged periods. These observations were supported by field observations of a 2-3 C drop in body temperature of telemetered muskrats during 40-50-min excursions from their lodge during the winter (MacArthur 1979). In order to minimize the extent of hypothermia during a swimming excursion, the muskrat has been observed to increase its body temperature during the 60 min prior to leaving the lodge (MacArthur 1979). Not only does this result in hyperthermia prior to submergence, it may also be accompanied by a period of grooming and waterproofing of the fur.
Several functions have been hypothesized for the Hardarian gland which in-
In the gerbil, an increased body temperature provokes autogrooming and Hardarian gland secretion release from the external nares (Thiessen and Kittrel 1980). This secretion is mixed with saliva and is spread over the face and body. Similar behavior was observed on muskrats in the communal holding cages. Prior to leaving the feeding platform and entering the water, individual animals would spend 30-40 min first rubbing their face, nose, and mouth with their paws, then rubbing their paws along the back of their head as well as the ventral and lateral sides of their body. Communal grooming commenced after the first muskrat began its auto-grooming, perhaps in response to pheromones released from the Hardarian gland (Thiessen 1977). This social activity was simple and consisted primarily of one muskrat rubbing its face and head along the back of conspecifics. Since muskrats generally live with several other individuals in a lodge during the winter (Schwartz and Schwartz 1959), communal grooming during the period of heat generation prior to entering the cold water could result in the full body spreading of lipids from Hardarian gland secretions.
Total removal of surface skin and fur lipids by shampooing dramatically reduced the muskrat's insulative ability and resulted in a significantly increased loss of body heat when submerged in cold water. Hardarian gland removal also resulted in a considerably larger reduction in body temperature than controls but not as great as total lipid removal. These results support the hypothesis that Hardarian gland secretions augment the thermoregulatory function of sebaceous gland lipid production in this semiaquatic mammal. It is possible that these lipids function by maintaining an air boundary between the skin and water. Support for this hypothesis is offered by a study in which all the air in the fur of muskrats was removed by rubbing the animal with a solution of the highly surface-active material "Aerosol OT" (diactyl-sodium sulfasiccinate). This treatment not only decreased the volume of muskrats' fur by about 21% but also appreciably reduced its heat-retaining capacity in cold water (Johansen 1962).
Removal of the Hardarian gland portion of fur lipids and consequent heat loss imply an associated increase in conductance of the fur. However, the calculated values for conductance were not significantly different between the Hardarianectomized and control groups. Indeed, both groups had conductance values very close to the predicted value based upon allometric equations (Herreid and Kessel 1967). It is possible that a difference in conductance of the fur between Hardarianectomized and control muskrats may be evident when the animals are submerged underwater as a result of lipid trapped air bubbles near the skin which increase the insulative property of the fur. The influence of fur lipids on conductance may not be evident when tested in cool air where the fur is piloerected rather than compressed. The thermal conductance of Hardarianectomized muskrats will soon be determined in water to test this hypothesis.
Mammalian skin is not completely impermeable to water and therefore accounts for a considerable proportion of total evaporative water loss. For example, Chew (1955) found that the deer mouse Peromyscus maniculatus sonoriensis had a cutaneous evaporative water loss that accounted for between 46% and 63% of the total evaporative water loss at 2 7 C. From calculations described in Material and methods, it was estimated that cutaneous water loss accounted for about 41% of the total evaporative water loss in muskrats, which was increased to 66% by shampooing and 52% by Hardarianectomy.
It is widely believed that the physical characteristics of the stratum corneum determine the rate of insensible water loss across the skin (Blank 1952;Mali 1956). However, as pointed out by Edwards and Haines (1978), an additional means by which cutaneous evaporative water loss could be increased would be a reduction of the effective boundary layer of air within and above the fur. These authors speculate that this would result in the steepening of the vapor pressure gradient between the evaporating surface and the ambient air. The importance of lipids in influencing the boundary layer and consequently retarding cutaneous water loss has not been adequately explored. Nicolaides suggested that the lipids that are secreted by the sebaceous glands may provide waxes, fatty acids, and alcohols that, when spread over the water at the skin surface, could form monolayers that might retard evaporation.
In addition, Quay (1965) noted that species of Dipodomys and some Perognathus have antero-posterior flattened hair shafts that form overlapping plates of hair fixed to one another by a lipid film. It is speculated that this may contribute to the prevention of cutaneous evaporative water loss. If, indeed, lipids retard water loss from the fur, one would expect shampooed and, to a lesser extent, Hardarianectomized muskrats to have an increased evaporative water loss over controls. Lipid removal from the fur of muskrats by shampooing did appear to increase evaporative water loss per unit of oxygen consumed, suggesting an increase in cutaneous evaporation. Hardarian gland removal also resulted in an increased water loss per unit of oxygen consumption. In addition, covering the fur with oil diminished the evaporative water loss. In this latter case, however, it is possible that the oily fur decreased the palatability of licking and perhaps saliva spreading as a thermoregulatory behavior. If so, this would account for a portion of the reduced evaporative water loss.
Even though the muskrat is a semiaquatic mammal with little apparent problem with water deprivation, it does encounter periods of heat stress during which it remains in the lodge isolated from its aquatic environment (MacArthur and Aleksiuk 1979). However, the importance of fur lipids for this semiaquatic animal may be primarily for retarding water penetration and heat loss while in cold water. Impaired water loss under hot, dry conditions may be simply a secondary effect of the fur lipids forming an air layer next to the skin. This latter effect may be of greater adaptive benefit to those species living in a more xeric environment (Quay 1965; Thiessen and Kittrell 1980). | v3-fos |
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