Datasets:

claran

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modular-s2orc

like 1

2018-04-03T02:48:24.968Z

1975-03-01T00:00:00.000Z

35169863

s2

Use of pH gradient plates for increasing the acid tolerance of salmonellae. Several strains of salmonellae survived higher concentrations of lactic acid after streaking on the surface of pH gradient plates. Most strains increased their acid tolerance by about 0.8 to 1.0 pH unit (9-to 10-fold), with Salmonella madelia showing the greatest differential, pH 5.2 in the wild strain and pH 4.2 after conditioning. The increased acid resistance was quickly lost after transferring to normal tryptic soy agar. Tests for survival in a liquid medium at pH values lower than those giving visible growth indicated that these pH values were bactericidal rather than bacteriostatic for both the wild and acid-conditioned strains. Several strains of salmonellae survived higher concentrations of lactic acid after streaking on the surface of pH gradient plates. Most strains increased their acid tolerance by about 0.8 to 1.0 pH unit (9to 10-fold), with Salmonella madelia showing the greatest differential, pH 5.2 in the wild strain and pH 4.2 after conditioning. The increased acid resistance was quickly lost after transferring to normal tryptic soy agar. Tests for survival in a liquid medium at pH values lower than those giving visible growth indicated that these pH values were bactericidal rather than bacteriostatic for both the wild and acid-conditioned strains. The minimum pH for growth of salmonellae has been studied by a number of workers. Stokes and Bayne (8) did not observe colony formation in trypticase soy agar buffered with phosphate-phosphoric acid at pH 5.0 with any of several dozen strains and serotypes of salmonellae. Even at pH 6.0 Salmonella gallinarum and S. pullorum grew very poorly. The optimum for all strains was pH 7.0, with many strains growing equally well at pH 8.0. On the other hand, in liquid egg white, Banwart and Ayres (1) found S. pullorum and several other strains to grow at a more rapid rate at pH 6.0 than at 7.0 and faster at 7.0 than at 8.0. Reduction in numbers occurred at pH values of 9.0 or above. Prost and Reimann (6) indicated that pH values above 9.0 and below 4.5 had a killing effect on salmonellae with destruction in a matter of minutes in lemon or lime juice (pH 2.3 to 2.5), whereas tomato juice (pH 4.3 to 4.5) was less bactericidal, with survivors found after 10 to 30 days. Chung and Goepfert (2) showed that the type of acid was important in determining the minimum pH for growth. Hydrochloric or citric acids required a pH of 4.05 to inhibit growth of S. anatum, S. tennessee, and S. senftenberg; lactic acid inhibited at pH 4.40, whereas acetic and propionic acids inhibited at pH 5.40 and 5.50, respectively. Levine and Fellers (5) also showed that the type of acid was important for determining minimum pH for growth, acetic acid being more toxic than lactic or hydrochloric acids. The bactericidal action of volatile fatty acids on S. typhimurium was shown to increase when the pH was lowered from 6.0 to 5.0 (3); there also appeared to be a decrease in bactericidal activity with increasing chain length of the acid. Many foods are naturally or artificially protected against microbial contamination. Smith and Palumbo (7) recently reported on the microbiology of Lebanon bologna manufacture. They found a considerable population of lactic acid bacteria during the fermentation, with a final pH of from 4.6 to 5.6, due most likely to lactic acid. Lactic acid was demonstrated to be the chief acid produced in Swedish sausage (4). Salmonellae are found in meat products and could be present in the raw materials for sausage manufacture. The study reported here was undertaken to determine if salmonellae could survive the acidity of fermented sausage and become adapted to growing at increasing lactic acid concentrations. MATERIALS AND METHODS Gradient plates. The two-layer gradient agar technique of Szybalski and Bryson (9) was used with square plastic petri dishes having bottom dish dimensions of 90 mm with a depth of 14 mm. The bottom layer was 25 ml of tryptic soy agar (TSA; Difco) poured while the lugs of one end of the dish rested on plastic tubing with a diameter of 6 mm. After solidification, the plates were placed on a level surface and a second layer of 27 ml of TSA containing 0.34 or 0.68% lactic acid was poured. The plates were then placed in a refrigerator for 20 to 24 h. Cultures and media. Salmonella serotypes were received from B. Blackburn of the National Animal Disease Laboratories, U.S. Department of Agriculture, Ames, Iowa, and were maintained on TSA slants. Cultures were streaked on the plates by means of a needle with a semicircular curve of about 2 mm diameter, using several back-and-forth strokes. Incu-bation was at 35 C for 20 to 24 h. pH measurements. The pH of the surface of the gradient plates was determined by placing Whatman no. 1 filter paper strips (10 by 35 mm) on appropriate portions of the equilibrated plates for 20 min. The strips were then placed in a 10-ml beaker, and 1.5 ml of distilled water was added. Minimum pH for growth. The lowest pH for growth in tryptic soy broth was determined by adjusting the pH with 20% lactic acid. The pH values did not change appreciably after autoclaving. The inoculum was 1 drop per 4 ml of a small amount of growth from the gradient plates or slants emulsified in 4 ml of H20. RESULTS The growth of the salmonellae on pH gradient plates is shown in Table 1. The cultures used as inocula were from 12-day-old TSA slants kept at room temperature. The pH values of 10-mm increments, as measured by the filter paper method, were 4.60, 4.55, 4.65, 4.75, 5.05, 5.35, 5.90, 6.40, and 6.75. The most acid-resistant culture was S. grumpensis, growing at a distance of 30 mm from the most acid end. The next most resistant was S. dublin (34 mm) followed by S. saint-paul and S. tournai (39 mm). These were in the 10-mm area, showing a pH of 4.75. All the other strains grew in the area with a pH of 5.05. Eleven of the salmonellae were further studied. Fresh slants were prepared from the original cultures used for the experiment of Table 1 and these (wild) cultures were compared with inocula taken directly off the gradient plates at the point of growth nearest the acid end (acid conditioned) ( Table 2). There was an appreciable increase in acid resistance with all cultures after growing on the gradient plates. The greatest differences of growth termini, indicating the greatest degree of acid conditioning, were with S. madelia (32 mm), S. braenderup (31 mm), S. cerro (29 mm), S. meleagridis (28 mm), and S. havana (25 mm). The least acid conditioning noted was with S. montevideo (7 mm). The change in growth-limiting pH of S. madelia appeared to be from 5.20 (50 to 60 mm) to 4.60 (20 to 30 mm), using the filter paper strip method of measuring surface pH values. A more accurate determination of the minimum pH values permitting initiation of growth of several of these salmonellae and S. typhimurium (similarly acid conditioned) was made in tryptic soy broth adjusted with 20% lactic acid to pH values of 4.2 to 5.3, in increments of 0.1 pH units. The pH values remained constant when measured after autoclaving and cooling (Table 3). S. madelia, which showed the great-TABLE 1. Growth of salmonellae and other Enterobacteriaceae on pH gradient platesa est increase in acid resistance in agar, was also the most acid resistant in broth, growing at pH 4.2; however, 2 days were required for growth to be evident. S. montevideo, which apparently showed the least acid conditioning in agar, showed an appreciable increase in acid tolerance in broth, with growth at pH 4.4 for the conditioned strain and 5.3 for the wild strain. Most of the salmonellae tested showed increases in acid tolerance of about 0.8 pH units. S. dublin (wild type) failed to grow at pH 5.3, the highest tested, although in another test it grew at 5.2 (wild) and 4.6 (acid conditioned). The conditioned strains quickly lost their acid VOL. 29, 1975 pH GRADIENT PLATES FOR SALMONELLAE ACID TOLERANCE resistance. After transferring two times on TSA slants, the conditioned strains were no more resistant than the wild. Tests for survival of the original inoculum in a Surface pH values (by the filter paper strip method) were 4.55 (0 to 10 mm from most acid end), 4.55 (10 to 20 mm), 4.60 (20 to 30 mm), 4.65 (30 to 40 mm), 4.95 (40 to 50 mm), 5.20 (50 to 60 mm), 5.80 (60 to 70 mm), 6.25 (70 to 80 mm), and 6.70 (80 to 90 mm). the tryptic soy broth were performed after 1 day of incubation on the two tubes, with pH values immediately below those showing visible turbidity. For these tests, 0.25 ml of the tryptic soy broth, previously inoculated with either wild or acid-conditioned strains, was placed into 4.5 ml of new broth and the tubes were reincubated for 3 days. The pH values of the new tubes, without further adjustment, were 5.40 for the original pH 4.20, 5.65 for the 4.40, 5.85 for the 4.60, and 6.05 for the 4.80. These pH values were high enough to permit growth of any of the salmonellae. All tests were negative, indicating that the effect of these pH values was bactericidal rather than bacteriostatic. DISCUSSION The results reported here indicate that salmonellae can become adapted to growing under more acid conditions, although this does not appear to be a genetic change, the characteristic being lost very soon after transferring to a neutral agar medium. Fermented foods such as Lebanon bologna ordinarily require several days for the pH to reach low enough levels to inhibit salmonellae. During this time salmonellae, if present, could conceivably become adapted to the acidity and persist in the finished product. (2) could not demonstrate any degree of increased acid tolerance in liquid media. This may indicate that the acid conditioning, as reported here, takes place only during aerobic growth as on the surface of pH gradient plates. This may be a reflection of the difference in metabolism of these cultures under aerobic conditions (tricarboxylic acid cycle) and in liquid, nonaerated systems (Meyerhof-Embden-Parnas system). LITERATURE CITED

v3-fos

2018-04-03T03:00:11.230Z

1975-12-01T00:00:00.000Z

17392313

s2

Heavy metal exposure from foods. The Food and Drug Administration has a continuing program of monitoring foods for their content of lead, cadmium, mercury, zinc, arsenic, and selenium to determine trends of increasing or decreasing levels. The monitoring protocol is that of the Total Diet Study, in which "market baskets" of typical foods and beverages consumed by 15- to 20-year-old American males are collected in various geographical locations at regular intervals during the year, divided into food classes, composited, and analyzed. Cadmium has the most widespread distribution of the six heavy metals and mercury the most limited. The analytical values for lead may be underestimated because of limitations of the methodology; these do not apply to the other five elements. A tabulation by year shows that the levels of these elements in foods do not vary significantly from one year to the next. Average intakes of lead, cadmium, and mercury are below the WHO/FAO tolerable intakes for adults; such tolerable intakes have not been established for arsenic and selenium. Increases in concentrations of these elements in foods would be considered undesirable, however. Determination of the heavy metal content of foods has been carried out by various laboratories of the Food and Drug Administration for a number of years. The data discussed were obtained from the Total Diet Survey program, or as it is better known, the Market Basket Survey. The program was originally designed to monitor the levels of various pesticides in foods and has been ongoing since 1965. In the last few years it has been expanded to include analyses for six heavy metals: lead, cadmium, mercury, zinc, arsenic, and selenium.t Lead, zinc, and selenium have been determined only since 1972, whereas data from earlier years are also available for the other three metals. A major purpose of this program has been to determine the levels of heavy metals in foods, or more specifically, to determine trends of increasing or decreasing levels of metals in various foods or food classes. The foods included in the Total Diet Study were determined by using 1965 diet survey data of the United States Department of Agriculture which indicate the food consumed by 15-to 20-yr-old *Food and Drug Administration, 200 C St. S.W., Washington, D.C. 20204. tIn this context, the term "heavy metals" is used in a colloquial sense and includes the toxic nonmetals arsenic and selenium. American males (1). Food consumption of this group was selected for study because the members of the group consume the largest number of calories of any age-sex group in the population, and the largest consumers of food generally have the highest exposure to food contaminants. However, it is recognized that there are possible exceptions to this generality if a food contaminant were present in only a few types of foods. By using this data base it has been found that over a 14-day period the average diet of young The foods are not analyzed individually but as part of a composite food class, as shown in Table 1. The actual composition of the garden fruits class is shown in Table 2. In all cases portions of the individual samples are retained. In actual practice, more than enough of each food item is collected for the requirements of the 14-day composite. If the level of a contaminant in a composite is unusually high the individual components of the composite will be analyzed to determine the source. Although it may be desirable to analyze all the foods individually, the costs would be very greatly increased. Qualitative evaluation of the data reveals that the pattern of distribution in foods differs substantially among the various metals. Cadmium has the most widespread distribution (Table 3). Specific sources of high concentrations of cadmium are shellfish and some vegetables such as spinach; however, when both food consumption and concentration of the metal are considered, a number of composites provide substantial portions of the cadmium intake. Among these food classes, cereals and grains provided the greatest percentage of total intake. Lead is also found in a large number of foods. However, fruits and vegetables are the most important sources. While they constitute only 23% of the total diet by weight, they account for 78% of the total lead intake (Table 3). Zinc was present in all the food classes examined. However, dairy products, meat-fish-poultry, and grains and cereals provide 77% of the zinc (Table 3), even though they account for only about half the diet by weight. Arsenic, which is reported as As20s, has a narrower distribution. In recent years, arsenic occurred most frequently in the meat-fish-poultry composites. Shellfish have been reported to contain generally higher levels, but they are not significant components of the average diet. Patterns of food consumption are such that meat-fish-poultry, dairy products, and grains and cereals are important dietary sources. Of the total arsenic in the diets studied in this program, 92% came from these three food classes (Table 4). With regard to selenium, essentially all selenium in the diet is attributable to the meat-fish-poultry group and the grain-cereal group. These two food classes provide over 99% of the selenium intake (Table 4). Mercury has the most fimited distribution of the metals studied. Virtually all dietary intake of mercury was found in the meat-fish-poultry class and in fish within that class where followup studies were performed (Table 4). This observation is further supported by Simpson et al. (2) in a report -4 Environmental. Health-Perspeeives. of a study on fish, other staple foods, and total diet composites. The report indicated that, except for fish, mercury levels in foods are generally so low as to be undetected by the usual flameless atomic absorption techniques; neutron activation analysis was necessary to detect any mercury present. The Market Basket surveys as currently performed give a good indication of general trends of distribution of the metals in foods. However, in some cases there may be greater intakes from some food classes than currently apparent because of the detection limits of the chemical methods employed. This problem becomes particularly apparent in the food classes that constitute a large percent of the total diet but may contain low concentrations of some of the metals: dairy products, meat-fish-poultry and beverages. Detailed data on the findings of heavy metals in various food classes are presented in Tables 5 and 6. In evaluating these data it has been recognized that the use of food composites creates difficulties in determining actual total quantities of metals present in foods. For example, the concentration of lead in many-ofthe composites is very-close to the detection limit of the method. Thus, analyses of many of the composites will yield the analytical report of trace or zero. How these traces or zeros are handled in calculations will substantially affect the magnitude of the value reported for total intake. This problem has been discussed in depth in a previous publication (3). A wide range of total lead intakes can be calculated based on various analytical assumptions (Table 7). Among the metals included in the Total Diet Study, this problem is most severe for lead. Cadmium, arsenic, and selenium present far fewer difficulties in this regard because of lower levels of detectability in relation to the levels that occur. The chemical methodology for zinc and mercury is sufficiently reliable at the levels determined that the estimated intakes for these metals are considered to be very accurate approximations of the actual values. Table 8 presents the actual values found in the 1973 Total Diet Survey and the effects of various assumptions on them. Although total exposure to lead may be underestimated, this is not as significant a problem for most of the metals determined in this study. Table 9 shows yearly values in the metal content of foods, as obtained from the Total Diet Surveys. Although the total dietary lead content is undoubtedly underestimated, the chemical methodology and handling of traces and zeros in calculations has remained constant, and the same is true for other metals. Thus comparisons of yearly changes are meaningful. These data, shown in Ta- In evaluating the toxicological significance of the various metals, the estimated intake from the Total Diet Study has been compared with the WHO/FAO provisional recommendations of tolerable intakes (4) for adults (Table 10). Concerning lead, two points are apparent. Because of underestimation of actual lead intake the per cent of the recommended level is far higher than the 14% which is calculated. However, on the basis of data presented in an earlier paper from our group it is known that the average lead intake is well below For cadmium and mercury the percent of tolerable intake is considered a good estimate. Cadmium is close enough to the tolerable intake so that further increases in the cadmium content of foods should be avoided. Thus far WHO/FAO tolerable intakes for arsenic and selenium have not been established. Selenium and zinc are both essential nutrients. The National Research Council of the USA recommends an intake of 15 mg zinc/day for adults (5). The teenage boy who consumes a large amount of food can easily meet this requirement. However, the recommended dietary allowances for pregnant and lactating women are 20 and 25 mg zinc per day, respectively. With lower calorie intakes and a higher requirement, foods must be carefully selected in order to avoid the problem of an undesirably low zinc intake. National Research Council recommendations on selenium intake have not been set (5). In summary, for the metals included in the Total Diet Study it is generally considered that while the food supply contains less than tolerable intakes of those toxic heavy metals for which recommendations exist, any increases in these trace metal concentrations of food are undesirable. ;68 --

v3-fos

2020-12-10T09:04:22.943Z

1975-05-01T00:00:00.000Z

237230391

s2

Egg Production, Shell Thickness, and Other Physiological Parameters of Laying Hens Affected by T-2 Toxin T-2 toxin has been reported to cause severe oral lesions and neural disturbances in young broiler chickens. T-2 toxin, when added at a level of 20 μg per g of feed, caused oral lesions but no abnormal neural disturbances in young broiler chickens. T-2 toxin, when added at a level of 20 μg per g of feed, caused oral lesions but no abnormal neural symptoms in laying hens. T-2 toxin had no effect on either hemoglobin, hematocrit values, erythrocyte count, plasma glucose, prothrombin times, or the sizes of the liver, spleen, pancreas, and heart. Lipid content of the liver was not altered. Feed consumption, however, was reduced, as were the total plasma protein and lipid concentrations and the total leukocyte count. Most important economically was the lowered egg production and a thinner egg shell. The timing and severity of the symptoms suggest that T-2 toxin causes primary oral lesions that reduce feed consumption with a consequent reduction in serum proteins and lipids, which culminate in decreased egg production. The leucopenia and thinner egg shell may be independent systemic effects of T-2 toxin in laying hens. T-2 toxin has been reported to cause severe oral lesions and neural disturbances in young broiler chickens. T-2 toxin, when added at a level of 20 jg per g of feed, caused oral lesions but no abnormal neural disturbances in young broiler chickens. T-2 toxin, when added at a level of 20 Aig per g of feed, caused oral lesions but no abnormal neural symptoms in laying hens. T-2 toxin had no effect on either hemoglobin, hematocrit values, erythrocyte count, plasma glucose, prothrombin times, or the sizes of the liver, spleen, pancreas, and heart. Lipid content of the liver was not altered. Feed consumption, however, was reduced, as were the total plasma protein and lipid concentrations and the total leukocyte count. Most important economically was the lowered egg production and a thinner egg shell. The timing and severity of the symptoms suggest that T-2 toxin causes primary oral lesions that reduce feed consumption with a consequent reduction in serum proteins and lipids, which culminate in decreased egg production. The leucopenia and thinner egg shell may be independent systemic effects of T-2 toxin in laying hens. T-2 toxin [4,15-diacetoxy-8-(3-methylbutyryloxy) -12,13 -epoxy -A9 -tricothecene-3-ol] is a mycotoxin originally isolated from a strain of Fusarium tricinctum (1). Both the toxin and the fungus have been implicated in moldy corn toxicosis of farm animals (7,11,13,22). The toxin induces severe inflammatory reactions in humans, rodents, and trout (17). In young broiler chickens the dose-related oral lesions are similar to the third or necrotic angina stage of alimentary toxic aleukia (ATA), a toxicosis of humans caused by grains infested with Fusarium (30). These lesions appear to be characteristic enough of the toxicosis in chickens to aid in the diagnosis of field cases (29). T-2 toxin consumption also provokes neural disturbances in young chickens suggestive of the profound neural abnormalities underlying ATA (28). The effect of T-2 toxin in adult chickens does not appear to have been studied previously. Our interest in the effect of T-2 toxin on laying hens was stimulated by a field case referred to us which was suggestive of T-2 toxicosis. In this paper we give a general description of T-2 toxicosis in laying hens. by the method of Burmeister (6) to give a crystalline product melting at 148 to 150 C. Animal husbandry. Mature laying hens (Dekalb 131), 30 weeks old, were assigned randomly to individual laying cages. They were housed in an environmentally controlled room with a temperature of 25 C and relative humidity of 20%. The birds were exposed to 15 h of light per day with a 15-min increase weekly. Feed consisted of a commercial layer ration free of all medications. T-2 toxicosis was induced by incorporating known amounts of crystalline T-2 toxin dissolved in 50% (vol/vol) aqueous ethanol into small portions of the diet which were dried before mixing into the remainder of the feed. Individual egg production was recorded daily, and individual body weights and feed consumption were recorded weekly. Experimental design. Twenty hens were selected on the basis of uniform egg production. Ten birds received the control diet and ten received the diet containing 20 Ag of T-2 toxin per g of feed for the same period. The experimental design was completely randomized. The data from the experimental and control birds were compared statistically by Student's t test (5). Blood and tissue analyses. A 5-ml blood sample was taken from the brachial vein of all birds at weekly intervals. The 5-ml sample was immediately mixed with 0.6 ml of 0.18 M sodium citrate and kept on ice until the analyses were performed. Plasma protein was measured by the biuret method (27), plasma glucose by the method of Dubowski (8), plasma lipid by the method of Friedman (10), and the prothrombin times were obtained with a Fibrometer (Baltimore Biological Laboratories, Cockeysville, Md.) using ho-mologous thromboplastin. At the end of the experiment hematocrit, hemoglobin, and erythrocyte and leukocyte counts were made. Hemoglobin was estimated according to Sunderman et al. (24), and the erythrocyte and leukocyte numbers were counted by standard hemocytometer procedures after staining with Natt-Herrick (21) stain modified to have brilliant cresol blue and methyl red instead of methyl violet (F. Craig, personal communication). Liver lipid was determined as outlined by Smith and Hamilton (23). Determination of egg parameters. Eggs were collected eight times daily and weighed immediately to negative water evaporation. Egg quality was evaluated by comparing the egg weight to the height of the thick white on a Haugh unit conversion chart (12). Egg shell thickness was measured at three points equidistant along the equator of the shell after drying the shells at 100 C for 2 h. Neurological examination. At weekly intervals all birds were subjected to a series of manipulations to observe whether the dietary T-2 toxin altered normal neural responses. Corneal reflexes were checked, the birds were manually placed on their backs to test their righting reflex, and the birds were manipulated in an effort to provoke the hysteroid seizures seen during T-2 toxicosis in young birds (28). Microbiological examination of the liver. Immediately after the birds were killed, the visceral organs were exposed. An exposed area of the liver was seared with a hot spatula and a sterile loop was inserted through the seared area. The material thus obtained was streaked on brain heart infusion agar plates (Difco) and incubated at 37 C for 7 days. The plates were examined at daily intervals for bacterial and fungal growth. Examination of the internal organs. At the end of the experiment the birds were killed by cervical dislocation. The internal organs were excised, trimmed of extraneous tissue, blotted, and weighed. Liver was frozen immediately for subsequent lipid analysis. RESULTS Oral lesions characteristic of T-2 toxicosis (30) appeared during the first week of toxin administration. The lesions appeared first on the sublingual and palatine areas and then on the tongue and in the corners of the mouth. The raised lesions were yellowish-white to gray in color, and they began as small necrotic foci that progressed to larger lesions about 1 cm in diameter. Although lesions appeared at three main areas of the mouth, the lesions in the corners of the mouth appeared more severe and occasionally impaired normal mouth movement. Among the first signs of T-2 toxicosis in laying hens was the drop in feed consumption; within 1 week consumption of the toxic diet was 25% less than the control (Fig. 1). Feed intake stabilized by the second and third weeks (Fig. 1). There was a significant (P < 0.05) weight loss after 1 week in birds receiving 20 Asg of T-2 toxin per g of diet (Table 1). Moveover, by the third week the group fed T-2 toxin lost approximately 10% of their initial body weight, whereas the control group maintained their body weight. Concomitant with the appearance of oral lesions and reduction in feed consumption was a significant decline in plasma protein (Fig. 2). After the first week of the experiment the plasma protein concentration was 15% lower and by the third week was 20% lower than plasma protein concentrations of control birds. Plasma lipid was decreased about 60% by the end of the third week ( ,'These values differ significantly (P < 0.05) from the control value for the particular column. (mg/100 ml) a Each dose was fed to 10 birds for 3 weeks, and each experimental value is the mean ± standard error of the mean. bThese values differ significantly (P < 0.05) from their corresponding control value. ters to T-2 toxin, i.e., hematocrit values, hemoglobin concentration, and erythrocyte numbers, were not significantly affected ( Table 2). The leukocyte number was significantly (P < 0.05) lowered by about 30% in birds receiving T-2 toxin. Prothrombin times were not altered significantly. These data suggest that T-2 toxin caused a leucopenia without anemia or impaired hemostasis in the laying hens. No significant change in any of the internal organs in response to dietary T-2 toxin was noted (Table 3). There was no effect on the lipid content of the liver, and microbiological examination of this organ revealed no bacterial or fungal contamination. Neurological examination of the hens revealed no neural disturbances like those seen in young broiler chickens. During the first week, no significant change in egg production was observed between laying hens receiving the dietary T-2 toxin and the control diet (Fig. 3). During the next 2 weeks egg production declined by about 20% in birds fed toxic feed. The egg weight and Haugh units were not significantly altered by the T-2 toxincontaminated feed, but the shell thickness was a Each dose was fed to 10 birds for 3 weeks, and each experimental value is the mean ± the standard error of the mean. Weights are expressed as grams/100 g of body weight. 643 VOL. 29,1975 significantly (P < 0.05) decreased (Table 4). The shells were so fragile that the pressure from a grease pencil used to mark the eggs for identification often caused cracks to occur. DISCUSSION The primary effect of T-2 toxicosis in laying hens appears to be an inflammatory reaction in the mouths of affected birds. This inflammatory reaction was first noted about 4 to 5 days after initiation of treatment. Lesions first manifested themselves as small gray to cream-colored areas on the upper beak and progressed to the distal portions of the tongue and corners of the mouth. The lesions appeared quite similar to those described in broiler chickens by Wyatt et al. (30) except that the lesions on the inside of the beaks were oval shaped and did not assume the V-shaped pattern seen in broilers. The oral lesions appear to reduce feed intake with a subsequent weight loss. Proteinemia and lipemia observed during T-2 toxicosis are probably a reflection of the lowered feed consumption without a concomitant immediate decrease in egg production, which draws down the hen's reserves of proteins and lipids. The thin-shelled eggs could result from lower consumption of calcium and phosphorous, although there might be an independent effect on egg shells. Certainly a mechanism independent of nutritional inadequacy would appear to be needed for the leucopenia observed. Lack of any significant effect on internal organs also supports the hypothesis that the primary effect of T-2 toxin is to cause an oral inflammation and not to act specifically on any target organ. Decrease in egg production and the occurrence of thin-shelled eggs suggest that T-2 toxin poses a threat to poultry health and the industry. Wyatt et al. (28) described outbreaks of disease in avians that were characterized by irritation and necrosis of the mouth, head, and feet. Subsequently we have encountered several apparent outbreaks of T-2 toxicosis in laying hens. One such outbreak involved 20,000 commercial layers. This disease was characterized by the sudden appearance of oral lesions like those observed in this study in a significant number of the birds, a 25% drop in egg production, and a high percentage of thin-shelled eggs that would not reach their normal markets. Unfortunately the feed had been consumed when the case was referred to us; hence no chemical analysis of the feed was possible. The birds returned to normal once a new supply of feed was obtained. These findings of decreased egg production and egg shell thickness may have considerable ecological importance if they can be extrapolated to wild birds. Much public and scientific concern has resulted from data linking certain commerically important pesticides to the production of thin-shelled eggs in some desirable avian species that are approaching extinction (4,15,16). Certainly grains and seeds become infected with molds under natural conditions, and T-2 toxin is one of many toxins likely produced under such conditions (3). It would be surprising if mycotoxicoses did not occur in wild birds, and these diseases may account for otherwise inexplicable variations in wild bird populations. A study of wildlife for signs of mycotoxicoses appears to be warranted. ATA was a major health problem in Russia for almost two decades (18). The toxic principle(s) responsible for the disease apparently was not isolated at the time of the disease outbreaks (3,19). More recent chemical and toxicological studies (2,20,30) have suggested that T-2 toxin may cause ATA. Similarities between ATA and T-2 toxicosis include dermal toxicity (1,19), inflammation about the nose and mouth (17), oral lesions (30), intradermal hemorrhages (17), and neural disturbances (9,18,28). The main piece of clinical evidence missing from the correlation of T-2 toxicosis with ATA is the devastation of the bone marrow and its hematological sequelae characteristic of ATA. The present finding of leucopenia during T-2 toxicosis of laying hens supplies part of the evidence needed to link the toxicosis to the damaging of bone marrow. Furthermore, Ueno et al. (25) reported cellular degeneration and karyorrhexis of the actively dividing cells of the bone marrow of mice receiving fatal dosages of culture filtrates of Fusarium strains known to produce T-2 toxin (26). It must be mentioned, however, that Kosuri et al. (14) observed leucocytosis in rats given rapidly lethal doses of T-2 toxin, so there may be a time factor involved in the effect on bone marrow.

v3-fos

2020-12-10T09:04:17.656Z

1975-12-01T00:00:00.000Z

237235394

s2

Accelerated Fermentation of Brewer's Wort by Saccharomyces carlsbergensis A rapid procedure for wort fermentation with Saccharomyces carlsbergensis at 12 C is described. Fermentation time was reduced from 7 to 4 days with normal inoculum by shaking. Increasing the inoculation to 5 to 10 times normal and shaking resulted in complete fermentation in 3 days. Maximum yeast population was reached rapidly with the large inocula, but fermentation proceeded at approximately the same rate when inoculations in excess of four times the normal were used. Similar results were obtained with both small-scale (100 ml) and microbrew (2.4 liters) fermentations. Many papers have been published concerning the reduction of the fermentation time and the attainment of maximum fermentation rate by an increase in inoculum and/or in temperature of incubation (1,7,9,11,16). Griffin (9) and Hudson (11) observed that the level of Saccharomyces cerevisiae in suspension was disproportionate to the amount of inoculum and that it was difficult to maintain higher yeast concentrations in the head. Others overcame this difficulty through the use of stirring (3,10). The yeast in suspension undergoes a different metabolic pattern than the sedimented yeast, probably because the metabolism of the suspended yeast is not diffusion limited (13). Analyses of the beer produced by accelerated fermentation indicated increases in the amount of higher alcohols (8,14) and the quantity of diacetyl (4). Little information has been published on the effects of changes in the amount of inoculum and stirring upon yeast metabolism in brewery wort with Saccharomyces carlsbergensis at 12 C. In the present study, a rapid, small-scale fermentation for use in studying the effects of changes in malting and mashing procedures has been developed. The utilization of reducing sugar and nitrogenous substances and the production of alcohol and yeast biomass were used as parameters to measure degree of fermentation. A comparison of this type of rapid fermentation was made with a larger-scale microbrew prepared in a similar manner. ). Prior to inoculation the yeast was grown in a shaken culture in wort supplemented with 1% (wt/vol) maltose at 12 C until a log culture was obtained. Different inocula, from 2.5 g/liter of fresh yeast weight, corresponding to 500 mg/liter of dry yeast weight (normal inoculum), to 10 times this amount, were used. Stationary and shaker fermentations (160 rpm) with normal inoculum were used as controls. Rapid fermentation is defined here as the use of five times the normal inoculum with shaking for 3 days. Fermentations were carried out at 12 C, using 100 ml of wort per 125-ml Erlenmeyer flask. Experiments were done in duplicate or triplicate. A rapid microbrew fermentation stirred with a magnetic stirrer (90 rpm) was carried out with five times normal inoculum and 2.4 liters of wort in a 3-liter Erlenmeyer flask. For comparison a stationary microbrew was prepared with normal inoculum and 2.2 liters of wort in a 2.75-liter tube fermenter. Hopped wort was prepared using 70%o Larker malt and 30% corn grits by the method of Burkhart et al. (5). Wort was stored frozen until needed, when it was thawed and filter sterilized. None of the wort used was aerated prior to inoculation. Maltose (1%) was added to the wort, and shaking was used to induce yeast growth. At the end of fermentation, the beer samples were centrifuged at 7,000 x g at 4 C for 30 min, and the yeast cells were washed once with 0.1 M NaCl and once with distilled water. They were then dried (at 80 C) to constant weight, and the weight of dry yeast per ml of wort was calculated. Beer and wort were analyzed for reducing sugar, formol nitrogen, dextrin, and alcohol content according to the methods of the American Society of Brewing Chemists (2). Total nitrogen in wort and beer was determined according to the procedure of 970 Johnson (12). Flavor evaluations were conducted with a taste panel of five members by comparing the test brew with a control, which was stationary microbrew. Two taste tests were run on separate days. RESULTS AND DISCUSSION The decreases in specific gravity of the stationary, shaken, and rapid fermentations are shown in Fig. 1. These data are compatible with the log phase of yeast growth (Fig. 2). The rate of yeast growth in the rapid fermentation was about three times that of the stationary fermentation during the first day. There was little difference between the stationary and shaken cultures in terms of specific gravity change or in yeast dry weight during the first day; however, there was a large difference in both parameters of these cultures at 2 days. Yeast growth reached a maximum after 4 days in the stationary and shaker fermentations and after 3 days in the rapid fermentation. The amount of log-phase yeast growth obtained from the rapid fermentation was about four times that of stationary fermentation. By comparison, the shaker fermentation showed a 2.4fold increase over the stationary fermentation. The relative increase in dry yeast matter obtained was greatest with the normal inoculum on the shaker. Shaking produced a 13-fold increase in yeast harvest, whereas the stationary method produced only a fivefold increase ( 2). This is in contrast to Cook's observation with S. carlsbergensis that shaking made little difference in the quantity of yeast produced (6). The lag and postexponential phase in the rapid fermentation did not exist or was too short to be detected with the sampling intervals used (Fig. 2). It has been speculated that the lack of a lag phase with large inocula is due to carryover of metabolites, possibly ribonucleic acids or their precursors, which increase during the lag phase and are necessary for cell growth and multiplication (15). Very little difference in the yeast growth, or in its chemical analysis, was produced by a yeast concentration 4 to 10 times the normal inoculum (Table 1). A relatively smaller increase in yeast production occurred with the larger inocula. Increase in nitrogen with an inoculum of 6 to 10 times normal may possibly be from yeast autolysis. The acidity of the beers increased slightly, as did the biomass, when up to four times the normal inoculum was employed. Beyond that it remained essentially constant. As expected, the pattern of disappearance of reducing sugars from wort during fermentation (Fig. 3) was closely related to the decrease in specific gravity (Fig. 1) (Fig. 6), and the increase in yeast growth early in fermentation (Fig. 2). Utilization of reducing sugar during fermentation reached a maximum after 3 days in the rapid fermentation and after 5 days in the shaker fermentation, whereas the stationary fermentation had not reached a maximum at 7 days. Reducing sugar utilization and yeast growth reached their maxima much more rapidly in the rapid fermentation, and from this point on the disappearance of reducing sugar became insignificant. from the medium of formol and total nitrogen, respectively. There appeared to be little or no lag phase in the uptake of formol nitrogen by any of the three types of culture, although there were obvious differences in the rates of uptake. There was also no lag phase in the uptake of total nitrogen by the rapid fermentation, but both the stationary and shaker cultures exhibited a lag. The large inoculum and improved availability of substrates and oxygen during the early hours of culture in the rapid fermentation permitted an immediate and rapid uptake of larger peptides and other nitrogenous compounds as well as the amino acids and smaller peptides. As might be expected, the production of alcohol (Fig. 6) coincides very closely with the disappearance of reducing sugar (Fig. 3). The amount of alcohol production in the stationary fermentation (7 days) compared favorably with that produced in 4 days in the shaken culture and that produced in slightly over 2 days in rapid fermentation. To determine the effects of scaling up the rapid fermentation by approximately 20-fold, a comparison was made between a normal stationary microbrew and one in which five times the normal inoculum was used with stirring ( Table 2). Stationary and rapid fermentations (100 ml) were also run for comparison. There was generally good agreement in the beer analyses and yeast growth between both types TIME IN DAYS FIG. 5. Changes in total nitrogen during wort fermentation. Symbols: 0, stationary fermentation; 0, shaken fermentation; A, rapid fermentation. of fermentations after 3 days, although the rapid microbrew was somewhat slower in maltose, formol, and total nitrogen uptake. The beer analyses and yeast growth in the stationary microbrew and stationary (100 ml) fermentation were comparable in pH, alcohol production, and utilization of reducing sugar, but the decreases in specific gravity, utilization of wort formol, and total nitrogen were higher in the stationary microbrew fermentation after 3 and 7 days. Yeast growth was also greater in the stationary microbrew after 3 days. The beer produced by these fermentations contained relatively low carbonation and somewhat more initial haze than either the stationary or the shaken fermentation products. Neither of these properties was of serious consequence, since they could be normalized by carbonation and chill proofing. The aroma and taste of the rapidly microbrewed beer were judged as acceptable or better than those of the stationary, microbrewed, control beer. The results described here show that fermentation time with S. carlsbergensis at 12 C can be reduced by about 4 days by use of five times the normal pitching rate and stirring. This is in general agreement with the data of Hudson (11), who found that four times the normal pitching rate with strains of S. cerevisiae at 18 C reduced fermentation time by about onehalf. Fermentations of the type described here provide a convenient tool for evaluating on a small scale the effects of wort composition on fermentation efficiency and beer properties. The results obtained with rapid microbrews prepared with larger inocula and stirring suggest an advantage in the use of rapid fermentations for pilot brewing studies. ACKNOWLEDGMENTS I wish to thank W. C. Burger and N. Prentice, research chemists with the Barley and Malt Laboratory, for their advice. The Barley and Malt Laboratory is supported in part by a grant from the Malting Barley Improvement Association.

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2020-12-10T09:04:20.428Z

1975-04-01T00:00:00.000Z

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Toxicity and Occurrence of Balansia on Grasses from Toxic Fescue Pastures Balansia epichloë (Weese) and B. henningsiana (Moell.) were isolated from grasses in toxic fescue pastures. B. epichloë, cultured in a synthetic medium, was toxic to chicken embryos. Thin-layer chromatography and ultraviolet absorption data indicated that in submerged culture the fungus produced compounds with the indole or ergoline nucleus. States, occasionally becomes toxic, and cattle grazing this grass develop a condition known as "fescue foot" (6,10). Clinical signs of this syndrome have been described (19). These signs are similar in many repects to the gangrenoustype alkaloid poisoning caused by the alkaloids from Claviceps purpurea as suggested by Woods et al. (18). Alkaloids from this ergot have been ruled out by other studies (6,13), and the etiological agent remains unknown. The toxic compound is generally considered to be a vasoconstrictor (5,13), and the sporadic and seasonal nature of the syndrome suggests that a mycotoxin might be involved. Several fungi, isolated from toxic fescue pastures, produce mycotoxins (9,11,20). These studies, however, did not prove that these fungi and their toxins caused the syndrome. In recent investigations of toxic fescue pastures in Georgia two species of Balansia were observed. This paper reports the isolation of these systemic phytopathogens from several species of grasses in these toxic pastures and on the in vitro toxicity of one species. Balansia epichloe (Wesse), obtained from ascospores, had not previously been cultured in the laboratory so we had to develop a medium which favored germination and growth. This medium consisted of: malt extract (Difco), 5 g; yeast extract (Difco), 20 g; glucose, 20 g; agar, 20 g; and distilled water, 1,000 ml. On this medium germination (60 to 85%) was complete after 3 days. The fungus was stored on agar slants of this medium at 4 C in capped test tubes. Subcultures and inocula were prepared from these tubes by macerating the contents of one tube in 10 ml of sterile, distilled water. For submerged culture of the fungus a synthetic medium was used: mannitol, 30 g; sucrose, 20 g; KH2PO4, 5 g; NH4NO3, 2 g; MgSO4-7H2O, 0.2 g; CaCl2-2H2O, 0.1 g; a trace element solution, 5 ml (H. J. Vogel, Microb. Genet. Bull. 13:42-43, 1956); thiamin, 0.1 ug/ml and distilled water, 1,000 ml. The inoculum (1 ml) was placed in a 2.8-liter Fernbach flask containing 250 ml of the synthetic medium, and incubated on a gyratory shaker (200 rpm, 1-cm circular orbit) for 28 days at 24 to 28 C. For routine analysis, the medium was separated from the mycelium by centrifugation (20,000 x g, 15 min) and filtered through glass wool, and proteins were removed by ultrafiltration with a hollow fiber concentrator (Amicon Corp., Lexington, Mass.). The dialysate was passed through a glass chromatographic column (18 by 3 cm ID) packed with Porapak Q (80 to 100 mesh; Waters Associates Inc., Milford, Mass.). Under a pressure of 10 lb/in2 the column was washed with 300 ml of distilled water and eluted with 300 ml of methanol. One portion of the methanol solution was used for chemical analysis; the other was used for toxicity studies. The methanol solution used for toxicity studies was evaporated to dryness on a rotary evaporator (40 C), and the residue was dissolved in 3 ml of CHCl,. The residue remaining after CHCl8 removal was dissolved in 3 ml of distilled water. The column bypass was freeze dried, and 1 g was dissolved in 3 ml of distilled water. These fractions were bioassayed for toxicity with fertile White Leghorn eggs (17) by injecting 0.01, 0.025, and 0.05 ml on the air cell before incubation. For controls the same volumes of uninoculated medium and CHC1, ex- tract of uninoculated medium were tested and found to be nontoxic (background motality ranged from 0 to 5%). The remainder of the methanol solution was concentrated and applied to thin-layer chromatography plates of silica gel GF 254 by the method of Agurell (2). Chloroform-methanol (4:1, vol/vol) was the developing solvent. After development, the plates were examined under ultraviolet light at 254 nm, and those bands showing a dark blue absorption were marked. The portion of the plate containing the spots was sprayed with a 4-dimethylaminocinnamaldehyde reagent (12). Ultraviolet absorption spectra in ethanol were obtained on the Beckmann spectrophotometer acta CIII. One toxic fescue pasture was observed for 1.5 years, and during July and August B. epichloe was noted sporulating on smut grass (Sporobolus poiretii [Roem. and Schult. ] Hitchc.). This weed is a serious problem in pastures in the southeastern United States (16). The fungus also was seen on fescue (Festuca sp.), love grass (Eragrostis hirsuta [Michx.] Nees), and panicum (Panicum anceps Michx.). Cattle were observed to graze all these grasses. This fungus was detected in the grasses by finding the black ergot-like pseudomorphic ascostromata on their adaxial leaf surfaces (Fig. 1) epichloe did not produce inflorescences. In these plants all the leaves in a clone showed either the asexual ephelidial stage (Ephelis state) or the black ascostromata. These fructifications were ephemeral. B. epichloe was toxic to the chichen embryo (Table 1). Toxicity resided in the CHClI, water-soluble, and column bypass fractions. Thin-layer chromatography of the methyl alcohol fraction revealed several bands that absorbed dark blue under ultraviolet light at 254 nm. When sprayed with 4-dimethylaminocinnamaldehyde reagent, several spots gave the characteristic color reaction for ergot-type alkaloids (2). Two of these major components (Rf 0.45) were scraped into glass funnels (fritted disk), and the silica gel was eluted with chloroform-methanol (1:1, vol/vol). The eluate was concentrated to dryness under a stream of nitrogen, and the residual material was taken up in ethyl alcohol. The ultraviolet absorption data of these two compounds ( Table 2) were suggestive of the indole (or ergoline) nucleus characteristic of the ergot-type alkaloids (3). We are investigating the structure and toxicity of these compounds. Balansia is a clavicipitaceous fungus, and like Claviceps might be expected to produce alkaloids. Some new ergot alkaloids have been reported from unrelated genera of fungi (1). These data suggest that in submerged culture B. epichloe can produce indole compounds in vitro. The morphology and nutritional requirements of the fungus in the synthetic culture medium will be reported elsewhere. The in vivo relationship of this fungus to the host's metabolism during various seasons of the year is unknown. Perhaps the production of toxic compounds is host or season dependent, since the variety of toxic compounds produced by a species of Claviceps is host dependent (1). This report is the first to indicate that B. epichloe produces toxic substances in vitro and suggests that the genus, because of its systemic nature, should be considered a potential hazard to animals grazing on parasitized forage grasses. This endophytic systemic parasite is a better candidate for the cause of fescue foot than the saprophytic fungi studied to date. That Balansia may be a problem in forages has been reported from India, where cattle and goats showed signs indistinguishable from ergot poisoning of Claviceps purpurea after grazing on lovethron grass (Andropogon aciculatus) parasitized with a species of Balansia (14). Claviceps sp. was not found in that study. The involvement of Balansia in the fescue foot syndrome is complicated by the general elusive nature of the fungus and fescue growth requirements. Cool temperatures favorable for the growth of fescue may suppress sporulation of the fungus. In the spring, we found only a few clones of fescue grass showing the ascostromata of the fungus. Earlier studies indicated that the fungus will not sporulate when grasses are kept at cool temperatures (lower than 20 C) but continue to grow and invade new tissue as its host grows (7). This suggests that parasitized grasses growing in their northern extremes or during cool seasons would not show signs of fungal sporulation, and thus go unnoticed. The fungi on warm-season grasses (smut grass, panicum, etc.) were heavily sporulating. Perhaps a more valid indicator for parasitism would be a test for chitin as proposed by Ride and Drysdale (15) or that suggested by Diehl (7,8). The significance of Balansia has been superficially examined (4,8). It parasitizes 10 tribes of the American Gramineae, many members of which are economically important forage crops (8). In more detailed investigations of this genus, we are studying its economic importance to the growth of forage grasses and to the animals grazing these parasitized grasses. LITERATURE CITED 1. Abe, M., and H. Iizuka. 1971. Alkaloid and steroid production by microorganisms, p. 175-200. In K. Sakaguchi, T. Uremura, and S. Kinoshita (ed.), Biochemical and industrial aspects of fermentation. Kodansha Ltd., Tokyo. 2. Agurell, S. 1965. Thin-layer chromatography and thinlayer electrophoretic analysis of ergot alkaloids. Relations between structure, Rm value and electrophoretic mobility in the clavine series. Acta Pharm. Suec.

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2020-12-10T09:04:17.560Z

1975-11-01T00:00:00.000Z

237231184

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Growth of Staphylococcus and Salmonella on Frankfurters With and Without Sodium Nitrite Conventional and nitrite-free frankfurters in loosely wrapped packages were compared as to their ability to support growth of Salmonella, Staphylococcus, and their naturally occurring spoilage flora at 7 C (simulating refrigerated storage) and 20 C (simulating possible temperature abuse). At 7 C Salmonella did not grow in either type of frankfurter; Staphylococcus and the natural spoilage flora sometimes grew more rapidly in the absence of nitrite, but the difference was not significant. At 20 C growth of Salmonella, Staphylococcus, and of the spoilage flora was, at most, only slightly faster on nitrite-free frankfurters. Salmonella was not suppressed in broth culture experiments at the pH and nitrite content found in frankfurters. Although either type of frankfurter can become hazardous due to growth of Salmonella or Staphylococcus, no unusual or additional hazard resulted from the omission of nitrite from frankfurters. That the incorporation of sodium nitrite in cured meats inhibits the growth of some foodpoisoning bacteria has long been known (16,22). Under some conditions it is also bactericidal (23,24). Nitrite also plays a role in the flavor and heat stable color of processed meats and originally may have been used primarily to impart these characteristics. In recent years there has been an increasing public consciousness of the use of additives in foods. Results of toxicological (17,18) and other investigations (1,9,27) have caused concern about the effect of additives, including nitrites, on the health of consumers. In response to this public interest, one large local food chain arranged for a meat processor to provide both nitrite-containing (cured or conventional) and nitrite-free (uncured) frankfurters for its retail meat counters. The cured frankfurters are vacuum-packed in plastic by the processor, whereas the uncured are shipped in bulk and loosely wrapped at the retail level. The uncured frankfurters have a gray appearance and slightly different taste. These unusual characteristics, and the lack of nitrite, prompted our interest in their microbiological safety. Our objective was to observe the growth characteristics ofStaphylococcus and Salmonella to determine whether they present a greater health hazard on nitrite-free frankfurters, if exposed to mishandling, than on similarly treated conventional frankfurters. A secondary objective was to determine whether deterioration caused by harmless spoilage organisms takes place more rapidly in nitrite-free frankfurters. MATERIALS AND METHODS The National Canners Association Laboratory, Berkeley, and the California State Public Health Laboratory, Berkeley, supplied the cultures of Staphylococcus aureus 413 and Salmonella enteriditis 2179, respectively. S. typhimurium Tm-1 was from our own collection. Experimental inocula of these strains were grown on a rotary shaker at 28 C for 60 h in Trypticase soy broth (BBL) containing 2% yeast extract (Difco). The cells were harvested by centrifugation, washed twice in distilled water, and resuspended in 100 ml of sterile demineralized water, giving a count of 109 or 1010/ml. They retained their viability during several months of storage at 7 C. Nitrite-free and conventional frankfurters were obtained from a local market or directly from the processor. Their pH ranged from 5.2 to 5.8, but was usually about 5.5. They were inoculated either internally, by injecting 0.5 ml of a 103/ml cell suspension of the test organism into each end of the frankfurter, using a 2.5-iml syringe with a 8-cm needle, or externally, by immersion in a water suspension containing 103 cells/ml for 2 min and draining until almost dripless. The inoculated frankfurters were placed in plastic pallets, which were then loosely wrapped with plastic film and incubated at either 7 or 20 C. Sampling times were 0, 2, 3, 4, 7, 10, and 15 days for 7 C incubated sausages and 0, 2, 4, 6, and 8 days for 20 C incubated sausages. At each sampling date, two whole frankfurters (about 45 g each) were aseptically removed. Separate counts were made on each as follows. 844 GROWTH OF STAPHYLOCOCCUS AND SALMONELLA 845 Staphylococcus. The samples were blended in Trypticase soy broth (BBL) containing 2% yeast extract (Difco), diluted in the same medium with 9.5% sodium chloride added, spread plated on Vogel Johnson Tellurite agar (BBL), and incubated at 37 C for 48 h (3). Salmonella. For a three-tube most probable number method, lactose broth was used as a blending, diluting, and pre-enrichment medium, brilliant green tetrathionate broth was used as an enrichment medium, and brilliant green sulfadiazine agar was used for a confirmatory test (10). Incubation was at 37 C. Total aerobic counts. These were done simultaneously with counts of Staphylococcus or Salmonella; thus, the blending media were as stated above. Subsequent dilutions were made immediately in Trypticase soy broth with 2% yeast extract, followed by spread plating on agar of the same composition and incubating at 28 C for 2 days. When the population increased, typical growth curves for Staphylococcus and for the total population (total counts) were drawn from the counts after various storage periods. The time required to reach an arbitrarily chosen population was taken as an end point for comparison of conventional with nitrite-free frankfurters. The counts on Salmonella were irregular because the most probable number method lacks precision, but it was possible to determine the approximate storage time at which the count first surpassed 105/g. When no growth occurred, the counts decreased and we compared the numbers of surviving organisms in the conventional and nitrite-free frankfurters. The direct effect of nitrite on aerobic growth of Salmonella in broth was determined in 250-ml nephelometer flasks. The broth contained Tryptone (Difco), 1%; yeast extract (Difco), 0.5%; NaCl, 0.3%; dextrose, 1%; and KH2PO4, 0.5%. The pH was adjusted to several levels in the range 4.6 to 7.5 with NaOH or phosphoric acid. A 10% stock solution of sodium nitrite sterilized through a Morton filter apparatus was added to give several final concentrations up to 8,000 gg/ml. Each flask, containing 50 ml of broth, was inoculated and incubated at 28 C for 48 h. Growth was measured with a Klett-Summerson photoelectric colorimeter equipped with a red filter (640 to 700 nm). Nitrite concentrations were determined by the method of Greiss (2). This method does not detect reaction products of the nitrite with the meat, although these may have physiological activity. RESULTS Staphylococcus. When inoculated frankfurters were incubated at 20 C, their count increased logarithmically to 10ff/g to 108/g within 2 to 8 days. Their count reached 106/g in 2 to 6 days without nitrite and in 2 to 8 days when cured (Table 1). There was no consistent difference between the conventional and the nitritefree frankfurters. Thus, omission of the nitrite did not permit any spectacular increase in the growth rate of the Staphylococcus on nitritefree as compared with conventional frankfurters. In one experiment (b, Table 1), growth on the conventional frankfurters was very slow and never reached 106/g. The reason for this is unknown, but a dependable inhibiting action of nitrite is not indicated as it was not seen in the other experiments. In similar experiments incubated at 7 C, growth was logarithmic after a lag period but much slower. As the count did not always reach 106/g during the 15-day storage period, a count of 104/g was taken as the end point for comparison. In three experiments (Table 2) growth was found on nitrite-free but not on the conventional frankfurters during the 15-day incubation period. In the three other experiments, growth was found both in the presence and the absence of nitrite. The lack of growth in the presence of nitrite probably reflects the fact that this temperature is very near the minimum growth temperature for Staphylococcus. In general, bacteria become unusually fastidious at temperatures near their minima (20); thus, the nitrite could prevent growth if it con- Salmonella. After about 2 days at 20 C, the count increased and the time required to reach a population of 105/g is shown in Table 3. Although variable, this time was not consistently less for the nitrite-free frankfurters than for the conventional. Growth was rapid enough on both types, however, to render them hazardous within a few days at 20 C. When frankfurters were inoculated similarly and stored at 7 C for 15 days, Salmonella did not grow, but survived at about the original level for the duration of the experiment ( Table 4). The counts were irregular and not in a pattern indicating any relationship between survival and the presence of nitrite in the frankfurters. Since Salmonella has not been observed to grow below 5.5 C (19,20), and the pH of these frankfurters (about 5.5) is not optimal for Salmonella, it is not surprising that no growth was observed at 7 C. Comparable survival but no growth has been observed at 5 C for Salmonella on surfaces of bologna (11). Total counts. Initial total aerobic counts on frankfurters, with and without nitrite, were between 102 and 104 cells/g but usually 103 cells/g. Throughout these experiments total aerobic counts were made simultaneously with those of surviving Staphylococcus and Salmonella, and always greatly exceeded the latter, usually by a factor of 100 or more. The total counts soon reached 106/g, which was chosen arbitrarily as an end point. The time required for nitrite-free frankfurters to reach this point was compared with that for conventional frankfurters (Table 5). These data are segregated according to whether the frankfurters were inoc- ulated internally or externally, but there seems to be no difference between these two treatments. The storage time before spoilage at 7 C is short compared with commercial practice. However, these frankfurters were not packed or treated as they would be commercially. Also, it has been reported (8) that organoleptic spoilage is not reached in frankfurters until the microbial population reaches 108/g. At both 7 and 20 C, the storage time required to reach a total count of 106/g was often less in the nitrite-free frankfurters, but the time difference was usually small and only exceeded 25% in 7 of the 22 trials (Table 5). It is concluded that the nitrite did not enhance the microbiological stability of the frankfurters to an important degree. Effect of thermal process on flora. It is known that the raw meat components of frankfurters bring to the sausage mix a rich and varied flora (21). Our total counts on two samples of the raw emulsion were 3 x 107 and 7 x 106 cells/g. The frankfurters were cooked for 1.25 h, with a maximum internal temperature of 70 C. After cooking, counts were from 3 x 104 to 102 cells/g in conventional frankfurters and 3 x 103 to 6 x 102 cells/g in nitrite-free frankfurters. This is a reduction in count of two to five orders of magnitude during cooking. Residual nitrite in frankfurters. The maximum legally allowable nitrite concentration is 156 j&g/g but this diminishes rapidly. In our tests, 39 gg/g remained after processing, 27 gg/g after 5 days of additional storage at 7 C, and 5 ,&g/g after 15 days at 7 C. This agrees with the more comprehensive data of Hustad et al. (14). Effect of pH on effective nitrite concentration. To observe the effect of nitrite on growth of Salmonella in a tryptone broth medium, S. enteriditis and S. typhimurium were inoculated into 50 ml of broth at levels of 8 and 40 cells/ml, respectively, and incubated at 28 C for 48 h. The nitrite concentration varied from 0 to 8,000 Zg/ml and the pH varied from 4.6 to 7.5. The inhibitory effect of nitrite was strongly dependent on pH and, at the concentration permitted in meat, nitrite was ineffective above pH 6 (Fig. 1). The nitrite concentration in the broth remained approximately constant during the course of the experiment, in contrast to that in the frankfurters. That pH influences the bacteriostatic effect of nitrite has been shown for Staphylococcus by others (5,6,23). DISCUSSION Each of the growth-survival experiments (Tables 1 through 5) was repeated several times with varying results. Nevertheless they present consistent patterns showing little difference between the conventional and the nitritefree frankfurters. Each lot of frankfurters was from a different commercial batch although from the same manufacturer. Also the conventional and nitrite-free frankfurters within an experiment were never from the same batch of meat emulsion. For these reasons, variation is to be expected. Staphylococcus and Salmonella are known to be present in the raw meat used in sausage manufacture (13,21,25,26). They are not usually found in frankfurters because they cannot survive the heat process (21). Also our data, showing that cooking reduces the total count by several orders of magnitude, indicate that Staphylococcus and Salmonella would suffer similar depletion and so could survive only after gross contamination ofthe raw meat emulsion. After cooking, the frankfurters can be contaminated on the packaging line, during warehousing and distribution, in the retail storage and preparation area, in retail supply cabinets, or in the home. Vacuum packaging only eliminates part of these chances for contamination. Salmonella is known to be carried by frankfurters (7). Since nitrite-free frankfurters are being sold locally and their sale and use is being advocated nationally, it is important to know whether the omission of nitrite will make them a more suitable substrate for Staphylococcus and Salmonella, and therefore more likely to carry these organisms to the consumer. Our data (Tables 1 and 3) show that these bacteria grow about equally on the conventional and the nitrite-free frankfurters when loosely wrapped for storage; hence nitrite does not inhibit growth under these conditions. There is other evidence that nitrite is not strongly inhibitory against Staphylococcus in frankfurters. S. aureus can multiply in the presence of extremely high concentrations of sodium nitrite under aerobic conditions and, even at a pH as low as pH 5.4, it can grow in the presence of 140 jig of nitrite per g (6). In our experiments, the plastic wrappings used during incubation did not exclude oxygen, and the nitrite concentration was far below 140 ,ug/g. Also, meat itself provides some protection to the microorganisms against the inhibitory effect of nitrite (16), thus minimizing any difference between growth on nitrite-free and on conventional frankfurters. Our broth culture experiments also showed that growth of Salmonella was not prevented 47 a- by the residual nitrite concentration (39 ,ug/ml) and pH (about 5.5) of our frankfurters (Fig. 1). Thus the nitrite concentration present in conventional frankfurters is insufficient to prevent growth of Staphylococcus and Salmonella, either in this type of frankfurter or in inoculated nutrient broth. Therefore, the omission of the nitrite does not present any new or unusual hazard from these organisms. Either type of frankfurter, if subjected to contamination by Staphylococcus or Salmonella followed by temperature abuse, can become hazardous. Frankfurters can be spoiled by nonhazardous spoilage organisms if they are subjected to temperature abuse. These organisms can survive the heat treatment in moderate numbers, and they can also be present as a result of postprocessing contamination. However, they do not grow faster to an important degree on the nitrite-free frankfurters as compared with the conventional (Table 5); hence, the omission of the nitrite will not result in a product that requires extra precautions to prevent spoilage. In experiments with Clostridium botulinum in bacon and frankfurters, the inhibitory action of nitrite depended on its level at the time of processing rather than its residual level during storage (3,12,14). This suggests that nitrite reacts with the meat to form other antibacterial compounds (22), and the effect of such compounds has been investigated (15). In our studies, frankfurters were inoculated with Salmonella and Staphylococcus after processing, thus simulating the probable route by which contamination could occur in commercial and home food handling. Under these conditions we found no evidence that nitrite, added at the maximum permitted level before processing, prevented growth of these organisms by itself or through the formation of antibacterial reaction products. ACKNOWLEDGMENT We express our appreciation to Gary M. McDonald for the nitrite determinations.

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2020-12-10T09:04:20.510Z

1975-07-01T00:00:00.000Z

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Effects of Trace Metals on the Production of Aflatoxins by Aspergillus parasiticus Certain metals added as salts to a defined basal culture medium influenced the level of aflatoxin production by Aspergillus parasiticus in the low microgramsper-milliliter range of the added metal. In many cases no change or a relatively small change in mat weight and final pH of the medium accompanied this effect. With zinc at added levels of 0 to 10 μg/ml in the medium, aflatoxin increased 30-to 1,000-fold with increasing of zinc, whereas mat weight increased less than threefold. At 25 μg of added zinc per ml, aflatoxin decreased, but mat weight did not. At an added level of 25 μg or less of the metal per ml, salts of iron, manganese, copper, cadmium, trivalent chromium, silver, and mercury partly or completely inhibited aflatoxin production, without influencing mat weight. Cottonseeds, peanuts, corn, and other agricultural commodities important in the food and feed industries may become contaminated with' aflatoxins, but the basic factors that influence aflatoxin production have not been completely studied (4). The toxin is a metabolite of Aspergillus flavus and of the very closely related species A. parasiticus. Effects of growth media on aflatoxin production by A. flavus have been studied mostly in comparisons during culture on various seeds and other natural substrates (4). More limited work has involved defined media containing various sources of carbon and nitrogen (3). Mateles and Adye (10) deleted molybdenum, boron, copper, iron, zinc and manganese, singly and in combination, from their growth medium and concluded that ". . . zinc is required for the production of aflatoxin. The slightly reduced yields obtained when iron or manganese were deleted were due to a visible reduction of growth, which was not observed when zinc was. deleted." Omission of copper, molybdenum, and boron did not reduce aflatoxin production. Lee et al. (5) also reported that zinc was specifically required for production of aflatoxins by A. flavus. An approximate maximum in mat weight was obtained with 0.4 ,ug of zinc per ml, whereas aflatoxin increased from a low level at this concentration to a maximum at about 2.0 jsg of zinc per ml. Davis et al. (2) concluded, "The influence of minerals on aflatoxin synthesis may be indirect 'Present address: U.S. Food and Drug Administration, Washington, D.C. 20204. through their essentiality for growth or more directly on the process of toxin synthesis per se. The influence of zinc and iron appeared to be indirect, whereas that of molybdenum appeared direct." Zinc and iron, at 0.5 to 1 ug of the sulfate per ml, stimulated both aflatoxin production and growth, i.e., mat weight; they also inhibited aflatoxin production, but not growth, at 10 ttg/ml. A minimum of 0.5 g of MgSO, per liter appeared essential for maximum production whereas a fifth of this amount was sufficient for maximum growth. The purpose of this work was to determine in synthetic culture whether salts of certain metals at low parts-per-million levels could influence the production of aflatoxins by A. parasiticus and, if so, whether this effect would be accompanied by changes in the overall growth of the fungus as measured by mat weight or in the finalplH of the growth medium. MATERIALS AND METHODS Organism. The isolate used was A. parasiticus NRRL 2999, from the USDA Northern Regional Research Laboratory, Peoria, Ill. The trace element composition of the basal medium was as indicated in the tables (see Tables 1-4). To make stock solutions of the standard trace metal salts, we dissolved FeSO4 .7HO, CuSO, .5HO, MnSO. and Ca(NO)2-4H20 in water. Zinc glycerophosphate was used to avoid zinc precipitation. Thus, 90 mg of this salt was dissolved in a minimum amount of glacial acetic acid and water, the solution was adjusted to pH 5.2, and the volume was made up to 25 ml. One ml of the zinc glycerophosphate solution in 1 liter of basal medium provided a final concentration of 1 Ag of zinc per ml. Cadmium, chromium, silver, cobalt, nickel, lead, and aluminum were added as nitrates, mercury as mercuric chloride, and ruthenium as the chloride. The complete salt medium was adjusted to pH 5.2 with glacial acetic acid. All water was passed through an ion-exchange column. Thirty milliliters of medium was dispensed into each of several 250-ml flasks. The flasks, previously acid washed, were stoppered with cotton plugs and autoclaved 15 min at 15 lb. of steam pressure. After cooling, they were inoculated by needle with spores of the fungus and incubated at 30 C without shaking for time periods as specified in table headings (see Tables 1-4). The treatments were replicated six times in individual flasks, except that Cr3+ and Ag+ treatments were replicated five times. Assays. After incubation, the final pH of the filtrate was measured, and the culture fluid was filtered through Whatman no. 1 paper. The mycelial mats were washed with 4 ml of distilled water, and the wash water was combined with the filtrate. The mycelial mats were dried for 3 days at 40 C and weighed. Aflatoxin analyses were performed on both filtrates and mats. A 25-ml portion of the pooled filtrates was extracted with 20 ml of CHCl, for 30 min in a 50-ml glass-stoppered centrifuge tube. Then it was centrifuged for 10 min at 2,000 rpm, and the aqueous phase was removed. The CHCl, phase was passed through a column of anhydrous sodium sulfate, and the column was rinsed with an additional 20 ml of CHCl. The chloroform extracts were evaporated to dryness, and the residues were saved for thin-layer chromatography (TLC). The combined mycelial mats from each treatment were placed into a blender cup and blended for 5 min at full speed with 50 ml of acetone-water (70:30). A 10-ml portion was transferred to a 50-ml glass-stoppered centrifuge tube, and 2 ml of lead acetate plus 14 ml of water was added to the tube. The precipitate that formed was sedimented by centrifugation for 10 min at 2,000 rpm. The supernatant was extracted with 20 ml of CHCl,, phase separation was aided by centrifugation, and the aqueous phase was discarded. Then the CHCl, phase was extracted with 2 ml of 0.1 N NaOH, shaking for 1 min. The sodium hydroxide was removed after centrifugation, and the CHCl, phase was passed through a column of anhydrous sodium sulfate. The column was rinsed with 20 ml of CHCl,, the combined CHCl3 effluents were evaporated to dryness, and the residue was saved for TLC. The residues were dissolved in 200 ml of CHCl,, and aliquots were applied to silica gel-prepared TLC plates (Schleicher and Schuell no. 1500). The plates were developed in unlined, unequilibrated tanks with 150 ml of acetone-chloroform (1:9). Developing time was about 45 min for a solvent movement of 12 cm. Quantitation was by comparison with reference standards of aflatoxins on the same TLC plate. Aflatoxin production was calculated as follows: Nanograms per flask = Nanograms aflatoxin in medium (= total milliliters of medium x ng/ml) number of replicates Nanograms aflatoxin in mats + (= total wet weight in grams x ng/ml number of replicates A test showed that the trace elements used in the culture flasks did not interfere with aflatoxin analyses. To each of a series of test tubes, a 10-ml sample of control medium with 240 ng of aflatoxin B1 per ml was transferred. Then each tube was spiked with a trace element at a concentration of 5 tig/ml. The trace elements used were iron, copper, zinc, manganese, cadmium, cobalt, lead, mercury, aluminum, and nickel. After extraction of the aflatoxin as described above, 10-ul amounts from each tube were spotted on TLC plates next to 10-Al aliquots of extract from the control medium with aflatoxin and no added trace elements. The plates were developed as described above. The intensities of the fluorescent spots from the control medium extract could not be distinguished from those from the spiked media extracts. Table 1 shows results with progressive additions of zinc, manganese, iron, and copper salts to the basal medium with a constant level of each of the remaining elements tested. At a zero-added level of zinc (Table 1), aflatoxin production was very low, mat weight was low, and final pH of the medium was low. RESULTS At zinc levels of 1 to 25 jg/ml, aflatoxin production was much higher, with a moderate depression at 25 yg/ml, whereas both mat weight and final pH were higher and nearly constant. The fact that at the 1 gg/ml level of added zinc the aflatoxin production was increased to about 1,000 times that with no zinc whereas mat weight was increased only 2.5 times suggests, in accord with previous reports (5,6,10), that zinc has a stimulating influence on aflatoxin production apart from its essentiality for overall growth. The ratio of aflatoxins in the mat to aflatoxins in the medium is not recorded here, because it did not vary consistently in this or other experiments. Thus, we used only the mat-plus-medium summation figures in all tables. In general, aflatoxin B1 > G1 > B2 > G2 throughout all zinc concentrations, a relationship also seen in later experiments with most other trace elements. With added manganese from 0 to 25 gg/ml (Table 1), aflatoxin production increased to its highest level at the 5 and 10 tg/ml levels and 53 VOL. 30, 1975 (Table 1), aflatoxin production was relatively high, mat weight was somewhat lower than at higher iron concentrations, and the final pH was 4.5. With iron in the range of 1 to 25 ,g/ml, aflatoxin production decreased progressively with increasing iron concentration while mat weight was constant around 240 mg and final pH was nearly constant around 7.0. At all levels of added copper from 1 to 25 ug/ml (Table 1), a depression of aflatoxin production occurred, even though mat weights and the final pH were unaffected. Thus, zinc, manganese, iron, and copper all depressed aflatoxin production at 25 gg/ml or at some ower level, but copper depressed it most. The aflatoxin-producing response of A. parasiticus to zinc (Table 1, experiment 1A) was verified in two similar experiments ( Table 2, experiments 1B and 1C). In them, the zinc-aflatoxin relation was much like that in experiment 1A; the response of the fungus to successive increments of zinc in the levels of 0 to 10 ug/ml was much greater in aflatoxin production than in growth, and the optimum zinc concentration for aflatoxin production was higher than that for growth. In the three experiments, zinc caused 30to 1,000-fold increases in aflatoxin levels with increases of less than threefold in growth. The decrease in level of aflatoxin between 7 and 14 days ( Table 2), was in accord with the literature (1,4). An extra confirmatory experiment on effects of zinc was run with half-strength amounts of each of four major salts as the only deviation from experiment 1A (Table 1). In this experiment, the total aflatoxin produced in micrograms at 0, 1, 5, 10, and 25 jg of zinc per ml was 0.47, 30.5, 126.2, 155.6, and 23.5 with mat weights of 100, 221, 223, 211, and 208 mg, and final pH values of 3.1, 2.8, 3.0, 3.4, and 6.0, respectively. These results, although marked by low pH, continued to indicate that zinc stimulates aflatoxin production more than mat weight. That is, between 0 and 5 Ag of zinc per ml, the total aflatoxins increased about 268fold, whereas mat weight only about doubled. As in the experiments reported above, the level of zinc for maximum aflatoxin production was higher than that for maximum growth. The maximum aflatoxin level was at 10 jg of zinc per ml. Cadmium at all levels added from 1 to 25 tig/ml depressed production of aflatoxin greatly, but without influencing mat weight or final pH (Table 3). Thus, its effects were similar quantitatively to those of copper. Trivalent chromium-depressed aflatoxin production at metal levels of 1 to 10 jg/ml, but was without major effect on mat weight or final pH (Table 3). TABEz 2. Effect of addition of zinc glycerophosphate to a basal medium on the production of aflatoxins, mat dry weight, and final pH of medium after 7-and 14-day incubation with A. parasiticus 2999a Silver up to 1 ug/ml depressed aflatoxin production without affecting mat weight or final pH (Table 3). At 5 gg/ml, it reduced aflatoxin to nondetectability and depressed mat weight greatly. Thus, at unusually low levels it effectively depressed aflatoxin production and growth. The effects of various metals at 5 gg/ml on aflatoxin production are shown in Table 4. Mercury depressed aflatoxin level greatly without decreasing mat weight or lowering the final pH, while aluminum and ruthenium also depressed aflatoxin production somewhat. Nickel, cobalt, and lead had little or no effect on any of the three measured test results. DISCUSSION The results here described agree with the conclusion of previous workers that zinc stimulates aflatoxin production (2,5,6,10). Also they agree with earlier findings that some trace metals at low concentrations may inhibit aflatoxin production (2). No certain explanation, however, can be offered for the mechanism of the effect shown here by the various metallic ions on the production of aflatoxins. Four of the more potent metals in the inhibition of aflatoxin production in the present work, i.e., copper, cadmium, silver, and mercury, are also fungicidal at higher concentrations. Each of these four is known for its reaction with sulfhydryl groups, and such reactions have been suggested as a cause of their fungicial action (9). In data similar to those reported here for the aflatoxins, Takao (11) has reported inhibition of riboflavin synthesis by copper, mercury, and silver. Aflatoxin contamination of the seeds at harvest appears to be very rare in the U.S. cotton crop and is localized especially in very limited areas. A. flavus, it might be reasoned by some individuals, is a ubiquitous fungus which infects cotton seeds very nearly universally before harvest but produces aflatoxins only in the presence of suitable amounts of trace metals. Actually, temperature seems to us to be a factor of dominant importance and mineral supply to the cotton plant of uncertain significance. Infection of the seeds at harvest with A. flavus is of very limited occurrence across the cotton belt and geographical localization of the boll rot seems most strongly associated with very high temperatures in the affected areas and with the fact that A. flavus is a fungus adapted to growth at high temperatures (7,8). TAmz 4. Effect of addition of salts of several metals at 5 Ag of metal per ml to a basal medium on the production of aflatoxins, mat dry weight, and final pH of medium after 7-day incubation with A. parasiticus 2999 a Fe, Cu, Zn, and Mn all present at 1 ug/ml in basal medium, also Ca at 10 ;g/ml. Aflatoxin per flask; ND, not detected.

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Influence of Carbohydrate and Nitrogen Source on Patulin Production by Penicillium patulum A strain of Penicillium patulum, isolated from cheddar cheese, produced patulin when grown on liquid media containing lactose and milk nitrogen sources. Patulin production was affected by the temperature of incubation, the type and amount of carbohydrate, and the type of nitrogen source present. Patulin levels generally were depressed by incubation at 5 C and low carbohydrate levels. Patulin was produced at low levels in the absence of sugars at 5 C when the mold was grown on milk nitrogen sources. No patulin was detected in cultures grown on 25% casein slurries or cheddar cheese, even though growth of the mold was extensive. Patulin, a metabolite of several Aspergillus and Penicillium species, is quite toxic to animals (1) and has been shown to be carcinogenic to rats in sublethal doses (4). Many types of substrates have been utilized to study the production of patulin by molds and to determine the optimum conditions for production. P. patulum Bainier, grown on Raulin-Thom broth, produced up to 1.3 mg of patulin/ml of medium after 2 weeks incubation at 25 C (15). P. expansum produced up to 0.48 mg of patulin/ml of yeast extract-sucrose broth after 2 weeks incubation at 25 C (10). The largest yields of patulin have been obtained by Norstadt and McCalla (11,12) using potato-dextrose broth in which P. urticae Bainier produced up to 2.7 mg of patulin/ml after 2 weeks incubation at 25 C. Glucose and iron salts were reported to stimulate patulin production, whereas yeast extract and corn steep liquor depressed its formation (9). Patulin production has also been limited by incubation at low temperatures. However, P. expansum and P. patulum have been reported to produce over 0.4 mg of patulin/ml of broth after 100 days incubation at 1.7 C (J. Lovett and R. G. Thompson, Abstr. Annu. Meet. Am. Soc. Microbiol. 1973, E71, p. 12). P. urticae has been observed to produce up to 0.25 mg of patulin/ml of broth after 3 weeks incubation at 5 C (12), and P. expansum has been reported to produce 0.7 mg/ml after 18 weeks at 0 C (20). Growth of patulin-producing molds may occur on foods and be accompanied by patulin production. Growth of P. expansum on apples has been reported to produce up to 125 tkg of patulin/g of apple tissue (20), up to 1 mg of I Published as paper no. 4003, Journal Series, Nebraska Agricultural Experiment Station, Lincoln. patulin/ml of apple sap (2), and up to 17.7 mg of patulin per apple (8). Patulin has also been found in commercial apple juices (18,23,24). However, growth of P. expansum on sausages and breads gave either no detectable patulin or a rapid disappearance of patulin (6,10,17). Lack of production of patulin or loss of patulin in these products was attributed to its reaction with sulfhydryl groups present in the substrates. No nutritional limitations of the foods for patulin production were indicated. Concern has been expressed about possible mycotoxin production on cheeses since mold growth can occur on cheese during ripening, curing, and refrigerated storage (3,7). In one study, up to 82% of the molds isolated from cheddar cheeses were observed to be Penicillium species, and 4% of these isolates were found to produce patulin (3). The work reported here describes a study of the effects of carbohydrate and nitrogen sources derived from milk on patulin production by a strain ofP. patulum isolated from cheddar cheese. MATERIALS AND METHODS Organism. A Penicillium isolate, obtained from cheddar cheese, was identified as P. patulum by using the keys of Raper and Thom (16). The identification was confirmed by D. I. Fennell of the Northern Regional Research Laboratory, Agricultural Research Service, U. S. Department of Agriculture, Peoria, Illinois. The organism was carried on potatodextrose slants stored at 5 C. Verification of patulin. Patulin production by the organism was confirmed by comparing crystalline patulin obtained from ethyl acetate extracts of potato-dextrose and Czapeks-Dox-glucose broth cultures of the organism with known patulin obtained from the laboratory of T. M. McCalla. This was done on the basis of melting point, infrared and ultraviolet absorption spectra, color reactions of patulin derivatives, and Rf values on thin-layer chromatography plates developed in benzene-methanol-acetic acid (90:5:5, vol/vol/vol) and toluene-ethyl acetateformic acid (60:30:10, vollvollvol). Derivatives of patulin were made using ammonia fumes and 4% phenylhydrazine and were compared with internal and external standards (19). Substrates. Several basal media were employed to study growth and patulin production by P. patulum. Potato-dextrose broth, prepared by the method of Norstadt and McCalla (11), was used as a control substrate. Czapeks-Dox broth was employed as a basal salts medium. It consisted of: KCl, 0.71 g; MgSO4-7H2O, 0.71 g; H2K2PO4, 1.43 g; FeSO4, 0.01 g; and distilled water, 1,000 ml. By using this basal medium, carbohydrate and nitrogen sources were compared. The carbohydrates glucose and lactose were studied. Stock solutions (10%) of the sugars were prepared and sterilized by filtration. The nitrogen sources, added to the basal salts solution before heat sterilization, were as follows: NaNO3, 4.29 g/liter; peptonized milk (Difco Laboratories, Detroit, Mich.), 26.8 g/liter; or acid-precipitated casein, 8.4 g/liter. After autoclaving at 121 C for 15 min, the solutions had a final pH of about 7.0. Carbohydrate solutions were then added in sufficient quantity to give levels of glucose or lactose of 3%. Filter-sterilized distilled water in the same quantity as the carbohydrate solutions was employed in the treatments having no carbohydrate. The final substrates thus obtained were: Czapeks-Dox-glucose (3% glucose-sodium nitrate, 3% glucose-peptonized milk, and 4% glucose-casein media), Czapeks-Dox-lactose (3% lactose-sodium nitrate, 3% lactose-peptonized milk, and 4% lactose-casein media), and basal saltspeptonized milk and basal salts-casein media. The peptonized milk contributed 0.75% lactose, as determined by a phenol colorimetric method (5), to media in which it was employed. Patulin production in systems with little or no carbohydrate but with high concentrations of casein was studied using two casein slurries. One slurry was prepared from acid-precipitated casein that had been washed three times in distilled water after removal of the whey; it still contained trace amounts of salts and lactose. The second was prepared from acid-precipitated casein that was washed three more times to make it essentially free from any carbohydrate and salts. It gave no browning reaction when heated in an oven to 140 C for 4 h. Each type of casein was used to make a 25% slurry in distilled water. After heating at 121 C for 5 min to kill vegetative cells and mold spores, the pH of the medium was about 5.0. Prior to sterilization, all of the substrates were dispensed in 50-ml quantities into 250-ml Erlenmeyer flasks. Each flask was inoculated with about 108 spores from a 7-to 10-day-old potato-dextrose slant culture ofP. patulum. The cultures were incubated for 2 weeks at 25 C or for 8.5 weeks at 5 C. Three replicates were done in duplicate for each treatment. The organism was also grown on cheddar cheese to determine whether patulin production might occur on cheese. Three-month-old cheddar cheese was shredded and aseptically added to sterile 250-ml Orlenmeyer flasks in 25-g lots. The cheese was inoculated with P. patulum in the same manner as the liquid substrates and was incubated for 2 weeks at 25 C or for 8.5 weeks at 5 C. Duplication and replication were the same as for liquid substrates. Extraction and analysis. After incubation, cultures were filtered through Whatman no. 4 fluted filter paper, and the filtrates were extracted with ethyl acetate by the method of Pohland et al. (14). Quantitation of patulin from ethyl acetate extracts was done using three methods. Patulin concentrations were estimated with thin-layer chromatography plates (20 by 20 cm, coated with 0.25-mmthick layer of silica gel GHR, Brinkmann Instruments Inc., Westbury, N. Y.). The plates were developed in toluene-ethyl acetate-formic acid, and the patulin concentrations were estimated by visual comparison with known standards of patulin after derivatization with phenylhydrazine (19). Patulin concentrations were also calculated using absorbance at 276 nm and an extinction coefficient of 14,125 and by a disk bioassay method employing Bacillus megaterium (21). Cultures grown on cheddar cheese were extracted by the method of Pohland and Allen (13). The extracts were examined for patulin by using thin-layer chromatography plates prepared as previously described and developed in benzene-methanol-acetic acid. RESULTS Patulin produced by P. patulum on potatodextrose broth and Czapeks broth had an identical melting point and ultraviolet and infrared absorption spectra as did standard patulin. A melting point of 110.5 C, an ultraviolet absorption maximum of 276 nm, and infrared absorption bands at 5.6, 6.0, and 6.1 ,um were obtained, which correlated with reported values (25). It also gave identical Rf values and color reactions as did standard patulin on thin-layer chromatography analysis. As shown in Table 1, maximum yields of patulin were obtained on potato-dextrose broth after two weeks of incubation of 25 C. Nearly 2.8 mg ofpatulin/ml of media was produced by a thin, heavily sporulated mycelium, which correlated well with the 2.7 mg of patulin/ml obtained by Norstadt ent also affected patulin production by P. patulum. Glucose supported toxin production better than lactose in the presence of NaNO3 at 25 C but essentially made no difference at 5 C. Cultures containing peptonized milk or casein plus glucose generally gave better yields of patulin than when lactose was present. An exception to this was the slightly higher levels of patulin obtained by a basal salts-peptonized milk-lactose medium at 25 C. Lactose cultures in this case yielded 253 ,ug of patulin/ml of media. Lactose in very low concentrations (0.75%) in peptonized milk supported patulin production, but in much smaller amounts than when substantial carbohydrate was present. These results support earlier findings that glucose is an optimal carbohydrate source for patulin production (9). Cultures containing little or no carbohydrate were unaffected by temperature; growth was scant and only very low amounts of patulin were produced at either temperature. The nitrogen source also affected the amount of patulin produced by P. patulum. Inorganic nitrogen in the presence of glucose supported large amounts of toxin production at 25 C, but it supported lesser amounts in the presence of lactose ( Table 1). The amount of patulin obtained from lactose and inorganic nitrogen was approximately the same as that obtained when milk nitrogen sources were present at both incubation temperatures. One exception was that only 84 ug of patulin/ml of basal salts-lactosepeptonized milk was obtained at 5 C incubation. Organic nitrogen from milk lowered patulin yields at 25 C, but it supported substantially more patulin production at 5 C than did inorganic nitrogen when used in combination with glucose. An exception was the basal salts-lactose-casein media, which actually supported more toxin production at 5 C than at 25 C incubation. Overall, casein in the basal salts medium supported more toxin production than did peptonized milk in the presence of glucose. Casein also supported some patulin production (4 gg/ml) by P. patulum in the absence of all carbohydrate. Mold growth on casein without carbohydrate in the basal salts medium was heavy at both 5 and 25 C, even though toxin production was very low. Mold growth on 25% slurries of unwashed and washed casein was heavy but produced no detectable patulin. Washed casein, free from any carbohydrates, did not support mold growth at 5 C. The organism grew abundantly on the shredded cheddar cheese at both 5 and 25 C. However, thin-layer chromatography examination of extracts of the moldy cheese did not detect the presence ofpatulin in any ofthe samples. DISCUSSION The results reported here indicate that lactose and casein supported less patulin production by P. patulum than did glucose and inorganic salts at 25 C. At 5 C, lactose supported a similar amount of patulin production as glucose. However, peptonized milk and casein supported more patulin production at 5 C with either glucose or lactose than the inorganic nitrogen did. In the absence of added carbohydrate at 5 C, both casein and peptonized milk supported patulin production in amounts similar to inorganic nitrogen combined with either carbohydrate. In all of these cases, the total amount of patulin produced was much less than that produced under optimum conditions at 25 C with glucose and inorganic nitrogen, even though growth of the mold was quite heavy. This suggests that dairy products such as natural cheeses, which are high in casein and low in total carbohydrate, might not be good substrates for patulin production, particularly if stored at low temperatures. On the other hand, in the presence of proper nutrients and given sufficient time, P. patulum produced substantial levels of patulin at low temperatures. Thus, refrigeration alone would not be sufficient to completely prevent patulin production in a food product. These data are in agreement with those of Sommer et al. (20). When the organism was grown on shredded cheddar cheese, the results supported these findings. Mold growth was abundant on cheese at both 5 and 25 C, but no patulin was detected in the extracts of any of the complete mold and cheese cultures. In previous studies with meats, breads, flours, and fruit juices, it has been suggested that patulin may react with sulfhydryl-containing compounds present in these foods and may be rendered nontoxic and nondetectable by physicochemical methods (10,17,19,22). This could have occurred in this study in the substrates containing organic nitrogen and in the cheese. On the other hand, no patulin was detected in washed mycelia grown on the 25% casein slurries, whereas patulin was detected in mycelia grown on the basal salts media containing casein. This suggests that casein alone, the lack of carbohydrate, the lack of certain salts, and/or the presence of inhibitory factors associated with high levels of casein may have prevented patulin synthesis even though mold growth was able to occur. Patulin production in a basal salts liquid medium by a strain of P. patulum isolated from cheddar cheese was affected by the incubation temperature, the amount and kind of carbohydrate, and the nitrogen source. An interaction between these factors apparently occurred. Growth and toxin production occurred at 5 C when provided lactose and nitrogen from milk. Thus, refrigerated storage alone would not prevent patulin production by P. patulum. The possibility exists that dairy products on which growth ofP. patulum has occurred may become contaminated with patulin. However, the amount of contamination would probably be low since, in general, these products would be expected to be unfavorable substrates for patulin production. This conclusion is supported by the fact that no patulin was detected in cheese that was heavily molded by P. patulum. Additionally, the presence of sulfhydryl groups in casein in products such as cheese would be expected to detoxify the small amounts of patulin that may be produced. Whether or not these reactions could be reversed to yield free patulin by such factors as low pH or the action of digestive enzymes is not known at this time.

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Survival of Salmonellae During Pepperoni Manufacture Survival of salmonellae in artificially contaminated beef-pork mixtures (approximately 104 salmonellae/g) was studied in pepperoni prepared by either a natural flora or lactic starter culture fermentation or in nonfermented sausages. The pepperoni did not become salmonellae free during the usual commercial 15 to 30-day drying period. Salmonella dublin was present in all products, fermented or unfermented, after 42 to 43 days of drying. At a lower level of contamination, 103/g, S. dublin could not be recovered from started culture fermented pepperoni after 14 days of drying but persisted in the natural florafermented sausage. S. typhimurium (initial count, 104/g) was absent after 42 days of drying when starter culture was used to ferment the pepperoni, but was still present in the natural flora-fermented and unfermented products. S. dublin, host adapted to cattle, or S. choleraesuis, host adapted to swine, had similar survival patterns in beef, pork, or beef-pork pepperoni. Heating salmonellaecontaminated beef-pork pepperoni (after fermentation but before drying) to an internal temperature of 60 C (trichinae inactivating) eliminated the food-borne pathogen from the sausage product. Dry and semidry fermented sausages have rarely been implicated in food poisoning. However, an outbreak of salmonellosis has been attributed to the presence ofSalmonella choleraesuis in Italian dry salami (5), and, in the United States, an incident of staphylococcal food poisoning was traced to Genoa sausage (2). In the latter case, the sausage contained coagulase-positive staphylococci with enterotoxin A; salmonellae were found also. Most workers have approached the study of the survival of salmonellae in fermented sausages by artificially contaminating the product at some step during manufacture and then following the number of survivors in samples taken during the remainder of the processing operations. The fate of salmonellae during the preparation and storage of"isterband," a Swedish fermented sausage (6), thuringer (3), dauerwurst (11), and Lebanon bologna (9) has been studied in this way. In 1972, pepperoni was implicated in a food poisoning outbreak; however, the causative agent was not identified (1). This food poisoning case prompted us to study the potential for survival of selected salmonellae during the processing of pepperoni. S. dublin, S. typhimurium, and S. choleraesuis were used as contaminants in different lots of pepperoni sausages. A 20-h culture of the test organisms grown at 37 C in tryptic soy broth (Difco) was diluted with distilled water to give the appropriate concentration of cells. The diluted culture was mixed into the meat by hand, and then the sausage mixture was stuffed into 55-mm fibrous casings. Fermentation of the pepperoni mixture was carried out either by encouraging the natural lactic microflora through the aging of meat containing 3% NaCl for 10 days at 5 C (7) or by the use of commercial Lactacel MC starter culture (Merck and Co., Rahway, N.J.), a mixture of Lactobacillus plantarum and Pediococcus cerevisiae. The starter culture was used according to the recommendation of the manufacturer except that a straight nitrate cure of 1.2 g of NaNO3/kg of meat was employed instead of nitrite. Unfermented pepperoni prepared from nonaged meat and without the addition of starter culture were processed by the procedures used for the fermented sausages. In those experiments that investigated the influence of a "trichinae cook" on the survival of salmonellae, the contaminated pepperoni were heated in a smokehouse set at 87.8 C (190 F), dry bulb, and 76.7 C (170 F), wet bulb. The sausages were removed when the internal temperature reached 60 C (140 F), quartered with a sterile knife, placed in plastic bags, and immersed immediately in an ice bath. The quartered sausages were kept refrigerated until assayed for salmonellae. Determination of the viable salmonellae count has been described in a previous publication (8). 760 SMITH ET AL. RESULTS The influence of Salmonella serotype as well as the type of meat used to prepare pepperoni were studied to determine the survival of salmonellae during processing. Beef, pork, or beefpork blends were prepared, and aliquots were contaminated with S. dublin (host adapted for cattle), S. typhimurium, or S. choleraesuis (host adapted for swine), respectively. The sausages were fermented by the natural lactic flora. These Salmonella serotypes were selected to determine whether a host-adapted serotype would behave differently from a non-hostadapted serotype in the three types of meat. However, there was very little difference in the survival patterns in pepperoni of the three Salmonella serotypes regardless of the meat type used. Since all commercial pepperoni are prepared from beef-pork mixtures (Palumbo et al., J. Food Sci., in press) and since there was no difference in Salmonella survival in pepperoni prepared from different meats, a beef-pork (1:1) mixture was used in all subsequent experimentation. The survival of S. dublin and S. typhimurium in starter culture-fermented, natural flora-fermented, and unfermented pepperoni made from a mixture containing equal amounts of beef and pork is shown in Tables 1, 2, and 3. The pepperoni that had starter culture incorporated into the meat mixture showed a decrease in pH from 6.1 to 4.5 after 1 day of fermentation at 35 C and a decrease in viable numbers of both test organisms (Table 1). No viable S. typhimurium were detected after 29 days of drying, whereas S. dublin was still detected after 43 days of drying. The pH decrease in sausages fermented by the natural flora was less pronounced than in those fermented by starter culture ( Table 2). The pH of the latter decreased from 6.1 to 5.1 in 2 days at 35 C. There was a decrease in the number of viable salmonellae during the fermentation period (Table 2), but after 42 days of drying viable S. dublin and S. typhimurium were still present. There was virtually no change in pH in the unfermented pepperoni after 2 days at 35 C; at 1 day, there was growth of both Salmonella serotypes followed by a decrease in growth on day 2 (Table 3). During drying the acid content increased, resulting in a slight lowering of the pH (from 6.0 to 5.7). There was no decrease in viable S. dublin within a drying period of 42 days; S. typhimurium decreased to a low level but was still detected at 42 days of drying (Table 3). S. dublin was shown to be more resistant to the processing conditions involved in pepperoni manufacture than S. typhimurium (Tables 1, 2, and 3). In the initial experiments, a relatively high number (about 4 x 10"/g) of S. dublin was added to the meat mixture used to make the pepperoni. When an initial inoculum of 1.7 x 103 S. dublinlg was used in natural flora-fermented pepperoni (pH 4.8 after a 2-day fermentation), there was still about 1 viable cell/g at the end of 42 days of drying. On the other hand, when the initial inoculum of S. dublin was 9.0 x 10'/g in starter culture-fermented pepperoni (pH 4.6 at 1 day of fermentation), no S. dublin could be recovered after 14 days of drying. Since pepperoni is usually made from porkbeef mixtures, the sausage or the pork must be given some treatment to inactivate trichinae which might be present. Trichinae can be inactivated by standardized freezing, heating, or drying. Large-scale industrial, freezing of pork for use in pepperoni is impractical and it has been demonstrated that drying is not completely reliable in destroying salmonellae in pepperoni (Tables 1, 2, and 3). Therefore, the fate of salmonellae added to pepperoni subjected to temperatures that would inactivate trichinae was investigated. The data in Table 4 indicate that heating the sausage to an internal temperature of 60 C (140 F) destroyed S. dublin remaining after the fermentation step (both starter culture and natural flora). The data indicate that salmonellae in unfermented pepperoni were destroyed by heating also. Salmonellae were not detected after 14 days ofdrying, indicating that there was no recovery of cells that might have been only heat injured. DISCUSSION Pepperoni is a highly spiced, fermented, dried sausage characteristically prepared from pork or pork and beef. The fermentation is similar to that of Lebanon bologna (7), but pepperoni is less acidic and is subjected to a long drying period. In a previous study, we found that salmonellae (at a level of about 104/g) was unable to survive the Lebanon bologna processing conditions except occasionally when natu- c The sausages were cooked to an internal temperature of 60 C (140 F). d Bacterial numbers lower than 0.03/g were considered to be zero. e After cooking, the sausages were placed in the dry room at 12 C and 60 to 65% RH. ral flora fermentation was used (8). The data obtained in this study indicated that salmonellae were not consistently killed by the processing conditions that were used in the manufacture of pepperoni. Fermentation with Lactacel MC starter culture was more effective than natural flora fermentation in destroying salmonellae. The same batch of meat was used to compare the effect of starter culture (Table 1) and natural lactic flora (Table 2) on the survival of S. dublin and S. typhimurium during processing. The pH of the pepperoni prepared with starter culture was lower than that prepared by the natural flora process, but viable S. dublin were still present after 42 days of drying with both types of fermentation. Viable S. typhimurium were present after 42 days of drying in pepperoni prepared with natural lactic flora but not with starter culture (Tables 1 and 2). When the initial inoculum of S. dublin was decreased approximately 50-fold (to about 103/g), no viable cells were found in pepperoni prepared with starter culture, but S. dublin was still present in sausages made by the natural flora fermentation (after 42 days of drying). Smith et al. (8) found that in Lebanon bologna contaminated with salmonellae starter culture fermentation was more effective than natural flora fermentation in eliminating the food pathogen from sausage. S. dublin, host adapted to cattle, orS. choleraesuis, host adapted to swine, behaved similarly in pork, beef, or beef-pork pepperoni. Host adaptation did not predispose the Salmonella serotypes to differential survival depending on the meat type used to prepare the sausage. The pH values of commercial pepperoni produced by nine different companies showed a wide range: 6.1 to 4.7. Three of the products ranged in pH from 5.6 to 6.1; three ranged from 5.1 to 5.3; and three ranged from 4.7 to 4.9 (Palumbo et al., J. Food Sci., in press). Since a sausage with a pH of 5.5 or above would not be considered a fermented product, a study of the survival of salmonellae in unfermented pepperoni was indicated. There was an increase in S. dublin during the processing period from an 'initial 4.1 x 104/g to 1.3 x 10c/g after 42 days of drying; S. typhimurium decreased to a very low value but was till detectable at 42 days of drying (Table 3). Unfermented pepperoni is marketed and presumably preferred by some consumers. The data presented in Table 3 suggest that Salmonella serotypes similar to S. dublin would probably survive in high numbers in such unfermented products and thus lead to a contaminated sausage. S. dublin was more resistant to destruction than S. typhimurium during the production of pepperoni (Tables 1, 2, and 3) and under the processing conditions for Lebanon bologna (8). Since all commercial pepperoni contain pork (Palumbo et al., J. Food Sci., in press), a trichinae-inactivating heating step at the end of fermentation and before drying was investigated to determine if this would result in the destruction of salmonellae. An internal temperature of 60 C (140 F) in the sausage was effective in destroying S. dublin (Table 4). Since salmonellae were not always killed during pepperoni processing (in the absence ofheating), apparently the safety of commerical pepperoni is due to other factors such as low contamination levels, postproduction heating (as in pizza), or by differences between commercial pepperoni processing methods and ours. Surkiewicz et al. (10) found that 28% of 560 fresh pork sausage samples were contaminated with low numbers of salmonellae, whereas only 0.4% of 735 fresh beef patties contained salmonellae (9). Therefore, pepperoni made from beef and pork mixtures could be expected to be occasionally contaminated with salmonellae. Commer-762 SMITH ET AL. cial companies normally hold their pepperoni in the dry room for 15 to 30 days (4), and since the level of salmonellae, if present at all, is usually very low, the number of contaminants might be reduced to undetectable levels by the end of the drying period. Our data indicated a large reduction of salmonellae during fermentation and drying. However, in cases of high contamination levels, an unsafe product could result. Inclusion of a trichinae heating step of 60 C in commerical pepperoni production would ensure a Salmonella-free product.

v3-fos

2018-04-03T05:38:00.600Z

1975-03-01T00:00:00.000Z

43697039

s2

Stability of Aflatoxin B, and Ochratoxin A in Brewing The stability of aflatoxin B, and ochratoxin A in brewing was investigated by adding the purified toxins to the raw materials at 1 and 10 yg/g levels during mashing in a conventional micro-brewing process. The results indicate that both toxins are stable to heat and are insensitive to cooker mash treatment. Both mycotoxins were partially removed in the mashing and brewing processes. About 14 to 18% and 27 to 28% of the added toxins were found in the final beers brewed from starting materials containing 1 and 10 Ag, respectively, of either toxin per g. The possible route of transmission of mycotoxins into beer is discussed. Since the discovery of aflatoxins, a series of potent carcinogens produced by Asperigillus flavus and A. parasiticus, a number of other fungal metabolites have been demonstrated to be toxic to animals (6,9). Among these toxins, ochratoxins, a series of nephrotoxins produced by several species of Aspergillia and Penicillia, have also attracted attention because of their high toxicity and the widespread occurrence to toxin-producing fungi (4). Aflatoxins and1 ochratoxins have been found in many agricultural commodities (4,6), including corn (13)(14)(15). Because of the unusual stability of these toxins in agricultural commodities, the question of whether they could carry through the food process has attracted considerable attention. Because of the natural occurrence of ochratoxin A (OA), the most potent toxin in the ochratoxin series, in barley and other cereals (10,12), scientists in the United States and Denmark have studied the possibility of transmitting OA into beer. Fischbach and Rodricks (8) reported that earlier work in the Food and Drug Administration had shown the retention of OA in the beer process, but a survey on OA and ochratoxin B in samples of beer and malted barley from each of the 130 breweries in the United States showed no detection of ochratoxins. These samples were tested by using an analytical method sensitive to 0.01 gg/g (10 jig/kg). Recent experiments by Krogh et al. (11) demonstrated, however, that OA was destroyed during the malting and brewing processing. To determine the stability of aflatoxin B, (Afla Bj) and OA in brewing, we added the purified toxins at two stages (to either malt or corn grits) during a micro-brewing process. This paper presents data on the stability of these two toxins in five major operations of brewing. MATERIALS AND METHODS Materials. Afla B, and OA were prepared by the methods of Chu (3) and Chu and Butz (5). Malt was prepared from Larker barley (Hordeum vulgare L.) that was grown in North Dakota in 1972. Portions (170 g, dry basis) were steeped in water at 16 C until the moisture content was 45%. The barley was allowed to grow at this temperature in the dark. The malting (growth) chamber is a modified drum type that slowly rotates 36 perforated cans containing the portions of barley. Moisture-conditioned air was circulated through the chamber and the samples during the 5-day malting period. The germinated barley was then kilned according to the following program: 9 h at 35 C, 7 h at 44 C, 5 h at 55 C, 5 h at 65 C, and 2 h at 85 C. Rootlets were removed after kilning, and the malt was ground in a Miag mill (Miag Co., Braunschweig, West Germany) prior to mashing. Corn (Zea mays L.) grits were obtained from the Krause Milling Co., Milwaukee, Wis. Adsorbosil-5 and Silica Gel G were obtained from Applied Science Laboratories (State College, Pa.) and the Brinkmann Instrument Co. (Westbury, N.Y.), respectively. All other solvents and chemicals were either analytical reagent grade or chemically pure. Micro-brewing procedures. The brewing procedure was that described by Burkhart et al. (2), in which 90% of the malt is used in the malt mash and 10% in the cooker mash. The essentials of the operation are as follows. (ii) Malt mash. Malt (189 g) and water (718 ml) were mixed at 38 C for 10 min. The mixture was allowed to stand at 38 C without stirring for 50 min for optimal hydrolysis of proteins. (iii) Combined mash. The cooled cooker mash was added, and the temperature was raised to 72 C. The mash was maintained at this temperature for 20 min for optimal carbohydrate hydrolysis. The mash temperature was then raised to 75 C, and the mixture was filtered. (iv) Wort. The entire filtrate (wort) was boiled with 1.5 g of hops for 90 min. Twenty minutes before the boiling was completed an additional 2.0 g of hops was added. The boiled wort was filtered and cooled to 12 C. Fermentation and storage. The wort was pitched with 5 g of moist cake yeast (1 g, dry basis) (Saccharomyces cerevisiae) and fermented in a covered, but not sealed, flask at 12 C for 8 days. A 64-mg amount of commercial chill-proofing preparation (crude papain) was added, and the beer was transferred to a storage flask and maintained at 0 C under 12 lb/in2 pressure of carbon dioxide for 14 days. The beer was then filtered and bottled under 12 lb/in2 carbon dioxide pressure. For pasteurization the bottled beer was kept at 60 C for 20 min and then cooled to room temperature in 15 min. The beer was stored thereafter at 12 C. Spiking of the purified toxins. For Afla Bl appropriate amounts of toxin in 5 ml of chloroform were added to the malt or corn grits at the beginning of the cooker and malt mashing stages at 0, 1, and 10 Mg/g levels (weight basis). The chloroform was removed by evacuation. For OA, the toxin was dissolved in 1 to 2 ml of 1% NaHCO. and added to cooker and malt mash directly before processing. Duplicate experiments were made for each level of toxins added. Extraction of brewing preparations and toxin analyses. Duplicate samples were analyzed at five stages during the brewing process: (i) the completed cooker mash; (ii) the malt mash at the end of the protein hydrolysis stage; (iii) the combined mash at the end of the carbohydrate hydrolysis stage; (iv) the wort after addition of hops and boiling; and (v) the bottled beer after pasteurization. The following procedures were used for toxin analysis. (i) Afla Bl. For Afla B,, official methods of analysis were followed (1). Samples of 100 g or 100 ml were extracted with chloroform according to Sect. 26.018 (a) (ref. 1). After evaporation of the chloroform from the extracts, the samples were redissolved in 1 to 2 ml of chloroform and chromatographed as described in Sect. 26.019 (a) (ref. 1). The chloroform elution fraction was evaporated to dryness, redissolved quantitatively in 1 to 5 ml of chloroform, and then chromatographed on a glass plate (20 by 20 cm) with a 250-um layer of Adsorbosin-5; methanol-chloroform (3:97) was the developing solvent. Quantitations were made by a fluorodensitometric method (5), except that a photo volt densitometer model III Turner fluorometer TLC scanner (G. K. Turner Associates, Palo Alto, Calif.) with primary and secondary filters of Turner no. 110-811 and 110-816 was used. (ii) OA. For OA determination, 100 g each of treated brewing product and a control was blended with 100 ml of 2% NaHCO, solution for 5 min and then centrifuged at 3,000 x g for 10 min. The supernatants were transferred to separatory funnels. The residues were blended twice with 150 ml of 1% NaHCO, for 5 min and centrifuged again as described above. All of the supernatants of each batch were then pooled and acidified with concentrated HCl to pH 2. Likewise, 100 ml of beer was directly acidified to pH 2.0 with HCl. The acidified solutions were extracted twice with equal volumes of chloroform in separatory funnels. The chloroform extract was then evaporated to dryness and dissolved quantitatively in 8 ml of chloroform for thin-layer chromatography analysis on Adsorbosil-5 as described before; the solvent system was benzene-acetic acid (9:1; 7). Quantitation was done on a Turner fluorometer as described for Afla B l- RESULTS AND DISCUSSION The validity of the present data rests primarily on the method of extraction and analysis; thus, control experiments, in which test mycotoxins were added to the brewing products after each process, with subsequent analysis of toxin content, were carried out. The results showed that the toxin recovery ranged from 78 to 91% for each step, with an overall average of 85%. Since the recovery yield falls into the range of mycotoxin analysis in other food (4), the analytical methods used in the present study might also be used for routine analy'sis of these mycotoxins in beer or related products. The presence of alcohol was removed by evaporation under vacuum, and the remaining material was analyzed for toxins. The results showed that both the treated (alcohol removed) and the untreated samples gave similar recoveries (86.6 versus 89.3%). The toxins were then directly extracted from the beer or acidified beer (for OA) with organic solvents before analyzing. The levels of Afla B, and OA in brewing at five stages are given in Table 1. In general, both mycotoxins were partially lost during the brewing. The patterns for the loss of both mycotoxins were similar; both were relatively stable in the cooker mash step but were more sensitive to later treatments, especially the protein hydrolysis, wort boiling, and final fermentation. Since both mycotoxins are relatively stable to heat (4,9,16), it is not surprising that more than 90% of STABILITY OF AFLA B, AND OA IN BREWING (14) Values in parentheses indicate a correction of 15% due to loss in the analysis. the added toxins were in the sample after cooker mash treatment. Removal of toxin from the product was very significant in the malt mash (with a loss of 12% for a 10 Mg/g sample and 24 to 27% for a 1 Ag/g sample), in boiled wort (20 to 30% loss of material added to the malt mash), and in the final fermentation (another 20 to 30% loss from the boiled wort step) steps. Since it has been observed that OA can be hydrolyzed by acid and some proteases to ochratoxin a and phenylalanine (4) and since ochratoxin a has been detected in the wort when OA-contaminated barley was used in the brewing (10), removal of OA in the brewing process may primarily be due to such hydrolysis. The mechanism of Afla Bl removal, however, is not known. The significant loss of both mycotoxins between the combined mash and boiled wort steps may be due to the nonspecific interaction or adsorption of both mycotoxins by the solid particles removed by the filtration process. Both mycotoxins were partially removed in brewing, but considerable amounts were transferred into the beer, especially if the contamination level was high. Approximately 25% of the added toxins were found in the final product when 10 ug of the toxin per g in the cereal was tested. At the lower testing level (1 Ag/g), recovery was about 15%. Our results on OA differ from those reported by Krogh et al. (11). They found significant loss in mashing (70% lost before boiled wort), and less than 10% of OA was recovered when barley, containing 1 to 5,ug of toxin per g, was used in the brewing. It is not known whether these contradictions are due to a difference in their brewing processes, in which lactic acid and bacterial enzymes were used, or to the method of extraction and analysis for which recovery yield in the control sample was not reported by the investigators. Inasmuch as mycotoxins in contaminated materials are not completely removed from the beer in brewing and might be converted to some unknown products which might also be toxic, the best protection would be to prevent the use of contaminated raw materials in brewing. In order that barley germinate and grow (malt) satisfactorily, and therefore meet the requirements of the malting industry, it must be stored under conditions which would not promote the growth of storage molds. Although Krogh et al. (11) demonstrated that when barley was heavily contaminated with OA it thus failed to germinate and would be rejected for malting, it is not known whether or not aflatoxin would also affect the malting process. Moreover, the material used as adjunct (the constituent other than malt in the cooker mash) functions primarily as a source of starch and other carbohydrates which are largely hydrolyzed to maltose during the "conversion" or carbohydrate hydrolyzing stage of mashing. Possibly these adjuncts (corn syrups, ground rice, and ground barley are commonly used), if they are already contaminated in the field or if they are stored under adverse conditions, could contribute mycotoxins to the mash.

v3-fos

2020-12-10T09:04:20.513Z

1975-02-01T00:00:00.000Z

237229825

s2

Influence of Modified Atmosphere Storage on Aflatoxin Production in High Moisture Corn Samples of freshly harvested corn and remoistened corn were inoculated with Aspergillus flavus and stored for 4 weeks at about 27 C in air and three modified atmospheres. Aflatoxins and fat acidity were determined weekly. Corn stored in the modified atmospheres did not accumulate over 15 μg of aflatoxin B1 per kg and 20 μg of total aflatoxins per kg. Corn from the high CO2 treatment (61.7% CO2, 8.7% O2, and 29.6% N2) was visibly molded at 4 weeks and had a higher fat acidity than the other treatments. In the N2 (99.7% N2 and 0.3% O2) and controlled atmosphere (13.5% CO2, 0.5% O2, 84.8% N2) treatments, a fermentation-like odor was detected. When the corn was removed from the modified atmospheres it deteriorated rapidly and was soon contaminated with aflatoxins. CO2, 8.7% 02, and 29.6% N2) was visibly molded at 4 weeks and had a higher fat acidity than the other treatments. In the N2 (99.7% N2 and 0.3% O) and controlled atmosphere (13.5% CO2, 0.5% 02, 84.8% N2) treatments, a fermentation-like odor was detected. When the corn was removed from the modified atmospheres it deteriorated rapidly and was soon contaminated with aflatoxins. Modified atmospheres have shown promise in controlling the Aspergillus flavus group and the subsequent aflatoxin production in stored peanuts. Sanders et al. (9) reported that little aflatoxin was found on inoculated peanuts maintained in an atmosphere of 60% CO2, 20% 02, and 20% N2, as compared with 206 pg of aflatoxin per g of peanuts stored in normal air at 25 C and 90% relative humidity (RH). The high CO2 atmosphere is similar to that recommended by Jay (4) for control of insects in stored products. Landers et al. (7) reported little aflatoxin production on peanuts with a 28.9% moisture content held in an atmosphere of 99% N2 and 1% 02. Pattee and Sessoms (8) reported that increases in fat acidity were highly correlated with visible A. flavus growth, and that fat acidity reached 60 mg of KOH per 100 g of peanut kernels before aflatoxins were detected. However, Landers et al. (7) found aflatoxins in peanuts with fat acidity lower than 60 mg of KOH per 100 g of kernels. Sauer and Christensen (10) observed increases in fat acidity in corn stored at moisture contents of 15.5 to 16.5% at 25 to 30 C. Recent studies by Jay et al. (6) and by Jay and Pearman (5) demonstrated that modified atmospheres created by purging storage structures with CO2 were effective in controlling insects in stored products. In laboratory studies Harein and Press (1) found that insects in stored peanuts could be controlled with N2 if less than 1% 02 was present. Controlled atmosphere (CA) generators have been used for many years for maintaining the quality of stored apples and for preserving alfalfa pellets. These generators consume either natural or bottled gas and remove the water vapor caused by combustion. The atmospheres produced usually contain 10 to 14% CO2 and less than 1% 02; the balance is principally N2 and argon, and occasionally some carbon monoxide if a high fuel-toair ratio prevails. In field tests with large CA generators, adult confused flour beetles, Tribolium confusum Jacquelin du Val, were controlled in 20,000 bu silos of wheat within 24 h (11). Aflatoxins have been of prime concern in peanuts and may occur in several other crops including corn. Trenk and Hartman (12) demonstrated that aflatoxins were produced in corn at moisture levels above 17.5% when temperature was 24 C or more. They also found that remoistened corn was subject to more rapid deterioration and subsequent aflatoxin formation than freshly harvested corn. Hodges et al. (2) found that fast cooling rates in bins were more effective in maintaining quality than slow cooling rates. They showed that slow cooling with initial temperatures above 70 F permitted rapid growth of A. flavus, heating, and some aflatoxin contamination in corn. Modified atmospheres to achieve control of both insects and the A. flavus group of fungi seems to be a relevant approach to these problems. Therefore, tests were designed to study the effects of high CO2, high N2, and an atmosphere from a commercially available CA generator on A. flavus infestation and aflatoxin production on high moisture corn. MATERIALS AND METHODS Cylinders of gas were used to supply the desired atmospheres to 3.8-liter jars containing corn. The gas flowed from the cylinders through pressure regulators and micrometer regulating valves connnected to flow meters, then through gas washing bottles containing glycerine-water mixtures calibrated to the initial RH of the inter-kernel atmosphere of the corn to temper the gas to the desired RH. The gas then passed through 7.6-m coils of copper tubing suspended in the 26.7 ± 1 C water bath connected to copper tubes (6.4 mm in outer diameter by 30 cm in length). These tubes entered through the lids, and the gas from these tubes diffused into the corn. The gas was exhausted to the atmosphere through an additional copper tube (6.4-mm outer diameter by 5.4 cm in length) mounted in the lid. This tube was also used to sample the gas composition in the jars. A bimetallic thermometer with a 22.9-cm stem and a humidity probe (Hygrodynamics, model 15-3001) were inserted through openings in the lid. In the first series of tests two replicates were run, one in each water bath. In the first series, freshly harvested Iowa-grown shelled corn having an initial moisture content of 29.4% was used. Before exposure to the gases, the corn was inoculated using a power atomizer with a 150-ml spore suspension of A. flavus Lk., isolate NRRL 5520 containing about 106 spores/ml giving 780,000 spores/liter of corn. The jars were then filled with the corn, sealed with tape, and placed in the water baths. Initial gas flow into the jars was 150 m/min and subsequent flow rate was reduced to 40 ml/min when the composition of the atmosphere in the jars approximated the atmospheres from the cylinders. In the first series the corn was exposed to four different atmospheres: (i) air with 0.03% C02, 21% 02, and 78% N2; (ii) the output product of a CA generator previously pumped into cylinders with about 13.5% CO2, 0.5% 02, and 84.8% N2; (iii) 99.7% N2 and 0.3% 02; (iv) a blended atmosphere having a composition of about 61.7% C02, 8.7% 02, and 29.6% N2. Two 0.4-liter samples were taken from each of four jars exposed to each of these four atmospheres after 1, 2, 3, and 4 weeks of exposure. One of the two 0.4-liter samples was oven dried at 65 C for 24 h. The duplicate sample was exposed to the atmosphere for 1 week in a room maintained at 26.7 + 1 C and 60 + 5% RH, then dried. The dried samples were held at 0 C until aflatoxin and fat acidity values were determined. Three replicates were run in a similar manner in the second series of tests. However, there were no continuous exposures for the entire 4-week period without sampling. Corn used in the second series of tests was Georgia grown and had been in cold storage for about 1 year. It had an initial moisture content of 14.2% and was tempered to 19.6% by the addition of water and thorough mixing in a drum tumbler contained in a cold room maintained at 5.6 C. The corn was tumbled for 1 h a day for 1 week and then was inoculated in the same way as in the first series with A. flavus spores. Moisture content of the corn in each container was determined initially and at the end of the 4-week exposures with a Motomoca model 919 moisture meter. Temperature and RH in each jar were taken daily Monday through Friday during the 4-week incubation. Also, gas samples were taken daily from each container with a 10-ml gas-tight syringe and were injected into a Fisher-Hamilton model 29 gas partitioner for analysis. This partitioner was equipped with a 0.5-ml gas sample loop and a dual column, dual detector system for separating the individual components. Output from the partitioner was integrated with a Vidor 6300 digital integrator. Aflatoxins from 50-g samples were extracted and determined using thin-layer chromatography according to the official AOAC method I (3). The fat acidity (mg of KOH required to neutralize free fatty acids from 100 g of corn) was determined using the official AOAC rapid method for corn (3). RESULTS Fat acidity and aflatoxin contents of corn after exposure to four different atmospheres for 1 to 4 weeks are given in Table 1. The fat acidity did not change greatly in corn exposed to CA or N2 in either test. When subsamples from these treatments were exposed to laboratory atmosphere for 1 week, however, fat acidity increased in all cases. In the modified atmospheres fat acidity was highest in the CO2 plus low 02 treatment in both series; and increased with the length of exposures. The corn in the freshly harvested series had little visible mold after 4 weeks in the CA or N2 atmospheres. A pleasant aromatic odor was detected, suggesting that fermentation was in progress. Corn in the CO2 plus low 02 atmosphere was visibly molded in both series and had an unpleasant odor after 4 weeks. The corn exposed to the normal air was visibly moldy after 1 week. Fat acidity and aflatoxin values were not determined after the corn was badly deteriorated in the air treatment. Aflatoxin synthesis was slight in the modified atmospheres in both series. Substantial increases occurred, however, when exposed to air. Table 2 presents the original gas compositions and the mean gas concentration in the effluent during the tests. The CO2 rose above 13% and the 02 concentration dropped below 10% in both of the air treatments in the first series due to the high rate of respiration and germination of the moist corn and microbial respiration. In the first series, corn exposed to gas from the CA generator or the N2 exhibited less than 1% rise in CO2 during the exposure period, whereas the Values are means of three replications. d Exposed to indicated atmosphere for stated length of time and then subsample exposed to the atmosphere for 1 week before sampling. ' Mean of four replications. ' No data taken due to deterioration of corn. CO2 concentration increased 3.6% in containers of corn exposed to the CO2 plus low 02 mixture, and the 02 concentration dropped 4.5%. In the second series (Table 2), the corn had a lower initial moisture content and corn exposed to air had a CO2 increase of 3.7%, while the 02 concentration dropped from 21 to 17.6%. Carbon dioxide increase in corn exposed to gas from a CA generator or to N2 was slight, whereas no CO2 increase was observed in corn exposed to CO2 plus low 02. An 02 reduction of less than 1% occurred in the CO2 plus low 02 exposure. Table 3 presents RH of the container air, initial and final moisture content of the corn, and temperature of the corn. Atmospheres of corn exposed to air had a higher RH in both series than containers exposed to other gases, with the exception of the exposure to CO2 plus low 02 in the first series in which the RH was about the same as that in containers exposed to air. The increases in RH and moisture were due to respiration. In the first series when corn was exposed to air, the moisture content rose from 29.4 to over 50%, whereas moisture in corn exposed to either CA or N2 rose only 3.4 to 4.2%. The moisture content of corn exposed to CO2 plus low 02 rose to 49.1%, or 19.7% during the exposure. Mean temperature was highest in corn exposed to air in the first series and ranged to over 30 C. Mean temperature of corn in this series exposed to other gases was about the same as that of the waterbaths (26.7 i 1 C) with the exception of the exposure to the CO2 plus low 02 mixture in which the mean was 27.6 C, and the highest temperature recorded was 28.3 C. In the second series the mean temperature of corn exposed to air was "Means calculated from 20 air samples from each container of each replication of each series; means in the first series are from two replications; means from the second series are from three replications. c Freshly harvested corn with 29.4% initial moisture. dRemoistened corn with 19.6% initial moisture. ' Corn held for 4-week period and sampled at the end of the 4-week exposure. l C or more higher than the other exposures. Temperatures in all exposures except air did not rise above 27.2 C. DISCUSSION None of the modified atmospheres tested allowed high levels of aflatoxin contamination. Subsamples of corn from all treatments exposed to normal atmosphere for 1 week, however, were generally contaminated with aflatoxins. Freshly harvested corn exposed to CO2 plus low 02, and the 4-week treatments with CA were not contaminated with aflatoxin upon exposure to air. Aflatoxins were formed in the corresponding treatments in the remoistened corn, this possibly reflects microbial activity or physioicgical differences between freshly harvested and remoistened corn. Appearance of remoistened corn did not seem to change much with exposure to CA or N, for 4 weeks. Freshly harvested corn in these treatments had undergone some fermentation but still appeared sound. The use of N, or CA treatments may produce corn desirable for feed. Further studies on the quality factors are needed to find if CA treatment would be useful as a means of holding corn temporarily before drying. If the gases from CA were mixed with enough 02 to block anaerobic fermentation, the fermentation problem might be eliminated and a residue-free, economical storage system could then be adopted. Experiments are planned using higher levels of 0..

v3-fos

2020-12-10T09:04:20.792Z

1975-07-01T00:00:00.000Z

237234945

s2

Presence of Two Virus-Like Particles in Penicillium citrinum Two icosahedral virus-like particles (28 and 19 nm in diameter, respectively) have been detected in sporogenic and asporogenic segregants of a strain of Penicillium citrinum. The distribution of the two particles differed among the two segregants. Virus-like particles have been detected and physicochemically characterized in various species of Penicillium (12). As reported previously, such particles are present in a wild strain of Penicillium citrinum Thom (3,5,15). This strain segregates spontaneously and at high frequency for small patches of white asporogenic mycelium which, in turn, may redevelop into normally green, sporogenic mycelium. We have been able to detect in this unstable strain the presence of two virus-like particles which can be distinguished by their dimensions and distribution in these two mycelial types. The organism was grown at 24 C on potatodextrose broth or on potato-dextrose agar, pH 7.0. Extracts were made from both mycelial types after 24 and 48 h of growth (1, 2) and were negatively stained with 2% (wt/vol) phosphotungstic acid (pH 7.2) containing dimethyl sulfoxide (13) and examined with a Siemens IA electron microscope operating at 60/80 kV. In these preparations two distinct virus-like particles were observed, one (Pcit-1) ranging from 26 to 28 nm in diameter and the other (Pcit-2) 17 to 19 nm. Both particles were clearly seen as icosahedrons and sometimes were observed as empty, single-capsid structures ( Fig. lb and 2b). Particles Pcit-1 and Pcit-2 differed in their relative distribution among the two types of mycelial segregants, in that Pcit-1 was predominantly seen in extracts from sporogenic mycelia (Fig. 2b) whereas Pcit2 was more abundant in similarly prepared extracts of white, asporogenic mycelia (Fig. lb). It should be stressed, however, that both particle types were present in all mycelia. These findings from negatively stained preparations were confirmed by observations of viruslike particles in thin sections of chemically fixed hyphal fragments prepared according to methods described previously (8). The particles were found normally associated with, or enclosed within, membranous structures in parts of the hyphae with prominent vacuolization or full degeneration ( Fig. la and 2a). In the virusinfected hyphae of P. citrinum membrane whorls, tubule formations and filamentous structures of unknown origin were frequently seen as well. The association of virus particles in fungi with membrane systems has been noted by other investigators (17,21; see also review 12) and might indicate an attempt of the fungus to delimit a potentially lytic virus into separate bodies. Two serologically distinct viral particles have been identified in Pencillium stoloniferum Thom (6,18,20) and three components of a single virus have been recognized in Penicillium chrysogenum Thom on the basis of their content of different double-stranded ribonucleic acid molecules (14). However, these particles, in contrast to those present in P. citrinum, are uniform in size. Pcit-2, moreover, appears to be the smallest virus-like particles detected so far in fungi. The two virus-like particles present in P. citrinum, if indeed viral in nature, may contain different, perhaps complementary, genomic determinants, and for either particle to replicate both particles have to infect the same hypha. There are precedents for multicomponent and interdependent viral systems in fungi and else- Virus-like particles for the most part are enclosed in a membrane. Tris(hydroxymethyl)aminomethane-1aziridinyl-phosphine oxide glutaraldehyde plus osmium fixation. The bar indicates 200 nm. (b) Negative staining of the sporogenic mycelial extract showing virus-like particles, 26 to 28 nm. Note the presence of empty capsid and the presence of a core-like structure in some particles (compare with Fig. la). Phosphotungstic acid plus dimethyl sulfoxide preparation. The bar corresponds to 50 nm. where (10,16,18). If this is in fact the case with the viruses of P. citrinum, the dissimilar distribution of particles in the two mycelial types, which show profound differences in sporulation efficiency, antibiotic production, and growth rate (3,15), suggests that these particles in disproportionate combination may somehow control fungal metabolism and differentiation. Some authors have already suggested a relationship between mycoviruses and extrachromosomal genetic factors (4,7,11,19) and we are currently investigating this prospect in P. citrinum. Preliminary experiments of curing have shown that cycloheximide promotes the reversion of the asporogenic mycelium into the sporogenic one with its own typical properties. In Saccharomyces cerevisiae Meyen ex Hansen, cycloheximide has been shown to be an effective curing agent of the "killer" determinant, an extrachromosomal genetic factor (9) that recent studies have strongly related to the presence of double-stranded ribonucleic acid containing virus-like particles (10). We are greatly indebted to Paul A. Lemke for his constructive criticism and helpful suggestions in preparing the manuscript. We wish to thank F. Cecere, G. Ignazzitto, M. Mari, and B. Pasquetti for their assistance.

v3-fos

2014-10-01T00:00:00.000Z

1975-08-01T00:00:00.000Z

14058252

s2

Effects of serum on mammary epithelial proliferation in vitro during mammary gland development. The proliferative response of mammary gland epithelium from nonpregnant, pregnant, and lactating mice to mammary serum factor and insulin was studied in vitro. Mammary gland epiithelium from nonpregnant and lactating animals has a delayed proliferative response to mammary serum factor and insulin when compared to the response of epithelium from pregnant animals. The results show that as the animals go through pregnancy into lactation the mammary gland epithelium becomes less responsive to mammary serum factor while it retains its responsiveness to insulin. The concentration of mammary serum factor in sera from animals at various physiological stages is constant. Sera from hypophysectomized rats, on the other hand, show a 50% drop in mammary serum factor activity. This loss of activity cannot be reversed by injecting prolactin, 17-beta-estradiol, or growth hormone into the hypophysectomized animals. A hypothesis that the mammary gland is composed of two proliferative epithelial populations is developed, and the possible role of prolactin in stimulating DNA synthesis is discussed. There have been described a number of tissue culture systems in which cellular proliferation is initiated by factors found in serum (1). Some of these studies have reported factors active in cell lines lacking particular differentiative functions, while others have reported factors that affect specific cell types or tissue types. That these factors may have some physiological role is suggested by the growing number of reports of peptide hormones such as somatomedin (2), erythropoietin (3), nerve growth factor (4), epidermal growth factor (5), and angiogenesis factor (6), which selectively promote cell proliferation in target tissues. Recently it has been reported that components of serum promote DNA synthesis in mammary epithelium in vitro (7 10). The effect is selective in that only the epithelial and not the stromal elements of the gland respond. This is due to serum components other than insulin, which is a known mitogen in this system (9). In this paper we describe further experiments on the biological response of mammary epithelium to mammary serum factor as the gland undergoes the changes of pregnancy and lactation, and we develop a hypothesis that the gland is composed of two proliferating epithelial cell populations. .4 nimals and Supplies C3H and BALB/C female mice at least 2 mo of age (Simonsen Laboratories, Gilroy, Calif.) were used in THE JOURNAL OF CELL BIOLOGY 9 VOLUME 66, 1975 9 pages 243-250 these studies. Timed pregnancies were determined by the vaginal plug method. 2-mo old male hypophysectomized rats were purchased from Simonsen Laboratories. 2 wk after surgery, bovine growth hormone (200 #g/day), ovine prolactin (200 #g/day), or I7-/3-estradioI (1 #g/day) was injected intraperitoneally into each animal for 3 consecutive days and the animals were bled on the 4th day. Tissue Culture Techniques The techniques used for organ culture of inguinal mammary glands of mice have been previously described (9). In brief, explants were cut from the inguinal mammary glands and placed on stainless steel screens floating in Medium 199 (Grand Island Biological Co., Grand Island, N. Y.) for 48 h. At this time, the medium was removed and the test medium was added for an additional 22 24 h of incubation. In some experiments the test medium was added at the start of the experiment. DNA Synthesis Determination DNA synthesis was measured by the incorporation of [SH]thymidine (Schwarz/Mann Div., Becton, Dickinson & Co., Orangeburg, N. Y., specific activity 17 Ci/mmol) into acid-insoluble materials during a 2-h pulse, 22 24 h after the addition of the test media. This technique has been described previously (9) and the amount of incorporation has been shown to correspond to the number of epithelial cells synthesizing DNA at the time. The incorporation was expressed as a percent stimulation over control values. The range of [SH]thymidine incorporation was from 1,600 to 3,000 cpm per mg ofstimulated tissue. Previous studies have demonstrated that tritiated thymidine in organ cultures of mammary gland is principally incorporated into epithelial cells and that the incorporation correlates with the mitotic index and the labeling index (9,11). Earlier work has shown that the incorporation of tritiated thymidine is dependent upon the amount of serum added to culture media (9). Where indicated, experiments were performed over a range of serum concentrations. Hormones and Sera The insulin solution (crystalline porcine insulin, 24.4 U/mg, which was a gift of Eli Lilly & Co., Indianapolis, Ind.) was prepared as previously described (9). Though rodent sera are most active in promoting growth in this system, it has been shown (9) that porcine sera are the most active of readily available sera, and therefore porcine serum was used in the experiments reported here. Sera were obtained from Grand Island Biological Co. or by bleeding laboratory animals. Time-Course of [3H]Thymidine Incorporation into Mammary Gland Explants at Various Physiological States When mammary gland epithelium from mature mice is incubated in Medium 199 containing insulin or serum, DNA synthesis is initiated (8,9,II,12). The nature of the response to insulin and serum differs both qualitatively and quantitatively depending upon the physiological state of the animal, that is, whether the animal is nonpregnant, pregnant, or lactating (8,9,13,14). As is illustrated in Fig. 1, when epithelium from nonpreg- nant animals is incubated in organ culture with insulin or serum, there is approximately a 20-h delay in augmented rates of DNA synthesis. On the other hand, such delay is not observed in mammary glands from pregnant animals. It has been shown that if nonpregnant animals are first primed with prolactin injections, the delay in DNA synthesis in vitro is abolished (10). This suggests that prolactin levels of the donor animal could be responsible for the differences in mammary epithelium sensitivity to serum and insulin in vitro. Since lactating animals are known to have a high level of endogenous prolactin, we compared the effects of serum and insulin on DNA synthesis of mammary epithelium from nonpregnant and pregnant animals with that found in lactating animals ( Fig. 1). In spite of the high endogenous levels of prolactin, epithelium from lactating animals behaved like that from nonpregnant animals where endogenous prolactin levels are low. It should be noted that the insulin response with each type of tissue differs quantitatively from that of serum (see next section), but that it follows the same basic qualitative pattern, that is, there is initial unresponsiveness (for 12 h or more) in explant tissues from both nonpregnant and lactating animals whereas the epithelium from pregnant animals responds rapidly (13). Similar findings have been reported by Oka et al. (14). Changes in Serum Sensitivity in Different Physiological States Mammary epithelium from nonpregnant animals responds at maximal rates of DNA synthesis to serum concentrations as low as 5% and the response is linearly dependent upon serum to a concentration of 5% (9). Both Topper's (10) and Turkington's (7) groups suggest that serum concentrations as high as 50% may be necessary to maximally stimulate DNA synthesis in mammary gland epithelium from pregnant animals. We compared the responsiveness of mammary epithelium from nonpregnant, pregnant, and lactating animals to concentrations of serum of up to 50% (Fig. 2). These experiments demonstrate that mammary gland epithelium from nonpregnant animals is more responsive to 5% serum than epithelium from pregnant and lactating animals. With epithelium from pregnant animals a concentration of 50% is required to give effects comparable to those of 5% on mammary epithelium of nonpregnant animals. Like that of epithelium from nonpregnant animals, the response of epithelium from lactating animals is not further stimulated by serum concentrations higher than 5%. These observations suggest that the qualitative difference in the kinetics of response to serum and insulin between epithelium from pregnant animals, on the one hand, and nonpregnant and lactating animals on the other, cannot be explained simply by exposure to endogenously high levels of prolactin. These observations show that pregnancy and lactation are associated with decreased responsiveness to mammary serum factor. To substantiate that this decreased responsiveness correlates with progression through pregnancy, the effect of serum on DNA synthesis of epithelium from animals at various stages of pregnancy and lactation was determined. The results in Fig. 3 A show that as animals progress through pregnancy, mammary epithelium becomes less responsive to 5% serum, while the effect of 50% serum remains rather constant until late pregnancy. During lactation there is extreme unresponsiveness to serum over a wide range of concentrations. To determine if during pregnancy there is a change in responsiveness of mammary epithelium to insulin as there is to serum, mammary epithelium from various stages of pregnancy was tested with low and high ( I or 5 #g/ml) concentrations of insulin (Fig. 3 B). The minimal concentration of insulin which gives maximal proliferation of mammary epithelium from nonpregnam animals is 1 #g/ml (9). As can be seen in Figs. I and 3 B, there is little change in responsiveness to insulin as the animal grows from pregnancy into lactation. Serum Concentrations of Mammary Serum Factor in Various Physiological States Progressive loss of epithelial responsiveness to mammary serum factor as pregnancy progresses The effect of serum and insulin on DNA synthesis in explants of mammary gland from mice at different physiological stages. (A) Explants were exposed to Medium 199 or Medium 199 plus either 5% or 50% dialyzed porcine serum (DPS). Explants from nonpregnant and lactating mice were pulsed with [SH]thymidine as in Fig. 1 after 40 h in the medium, and the explants from pregnant mice were pulsed after 24 h in the medium. Acid-insoluble counts were determined and expressed as percent stimulation of control. (B) Explants were exposed to Medium 199 or Medium 199 supplemented with 1 #g/ml or 5 ~g/ml of insulin (I). Experimental procedures were the same as in Fig. 3 A. could be due to saturation of the epithelial cell by exposure to high endogenous levels of mammary serum factor during pregnancy. To test this possibility, sera from nonpregnant, pregnant, and lactating animals were obtained and tested (Table I). Though the levels of mammary serum factor increase slightly at midpregnancy, this amount of change over the control is within the variability found within this system. These observations suggest that there is probably very little, if any, change in the concentration of mammary serum factor in serum during pregnancy. To determine if the factor responsible could be somatomedin or could be mediated by the pituitary gland, young rats were hypophysectomized, bled 2 wk later, and the sera were tested in this system. Some hypophysectomized animals were injected with bovine growth hormone, ovine prolactin, or 17-~3-estradiol before exsanguination. Table II shows that hypophysectomy decreases mammary serum factor concentrations by about 50% and that injection of growth hormones or other hormones does not restore the lost activity. The failure of bovine growth hormone to restore activity, as it does for somatomedin, suggests that the factor responsible for the serum effect on growth is not somatomedin. The results of hypophysectomy on mammary serum factor levels and the known effects of prolactin on proliferation of the mammary gland when administered to nonpregnant animals sug-gest that prolactin could play a role in mammary serum effects. However, the failure of prolactin injections to restore mammary serum factor activity, and the lack of a significant effect of prolactin on mammary epithelial proliferation in organ culture (Table liD, suggests that prolactin alone may not be sufficient to promote growth in this gland. It is possible that prolactin-primed tissue becomes responsive to mammary serum factor or Table II. that mammary serum factor interacts with prolactin to promote the growth effect. To test these possibilities, mammary gland tissue from nonpregnant animals was preincubated for 48 h in various concentrations of prolactin before exposure to serum, or various concentrations of prolactin and serum were added simultaneously to explants. Preincubation in prolactin does not alter the response to serum (Table IV). However, when prolactin is added simultaneously with serum there is an alteration in serum-promoted growth (Table V). Over a large range of concentrations (0.01-1 #g/ml), prolactin has no effect on mammary serum factor-mediated DNA synthesis. At higher concentrations (5-20 p.g/ml), prolactin Table 11. Table I1. * PS, porcine serum. Effects of insulin and prolactin on DNA synthesis in mammary gland explants from nonpregnant mice. Explants were preincubated in Medium 199 alone for 48 h and then Medium 199 plus insulin (5 p-g/ml) plus various concentrations of prolactin. Explants were pulsed as in Table I1. * 1, insulin. appears to block the mammary serum factormediated DNA synthesis. When prolactin is added simultaneously with insulin there is no altered effect on DNA synthesis (Table VI). It appears that prolactin selectively interferes with the serummediated DNA synthesis and not the insulinmediated DNA synthesis. DISCUSSION These experiments demonstrate that there is a factor(s) in sera, mammary serum factor, which promotes the proliferation of mammary explant epithelium and that the epithelium of the gland becomes increasingly less responsive to the factor(s) during pregnancy and lactation. Epithelium from nonpregnant animals is most responsive to mammary serum factor, but late in pregnancy increasing concentrations are required to give maximal stimulation. During lactation, even concentrations as high as 50% give only minimal responses. Responsiveness to insulin, another epithelial proliferation-promotion agent, on the other hand, is retained through pregnancy and on into lactation. The qualitative nature of the response to serum and insulin also differs in the various physiological states. There is approximately a 20-h delay in augmented rates of DNA synthesis in explants from both nonpregnant and lactating animals (1 I, 14). Explants from pregnant animals respond with increased rates of DNA synthesis within a few hours of culture in the presence of insulin or serum. The difference in response between tissues from nonpregnant and pregnant animals has been attributed to prolactin, in that Oka and Topper (I0) have shown that explants from animals previously injected with prolactin do not have a delay in their response to insulin or serum. However, the results reported here on lactating animals suggest that factors other than exposure to prolactin alone must alter responsiveness to insulin and mammary serum factor. At times of high endogenous levels of prolactin there is the same delay in response to both agents that is found in tissues from nonpregnant animals where there are low endogenous levels of prolactin. Differences in response to mammary serum factor and insulin may be more related to shifts in the composition of the epithelial cell population comprising the gland rather than to exposure to prolactin per se. We know that the growth-promoting effect of serum ahd insulin is exclusively on the epithelial cells of the mammary gland (9). These agents appear to affect different groups of cells in that if each agent alone is added to organ culture at maximally effective concentration, a limited percentage of epithelial cells will initiate DNA synthesis. When the two agents are combined there are additive effects on the numbers of cells synthesizing DNA (9). These observations, coupled with the demonstration of the retention of insulin responsiveness with changes in responsiveness to mammary serum factor as the gland progresses from pregnancy into lactation, suggest that there are at least two populations of mammary gland epithelial cells. These populations are operationally defined by their response to insulin and serum. One population is present at all stages of development and is responsive to insulin; the other, a serum-responsive population, decreases as pregnancy progresses. There are probably a number of schemes which could explain the two cell populations and their changes, for example: (a) a stem-cell scheme in which the insulin population continues to produce insulin-responsive cells whereas the stem-cell population responsive to mammary serum factor can only go through a limited number of cell cycles; (b) a scheme whereby there is competition between one or more peptide hormones and mammary serum factor for active site in or on the epithelial cell population; or (c) a scheme whereby cells which undergo proliferation and respond to the hormones of pregnancy produce daughter cells lacking mammary serum factor-responsive sites. It may be possible to test these latter possibilities. The in vitro competition experiments reported here lend some credence to the second possibility, even though the concentration of prolactin required to see this effect was extremely high. To better understand the biological function of mammary serum factor and how it interacts with the hormones of pregnancy requires better characterization of the factor(s). Preliminary studies indicate that the mammary serum factor is heat stable, and at neutral pH is associated with high molecular weight material. It is present in highest concentrations in rodent serum when the test system used is rodent mammary gland (9). Preliminary investigations on partially purified materials indicate that it is an acidic protein. 1 The origin of the factor is not known. Serum from animals previously hypophysectomized loses 50% of its activity for promoting DNA synthesis in mammary gland epithelium. Injections in vivo of growth hormone, prolactin, and estrogen do not restore the activity to the serum. These observations suggest that the pituitary may be involved in the serum effect, as has been suggested by the work of Gospodarowicz (15) where brain and pituitary extracts promote the growth of 3T3 cells. The fact that growth hormone does not restore activity suggests that the factor under study is probably not somatomedin. The failure of prolactin to restore activity suggests that prolactin itself is probably not the operative component. This observation plus the demonstration that in vitro prolactin alone does not promote mammary gland proliferation suggests that, to be operative, prolactin may have to be presented to the tissue in the presence of other factors, or at a time in development in which a particular cell population has been produced.

v3-fos

2020-12-10T09:04:17.328Z

1975-10-01T00:00:00.000Z

237234417

s2

Psychrophilic Microorganisms from Areas Associated with the Viking Spacecraft Microorganisms capable of growth at 7 C were enumerated and isolated from soil samples from the manufacture and assembly areas of the Viking spacecraft. Populations ranging from 4.2 × 103 to 7.7 × 106/g of soil were isolated from the 15 soil samples examined. Temperature requirements were determined, and those growing at 3 C, but not at 32 C, were designated as obligate psychrophiles in this investigation. Populations of soil bacteria, including aerobic sporeformers, ranging from 1.5 × 102 to 9.8 × 105/g were capable of growth at 3 C, but not at 32 C. Bacterial isolates were identified to major generic groups. No psychrophilic sporeformers were isolated from soil from the manufacture area, but psychrophilic sporeformers ranged from 0 to 6.1 × 103/g from soil from the assembly area. One purpose of the United States planetary quarantine policy is to determine guidelines for the prevention of contamination of Mars with terrestrial microorganisms which might grow in the Martian environment (6). For this reason, it is essential that various groups of microorganisms associated with planetary vehicles destined for Mars be studied in this respect. A great deal of previous research has been conducted on organisms isolated from the manufacture and assembly areas of interplanetary spacecraft, but most of these studies dealt primarily with mesophilic bacteria and the heat resistance of mesophilic sporeformers. The Viking spacecraft will be decontaminated by means of dry heat (13). The standard procedures for the microbiological assay of such spacecraft environments call for the exclusive use of 32 C as the incubation temperature (11). This procedure limits the assay to detection of mesophilic organisms, which make up the majority of microorganisms found on spacecraft surfaces (15). Although it is generally agreed that psychrophiles may not be the most heat resistant of the microorganisms, they should not be excluded from investigations related to planetary quarantine, because these may include organisms with the physiological characteristics to grow in the hostile environment of Mars (7). Also, it is known that some Bacillus spp. and Clostridium spp. can grow at low temperatures (9,17), and sporeformers are the more heat-resistant microorganisms. It is recognized that there are numerous definitions of psychrophilic organisms, including those based on optimum growth temperature and those based on possible growth ranges (8). This present study is not intended to debate the definition of psychrophiles but to demonstrate that the present incubation temperature of 32 C used in the microbial monitoring of spacecraft environments might be excluding propulations ofmicroorganisms of significance to planetary quarantine. The Viking spacecraft is scheduled to be launched to Mars in 1975; consequently, the primary objective of this investigation was to determine the presence and concentration of psychrophilic microorganisms in various environmental areas associated with the spacecraft. MATERIALS AND METHODS Selection of samples. Soil samples were obtained from three sites where the Viking spacecraft is manufactured (Denver, Colo., M), and 12 sites where the spacecraft is housed in preparation for launching (Cape Canaveral, C). Surface soil samples no deeper than 6 inches (ca. 15 cm) were taken from areas around main entrances through which dust contamination might enter the buildings. Isolation of microorganisms. Immediately after being returned to the laboratory, each sample was thoroughly mixed, and 10-g portions of each were decimally diluted in 1.0% peptone to a final dilution of 10-6 prior to plating. The first bottle (90 ml) in each dilution series contained glass beads, the bottle was sonicated for better dispersal of soil particles (14). Subsequent dilution tubes (9 ml) were mixed on a Vortex mixer to assure thorough mixing. Amounts of 0.1 ml were transferred to the surface of Trypticase soy agar (Baltimore Biological Laboratories [BBLI) and Mycophil agar (BBL), pH 4.0, and spread with glass spreading rods. Because all isolations included incubation of samples at 7 C, media used throughout this investigation were stored at 7 C for at least 24 h prior to use. To prevent possible damage to psychrotolerant organisms by addition of molten agar, the spread-plate technique was employed in all counts. All manipulations were performed in a laminar flow cabinet (Envirco MiniBench, model MBO-48, Albuquerque, N.M.). Duplicate plates were prepared for aerobic, anaerobic, and fungal counts. The plates were placed in the 7 C incubator (Freas model 805) immediately after inoculation, and only the anaerobic plates were allowed to reach room temperature during the manipulations. The anaerobic plates were rechilled after inoculation, placed in Brewer Anaerobic Jars with GasPaks and anaerobic indicators (BBL), and plated in the 7 C incubator as soon as anaerobic conditions were achieved as shown by the indicator. A freshly inoculated Trypticase soy agar slant of Alcaligenes fecalis (NASA standard test organism, Center for Disease Control, Phoenix, Ariz.) was placed in each anaerobe jar as a biological indicator of anaerobiosis. In no case did this control organism grow in the anaerobe systems. All incubators were monitored with maximum-minimum registering thermometers (Taylor model 5458), which were checked daily. Slight increases in temperature occurred only during times when samples were being added to or removed from the incubators. Temperature studies. Colonies from plates having 30 to 300 colonies after 14 days of incubation were transferred to Trypticase soy agar slants for incubation at 3 C (10 to 14 days), 24 C (3 to 5 days), and 32 C (48 h). After growth had occurred, the results were recorded, and organisms showing growth at 3 C, but not at 32 C, were classified as psychrophilic, according to the definition used in this investigation. Identification of isolates. All isolates from the temperature studies were examined individually by staining and biochemical testing. From these results, the temperature studies, and colonial characteristics, the organisms were identified to major generic groups. The isolates were stained for their Gram reaction, tested for motility by phase-contrast microscopy, tested for oxygen requirements, and subjected to numerous biochemical tests (4). Organisms thought to be sporeformers were grown on AK-2 sporulating agar (BBL) at either 7 C (10 to 14 days) or 24 C (2 to 3 days). These were then stained to demonstrate production of spores. Mwcrococcaceae were identified according to the method of Baird-Parker (1). The gram-positive rods were identified as sporeformers (Bacillus) or nonsporeformers. On the basis of the tests performed, the latter group was designated as the Corynebacterium-Brevibacterium group. The gram-negative rods were placed into one of two groups. The nonpigmented ones were placed into the Alcaligenes-Acinetobacter group and the pigmented ones into the Flavobacterium-Cytophaga group, although the taxonomic relationship of this group is still uncertain (16). The fungi were identified to genus according to the methods of Barnett and Hunter (2) and Barron (3), and the yeasts were identified following the method of Lodder (10). This was performed with the assistance of John Brandsberg, Center for Disease Control, Kansas City, Kansas. RESULTS Population studies. Viable counts of microorganisms growing at 7 C from soils from the manufacture and assembly areas of the Viking spacecraft are presented in Table 1. Viable counts from Athe manufacture area are approximately 2 logs higher than those from the assembly area. In all but one sample (C-4) the aerobic bacterial counts were the highest, with sample C-4 containing a higher population of anaerobes. In 10 of the 15 samples the anaerobic counts were higher than the fungal counts. Temperature studies. One of the means of grouping the various isolates for identification included their ability to grow at 3 C (10 to 14 days), 24 C (3 to 5 days), and 32 C (48 h). Organisms showing growth at 3 C, but not at 32 C, in the designated time are defined as psychrophiles. Many of these did show growth at 24 C ( Table 2). Since many investigators prefer a more rigid definition of psychrophiles, results in Table 2 also show the percentage of organisms that grew at 3 C but not at the other two temperatures. Even though the total population is higher in the samples from Denver, the percentage of psychrophiles is higher in the samples from Cape Canaveral. This is espe- cially true in the group that includes organisms growing at 3 C but not at 24 or 32 C. The results given for percentage of obligate psychrophiles from these soil samples may appear high, but it must be remembered that they are based upon counts in which the organisms were orginally isolated at 7 C. Identification. Anaerobic isolates from the manufacture area were stained and found to consist of approximately 70 to 80% sporeformers and 20 to 30% gram-positive non-sporeforming rods. Samples from Cape Canaveral yielded almost 100% sporeformers. During further testing, it was determined that these were facultative aerobes belonging to the genus Bacillus. It was also determined that many of the aerobic isolates were facultative. From these results, it was decided to exclude the anaerobes from further investigation because obligate anaerobes were not demonstrated. The fungi were indentified to genus, and these results are presented in Table 3. Molds were not identified to species. The yeasts from the manufacture area were all determined to be Cryptococcus albidus. Those from Cape Canaveral were identified as either C. albidus, Cryptococcus laurentii, Rhodotorula rubra, or Rhodotorula minuta. The aerobic bacteria were identified to major generic groups including Micrococcus, Bacillus, Corynebacterium-Brevibacterium, Alcaligenes-Acinetobacter, andFlavobacterium-Cytophaga. Different isolates within each major group were recognized, and representatives of these have been lyophilized. The results of these determinations are presented in Table 4. The fungi isolated on these plates were not identified but were included in the counts. Samples from the manufacture area appear to contain primarily members ofthe Corynebacterium-Brevibacterium group and members of the genus Micrococcus, especially subgroup 8. Of interest is the fact that these samples contain only low percentages of aerobic sporeformers. In contrast to this, samples from Cape Canaveral contain primarily members of the genus Bacillus and the Corynebacterium-Brevibacterium group. Percentages given in Table 4 are calculated from the aerobic counts of Table 1. The actual number of Bacillus per gram of soil is similar when the manufacture area samples are compared to the Canaveral samples. Even though the percentage ofBacillus in the aerobic population of the manufacture area samples is quite low, M-3 contained the largest population of aerobic sporeformers. Yeasts and molds 1 3 Unable to subculture 3 1 a Percentage of aerobic count from Table 1. Distribution of obligate psychrophiles. From the results of the temperature studies and the distribution of major groups of isolates, the percentage of psychrophiles (growth at 3 C, but not at 32 C) within each group was determined ( DISCUSSION Attempts to prevent the contamination of interplanetary spacecraft intended to enter the atmosphere of Mars have at least a twofold purpose. One is to prevent the contamination of the Martian surface with terrestrial organisms which might alter the state of the planet; the other is to assure that terrestrial contaminants do not interfere with the life detection experiments on the spacecraft (6). It is accepted that the conditions on the Martian surface are not such that mesophilic organisms will be exposed to a temperature approaching 32 C (12), and the Viking Lander Biological Instrument will be maintained at a temperature of approximately 15 C (H. P. Klein, personal communication). Standard NASA procedures for the microbial monitoring of spacecraft specifies incubation at the single temperature of 32 C (11). The majority of contaminants found in the spacecraft are mesophilic organisms (15), but the use of this single temperature could possibly exclude organisms better adapted to grow in the cold environment of Mars or in the Viking Lander Biological Instrument. This present investigation has demonstrated the presence of relatively large soil populations of organisms which would not be detected by the present microbial monitoring procedures for spacecraft environments. This estimate might be low because of the use of a single isolation temperature of 7 C. Of the populations just described, the sporeforming rods are probably the most important group, because the Viking spacecraft will be subjected to dry-heat decontamination prior to launch. The soil samples from Denver showed no psychrophilic sporeformers, whereas the samples from Cape Canaveral showed an average of 2.1 x 103 psychrophilic sporeformers/g of soil. These results demonstrate the presence of a population that may have been excluded by present monitoring procedures.

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2020-12-10T09:04:20.886Z

1975-03-01T00:00:00.000Z

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Stability of Aflatoxin B1 and Ochratoxin A in Brewing The stability of aflatoxin B1 and ochratoxin A in brewing was investigated by adding the purified toxins to the raw materials at 1 and 10 μg/g levels during mashing in a conventional micro-brewing process. The results indicate that both toxins are stable to heat and are insensitive to cooker mash treatment. Both mycotoxins were partially removed in the mashing and brewing processes. About 14 to 18% and 27 to 28% of the added toxins were found in the final beers brewed from starting materials containing 1 and 10 μg, respectively, of either toxin per g. The possible route of transmission of mycotoxins into beer is discussed. Since the discovery of aflatoxins, a series of potent carcinogens produced by Asperigillus flavus and A. parasiticus, a number of other fungal metabolites have been demonstrated to be toxic to animals (6,9). Among these toxins, ochratoxins, a series of nephrotoxins produced by several species of Aspergillia and Penicillia, have also attracted attention because of their high toxicity and the widespread occurrence to toxin-producing fungi (4). Aflatoxins and1 ochratoxins have been found in many agricultural commodities (4,6), including corn (13)(14)(15). Because of the unusual stability of these toxins in agricultural commodities, the question of whether they could carry through the food process has attracted considerable attention. Because of the natural occurrence of ochratoxin A (OA), the most potent toxin in the ochratoxin series, in barley and other cereals (10,12), scientists in the United States and Denmark have studied the possibility of transmitting OA into beer. Fischbach and Rodricks (8) reported that earlier work in the Food and Drug Administration had shown the retention of OA in the beer process, but a survey on OA and ochratoxin B in samples of beer and malted barley from each of the 130 breweries in the United States showed no detection of ochratoxins. These samples were tested by using an analytical method sensitive to 0.01 gg/g (10 jig/kg). Recent experiments by Krogh et al. (11) demonstrated, however, that OA was destroyed during the malting and brewing processing. To determine the stability of aflatoxin B, (Afla Bj) and OA in brewing, we added the purified toxins at two stages (to either malt or corn grits) during a micro-brewing process. This paper presents data on the stability of these two toxins in five major operations of brewing. MATERIALS AND METHODS Materials. Afla B, and OA were prepared by the methods of Chu (3) and Chu and Butz (5). Malt was prepared from Larker barley (Hordeum vulgare L.) that was grown in North Dakota in 1972. Portions (170 g, dry basis) were steeped in water at 16 C until the moisture content was 45%. The barley was allowed to grow at this temperature in the dark. The malting (growth) chamber is a modified drum type that slowly rotates 36 perforated cans containing the portions of barley. Moisture-conditioned air was circulated through the chamber and the samples during the 5-day malting period. The germinated barley was then kilned according to the following program: 9 h at 35 C, 7 h at 44 C, 5 h at 55 C, 5 h at 65 C, and 2 h at 85 C. Rootlets were removed after kilning, and the malt was ground in a Miag mill (Miag Co., Braunschweig, West Germany) prior to mashing. Corn (Zea mays L.) grits were obtained from the Krause Milling Co., Milwaukee, Wis. Adsorbosil-5 and Silica Gel G were obtained from Applied Science Laboratories (State College, Pa.) and the Brinkmann Instrument Co. (Westbury, N.Y.), respectively. All other solvents and chemicals were either analytical reagent grade or chemically pure. Micro-brewing procedures. The brewing procedure was that described by Burkhart et al. (2), in which 90% of the malt is used in the malt mash and 10% in the cooker mash. The essentials of the operation are as follows. (ii) Malt mash. Malt (189 g) and water (718 ml) were mixed at 38 C for 10 min. The mixture was 313 allowed to stand at 38 C without stirring for 50 min for optimal hydrolysis of proteins. (iii) Combined mash. The cooled cooker mash was added, and the temperature was raised to 72 C. The mash was maintained at this temperature for 20 min for optimal carbohydrate hydrolysis. The mash temperature was then raised to 75 C, and the mixture was filtered. (iv) Wort. The entire filtrate (wort) was boiled with 1.5 g of hops for 90 min. Twenty minutes before the boiling was completed an additional 2.0 g of hops was added. The boiled wort was filtered and cooled to 12 C. Fermentation and storage. The wort was pitched with 5 g of moist cake yeast (1 g, dry basis) (Saccharomyces cerevisiae) and fermented in a covered, but not sealed, flask at 12 C for 8 days. A 64-mg amount of commercial chill-proofing preparation (crude papain) was added, and the beer was transferred to a storage flask and maintained at 0 C under 12 lb/in2 pressure of carbon dioxide for 14 days. The beer was then filtered and bottled under 12 lb/in2 carbon dioxide pressure. For pasteurization the bottled beer was kept at 60 C for 20 min and then cooled to room temperature in 15 min. The beer was stored thereafter at 12 C. Spiking of the purified toxins. For Afla Bl appropriate amounts of toxin in 5 ml of chloroform were added to the malt or corn grits at the beginning of the cooker and malt mashing stages at 0, 1, and 10 Mg/g levels (weight basis). The chloroform was removed by evacuation. For OA, the toxin was dissolved in 1 to 2 ml of 1% NaHCO. and added to cooker and malt mash directly before processing. Duplicate experiments were made for each level of toxins added. Extraction of brewing preparations and toxin analyses. Duplicate samples were analyzed at five stages during the brewing process: (i) the completed cooker mash; (ii) the malt mash at the end of the protein hydrolysis stage; (iii) the combined mash at the end of the carbohydrate hydrolysis stage; (iv) the wort after addition of hops and boiling; and (v) the bottled beer after pasteurization. The following procedures were used for toxin analysis. (i) Afla Bl. For Afla B,, official methods of analysis were followed (1). Samples of 100 g or 100 ml were extracted with chloroform according to Sect. 26.018 (a) (ref. 1). After evaporation of the chloroform from the extracts, the samples were redissolved in 1 to 2 ml of chloroform and chromatographed as described in Sect. 26.019 (a) (ref. 1). The chloroform elution fraction was evaporated to dryness, redissolved quantitatively in 1 to 5 ml of chloroform, and then chromatographed on a glass plate (20 by 20 cm) with a 250-um layer of Adsorbosin-5; methanol-chloroform (3:97) was the developing solvent. Quantitations were made by a fluorodensitometric method (5), except that a photo volt densitometer model III Turner fluorometer TLC scanner (G. K. Turner Associates, Palo Alto, Calif.) with primary and secondary filters of Turner no. 110-811 and 110-816 was used. (ii) OA. For OA determination, 100 g each of treated brewing product and a control was blended with 100 ml of 2% NaHCO, solution for 5 min and then centrifuged at 3,000 x g for 10 min. The supernatants were transferred to separatory funnels. The residues were blended twice with 150 ml of 1% NaHCO, for 5 min and centrifuged again as described above. All of the supernatants of each batch were then pooled and acidified with concentrated HCl to pH 2. Likewise, 100 ml of beer was directly acidified to pH 2.0 with HCl. The acidified solutions were extracted twice with equal volumes of chloroform in separatory funnels. The chloroform extract was then evaporated to dryness and dissolved quantitatively in 8 ml of chloroform for thin-layer chromatography analysis on Adsorbosil-5 as described before; the solvent system was benzene-acetic acid (9:1; 7). Quantitation was done on a Turner fluorometer as described for Afla B l- RESULTS AND DISCUSSION The validity of the present data rests primarily on the method of extraction and analysis; thus, control experiments, in which test mycotoxins were added to the brewing products after each process, with subsequent analysis of toxin content, were carried out. The results showed that the toxin recovery ranged from 78 to 91% for each step, with an overall average of 85%. Since the recovery yield falls into the range of mycotoxin analysis in other food (4), the analytical methods used in the present study might also be used for routine analy'sis of these mycotoxins in beer or related products. The presence of alcohol was removed by evaporation under vacuum, and the remaining material was analyzed for toxins. The results showed that both the treated (alcohol removed) and the untreated samples gave similar recoveries (86.6 versus 89.3%). The toxins were then directly extracted from the beer or acidified beer (for OA) with organic solvents before analyzing. The levels of Afla B, and OA in brewing at five stages are given in Table 1. In general, both mycotoxins were partially lost during the brewing. The patterns for the loss of both mycotoxins were similar; both were relatively stable in the cooker mash step but were more sensitive to later treatments, especially the protein hydrolysis, wort boiling, and final fermentation. Since both mycotoxins are relatively stable to heat (4,9,16), it is not surprising that more than 90% of (14) Values in parentheses indicate a correction of 15% due to loss in the analysis. the added toxins were in the sample after cooker mash treatment. Removal of toxin from the product was very significant in the malt mash (with a loss of 12% for a 10 Mg/g sample and 24 to 27% for a 1 Ag/g sample), in boiled wort (20 to 30% loss of material added to the malt mash), and in the final fermentation (another 20 to 30% loss from the boiled wort step) steps. Since it has been observed that OA can be hydrolyzed by acid and some proteases to ochratoxin a and phenylalanine (4) and since ochratoxin a has been detected in the wort when OA-contaminated barley was used in the brewing (10), removal of OA in the brewing process may primarily be due to such hydrolysis. The mechanism of Afla Bl removal, however, is not known. The significant loss of both mycotoxins between the combined mash and boiled wort steps may be due to the nonspecific interaction or adsorption of both mycotoxins by the solid particles removed by the filtration process. Both mycotoxins were partially removed in brewing, but considerable amounts were transferred into the beer, especially if the contamination level was high. Approximately 25% of the added toxins were found in the final product when 10 ug of the toxin per g in the cereal was tested. At the lower testing level (1 Ag/g), recovery was about 15%. Our results on OA differ from those reported by Krogh et al. (11). They found significant loss in mashing (70% lost before boiled wort), and less than 10% of OA was recovered when barley, containing 1 to 5,ug of toxin per g, was used in the brewing. It is not known whether these contradictions are due to a difference in their brewing processes, in which lactic acid and bacterial enzymes were used, or to the method of extraction and analysis for which recovery yield in the control sample was not reported by the investigators. Inasmuch as mycotoxins in contaminated materials are not completely removed from the beer in brewing and might be converted to some unknown products which might also be toxic, the best protection would be to prevent the use of contaminated raw materials in brewing. In order that barley germinate and grow (malt) satisfactorily, and therefore meet the requirements of the malting industry, it must be stored under conditions which would not promote the growth of storage molds. Although Krogh et al. (11) demonstrated that when barley was heavily contaminated with OA it thus failed to germinate and would be rejected for malting, it is not known whether or not aflatoxin would also affect the malting process. Moreover, the material used as adjunct (the constituent other than malt in the cooker mash) functions primarily as a source of starch and other carbohydrates which are largely hydrolyzed to maltose during the "conversion" or carbohydrate hydrolyzing stage of mashing. Possibly these adjuncts (corn syrups, ground rice, and ground barley are commonly used), if they are already contaminated in the field or if they are stored under adverse conditions, could contribute mycotoxins to the mash.

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2020-12-10T09:04:20.739Z

1975-01-01T00:00:00.000Z

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Effect of pH and Sodium Chloride on Growth of Bacillus cereus in Laboratory Media and Certain Foods The effects of NaCl concentration, pH, and water activity (aw) on the ability of vegetative cells of Bacillus cereus to initiate aerobic growth in brain heart infusion broth at 30 C were studied in a factorial design experiment. By using multiple regression techniques, equations were derived which related the decimal reduction of the bacterial population to the concentration of NaCl and pH of broth to which the population was exposed. From these equations, the percentage of inoculated cells capable of initiating growth could be calculated. The reliability of these equations in foods was tested in laboratory-processed meat and rice media. The foods were less inhibitory than the broths, so that accurate prediction of growth initiation in foods was not possible by using the developed formulas. The impact of this type of quantitative study on the development of specific microbial standards for foods is discussed. When the NaCl concentration is increased, the aw is decreased and, with increased deviation of pH from optimum, more concentrated inoculum of B. cereus cells is needed to assure initiation of growth in culture media and foods. Bacillus cereus is a sporeforming bacterium which is widely distributed in nature. It has been recognized as a causative agent of food poisoning for almost 25 years. The symptoms are relatively mild and the duration is short. All kinds of foods can serve as vehicles, but typical ones are meat and certain meat products, as well as different types of puddings (10). Most epidemics reported have happened in Europe. In the United States B. cereus is rarely reported as the cause of foodborne disease: between the years 1968 and 1973, only seven outbreaks were reported (23). It has been shown recently that one or more substances produced during the exponential and late exponential phases of growth can induce fluid accumulation in rabbit ileal loop (21), a necrotic reaction in guinea pig skin (8), and an altered vascular permeability in rabbit skin (9). These substances are produced also by strains of B. thuringiensis and B. mycoides (9), and they are different from lecithinase C and hemolysin and yet indistinguishable from B. cereus lethal toxin (8). Whether these substances are responsible for human food poisoning still has to be proven. To evaluate the microbiological safety of a particular food, it is very important to know the percentage of inoculated cells capable of initiating demonstrable growth in that food environment. By using different preservation methods, this fraction of cells can be decreased to maintain a certain standard limit, allowing us to consider the food environment as safe. The effect of heat treatment on decreasing bacterial populations has been studied extensively. On the basis of studies done mainly with Clostridium botulinum spores, the canning industry has adopted the 12-D concept for low acid foods (16). This 1/1012 probability of a spore surviving canning is considered as a minimal safety standard. Decimal reduction values for the effects of other preservation methods, except heating and radiation, are not generally found or used. Genigeorgis et al. (5) explored the possibility of applying derived equations to predict the probability that one staphylococcal cell will initiate growth in media at various pH values and NaCl concentrations. Information concerning the effects of NaCl concentration and pH on the growth of B. cereus is limited (10,22). The purpose of this study is to obtain additional information of the effect of these parameters on the aerobic growth of B. cereus in laboratory media. Equations were 68 developed which predict the decimal reduction of populations of various B. cereus strains exposed to such laboratory media. Practical applications of the equations were tested eventually in processed meat and rice environments. MATERIALS AND METHODS Preparation of experimental broths. The broths based on brain heart infusion (BHI) broth (Difco) were prepared as described previously (5). Inoculation and incubation of broths. The following five B. cereus strains were used: ATCC 9139, ATCC 14579 and 2006 obtained from G. York, Department of Food Science and Technology, University of California, Davis, and 01552 and 5063 obtained from R. Wood and T. Midura of the California State Department of Public Health, Berkeley. The latter two strains had been isolated from food poisoning outbreaks. Lyophilized stock cultures of the test organisms on porcelain beads were prepared according to a slight modification of the method of Hunt et al. (11). In the experiments, overnight BHI broth cultures of the strains were inoculated into 25 ml of BHI broth containing 0.25% Tween 80 (Difco). The fresh cultures were incubated at 30 C on a reciprocal shaker for 4 h. The cultures were then centrifuged, the cells were washed once with saline containing 0.5% peptone (Difco), and the concentration of cells was adjusted to an optical density of 1.4 to 1.7 at 615 nm with a Spectronic-20 colorimeter (Bausch & Lomb). Nine tubes, each containing 9 ml of broth, were prepared from each type of experimental broth. A 1-ml amount of the cell suspension was added to the first tube, and nine 10-fold serial dilutions of the suspensions were prepared. Three portions of 2 ml each were transferred with a sterile syringe from each of the nine tubes to 2-ml screw-cap vials. The caps were put on loosely and the vials were placed in 3-lb (ca. 1.4-kg) coffee cans (15.5-cm diameter by 17-cm height). In each can a vessel containing an NaCl solution of the same strength as the broth was placed. The cans were closed with their plastic lids, and the broths were incubated at 30 C for 10 days. Every other day vials with growth (turbidity) were removed and recorded. From the presence or absence of growth in the 27 vials prepared for each NaCl-pH combination, the most probable number of cells which had initiated growth was calculated from the tables of Fisher and Yates (4). The number of B. cereus cells present in the cell suspension used as inoculum was determined by plating on BHI agar (Difco) in duplicate. This number was always between 1.5 x 106 and 1.8 x 10'. Statistical methods. The experiments, arranged in a factorial design (20), involved five B. cereus strains, four NaCl concentrations (0, 2.5, 5, and 7.5%), and four pH values (4.6, 6.1, 7.5, and 8.8). For the statistical evaluation of the effects of NaCl and pH and their interaction upon the growth of B. cereus, the logarithm (log) of the ratio R,-RG was calculated for each broth and strain combination, where R, was the number of cells in the inoculum and R, the number initiating growth. This log represented the number of decimal reductions of a bacterial population resulting from its exposure to a particular environment. The logs determined in the factorial experiments were evaluated by using the biomedical computer program (2) for multiple regression analysis. Equations representing the trend surface were constructed for each individual strain and for pooled data. Each equation related the effects of NaCl and pH levels on log decrease for that strain. Preparation of experimental foods. (i) Meat. Cooked meats with different pH values and brine concentrations were prepared as described previously (6) and kept in the refrigerator until use. (ii) Rice. Commercial enriched long grain rice (Town House) was washed with water and cooked for 15 min (until all cooking water had evaporated). The rice was allowed to cool to room temperature, and then it was divided into two lots, to one of which 5% (wt/wt) NaCl was added. The two lots of rice were homogenized in mortars. After overnight refrigeration, both lots were again homogenized and 0.2% (wt/wt) glucono-delta-lactone was added to decrease pH of certain samples. The rice media were packed, pasteurized, and stored in a manner similar to that of the experimental meats (6). Inoculation and incubation of experimental foods. Small food disks used as growth media in the experiments were made by means of 9-mm diameter sterile cork borers and were placed in a sterile standard plastic petri dish. B. cereus cell suspensions were prepared in a similar way as in the broth trials. Nine 10-fold serial microbial dilutions in sterile 0.1% (wt/vol) peptone were prepared. Portions of 0.01 ml from each dilution were inoculated on three food disks with a sterile microsyringe. The petri dishes containing the disks were then placed in 3-lb coffee cans. In each can there was a vessel containing a brine of the same NaCl concentration as that of the food sample. Incubation was similar to the broths. After the appropriate incubation time, impression smears from each inoculated disk and uninoculated control disks were prepared on standard microscopic slides. The smears were examined for B. cereus growth with a Carl Zeiss phase contrast microscope. The most probable number of cells initiating growth was calculated in a similar way as in the. broth experiments. Physicochemical analysis of the growth media. Water content, NaCl and brine concentration, and pH of the experimental media were determined by methods previously described (6). Water activities (a.) of the experimental broths as well as food samples were measured by a model electric hygrometer (Hydrodynamics, Inc., Silver Spring, Md.) equipped with a gray band hydrosensor (range a, 0.81 to 0.99). Recalibration of the sensors was based on the use of NaCl solutions of different known molalities and a. values at 25 C (17). A 30-g sample of material was put into a 200-ml Kerr mason jar that was allowed to stand at 25 C for 24 h for humidity equilibrium before the dial reading was taken. Two unused sensors were utilized. Water activities of the experimental broths were also determined by an equilibrium moisture absorption technique as described by Vos and Labuza (24), based on the use of avicel microcrystalline cellulose. RESULTS The raw data of the combined effects of pH and NaCl of the broths on the log (decimal) decrease of the populations of the five B. cereus strains exposed to various broth environments are presented in Fig. 1. From the multiple regression analysis of the data, the following five equations were derived for the strains used, When the data of all the strains were pooled, the summary equation was as follows: Ye = 44.26 -0.80 (salt) -11.88 (pH) + 0.03 (salt)2 + 0.79 (pH)2 + 0.21 (salt x pH). Statistical analysis of the data indicated significant effects of (salt), (pH), (pH)2, and (salt x pH) upon the magnitude of log decrease. Although the effect of the term (salt)2 on the log decrease was not statistically significant, it has been retained in the equations for symmetry. By using the equations given above, response curves were constructed for each strain and for the pooled data relating pH, NaCl, and log confidence contours for a specified log reduction can be obtained by using Ye ± standard error, and approximate 95% confidence contours by using Ye ± 2 standard errors. A response curve and the approximate 68 and 95% confidence limits for 4-log reduction of B. cereus strain ATCC 14579 are presented in Fig. 3. To test the above-developed equations in foods, the log decreases of bacteria populations caused by meat and rice environments with different pH values and NaCl concentrations were determined for B. cereus strains 5065 and 01552 ( Table 1). The a, of the BHI broths with 0, 2.5, 5.0, 7.5, and 10% brine were found to be 1. Response curve (0) and approximate 68 (0) and 95% (U) confidence limits for 4-log reduction of B. cereus strain ATCC 14579 exposed to different pH and NaCl combinations. DISCUSSION The range of pH permitting growth of B. cereus in laboratory media has been reported to be pH 4.9 to 9.3 (10). The few articles (13)(14)(15) dealing with growth in foods of different acidity give the minimal pH for growth varying from pH 4.5 to 5.15. According to existing data (12,19,22) B. cereus is able to grow in 7% NaCl but not in 10%. Minimal a, value allowing growth is reported to be 0.950 (18). Previous data agree generally with the findings of the present study. Initiation of B. cereus growth in a meat environment at a pH as low as 4.35 has been observed for the first time. However, a high inoculum (106 cells in 0.01 ml) was used, thus giving greater probability of initiating growth. The maximal pH used in the study was 8.8. Though this pH was found to be relatively noninhibitory to growth of B. cereus, its significance to food protection is minimized by the fact that very few foods have such a high pH. In this study the effects of NaCl and pH on the log reduction of B. cereus populations ex-posed to different environments were studied. The quantitative approach used permits prediction of the percentage of inoculated cells capable of initiating growth, when inoculum and environmental parameters (% NaCl, pH) are known. This predictive potential is important in the development of microbiological standards for different types of foods. Equations were derived relating NaCl concentration and pH of BHI broth medium to log reduction of a B. cereus population exposed to this environment. The probability that one cell can initiate growth can be calculated from these equations. For example, for a broth at pH 5.5 with 2% NaCl, the log reduction for strain 5065 is 3.88, the antilog is 7585, and the probability of initiating growth is that 1:7585 or 0.0134% of inoculated cells will be capable of initiating growth. If salt concentration is increased to 4%, then 0.0011% of inoculated cells will be capable of initiating growth. Similar calculations can be done to any NaCl-pH combination. Growth characteristics of B. cereus do not differ qualitatively from the general bacterial behavior pattern when exposed to different NaCl and pH conditions. These shapes of response curves resemble those found by Genigeorgis et al. (5) in their study on growth of Staphylococcus aureus. The general findings can be summarized as follows. (i) The effects of pH and NaCl on growth of B. cereus vary with strain and growth medium used. (ii) There is a decreased rate of growth of B. cereus when exposed to media with NaCl concentrations increasing from 0 to 10%. (iii) When we increase the NaCl concentration of a medium, more concentrated inoculum is needed to assure initiation of growth. The same is true for pH when the values drift away from pH 6.5 to 7. (iv) High NaCl concentrations and extreme pH values prevent growth. (v) Smaller concentrations of NaCl are required to inhibit initiation of growth at pH values remote from optimum. Food served as vehicles in epidemics of B. cereus food poisonings are usually highly contaminated. Microbiological examinations have regularly revealed contamination levels of 106 to 107 cells per g of food involved in food poisonings (10). The amount of contaminated food eaten by an individual before getting ill is, however, infrequently mentioned in reports concerning food poisoning epidemics caused by B. cereus. Generally, low attack rates for the sources of outbreaks as well as experimental feeding trials (10) indicate that a relatively large amount of a contaminated food must be consumed in order to produce symptoms. Because of the relatively low prevalence of reported food poisoning cases caused by B. cereus and the mild nature of the disease, no standards have generally been established for foods. To prevent B. cereus growth to as high levels as mentioned above, the growth environment should be inhibitory enough to reduce the probability of growth initiation at least by a factor of 104. The response curve for 4-log decrease for a population of B. cereus strain ATCC 14579 in BHI broth is presented in Fig. 3. Any NaCl-pH combination above the response curve causes the desired minimal inhibition. Figure 3 indicates also the approximate 68 and 95% confidence contours of the response curve. The curves are specific for this strain and growth environment and, as such, cannot be applied to foods. To test the reliability of the formulas developed for BHI broths in food environments, experiments were made in which laboratoryprocessed meats and rices served as growth media. These types of foods have frequently been reported as vehicles in B. cereus food poisoning epidemics (10,23). The data collected (Table 1) indicate that the food environments tested are remarkably less inhibitory than BHI broths. Thus, the equations will give too high log reduction values if applied directly in foods. For instance, strain 5065 inoculated into meat with 4.1% NaCl concentration in brine and pH 6.1 had a log decrease of 4.80 instead of 0.07 measured. Similarly, for the same strain, a meat environment of 4.6% brine concentration and pH 7.85 would have a log decrease of 3.98 instead of 1.76 measured. Rice also appears to be a better growth medium than BHI broth. Sample number two (Table 1) had 0% salt and pH 5.0. The equations for strains 01552 and 5065 and such a salt-pH combination produced "expected" log decreases of 5.03 and 4.79, respectively, instead of 1.02 and 3.24 log decreases obtained experimentally in rice. Genigeorgis et al. (7) have also obtained less inhibition of staphylococci inoculated in processed meats than the predicted level of inhibition based on formulas derived from studies on BHI broths. To make accurate predictions of the probability of growth initiation in a certain food, the formulas should be developed for that particular food item as bacterial growth media. Glucono-delta-lactone was used as an acidulant for the food samples and HCl was used for the broths. However, this fact cannot explain the differences between inhibitory capabilities of the environments. At least in the case of Salmonellae, HCl has been shown to permit growth at lower pH values than gluconic acid (1). Foodborne bacterial pathogens in general grow at a, levels of 0.83 to 0.999 (22). In this high range, measurements by the electric hygrometer having a moisture-sensitive, saltcoated probe are considered to be inaccurate, especially when the probe gets older (3). In these experiments two sensors were used, one new and the other several years old but unused. When calibrated before use, no significant differences could be found between the results. Shape of the standard curves was, however, different. The new sensor gave remarkably vaster ranges for dial readings at a, values over 0.9, thus giving better accuracy. New hygrometer probes are considered to be accurate within i 0.005 a, inside their specific ranges, that in this case (a gray band sensor) ends when a, = 0.99. Water activities greater than that were reported as a, = 1.000. The water activities measured by equilibrium moisture absorption of the microcrystalline cellulose method were generally 0.01 a, units lower than those given by electrical hygrometer. This trend does not agree with the results of Vos and Labuza (24), who got about equal values by both methods. The lowest limit of a, = 0.950 for growth of a vegetative B. cereus cell has been stated (18). The results of this study agree with the reported information. The lowest aw value that permitted growth was 0.955 when measured by an electric hygrometer.

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2018-04-03T02:15:27.875Z

1975-09-01T00:00:00.000Z

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Influence of wine composition on the heat resistance of potential spoilage organisms. Pasteurization studies were conducted on 29 yeast and five lactic acid bacteria. In general the yeasts were more heat resistant in wine than were the bacteria. The one exception was a strain of Lactobacillus fructivorans that gave an average D-value of 1.7 min at 60 C. Alcohol was the wine constituent that had the greatest effect on resistance; D-values for all test species were inversely related to the ethanol concentration. The response of organisms to other factors such as pH, sugar, and sulfur dioxide varied with the species. Bottled wines that have been sweetened or which contain some residual fermentable carbohydrate are susceptible to spoilage by yeasts and lactic acid bacteria (7,8,12). Multiplication of these organisms often results in off-flavors as well as objectionable turbidity and sediment (1). One of the methods used to prevent spoilage is pasteurization. Although a very old procedure, surprisingly few quantitative data are available regarding the conditions necessary to impart biological stability to wine. As a result, the heat processes that are presently used differ markedly in severity. Some wineries, for example, flash pasteurize at 80 C or higher; others use hot bottling procedures in which the wine is filled into bottles at temperatures ranging from 45 to over 70 C. In addition to the need for minimum process data, impetus for this research was provided by the so-called "mod" or "pop" wines that have been introduced in recent years. It seemed that the different composition of these wines might have a significant effect on their pasteurization requirements. Media. Yeasts were propagated in 100 Brix Thompson seedless grape juice. Potato dextrose agar, pH 5.6, was the plating medium used for viable counts. The lactic acid bacteria were grown in tryptoneglucose-yeast extract-salts broth (11) supplemented with 10% tomato juice serum and 0.5% fructose. (The latter ingredients were needed for growth ofLeuconostoc oenos and Lactobacillus fructivorans.) For plating, 2% agar was added to the medium. Heat resistance. Heating was conducted in flame-sealed capillary tubes (4,10). In a typical trial, stationary-phase cells were centrifuged from the culture medium, then suspended in the test 36 solution, usually a wine. A Hamilton syringe with a 500-iul capacity was used to transfer 20-Al volumes of the cell suspension to capillary tubes (1.6 by 50 mm); a Hamilton repeating dispenser facilitated this operation. The tubes, held in Thomas pinch clamps, were submerged in a constant-temperature water bath. After heating for a given time period, the capillaries were cooled in 95% ethanol. (A dual function of the alcohol was to destroy many of the microorganisms that might be present on the surface of the tubes. Ethanol-resistant organisms apparently were eliminated by subsequent dilutions; contamination was not a problem.) Five tubes from a given treatment were pooled in a dilution bottle containing 5 ml of 0.1% peptone solution. After crushing the tubes with a sterile glass rod, appropriate decimal dilutions for plating were made in 0.1% peptone solution. The yeasts were incubated at 28 C, and the lactic acid bacteria were incubated at 32 C. The incubation period ranged from 2 to 7 days, depending upon the culture. An experimental wine prepared in our pilot plant from Niagara grapes served as our model in most experiments. This 1971 vintage wine contained 12.1% ethanol, total acid of 0.82% (expressed as tartaric), 20 mg of tannin per 100 ml, less than 5 mg of free SO2 per liter, and less than 0.1% reducing sugar. Its pH was 3.2. The methods for these analyses are described by Amerine and Ough (2). Modifications of the wine were made in such a way as to minimize changes in the concentration of the various constituents. Thus solutions used to adjust pH and the concentration of glucose were prepared by dissolving the reagents directly into samples of the wine. RESULTS AND DISCUSSION Species resistance. During the course of this study, the resistance of 29 yeasts heated in the Niagara wine was determined. Many of the cultures were strains ofSaccharomyces cerevisiae used in commerical wine making; others on May 5, 2020 by guest http://aem.asm.org/ Downloaded from were flor yeasts, species of Saccharomyces involved in the sherry fermentation. The average surival figures are summarized in a histogram ( Fig. 1). It can be seen that most of the yeasts yielded D-values of under 0.6 min when heated at 49 C. The most heat-resistant yeast, a strain of S. cerevisiae, gave an average D-value in five experiments of 1.6 min. The single nonwine yeast examined, Candida utilis NRRL Y-900, gave a D-value of under 0.1 min at 49 C. The initial studies with lactic acid bacteria were conducted with the first four species listed in Table 1. Because they were rapidly killed at 45 C, a temperature 40 lower than that used for the yeasts, it first was concluded that the lactic acid bacteria as a group possessed considerably less resistance than yeasts. L. oenos ML 34, an important malo-lactic strain (6), was especially sensitive; 43 C was a lethal temperature for it. These results are in agreement with those of Murdock et al. (5) whose studies with orange juice also indicated leuconostocs to be most heat sensitive, followed by the lactobacilli and then the yeasts. Our hypothesis about the lactics had to be revised, however, when we later isolated a strain of L. fructivorans from a spoilage outbreak involving a premixed Bloody Mary cock- tail. This organism was considerably more heat resistant than any of the yeasts: it was difficult to detect destruction of it at temperatures below 58C. L. fructivorans is relatively alcohol tolerant and our isolate grew rapidly in broth containing over 12% by volume. This raised the question as to whether the low heat resistance of the other bacteria was related to an alcohol sensitivity. Perhaps the shock of being exposed to alcohol for the first time was partly responsible for their death. To test this hypothesis, an attempt was made to select for more heat-resistant strains by growing the lactic acid bacteria in broth containing ethanol. As the results with Lactobacillus plantarum illustrate (Table 2), we were not successful. Ethanol-grown cells did not exhibit significantly greater heat resistance than those propagated in the standard medium. Ethanol. Of the different wine constituents that were studied, alcohol had the greatest effect on heat resistance ( Table 3). As illustrated here, the resistance of both lactics and yeasts was reduced. The flor yeast was affected to a greater extent than L. fructivorans: the Dvalue for it was over 50-fold greater in the absence of ethanol than when heated in the presence of 12%; the lactic exhibited a difference of less than fourfold. Thermal death time curves have been plotted (Fig. 2). The parallel slopes show that these differences in concentration did not affect the organism's response to a change in temperature. In other words, the zvalues for these organisms were not affected by alcohol. Studies by other workers also have shown ethanol to reduce the heat resistance of yeasts (3,4,9). Schanderl (9) observed that differences in alcohol concentration of only 3% had a marked effect on survival, which is in agreement with our findings. Carroll and Lopez (3), on the other hand, obtained little difference in thermal destruction of S. cerevisiae when heated in buffers containing 6 and 10% ethanol. pH. The effect of pH on heat resistance was determined by adjusting the reaction of Niagara wine with NaOH or tartaric acid. The study was limited to the pH range of 3 to 4 since these are common values for wine. The results ( Table 4) indicated that individual species respond differently to hydrogen ion concentration. Thus the flor yeast gave comparable survivals over this range while L. fructivorans showed an increase in D-value as the pH was raised. Data obtained with L. plantarum B246 (not shown) indicated comparable survivals between pH 3.4 to 4.0 and significantly lower figures at pH 3.0 and 3.2. Sugar. Glucose is another common wine con- stituent to which individual species responded differently. Thus the addition of 10% to Niagara wine increased the D-value of L. fructivorans almost threefold but afforded only slight protection to L. plantarum and the flor yeast (Table 5). These results suggest, of course, that the pasteurization requirements for a dessert or a pop wine might be somewhat greater than for, a dry, table wine. Sulfur dioxide. In the studies with SO2, freshly prepared solutions of potassium metabisulfite were mixed with the wine just prior to filling the capillary tubes. It is assumed, however, that the actual amount of free S02 was lower than the calculated value since some would react with aldehydes and other wine constituents. The results ( Table 6) again showed a variable response depending upon the organism: SO2 appreciably reduced the resistance of L. fructivorans, whereas an effect on the yeasts could not be detected. We were not too sur- Other constituents. In addition to the more obvious variables already described, it seemed that wines might contain other substances that would influence heat resistance of potential spoilage organisms. For example, certain tannins or other polyphenolic compounds, natural constituents of the grape, might either protect organisms or cause them to be more heat sensitive. One approach in the search for other factors was to compare heat resistance in various concentrations of the Niagara wine. In these studies the wine was diluted with a solution of 12.1% aqueous ethanol so that the concentration of all constituents except alcohol would be decreased. The results (Table 7) suggest that a small amount of protection was provided by some ingredient(s) of the wine. Thus the survivals ofS. cerevisiae and L. fructivorans, averages of two experiments, were slightly lower when heated in the straight aqueous ethanol solution. In other trials resistance was compared in wines fermented from different grape varieties. To eliminate the influence of ethanol, after determining the amount present, small quantities of water were added so that all of the wines ended up with the same concentration of ethanol, 11%. Again the results failed to reveal the presence of other wine constituents that had more than a modest effect on heat resistance (Table 8). It is suspected that the Delaware wine actually permitted somewhat higher survivals since the averages of two trials agreed very well. So far, however, it has not been possible to relate increased resistance to some intrinsic property of the Delaware wine such as pH, total acid, or free SO2. It is concluded from these studies that poten- tial spoilage organisms often possess little resistance when heated in a traditional table wine and that many wines may receive a more severe pasteurization than is actually required. The data also show that some formula modifications would increase the process requirements, in particular, the lowering of the alcohol content, the raising of the pH, and the addition of sugar. The single organism encountered in this study that possessed relatively high heat resistance, L. fructivorans, would not present a spoilage problem for many wines because we have found that it will not initiate growth in the tryptone-glucose-yeast extract salts broth when acidified to pH 3.8. Most wines have a pH lower than this. It is also possible that L. fructivorans would be inhibited by the levels of free S02 that generally are present in commercial wines.

v3-fos

2018-04-03T01:06:46.011Z

1975-01-01T00:00:00.000Z

20906712

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Incidence of Aflatoxin in California Almonds In a survey of California almonds, aflatoxin was found in 14% of 74 samples of unsorted, in-shell almonds as received by the processor in 1972, but it occurred at very low levels (below 20 parts per billion [ppb]) in 90% of the contaminated samples. The overall proportion of individual nuts contaminated was especially low and is estimated with 95% probability to have been in the range of 1 nut/55,300 nuts to 1 nut/14,700 nuts. Aflatoxin contamination is not restricted to any particular section of the almond-growing region of California. Commercial sorting procedures are effective in removing most aflatoxin-contaminated nutmeats, since none of 26 samples of processed, whole nutmeats contained aflatoxin. In contrast, 13 of 27 samples of diced almonds were contaminated, but nine of these 13 samples contained less than 20 ppb. Only one of 25 samples of sliced nutmeats contained aflatoxin (4 ppb). Thus, aflatoxin incidence in almonds varies greatly with the category of finished product. The apparent high incidence in diced nutmeats is probably due mostly to the more uniform distribution of aflatoxin occurring in this product (because of its small particle size) than that occurring in the other products. Sample size requirements for monitoring aflatoxin in almonds are discussed. In a survey of California almonds, aflatoxin was found in 14% of 74 samples of unsorted, in-shell almonds as received by the processor in 1972, but it occurred at very low levels (below 20 parts per billion [ppb]) in 90% of the contaminated samples. The overall proportion of individual nuts contaminated was especially low and is estimated with 95% probability to have been in the range of 1 nut/55,300 nuts to 1 nut/14,700 nuts. Aflatoxin contamination is not restricted to any particular section of the almond-growing region of California. Commercial sorting procedures are effective in removing most aflatoxin-contaminated nutmeats, since none of 26 samples of processed, whole nutmeats contained aflatoxin. In contrast, 13 of 27 samples of diced almonds were contaminated, but nine of these 13 samples contained less than 20 ppb. Only one of 25 samples of sliced nutmeats contained aflatoxin (4 ppb). Thus, aflatoxin incidence in almonds varies greatly with the category of finished product. The apparent high incidence in diced nutmeats is probably due mostly to the more uniform distribution of aflatoxin occurring in this product (because of its small particle size) than that occurring in the other products. Sample size requirements for monitoring aflatoxin in almonds are discussed. Aflatoxins may occur in food products if certain molds, namely Aspergillus flavus or A. parasiticus, develop on them under appropriate conditions. These molds, like other storage fungi, seem especially likely to be a problem when seeds or nuts are at an intermediate moisture level, i.e., below that required by field fungi and above that inhibitory to fungal growth (4). The minimum moisture level for aflatoxin production at 30 C by A. flavus is equal to the moisture content of a product in equilibrium with 83% relative humidity or higher, depending on the nature of the substrate and the duration of storage (6). For starchy cereal seeds such as maize and wheat, the limiting moisture level for growth of A. flavus is about 18.5% (4), whereas in oily seeds such as peanuts it is 8 (4) or 9% (7). For almonds (Prunus amygdalus), the limiting moisture content for growth ofA. flavus is likely to be similar to that found for peanuts, due to the similarity in composition of these nuts. Infection of tree nuts with aflatoxigenic molds probably occurs most often in the field before and/or during harvest while the kernels are still moist. Tree nuts are generally exposed to field dirt and to possible physical damage by modem I Present address: Santa Clara County Department of Public Health, Occupational Health Chemistry, San Jose, Calif. 95128. methods of mechanical harvesting, which involve knocking the nuts to the ground and later collecting them with a machine that brushes them onto a conveyor belt traveling in front of a collecting bin (8). Insect damage before and after harvest might contribute to the invasion and development of molds. The exact conditions responsible for the contamination of tree nuts remain to be established. Since aflatoxins are highly toxic to most animals and carcinogenic to at least some animal species (12), their presence in various food and feed crops poses a serious threat to the safety of our foods. Foods subject to aflatoxin contamination have been regulated by the U.S. Food and Drug Administration (FDA) under the Federal Food, Drug, and Cosmetic Act, as amended 1972, Section 402a, which relates to adulterated foods (3). Although no legal tolerance has been established as yet for these toxic compounds in foods, the FDA has set an informal guideline level of 20 ppb of aflatoxin, beyond which they will seize a product (1). The guideline level applies to the total amount of aflatoxins, which by analytical methods used routinely by the FDA includes aflatoxins B1, B2, G1, and G2 (2). In 1971, the FDA alerted the almond industry of California that the results of a recent FDA study indicated that almonds, as well as other tree nuts, are sometimes contaminated with aflatoxins. Therefore, the present study was undertaken with the support of the California almond industry to determine the incidence of aflatoxin in almonds and to determine the effects of commercial handling and processing practices on aflatoxin incidence. This study includes almonds as they arrive from the grower, almonds at various stages of sorting in the processing plant, and almonds as finished products marketed by the processor. Since studies on aflatoxin in peanuts have shown that only a few nuts out of many thousands need to be contaminated to be a serious problem (5,(9)(10)(11), it was assumed that very large samples of almonds would be required for representative sampling and meaningful analysis. The sample size chosen represents a compromise between the estimated appropriate size and a size that is practicable in regards to cost and analysis. MATERIALS AND METHODS Samples. Samples from the 1972 almond crop were collected throughout the harvesting and processing season. The following general categories of almonds were sampled: (i) unsorted in-shell nuts, representing incoming almonds as received by the processor; (ii) "in-process nuts," representing nutmeats at various stages of sorting by the processor; and (iii) processed nutmeats, representing various finished products sold for food use. This last category consisted of diced, sliced, and whole nutmeats, including the whole nutmeat samples from the final stage of the in-process or sorting study. Only Nonpareil variety almonds were used for the study of unsorted in-shell nuts and of whole nutmeats, because this variety represents the major portion of the California almond crop and because this variety seems most likely to have mold damage due to its extra soft, thin shell. With sliced and diced nutmeats it was not possible to limit samples to varieties, since they are usually prepared from mixed varieties. The nut samples were obtained from six almond processors of large and medium size, who together handle most of the California almond crop. The unsorted in-shell nuts and the in-process nutmeats were obtained from all six processors, whereas the sliced and diced nutmeats were collected from only three processors. The geographical origin of each in-shell sample was recorded as it was collected. Altogether 223 samples of in-shell nuts and nutmeats were assayed. Sample handling and preparation. Samples were collected in plastic bags and stored at 32 to 34 F (0 to 1 C) as soon as received at the laboratory to arrest any aflatoxin formation. Storage at low temperature was probably not critical, because all samples received were dry enough (e.g., below 7% moisture) to be fairly safe from mold and aflatoxin development, at least over short periods of storage. The samples were removed from cold storage 1 to several days before being prepared for assay to allow them to reach room temperature and thereby avoid condensation on the nuts. In-shell samples were sorted by hand to remove foreign materials, i.e., loose hulls, loosely attached hulls, rocks, sticks, etc. Removal of the variable amounts of foreign materials made the samples more comparable, was necessary to protect the blades of the equipment used for sample preparation, and avoided interference in the analysis by certain constituents of the hulls. In all but a few cases, there was sufficient sample available to permit 18.0 lb (ca. 8.2 kg) of in-shell nuts or 15.0 lb (ca. 6.8 kg) of nutmeats to be cut and blended in a Hobart vertical cutter-mixer (VCM) (25-qt [about 22.3 liters] VCM). A fine, homogeneous meal was prepared with the VCM by intermittent cutting (e.g., 15 s at a time) at slow speed for 1 min and then at high speed for another 1 or 1.5 min. Allowing the samples to cool between the periodic cuttings avoided problems of overheating, oiling-out, and compacting of the product. In a few cases, it was necessary to use Celite Hyflo Supercel (Johns Mansville) as a cutting aid to attain a finely cut, free-flowing meal. Using a sharp wave-cut blade of the type available for the Hobart VCM contributed to the preparation of a fine nut meal without aid of the diatomaceous earth. Analysis. Aflatoxins B,, B,, G,, and G, were determined by a procedure very similar to Method I for aflatoxins in peanuts given in the Official Methods of Analysis of the AOAC (2). Method I has been found by the FDA and ourselves to be the most reliable method for tree nuts of the several official aflatoxin methods. The analysis was carried out under gold fluorescent lighting to avoid possible loss of the ultraviolet-sensitive aflatoxins. A weighed portion of the finely cut samples of nutmeats (50.0 g) or in-shell nuts (75.0 g, which is equivalent to about 50.0 g of nutmeats) was mixed with 15 g of Celite Hyflo Supercel, 25 ml of water, and 250 ml of chloroform in a 500-ml, glass-stoppered (g.s.) Erlenmeyer flask. If cutting aid was present in the sample, the sample size was increased proportionately. After shaking the sealed flask and its contents on a Burrell wrist-action shaker for 30 min, the extract was filtered rapidly through a Buchner funnel containing filter paper (Schleicher and Schuell no. 595) coated with 10 g of Celite Hyflo Supercel. A 50-ml sample of the filtrate was chromatographed on a silica gel column, and the eluate was analyzed by thin-layer chromatography according to the AOAC procedure. The aflatoxins were estimated quantitatively against aflatoxin standards obtained from the Southern Regional Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, New Orleans, La. Analysis of subsamples. The method of analyzing the 50 g of subsamples used to study aflatoxin distribution differed from the usual method slightly in sample preparation and extraction. Thus, 50 g of the sample (whole nutmeats or diced nutmeats) was used as received. The sample together with 15 g of Celite Hyflo Supercel was ground in a Waring blender (1-qt jar [about 0.946 liters]) with 250 ml of chloroform for 1 min. The mixture was transferred to a 500-ml g.s. flask, 25 ml of distilled water was added, and the 49 VOL. 29, 1975 on March 22, 2020 by guest http://aem.asm.org/ Downloaded from extraction was continued on a wrist-action shaker for 30 min. The extract was filtered and analyzed as described in the method used for the survey. RESULTS AND DISCUSSION Unsorted in-shell nuts. The survey of incoming in-shell nuts was expected to give some idea of the incidence and level of aflatoxin in Nonpareil almonds as received at the processing plant. Although the overall concentration of aflatoxin in these nuts appears to have been fairly low, aflatoxin was by no means a rare contaminant. About 14% of the samples (10 of 74) were contaminated with aflatoxin at 1 ppb or more. The amounts of aflatoxin found in the positive samples are shown in Table 1. It should be noted that the concentrations in this table are expressed on a total weight basis (i.e., kernel plus shell). Whereas only one sample of in-shell nuts exceeded the present guideline of 20 ppb (edible portion), four other samples contained 5 ppb or more of aflatoxin. Since aflatoxin incidence in in-shell almonds might be related to orchard location, the geographical origin of each contaminated sample was examined. The 10 almond samples containing aflatoxin originated in all sections (i.e., northern, central, and southern sections) of the growing area in the Central Valley of California. Although a more extensive study might show some correlation of aflatoxin incidence to climatic conditions or cultural practices within the almond-growing area of California, it is evident that the aflatoxin problem is not restricted to any one district of California. Estimation of proportion of contaminated nuts. It must be emphasized that, although the proportion of samples found contaminated was substantial (14%), the proportion of individual nuts contaminated was probably exceedingly small, assuming aflatoxin incidence in almonds is similar to that found in peanuts (5,10). The proportion of contaminated nuts can be estimated by statistical analysis. The probability of a contaminated sample can be estimated by the Poisson distribution: where: k = number of nuts in the sample and p = proportion of contaminated nuts. A point estimate of the proportion of contaminated nuts (p) can be obtained by equating this function (1) to the proportion of positive samples (x) actually found in n samples. x/n = 1e -kp solving for p: Since (1) defines the probability of one of two possible outcomes, it is appropriate to use the standard formulas for binomial confidence intervals on this function. The resulting upper and lower limits are each solved for p. The average proportion of contaminated nuts (p) and its 95% confidence intervals (upper and lower limits) can be estimated for the unsorted in-shell nuts, if it is assumed that all samples had 240 in-shell nuts/lb (or per 453.6 g). Thus, it is estimated that the average proportion of nuts contaminated with aflatoxin for the unsorted in-shell nuts is 3.78 x 10-s and that the lower and upper 95% confidence limits are 1.8 x 10-s and 6.7 x 10-5, respectively. The reciprocal of the proportion of contaminated nuts, which gives the number of good nuts per contaminated nut, provides a better visual picture of the small porportion of nuts that are contaminated. Thus, it is estimated that on the average only one nut in 26,500 nuts (about one nut in 110 lb [about 49.9 kg] of in-shell almonds) is contaminated; the 95% confidence interval is one nut per 55,300 nuts to one nut per 14,700 nuts. In-process nuts. The survey of nutmeats taken at various stages of commercial sorting and grading shows that normal sorting procedures, which are based on physical appearance, reduce the incidence and levels of aflatoxin in almonds. The results show that manual sorting tends to remove the aflatoxin-contaminated kernels from the sorted (select) kernels, resulting in their concentration in the rejected kernels ( of the 16 samples of hand-rejected kernels (oil stock) were contaminated. Furthermore, nine of the 10 positive samples of manually rejected kernels contained aflatoxin in excess of the guideline level of 20 ppb. The presence of measurable amounts of aflatoxin in only two of the 16 samples (12%) of the unsorted kernels indicates an incidence of aflatoxin that is in fair agreement with that found in the unsorted, in-shell nuts from the growers (14%). In fact, by statistical analysis similar to that used for in-shell nuts, assuming 400 kernels/lb, the average proportion (p) of contaminated kernels in the unsorted kernels is estimated to be roughly the same as that in the incoming in-shell nuts (2.23 x 10-5 versus 3.78 X . Hand sorting appears to be more effective than electronic sorting in removing aflatoxincontaminated kernels. Not only was the number of contaminated samples highest in the handsorted rejects, but the average aflatoxin concentration was also highest in these rejects. The difference in aflatoxin incidence in hand-sorted rejects and electronically sorted rejects could result from the fact that the kernels rejected by electronic sorting contain a high proportion of broken and sheller-damaged kernels, which dilute the aflatoxin-contaminated kernels that are associated with the "seriously damaged" (moldy and insect-damaged) kernels. Too few samples were analyzed to prove statistically that electronic sorting tends to remove aflatoxin-contaminated nuts. Since there is a tolerance for seriously damaged kernels in the United States standards for grades of shelled almonds, some samples of finished product eventually may be contaminated with aflatoxin. The higher the tolerance for seriously damaged kernels, the more likely this will occur. There is little chance of the most seriously damaged kernels being in the finished product, but the present study did not attempt to relate type or degree of defect with aflatoxin. On the basis of the data obtained, it appears that one does not have a very good chance of finding a positive (contaminated) sample in sorted whole nutmeats unless one uses a sample much larger than 15 lb (6.8 kg). For example, if the sorted nutmeats contain 1% of the seriously damaged kernels that are equivalent to those rejected by hand sorting, the probability of finding one or more contaminated samples in the 26 samples of sorted nutmeats used in this study is only 0.225. That is, on the basis of the data (i.e., p = 1.63 x 10-4 for hand-rejected kernels; 1% tolerance for seriously damaged kernels) it can be estimated that 77.5% of the time one can examine 26 samples of 15 lb (6.8 kg) each without finding any contamination. Processed (finished product) samples. The survey of processed nutmeats was made to determine the frequency and amount of aflatoxin contamination in various finished products available to the consumer. The products sampled for this part of the study were from the following basic categories: whole almonds, sliced almonds, and diced almonds. Aflatoxin occurs to a much greater extent in diced almonds than in sliced or whole almonds (Table 3). Thus, whereas none of the 26 samples of whole nutmeats and only one of the 25 samples of sliced nutmeats were contaminated, 13 of the 27 samples (48%) of the diced nutmeats contained aflatoxin. However, only four of these 13 contaminated, diced samples were above the existing FDA guideline of 20 ppb. Although blanching may tend to lower aflatoxin concentration, this factor was not considered in the analysis of the data because of the small number of samples involved. A higher tolerance for seriously damaged kernels used for dicing than for any USDA grade of whole or broken almonds undoubtedly contributes to this finding. To exemplify the situation, a statistical estimate can be made of the proportion of contaminated kernels (whole) in the diced nuts on the basis of the proportion of contaminated samples that were found. Thus, if it is assumed that all diced nut samples were contaminated at the same level and that there were 400 kernels/lb in the raw material from which they were prepared, it can be estimated that the average proportion of contaminated kernels in the diced nuts is 1.09 x 10-' with 95% confidence limits of 5.6 x 10-1 and 1.9 x 10-g. That is, on the average there was one contaminated kernel in every 9,200 kernels used for dicing, and the limits for 95% confidence were a lower limit of 1 nut/17,900 nuts and an upper limit of 1 nut/5,300 nuts. Since no samples of sorted, whole kernels were contaminated, it is not possible to make a similar statistical estimate for whole almonds. However, a statistical estimate of the upper limit for a 95% confidence interval of the average proportion of contaminated kernels can be made for whole almonds, and it is 1.9 x 10-I or no more than 1 nut/52,600 nuts. Since the size of the sample required to include a contaminated kernel is inversely related to the proportion of kernels contaminated, it is evident that much larger samples must be used to survey aflatoxin in whole nutmeats than in diced nutmeats. Distribution study. The most important factor influencing incidence and sample size requirements is distribution (i.e., degree of nonuniformity) of the contaminant in the product. Aflatoxin distribution undoubtedly varies among the different product categories. Thus, distribution is likely to improve as particle size of the product is reduced, so that aflatoxin should be distributed more uniformly in diced nutmeats than in whole nutmeats. Therefore, in several cases the unground materials remaining from the above surveys of aflatoxin were subsampled to examine aflatoxin distribution in whole and diced nutmeats. The size requirements for representative sampling can be estimated on the basis of such distribution studies; however, it was not possible to make a reliable estimate from this study, because it was limited by the amount and nature of the material remaining from the surveys. Nevertheless, this cursory study did demonstrate a vast difference between aflatoxin distribution in whole nutmeats and that in diced nutmeats. For example, the whole nutmeats remaining from two in-process samples found to contain aflatoxin (28 and 47 ppb) were subsampled for distribution analysis. In each case none of the five subsamples (50 g each) contained aflatoxin. The remaining unground, diced nutmeats of three positive samples (5,75, and 119 ppb) were similarity analyzed, with three, five, and seven subsamples, respectively. In contrast to the results with whole nutmeats, all subsamples of the diced nutmeats contained aflatoxin, although great variation in amount did exist (Table 4). Aflatoxin varied from a trace to a level higher than that found in the 15-lb (6.8-kg) analytical sample. Furthermore, the relative amounts of the four aflatoxins (B1, B2, G,, G2) differed among the subsamples. Nevertheless, aflatoxin was distributed more uniformly in diced nutmeats than in whole nutmeats. On the basis of the distribution study, it is evident that the likelihood of finding a contaminated sample in a given lot is much more dependent on aflatoxin distribution than on the proportion of contaminated kernels, which is likely related to the tolerance for damaged nuts. In the survey, the higher proportion of contaminated samples found with diced nutmeats than with whole nutmeats was due primarily to more uniform distribution of aflatoxin in the small nut pieces and only secondarily to the presence of a higher proportion of contaminated kernels (i.e., from the higher tolerance for damaged nuts). Because of the difference between aflatoxin distribution in diced nutmeats and in whole nutmeats, much smaller samples of diced nutmeats than whole nutmeats should be re- on March 22, 2020 by guest http://aem.asm.org/ Downloaded from quired to monitor aflatoxin. Whereas 15 lb (6.8 kg) may be an adequate size for monitoring aflatoxin in diced nutmeats, it is estimated that a sample of 10 to 100 times this size would be necessary for equal assurance of properly evaluating a lot of whole nutmeats. Further study is necessary before any precise estimate can be made of sampling requirements for monitoring aflatoxin in almonds.

v3-fos

2018-04-03T05:16:36.127Z

1975-10-01T00:00:00.000Z

42217781

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Toxigenic Aspergillus and Penicillium Isolates from Weevil-Damaged Chestnuts Aspergillus and Penicillium were among the most common genera of fungi isolated on malt-salt agar from weevil-damaged Chinese chestnut kernels (16.8 and 40.7% occurrence, respectively). Chloroform extracts of 21 of 50 Aspergillus isolates and 18 of 50 representative Penicillium isolates, grown for 4 weeks at 21.1 C on artificial medium, were toxic to day-old cockerels. Twelve of the toxic Aspergillus isolates were identified as A. wentii, eight as A. flavus, and one as A. flavus var. columnaris. Nine of the toxic Penicillium isolates were identified as P. terrestre, three as P. steckii, two each as P. citrinum and P. funiculosum, and one each as P. herquei (Series) and P. roqueforti (Series). Acute diarrhea was associated with the toxicity of A. wentii and muscular tremors with the toxicity of P. terrestre, one isolate of P. steckii, and one ofP. funiculosum. Because of its resistance to Endothia blight (1), the Chinese chestnut (Castanea mollisima Blume) was introduced into the United States for hybridizing with the nearly extinct American chestnut (C. dentata [Marsh.] Borkle). Annual chestnut production in the state of Georgia is about 60,000 to 70,000 lb (ca. 27,215.52 to 31,751.44 kg) (personal communications, R. L. Livingston, University of Georgia) and is marketed principally in the Appalachian area of the eastern United States-roughly, the center of the original range of the American chestnut. Production estimates for other states are unavailable. Chestnuts are a perishable commodity, easily spoiled by fungi and insects. Mature nuts are allowed to drop from trees and may lie for several days or weeks until gathered. Decay development may begin while they are on the ground (5). Commercially, chestnuts may be held in refrigerated storage for several months before marketing. Losses due to fungi frequently occur, particularly at the consumer level (15). In experimental storage studies Hammar (6) found that spoilage ranged from 5 to 10% after 1 month and 15 to 60% after 7 months at 2 C. Wright (16) reported that 62% of kernels examined soon after harvest contained visible fungus infections. The most common fungi isolated from decayed tissues were Phoma castaneae Oud. and Pestalotia spp. Of minor importance were species of Phompsis, Penicillium, Alternaria, Fusarium, Rhizopus, and others. Researchers in Italy and France have found that the most common genera of decay fungi isolated from European chestnut (Castanea sativa Mill.) kernels in storage were Penicillium, Fusarium, Phoma, Aspergillus (A. niger), and Rhizopus (2,8,12). There have been no reports, to our knowledge, on the mycoflora of weevil-damaged chestnuts. The chestnut weevil (Curculio sayi Gyllenhal), commonly called the small chestnut weevil, is a major threat to chestnut production because it attacks the nut kernels (9). Adults infest trees from April to late June and deposit eggs in nearly mature nuts during August and September. The larvae feed on kernel tissues, then emerge by cutting through the shell. Infested nuts may contain several weevil larvae, or, if larvae have already emerged, may contain weevil burrows filled with excrement. Weevil-damaged nuts are likely to harbor a wide variety of mycoflora and be subject to spoilage. Nuts from which weevils have emerged are generally culled from the packing operations by flotation. Nuts containing weevils, however, are not separated by the flotation process. Weevils then emerge while chestnuts are in storage or transit, and damaged nuts enter the market channels. Although such nuts are generally discarded by the consumer, some might be incorporated into processed chestnut products or food combinations. The potential for consumption of spoiled chestnuts is increased by the absence of visible mold on many kernels with incipient fungal infections. Fungi ofthe generaPenicillium andAspergillus have been associated with toxin production on many agricultural commodities (14,17). Other fungi, especially those of the genera Alternaria and Fusarium, have also been re-ported as toxin producers (3,4). This study was to determine the incidence of some mycotoxinproducing fungi on weevil-damaged chestnuts and was limited to Penicillium, the most frequently isolated genus, and to Aspergillus, the genus most often associated with mycotoxin contamination of foodstuffs. MATERIALS AND METHODS Isolation of fungi. Freshly gathered chestnuts were obtained in October 1972 and 1973 from orchards in central Georgia. Chestnuts with weevilemergence holes were selected and stored for 1 week at 3 C. Kernel pieces containing sections of weevil burrows were surface sterilized for 3 min in 0.5% sodium hypochlorite solution containing 3% ethyl alcohol, rinsed in sterile water, and plated on maltsalt agar. Fungus colonies developing from kernel pieces after 3 weeks at 21 C were classified by genera. Those not readily indentifiable were placed in a miscellaneous category. All Aspergillus and Penicillium colonies were transferred to potato-dextrose agar slants by mass transfer, allowed to grow at 21 C for 2 weeks, and stored at 3 C. Bioassay for toxicity. Cultures for bioassay were grown on shredded wheat (7) or on fresh or on autoclaved medium. Autoclaved chestnut medium, prepared in a 500-ml flask, consisted of 50 g of quartered, fresh chestnuts and 10 ml of water, which were autoclaved at 15 lblin2 and 121 C. Fresh chestnut medium was prepared by adding 50 g of surfacesterilized quartered kernels to an autoclaved flask containing 10 ml of water. Media were inoculated by mass transfer of spores from the potato-dextrose agar slants, and cultures were grown for 4 weeks at 21 C. Cultures were extracted for bioassay by the method based on that of Kirksey and Cole (7). Cultures were blended with 200 ml of chloroform in a Waring blender for 45 s, and the homogenates were filtered through a 1-cm pad of sodium sulfate on a 90-mm Buchner funnel. Chloroform filtrates were transferred to 150-ml beakers containing 5.5 ml of corn oil and placed on a steam plate for 3 h to completely evaporate the chloroform. Five 1-day-old DeKalb 151 cockerels were dosed by crop intubation with 1 ml of corn oil which contained extract. Checks were dosed with corn oil to which only pure chloroform had been added and then evaporated. Cockerel mortality, expressed as survival ratios, and any clinical symptoms were recorded over a 5day observation period. If mortality was over 50% or if survivors exhibited unusual clinical symptoms one or two additional bioassays were conducted. If confirmatory tests were also positive, the extracts were considered toxic. Cultures were rated for degree of toxicity: less than 50% mortality but with debilitated survivors equals low toxicity; more than 50% but less than 90% mortality equals moderate toxicity; and over 90% mortality equals high toxicity. Identification of toxic isolates. Cultures which produced toxic extracts were transferred to diagnostic media (10) for taxonomic identification. All taxo-nomic identities at the species level were considered definitive if major cultural and microscopy characteristics of an isolate agreed with published descriptions (10,11). When one or more characteristics of an isolate were at variance with descriptions, identification was at series level only. All Aspergillus isolates and only those Penicillium isolates shown to be toxic were identified at species level. RESULTS Penicillium spp. were the fungi most frequently isolated (40.7% occurrence) from weevil-damaged chestnuts (Table 1). Next, in order of frequency of occurrence, were Rhizopus, Alternaria, and Aspergillus, each comprising about 17% ofthe total mycoflora isolated. Fusarium constituted 6.4% of the colonies isolated, and fungi of unidentified and miscellaneous genera constituted 1.3%. Twenty-one of the 50 Aspergillus cultures isolated from chestnuts were toxic to day-old cockerels ( Table 2). Most of the isolates were A. A. wentii caused acute diarrhea, loss of appeflavus isolates and one of the fourA. flavus var. tite, and general debilitation or mortality. The columnaris isolates were toxic ( ity, each causing a cumulative average mortalcaused over 90% mortality, and isolates CA 14, ity of less than 50% ( Table 3). The remainder of CA 22, CA 48, and CA 52 were moderately toxic the A. wentii isolates (CA 9, CA 15, CA 19, CA (Table 3). 43, CA 45, and CA 51) were moderately toxic, None of the A. oryzae or A. niger cultures causing over 50% but less than 90% mortality. isolates from chestnuts were toxic ( Table 2). The remaining toxic Aspergillus cultures be-Eighteen of the 50 bioassayed Penicillium Table 2). Twelve of these 18 were associated with sustained muscular tremors and, in some cases, convulsions before death. Of the toxic Penicillium isolates, nine identified as P. terrestre (Series) were tremorgenic and of varying toxicity ( Table 3). Three of the toxic Penicillium isolates were P. steckii and were moderately to highly toxic. Two were P. citrinum and two were P. funiculosum, all but one were highly toxic. The P. funiculosum isolates were tremorgenic. One isolate of P. herquei (low toxicity) and one of P. roqueforti (moderately toxic) were also identified. Selected isolates of each major group of toxic fungi were cultured on autoclaved and on surface-disinfected fresh chestnuts. Of the three A. wentii cultures tested, CA 8 extracts from either autoclaved or fresh chestnuts were not toxic, CA 10 extracts from autoclaved but not from fresh chestnuts were toxic, and CA 13 extracts from both media were toxic ( Table 4). Six of seven A. flavus isolates tested (including A. flavus var. columnaris) produced toxin on autoclaved and on fresh chestnuts, and one (CA 46) was toxic on autoclaved chestnuts only. With the exception of P. herquei (CP 35), all extracts of Penicillium isolates grown on autoclaved chestnuts were toxic to day-old cockerels. On fresh chestnut medium, one isolate each (of two tested) of P. terrestre (CP 39), P. steckii (CP 18), and P. citrinum (CP 41) was toxic. The one isolate tested ofP. funiculosum (CP 25) was of low toxicity, and P. herquei (CP 35) and P. roqueforti (CP 14) were not toxic when grown on fresh chestnuts. DISCUSSION A high percentage ofPenicillium and Aspergillus isolates from weevil-damaged Chinese chestnuts were capable of producing mycotoxins. Forty-two percent of all aspergilli were toxic, and seven of 10 representative isolates tested produced toxin on inoculated, fresh chestnuts. Similarly, 36% of the penicillia bioassayed produced toxin on artificial media, and four of nine tested produced the toxins on fresh chestnuts. The organisms studied were fungi established in dehydrated or discolored tissues adjoining insect-damaged areas. No mycotoxins have been found on market chestnuts; however, a potential exists for toxin production in the event fungal development occurs on kernel tissues. The presence of surface contaminants also presents a potential problem if chestnut quality deteriorates in the market or in storage. The potential presence of mycotoxins in weevil-damaged chestnut kernels suggest the need for effective weevil-eradication programs in the orchards and for fastidious, quality control measures after harvest. Most A. wentii isolates lost a degree of toxicity during the course of this study. Initial subcultures of original isolates were highly toxic. Subcultures taken from original isolates in storage for 6 to 8 months were less toxic although diarrheagenic symptoms were strong. Prolonged storage of these toxigenic fungi on artificial medium or repeated subculturing may have resulted in mutations or in metabolic changes affecting toxicity. Further research is needed to test the capability offungi other than the aspergilli and penicillia present on weevil-damaged chestnuts. Genera such as Alternaria, Fusarium, and others have been associated with mycotoxicity. Research is now in progress to identify the toxins produced by the fungi reported in this study. The A. wentii toxin has been isolated and identified as emodin (13). Preliminary analyses suggest that aflatoxins and citrinin are the toxins responsible for the activity of A. flavus VOL. 30, 1975 on May 7, 2020 by guest http://aem.asm.org/ Downloaded from

v3-fos

2020-12-10T09:04:20.948Z

1975-07-01T00:00:00.000Z

237234851

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Effect of pH on Sporulation of Bacillus Stearothermophilus An improved broth medium was developed for high growth yields of Bacillus subtilis var. niger NCIB 8649, Bacillus cereus NCIB 9373, and Bacillus stearothermophilus NCIB 8919 and ATCC 7953. Sporulation was abundant (1.1 × 108B. subtilis var. niger and 9.2 × 107B. cereus per ml) at an initial pH of 7.0. Sporulation of both strains of B. stearothermophilus took place (1.9 × 107 and 2.4 × 107/ml, respectively) in this medium when initial pH values of 7.7 to 8.7 were used. An improved broth medium was developed for high growth yields of Bacillus subtilis var. niger NCIB 8649, Bacillus cereus NCIB 9373, and Bacillus stearothermophilus NCIB 8919 and ATCC 7953. Sporulation was abundant (1.1 x 108 B. subtilis var. niger and 9.2 x 107 B. cereus per ml) at an initial pH of 7.0. Sporulation of both strains of B. stearothermophilus took place (1.9 x 107 and 2.4 x 107/ml, respectively) in this medium when initial pH values of 7.7 to 8.7 were used. Bacillus stearothermophilus sporulates poorly in most liquid bacteriological media; attempts have been made, therefore, to develop media in which a significant number of spores are produced, but little work has been done on the environmental and pH aspects of sporulation. Several workers (3,5,7,8,11) developed special media in which B. stearothermophilus sporulates, yet the degree of sporulation obtained in this laboratory, even with these special media, was not consistently high and abundant. In the experiments reported here, a reproducible method for obtaining high yields of spores of B. stearothermophilus in an improved broth medium suggested by Thompson and Thames (11) Inocula were prepared by washing the growth from an appropriate culture into 100 ml of the medium described by Thompson and Thames (11). After 12 h of incubation, cells were removed by centrifugation and used for inoculation of the sporulation media. Sporulation media. Ten milliliters of inoculum with an optical density of 0.70 at 580 nm was added to a 250-ml flask containing 150 ml of sporulation medium. The sporulation medium consisted of: peptone (Oxoid), 0.30%; tryptone (Difco), 0.25%; yeast extract (Difco), 0.40%; beef extract (Difco), 0.25%; KHPO4, 0.20%; and MnSO4, 10 ,g/ml. The pH was adjusted from 5.0 to 9.50 with 0.1 N NaOH or 0.1 N HP04, and the media were autoclaved (121 C) for 20 min. After sterilization the pH ranged from 5.20 to 9.50. The addition of inocula did not change the pH of the medium. In modifying the medium suggested by Thompson and Thames (11) various components were omitted or added, one at a time, to determine their effect on sporulation of these strains of B. stearothermophilus. The components were also tested in different concentrations and at different initial pH values to determine the optimal concentrations and pH values stimulatory to sporulation. In the final selection of the concentration of the individual compounds of the medium, the choice was usually made to favor increased sporulation in B. stearothermophilus 7953, which normally produced the lower number of spores; a solid medium with the above composition at pH 8.0 gave a good crop of spores, which was not counted. Measurements. A PYE (PYE Unicam Ltd., Cambridge, England) pH meter equipped with a digital recorder was used to automatically record the pH of the growing culture at 20-min intervals. Plating and microscopic examinations were used to estimate sporulation and compare viable with total numbers. For plating, 25-ml samples of each culture were transferred to stoppered flasks and heated to 110 C for 5 min after temperature equilibration (5 min) for B. stearothermophilus (11), or to 78 to 80 C for 20 min for B. cereus and B. subtilis var. niger (4), to destroy vegetative cells. Thus, only spores were determined by outgrowth in triplicate on tryptone-glucose extract agar. Dilutions were made in distilled water and viable counts were made after incubation for 48 h. The percentage of sporulation of an organism has been determined on the basis of the number of colonies obtained before and after a given heat treatment; however, this method may be inaccurate for certain sporeforming organisms, because in some instances there is a heat activation requirement for spores before they will undergo optimal germination and outgrowth. The total colony counts of both species of B. stearothermophilus spores were the same before and after heat treatment at 110 C for 5 min. This suggested that the heat treatment did not stimulate additional germination and outgrowth of the spores. For this reason, the results presented here are reported as the percentage of total cells. A second reason for this procedure was that the low number of spores produced by these strains in some media made it impossible to accurately measure the total sporulation by direct microscopic counts. Microscopic estimations of sporulation in wet mounts were made by use of phase-contrast micrscopy; the percentage of sporulation was calculated on the basis of 250 organisms. RESULTS Changes in pH of the sporulation medium with an initial pH 7.0 + 0.1 for four species of bacilli and the points at which sporulation started are presented in Fig. 1. After a lag period, nearly 3.5 h for all four species, a sudden fall in pH of the medium was observed, followed by a sharp rise in pH of the medium in cultures of B. subtilis var. niger and B. cereus. Observed changes in pH for B. cereus and B. subtilis var. niger are typical of Bacillus spp. cultured in complex media (6,7,10). The presence of spores was detected in these two species, but in B. stearothermophilus the pH did not rise and few spores were detected. Changes in the pH of the medium with an initial pH of 8.5 for both strains of B. stearothermophilus and the points at which sporulation started are presented in Fig. 2. The patterns of changes in pH were similar to the other Bacillus spp., but the pH was never less than 6.8 in this medium. The percentage of sporulation of two B. stearothermophilus strains in media of different initial pH values is ploted in Fig. 3. Below pH 5.0 and above pH 9.0 the growth of both strains of B. stearothermophilus was suppressed. Between these two values there was little difference in the amount of 8.Or growth (vegetative cells plus spores) after 16 h of incubation. Maximum sporulation (about 88%) occurred between pH 7.7 to 8.7. Aeration by shaking did not increase the amount of growth and had no effect on the percentage of sporulation. DISCUSSION It has been shown (6, 10) that during the exponential period of growth of B. cereus T pyruvic and acetic acids are released, bringing about an initial fall in the pH of the culture. When sporulation began, the acids were utilized and the pH rose. The medium used in the present work did not contain added glucose; however, it is possible that some of the ingredients (particularly yeast extract) contained car- bohydrate from which acids were formed. The pattern of events described for B. cereus T (6, 10) would explain the cause of the changes in the pH of the medium which occurred in the present investigation. When B. stearothermophilus ATCC 7953 and B. stearothermophilus NCIB 8919 were cultured in the medium at an initial pH of 7.0 there was a decrease in pH of the culture to about 5.5, after which the pH remained unchanged; few spores were produced. When grown in a medium that initially was adjusted to pH 7.7 to 8.7, however, the pattern of changes in pH of the culture was similar to those observed with the other species of bacilli, and sporulation of the B. stearothermophilus occurred. This indicates that the enzymes of B. stearothermophilus which are responsible for utilizing organic acids during spore production may not be induced or may be inactive at low pH values produced during growth of vegetative cells, and, therefore, sporulation does not occur. It may be that at pH 7.7 to 8.7 these enzymes are induced, become activated, or are not inactivated, and sporulation occurs. Alternatively, it is possible that the release and accumulation of cell wall lytic enzymes or antibiotics (1,9) produced during growth and at the beginning of sporulation are more active at low pH, causing damage to the remaining vegetative cells before sporulation begins.

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2018-04-03T00:05:53.431Z

1975-03-01T00:00:00.000Z

8217406

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Quantitative Method for the Gas Chromatographic Analysis of Short-Chain Monocarboxylic and Dicarboxylic Acids in Fermentation Media A method for the preparation and gas chromatographic analysis of the butyl esters of volatile (C,-C7) and nonvolatile (lactic, succinic, and fumaric) acids in microbial fermentation media is presented. Butyl esters were prepared from the dry salts of the acids. The esters were separated by temperature programming on a column of Chromosorb W coated with Dexsil 300 GC liquid phase and analyzed with a flame ionization detector. Apparent recoveries with butanol-HCl or butanol-H2SO as butylating agents were 80 to 90% for most acids. Chromatographic profiles of the butyl esters demonstrated that both volatile and nonvolatile acids can be detected and separated in 24 min on a single column. Standard calibration curves (peak area versus concentration) of the butyl esters were linear in the range of 5 to 40 ,mol of acid per ml. The advantages of using an internal standard (heptanoic acid) for quantitating fatty acids in a mixture are given. Chromatograms of butylated fermentation media in which rumen anaerobic bacteria were grown illustrated that this method is useful for determining short-chain volatile and nonvolatile acids of taxonomic significance. microbial fermentation media is presented. Butyl esters were prepared from the dry salts of the acids. The esters were separated by temperature programming on a column of Chromosorb W coated with Dexsil 300 GC liquid phase and analyzed with a flame ionization detector. Apparent recoveries with butanol-HCl or butanol-H2SO as butylating agents were 80 to 90% for most acids. Chromatographic profiles of the butyl esters demonstrated that both volatile and nonvolatile acids can be detected and separated in 24 min on a single column. Standard calibration curves (peak area versus concentration) of the butyl esters were linear in the range of 5 to 40 ,mol of acid per ml. The advantages of using an internal standard (heptanoic acid) for quantitating fatty acids in a mixture are given. Chromatograms of butylated fermentation media in which rumen anaerobic bacteria were grown illustrated that this method is useful for determining short-chain volatile and nonvolatile acids of taxonomic significance. An important feature in the identification of anaerobic bacteria is an analysis of the acid fermentation products formed in culture media (9). These volatile (C1-C,) and nonvolatile (lactic, fumaric, and succinic) acids have been analyzed by various gas-liquid chromatographic methods. Problems arising from direct chromatography of the volatile components include free acids interacting with metal columns and adsorbing to polar supports and stationary phases, resulting in poor quantitation due to sample loss, peak tailing, and ghosting (10,13). Moreover, many stationary phases have low thermal stabilities and tend to desorb ("bleed") from supports with repeated use. In a report by Lambert and Moss (11) a procedure was described for the preparation and analysis of the butyl esters of short-chain volatile and nonvolatile acids on a Chromosorb-W support coated with a new, highly stable stationary phase, Dexsil 300 GC. This method is useful since it eliminates those problems associated with direct analysis of free acids, and both volatile and nonvolatile acids can be separated on a single column. The procedure described by Lambert and Moss, however, includes solvent extraction and evaporation steps which could lead to considerable loss of free acids from samples (5,8). In this publication we report a modified and simplified Lambert and Moss method for butylation of the sodium salts of carboxylic acids which provides for chromatography of multiple samples of fermentation media. When acids were esterified from samples and compared with authentic butyl esters, the apparent recoveries were 80 to 90% for most acids. Furthermore, in these studies the addition of an internal standard (heptanoic acid) to samples facilitated quantitation of the volatile and nonvolatile acids. MATERIALS AND METHODS Gas chromatograph and auxiliary equipment. The gas chromatograph used was a Hewlett-Packard model 5754B equipped with a hydrogen flame ionization detector. Additional components used with this instrument were also Hewlett-Packard equipment and consisted of the following: (i) an autosampler (model 7670A) with operating controls and injection units set for single analysis per sample and minimum wash cycle; (ii) a strip-chart recorder (model 7123A) of 100 mV full-scale deflection set for a chart speed of 0.5 inch (1.3 cm)/min, and (iii) a digital integrator (model 3373B) set for minimum input sensitivity SHORT-CHAIN MONO-AND DICARBOXYLIC ACIDS acid butyl esters were separated on a coiled glass column (0.25 inch by 6 feet [0.64 by 182.9 cm ]) packed with Chromosorb W (80 to 100 mesh, HP, DMCS, AW) coated with 10% Dexsil 300 GC (Analabs, North Haven, Conn.). Dexsil 300 GC is a recently developed (Olin Corp.) polycarboranesiloxane polymer stationary phase of moderate polarity which is stabilized against thermal degradation (maximum temperature limit, 450 to 500 C). Physical and chemical properties of this material have been given by Finch (7). The coated packing was loaded into columns by gentle tapping with a vibrator. Silanized glass wool was used to plug the column ends. The column was conditioned prior to use by heating to 250 C for 24 h and under a flow of nitrogen gas. Sample application was made by on-column injection. The following additional chromatographic conditions were employed throughout this study: (i) gas flow rates of 84, 260, and 50 ml/min for hydrogen, air, and nitrogen, respectively; (ii) injection port temperature, 230 C; (iii) detector temperature, 270 C; and (iv) temperature program, initial column bath was set at 50 C for 3 min followed by 10 C/min temperature increase to 250 C. The total time for chromatographic separation and integration of each sample was 24 min. A 5-min column bath cooling period followed after the last ester (dibutyl fumarate) was detected and integrated. Sample preparation, butylation procedure, and recovery of esters. A standard mixture containing 40 gmol each of the following acids per ml was prepared in distilled water, and the pH was adjusted to 9 to 10 with 10 N NaOH: formic, acetic, propionic, isobutyric, butyric, isovaleric, valeric, caproic, heptanoic, lactic, fumaric, and succinic. Dilutions of the standard mixture were made to obtain additional samples containing 5, 10, 15, 20, and 30 Mmolml. (All samples were made alkaline to convert the free acids to the ionized species and thus prevent their loss during subsequent lyophilization.) Samples (1 ml) of each fatty acid mixture (or individual fatty acids) were placed in culture tubes (13 by 100 mm) (Kimble Products), frozen in an alcohol-dry ice bath, and dried overnight on the shelf unit of a continuous freeze dryer (New Brunswick, model V-13). To the dry salts of the acids were added 0.8 ml of chloroform (or hexane) and 0.2 ml of 1-butanol saturated with anhydrous HCl. (Butanol was saturated with anhydrous HC1 by bubbling until a pH of 1 or less was achieved. The butanol-HCl mixture will remain saturated in a stoppered bottle for about 1 month.) Butanol (0.2 ml)-HSO. (0.05 ml) and 0.2 ml of boron trifluoridebutanol (14% wt/vol; Applied Science) were also compared as butylating agents. After mixing on a Vortex spinner, the tubes were tightly capped (Teflon-lined, screw cap), and the mixture was heated at 80 C in a temperature block (Lab-Line Instruments, Melrose Park, Ill.) for 2 h. Tubes were then cooled to room temperature and 0.2 ml of trifluoroacetic anhydride (TFA; Sigma Chemical Co.) was added to each; the solution was mixed and allowed to react for 1 h. The TFA was used to (i) react with: hydroxy acids (e.g., lactic) forming the trifluoroacetyl esters and (ii) react with any excess butanol in the reaction mixture. Samples were then washed twice with 1-ml aliquots of distilled water to remove excess TFA reagent, and the water layer was discarded. The chloroform layer (adjusted to 1 ml) which contained the butyl esters was placed into 1-ml vials with Teflon-lined aluminum seals (Wheaton Glass Co., Millville, N.J.). Two microliters of sample was injected and analyzed by gas chromatography. Cultures of rumen anaerobic bacteria were centrifuged to remove cells, and the supernatant (1 ml) was treated in the same way as the standard acid mixture. Authentic butyl esters of the various acids were made up in chloroform (5 to 40 umol/ml concentration) and analyzed either singly or as a mixture. Internal standardization and calculations. Calibration with an internal standard was performed by the addition of a known amount of heptanoic acid to standard mixtures of fatty acids or fermentation media samples before freeze drying and esterification. The formula used for calculating the quantity (micromoles per milliliter) of a sample unknown, containing an internal standard, is given by: (A.) (C,)/(RRF)-(A,), where A. and A, are areas of the unknown component in a sample and internal standard, respectively; C, is the concentration of internal standard in micromoles per milliliter; and RRF (relative response factor) is the ratio of AC,-AC,, where A, and C, are the area and concentration of prepared fatty acid butyl esters prepared from a standard calibration mixture containing 5 to 40 gmol each of C1-C7, lactic, fumaric, and succinic acids per ml. The percent recoveries of prepared butyl esters were calculated by comparing peak areas (in microvolts per second integrator units) with those of authentic esters. Mean peak areas and standard deviation of samples run in triplicate were also determined for each butyl ester. Monocarboxylic (C1-C7, iso C,, iso C., and lactic) and dicarboxylic (fumaric and succinic) acids were obtained as the free acid from Eastman Kodak Co. Cultures were centrifuged to remove cells, and the fermentation medium was processed as described above for preparation and recovery of butyl esters. Uninoculated media samples served as controls. RESULTS AND DISCUSSION Effects of butylating agents on esterification of acids. Data on esterification and apparent recovery of various volatile and nonvolatile acids using different butylating agents are shown in Table 1. With butanol-HCl, recovery of C l-C, and lactic acids (relative to authentic butyl esters) varied from 80 to 95% (see also Table 2); recoveries for dibutyl succinate and dibutyl fumarate, however, were 78 and 68%, respectively. Using butanol-H 2SO as the butylating agent, recoveries of acids were comparable to those with butanol-HCl, except that only 30% of the formic acid was recovered as the butyl ester. The low recovery of butyl formate with butanol-H2SO4 may be due to oxidation of the formic acid to CO2 and water by the concentrated sulfuric acid. In contrast, esterification of similar acid mixtures with BF,- butanol resulted in butyl ester recoveries varying from 25 to 95%; poorest recoveries were obtained for succinic and fumaric acids. Several aspects of the butylation reaction with butanol-HCl or butanol-HSO have been considered. Fatty acids can be reacted and extracted in hexane as well as in chloroform with no appreciable difference in recovery or alteration of the chromatographic profile. Diethyl ether, however, is not a desirable reaction solvent because of its high volatility (boiling point, 35 C). Maximum butylation of fatty acid mixtures at concentrations of 5 to 40 umol/ml each was achieved (particularly for formic and acetic acids) when the reaction was carried out for 2 h at 80 C. We have observed that recoveries of formic, acetic, and fumaric butyl esters were at least 5 to 10% lower when the butylation reaction was carried out for 30 to 60 min. Extending the heat step reaction beyond 3 h resulted in reduced recoveries (30 to 40% less) of the C1-C7 acids. It is necessary to allow the mixture to react with TFA to remove excess butanol, since butanol adsorbs tenaciously to the Dexsil column and interferes with elution of the butyl esters. TFA also reacts rapidly with the hydroxyl group of lactic acid forming the trifluoroacetyl ester of butyl lactate. In this respect, TFA esterification of lactic acid, presumably as the TFA and butyl derivative, was 10 to 15% (based on recovery) lower when the TFA was allowed to react for 15 to 30 min. However, authentic trifluoroacetyl butyl lactate (synthesized from TFA and butyl lactate, and structure confirmed by nuclear magnetic resonance spectroscopy and elemental analysis) co-chromatographs with butyl lactate under the conditions used in this study. In addition to having the same retention time, butyl lactate and trifluoroacetyl butyl lactate have similar response factors. These esters, therefore, were chromatographically indistinguishable. It is also likely that a mixture of butyl lactate and trifluoroacetyl butyl lactate is formed during the esterification reactions. For purposes of lactate quantitation, however, it is not necessary to know which derivative is formed, since the response factors for these esters are similar. Although pyridine has been used as a catalyst to enhance trifluoroacetylation of hydroxyl groups on acids (2), our studies indicate that pyridine is not necessary for this reaction. In fact, addition of pyridine (10 to 50 ,ul) to the reaction mixture reduced recoveries of butyl lactate (15 to 70% less) and most other butylated short-chain acids (10 to 30% less), depending upon the butylating agent used. It is common practice to extract the watersoluble, short-chain, volatile and nonvolatile acids into organic solvents (diethyl ether or chloroform) from acidified aqueous media. However, poor solvent extraction and recovery of volatile (e.g., formic and acetic) and nonvolatile (lactic and succinic) acids is a frequently reported observation (6,8). Hankinson et al. (8) noted that only continuous shaking of aqueous samples with a mixture of diethyl ether and petroleum ether resulted in recovery of 57% of the formic acid and 80 to 100% of the other volatile acids present in milk. Doelle and Manderson (6) determined that it was necessary to extract these acids from acidified microbial fermentation media for 2 to 3 h with diethyl ether to recover 90 to 100% of the volatile acids. In this latter study, losses of 10 to 20% occurred due to evaporation of the mixture, whereas distillation resulted in losses of 80, 60, and 20% for acetic, propionic, and isobutyric acids, respectively. Our experience with rapid solvent extraction procedures of acids from aqueous media confirms observations of others (1); only 10 to 30% of the formic and acetic acids and 60 to 80% of the C,-C. acids and lactic, succinic, and fumaric acids are recovered with diethyl ether or chloroform. Neither the use of hot solvents nor saturation of samples with salts significantly improves the extraction and recovery of most acids. If the extracted sample is evaporated to a smaller volume (e.g., 0.1 to 0.2 ml), we lose approximately 60% of the formic acid and 20 to 30% of the other volatile acids. Our method of lyophilizing media yields a greater recovery of fatty acids and, therefore, has an advantage over extractions and distillation techniques. Chromatographic profiles and calibration curves of acid butyl esters. Typical gas chromatographic separations of a mixture of authentic butyl esters and those prepared from the salts of acids are shown in Fig. 1. The chromatographic profile of authentic and prepared esters of volatile (C -C7) and nonvolatile (lactic, succinic, and fumaric) acids are superimposable, and individual acids were baseline separated on the Dexsil-Chromosorb W column (Fig. la and c). Only lactic and isovaleric did not separate as well, and thus integration and quantitation of the lactic and isovaleric acid peaks were affected. The butyl ester of 2-methyl butyric acid could not be separated from that of isovaleric acid. Retention times of the prepared esters are given in Table 2. A comparison of elution times between authentic and prepared butyl esters indicated they were highly reproducible and usually did not vary by more than 0.5 to 1.0% at the concentrations of acid tested. The profile of acids shown in Fig. 1 has been maintained for the last 6 months on the same preparation of column material (over 1,000 injections), demonstrating the remarkable stability of the Dexsil 300 GC stationary phase with repetitive column temperature programming. Baseline shifting or peak broadening, which may be indicative of desorption or breakdown of the liquid phase, was not observed. The reagent blank chromatogram in Fig. lb shows that reagents or by-products of the butylation reaction contribute a minor peak (between 5 and 6 min) to the butyl formate peak; another peak elutes at about 9.5 min but does not interfere with the separation of any other butyl ester. Unknown peaks (U1, U2, and U,) in the chromatogram of the prepared butyl esters of acids (Fig. lc) were due to trace components contaminating the commercially available succinic and fumaric acids. Similarly, separation of the butyl esters of valeric and caproic acids individually produced minor peaks (trace amounts) which chromatographed under butyl propionate and butyl butyrate, respectively. All other purchased acids and authentic butyl esters were chromatographically pure and free of detectable contaminating components at the concentrations employed. Standard curves relating peak areas and concentration of fatty acid are given in Fig. 2 problems associated with the analysis of fatty acids in a sample arise from the fact that usually an external standard (mixture of fatty acids) and a sample are run as separate injections at different times. An internal standard compensates for variations between runs in column temperatures, gas flow rates, detector sensitivity, sample preparation, and injection of different amounts of sample, since both sample and the internal standard are co-chromatographed and thus are influenced by these factors to the same relative extent. Heptanoic acid (as the butyl ester) fulfills several criteria for use as an internal standard for determining those fermentation acids considered here; it is (i) chemically similar to the sample species but normally not present in such samples, (ii) available as a pure and stable compound which may be accurately added to the sample, (iii) nonreactive with other acid components in a sample, and (iv) resolved from other acid peaks but similar in its retention time. Table 3 gives a list of the calculated RRFs of various prepared acid butyl esters relative to that of the internal standard, butyl heptanoate (added to each concentration mixture as heptanoic acid). These values show that the RRFs vary with concentration by 0.4 to 9% for most esterified acids. Butyl lactate, however, varied as much as i 30% (standard deviation, 0.122). The apparently low RRF for butyl lactate at sample concentrations of 5 and 10 gmol/ml is partly explained by the fact that the butyl lactate peak is not completely resolved from butyl isovalerate, and consequently there is interference with integration of this peak. In most fermentation media samples, isovaleric acid is a minor component and would not interfere with quantitation of butyl lactate. In our experience with microbial fermentation media, we have made use of an average RRF (see Table 3) and observed that most acids in a sample can be accurately quantitated to within 5 to 10% when they are present in concentrations ranging from 5 to 40 ,mol/ml. Application of the methods described in this paper to samples of microbial fermentation media are illustrated in Fig. 3 and Table 4. Chromatograms of fermentation products produced by rumen bacterial species in a rumen fluid-glucose medium are shown in Fig. 3, and quantitation of acids as the butyl esters using an internal standard is given in Table 4. Similar fermentation patterns (major and minor products) for these bacterial strains have been described by Bryant (3) and illustrated in the VPI Anaerobe Laboratory Manual (9). These data indicate that acids (C1-C , lactate, and succinic) as the butyl esters can be detected and quantitated with little interference from other components of the medium. The methods described in this paper are suitable, therefore, for analysis of acid fermentation products produced by anaerobic bacteria for purposes of identification. Preparation and analysis of the butyl esters of monocarboxylic and dicarboxylic acids have also been useful to determine milk fatty acids (14) and to identify species of Pseudomonas (12) a Products formed were determined from duplicate cultures of each strain. Calculations are based on the internal standard, heptanoic acid, and are corrected for acids in uninoculated (control) media. Abbreviations: F, formic; A, acetic; P, propionic; B, butyric; L, lactic; iV, isovaleric; V, valeric; C, caproic; S, succinic; Fu, fumaric. b Uppercase letters refer to acids formed in amounts of 10 Mmoles/ml of medium or greater, whereas lowercase letters refer to amounts less than 10 jimol/ml. Products formed in less than 0.5 jsmollml amounts are not given. LITERATURE CITED

v3-fos

2018-04-03T03:12:58.967Z

1975-11-01T00:00:00.000Z

1216976

s2

Survival of salmonellae during pepperoni manufacture. Survival of salmonellae in artificially contaminated beef-pork mixtures (approximately 10(4) salmonellae/g) was studied in pepperoni prepared by either a natural flora or lactic starter culture fermentation or in nonfermented sausages. The pepperoni did not become salmonellae free during the usual commercial 15 to 30-day drying period. Salmonella dublin was present in all products, fermented or unfermented, after 42 to 43 days of drying. At a lower level of contamination, 10(3)/g, S. dublin could not be recovered from starter culture-fermented pepperoni after 14 days of drying but persisted in the natural flora-fermented sausage. S. typhimurium (initial count, 10(4)/g) was absent after 42 days of drying when starter culture was used to ferment the pepperoni, but was still present in the natural flora-fermented and unfermented products. S. dublin, host adapted to cattle, or S. choleraesuis, host adapted to swine, had similar survival patterns in beef pork, or beef-pork pepperoni. Heating salmonellae contaminated beef-pork pepperoni (after fermantation but before drying) to an internal temperature of 60 C (trichinae inactivating) eliminated the food-borne pathogen from the sausage product. Dry and semidry fermented sausages have rarely been implicated in food poisoning. However, an outbreak of salmonellosis has been attributed to the presence ofSalmonella choleraesuis in Italian dry salami (5), and, in the United States, an incident of staphylococcal food poisoning was traced to Genoa sausage (2). In the latter case, the sausage contained coagulase-positive staphylococci with enterotoxin A; salmonellae were found also. Most workers have approached the study of the survival of salmonellae in fermented sausages by artificially contaminating the product at some step during manufacture and then following the number of survivors in samples taken during the remainder of the processing operations. The fate of salmonellae during the preparation and storage of"isterband," a Swedish fermented sausage (6), thuringer (3), dauerwurst (11), and Lebanon bologna (9) has been studied in this way. In 1972, pepperoni was implicated in a food poisoning outbreak; however, the causative agent was not identified (1). This food poisoning case prompted us to study the potential for survival of selected salmonellae during the processing of pepperoni. S. dublin, S. typhimurium, and S. choleraesuis were used as contaminants in different lots of pepperoni sausages. A 20-h culture of the test organisms grown at 37 C in tryptic soy broth (Difco) was diluted with distilled water to give the appropriate concentration of cells. The diluted culture was mixed into the meat by hand, and then the sausage mixture was stuffed into 55-mm fibrous casings. Fermentation of the pepperoni mixture was carried out either by encouraging the natural lactic microflora through the aging of meat containing 3% NaCl for 10 days at 5 C (7) or by the use of commercial Lactacel MC starter culture (Merck and Co., Rahway, N.J.), a mixture of Lactobacillus plantarum and Pediococcus cerevisiae. The starter culture was used according to the recommendation of the manufacturer except that a straight nitrate cure of 1.2 g of NaNO3/kg of meat was employed instead of nitrite. Unfermented pepperoni prepared from nonaged meat and without the addition of starter culture were processed by the procedures used for the fermented sausages. In those experiments that investigated the influence of a "trichinae cook" on the survival of salmonellae, the contaminated pepperoni were heated in a smokehouse set at 87.8 C (190 F), dry bulb, and 76.7 C (170 F), wet bulb. The sausages were removed when the internal temperature reached 60 C (140 F), quartered with a sterile knife, placed in plastic bags, and immersed immediately in an ice bath. The quartered sausages were kept refrigerated until assayed for salmonellae. Determination of the viable salmonellae count has been described in a previous publication (8). 760 SMITH ET AL. RESULTS The influence of Salmonella serotype as well as the type of meat used to prepare pepperoni were studied to determine the survival of salmonellae during processing. Beef, pork, or beefpork blends were prepared, and aliquots were contaminated with S. dublin (host adapted for cattle), S. typhimurium, or S. choleraesuis (host adapted for swine), respectively. The sausages were fermented by the natural lactic flora. These Salmonella serotypes were selected to determine whether a host-adapted serotype would behave differently from a non-hostadapted serotype in the three types of meat. APPL. MICROBIOL. However, there was very little difference in the survival patterns in pepperoni of the three Salmonella serotypes regardless of the meat type used. Since all commercial pepperoni are prepared from beef-pork mixtures (Palumbo et al., J. Food Sci., in press) and since there was no difference in Salmonella survival in pepperoni prepared from different meats, a beef-pork (1:1) mixture was used in all subsequent experimentation. The survival of S. dublin and S. typhimurium in starter culture-fermented, natural flora-fermented, and unfermented pepperoni made from a mixture containing equal amounts of beef and pork is shown in Tables 1, 2, and 3. The pepperoni that had starter culture incorporated into the meat mixture showed a decrease in pH from 6.1 to 4.5 after 1 day of fermentation at 35 C and a decrease in viable numbers of both test organisms (Table 1). No viable S. typhimurium were detected after 29 days of drying, whereas S. dublin was still detected after 43 days of drying. The pH decrease in sausages fermented by the natural flora was less pronounced than in those fermented by starter culture ( Table 2). The pH of the latter decreased from 6.1 to 5.1 in 2 days at 35 C. There was a decrease in the number of viable salmonellae during the fermentation period (Table 2), but after 42 days of drying viable S. dublin and S. typhimurium were still present. There was virtually no change in pH in the unfermented pepperoni after 2 days at 35 C; at 1 day, there was growth of both Salmonella serotypes followed by a decrease in growth on day 2 (Table 3). During drying the acid content increased, resulting in a slight lowering of the pH (from 6.0 to 5.7). There was no decrease in viable S. dublin within a drying period of 42 days; S. typhimurium decreased to a low level but was still detected at 42 days of drying (Table 3). S. dublin was shown to be more resistant to the processing conditions involved in pepperoni manufacture than S. typhimurium (Tables 1, 2, and 3). In the initial experiments, a relatively high number (about 4 x 10"/g) of S. dublin was added to the meat mixture used to make the pepperoni. When an initial inoculum of 1.7 x 103 S. dublinlg was used in natural flora-fermented pepperoni (pH 4.8 after a 2-day fermentation), there was still about 1 viable cell/g at the end of 42 days of drying. On the other hand, when the initial inoculum of S. dublin was 9.0 x 10'/g in starter culture-fermented pepperoni (pH 4.6 at 1 day of fermentation), no S. dublin could be recovered after 14 days of drying. Since pepperoni is usually made from porkbeef mixtures, the sausage or the pork must be given some treatment to inactivate trichinae which might be present. Trichinae can be inactivated by standardized freezing, heating, or drying. Large-scale industrial, freezing of pork for use in pepperoni is impractical and it has been demonstrated that drying is not completely reliable in destroying salmonellae in pepperoni (Tables 1, 2, and 3). Therefore, the fate of salmonellae added to pepperoni subjected to temperatures that would inactivate trichinae was investigated. The data in Table 4 indicate that heating the sausage to an internal temperature of 60 C (140 F) destroyed S. dublin remaining after the fermentation step (both starter culture and natural flora). The data indicate that salmonellae in unfermented pepperoni were destroyed by heating also. Salmonellae were not detected after 14 days ofdrying, indicating that there was no recovery of cells that might have been only heat injured. DISCUSSION Pepperoni is a highly spiced, fermented, dried sausage characteristically prepared from pork or pork and beef. The fermentation is similar to that of Lebanon bologna (7), but pepperoni is less acidic and is subjected to a long drying period. In a previous study, we found that salmonellae (at a level of about 104/g) was unable to survive the Lebanon bologna processing conditions except occasionally when natu- c The sausages were cooked to an internal temperature of 60 C (140 F). d Bacterial numbers lower than 0.03/g were considered to be zero. e After cooking, the sausages were placed in the dry room at 12 C and 60 to 65% RH. ral flora fermentation was used (8). The data obtained in this study indicated that salmonellae were not consistently killed by the processing conditions that were used in the manufacture of pepperoni. Fermentation with Lactacel MC starter culture was more effective than natural flora fermentation in destroying salmonellae. The same batch of meat was used to compare the effect of starter culture (Table 1) and natural lactic flora (Table 2) on the survival of S. dublin and S. typhimurium during processing. The pH of the pepperoni prepared with starter culture was lower than that prepared by the natural flora process, but viable S. dublin were still present after 42 days of drying with both types of fermentation. Viable S. typhimurium were present after 42 days of drying in pepperoni prepared with natural lactic flora but not with starter culture (Tables 1 and 2). When the initial inoculum of S. dublin was decreased approximately 50-fold (to about 103/g), no viable cells were found in pepperoni prepared with starter culture, but S. dublin was still present in sausages made by the natural flora fermentation (after 42 days of drying). Smith et al. (8) found that in Lebanon bologna contaminated with salmonellae starter culture fermentation was more effective than natural flora fermentation in eliminating the food pathogen from sausage. S. dublin, host adapted to cattle, orS. choleraesuis, host adapted to swine, behaved similarly in pork, beef, or beef-pork pepperoni. Host adaptation did not predispose the Salmonella serotypes to differential survival depending on the meat type used to prepare the sausage. The pH values of commercial pepperoni produced by nine different companies showed a wide range: 6.1 to 4.7. Three of the products ranged in pH from 5.6 to 6.1; three ranged from 5.1 to 5.3; and three ranged from 4.7 to 4.9 (Palumbo et al., J. Food Sci., in press). Since a sausage with a pH of 5.5 or above would not be considered a fermented product, a study of the survival of salmonellae in unfermented pepperoni was indicated. There was an increase in S. dublin during the processing period from an 'initial 4.1 x 104/g to 1.3 x 10c/g after 42 days of drying; S. typhimurium decreased to a very low value but was till detectable at 42 days of drying (Table 3). Unfermented pepperoni is marketed and presumably preferred by some consumers. The data presented in Table 3 suggest that Salmonella serotypes similar to S. dublin would probably survive in high numbers in such unfermented products and thus lead to a contaminated sausage. S. dublin was more resistant to destruction than S. typhimurium during the production of pepperoni (Tables 1, 2, and 3) and under the processing conditions for Lebanon bologna (8). Since all commercial pepperoni contain pork (Palumbo et al., J. Food Sci., in press), a trichinae-inactivating heating step at the end of fermentation and before drying was investigated to determine if this would result in the destruction of salmonellae. An internal temperature of 60 C (140 F) in the sausage was effective in destroying S. dublin (Table 4). Since salmonellae were not always killed during pepperoni processing (in the absence ofheating), apparently the safety of commerical pepperoni is due to other factors such as low contamination levels, postproduction heating (as in pizza), or by differences between commercial pepperoni processing methods and ours. Surkiewicz et al. (10) found that 28% of 560 fresh pork sausage samples were contaminated with low numbers of salmonellae, whereas only 0.4% of 735 fresh beef patties contained salmonellae (9). Therefore, pepperoni made from beef and pork mixtures could be expected to be occasionally contaminated with salmonellae. Commer-762 SMITH ET AL. on March 18, 2020 by guest http://aem.asm.org/ Downloaded from cial companies normally hold their pepperoni in the dry room for 15 to 30 days (4), and since the level of salmonellae, if present at all, is usually very low, the number of contaminants might be reduced to undetectable levels by the end of the drying period. Our data indicated a large reduction of salmonellae during fermentation and drying. However, in cases of high contamination levels, an unsafe product could result. Inclusion of a trichinae heating step of 60 C in commerical pepperoni production would ensure a Salmonella-free product.

v3-fos

2018-04-03T03:23:37.912Z

1975-06-01T00:00:00.000Z

2752819

s2

Extracellular polysaccharide from the black yeast NRRL Y-6272: improved methods for preparing a high-viscosity, pigment-free product. When the extracellular polysaccharide from the black yeast NRRL Y-6272, composed of two parts N-acetyl-D-glucosamine and one part N-acetyl-D-glucosaminuronic acid, is isolated at maximum culture viscosity, adhering black pigment gives the polysaccharide preparations a gray-to-black appearance. Precipitation of the polysaccharide from cell-free culture supernatants with either ethanol of hexadecyltrimethylammonium bromide failed to remove the pigment. Various other methods were therefore tried for obtaining a high-viscosity polysaccharide product free of pigment. By systematically varying ingredients of defined and semidefined media, an improved medium was found that not only gave polysaccharide preparations of increased viscosity, but also increased yield. A key ingredient in this medium is L-asparagine. Also, adding autoclaved bovine serum albumin or egg albumin to this medium at the time of inoculation allowed a pigment-free polysaccharide to be isolated by standard procedures. None of several other proteins of synthetic polyamides tested were as effective as bovine serum albumin or egg albumin. In an alternate approach, pink mutants obtained by irradiation of the parent black strain with ultraviolet light, apparently produce the same extracellular polysaccharide free of any pigment but in lower yields or inferior in quality. The black yeast strain NRRL Y-6272 (15) produces an acidic extracellular polysaccharide that contains both N-acetyl-D-glucosamine and N-acetyl-D-glucosaminuronic acid (15,27). This polysaccharide appears to have industrial potential, since it contains both acidic (carboxyl) and masked basic groups (N-acetyl) and since it disperses readily in water to give extremely viscous solutions. When the polysaccharide was isolated at maximum viscosity of the culture after 4 days of incubation by precipitation with either ethanol or hexadecyltrimethylammonium bromide (28), the product was colored gray to black by adhering pigment(s). When the polysaccharide. was isolated after 10 days of incubation, however, all the pigment was sedimented along with the cells when the culture was centrifuged (15). Incubation for 6 days beyond the time of maximum culture viscosity (5,000 to 6,000 centipoises) not only was impractically long, but also resulted in an irreversible decrease in viscosity to 2,000 to 3,000 centipoises. Just as with pullulan (35), pigment can be removed from polysaccharide Y-6272 by the Sevag fractionation technique (32). This technique, however, is impractical because it has to be applied numerous times for complete pigment removal and because yield of pigment-free polysaccharide is low (35) (up to 15% of the polysaccharide is lost with each application). We have found that the pigment contaminating polysaccharide Y-6272 is melanin-like, except for its apparent solubility in water. Reportedly, melanin binds many compounds (2) including chitin (4) and pullulan (35). In seeking a practical method for obtaining not only a pigment-free polysaccharide, but also a product of high viscosity and in an apparent narrow range of molecular weight distribution, we tried to find: (i) methods Culture media. All concentrations are for the whole medium. All parts were autoclaved separately at 121 C for 15 min, unless otherwise noted, and combined aseptically. Medium no. 1 (15) consisted of: part A, yeast extract (1%) and tryptone (3%), pH adjusted to 7.0; part B, glucose (5%). Medium no. 2 (11,12) consisted of: part A, KHPO4 (0.9%) and glucose (5%); part B, Wickerham's nitrogen base (34) (10 times more concentrated than that used for carbon assimilation tests). Part B was filter sterilized (Seitz) and 7.5 ml was added aseptically per 67.5 ml of part A. Medium no. 3 consisted of: part A, K2HPO4 (0.5%), MgSO4 (0.1%), ZnSO4 (200 Ag/75 ml of whole medium), and Vico P-300 (0.37%); part B, L-asparagine (0.75%); and part C, D-glucose (5%). Parts A and B were adjusted to pH 7.1 before autoclaving. Culture maintenance and propagation. Stock cultures of strain Y-6272 were maintained on yeastmalt agar slants (14) which, after being streaked, were held at 25 C for 2 days and then stored at 4 C. Stock cultures were transferred at 1-month intervals. For experimental polysaccharide production, 300-ml Erlenmeyer flasks containing 75 ml of media (plus inoculum) were incubated at 25 C on a rotary shaker (200 strokes/min, 2-inch eccentricity). To develop preinoculum, the stock culture was first transferred to fresh yeast-malt agar slants, incubated for 2 days, and then inoculated into yeast-malt broth (0.3% yeast extract, 0.3% malt extract, 0.5% peptone, and 1.0% D-glucose). After 2 days of incubation, this preinoculum (7.5 ml or 10% by volume) was transferred to an inoculum flask containing 75 ml of test medium, and the culture was shaken for 1 day. Then this inoculum (3.75 ml or 5% by volume) was transferred to production flasks containing 75 ml of the test medium. With the native strain, the culture liquor generally reaches maximum viscosity (in media 1 and 3) after shaking for 4 days and is highly cohesive and ropy like egg white. Recovery of polysaccharide Y-6272. Four-day cultures, having high viscosity (4,000 to 6,000 centipoises; Brookfield viscometer, model LVT, spindle no. 4, 30 rpm), were diluted with 3 volumes of water to reduce the viscosity (-175 centipoises) and then centrifuged (35,000 x g, 30 min) to remove cells. By addition of KCl (1%) and ethanol (95%, 2 volumes), the total polysaccharide separated as a stringy precipitate that wound around the stirrer. After this precipitate was dissolved in water, hexadecyltrimethylammonium bromide (1%, Eastman Organic Chemical) (28) was added to remove the major fraction (acidic), again as a tough stringy precipitate that wound around the stirrer; the minor (neutral) fraction remained in solution. The acidic fraction was dissolved in 2 M KCl and, after dilution by addition of 1 volume of water, was reprecipitated by addition of 2 volumes of ethanol. The acidic polysaccharide (in K salt form) was then redissolved in deionized water and dialyzed until free of extraneous salt. The dialyzed solution was adjusted to pH 6.25 with dilute KOH and then freeze dried. RESULTS Characteristics of the soluble pigment. The absorption spectrum of the pigment in cell-free supernatants, which also contain the extracellular polysaccharide, has no unique features; the optical density at 400 nm was used as a measure of the amount of soluble pigment, as has been done for the black yeast Phialophora jeanselmei (11,12). Various physical, chemical, and enzymatic procedures were ineffective for separating pigment from polysaccharide. Physical treatments tried were: dialysis; addition of Celite, diethylaminoethyl-cellulose, or activated carbon followed by centrifugation; precipitation of the polysaccharide with ethanol from neutral, acidic, or basic solutions; precipitation with hexadecyltrimethylammonium bromide; and extraction with several organic solvents. Chemical treatments included the following. Addition of ferric salts, although expected to precipitate melanin (16), formed a waterinsoluble polysaccharide-pigment complex; acidification with HCl precipitated pigment along with polysaccharide; NaOCl not only bleached the pigment to straw color, but also degraded the polysaccharide. Neither Pronase nor papain was effective in freeing polysaccharide from pigment. In its behavior underthese various treatments, the pigment contaminant of polysaccharide Y-6272 appears to be melanin-like (13,16,30) except for its apparent water solubility. Several compounds that were previously reported to inhibit tyrosinase or melanin formation in other organisms (7,23,24), when added to the culture medium of Y-6272, adversely affected either growth of the organism or poly-saccharide biosynthesis. Attempts were also unsuccessful to inhibit pigment formation by complexing suspected phenolic precursors (18,19) with such synthetic polymers as PVP. Factors involved in pigment and polysaccharide formation. A chemically defined medium (no. 2) (11, 12) was selected, and the effect of systematic addition of other ingredients was observed. Growth of Y-6272 on this defined medium was slow; the yeastlike cells were brown; cell-free culture supernatants were straw colored and contained little polysaccharide, as indicated by both the low viscosity of culture liquors and the low yield of isolated material. Adding L-asparagine to the medium caused black pigment to occur in both the cells and cell-free culture supernatants and increased viscosity. Whereas addition of filter (Millipore Corp., 0.22-Am pore size)-sterilized BSA (11,12), with L-asparagine either present or absent, resulted in highly pigmented cell-free culture supernatants of low viscosity, addition of heat-sterilized BSA plus asparagine resulted in moderate viscosity with less pigment than with asparagine alone. Addition of L-asparagine evidently stimulates polysaccharide Y-6272 biosynthesis, and addition of BSA, when autoclaved, diminishes soluble extracellular pigment. The stimulative effect of L-asparagine on polysaccharide production noted with defined medium (no. 2) was an important factor in the development of the more practical medium, no. 3. Among the various media compositions we have tested, medium no. 3 was best for production of polysaccharide Y-6272 from D-glucose at 25 C. The viscosity of cultures grown on medium no. 3 is 5,000 to 6,000 centipoises in contrast to 2,000 to 3,000 centipoises obtained with medium no. 1 (15). When grown on either medium no. 1 or 3, cultures reach maximum viscosity in 4 days with essentially no change in pH (6.8 to 7.2). When shaking was continued to 10 days, cultures on medium no. 3 retained their viscosity and soluble pigment, whereas for those on medium no. 1 viscosity decreased and pigment became insoluble. To prepare both pigment-free and high-quality polysaccharide, further information on the combined roles of L-asparagine and BSA seemed necessary. Effect of BSA on soluble pigment. After being heated under sterilizing conditions, BSA lowers the amount of pigment in cell-free supernatants of Y-6272 grown on medium no. 3, as well as on medium no. 2. The BSA may either be autoclaved with part A of the medium or heat sterilized separately and then added aseptically to the other sterile ingredients. In either case, however, the BSA is more effective when present in the medium at the time of inoculation rather than when autoclaved and added aseptically to the culture 2 or 4 days after inoculation. This effect is shown by optical density measurement of soluble pigment in cell-free supernatants when fermentation is terminated at 4 days (Fig. 1A). Heat-sterilized BSA, whether present at inoculation or added later to the growing culture, does not affect viscosity (Fig. 1B). In contrast to the effect of heat-sterilized BSA, filter-sterilized BSA stimulates formation of soluble pigment (Fig. 1A) and lowers viscosity (Fig. 1B) throughout the course of fermentation. Thus the polysaccharide having the most favorable quality, as compared with the control, is obtained under the conditions of points la and lb in Fig. 1A and 1B, respectively. The optimum concentration of BSA and length of autoclaving (121 C) are 0.2% (wt/vol) and 5 to 15 min, respectively. After culture fluids were centrifuged, the cell pad of cultures of Y-6272 to which heat-sterilized BSA had been added before inoculation had two visible layers: a large one at the bottom, which is a mixture of black yeast cells and insoluble BSA (white chunks), and a small gray-black one on top of the cell layer. This gray-black layer does not contain yeast cells and appears to be a pigment-BSA material. Control samples without added BSA do not have the top gray-black layer. We have found that filtration (Millipore Corp., 0.65-,um pore size) is also effective in reducing the level of pigment in culture supernatants, primarily by removal of the last traces of suspended pigmented cells and debris. Filters of pore size less than 0.45 um remove the polysaccharide and readily become clogged. Filtration of whole-culture broth is not practical, because filters rapidly become clogged with cells. Effect of various proteins and synthetic polyamides on soluble pigment. To test their applicability as agents for removing soluble pigment, proteins other than BSA were added to part A of culture medium no. 3 before autoclaving (15 min) ( Table 1). Of the materials tested, only EA was as effective as BSA, whereas almost all the materials tested actually increased the amounts of pigment in culture supernatants. After autoclaving, BSA and EA became insoluble. The amount of insoluble protein, as observed visually, was related in- versely to the amount of soluble pigment in polysaccharide preparations; i.e., test media with the greatest amounts of insoluble protein gave the lowest amounts of pigment in cell-free supernatants. The optimum length of autoclaving (15 min) for both BSA and EA was established experimentally; however, for the other proteins optimum conditions were not sought. The yield of polysaccharide was not affected by the addition of either BSA or EA; when Promine-D, gelatine, or Plasdone was added, the apparently high yield probably was due to large amounts of contaminating pigment ( Table 1). The synthetic polyamide PVP, when added in an insoluble form (Polyclar AT), remained insoluble in the medium and appeared to be inert. Addition of PVP in a soluble form (Plasdone-C) resulted in low viscosity and high pigmentation of cell-free supernatants ( Table 1). Pink mutant strains. After irradiation of the black parent strain Y-6272 with ultraviolet light, two mutants were selected that are pink initially when streaked on yeast-malt agar slants (14). When these mutants were stored on slants at 4 C for 14 days, mutant Y-6272 pink (colony 10-2) remained pink, whereas Y-6272 brown (colony 10-1) gradually turned brownblack. After growth in medium no. 3, cell-free supernatants of both mutant strains were straw colored, and the extracellular polysaccharide when isolated was colorless. All the pink coloration remains with the cells. To test whether L-aspartic acid, L-glutamic acid, or L-glutamine is a better nitrogen source than L-asparagine for polysaccharide production, the parent and two mutant strains were compared after 4 days of culture growth on medium no. 3 supplemented by each of these amino acids (Fig. 2). The samples fall into two distinct categories: (i) those from both mutants grown on L-asparagine or L-glutamine, additives which apparently provide unfavorable nutritive conditions (Fig. 2, lower right); and (ii) all the others, which fall on the same viscosity-yield line and for which nutritive conditions apparently were more favorable. After isolation, all samples of group (ii) gave nearly the same viscosity on a dry-weight basis and so are similar in nature but differ in yield. The addition of L-asparagine, as compared with the other nitrogen additives tried, doubles the yield of polysaccharide with both the native and mutant strains (Fig. 2); however, the product from the two mutant strains appears to be degraded since the viscosity of these samples (10 and 85 centipoises) is much lower than that from the native strain (6,500 centipoises). Analysis of the isolated polysaccharides by the NaOCl-amylose-KI method (26) for amino sugars, by the phenol-sulfuric acid assay for neutral sugars (9), and by paper chromatography of acid hydrolysates indicates that all three strains produce the N-acetyl-D-glucosamine-N-acetyl-D-glucosaminuronic acid polysaccharide as the major product regardless of nitrogen additive; however, the mutant strains produce more neutral polysaccharide(s) composed mainly of mannose and glucose. DISCUSSION Production of both black pigment and extracellular polysaccharide by the unidentified black yeast NRRL Y-6272 appears to be closely interrelated. The addition of known inhibitors of tyrosinase or melanin formation, culture conditions that preclude black pigment formation by the parent strain, either diminished or eliminated polysaccharide production. Mutant strains induced by ultraviolet irradiation, which contain pink but not black pigment, apparently produce a main (acidic) polysaccharide product that is compositionally the same as that from the black parent strain but varies in viscosity with the nitrogen source in the growth medium. Except for its water solubility, the black pigment appears to be melanin-like, as indicated by its chemical and physical behavior. Failure to remove the pigment by dialysis indicates either that the molecular weight is so large that the pigment cannot pass through the membrane, or the pigment itself is small but interacts with the polysaccharide so strongly that the pigment-polysaccharide will not dialyze out. Removal of pigment from polysaccharide Y-6272 by the Sevag technique (32), generally used to separate noncovalently bound protein from polysaccharides, suggests that contaminating pigment is physically associated and has some characteristics of a protein. The low recovery of polysaccharide Y-6272 with the Sevag technique appears to be due mainly to its loss into the emulsion layer from which a considerable amount of polysaccharide can be removed. Characterization of fractions of this recovered polysaccharide indicates that most of it is equivalent to the main product. Loss of some polysaccharide would be expected if a small percentage of the polysaccharide molecules are firmly complexed with pigment and so are removed by the Sevag technique. Further indication that the pigment has some proteinaceous character is the stimulation of soluble pigment formation by addition of soluble proteins, such as BSA, to culture media. With the black yeast P. jeanselmei (11,12), addition of soluble BSA solubilized the black pigment, normally found in mycelia, by attachment to the BSA molecule. These same studies also showed that the size of the BSA-pigment complex increased with incubation time until it was so large that the complex precipitated from solution. Similar behavior may take place when Y-6272 is grown on medium no. 1 since the soluble pigment seen at 4 days is removed by centrifugation when incubation is extended to 10 days. In this instance, soluble proteins in the medium may act like BSA. Failure to remove pigment from polysaccharide Y-6272 by treatment with Pronase or papain before isolation by alcohol precipitation may indicate either that the pigment is inhibitory to the enzymes or the proteinaceous portion of the pigment has been modified sufficiently to resist enzymatic cleavage. The mechanism of action of BSA and EA is not established by our observations, but apparently autoclaving medium no. 3 for 15 min places BSA and EA in an optimum physical state for acting as an insoluble anchor to which pigment can be attached preferentially. There does not seem to be any correlation between the amount of tyrosine in the various proteins tried and pigment removal since gelatine and Protamine-D, which have no tyrosine (31), stimulate extracellular pigment formation (see Table 1). Proteins, such as BSA, may be involved in the regulation of melanin biosynthesis (20). The correlation between low viscosity and high pigmentation of polysaccharide supernatants is not due to the precipitation of the polysaccharide by the added protein, since we found that pH of culture fluids remains near neutrality (6.8 to 7.2) throughout the 4-day culturing period and that pH must reach pH 4.5 or below for BSA to carry a positive charge and thus be able to precipitate acidic polysaccharide. This behavior is similar to that reported for an a-1,4-linked poly-N-acetylgalactosaminuronic acid (Vi antigen) polymer (33) and is the basis for a turbidimetric assay of the Vi antigen and other acid polysaccharides (8,25). The lowering of viscosity by added protein may be either due to the stimulation of biosynthesis of pigment, which binds the polysaccharide, or to the stimulation of an enzyme, such as the Vi antigen-degrading enzyme (1), which depolymerizes the polysaccharide. The stimulation of polysaccharide Y-6272 production by addition of L-asparagine is not completely understood, especially in view of our finding that L-glutamine, not L-asparagine, is involved in the enzymatic biosynthesis of hexosamine; i.e., L-glutamine, but not L-asparagine, is a substrate in the assay of fructose 6-phosphate amidotransferase (EC 2.6.1.16) in washed-cell homogenates. Two hypotheses have been proposed to account for the effect of L-asparagine: (i) L-asparagine may relate to the biosynthesis of a glycoprotein that is vitally related to polysaccharide synthesis, possibly as a bridge between protein and carbohydrate (17,29); (ii) L-asparagine may induce in the microorganism a morphological change (5) that must take place before polysaccharide production can begin. The inability to classify Y-6272 taxonomically is due mainly to our failure to induce sporulation. By an alternate approach of seeking taxonomic relationship through comparison of composition of polysaccharide products, we isolated extracellular polysaccharide from strains of a number of classified black yeasts. None of these produced extracellular polysaccharide comparable to that of Y-6272. The majority of strains were Aureobasidium pullulans, which produced glucans rather than amino sugar polysaccharides.

v3-fos

2018-04-03T05:03:31.112Z

1975-06-01T00:00:00.000Z

41465182

s2

Basis for the resistance of several algae to microbial decomposition. The basis for the resistance of certain algae to microbial decomposition in natural waters was investigated using Pediastrum duplex, Staurastrum sp., and Fischerella muscicola as test organisms. Enzyme preparations previously found to convert susceptible algae into spheroplasts had no such effect on the resistant species, although glucose and galacturonic acid was released from P. duplex walls. Little protein or lipid but considerable carbohydrate was found in the walls of the refractory organisms, but resistance was not correlated with the presence of a unique sugar monomer. A substance present in Staurastrum sp. walls was characterized as lignin or lignin-like on the basis of its extraction characteristics, infrared spectrum, pyrolysis pattern, and content of an aromatic building block. Sporopollenin was found in P. duplex, and cellulose in Staurastrum sp. Cell walls of the algae were fractionated, and the fractions least susceptible to microbial degradation were the sporopollenin of P. duplex, the polyaromatic component of Staurastrum sp., and two F. muscicola fractions containing several sugar monomers. The sporopollenin content of P. duplex, the content of lignin or a related constituent of Staurastrum sp., and the resistance of the algae to microbial attack increased with age. It is suggested that resistance results from the presence of sporopollenin in P. duplex, a lignin-like material in Staurastrum sp., and possibly heteropolysaccharides in F. muscicola. The basis for the resistance of certain algae to microbial decomposition in natural waters was investigated using Pediastrum duplex, Staurastrum sp., and Fischerella muscicola as test organisms. Enzyme preparations previously found to convert susceptible algae into spheroplasts had no such effect on the resistant species, although glucose and galacturonic acid were released from P. duplex walls. Little protein or lipid but considerable carbohydrate was found in the walls of the refractory organisms, but resistance was not correlated with the presence of a unique sugar monomer. A substance present in Staurastrum sp. walls was characterized as lignin or lignin-like on the basis of its extraction characteristics, infrared spectrum, pyrolysis pattern, and content of an aromatic building block. Sporopollenin was found in P. duplex, and cellulose in Staurastrum sp. Cell walls of the algae were fractionated, and the fractions least susceptible to microbial degradation were the sporopollenin of P. duplex, the polyaromatic component of Staurastrum sp., and two F. muscicola fractions containing several sugar monomers. The sporopollenin content of P. duplex, the content of lignin or a related constituent of Staurastrum sp., and the resistance of the algae to microbial attack increased with age. It is suggested that resistance results from the presence of sporopollenin in P. duplex, a lignin-like material in Staurastrum sp., and possibly heteropolysaccharides in F. muscicola. The cell wall probably is a major determinant of the resistance or susceptibility of algae to microbial decomposition in natural ecosystems inasmuch as it represents a key obstructing structure which must be disrupted, either mechanically or enzymatically, before an attacking organism has access to the contents of the algal protoplast and causes death of the organism. Although considerable work has been done to determine which components of the cell walls of fungi render these organisms refractory to microbial decomposition (3,5), little is known about the components of algal cell walls which protect them from possible attack in nature. In a study of the susceptibility of several algae to microbial degradation in natural waters, it was observed that Staurastrum sp., Fischerella muscicola, and Pediastrum duplex were particularly resistant to attack under conditions where other algae were readily destroyed and their contents liberated (Gunnison and Alexander, Limnol. Oceanogr., in press). The present investigation was initiated to determine which components in the cell walls were respon-I Present address: U. S. Army Corps of Engineers, Waterways Experiment Station, Vicksburg, Miss. 39180. sible for the resistance to microbial degradation. GUNNISON AND ALEXANDER amino acid, lipid, uronic acid, and sugar content of the walls were determined in the same manner as described for susceptible walls (Gunnison and Alexander, Can. J. Microbiol., in press). The walls were fractionated by a procedure modified from that of Boroughs and Bonner (6). A 50-mg sample of walls was treated for 2 h with 20 ml of 0.2% ammonium oxalate at room temperature, and the residue (designated I-1) was removed by centrifugation. The soluble fraction was designated S-1. Fraction I-1 was washed with distilled water and then treated with 20 ml of 0.05 N HCl at 85 C for 4 h. The insoluble residue was removed by centrifugation and discarded, the solubilized material being designated fraction S-2. A separate 100-mg portion of walls was incubated for 4 h at 4 C in 20 ml of 4% NaOH, and the soluble fraction (S-3) was separated from the unextracted material (fraction F-2) by centrifugation. Fraction F-2 was then suspended for 4 h in 17.5% NaOH at 4 C, and the suspension was separated into a soluble (S-4) and an insoluble fraction (I-3) by centrifugation. Fractions S-1, S-2, S-3, S-4, and I-3 are stated to contain pectates, protopectin, polyuronide hemicelluloses, noncellulosic polysaccharides, and a-cellulose, respectively (6). All materials were extracted twice in 20-ml volumes of extraction solution. Each insoluble residue was collected by centrifugation at 31,000 x g and washed once in 20 ml of distilled water. The two extracts and the water used to wash the insoluble residue derived from a given extracting solution were pooled. The extracts were taken to dryness in a rotary evaporator (Rinco Rotavapor). Fraction S-1 was then dissolved in 8.0 ml of distilled water. Prior to concentration, fractions S-2, S-3, and S-4 were neutralized, and after concentration, they were suspended in distilled water, dialyzed against distilled water at 4 C for 12 h, reconcentrated, and then again suspended in distilled water. Fraction I-3 was washed 10 times in distilled water and then lyophilized. For analysis of the fractions, either 10 ml of a soluble fraction or 10 to 15 mg of fraction I-3 was hydrolyzed in a sealed ampoule for 12 h with 2 ml of 6 N HCl at 105 C. After removal of excess HCl at 50 C under vacuum, the hydrolyzate was examined for total carbohydrate, reducing sugars, and sugar monomers, the latter by thin-layer chromatography (Gunnison and Alexander, Can. J. Microbiol., in press). Protein content was assessed after hydrolysis of the fraction for 60 min with 1 N NaOH at room temperature followed by neutralization with HCl; protein was determined by the Folin phenol method (13). A lignin-like material was extracted from Staurastrum sp. walls and from pine wood sawdust by the H2SO4 extraction method of the Technical Association of Pulp and Paper Industries (18). Sporopollenin was prepared from P. duplex by the sequential extraction method of Zetsche and Vicari (23). For infrared analysis, 2 to 4 mg of the material was made into a pellet with 40 mg of KBr, and the spectra were obtained with an infrared spectrophotometer, model IR-10 (Beckman Instruments, Fullerton, Calif.). The quantity of apparent lignin and sporopollenin in the wall preparations was established by determining the residue remaining after the extraction. For pyrolysis-gas liquid chromatographic analysis, 100 Ag of the lignin preparation was applied to a platinum pyrolysis coil; the latter was subsequently fired at 800 C in a model A-25 Pyrolyzer Accessory (Wilkens Instruments, Walnut Creek, Calif.). Separation of the resulting products was accomplished by gas chromatography using a stainless-steel column (3 mm diam by 2 m long) packed with Chromosorb W, 60 to 80 mesh (Johns Mansville, New York), as a solid support for the SE-52 liquid phase (Applied Science Laboratories, State College, Pa.). The products of pyrolysis were carried onto the column with N2 flowing at a rate of 20 ml/min. The column was held at 50 C for the first 6 min after pyrolyzer firing, and it was then subjected to a programmed temperature rise of 8.6 C/min until 185 C was reached. A flame ionization detector was used to monitor the exit of products from the column. Analysis of the fused lignin for phenylcarboxylic acids by thin-layer chromatography was performed on precoated plates of silica gel containing a fluorescent indicator (Eastman Kodak Company, Rochester, N.Y.) using benzene-methanol-acetic acid (90:16:8) as the solvent system (17). The solution of fused lignin was analyzed for catechols by gas chromatography using a modification of the procedure of Helling and Bollag (10) in which 10% DC 200 (Applied Science Laboratories) on Chromosorb W, 80 to 100 mesh (Johns Mansville), was used as the stationary phase. For gas chromatography, the injector, column, and detector temperatures were 260, 230, and 290 C, respectively. The flow rate of the carrier gas, N2, was 60 ml/min. To obtain 14C-labeled walls, the cultures were harvested after 14 days, and the algal cells collected from 8 liters of medium were placed in each of two 2-liter Erlenmeyer flasks. To these flasks was added 1.5 liters of the culture supernatant which had been adjusted to pH 8.3, and 0.5 ml of NaH14CO (New England Nuclear, Boston, Mass.) having an activity of 1 mCi/ml was then introduced. The algal suspensions were incubated on a rotary shaker at 150 rpm at 22 C under light at 5,400-lux intensity, and the labeled cells were harvested after 7 days, washed in 0.01 M phosphate buffer (pH 7.0), and lyophilized. Cell walls and wall fractions were prepared as described above. To determine the resistance of labeled walls and wall fractions to microbial attack, 50 mg of walls, 10 to 90 mg of a wall fraction, 21 mg of lignin, or 18 mg of sporopollenin was suspended in distilled water to a final volume of 10 ml. To each of two 250-ml Erlenmeyer flasks were added 5.0 ml of a suspension of the walls or an isolated fraction, 35 ml of the salts solution, and 0.25 g of soil (Williamson silt loam) contained in 2.5 ml of distilled water. In some instances, 2 ACi of [I4C ]glucose rather than walls or a wall fraction was used as the sole carbon source. The flasks were incubated at 25 C on a shaker operating at 120 strokes/min. Samples were taken at regular intervals, and the loss rate of labeled carbon was determined by a modification of the method of (11). For this purpose, labeled CO2 that remained in the reaction flask at the sampling time was driven off by acidification with 0.5 ml of 2 N HCl, and 0.50-ml amounts of the acidified reaction mixture were placed in scintillation vials with 10 ml of scintillation solution (7). The radioactivity was then determined in a scintillation counter, model Mark II (Nuclear Chicago, Chicago, Ill.). RESULTS Enzymatic digestion of cell walls. The sources of the various enzyme preparations used have already been described (Gunnison and Alexander, Can. J. Microbiol., in press). Chitinase, ,B-glucuronidase, hemicellulase, lipase, lysozyme, Pronase, and lytic enzyme preparations of Streptomyces isolates G4 and G7 had no effect on walls of these three resistant algae. Similarly, cellulase (free of polygalacturonase activity) was without effect on walls of F. muscicola and Staurastrum sp., and the polygalacturonase preparation did not digest F. muscicola walls. On the other hand, carbohydrate was released from the walls of P. duplex by cellulase and polygalacturonase preparations. Most of the solubilized carbohydrate appeared to be glucose, although the Bitter and Muir (4) test showed the presence of some galacturonic acid (Table 1). Essentially all of the wall material solubilized enzymatically could be accounted for as glucose and galacturonic acid. The polygalacturonase preparation had cellulase activity as determined by the formation of glucose from cellulose, but it released no sugar from ,B-1,2 glucan, ,B-1,6 glucan, laminarin, or starch. The polygalacturonase preparation also released small amounts of carbohydrate from Staurastrum sp., but neither reducing sugars nor specific products were found in the hydrolyzates. By contrast, incubation of these two enzyme preparations with intact algae, using the methods previously shown to lead to sphero- plast formation with susceptible species (Gunnison and Alexander, Can. J. Microbiol., in press), did not result in the conversion of Staurastrum sp. or P. duplex cells into spheroplasts. Composition of the wails. The amino acid content of walls of two of the algae is given in Table 2. No amino acid was dominant, and the quantitative composition differed among the two species. It is interesting that the results for the green alga, P. duplex, were quite similar qualitatively and quantitatively to those found in Cylindrospermum sp. (Gunnison and Alexander, Can. J. Microbiol., in press), a bluegreen alga that is decomposed readily by microorganisms. Amino acids were not detected in Staurastrum sp. cell walls, but it is possible that levels below the sensitivity of the ninhydrin method used (15) were present. The protein, carbohydrate, uronic acid, and lipid composition of the cell walls is presented in Table 3. The wall of Staurastrum sp. is dominated by carbohydrate and uronic acids, and no lipids were found. Carbohydrates are also prominent in the walls of F. muscicola and P. duplex, but their abundance is not as great as in Staurastrum sp. The data for carbohydrate and uronic acid content of F. muscicola are anomalous because of the high recoveries, and it is likely that interfering substances affected the analytical determinations. A comparison of the sugar and uronic acid monomers found in acid hydrolyzates of the cell walls is presented in Table 3. Glucose was abundant in the three organisms, but different though F. muscicola and P. duplex contained this sugar. Mannose was present only in two of the organisms. These data provide no insight into why the algae are resistant since a unique composition is not indicated. Composition of wall fractions. The values obtained from chemical analyses of the wall fractions are presented in Table 4. It is evident that only a small percentage of the Straurastrum sp. wall was solubilized by the ammonium oxalate, HC1, and NaOH treatments which were used to obtain fractions S-1, S-2, S-3, and S-4. Fraction I-3, which had residual wall constituents not solubilized by NaOH, contained 78% of the Staurastrum sp. material which was originally subjected to the alkali treatments, and a large portion of this residue was not hydrolyzed by treatment with 2 N HC1. Uronic acids made up a significant part of fractions S-1, S-2, and S-3 of Staurastrum sp., and these may be in the form of pectic substances since the only uronic acid found was galacturonic acid. A lignin-like material was found in fractions I-3 and S-4, the quantity of this substance in the two fractions being equivalent to 5.6 and 0.3%, respectively, of the wall weight. This lignin-like substance was not observed in the other fractions or in the Most of the material subjected to fractionation appeared in either the HCl-soluble S-2 or the NaOH-insoluble I-3 fractions (Table 4). These two fractions were quite similar in the kinds and amounts of materials which they contained, although alkaline hydrolysis of the two fractions followed by the Folin protein determination showed that fractions S-2 and I-3 had 0.2% and 3.1%, respectively, of the wall weight in the form of protein. The major components in hydrolyzates of fractions S-2 and 1-3 were reducing sugars. Alkaline hydrolysis followed by protein determination (13) showed that; fractions S-1, S-3, and S-4 contained an amount of protein equal to 0.2, 0.1, and 0.2%, respectively, of the total wall weight. Fractionation of the walls of P. duplex yielded results similar to those expected in the fractionation of walls of higher plants, for which the method was devised (6). Galacturonic acid was an important component of fractions S-1 and S-2, whereas the only reducing sugar in S-2 was xylose. Because the anthrone test that revealed the abundance of soluble materials in S-2 is primarily a test for carbohydrates, xylose was probably the principle monomer responsible for the 6.7% value. Fractions I-3 and S-4 contained sporopollenin in amounts equal to 2.4 and 1.2% of the wall weight, but none of the other fractions and neither of the other two algae contained sporopollenin. After hydrolysis, fraction S-3 was found to have a reducing sugar component (fucose) in nearly four times the abundance of the uronic acid (galacturonic acid). Fraction S-4 had the monomeric composition which would be expected of a polyuronide hemicellulose, whereas fraction I-3 consisted almost entirely of glucose. Part of fraction I-3 could not by hydrolyzed with acid, a substantial portion of this unhydrolyzed material probably being sporopollenin. Alkaline hydrolysis of the fractions followed by the Folin protein test showed that fractions S-1, S-2, S-3, S-4, and 1-3 of P. duplex contained 0.1, 0.3, 0.2, 0.1, and 0% of the total wall weight as protein, respectively. X-ray diffraction analysis. To provide further evidence for the occurrence of cellulose, X-ray diffraction analysis was performed on dried slide mounts of the I-3 fractions of the algae and of MN cellulose 300 (Brinkman Instruments, Westbury, N.Y.) using the method of Gunnison and Alexander (Can. J. Microbiol., in press). The X-ray patterns are depicted in Fig. 1. Authentic cellulose showed broad peaks with maxima at 15 and 22 degrees 20 and a small deflection having its maximum at 34 degrees 20. 1-3 fractions of F. muscicola and P. duplex gave none of these patterns, thus indicating that cellulose was either not present or was masked by other components in these preparations. The I-3 fraction of Staurastrum sp. exhibited a 22-degree peak in the same region as the cellulose standard, suggesting that this polymer was present. Resistant components of cell walls. Sporopollenin was isolated by the same procedure from the spores of Lycopodium clavatum (obtained from J. Russell, Cornell University), the "classic source of sporopollenin" (8), and from the walls of P. duplex. The infrared spectra of these preparations and of the acetolyzed derivative of P. duplex cell walls prepared by the method of Atkinson et al. (2) are given in Fig. 2. The spectra of the sporopollenins obtained from the walls of P. duplex and from L. clavatum are nearly identical. Almost the same spectrum was obtained using acetolyzed P. duplex walls. Since sporopollenin is the only known polymer of biological origin which can withstand the acetolysis process (8,21) and because the infrared spectrum of P. duplex is the same as that of L. clavatum, the preparation derived from P. duplex is considered to be sporopollenin. Analysis of the walls obtained from a 21-day-old P. duplex culture showed they contained 3.3% sporopollenin. Lignin-like material was obtained from Staurastrum sp. in an amount equal to 5.6% of the wall weight. The pyrolysis-gas chromatograms of lignin from Staurastrum sp. and pine wood meal are shown in Fig. 3. The patterns are nearly identical, except that the Staurastrum sp. preparation yielded a smaller quantity of products per unit of material pyrolyzed and only the algal preparation showed a small peak at about 4 min after initiation of pyrolysis. Infrared spectra of the two lignins were nearly identical except for a small shift of pattern between 1,400 and 1,800 cm-1 and slight differences in the peaks occurring in the region between 800 and 1,400 cm-1 (Fig. 4). The authentic and the algal lignins were subjected to alkaline fusion by a slight modification of the method of Rassow and Zickmann (20). To 2.5 g of KOH and 2.5 ml of distilled water in a nickel crucible were added 10 mg of the preparation and 90 mg of zinc dust. The mixture was heated on a steam bath either for 10 min or until the KOH had dissolved. The mixture was brought to 250 C in a furnace, at which time 5.0 ml of a 50% KOH solution in distilled water and an additional 0.75 g of zinc dust were added. Heating at 250 C was continued for 1.5 h, after which the mixture was cooled to room temperature and dissolved in 40 ml of distilled water. The alkali-fused lignin was acidified to pH 2.5 with 6 N HCl, and the mixture was dried on a rotary evaporator and then dissolved in 5.0 ml of distilled water. Residual KCI was removed by filtration. Using the procedures described above for analysis of the fused lignin, it was observed that protocatechuic acid and the products of alkaline fusion of the authentic and the algal lignins had RF values of 0.39 by thin-layer chromatography and retention times of 39 s by gas chromatography. Thus, the Staurastrum sp. preparation apparently possessed a phenyl structure, a characteristic of lignins (20). Decomposition of labeled walls and wall fractions. The degradation of Staurastrum sp. is presented in Fig. 5. Although the lignin preparation obtained from this alga was subject to microbial decomposition, it was attacked more slowly than the original walls and all of the other fractions. The lignin-like material accounted for less than 30% of S-4; its presence may explain the slower rate of attack on S-4 than the other fractions, but the degree of protection is obviously not great. 1-3 was readily metabolized, possibly the result of its content of cellulose and the lack of influence of the small quantity (less than 4%) of lignin-like material present. Fractions S-1, S-2, and S-3 were rapidly degraded, and less than 25% of the original 14C label remained after 24 days. Since the quantity of '4C left in the fractions after 24 days was directly correlated with their content of apparent lignin, it is possible that the constituents metabolized in the first week are largely wall material could be obtained. The cells were Ys then suspended in 100 ml of buffer. One-half of )osition of labeled Stauthe suspension was used to prepare algal lawns ractions. as described by Gunnison and Alexander (Limpolysaccharides, leaving a refractory lignin-rich substance behind. The decomposition of F. muscicola walls and wall fractions is depicted in Fig. 6. It is evident that S-1, S-3, and S-4 were rapidly degraded, each losing more than 50% of the original "C' label in 24 days. The rates of decomposition of fractions S-2 and I-3 were quite similar, and they were much more resistant to decomposition than the other fractions. The unfractionated wall material was especially refractory, and more than 80% of the original label was still present after 24 days. It is noteworthy that the rates of decomposition of S-2, I-3, and unfractionated walls were quite similar after the first few days, suggesting a possible role for components of S-2 and I-3 in shielding the walls from microbial attack. The rate of metabolism of [14C ]glucose was determined to illustrate the rate of oxidation of a good carbon source for microorganisms. The decomposition of labeled walls and wall fractions of P. duplex is shown in Fig. 7. The sporopollenin component was not attacked at all in the 24-day interval since all of its 14C label was recovered at the end of the test period. Fraction S-4 was more extensively degraded, but 50% of its original label was still present after 24 days. Unfractionated walls were even nol. Oceanogr., in press), whereas the remainder was lyophilized and used to prepare cell walls. The lawns were examined for their susceptibility to microbial attack by the method of Gunnison and Alexander (Limnol. Oceanogr., in press), and the lignin content of Staurastrum sp. walls and the abundance of sporopollenin in P. duplex walls were determined. The data demonstrate a relationship between the lignin content of Staurastrum sp. walls and the decomposability of the alga ( Table 5). The correlation is not linear because the major increase in lignin content occurred between 5 and 12 days, during which time the alga remained moderately susceptible, whereas the lignin level increased only slightly between 12 and 21 days during which period the cell became markedly resistant. The results with the sewage inoculum, in which a decrease in resistance occurred between day 5 and day 12, are anomalous. Studies of the decompositon of P. duplex cells of various ages strongly suggest a relationship between the sporopollenin content of the wall and the decomposability of the alga. Although the 5-day-old cells were highly to moderately susceptible to degradation, 12-day-old cells were quite resistant (Table 5). During the 3-day period, the sporopollenin content of the walls rose from 1.2 to 4.2%. No difference in resistance was observed between the 12-and 21-day-old cells, and the sporopollenin content during this period remained reasonably constant. DISCUSSION Cellulase and polygalacturonase preparations act on Chlamydomonas reinhardtii and Ulothrix fimbrata, two algae that are readily susceptible to attack in natural waters, and the preparations extensively degrade the algal walls so that the cells are converted to spheroplasts (Gunnison and Alexander, Can. J. Microbiol., in press). By contrast, the cellulase and polygalacturonase preparations exhibited low activity against the resistant algae. These data thus correlate with the resistance of the organisms in freshwaters (Gunnison and Alexander, Limnol. Oceanogr., in press). The observation that moderate digestion of P. duplex walls was affected by the two enzyme preparations, as contrasted with the small influence on intact cells, suggests that enzymatic hydrolysis of the surface of this organism is facilitated by the exposure of areas of the wall other than the outer surface. Inasmuch as such exposures probably occur in nature only when the cell is perforated mechanically or when autolysis occurs, the polysaccharidase activity probably is not often important in governing the persistence of the alga. Although the polygalacturonase preparation was not free of cellulase, the release of galacturonic acid from P. duplex walls which were incubated with this preparation suggests that the walls contain a polygalacturonic acid, the latter possibly serving to hold together either fibrils or layers of cellulose. The failure of lysozyme to digest walls of these resistant species is in contrast with the susceptibility of Cylindrospermum, a lysozyme-sensitive blue-green alga quite susceptible to microbial decomposition in freshwaters (12; Gunnison and Alexander, Limnol. Oceanogr., in press). The walls of Staurastrum sp. contain a component which appears to be similar if not identical with lignin, a polyaromatic known for its resistance. The slow decomposition of the purified microbial lignin or lignin-like material, as compared with the rate of degradation of the other fractions or the unfractionated walls, suggests that the polyaromatic may be responsible for the slow rate of decompositon of the alga in nature. The absence of a linear correlation The data strongly suggest but do not show unequivocally that the material isolated from Staurastrum sp. walls is in fact lignin. The limited amount of information on the possible occurrence of lignin in algae is usually interpreted to mean that it is not present (17), although some data exist to the contrary (9). On the other hand, lignin in a group as primitive in the evolutionary sequence as the algae might differ substantially from the polymer characteristic of higher plants. F. muscicola was resistant not only in natural waters but its walls also were totally unaffected by the enzymes used. Nevertheless, three of the cell wall fractions were easily degraded, so that they probably do not contain the substance conferring resistance. Fractions S-2 and I-3 decomposed quite slowly and at approximately the same rate, and hence they may contain the refractory constituent. S-2 and I-3 had approximately the same monomer composition, and it thus seems reasonable to believe that resistance of the walls and of the alga are related to constituents of fractions S-2 and I-3. Although these fractions were metabolized to a slight degree before the rate of decomposition declined to almost that of the original walls, this initial attack may have been at the expense of inaccessible components made available by the fractionation procedure. Because acid hydrolyzates of these two fractions contained several different sugars in similar amounts as well as galacturonic acid, it is tempting to postulate that the resistance is attributable to the presence of a heteropolysaccharide. Sporopollenin but none of the other fractions of the walls of P. duplex was completely refractory to microbial attack. Thus, sporopollenin seems to be responsible for the resistance of the walls and presumably the persistence of the alga. Evidence exists that the sporopollenin is localized in the outermost layers of the wall of Pediastrum (2,16). Silica is also present on the surface of Pediastrum walls (14), and the silica may likewise protect other components of the wall and thus shield the cell contents from microbial attack. In view of the digestion of the walls and the release of glucose by the cellulase preparation, cellulose is apparently present in P. duplex. This polymer may be located in fraction I-3, which yielded large quantities of glucose on acid hydrolysis. The results demonstrate that the biochemical bases for resistance of at least certain algae to microbial attack in natural habitats can be attributed to discrete structural entities. In addition, resistance of the wall to decomposition has an important effect on the rate of mineralization of its several constituents. Why polyaromatics and heteropolysaccharides may contribute to the longevity of the algae has already been discussed (1). Whether these constituents are important to the survival of other species and if different wall components account for the resistance of other algae to microbial decomposition require additonal investigation.

v3-fos

2018-04-03T06:16:05.627Z

1975-07-01T00:00:00.000Z

46160844

s2

Presence of Two Virus-Like Particles in Penicillium citrinum Two icosahedral virus-like particles (28 and 19 nm in diameter, respectively) have been detected in sporogenic and asporogenic segregants of a strain of Penicillium citrinum. The distribution of the two particles differed among the two segregants. Virus-like particles have been detected and physicochemically characterized in various species of Penicillium (12). As reported previously, such particles are present in a wild strain of Penicillium citrinum Thom (3,5,15). This strain segregates spontaneously and at high frequency for small patches of white asporogenic mycelium which, in turn, may redevelop into normally green, sporogenic mycelium. We have been able to detect in this unstable strain the presence of two virus-like particles which can be distinguished by their dimensions and distribution in these two mycelial types. The organism was grown at 24 C on potatodextrose broth or on potato-dextrose agar, pH 7.0. Extracts were made from both mycelial types after 24 and 48 h of growth (1, 2) and were negatively stained with 2% (wt/vol) phosphotungstic acid (pH 7.2) containing dimethyl sulfoxide (13) and examined with a Siemens IA electron microscope operating at 60/80 kV. In these preparations two distinct virus-like particles were observed, one (Pcit-1) ranging from 26 to 28 nm in diameter and the other (Pcit-2) 17 to 19 nm. Both particles were clearly seen as icosahedrons and sometimes were observed as empty, single-capsid structures ( Fig. lb and 2b). Particles Pcit-1 and Pcit-2 differed in their relative distribution among the two types of mycelial segregants, in that Pcit-1 was predominantly seen in extracts from sporogenic mycelia (Fig. 2b) whereas Pcit2 was more abundant in similarly prepared extracts of white, asporogenic mycelia (Fig. lb). It should be stressed, however, that both particle types were present in all mycelia. These findings from negatively stained preparations were confirmed by observations of viruslike particles in thin sections of chemically fixed hyphal fragments prepared according to methods described previously (8). The particles were found normally associated with, or enclosed within, membranous structures in parts of the hyphae with prominent vacuolization or full degeneration ( Fig. la and 2a). In the virusinfected hyphae of P. citrinum membrane whorls, tubule formations and filamentous structures of unknown origin were frequently seen as well. The association of virus particles in fungi with membrane systems has been noted by other investigators (17,21; see also review 12) and might indicate an attempt of the fungus to delimit a potentially lytic virus into separate bodies. Two serologically distinct viral particles have been identified in Pencillium stoloniferum Thom (6,18,20) and three components of a single virus have been recognized in Penicillium chrysogenum Thom on the basis of their content of different double-stranded ribonucleic acid molecules (14). However, these particles, in contrast to those present in P. citrinum, are uniform in size. Pcit-2, moreover, appears to be the smallest virus-like particles detected so far in fungi. The two virus-like particles present in P. citrinum, if indeed viral in nature, may contain different, perhaps complementary, genomic determinants, and for either particle to replicate both particles have to infect the same hypha. There are precedents for multicomponent and interdependent viral systems in fungi and else- where (10,16,18). If this is in fact the case with the viruses of P. citrinum, the dissimilar distribution of particles in the two mycelial types, which show profound differences in sporulation efficiency, antibiotic production, and growth rate (3,15), suggests that these particles in disproportionate combination may somehow control fungal metabolism and differentiation. Some authors have already suggested a relationship between mycoviruses and extrachromosomal genetic factors (4,7,11,19) and we are currently investigating this prospect in P. citrinum. Preliminary experiments of curing have shown that cycloheximide promotes the reversion of the asporogenic mycelium into the sporogenic one with its own typical properties. In Saccharomyces cerevisiae Meyen ex Hansen, cycloheximide has been shown to be an effective curing agent of the "killer" determinant, an extrachromosomal genetic factor (9) that recent studies have strongly related to the presence of double-stranded ribonucleic acid containing virus-like particles (10). We are greatly indebted to Paul A. Lemke for his constructive criticism and helpful suggestions in preparing the manuscript. We wish to thank F. Cecere, G. Ignazzitto, M. Mari, and B. Pasquetti for their assistance.

v3-fos

2018-12-12T12:37:05.683Z

1976-01-01T00:00:00.000Z

155170217

s2

Milo stover and forage sorghum silages for growing heifers 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 1976 Kansas State University Agricultural Experiment Station and Cooperative Extension Service. Introduction Milo stover silage and dehydrated milo stover pellets were compared with forage sorghum silage in two previous heifer growing trials at this station (Prog. Rept. 210, Kan. Agr. Expt. Sta., 1974 andProg. Rept. 230, Kan. Aqr. Expt. Sta., 1975). Results showed: (1) milo stover had a feeding value of 63 to 67% that of forage sorghum, (2) cattle consumed 12 to 14% less milo stover silage than forage sorghum silage, and (3) growing calves fed milo stover silage as the major energy source should gain about 1.0 lb. per day and require about 10 to 14 lb. of dry matter per lb. of gain, less than acceptable performance for most cattle feeders. Could milo stover provide only a part of the energy in growing rations? Our objective in this trial was to measure performances obtained with various percentages of milo stover and forage sorghum silages. Experimental Procedure Milo stover and forage sorghum (high-grain variety) each was obtained from a single source in October, 1974. The forage harvester was equipped with a two-inch recutter screen and both forages were ensiled in upright concrete stave silos (10 ft. x 50 ft.). Moisture content of the milo stover was about 65%; that of the forage sorghum, about 30%. Compositions of the four experimental rations and their supplements are shown in table 18.1. All rations were formulated to be equal in crude protein (12.5%), minerals, vitamins and additives. Rations were mixed twice daily and fed free-choice, Initial and final weights of the heifers were taken after they went 15 hours without feed or water. Results Dry matter (%) and crude protein (% on a dry matter basis) for the milo stover were 33.6 and 4.25; for the forage sorghum silage, 29.8 and 7.1. Heifer performances are shown in table 18.2. Heifers fed the 100% forage sorghum silage ration gained faster (P<.O5) and more efficiently (P<.05) than heifers fed any of the other three rations. Heifers receiving 100% milo stover silage had the slowest (P<.05) and least efficient (P<.05) gain. As forage sorghum increased and milo stover decreased in the ration, rate of gain increased and feed required per lb. of gain decreased. Dry matter consumption tended to increase as forage sorghum replaced milo stover. Light-weight and heavy-weight calves had similar gains, but light-weight calves gained more efficiently (7.98 lbs. vs. 9.60 lbs. of feed per lb. of gain). Estimated net energies for the two silages were calculated from gains and feed intakes obtained from the 100% milo stover and 100% forage sorghum silage rations. The estimates gave predicted daily gains for heifers fed the 67% and 33% milo stover rations to be 1.29 and 1.58 lbs., respectively, but actual daily gains were 1.50 and 1.66 lbs., respectively. These results suggest that milo stover silage may have greater value than expected when it is fed in combination with a higher-energy forage.

v3-fos

2014-10-01T00:00:00.000Z

1976-01-15T00:00:00.000Z

16687185

s2

Comments on optimization of cattle breeding schemes: beef breeds for suckling herds. A review Optimization of selection of beef breeds for suckling herds is little developed withm the European Economic Community (E.E.C.) because of the small proportion of suckling cows and the very different systems of management of the latter. These various situations of selection are defined by the female populations involved and their genetic improvement objectives. We have distinguished between 3 types of populations : specialized beef breed herds, hardy herds used in crossing for their maternal abilities, herds composed of F1 females : dairy X beef. The selection goals of beef breeds necessary for these different systems depend on the fate of the female offspring of the chosen (selected) sires. - Optimization of selection requires a better knowledge of the genetic parameters concerning breeding qualities of beef breeds both for their paternal contribution (direct effects) and for their maternal contribution (direct and maternal effects). According to the performances recorded in the Charolais breed, a general reduction of breeding qualities and notably of fitness can be observed, as well as an increasing muscle development. The reduction of calving ability is due both to an increase in the size of the animals and to an improvement of their musculature. With respect to mothering ability, the antagonism generally observed between direct and maternal effects would rather be of environmental than of genetic origin. In addition, selection on muscle development in Europe is made in conditions where natural selection on breeding qualities are less and less involved. As the artificial insemination is proportionally more developed in Europe than anywhere, it is possible to set up integrated selection schemes on breeding qualities of beef breeds. Artificial insemination is used to optimize the choice of breeding animals (selection on progeny) and to accelerate the diffusion of genetic change to all the herds (natural mating). Optimization of these schemes is confronted with difficulties arising from the distribution of the costs (supported by the AI) and the returns (obtained by natural matings). Beyond this approach, it would be advisable to consider the improvement of beef cattle for suckling herds within the enlarged scope of the E.E.C. and for the coming the very different systems of management of the latter. These various situations of selection are defined by the female populations involved and their genetic improvement objectives. We have distinguished between 3 types of populations : specialized beef breed herds, hardy herds used in crossing for their maternal abilities, herds composed of F 1 females : dairy X beef. The selection goals of beef breeds necessary for these different systems depend on the fate of the female offspring of the chosen (selected) sires. -Optimization of selection requires a better knowledge of the genetic parameters concerning breeding qualities of beef breeds both for their paternal contribution (direct effects) and for their maternal contribution (direct and maternal effects). According to the performances recorded in the Charolais breed, a general reduction of breeding qualities and notably of fitness can be observed, as well as an increasing muscle development. The reduction of calving ability is due both to an increase in the size of the animals and to an improvement of their musculature. With respect to mothering ability, the antagonism generally observed between direct and maternal effects would rather be of environmental than of genetic origin. In addition, selection on muscle development in Europe is made in conditions where natural selection on breeding qualities are less and less involved. As the artificial insemination is proportionally more developed in Europe than anywhere, it is possible to set up integrated selection schemes on breeding qualities of beef breeds. Artificial insemination is used to optimize the choice of breeding animals (selection on progeny) and to accelerate the diffusion of genetic change to all the herds (natural mating). Optimization of these schemes is confronted with difficulties arising from the distribution of the costs (supported by the AI) and the returns (obtained by natural matings). Beyond this approach, it would be advisable to consider the improvement of beef cattle for suckling herds within the enlarged scope of the E.E.C. and for the coming io or 20 years. Optimum selection systems risk might to exceed the means, i.e. the actions undertaken within each breed or each country ; one of the objectives of the European Economic Community would be perhaps to facilitate the selection of this future breeding stock. In the European Economic Community (E. E.C.), the proportion of suckling cow herds is small. The situations observed in the various countries are extremely varied and the number of artificial inseminations is very often restricted in these herds as compared to dairy herds. For that reason, the studies made to define optimum selection schemes have not progressed as much as those concerning the dairy schemes and beef schemes for industrial crossing. We are therefore only going to point out the conditions and objectives of selection for suckling cow herds as well as the genetic parameters pertaining to their principal abilities ; we will thereaftcr attempt to analyse how to complete and coordinate these parameters with the aim of optimizing the selection for suckling herds. i. Specialized beef breed herds. They are composed of three main ethnic groups : british breeds of small size and characterized by very early fattening (Aberdeen-Angus, Hereford), continental breeds traditionally exploited for beef production in suckling cow herds (Charolaise and Limousine), local and hardy breeds characterized by numerous abilities and which, after suppression of milking and utilization of the animals for draught, have been converted into beef breeds (Piemontaise). 2 . Herds of hardy breeds which have not been used as mentionned above ; the purebred or crossbred females are used in terminal-like crossing with sires of beef breeds (A ubrac, Gasconne and Sarde). 3 . Herds composed of F1 females)) : « dairy X beef from industrial crossing in dairy herds. This type of herds belongs to the same category as the previous one as the females come from hardy breeds formerly subjected to milking. The development of beef production in Western Europe then appears as a by-product of dairy selection (ArrorrYMa, i 97 2 ; MÉNISSIER el al., 1974). The reduction of milk production primarily leads to development of crossing between milked females and beef sires, then suppression of milking and extension of the industrial crossing (V I SSAC, 1975). As far as the production conditions are concerned, the herds corresponding to the former and the latter types (specialized beef breeds and F, females) are generally exploited in regions with a high grassland production; conversely, the hardy breeds are currently used in areas with a poor grass production and their ability of exploiting these environmental conditions becomes reponderant (C ASU et al., 197 5). As a matter of fact the selection objectives in these various stiuations will depend on the fate of the female offsprings of the sires from the breeds involved (M!NISSIER et al., 1974 ). This fate is characterized by the proportion of females maintained for reproduction, by the length of the reproductive life of these females and by the utilization of the products of this female progeny for reproduction (table I ). If the male progeny is always intended for slaughter, the utilization of the reproductive performances and maternal abilities of the female offsprings varies considerably. In the case of male lines of terminal crossing, these traits are not used at all, whereas in female lines of terminal crossing or maternal strains, they are used to a maximum. The importance of maternal performances depends on two factors : -Primarily, on the breeding system : the maternal performances are less important in purebreeding systems (or rotational crossbreeding) where the reproduction of the females merely ensures the maintenance of the stock of this population, most of the females being then fattened like the males. Conversely, in systems including terminal crossing, just the strictly necessary amounts of females for terminal crossing are produced for the renewal of the herd ; generally, their reproductive abilities are exploited to a maximum. -Secondly, on the carcass value of the female and its variation with age : in the continental beef breeds, slaughtering of young females after some few reproductive cycles is not only accepted but also often advised because of their high carcass value (extreme cases in double muscle beef breeds). Conversely, the females of British beef herds or of hardy breeds are used as long as possible for reproduction. Since some traits of the females are related to age (sexual precocity, calving ability) the relative importance to be imputed to these traits in the selection will naturally vary according to the proportion of heifers used in the herds. Moreover, in the case of traits being expressed according to one or several thresholds, the objectives of the selection of breeds intended for crossing will depend on the relationships between the components of the traits related to the mother and those related to the calf, i.e. on the complementary (calving ability and pre-weaning growth). This presentation of the selection objectives has been made with reference to the situation existing within the E.E.C. (populations, types and systems of production). A more theoretical and global approach might be obtained by applying systems or models for the analysis of the economic efficiency to these different situations; these models of analysis have already supplied valuable results in the case of similar production systems (L EIGH et al., I9!z; LONG, I9!z; C ARTWRIGHT et F I T ZHUGH et al., 1975 ;LONG et al., 1975 ;MORRIS et al., I9!j ;PARKING et al., 1975 ;3 W ILTON et MORRIS, 1975 ). Independently of the human constraints, the application of these models would allow to define more accurately the relationships between the different traits and the parameters used (see the communication of C ART wRIGHT). II. -GENETIC VARIABILITY OF THE PRINCIPAL MATERNAL TRAITS After determination of the importance of the traits relatively to the paternal contributions (direct effects) and maternal contributions (direct and maternel effects), the estimation of the overall genetic value assumes knowledge of the genetic parameters concerning these different contributions. In the light of our most recent results about the Charolais breed, we are only going to present the most interesting aspects of the maternal contributions, those concerning the paternal contribution having been developed elsewhere (see communications of Four.r.EY and L IN nH>:). The following points will be considered successively : numerical productivity, calving ability, mothering ability. A. -Numerical productivity (or number of calves weaned per female kept for reproduction) The heritability of this criterion is generally low (h 2 = o to 5 p. ioo) as for most of the reproductive performances (M AIJALA , 19 6 4 ) ; even in the case of heifers tested at the station, we only observe ¢ p. ioo (table z). As a matter of fact, the numerical productivity represents a synthetic complex criterion resulting from the incidence of very different physiological functions : conception, gestation and viability of the calves. Separately, these traits may have different heritabilities (tabl 2 ) : those connected with conception or gestation being more heritable than the maternal component of the survival rate of the calves. In Charolais heifers, where we have reduced as much as possible the variance depending on the environment, the heritability of conception or gestation rates ranges around 10 p. 100 . This rather high value has been confirmed by the findings of D EARBORN et al. ( 1973 ) for British breeds (h 2 = o.z2 ! 0 . 17 ). As compared to growth and conformation at 1 8 months of these heifers (table 3 ), fertility is phenotypically independent (ADG 9 to 1 8 months) or positively related (weight at i months) to growth ; On the other hand it appears to be genetically in opposition to these criteria (&mdash; 0 . 07 to -0 . 53 ). Analogous trends can be observed with respect to conformation and especially with respect to muscle development and subjective judgment of the breed qualities (the two latter criteria are related genetically : rg = 0 .6 2 ). These genetic relationships might confirm the opposition betweeu muscle development and breeding qualities generally observed between genotypes. A more thorough analysis of these relationships should be made. Even, when distinguishing between the mortality of the calves during their foetal life, at birth and after, we did not notice any genetic variability for the maternal component. This result does not exclude the probable existence of direct genetic effects (V AN D IETEX , I9 6 3 ; I CLOPPENB U R G, 1966; 1VIAIJALA, I96:! ; BAR-ANA N , Ic)!2 ; P H IL I PSON, 1975 ); besides, the genetic variability of the latter is low and partly related to calving difficulties (direct effect). Independently of the mortality, we have observed some heritability of the maternal component vigour of the calves at birth (h 2 = 27 to 37 p. ioo) ; besides, it is genetically related to calving easiness (r a = + 0. 3 to + 0 . 5 ). The recent results of P HILIPSON ( 1975 ) about the mortinatality in dairy herds, confirme the low heritabilities of the direct effects (h 2 = 0 . 01 ) and maternal effects (h 2 = 0 . 01 to 0 . 0 8) as well as the absence of genetic correlation between these effects (rg = + 0 . 02 ). The viability of the calves is one of the criteria that should be particularly considered in selection, both for industrial crossing as well as for suckling herds, in relation with the degree of maturity at birth. B. -Calving ability (as maternal trait) The heritability of the maternal contribution of this criterion is relatively higher than that of the previous criteria (B R tNxs et al., 1973 ;C OUTEAUDIER et al., 197 1 ;HAasEa, 1975). In the Charolais breed we have obtained from 15 to 20 p. 100 (table 4 ) these values are higher than those generally estimated for direct effects (h 2 = 0 . 05 about; MA NISSIER , i 97 .1), but they are mostly obtained under conditions where the environmental variability is reduced (Stations or experimental herds) and where the genetic variability is expressed to a maximum (calving of the heifers). This maternal ability is directly related with the size of the calf ( l' g = + 0.6! and rp = + 0 . 59 ) and much less with the gestation length (r a = -0 . 07 and r z , = -)-0 . 21 ). Although these two traits have a maternal component as heritable as that of calving ability, they are more subjected to direct effects than to maternal effects (P HILIPSON , 1975 ) and depend more on the genotype of the calf than on that of the mother. Furthermore, the genetic correlation of their direct and maternal effects is naught or negative (table 7 ; KocH, 1972 ;PHILIPSO N , 1972 ) which might be the expression of a competition between the mother and the foetus regarding their requirements in the case of animals with a high growth potential. The genetic opposition is less evident in the case of calving ability (rg = &mdash; o.i 9 ; P H I LIPSON , i9!5), but let us recall with respect to this that a greater number of traits are involved (MÉ NISSIER , 1975 ). The morphological traits of the mother at calving (weight and pelvic opening) are the most heritable criteria (table 4 and C OUTEAUDIER et al., 1971 ). With respect to the weight at calving, there is no, or a slightly negative, phenotypic correlation with calving difficulties, whereas there is a very high positive genetic correlation (rg = !-0 . 5 to + 0 .8). The relationships with growth and the conformation of the heifers at i8 months confirm this genetic opposition (table 5 ). As a matter of fact, several phenomena have to be mentioned. Primarily, the increase in size of the mothers (weight) is connected with an increase in birth weight of the calves. Proportionally, this increase would be greater than that of the size of the mothers (M ONTEIRO , ig6g) or of their pelvic opening related with the larger size of the dams (T AYLOR et al., i 97 g) ; consequently, there would be more calving difficulties in large-sized breeds. Increase in calf weight/mother weight ratio would signify a higher maturity of the calves at birth ; there is a apparently discrepancy between this and the observations of F ITZHUGH and T AYLOR ( 1971 ). In addition, we do not know the respective share of the direct effect and maternal effect in the increase of weight at birth. The second phenomenon to be mentioned for the increase of calving difficulties, is the improvement of muscle development. We have noticed that muscle development causes more difficulties at calving (r e = -!-o. 51 ) without however increasing the weight of the calves (r e = + 0 . 01 ). The effect of muscle development consists probably more in changing the morphology of the calves and especially in reducing the pelvic opening of the mothers relatively to their size. This phenomenon has been described in connection with studies on the double muscle trait (V ISSAC et al., i9!3 ; MA NISSIER , 1974 a). In beef breeds, calving difficulties might be a consequence of their size and/or of their muscle development (M ÉNISSIER et al., r9!q b). It would be necessary to examine more thoroughly the incidence of their present selection on this ability. C. -Mothering ability The variability of the maternal contribution on the preweaning growth of the calves is better known than in the case of the previous abilities ; 15 to 30 p. 100 of this variability are of genetic origin (table 6 and K OCH , 1972 ). It is also a complex criterion since it closely depends on the maternal environment ; for example, milk production of beef breeds accounts for 20 to 70 p. ioo of the variance of the calves preweaning growth (B IBE et H IVER T, 1975 ). A rather great number of authors (table 7 ) have estimated for the growth of the calves, the respective share of genetic variability in the direct effects and maternal efiects : the heritability of the maternal effects often exceeds that of the direct effects. An antoganism between these two types of effects is generally observed. If we do not take into account the inaccuracy in the estimations of the covariance and the omission of the dominance effects, this negative relationships would be of environmental rather than of genetic origin, indeed, young females placed in a very favourable environment (on account of the age of the mother, year or season, herd management) and which have shown a high early growth, produce thereafter lighter calves at weaning than heifers having exhibited normal growth (CHRISTIAN et al., I9 6g ; V OGT et M ARLOWE , I9 66 ; T OTU -SEK , I9 6H ; K OCH , I9 6 9 ; M ANGUS et BRINKS, 1971 ;K RESS et BURFENING, 1972 ) ; This might be due to a deterioration of their dairy abilities (S WANSON , 19 6 0 and 19 6 7 ). If the preweaning growth superiority of heifers is of genetic origin (paternal or maternal lines for instance, K RESS et B URFENING , 1972 ;M ANGUS et BRINKS, 1971 ), the deteriorating effect on mothering ability is less obvious. As regards the preweaning growth of Charolais heifers, we have not found any evident opposition (table 8) ; only conformation score appears to be negatively related to milk production, without any repercussion on the growth of the calves. Similar trends have been reported by F REY et al. ( 1972 ) for Aberdeen-Angus. Thus, all results pertaining to the genetic variability show the necessity of pursuing the analysis of the variations and covariations in the direct and maternal effects in order to optimize selection. The most obvious genetic oppositions appear between muscle development and each of the maternal components : this corresponds to the observations that can be made both in France and in the whole World on the abilities of the various strains of Charolais « individualized » since the beginning of the century : increase in muscle development leads to reduction of fitness and adaptation traits (V ISSAC et al., 1973 III. -SELECTION SCHEMES FOR SUCKLING COW HERDS Up to the present time these schemes concerned herds kept in rather extensive systems with natural mating. Mass selection of males and females on conformation and growth criteria (especially weaning weight) appeared to be sufficient, as the extensive conditions favoured natural selection with respect to the fitness trait. The situation in the E.E.C. differs for two reasons : -Primarily, the selection concerns in particular the muscle development with animals from small family farms (well supervised herds) and with more intensive systems (management and feeding). In these conditions the natural selection on breeding qualities intervenes less and less. -In addition, 20 to 6 0 p. I oo of these herds according to cases, are subjected to artificial insemination. It therefore seems logic to use this technique, on the one hand, in order to optimize the choice of the selected animals on the basis of their breeding qualities (selection on progeny) and, on the other, in order to accelerate the diffusion of genetic change to all suckling herds. I . -Choice of the breeding animals For that purpose, integrated selection schemes of French beef breeds for production of breeding females have been developed the last few years (V ISSA C, 1970 ;MÉ NI S SIER et al., 197 4 ;VISSAC et ME K issiER, Ic!!4 ;ME N ISSI ER , 1975 ;B OYAZOGLU , 1975 ). These schemes (fig. I and table 9 ) are based on two mains steps : a) The most original step is the progeny testing on breeding qualities on daughters of A.I. sires. This testing was made with a sample of 20 purebred daughters/sire, kept for two years at the station, from weaning till the second gestation. Not only their growth, but also their fertility, calving ability and maternal mothering ability at first calving when 2 years old, were estimated. Although it represents one of the selection objectives, this early first calving was retained in particular in order to reduce the duration of progeny testing and to permit a better expression of the genetic variability of the maternal abilities. This assumes a good repeatability of these abilities (the value of which is perhaps not always as high as claimed : C UNNINGHAM et HENDERSON , I96 g ; BOSTON et Cbl., 1975). b) The second step consists in a combined choice on ancestry and individual value of the young bulls, after having planified rational matings on a nucleus of elite cows (or rc sire mothers »). The choice of sire mothers distributed in a great number of herds, is made from performances recorded on the farm. At the level of the overall controlled population, these performances are generally elaborated in form of u female index n (or estimation of the «Most Probably Producing Ability u ; R EGIS , 1974 ), thus leading to rather large selection pressures. The young males produced are subjected to performance testing before they are chosen for the subsequent steps. In this scheme, the efficiency of the choice of breeding animals depends on the quality of the estimations of their genotypic value at each stage. It might be improved by a better combination of the available informations (the taking into account of the oppositions between direct and maternal effects for preweaning growth, for instance ; MA NGU S et BRINKS, 1971 ) as well as by the utilization of early selection criteria. These criteria might as well be chromosomal aberrations or hormonal levels (LH and prolificacy, oestrogenes and mothering ability) as morphological traits such as the pelvic opening of the young bulls (MÉ NISSIER et V ISSAC , 1971 ) relatively to their size (T AYL OR El al., 1975). Lastly, the optimization of such selection schemes requires a rationalization of the utilization of breeding animals chosen at each step ; with the aim of obtaining a rapid diffusion of genetic change to the overall population. In particular, the sires selected by this scheme, after progeny testing, should in priority be kept for planified matings (by AI) with the elite females, in order, on the one hand, to procreate the following generation with the best elite mothers and, on the other, to produce young males with the other elite cows for the natural services in the recorded or unrecorded commercial herds. Before being used, these young males should be performance tested. Such an integration of the selection schemes at the level of the population, is difficult at the present time because of the difficulty in the distribution of the costs (supported by the AI) and the returns (obtained by natural matings). CONCLUSIONS With the aim of determining an optimum breeding scheme for beef herds, the various local and national situations with their specific restrictions chiefly concerning the environments, the production systems and market requirements, the sociological place of traditional breeders and pro-ducers, should be considered. In this limited but realistic context, partial results have been obtained by several teams of research workers : breeding goals and production systems (U.S. A., Canada, United Kingdom, France, ...) as well as the genetic variability traits, for example, calving ability (France, ...) and preweaning growth (U.S. A., ...). It would now be necessary to confront the results in order to elaborate optimum selection models fitted to the existing situation. Beyond this approach, it is also important to consider the improvement of beef cattle for suckling herds in the enlarged scope of the European Community and for the coming io or 20 years. It appears that suckling herds will chiefly develop from beef crosses coming from the dairy herds (grassland and cultivated lowland areas with medium sized farms) and in difficult environments of mountainous or arid zones from adapted breeding types. We have now to find the optimum production system, the place of beef breeds in each of them, the breeding goals of these breeds and hardy breeds, using analysis models of efficiency from beef production. These goals and optimum breeding schemes which will result from such analysis (selection of maternal lines for example) may be different from those of existing breeds in purebreeding with an average early culling. To reach these goals, it might be interesting to select for special strains from a synthetic. population (from a gene pool) Creation of such adapted beef strains, overstepping the schemes of each breed in the imposed local and national context, is naturally a too large enterprise considering the facilities and responsabilities of each country. To be achieved such a goal, could be a stimulating challenge for European Economic Community. ACKNOWLEDGEMENTS The author is indebted to B. V ISSAC and J. L. F OULLEY for their helpful suggestions and comments during the preparation of this paper, and to Mrs Kristen RP RAT for her valuable assistance in translation.

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Properties and applications of pyrethroids. Improved understanding of the factors determining the insecticidal activity, the mammalian toxicity, and the stability in air and light of natural and synthetic pyrethroids has led to a series of new compounds with a very favorable combination of properties. Their characteristics include outstanding potency to insects, low toxicity to mammals associated with rapid metabolic breakdown and, in appropriate cases, adequate stability on plant surfaces even in bright sunlight. Initial tests indicate that even the more stable compounds are degraded rapidly in soil, so if the trials at present in progress reveal no toxicological or environmental hazards, within a few years synthetic pyrethroids should be available to control a wide range of domestic, veterinary, horticultural, agricultural, and forest pests at low rates of application. Introduction The natural pyrethrins and related synthetic compounds have traditionally been recognized as excellent insecticides, harmless to mammals but too unstable and expensive to control pests of agricultural crops and forests efficiently and economically (1)(2)(3)(4)(5)(6)(7)(8). This limited potential has changed dramatically in the past two to three years, for workers in Great Britain (9,10) and Japan (11)(12)(13) have shown that the photolabile centers of the molecular framework of pyrethroids may be replaced with alternative groups giving much greater overall stability in air and light, while the features essential for the great insecticidal activity and low mammalian toxicity are retained. As a consequence of these advances, a range of new insecticides is being developed. It now seems probable that by 1980, synthetic pyrethroids, with their combination of favorable properties, may have an importance comparable with organochlorine compounds, organophosphates, and carbamates as practical insecticides used on a large scale. Much evaluation, at present in progress, of the toxicological and environmental properties of the new compounds remains to be completed. However, preliminary results from these studies are favorable. *Department of Insectides and Fungicides, Rothamsted Experimental Station, Harpenden, Hertfordshire, AL5 2JQ. Great Britain. In summary, some characteristics of recent synthetic pyrethroids are: (1) greater stability in field than some organophosphates and carbamates, e.g., active after 2 weeks on leaf surface in bright sunlight; (2) rapid metabolism and elimination from mammalian systems, giving low toxicity; (3) limited persistence in soil (weeks, not years); (4) greater potency than other insecticides; low application rates which minimize environmental contamination and offset higher costs. It is therefore appropriate to discuss the new insecticides at a conference in 1975 on the current and future uses and potential human health effects of new approaches to insect pest control. Present Situation In contrast to the large quantities of organochlorine, organophosphate, and carbamate insecticides now used, the total consumption of natural and synthetic pyrethroids is below 500 tons (14) and comparable to that of nicotine, the only other important natural insecticide. However, the natural and the more active synthetic pyrethroids are generally more potent than other classes of insecticides (8) and, unless precluded by instability, or by lack of systemic properties, kill a wide range of insects efficiently by contact or as stomach poisons (15). Such broad activity may affect beneficial insects and predators as well as target species, but the instability, outstanding activity, April 1976 bSubstituents and configuration of 3-vinyl side chain on 2,2-dimethylcyclopropane carboxylate. C(±)-4-Chloro-a-isopropylphenylacetic acid. and low mammalian toxicity of pyrethroids can frequently be exploited to achieve effective and selective control not possible with more stable insecticides. Bioresmethrin (7,16) for example, which is a very potent insecticide, but has very low mammalian toxicity [ca. 8000 mg/kg oral toxicity, female rats (17,18)] is especially suitable for immediate preharvest treatment of food crops (19). These and other uses for the pyrethroids available at present are well documented (20). In this paper, therefore, new applications made possible by the greater stability, still combined with low mammalian toxicity, of the more recent compounds, will be emphasized. First, the development of the present range of pyrethroids will be discussed. Structure and Insecticidal Activity A typical extract of natural pyrethrum from Chrysanthemum cinerariaefolium contains pyrethrins, cinerins, and jasmolins. Pyrethrin I is the most important natural constituent for killing insects (7,21) while pyrethrin II provides much of the valued rapid knock-down action against flying insects (22). The structure of pyrethrin I is an appropriate basis for discussing the molecular features necessary for insecticidal activity, and for considering how these may be modified to increase potency, decrease toxicity to mammals and improve stability. This will illustrate the guiding principles which have led to the present range of synthetic pyrethroids. The insecticidal activity of pyrethrin I depends on the overall structure of the ester, in particular on methyl groups at C-2 on the cyclopropane ring (23) maintained in a definite stereochemical disposition with respect to an unsaturated side chain at C-3 and the ester link at C-1 (24)(25)(26). Without steric constraint, the ester probably adopts an S-trans conformation (2,25,26) and, supported by the near-planar cyclopentenolone ring, the cis-pentadienyl side chain can adopt only certain orientations in space with respect to the features of the acid structure discussed above. High activity is related to the ability of the molecule to adopt an appropriate shape or conformation at the site of action, and this will be particularly influenced by the absolute configurations of the asymmetric centers at C-1 of the cyclopropane ring and at C-4 of the cyclopentenolone ring. In pyrethrin I, these configurations are, respectively, R and S (27,28), axid inverting either diminishes or eliminates insecticidal activity. A number of synthetic pyrethroids have been derived from the structures of the natural esters. The first important synthetic compound, still used today, was allethrin, developed by Schechter, Green, and La Forge in 1949 (29), by shortening and simplifying the pentadienyl side chain of the natural esters (4,5). Allethrolone, the alcoholic component, is now resolved on a commercial scale, (30)(31)(32), gives Sbioallethrin, corresponding to pyrethrin I in stereochemical form. The quantity of allethrin, bioallethrin, and S-bioallethrin manufactured exceeds that of any other synthetic pyrethroid. When supplies of natural pyrethrum are restricted, allethrin is used as a substitute. Much allethrin is also used in mosquito coils, where its volatility and thermal stability, greater than the natural esters, is an advantage. S-Bioallethrin (33) is an outstandingly rapid knockdown agent against some insects, but like bioallethrin and allethrin lacks the broad spectrum of activity shown by the natural pyrethrins (7). Neopynamin (tetramethrin) (34), reported in 1964, was the next synthetic pyrethroid produced commercially. The alcoholic component is not directly related by structure to other synthetic pyrethroids, and although neopynamin knocks insects down rapidly when incorporated into aerosols and comparable formulations, it is not generally a good killing agent (7,35). In 1966, (16,36,3 7), the first compounds that had properties superior to those of the natural ester were synthesized. The discovery of these esters of 5benzyl-3-furylmethyl alcohol suggested that continued investigation of this group of compounds would be likely to lead to further practical insecticides. The furan alcohol was found by a systematic examination of the features essential for insecticidal activity in cyclopentenolone pyrethroids; the furylmethyl component, which, unlike the natural alcohol, had no asymmetric center, reproduced the stereochemistry of the cyclopentenolone nucleus and the phenyl group the function of the unsaturated olefinic side chain. An additional valuable property of the furylmethyl esters, especially that from (+ )-trans-chrysanthemic acid (bioresmethrin) was that the great insecticidal activity was combined with very low mammalian toxicity (7). These favorable properties merited commercial production of resmethrin and bioresmethrin. The same furan alcohol is a component of other practical pyrethroids. The ethanochrysanthemate (K-othrin, RU 11,679) (38-40) has even greater insecticidal activity against some insect species than bioresmethrin, though the mammalian toxicity is also higher (7). Kadethrin (41) (RU 15,525) has a thiolactone ring cis to the ester function and knocks down insects more rapidly than any other compound yet reported. The change in biological properties from K-othrin to Kadethrin with minor alteration of chemical and stereochemical features illustrates well the potential for developing a wide range of insect control agents in the pyrethroid group. Two other more volatile pyrethroids derived from furan alcohols with propargyl side chains, proparthrin and prothrin (35,(42)(43)(44) have been developed in Japan as useful constituents of aerially dispersed insecticidal sprays, but the propargyl side chain is less effective than benzyl in giving the esters a wide spectrum of activity (44). A further advance in developing synthetic pyrethroids was made independently in Great Britain and Japan in 1969 when it was recognized that mr-substituted derivatives of benzene could reproduce the stereochemistry of the furylmethyl unit and a phenoxy group could replace the benzyl side chain (7,9,11). 3-Phenoxybenzyl alcohol gives esters highly active against insects with a smaller range of acids than 5-benzyl-3-furylmethyl alcohol-for example, the cis-thiolactone ester corresponding to Kadethrin is not a good knockdown agent (45). The chrysanthemate (phenothrin) is one third to one half as active as the 5-benzyl-3furylmethyl ester to most insect species (7,11). However, 3-phenoxybenzyl alcohol is accessible by a variety of routes and is less expensive, compensating in some circumstances for the lower insecticidal activity of its esters. At Rothamsted Experimental Station in 1972 an exceptionally valuable combination of properties was found in the esters (permethrin) of 3-phenoxybenzyl alcohol with the cis-and transdichlorovinyl acids (9,10,46,47) analogs of chrysanthemic acid with chlorine in place of methyl in the isobutenyl side chain. Not only was permethrin more active against many insect species than would have been predicted from experience with other esters of the acidic and alcoholic components, but it was also very much more stable in air and light than other potent pyrethroids and had lower mammalian toxicity than the 5-benzyl-3-furylmethyl esters of the same acid (9,17,18,48). Although all the synthetic pyrethroids discussed so far, from allethrin to resmethrin and related compounds have been valuable additions to the armory of pyrethroidlike insecticides, they did not greatly extend the range of applications established for the natural pyrethrins, their main advantage being that some, like bioresmethrin, provided an even greater margin of safety. Permethrin, however, retains most of the favorable characteristics of earlier synthetic compounds and is, in addition, stable enough to control insects in the field as efficiently as established organophosphates, carbamates, and organochlorine compounds, many of which it surpasses in potency. A further outstanding advance (12,13) was disclosed by Japanese workers in 1974. With a suitably substituted aromatic ring in a steric position corresponding to the unsaturated side chain in chrysantheniates, the methyl groups adjacent to the ester link need not be supported on a cyclopropane ring and a-isopropylphenylacetates of established pyrethroid alcohols such as 5benzyl-3-furylmethyl and 3-phenoxybenzyl alcohols have insecticidal activity comparable with the dimethylcyclopropane esters. The two series of compounds probably owe their insecticidal activity to common structural features, because both the Sisopropylphenylacetic acids and 1-R-dimethylcyclopropanecarboxylic acids, which correspond with one another in absolute configuration, give more esters than their optical isomers (12,49). Insufficient data are as yet published to permit pre-cise comparison of the insecticidal activity attainable with the traditional cyclopropane carboxylates with that of corresponding a-isopropyl arylacetates, but the most active diahalovinylcyclopropanecarboxylates appear to be two to three times more potent to some insect species than the S-aisopropyl 4-chlorophenylacetates, for which there are most results. The discovery of the potency of esters of these acids without cyclopropane functions enormously increases the scope for developing useful synthetic insecticides related to the pyrethrins. Finally esters derived from 3-phenoxybenzaldehyde cyanhydrin (50) have been shown to be two to three times more active than those from 3-phenoxybenzyl alcohol. The dihalovinylcyclopropane ester of 3-phenoxybenzaldehyde cyanhydrin (NRDC 149) is more active (P. E. Burt, A. W. Farnham, P. H. Needham, personal communications) than the a-isopropyl 4-chlorophenyl acetate (so relative potencies of the esters are similar to those previously found with other alcohols). By using 1-R, cis-2,2-dimethyl-3(2,2-dibromovinyl)cyclopropanecarboxylic acid as resolving agent (51), the two optically isomeric cyanhydrins were separated. The crystalline isomer derived from the S-cyanohydrin (NRDC 161) (51,52) has quite exceptional insecticidal activity (on a molar basis, approximately 1700 times that of pyrethrin I to normal susceptible houseflies and at a level of ca. 0.03 mg/kg to other insect species), demonstrating the great potential latent in the pyrethroid group (26). To summarize this discussion of the development of practical pyrethroids, Table 1 shows the relative insecticidal activities of some of the more recent compounds to houseflies and American cockroaches. The data are typical of results obtained by topical application of measured drops of insecticide solution to other species. References to Environmental Health Perspectives the relative potencies of other natural and synthetic pyrethroids are given in earlier publications (7,8). Future Developments The wide variety of structural features and high level of insecticidal activity of natural and synthetic pyrethroids shows the scope for developing many potent new compounds. If limited to applications where instability in light was not a disadvantage, few new uses for these compounds could be foreseen. However, the discovery, in the past few years, of structural variations giving increased stability in light has greatly extended the scope of the group. Relative stabilities for some pyrethroids under typical conditions of exposure to air and light in the field are shown in Table 2 to illustrate the principles now recognized for designing more photo-stable compounds. The photochemical degradation products from chrysanthemates (53,54) indicate that the isobutenyl side chain is attacked and the pentadienyl side chain of pyrethrins I and II is another reactive position; the presence of at least two photosensitive centers in the same molecule explains the observed instability in air and light of pyrethrin I. In 5-benzyl-3-furylmethvl esters the photochemical decomposition products show that the furan ring reacts as a dienophile with oxygen (55,56) so that like pyrethrin I, resmethrin has two sensitive positions and is unstable. In phenothrin (11), a stable aromatic center replaces the furan ring but the isobutenyl side chain remains, so that stability is still limited. Substituting chlorine for the methyl groups on the isobutenyl side chain of chrysanthemic acid increases insecticidal activity and inhibits photochemical attack, so that in NRDC 139 (48) only the furan ring remains for photochemical reaction, but this is sufficient to confer instability. However when all sites at which photochemical reaction is initiated are removed, in permethrin, the compound is adequately stable, as results discussed below demonstrate. 3-Phenoxybenzyl a-isopropyl aryl acetates similarly lack photosensitive centers (12,13) so that these two series of very potent pyrethroid insecticides are adequately stable to give residual control of pests in the field. Which particular compounds are most suitable for further development depends on a combination of factors, such as cost of production, insecticidal potency and spectrum of activity, stability required to give adequate control of the target pest and toxicity to mammals and other nontarget organisms. Physical Properties of Pyrethroids and Other Insecticides Some insight into the effects on the environment of introducing more stable pyrethroids to control insect pests in field and forest may be gained by comparing the physical properties of the newer compounds with those of established insecticides and extrapolating from experience with the known compounds. Table 3 lists the octanol-water partition coefficients of some representative insecticides (G. G. Briggs, personal communication; 57,58). Both behavior in the environment and type of action exerted in mammalian and insect systems will be influenced by this property. The three pyrethroids, bioresmethrin, permethrin, and Kothrin are lipophilic compounds, and resemble, in The less desirable properties of chlorinated hydrocarbon insecticides are associated with their lipophilicity and inert chemical structures so that they are stored in the fatty tissues of organisms rather than being metabolized and eliminated. Although pyrethroids are similar to the chlorinated hydrocarbons in some of their physical properties, much evidence from research in the past decade (59)(60)(61)(62)(63)(64)(65)(66)(67)(68) suggests that the structural features of pyrethroids render them susceptible to mammalian detoxification systems, so that by one or more of several possible pathways they are rapidly converted to more polar compounds and are excreted in the urine or feces. This explains their low toxicity to mammals and probably their selective potency to insects which are not able to detoxify them and prevent penetration to the nervous system on which pyrethroids act. In contrast, Barnes, Verschoyle, and White (18) consider that observations on rats poisoned with pyrethroids indicate that, although the nervous systems of these mammals may also be sensitive to their toxic effects, such compounds are very rapidly metabolized after absorption, preventing an effective concentration being reached at the sensitive sites of mammals. The mammalian toxicity of a pyrethroid is related to the facility and speed with which the compound is metabolized. Typical detoxification routes are reactions at methyl and methylene groups [eq. [eqs. (6), (7)]. This hydroxylation reaction will be IN suppressed on aromatic rings deactivated byhalogen substituents as in the chlorinated hydrocarbon insecticides and elsewhere, but phenoxy groups seem (1) especially susceptible (65,68). This may contribute to the extremely low mammalian toxicity ofphenothrin (11,65) and, in the important instance of permethrin, partly explain the lower oral toxicity which Barnes and Verscholyle (9,18) found for (2) both trans and cis isomers compared with the corresponding isomers of the 5-benzyl-3-furylmethyl esters. An interesting recent observation (68) is that when oxidation of the trans-methyl group in the (3) isobutenyl side chain of chrysanthemates, the first Environmental Health Perspectives metabolic reaction established for pyrethroids (59,62) is not possible, as in dichlorovinylcyclopropane esters, one of the adjacent gemdimethyl groups on the cyclopropane ring is attacked eq. (1) No reports have yet been published of the mammalian toxicity or the nature of the metabolic products from the a-isopropyl aryl acetates, although from general considerations of chemical reactivity, ester cleavage should be relatively hindered in this series of compounds. Insecticidal Properties and Applications of Pyrethroids The outstanding potency and other favorable properties of pyrethroids are especially significant with the more stable compounds potentially available (69). Table 4 shows the potency of permethrin and of other common insecticides against the adult desert locust (70) and the rat. The ratios -in the third column indicate the relative effectiveness and safety of the compounds, and permethrin is at least twenty times superior to the organophosphate, sumithion, which most closely approaches it. Other insect species give similar results (47,71), so if larger-scale production and low application rates make general and economic use of pyrethroids possible, these compounds should contribute to pest control at levels which will diminish appreciably the risk of contaminating the environment. As an example, Aedes taeniorhynchus was controlled satisfactorily with only 1.5 g/ha of even the unstable pyrethroid bioresmethrin (72). Lepidoptera, especially the larval stages, are serious pests affecting economically important crops such as cotton, maize, tobacco, rice, sugar beet, and sugar cane throughout the world and enormous quantities of insecticides are used to control them ( Table 5). The natural pyrethrins are very active against lepidopterous larvae but in the past instability, even when specially formulated has precluded use in practice. The synthetic pyrethroids are even more potent than the natural esters to these insects; Ford, Reay, and Watts (73) have projected that efficient and economic control of Spodoptera littoralis is possible with ultra-low volume applications of permethrin. Synthetic pyrethroids, including permethrin, are extremely active against the third instar larvae of an insecticide-resistant strain of the tobacco budworm Heliothis virescens (F.) (F. W. Plapp, personal com- Toxaphene 100 + 69 1 DDT 100 + 250 2 Chlordane 50 500 10 Carbaryl 31 540 17 Malathion 31 1400 45 DNOC 13 45 4 BHC 9 190 21 Aldrin 8 44 6 Suniithion 6 375 62 Diazinon 5 185 37 Dichlorvos 2 65 33 Parathion 1 munication). In laboratory tests, permethrin was more toxic than methyl parathion to this insect, to the bollworm. H. zea (Boddie) and to the boll weevil, Anthonomus grandes Boheman. In the field, it controlled Heliothis spp. as well as did methomyl and monocrotophos (74), so the prospects for practical use against cotton pests are promising. The a-isopropyl acetate, S-5439, also has very good residual activity against the tobacco cutworm (army worm) (Spodoptera litura Fabricius) (13), and the corresponding a-cyano ester S-5602 is more than twice as toxic as lannate in a pot test to this insect. Table 6 shows the relative stabilities and toxicities to codling moth larvae of permethrin and other insecticides (J.G. Allen, East Malling Research Station, personal communication) and toxicities to female rats of these compounds. The data show that under practical conditions permethrin has much greater activity, low mammalian April 1976 toxicity and a useful stability (estimated by bioassay) more than twice as long as the next most active compound, methidathion. As a spray (250 ppm) on cabbage leaves permethrin gave a greater than 50% mortality ofPieris brassicae (Lep) larvae (IV instar) for 19 days, azinphosmethyl (Gusathion, Guthion) for 9 days and bioresmethrin for 2 days (C.N.E. Ruscoe, Jealotts' Hill Research Station, personal communication). Permethrin also shows promising activity against other insect pests. It gave excellent control under field conditions of larvae and pupae of the mosquito Culex peus Speiser at 0.025-0.05 lb/acre and good control of Aedes nigromaculis Ludlow at 0.01-0.025 lb/acre (75). An unexpected result was demonstrated at Rothamsted Experimental Station, where permethrin as a dressing on wheat seeds gave protection without phytotoxic effects against wheat bulb fly larvae (Table 7) comparable with the best organophosphorus insecticide, isophenphos. Under these conditions permethrin probably remained in the waxy coating on the seed from December to March. This is the first successful seed treatment against this pest by other than organochlorine compounds or organophosphates (D. C. Griffiths, personal communication). Such persistent activity as a seed coating does not imply long life for permethrin in soils however, where microorganisms would be expected to decompose it rapidly. In another investigation, F. T. Phillips, P. Etheridge, and G. C. Scott (personal communication) found that permethrin, microencapsulated to delay toxic action, was as effective against two species of leaf cutting ants (Atta sexdens and Atta cephalotes) at 0.005% as pirimiphos methyl at 0.1%. bEffective life of insecticides on apple surface. Although control of agricultural pests, discussed above, will probably most greatly extend the use of more stable pyrethroids there may be further applications in the domestic, horticultural and veterinary fields (76). Preliminary results indicate that in hand spray applications to the entire body surface of lactating dairy animals, at a level needed for adequate fly control, residues of permethrin in milk are unlikely to pose a problem; permethrin also shows a wide range of activity against veterinary parasites (Wellcome Research Laboratories, Berkhamsted, personal communications). Wickham and Chadwick (20) discuss new applications for pyrethroids including permethrin, which they found to have a quite remarkable residual life on plywood. A deposit of 300 mg/M2 on plywood gave 100% kill of Blatella germanica after 12 months exposure in the sun on the windowsills of a laboratory. They note that few compounds in the organophosphorus group last 2 weeks under such circumstances, and the results suggest a promising residual use against mosquitoes, stored products, insects and cockroaches particularly as the compound has low mammalian toxicity and is also apparently biodegradable. Although only limited results are available so far on the effects of permethrin on wildlife, no lethal effect was observed when male Japanese quail received 5 g of permethrin/kg-day for 3 consecutive days. (Pest Infestation Control Laboratory, Worplesdon, personal communication). Synergists Synergists (77,78) such as piperonyl butoxide are valuable in extending the economic use of the Environmental Health Perspectives natural pyrethrins and some synthetic pyrethroids. However, resmethrin, permethrin and related compounds are relatively less well synergized (7,69), and producing a formulation for agricultural use, where the synergist and toxicant must be presented together to the target pest, is relatively difficult. Complications also arise from the necessity to establish safety under all conditions for the toxicant and synergist, so clearance trials are more expensive. For these reasons, unless much more effective and stable synergists are discovered, which can be appropriately formulated for the field, more extensive use of these adjuvants is unlikely. Resistance Where natural and synthetic pyrethroids have been used intensively, especially to control insects resistant to most of the commonly used insecticides (for example, flies in Denmark) (79) resistance to all insecticides, including pyrethroids has developed. Dyte (80) has discussed the problems associated with resistance in stored products pests of which some laboratory selected strains are tolerant to pyrethroids. Although resistance in pests of agricultural crops is less well documented, there is no reason to suppose that the mechanisms involved will not extend to any pest continually exposed to a toxicant new, or well established. Keiding (81) showed that resistance genes have a seemingly unlimited persistence which precludes reuse of insecticides against populations apparently susceptible again. Therefore, if more stable pyrethroids are introduced to control pests in new situations, research on treatment regimes likely to minimize the rate of resistance development is essential. (R. M. Sawicki, personal communication) Summary and Conclusions Knowledge of the relationship between chemical structure and insecticidal activity, toxicity to mammals and photostability of pyr 3throids has developed rapidly in the past decade. This has been applied to designing lipophilic insecticides with, in some respects, more favorable combinations of properties than are possessed by any other naturally occurring or synthetic group of compounds; further application and extension of these principles should provide a wide range of compounds with properties appropriate for controlling many pests under any given circumstances. However, although preliminary examination of the environmental properties and behavior in nontarget organisms has not yet disclosed any significant or insuperable problems (82), much more evaluation remains to be completed, and industrial processes to produce the compounds economically must be developed. Great caution is necessary lest overenthusiastic initial application lead to rapid development of resistant field species which prudent deployment might avoid. In favorable circumstances however, synthetic pyrethroids should help to control more insect pests in the future with smaller hazard to man and the environment than earlier, widely used pesticides.

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Chemistry of rubber processing and disposal. The major chemical changes during the processing of rubber occur with the breakdown in mastication and during vulcanization of the molded tire. There is little chemical change during the compounding, calendering, extrusion, and molding steps. Reclaiming is the process of converting scrap rubber into an unsaturated, processible product that can be vulcanized with sulfur. Pyrolysis of scrap rubber yields a complex mixture of liquids, gas, and residue in varying ratios dependent on the nature of the scrap and the conditions of pyrolysis. The literature has little information on the chemistry of rubber processing and disposal; it has been necessary to draw from various sources to develop a picture of the chemical events that may occur. This review is limited to natural rubber and to the important synthetic rubbers of the tire industry. Processing Rubber processing consists of operations performed on materials or systems to increase their utility through chemical reaction, flow, or permanent change in a physical property (1). The major processing steps wherein most chemical changes occur in tire production are: polymer breakdown by mastication on a mill or in a Banbury, extrusion, calendering, and curing. The tire building process and molding in the curing operation do not normally involve chemical reactions. Mastication In 1820 Hancock discovered the effect of mastication on rubber. The process has become basic in the commercial processing of rubber. Mastication gradually renders the rubber more plastic and decreases its solution viscosity. It is now known that the high molecular wveight of rubber is reduced during this degradation. Even the molecular weight to one-tenth its initial level. Hancock had softened rubber in an oven and later had used steam for the purpose. Over the years it has been deterimined that heat alone is not the cause, for cold mastication is even more effective than warm milling, and both hot and cold milling are more effective than heat alone. The autoxidation of natural rubber was first studied in 1861, when the deterioration of gutta percha was attributed to oxidation. Progress on the subject was complicated by normal antioxidants carried with rubber from the tree and traces of different impurities that act as catalysts (2). It was further complicated by the action of ultraviolet light in accelerating oxidation. The harmful proportion of oxygen is extremely slight; less than 17o in a vulcanizate produces quite obvious changes. Fortunately, crude rubber, which has a greater sensitivity toward oxygen, is normally well supplied with natural antioxidants. In 1921 workers at the Research Association of British Rubber Manufacturers reported that the softening effect produced by milling can also be observed by heating rubber in air or oxygen. There was little softening in the absence of oxygen (Fig. 1). Repeated passes of natural rubber through a tight mill showed a significant drop in viscosity (Fig. 2). Concurrent work by Busse and by Cotton (3)(4)(5)(6) identified the dependence of softening during mastication on the presence of air, oxygen, or ozone. Volatile aldehydes were evolved during mastication in air, and in 140 min natural rubber increased in weight by 0.32%. These workers attributed part of the increase in nitrogen to air dissolved in the sample (Fig. 2). Oxidation is held responsible for "hot" breakdown in the presence of oxygen (Fig. 4). At low temperatures, the chain is ruptured at primary bonds through stresses set up in mastication. The rubber is compressed, stretched, squeezed, sheared in rapid succession and free radicals are produced. These recombine, unless some other substance (e.g., quinones, thiols, disulfides) is present to serve as a free-radial acceptor. Mastication in an inert atmosphere leads to breakdown as long as a radical acceptor is present [Eqs. (1)- (4)]. This was shown with 1% thiophenol [Eq. (4)].' Radical acceptors are effective only in "cold" mastication in the absence of oxygen and depend on the mechanical rupture which is significant only at lower Environmental Health Perspectives 0i 80 temperatures. As the rubber becomes softer, fewer ruptures occur. R-R -2R * (1) N2 2R *-> RR (2) 2R+O02-*2RO2 N2 R * + HS-CGH5 -> RH + SC6H5 (4) It is further possible that addition products react with other fragments to build up, rather than decrease the chain length or to crosslink to form gel. Some chemical plasticizers (peptizers) accelerate breakdown. These are not radical acceptors but are oxidation catalysts (copper naphthenate) and are normally effective only above 1000C. An interesting application of oxidative softening has been used in the German synthetic rubber industry (8). Immediately before it was used, Buna S was exposed in crumb or noodle form to an oxygen atmosphere at 130-135°C. The rubber softened during this step and could be brought to the plasticity needed by the users. The softened rubber had to be used within a short time (24 hr); otherwise it rapidly became stiffer than the original rubber and could not be resoftened. Here again, the softening was dependent on oxygen; it would not proceed in its absence (8). When butadiene-styrene rubber (GR-S) was introduced during World War II, its response to milling was found to be different from that of natural rubber, and factory and labor problems arose. While GR-S had a lower initial plasticity than natural rubber, it softened more slowly during conventional milling. GR-S can be softened by oven aging but, as in the case of the Buna S, once this process starts it continues to a resinous state. Here the processes of chain scission and crosslinking compete at rates dependent on temperature. The various rubbers have shown that the greater the unsaturation, the greater the sensitivity toward oxidation. Natural rubber retains some natural antioxidants from the tree and is storage-stable, but antioxidants must be added during the manufacture of synthetic rubber for shelf and processing stability. Among the synthetic rubbers, the butadiene and isoprene polymers are more sensitive to oxidation than Butyl rubber (containing iso-prene) and much more than ethylene-propylene-based rubbers. Natural rubber and Butyl rubber undergo chain scission to weak stocks with surface tack. Chemical analysis shows that every possible oxygen-containing functional group is present. Butadiene-styrene and butadiene-acrylonitrile rubbers undergo chain crosslinking, becoming increasingly boardy, inflexible, "short", and free of tackiness. A field receiving considerable study is the formation of interpolymers through the action of mastication (9,10). In a nitrogen atmosphere, interpolymers have been made from natural and synthetic rubber as well as copolymers of these with phenolic resins. For instance, when Neoprene and natural rubber are masticated in the usual way, a simple mixture occurs. In a nitrogen atmosphere, the rubber becomes bound to the Neoprene so that the product responds to a typical Neoprene cure, although the mixture does not. Vulcanization Vulcanization is the process of treating natural or synthetic rubber with a chemical, usually sulfur, a sulfur-containing compound, or a peroxide, so as to impart improved properties, such as better elasticity, lower plasticity, or more resistance to solvent action. Few rubber products are used in the unvulcanized state, and only a limited amount of rubber goes into pure gum stocks. Therefore, the blending of rubbers, alone or in mixtures, with reinforcing agents, fillers, and cure systems must be carefully controlled in the manufacture of high quality rubber products. The classic procedure for compounding rubber has mixed baled rubber on a mill or in an enclosed mixer. As synthetic rubber has become available at a practical price in powder, solution, or latex form, novel methods of incorporating compounding ingredients have been investigated, but the established routes of mixing conventional rubbers will be changed only if some outstanding price or property advantage results. Sulfur and sulfur-liberating compounds are most often involved in commercial vulcanization but crosslinking can also be obtained with radiation and with compounds that decompose at curing temperatures to yield free radicals, oxidants of appropriate resonance structure, or Curing agent Table 1. The interaction of polymers with curing agents is possible because of the reactivity conferred by the ethylenic unsaturation. The ally-Natural rubber, isoprene rubber: lic or hydrogen atoms (marked with the double asterisk) are the most reactive; those bearing a single asterisk are less so. Natural rubber is more rapidly vulcanized than are the butadiene polymers because of the activating effect of the methyl group. It is essential that only a limited amount of cure occur during the mixing, storage, and tire-building steps. The crosslinking or vulcanization must begin after the item is molded and it must stop when the desired state of cure is reached. Thus, the compounding of a vulcanizable rubber for commercial use includes the addition of supplementary materials along with the curing agent. These serve a number of roles in optimizing final properties. Cure accelerators or cure retarders are added to control the rate of cure, adapting it to give the desired state of cure and the best final properties at the temperature achieved. These additives may be inorganic, organic, or mixtures. They do not significantly change the chemical modification achieved during vulcanization but, instead, control the rate. A change in the curing system in addition to altering the nature and balance of the reactions occuring during vulcanization can also affect the oxidation stability of the network. Choice of crosslinking system can vary combinations of C-C and C-Se-C bonds. In the more complex case of synthetic rubbers, the crosslinking efficiency increases with vinyl content, apparently due to the markedly different reactivity of 1,2 as compared with 1,4 units. Thus the network structure of synthetics becomes more complex. In addition to the vulcanization systems, a commercially compounded stock may contain a number of other ingredients, depending on the ultimate use of the product (11) : (1) processing aids (plasticizers, such as oils, softeners, or peptizers); (2) antioxidants to prolong product life; (3) antiozonants to reduce ozone damage; (4) reinforcing agents, normally carbon black; (5) fillers. The reinforcing action of carbon black can be improved by high-temperature mastication and by adding certain chemicals (e.g., sulfur, organic sulfur compounds, dinitrosobenzene, quinone dioxime) to the mix (12). An increasing number of bonds form between the rubber and the carbon black surface. It is difficult to measure precisely the amount of interaction between the rubber and black without being confused by crosslinking of the rubber itself. Polymeric free radicals on the surface of the black, possibly through surface groups such as quinones or the polynuclear aromatic layer molecules, may be formed during mastication or thermal or oxidative aging. These make direct observation of the filler-rubber interaction most difficult. Extrusion and Calendering Extrusion and calendering are really modifications of the mill mixing step wherein the compounded rubber is exposed to heat and air for a relatively short time. By this time the stock is fully compounded, and the presence of antioxidants and antiozonants significantly reduces the sensitivity to oxygen. Reclaimed Rubber Rubber is reclaimed by being ground as fine as is economically feasible, separated from fi-Environmental Health Perspectives ber and metal, if possible, and heated in steam in the presence of reclaiming agents (acids, alkalies, inorganic salts). Six routes are available for commercial use, depending on the type of scrap available and the economics of the reclaiming process compared with the cost of new rubbers (9). Reclaiming cannot be considered the reverse of vulcanization, for none of the combined sulfur is removed from the compound as "devulcanization" occurs. It has been postulated that a break develops in the crosslinked molecule to form a shorter chain. At the same time, additional double bonds develop and become available for further sulfur crosslinking. This new vulcanization will normally attack the rubber molecule at the double bond. Thus, the vulcanized scrap rubber has a different chemical make-up from cured virgin rubber. The production of a uniform reclaim depends on the fineness of the ground rubber (increasingly costly) and whether the metal and/or fiber can be removed prior to the reclaiming step itself. Prior to World War II, natural rubber predominated in scrap being reclaimed. The introduction of an increasing number of synthetic rubbers into tires has greatly complicated the picture requiring the development of new reclaim processes. This has further been complicated as new fibers (nylon, polyester, glass, steel) replaced cotton and rayon. In recent years the price and availability of natural and synthetic rubbers and the increasing cost of reclaim rubber have severely limited the role of reclaim in tire production and the output of reclaim has decreased. Possibly, reclaimed rubber may assume a new importance as feed stock becomes less available and more costly, but a tight supply situation must be encountered to force such a change. Disposal There are several possible ways of disposing of scrap rubber (13,14). Those not involving any chemical reaction are: burial, whole or in chopped form; grinding for use to improve soil; discarding in reefs for fish protection; grinding for use as a filler in rubber compounds; dissolving in oil for subsequent carbon black manufacture. Methods in which no chemical product is recovered are disposed of by burning or by burning in a special furnace for production of steam. A third method, pyrolysis for production of gas, oil, or black, is the one method of tire disposal which is related to the subject of this paper; the other routes do not involve chemical changes. A joint program between Firestone and the Bureau of Mines explored the pyrolysis of scrap tires under a range of conditions selected to maximize gas or oil. The tires were shredded to various sizes ranging from 35 mesh to 3-in. pieces, and pyrolysis was conducted in the presence and absence of fabric and beads. The Bureau of Mines-American Gas Association pilot plant was equipped with a retort in an electric furnace and a series of traps to separate readily condensable liquids fromgaseous products (15). Pyrolysis temperatures ranged from 140 to 926°C; a temperature of 5000C was chosen for high yields of liquid, and 9000C was used for increased gas yields ( creased with increasing pyrolysis temperature; the yield of heavy oil decreased at the same time, and the amount of gas increased. The lower yield of residue from truck tires pyrolyzed at 5000C (Table 3) was attributed to the greater content of natural rubber. The black residue was found to contain 7.0-15.3% ash, significantly higher than carbon blacks used in tire manufacture, and the quality was inferior. The light oil condensed at -70°C was subjected to gas chromatographic analysis, and 61 components were identified. Most of the components were eluted prior to 2,4-dimethylpentane. Oil from the 500°C run eluated relatively more saturated and less unsaturated hydrocarbons than did the higher temperature runs (Table 4). The heavy oils were submitted to a spectrometric analysis and a number of probable components were proposed. Alkylbenzenes and alkylnaphthenes were the predominant components. Probable compounds identified from mass spectra included alkylbenzenes, phenols, 3-ring aromatics, biphenyl, acenaphthene, alkylnaphthalenes, phenols, 3-ring aromatics, biphenyl, acenaphthene, alkylnaphthalenes, indenes, styrene, alkylstyrenes, and/or indans. Product from a 9000C pyrolysis contained from aromatic compounds than did the fraction from 500°C runs (Table 5).

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Estimating crossbreeding parameters when two breeds utilize common breeding animals alternative for analyzing crossbreeding The Poisson distribution was used to examine the precision attainable in bull progeny testing for traits like calving difficulty as the following parameters were varied : po the basic incidence level in the population, n, the progeny group size, and a and the probabilities of two kinds of erroneous conclusion. The results showed that, in all circumstances, a group size of 250-300 progeny gave a good compromise between cost and precision. There was little to choose between testing based on calvings in heifers and in cows. As the basic frequency increases, more progeny are required for a given degree of precision. Thus testing is more expensive in breeds with high rates of difficult calving than in those with low rates. The purpose of this study was to examine the influence of errors in estimates of genetic covariances on the efficiency of a cow selection index. The indexes utilised information obtained from the cow’s own three records, or these three records together with the records of groups of paternal half-sisters. The efficiency was studied by correlating the estimates with indexes based on genetic parameters estimated with errors. The efficiency of indexes calculated from genetic covariances estimated with an error decrea-sed as the error itself increased. Large losses of efficiency, however, only occurred with very serious under- and over-estimations. Moderate errors were found not to affect efficiency substantially particularly when the index contained large amounts of information. Three alternative procedures for analyzing crossbreeding experiments in pigsan indirect method, least squares, and maximum likelihoodwere compared as applied to two years test results from a German Federal Crossbreeding Experiment. The results are : i. Estimates of error mean squares obtained in the least squares analysis were about 5 -15 p. 100 smaller than those obtained in the indirect analysis. , 2 . Standard errors of estimates of cross effects obtained from direct procedures (LS, ML) were slightly greater than those from the indirect analysis. 3 . Correlations between rankings of crosses in two years were slightly higher for carcass characteristics when least squares or maximum likelihood constants were used instead of constants from the indirect method but this was not true for days fattened. 4 . Correlations between rankings of crosses under alternative procedures within each year are rather high (r ! .90) for carcass traits, but not for fattening traits. 5 . Least squares and minimum likelihood methods rank crosses with similar precision. The Poisson distribution was used to examine the precision attainable in bull progeny testing for traits like calving difficulty as the following parameters were varied : p o the basic incidence level in the population, n, the progeny group size, and a and the probabilities of two kinds of erroneous conclusion. The results showed that, in all circumstances, a group size of 250 -300 progeny gave a good compromise between cost and precision. There was little to choose between testing based on calvings in heifers and in cows. As the basic frequency increases, more progeny are required for a given degree of precision. Thus testing is more expensive in breeds with high rates of difficult calving than in those with low rates. Department of Animal Genetics and Breeding, Agricultural University of Norway, As-NLH, Norway. Semen from elite-bulls of Swedish Red (SRB) and of Norwegian Red (NRF) breeds has for some times been exchanged. The elite-bulls are the sires of the next generation of young bulls. The young bulls used within one of these cattle populations would therefore include both full and half bloods individuals. A batch of these young bulls are progeny tested in a regulary way. The sire evaluation method used within both breeding populations is assumed to eliminate non-genetic factors properly. By combining linear functions of the sire proves from both populations estimates of crossbreeding parameters of interest can be obtained. The crossbreeding parameters of interest can be given as follows : The proves of these young bulls tested within one of these breeding populations have expectations that are linear functions of G s , G N and H. The first letter of the subscript denotes the origin of the sire of the young bull and the second letter of the subscript denotes the breed of the test population. The following relations can be obtained : where 1 attached with subscripts are mean performance of different breed groups tested in different population. Thus, it follows that : Henceforth, the crossbreeding parameters of interest can be estimated by combining the proves of the young bulls used in these two cooperative breeding populations. Communications libres THE INFLUENCE OF ERRORS IN GENETIC COVARIANCES ON THE EFFICIENCY OF A COW INDEX A. ZARNECKI. Academy of Agriculture, Department of Genetics and Animal Breeding, Cmcow, Poland. The purpose of this study was to examine the influence of errors in estimates of genetic covariances on the efficiency of a cow selection index. The indexes utilised information obtained from the cow's own three records, or these three records together with the records of groups of paternal half-sisters. The efficiency was studied by correlating the estimates with indexes based on genetic parameters estimated with errors. The efficiency of indexes calculated from genetic covariances estimated with an error decreased as the error itself increased. Large losses of efficiency, however, only occurred with very serious underand over-estimations. Moderate errors were found not to affect efficiency substantially particularly when the index contained large amounts of information.

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A cheap method of progeny testing a.i. bulls for milk protein Sampling and selection for production and management traits in dairy cattle is a complex problem. In the scope of modern breeding plans (scheme I) it is stated that only the best 25 p. 100 of the bulls tested on production (+-!-f- 10 p. 100 and -!--!- 15 p. 100) are worthwhile to be ranked according to management traits. A uniform and clear presentation of progeny test results is proposed, namely the breeding value expressed as a deviation from the average (kg milk, 305 days, first lactation). Management traits can be subdivided in categories like reproduction traits (stillbirth, ferti- lity, and so on) milking traits (ease of milking, udder, mastitis) and conformation traits. Accurate sampling is necessary but also a clear and uniform system of handling and classification along the same lines as for production (scheme 2). A final ranking of the top classes (SS and SD) is necessary. A proposal of a sire summary (scheme 3) is given. It is worthwhile to study this subject more closely in collaboration with the International Dairy Federation (I.F.D.) to find some gene-ral guidelines for handling and selection of production and for management traits. dystocia can be minimized by selecting sires for production, evaluating them for ease of calving, mating heifers to sires whose offspring are born easily without direct selection against dystocia. Selection against mastitis might be effective, but management practices can reduce the incidence of mastitis. Selection for udder and leg structure may be necessary in order to avoid problems concerning economical food production. Speed of milking has economic importance and it responds to selection. The amount of emphasis for the selection of milking speed and the most efficient way to apply this has not been clarified. Additional study of the genetics of body-weight change is necessary in order to maximize feed conversion. Genetic control of metabolic disorders in not now feasible ; management control is necessary. Dairymen desire amenable cows, but heritability estimates for this trait are low. Measures of temperament need improvement. The economic worth of all management traits should be further quantified. heritabilities of one : milk yield 100, protein/fat-ratio and The ; the most the the of the of daughters of 70 p. in progeny were 35, 3T, 35! 53, 42, 47 and 42 respectively. In the number of mean zo-3o p. ioo of needed to reach the same accuracy with a monthly sample of i 6 daughters during lactation. Progeny tests based on at least ten daughters were obtained from 136, 134 and 156 bulls each year respectively. It as though with a relatively small number of samples the progeny testing of bulls can be managed. In the progeny testing results the correlation of milk yield with fat-p. 100 was -0.42, with protein-p. 100 with fat yield o.81, and with protein yield o.91. The correlation between fat-p. 100 and protein-p. 100 was 0.44, and between fat yield and protein yield o.84. The genetic correlations calculated from single observations were similar to these both for signs and values. Metabolites and enzymes in the blood which are normally used for clinical diagnoses (total bilirubin, glucose, cholesterol, GOT, CK LDH and LDH-isoenzymes), were regularly determined in a high yielding dairy herd, during all stages of lactation. The correlations between serum levels and the dairy traits were analysed. analysing from of cows of disease. influence season for adjustment biases by differences in age and stage of lactation intraclass correlations for different stages lactation age classes. Numerous significant correlations between the serum levels and the milk yield (milk kg, FCM, fat-p. 100, fat kg) at different stages of lactation were found (r = 0.23 o.61). For all parameters, rank correlations were calculated. The ranking was determined by the deviations of the measured serum levels from the regression line relating milk yield and serum level. Significant rank correlations between glucose and GOT, cholesterol and glucose and between GOT and LDH-isoenzymes i !- 2 were detected. The correlations between the serum levels of two successive lactations were calculated as measurements of the repeatability of the different parameters. Some parameters showed close correlations. In all stages of lactation glucose had the closest correlations of all the parameters examined (up to 0.997). The serum levels of glucose and cholesterol samples, taken 8 weeks before parturition when they are nearly uninfluenced by the milk yield, were about 10 p. 100 higher in animals selected than in animals culled during second lactation. However, the difference is not significant. The other parameters showed no differences between selected and culled animals. Primary selection emphasis should be on milk production and milk constituents. Management traits contributing to ease or economy of production should be selected only if economic importance and phenotypic and genetic relationships to productive traits justify such selection. Breeding efficiency is economically important but genetic improvement within breeds is difficult. Evidence indicates dystocia can be minimized by selecting sires for production, evaluating them for ease of calving, mating heifers to sires whose offspring are born easily without direct selection against dystocia. Selection against mastitis might be effective, but management practices can reduce the incidence of mastitis. Selection for udder and leg structure may be necessary in order to avoid problems concerning economical food production. Speed of milking has economic importance and it responds to selection. The amount of emphasis for the selection of milking speed and the most efficient way to apply this has not been clarified. Additional study of the genetics of body-weight change is necessary in order to maximize feed conversion. Genetic control of metabolic disorders in not now feasible ; management control is necessary. Dairymen desire amenable cows, but heritability estimates for this trait are low. Measures of temperament need improvement. The economic worth of all management traits should be further quantified. In order to develop a cheap method for progeny testing of bulls for milk protein content and yield, about 1 6 6oo milk samples were collected of firstcalving daughters of bulls ( I sampledaughter) in 1971 -1 973 . In Io7I the protein determinations were made with Promilk in one labo-ratory and with 1RMA in another. In 1972 -1973 the samples were investigated with Prot-o-Mat in two laboratories. In each year there appeared significant differencies in protein-p. 100 between laboratories but these grew continuously smaller. These differencies like the effects of days after calving, of average herd milk yield and of month of test day were eliminated with statistical corrections prior to estimating the heritabilities and progeny testing, Daughter's fat-p. ioo, milk yield of test day, protein/fat-ratio and fat and protein yields of test day were computed in the same way. The heritabilities of different traits on the basis of one test day were as follows : milk yield 25 p. 100 , fat-p. 100 2 8 p. ioo, protein-p. 100 25 p. 100 , protein/fat-ratio i 7 P . 100 , fat yield 21 p. zoo, protein yield 19 p. 100 and fat + protein yield 21 p. 100 . The estimates varied slightly in each year ; it seems that the most reliable estimates were from the year 1973 . On the basis of the mentioned heritabilities the number of daughters required for a repeatability of 70 p. 100 in progeny testing were 35, 3T, 35! 53, 4 2, 47 and 42 respectively. In the number of samples these mean zo-3 o p. ioo of what is needed to reach the same accuracy with a monthly sample of i 6 daughters during lactation. Progeny tests based on at least ten daughters were obtained from 13 6, 134 and 15 6 bulls each year respectively. It seems as though with a relatively small number of samples the progeny testing of bulls can be managed. In the progeny testing results the correlation of milk yield with fat-p. 100 was -0 . 42 , with protein-p. 100 -0 . 29 , with fat yield o.8 1 , and with protein yield o. 91 . The correlation between fat-p. 100 and protein-p. 100 Metabolites and enzymes in the blood which are normally used for clinical diagnoses (total bilirubin, glucose, cholesterol, GOT, CK LDH and LDH-isoenzymes), were regularly determined in a high yielding dairy herd, during all stages of lactation. The correlations between serum levels and the dairy traits were analysed. What we are really looking for by analysing these correlations is to find indicators for the individual physiological reactions of dairy cows on the stress caused by a very high milk production. We think that physiological differences between high yielding cows do exist. There might be cows with a very stabile organism which easily give a high yield, and others which are suffering much more by the stress caused by the production of the same amount of milk. The investigations were carried out during a period of two years in the dairy herd of the University of Munich consisting of about 100 Holstein-Friesian cows. The average milk yield amounted to 7 ooo kg milk per year with 3 .8 7 p. 100 butterfat. Each cow was bled 8 weeks before parturition, i week before parturition, 6-24 hours after parturition, 1 , 2 , 3 , 5 weeks after parturition and afterwards in intervals of 8 weeks. The serum levels of all examined blood constituents were significantly influenced by diseases, age, season and stage of lactation. The influence of diseases was eliminated by only analysing data taken from samples of cows which showed no signs of disease. The influence of season was accounted for by adjustment factors. To avoid biases caused by differences in age and stage of lactation intraclass correlations for different stages of lactation were calculated within age classes. Numerous significant correlations between the serum levels and the milk yield (milk kg, FCM, fat-p. 100 , fat kg) at different stages of lactation were found (r = 0 . 23 -o.61). For all parameters, rank correlations were calculated. The ranking was determined by the deviations of the measured serum levels from the regression line relating milk yield and serum level. Significant rank correlations between glucose and GOT, cholesterol and glucose and between GOT and LDH-isoenzymes i !-2 were detected. The correlations between the serum levels of two successive lactations were calculated as measurements of the repeatability of the different parameters. Some parameters showed close correlations. In all stages of lactation glucose had the closest correlations of all the parameters examined (up to 0 . 997 ). The serum levels of glucose and cholesterol samples, taken 8 weeks before parturition when they are nearly uninfluenced by the milk yield, were about 10 p. 100 higher in animals selected than in animals culled during second lactation. However, the difference is not significant. The other parameters showed no differences between selected and culled animals.

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Experimental results on crossbreeding involving beef (Piedmont and Romagna) and dairy cattle (Friesian) in italy Two trials have been carried out in order to evaluate the ability for beef production of (Double-muscled » Piedmont x Friesian and Romagna x Friesian crosses, males and females. Compared with Friesian purebred young bulls, the Fl male offsprings from both crossings showed higher level performances for all the traits considered. Female crosses exhibited good carcass traits. On the whole, Friesian purebred young bulls gave the lowest performances. The P X F female INTRODUCTION The results here presented are part of a larger experimental project currently in progress at our Institute with the purpose of finding out the possibilities of improving beef production in cattle by exploiting two-breed crosses. The present study will report some data collected in two different experimental trials, carried out on two F l progenies, males and females, from different crosses. In the first experiment, the performance traits, relating to quality and quantity of beef production in young bulls and heifers resulting from crossing « Double-muscled n Piedmont X Italian Friesian (P X F) breeds, were investigated. In the second experiment, a research was carried out on F, crosses between Romagna and Italian Friesian (R x F) breeds. MATERIALS AND METHODS The animals used in the first experiment were Z i male Piedmont X Friesian crosses (P X F S), 10 females from the same crossing (P X F !!) and 21 male Friesian purebred calves (F 0101 ). All the crossbreds dropped in the same research farm were used in this experiment ; the straightbred Friesians, on the contrary, were chosen at random from those born in the same period. In crossbreds, the double muscled condition resulted to be latent at birth and appeared later in development. It occurred, however, in the affected calves, with a varying intensity. This condition was never found very marked and appeared clearer in males. The animals were raised in open shelters and loose housed in separate lots. The experiment started with the beginning of the post-weaning period, when the calves were 8 0 to 8 5 days old. The pre-weaning period acted as a pre-experimental test. Diets included hay over the first few months and afterwards corn silage. Throughout the whole experimental period also concentrates were added, as usually practiced. Owing to the fact that the animals were loose housed, feed intake and feed conversion were measured on average within each group. Hence, no statistical and mathematical analysis has been made out of these data. Once the animals had reached a sufficient finishing degree, they were slaughtered. After a 24 hrs pre-refrigeration period, thirty right sides were selected at random ( 10 from each group) and stored in a freezer for 6 days. After this time, the right sides were submitted to dissection and complete separation tests (muscle, fat and bone). In a subsequent experiment the animals used were 10 male F, calves from Ronaagna X Friesian cross (R X F aa), 10 females from the same crossing (R X F !!) and 10 male Friesian purebred calves (F öö). This second trial was conducted in a different experimental farm where the animals were conventionally reared in barns and kept individually tied. In this manner it was possible to determine food intake and feed utilization for each animal. All the animals were chosen at random in the same farm among those born in a month period. Also this experiment started when the calves were on average 79 to 8 5 days old and concerned the post-weaning period. Diets included a small quantity of hay and concentrates according to live-weights. As the animals appeared to have attained a good finishing degree, they were slaughtered. After a preliminary refrigeration of z hrs, the cold carcasses were weighed and all the right sides stored in a freezer for 6 days. After this period, these sides were submitted to dissection and complete separation, in order to evaluate the major wholesale cuts and separable components. RESULTS A. -First experiment (Piedmont X Friesian) The results from the first experiment are given in table i. In the comparison between Piedmont x Friesian crossbred males and females (i vs 2 ), the formers present a significantly higher daily gain and a slightly better feed conversion rate. Crossbred females, however, are sufficiently finished for slaughter in a shorter time, though at much lower weights. Slaughtering tests show significant advantages in favour of crossbred males, since they are superior to females with respect to dressing percentage, although this difference does not reach significance. This appears to be due mainly to the greater weight of viscera in females (P < o.oi). Complete separation and dissection tests on the right sides, too, show crossbred males to have, on the whole, better carcasses than females, in that they present right sides much heavier and richer in percentage of lean (P < 0 . 05 ) ; females, on the contrary, produce fatter carcasses !(P < o.oi), but relatively more developed hindquarters, which yield higher quality cuts. From the comparison between crossbred and straightbred males ( I vs 3 ), it turns out that crossbreeding has allowed to improve beef production at all levels. Crossbreds, in fact, are found to have higher daily gains than pure Friesians (P < o.oi) and more favourable feed conversion rates. Furthermore, crossbreds reach a finishing degree for slaughtering at much heavier weights, though in the same period of time as pure Friesians, and, consequently, they have heavier carcasses (P < o.oi). P X F offsprings give significantly higher dressing percentages ; head, feet, hide, pluck, stomachs and intestines are, in fact, heavier in purebred Friesians. All these differences are highly significant (P < o.oi). As concerns complete separation tests on the right sides, P X F produce higher quality carcasses than pure Friesians. As a matter of fact, crossbred right sides appear to have a higher percentage of lean, a lower percentage of fat and a smaller incidence of bone. Only this latter difference, however, exceeds the minimum value of statistical significance. The results of the second experiment are shown in table 2 . From the comparison between R X F males and females ( I vs 2 ), the formers show a greater ability for beef production, in that they are superior in growth and feed conversion rate. Females appear to be finished for slaughter in a shorter time, but at much lower weights. All these differences are highly significant (P < o.oi). Male crossbreds are superior in dressing percentage to females. Head, feet, and hide are heavier in males, whereas viscera in females. The different levels of significance are reported in table. Males exhibit the best carcasses, in that, besides being far heavier, they yield a higher percentage of lean and a lower percentage of fat (P < o.oi). Females, however, show a lower incidence of bone, although this difference does not reach significance. On the other hand, even if crossbred female carcasses are much lower in weight, they present proportionally more developed hindquarters, which yield higher quality cuts. In this experiment too, females result to have a larger amount of kidney fat (P < o.oi). By comparing R X F male offsprings with straightbred male Friesians ( I vs 3 ), it turns out that the two-breed crossing has improved beef production at all levels, even though not so remarkably as in the first experiment. Male crosses, in fact, are found to be superior in daily gains to pure Friesians (P < 0 . 05 ), but to have a rather identical feed conversion. Furthermore, crossbreds are significantly superior in dressing percentage ; head, feet and hide are heavier in F, offsprings, viscera in purebred Friesians (P < o.oi). Dissection tests do not reveal any significant differences, whereas the composition of the right side shows that the use of the Romagna breed has allowed to increase the contents of lean of the carcass (P < 0 . 05 ), with a consequent decrease in fat percentage. DISCUSSION The results obtained show that « Double-muscled n Piedmont x Friesian and Romagna X Friesian crosses, both males and females, possess a high degree of specialization for beef production. F l male offsprings, however, in both experiments; provided higher performances than F, females, in that they were superior in daily gain, feed conversion and dressing percent. Moreover, they produced higher quality carcasses, i. e. with a greater lean content. Crossbred females, though slaughtered after a shorter growth and fattening period, presented much lighter and excessively fat carcasses. In P X F offsprings, the total feed consumed&mdash;expressed in Sc. FU -necessary to produce I kg of lean was found to be of 10 . 7 for males and 10 . 2 for females. In R X F offsprings the same relation was 9 .8 for males and Io.! for females. From the comparison between crossbred and straightbred F y iesian males, it turns out that two-breed crossing has improved the ability for beef production of pure Friesians. The P X F crossing has increased daily gain, and remarkably improved feed conversion. An improving effect, but not so remarkable, has been detected for the R X F crossing, where we have a superior daily gain, but the same feed conversion rate as pure Friesians. Crossbred males from both trials exhibited heavier carcasses and higher dressing percentages. The crossbreds in the second experiment showed lower dressing percentages than the crossbreds used in the first experiment, and this appears to be caused by the higher weights of head, feet and especially hide in Romagna cattle, compared with Piedmont cattle. Similar findings were reported by another Author (BONSEMBIANT!, 1974 ) from trials in which crossbreds of the same crosses were involved. The P X F crossing, contrary to all expectations, has not so largely improved carcass quality. All the same, crossbred males present a higher proportion of lean and a lower proportion of fat in the right side with respect to straightbred F y iesians, even though these differences do not reach significance. P x F offsprings, in addition, have a lower incidence of bone and this difference do reach significance (P < 0.05). Also the R X F crossing has improved carcass quality, in that the higher percentage of lean and the lower percentage of fat result significant (P < 0 . 05 ). In both trials, the relation between total feed consumedexpressed in Sc. FU and lean obtained (Sc. FU/kg lean) is found to be more favourable in crossbred males than in Friesian males ( 10 . 7 vs 12 . 5 in the first experiment; 9 .8 vs 9 . 95 in the second). These data indirectly confirm the results from the Sc. FU/kg gain ratio. The data obtained point out that « Double-muscled » Piedmont X Friesian and Romagna X Friesian male crosses are highly specialized for beef production. A higher carcass quality could have been expected from P X F males, but probably they had not the appropriate rate of maturity for slaughter. These findings, however, are in agreement with those referred in our previous study (S ANTORO , Z ACI mNt and Q UA -R ANTINI, 1 973). Crossbred females too, show on the whole a good specialization degree for beef production. Even if they did not provide so high performances as crossbred males, F l females, however, did not result inferior to Friesian males in some of the traits considered. Although female crosses resulted to possess a good ability for beef production, we consider, however, that they could be used with greater profit for threebreed crossings. In our opinion, in fact, F, heifers should rather be mated to a sire of a third different breed, highly specialized in beef production, so as to obtain even more productive F 2 crosses. In this view, the Romagna breed, though not so highly specialized as the Piedmont, being provided with a high rusticity degree and a good grazing capacityas we reported in previous studies (M ON E T T I and SA N TO R O, y Deux essais ont été effectués visant à évaluer l'aptitude à la production de viande chez des métis Piémontais de la cuisse (« culard ») x Frisonne et Romagnole x Frisonne, mâles et femelles. Par rapport aux Frisons mâles de race pure, les F, mâles des deux croisements ont donné des résultats meilleurs et de loin pour tous les caractères considérés. Les femelles des deux croisements différents ont montré des carcasses plus grasses que les mâles. Les mâles de la race Frisonne ont offert dans l'ensemble les plus mauvais résultats. Les femelles P x F ont donné de meilleurs rendements à l'abattage que les femelles R X F. Malgré une bonne aptitude à la production de viande présentée en définitive par les femelles, un programme de croisement de trois races avec utilisation de génisses F, pour la production des croisements F 2 encore plus productifs est toutefois jugé préférable. Sono state eBettuate due prove al fine di valutare 1'attitudíne alla produzione della carne in meticci Piemontese « della coscia » x Fvisona e Romagnola x Fvisona, maschi e femmine. A confronto dei Frisoni maschi dí razza pura, i maschi F! provenienti dai due diversi incroci hanno dato risultati di gran lunga migliori per tutti i caratteri considerati. Le femmine meticce hanno offerto a1cune buone caratteristiche delle carcasse. I maschi di razza Frisona hanno fatto regisstrare, nel complesso, i peggiori risultati. Le femmine P X F hanno dato migliori rese di macellazione delle femmine R x F. Anche se 1e femmine hanno presentato, in dennitiva, una buona attitudine per la produzione della carne, si ritiene che un programma d'incrocio a tre razzeimpiegando 1e manze F i per produrre meticci F 2 -sia comunque preferibile.

v3-fos

2018-04-03T04:30:05.165Z

1976-07-01T00:00:00.000Z

39115640

s2

Brucellosis in Veterinary Surgeons in Wales Brucella abortus is the one species in the genus Brucella enzootic in Britain. Its natural host is the cow, and man, and several domestic and wild animals are infected directly or indirectly from the cow. The cost of bovine brucellosis in economic terms has long been recognised. A recent national survey showed that 25 to 30 per cent of dairy herds were infected (Animal Disease Surveys, 1964) and now, belatedly, a programme of eradication by slaughter has begun, with promise of an early conclusion. Brucellosis in humans is primarily an occupational disease and veterinary surgeons are among those especially at risk (Barrett and Rickards, 1953). The cow's placenta, rich in erythritol, is an ideal culture medium for Brucella abortus (Williams et al., 1962). It can penetrate the abraded skin, and cleansing or removing retained products of conception after abortion or calving therefore offers the greatest risk. Though in the calving season the veterinary surgeon confronted with an abortion storm may be asked to cleanse three or more times in a day, he has no real means of self-protection, for disposable shoulder-length gloves are liable to tear and some practitioners even refuse to wear them, on the grounds that they impair the sense of touch needed to separate placental cotyledons from the uterine wall. When infected cows give birth to healthy calves, help with calving calls for strenuous effort; gloves are then a hindrance, and almost invariably they tear. Inhalation of Brucella organisms and conjunctival contamination can also introduce infection but in routine veterinary practice / masks and eye shields are rarely worn. promise of an early conclusion. Brucellosis in humans is primarily an occupational disease and veterinary surgeons are among those especially at risk (Barrett and Rickards, 1953). The cow's placenta, rich in erythritol, is an ideal culture medium for Brucella abortus (Williams et al., 1962). It can penetrate the abraded skin, and cleansing or removing retained products of conception after abortion or calving therefore offers the greatest risk. Though in the calving season the veterinary surgeon confronted with an abortion storm may be asked to cleanse three or more times in a day, he has no real means of self-protection, for disposable shoulder-length gloves are liable to tear and some practitioners even refuse to wear them, on the grounds that they impair the sense of touch needed to separate placental cotyledons from the uterine wall. When infected cows give birth to healthy calves, help with calving calls for strenuous effort; gloves are then a hindrance, and almost invariably they tear. Inhalation of Brucella organisms and conjunctival contamination can also introduce infection but in routine veterinary practice masks and eye shields are rarely worn. A live vaccine of the S 19 strain of Br. abortus, widely used since 1944, is claimed to halve the incidence of disease in immature animals (Morgan, 1970). An inactivated vaccine of strain 45/20, barred until recently in areas designated for eradication, is also effective. Brucella S 19 administration is a tedious repetitive task for veterinary surgeons. Individual doses, freeze-dried, are dispensed in sealed vacuum containers and, if air as well as diluent is added, a fine spray may follow the needle as it is withdrawn, soiling skin or conjunctiva. There is also a risk of self-inoculation. Loaded syringes are often carried carelessly and unguarded, the breast pocket and mouth serving as convenient repositories between injections. Wary practitioners, too, have cause to fear when calves are not properly restrained. 351 Most veterinary surgeons employed by Government have served in practice. Field officers take blood and vaginal smears from suspect cows, and investigators in laboratories examine plate isolations and carcasses. Thus, taking the risk overall, raw milk consumed on the farm is an unlikely source of brucellosis in veterinary surgeons. At the Annual Congress of the British Veterinary Association in 1966 members were warned that the profession was heavily infected with Brucella organisms (Kerr et al., 1966a) and they were urged to consider seriously this hazard to their health. Prompted by these and other expressions of concern, clinical surveys have since been carried out in County Cork (Foley andCorridan, 1968), in Northern Ireland (McDevitt, 1970b) and in Worcestershire (Henderson et al., 1975) but the limitations of partly retrospective enquiries into the incidence of brucellosis in veterinary surgeons must be recognised. In occupational brucellosis serological tests are unhelpful for they are often positive in health. Attempts to isolate the organism, even in severe acute infection, usually fail and in chronic brucellosis especially the criteria for a confident clinical diagnosis may not be fulfilled. Characteristically, prolonged or recurring lassitude is associated with sweats, frontal headache and pain in back or limbs but lassitude may be the only symptom or there may be other unusual features with an uncertain overlay of anxiety or depression. Veterinary surgeons, furthermore, are hardy and reluctant patients, preferring sometimes to treat themselves. Method Since 1966, 105 veterinary surgeons have been interviewed and examined and the great majority are still under observation. Another committed suicide five days before his arranged first appointment, and details of his illness were obtained. When first seen, 90 were practitioners, 12 were administrators and field officers for the Ministry of Agriculture, Fisheries and Food, and 3 worked in veterinary investigation centres; 3 practitioners were female. At each visit blood was examined for Br. abortus antibodies by the phenol saline agglutination test (PS), the mercapto ethanol test (ME), the anti-human globulin test (AHG) and the complement fixation test (CF) (Bradstreet et al., 1970), and other investigations were requested when appropriate. To appreciate some of the hazards of large animal practice, veterinary surgeons were also accompanied during their work. Blood samples were obtained from 91 doctors to form a control group for the interpretation of serological tests. The 84 males were asked whether they had suffered from epididymo-orchitis not due to mumps while in practice but no other clinical details were sought. 352 Results When first examined, 71 veterinary surgeons were well and they have remained well throughout the period of the enquiry; 14 presented with a variety of illnesses, mostly minor, although after six years, one died of senility and heart failure and another of hepatic cirrhosis. Twenty-two, 16 in the former group and 6 in the latter, described earlier episodes of ill-health, suggesting a probable diagnosis of brucellosis. Most had first complained of symptoms soon after starting in veterinary practice. They had stayed at work and after months or years had recovered completely. Brucellosis During the Enquiry A diagnosis of acute or chronic brucellosis was made in 13 patients during the enquiry. In 5, symptoms had recurred for four to twelve years but in none were they sufficiently severe to cause long absences from work. Another, a retired practitioner, was admitted with brucellosis in relapse but the most severe observed long illness remained undiagnosed until epididymo-orchitis was a late complication. The one veterinary surgeon with brucellosis whose spleen was palpable owned only to a vague unaccountable lassitude, insomnia, anorexia and weight loss, present for six months. In four patients with acute brucellosis disability was also relatively mild. A fifth had epididymo-orchitis and was therefore one of two with this complication during the enquiry. A veterinary investigations officer aged 46 years presented 14 months after first complaining of backache and pain in the knees and left shoulder. Tiredness he had blamed on pressure of work and headache on sinusitis. He had also become irritable, anxious, depressed and forgetful forgetting his destination when called to a farm and failing to recognise hitherto familiar landmarks. Four months before he had attended a veterinary congress in Exeter and on his return could not recall the journey or an occasion when he dined with friends. When reminded by them it served only to increase his anxiety. He now had insomnia and his sleep was also disturbed by nightmares of frightening intensity. On one Saturday he had sat at home intending to read. Two hours later his wife saw that the page of his book had not been turned and helped him to bed. He soon awoke, agitated, confused and unaware of his surroundings. Urgent psychiatric advice was obtained and tranquillisers and antidepressants were prescribed. A diagnosis of brucellosis was not considered. After seven weeks he returned to work believing he was better but again complained of lassitude, headache and muscle pains. For nine days before attending, his right testicle had been painful and swollen. He denied sweating, but according to his wife for months he had sweated freely in bed. He looked anxious, tired, and ill. The upper pole of the right epididymis was swollen and tender. His spleen could not be felt but Brucella antibody tests were positive PS 40 ME 40 AHG> 5120 CF 80. (The reciprocals of the dilutions are shown.) Treated with ? 354 tetracycline he recovered and when recalled two and a half years later, he was well: PS 0 ME 0 AHG 40 CF 40. A young assistant in practice was a victim of mild uncomplicated acute brucellosis. When first seen he denied symptoms but Brucella antibody tests were strongly positive PS 1280 ME 1280 AHG 5120 CF 320. A fortnight before, he had been unwell for a week with headache, pain in both knees, sweats, and attacks of shivering, but he had recovered without treatment. Four months before splenectomy had been performed for traumatic rupture, the result of a road traffic accident. Three months after examination, though still at work, he was again complaining of drenching sweats, especially at night. Liver biopsy showed numerous granulomas and intrasinusoidal collections of lymphocytes (Figs 1, 2, 3). A few weeks later he returned to Ireland and has since been well. Seven and a half years later the results of Brucella antibody tests were as follows: PS 20 ME 20 AHG 20 CF 20. Brucellosis and Suicide A practitioner, aged 47 years, stayed in bed for one day with 'influenza' and then insisted on returning to work. He remained unwell. Backache which had recurred for several years was unusually severe. He became increasingly tired, with chills, sweats and intractable insomnia. After a month he looked pale and drawn and though advised to take a holiday he refused. He was now severely depressed and told his wife of other veterinary surgeons whom he believed had been forced to retire as a result of brucellosis. In the seventh week, after learning that his own Brucella antibody tests were positive and agreeing to attend in consultation, he took his own life. Brucellosis as an Unlikely Diagnosis In seven veterinary surgeons, six with positive antibody tests, brucellosis was considered an unlikely diagnosis although it could not be ruled out with certainty as a contributory cause of symptoms. Three had recurring mild anxiety and depression. A fourth for six years had complained of severe pain in his back and limbs. He had no other symptoms of disease and there were no abnormal signs. Recurring lassitude, sweats and fever in a fifth were invariably associated with tracheitis, suggesting that brucellosis was not the cause. A veterinary surgeon, aged 47 years, was tired and depressed, complaining of vague muscle pains. Two years earlier he had been admitted with severe psittacosis pneumonia which was followed by cardiac infarction. He had then returned too soon to a busy practice. In a seventh patient, with early rheumatoid disease, the cause of splenomegaly was uncertain. Tetracycline was prescribed but a year later his spleen was still enlarged. After eight years he had left a large animal practice because of severe rheumatoid deformity of his hands. His spleen was no longer palpable. Ep ididymo-orchitis Two veterinary surgeons presented with brucellosis complicated by unilateral epididymo-orchitis and four others recalled this complication of brucellosis in earlier years. Two had suffered a relapse. Two of 84 doctors questioned had had epididymo-orchitis. In one a diagnosis of brucellosis had been made. In the other, the diagnosis had not been considered but, fifteen months after his illness, tests for Brucella antibodies were positive: PS 160 ME 160 AHG 320 CF 0. Splenomegaly Marked enlargement of the spleen, four fingersbreadth, of one veterinary surgeon was due to haemochromatosis, hitherto undiagnosed. The spleen was enlarged one to two fingersbreadth in six others when first examined. All showed serological evidence of Brucella infection. Splenomegaly in one, with symptoms of brucellosis, disappeared in response to treatment. Another, attending seven years after effective treatment, still had an enlarged spleen but on re-examination one year later it was no longer palpable. In a patient with progressive rheumatoid disease the role of brucellosis as a cause of transient splenomegaly was uncertain. Three veterinary surgeons denied all symptoms: the spleen in one was felt once only; in two it was still palpable after five years, and liver biopsy in one of these showed no evidence of cirrhosis. 356 Alcohol The veterinary surgeon with 'idiopathic' haemochromatosis consumed eight pints of beer a day. One veterinary surgeon died of alcoholic cirrhosis during the enquiry. Three others have needed treatment for alcoholism, two in hospital during episodes of acute psychosis. Two who first took alcohol to alleviate evening fatigue, depression and other symptoms of brucellosis, have recovered, and one firmly believes that brucellosis was the main cause of his addiction. Cleansing Rashes Fifty-eight veterinary surgeons described rashes after cleansings or calvings: in 12 a diffuse erythema, in 13 discrete papules, and in 3 3 pustules as well as papules; 7 attributed their rashes to Brucella allergy resulting from direct contact with cows, though on no occasion had bovine infection been confirmed; 7 blamed repeated exposure to bactericides and detergents. Salmonella dublin was isolated from pustules on the arms of four veterinary surgeons attending routinely, one on a second occasion after an interval of three years. The herd source of four of these infections was confirmed. One veterinary surgeon had worn a shoulder length disposable glove but it had torn and three had tried to achieve asepsis by washing repeatedly with a solution of povidone iodine. Accidental Self-inoculation with Brucella Strains S 19 and 45/20 Sixty-two veterinary surgeons recalled pricking or scratching their hands with needles containing Brucella S 19. Three, from syringes in dust jacket pockets, had injected an arm in the axilla, the praecordium on bending, and a thigh on climbing a fence. One had found the helping hand of a farmer. A lasting tender subcutaneous nodule was a common sequel but in nine veterinary surgeons the local and systemic effects of Brucella S 19 were more severe. A veterinary surgeon, age 49 years, carelessly, by his own admission, injected Brucella S 19 into the base of his left thumb (Fig. 4). Within an hour his hand was swollen, he felt faint and weak and complained of intense headache. Tetracycline Was prescribed, the thumb was incised and the limb immobilised. For two weeks he was delirious and sweated profusely. After five weeks tetracycline was replaced with co-trimoxazole and he then improved but for four months a sinus continued to discharge. Now, after five years, there is marked thenar wasting (Fig. 5). A veterinary surgeon, age 36 years, jogged by a calf, inserted a needle into the base of the middle finger of his left hand. Within three hours he was feverish and his hand was painful and swollen. He was treated promptly with penicillin, streptomycin and chloramphenicol and on the fifth day the finger was incised. Chlortetracycline was then added, and the limb was immobilised. Skin necrosis occurred over the proximal third of the finger, exposing flexor tendons (Fig. 6) and skin grafting was performed but was unsuccessful. The proximal inter-357 phalangeal joint became fixed in flexion and six months after the accident the finger was amputated. One of four veterinary surgeons who had directed Brucella S 19 into an eye attended with acute conjunctivitis. He had no symptoms of generalised infection. Three veterinary surgeons described accidents with Brucella 45/20. One reported 358 Fig. 4. The hand of a veterinary surgeon ten days after self inoculation with Brucella S 19. Fig. 4. The hand of a veterinary surgeon ten days after self inoculation with Brucella S 19. from Ireland that skin on a finger had sloughed, exposing bone. Grafting and amputation were considered but the finger healed. the results of laboratory tests Anaemia in one veterinary surgeon was due to hiatus hernia. Eosinophilia in another, persisting for two years, was unexplained. Leucopenia was not observed. The erythrocyte sedimentation rate was 46 mm in one hour (Westergren) in one 359 Fig. 6. Necrosis of skin due to Brucella S 19, exposing the tendons of flexor digitorum profundus and flexor digitorum sublimus. The finger was later amputated. Fig. 6. Necrosis of skin due to Brucella S 19, exposing the tendons of flexor digitorum profundus and flexor digitorum sublimus. The finger was later amputated. patient with brucellosis in relapse. It was also raised in two patients with rheumatoid arthritis but was normal in all other subjects. Brucella Antibody Tests Positive results were obtained in 97 of the 105 veterinary surgeons examined. Fifty-five of 71, well throughout the enquiry, could not recall an earlier illness to suggest a diagnosis of brucellosis. Of these, 22 had positive results in a titre of 1/80 or above in the phenol saline test, 21 in the mercapto ethanol test, 46 in the AHG test and 30 in the complement fixation test. However, in 89 of 91 doctors, Brucella antibody tests were negative. In one, the AHG test alone was positive in low dilution, PS 0 ME 0 AHG 40 CF 0. In another, whose positive results have already been described, they were obtained 15 months after an attack of epididymo-orchitis. During the period of the enquiry a number of veterinary surgeons moved to other practices. Thirty-nine, examined before 1971, had remained in the district and were still visiting the same farms when recalled in 1975. Raised antibody titres (1/80 or above) before 1971 and in 1975 were as follows: 23 reducing to 2 for the phenol saline test, 19 reducing to 2 for the mercapto-ethanol test, 39 reducing to 24 for the AHG test and 31 reducing to 21 for the complement fixation test. The AHG test was initially positive in all and the complement fixation test was negative in only one. In 1975 the AHG test was negative in 11 and the complement fixation test was negative in 12. Liver Biopsy Biopsy sections from one patient with mild acute brucellosis contained histiocytic and giant cell granulomas (Figs 1 and 2) (Hunt and Both well, 1967) and foci of intrasinusoidal collections of lymphocytes (Fig. 3). Another patient was a retired veterinary surgeon with brucellosis in relapse. Microgranulomas were seen (Weinbren, 1975). In one veterinary surgeon with recurring anxiety and depression the role of brucellosis as a cause of symptoms was uncertain. Liver biopsy showed one large giant cell granuloma, an intrasinusoidal collection of lymphocytes and scanty foci of necrotic parenchymal cells. Biopsies in seven others showed scanty or single microgranulomas with lipogranulomas in four. Five of the seven had received prolonged antibiotic treatment and in none was there clear clinical evidence of disease when biopsy was performed. Hepatosplenomegaly in one symptomless veterinary surgeon was attributed to haemochromatosis. DISCUSSION Antibody tests in brucellosis reflect changes in immunoglobulins IgG, IgA and IgM (Coghlan and Weir, 1967) and the suggestion that IgG antibodies are present only during active infection (Reddin et al., 1965;Kerr et al., 1966b), the cause of much of the anxiety expressed at the British Veterinary Association Congress in 360 1966, has not been confirmed (Kerr et al., 1966a). Thus, the antiglobulin and complement fixation tests for IgG are often positive in health (McDevitt, 1970a), as in the majority of veterinary surgeons in the present series. It has also been shown that, rarely, they are negative (Payne, 1974) or positive only in low dilution (Poole, 1975) when infection is proved by blood culture. When antibody levels are persistently raised, continuing exposure to Brucella organisms or vaccines may be the cause. Br. S 19 is still widely used and the fact that practitioners in south west Wales who still visit the same farms show a fall in antibody titres may be an encouraging indication of progress in the eradication scheme. In reports on brucellosis in veterinary surgeons no clear picture of its clinical importance has emerged. Though rarely fatal, it caused one death from suicide in the present series. Epididymo-orchitis is a common complication but others, more serious, are rare. One overworked Government Officer presented with acute delirium, and only with hindsight was it properly attributed to brucellosis. Depression in this disease is a known cause of alcoholism (Spink, 1956), as in one or possibly two veterinary surgeons in the present series. Hepatic cirrhosis is described (McCullough and Eisele, 1951) but in one patient who died of cirrhosis its cause was alcohol which may also have been a contributory cause of haemochromatosis in another. Liver biopsy in 23 veterinary surgeons reassuringly showed no evidence of cirrhosis. A high incidence of brucellosis in rural Britain has been questioned (Henderson, 1967). In veterinary surgeons especially it is wrongly diagnosed if undue emphasis is placed on laboratory tests. Furthermore, splenomegaly can occur without symptoms and it can persist long after effective cure. But brucellosis is not always severe or disabling and mild symptoms are perhaps less likely to be misinterpreted if surveillance is maintained. A clinical diagnosis was made in a third of the veterinary surgeons in the present series. In many the illness was mild and eventual recovery was the rule. The risks from self inoculation with Brucella S 19 are known (Spink and Thompson, 1953) and are often publicised in veterinary journals (Harvey, 1969). Accidents, however, are not always the result of negligence. Calf vaccination is believed to be an essential prerequisite to effective eradication and Brucella 45/20 is not a safe alternative (Price, 1973). Rashes after cleansings and calvings have been blamed on Brucella allergy (Huddleson, 1943), an attractive hypothesis without proof. There is proof that a papular and pustular dermatitis can be caused by Listeria monocytogenes (Dijkstra, 1959;Owen et al., 1960), Salmonella dublin (Williams, 1969), S. typhimurium (McCoy, 1969) and S. abortus equi (Clarenburg, 1964). Veterinary obstetricians are therefore vectors of common bovine pathogens, which is an indictment of modern materials and techniques. Most brands of disposable shoulder-length gloves, though accepted by a tolerant profession, are liable to tear, and ritual washing with povidone iodine does not create an effective bactericidal s barrier. Asepsis in large animal practice, however, is illusive for other immutable reasons and brucellosis is an occupational hazard that will remain until eradication is achieved. As a rare exotic illness it will then present an even greater diagnostic challenge.

v3-fos

2018-04-03T00:37:07.813Z

1976-07-01T00:00:00.000Z

3609342

s2

Variant infectious bronchitis virus isolated from Indiana chickens. Infectious bronchitis virus (IBV) associated with a catarrhal tracheitis, sudden decline in egg production, and reduced shell quality was isolated from an Indiana White Leghorn breeder flock. It was found to be serologically different from Massachusetts, Connecticut, Iowa 97, Iowa 609, Florida, Arkansas 99, JMK, Holte, Gray and SE 17 IBV serotypes. Two different Massachusetts vaccine strains protected chickens from respiratory signs but not against virus infection using the isolant for challenge in laboratory trials. The isolant was passed through a 0.22 μ. filter. It was heat (56° C), acid pH (3.0), ether and chloroform labile. In embryos it produced deaths or lesions of infectious bronchitis in one to five days after inoculation. It is suggested that this IBV isolant be designated Indiana-type. INTRODUCTION I NFECTIOUS bronchitis (IB) is a highly contagious, coronavirus-induced disease of chickens affecting the respiratory tract and reproductive performance. For many years IB vaccination has become standard practice using either a Massachusetts-type strain alone or combined with the Connecticut-type during the growing period of laying chickens. However, many variant strains have been isolated which are immunologically different from the vaccine virus used (Hofstad, 1958;Hopkins, 1969;Jungherr etal., 1956;Winterfield and Hitchner, 1962;Winterfield et at, 1964Winterfield and Fadly, 1971). This paper describes the isolation of a variant IB virus from a flock which experienced production problems. MATERIALS AND METHODS Ten-to-11-day-old susceptible chicken embryos were inoculated via the chorioallantoic (CA) cavity with suspensions of tracheal mucous or infective CA fluids in nutrient broth containing antibiotics. Titrations were performed by serially diluting (10-fold) CA fluids and inoculating five embryos per dilution. All embryos were candled daily. Deaths occurring within 48 hours after inoculation were considered non-specific except when using the isolant which caused embryo deaths as early as 24 hours. Between 2 and 7 days post-inoculation (PI), the embryos were examined for typical signs of (IBV) infection (National Academy of Science-National Research Council, 1971). CA fluids from stunted or dead embryos were examined for bacterial sterility and frozen at -65° C. After initial isolation, CA fluids were harvested 24 hours P.I., tested, and stored. EID 50 virus endpoints were calculated according to the method of Reed and Muench (1938). CA fluid containing the Indiana virus isolate was filtered through a 0.22 |x. millipore filter. This material was then diluted 1:10 and injected into the CA cavity of 10-day-old SPF fertile embryos and observed for lesions or deaths twenty-four hours P.I. Serums possessing high titers of virus neutralizing antibodies were obtained by infecting isolated groups of SPF chickens intratracheally with one serotype of IBV and bleeding them 4 weeks later. The following IB virus types were used: Massachusetts, Connecticut, Iowa 97, Iowa 609, JMK, Florida, Holte, Gray, SE17, and Indiana isolant. Antiserum derived from the Indiana virus was also tested against an Arkansas 99 serotype. The serums from one group were pooled, tested for steril-ity, inactivated in a 56° C. water bath for 30 minutes, and frozen until needed. Indiana virus and the respective serums were tested for neutralizing capabilities against homologous and heterologous immune serums and viruses. Homologous virus and immune serums of the other types were also tested for control purposes. Each trial consisted of a virus titration control and a titration of the virus with the dilutions mixed 1:1 with the respective serum. Serum virus mixtures were incubated at room temperature for 30 to 60 minutes prior to inoculation. EID 50 endpoints were calculated and log 10 neutralization indices (NFs) were determined from these endpoints seven days P.I. In trial 1, sixty Hubbard broiler-type chickens were reared in isolation until four weeks of age and then were vaccinated with a commercial Massachusetts-type (M41) virus using a Root-Lowell sprayer at the low output. A virus titer of 4.17 log 10 EID 50 per ml. was used with the diluent being 80% distilled water and 20% glycerine. Exposure time to the spray was ten seconds. In the second trial, eighty, one-day-old Hubbard broiler-type chicks with Massachusetts-type parental immunity were vaccinated by eyedrop with 10 4 EID so per chick of the Holland-72 Massachusetts virus. Nonvaccinated controls were maintained separately in adjacent, but isolated, quarters. Low egg-passage virus types were used for challenge. Indiana and M41IBV, were diluted with nutrient broth so as to provide each bird with 10 3 EID 50 , given by eyedrop 4 weeks post-vaccination. Ninety-six hours postchallenge, the chickens were examined for rales and swabs of tracheas and clefts were taken for virus isolation. These swabs were placed in 3 ml. of nutrient broth containing antibiotics and frozen until isolation attempts were made. In heat stability trials, vials of Indiana virus were immersed in a 56° C. water bath for varying fifteen minute intervals allowing for a two-minute temperature acclimation period. Titration of this fluid was then performed in embryos after cooling. The effect of acid pH on the agent was determined by addition of hydrochloric acid to infective CA fluid to attain a pH of approximately 3. After thirty minutes incubation at room temperature, the fluid was titrated. Ether susceptibility was tested by adding 0.2 ml. of pure diethyl ether to 1.0 ml. of CA fluid containing Indiana IBV. This was then mixed and placed in a refrigerator at 4° C. for 18 hours before being poured into a petri dish at room temperature to allow evaporation of the ether. The remaining fluid was then titrated. Analytical grade chloroform, 0.05 ml., was added to 1.0 ml. of CA fluid from an infective high titer pool of the isolated agent. This combination was shaken vigorously by hand for ten minutes; the chloroform allowed to settle and the fluid at the surface was then assayed. In each treatment above, untreated controls were maintained and titrated concurrently. RESULTS White Leghorn chickens from three different houses on an Indiana poultry farm were submitted to the diagnostic laboratory. A sharp decrease in egg production and increased mortality without apparent clinical signs was reported. Each house contained approximately 12,000 females and 1,000 males. All birds had been vaccinated with commercial IB vaccine virus strains (Massachusetts and Connecticut types) 3 times during the growing period. The older birds (53 weeks) in House A, showed a more drastic decline in production, 20% in two days, than either House B (40 weeks old), or House C (42 weeks old) which dropped five to ten percent in production in a week. Egg shell quality problems occurred two weeks after the initial production drop. The birds did not achieve normal production after this period of illness. Upon necropsy, approximately 1 week after the production drop, affected birds exhibited a catarrhal tracheitis, airsacculitis, and inactive ovaries. Virus isolation attempts from these birds were unyielding since the tracheal swab material was heavily contaminated with antibiotic resistant bacteria. However, swabs were then taken from several birds showing no respiratory signs and pooled for virus recovery attempts. Upon initial passage in embryos, only one of ten embryos showed stunting and dwarfing. CA fluid harvested from this embryo was then passed a second time in embryos, after a ten-fold dilution, and within 48 hours, a substantial number of embryos died. Table 1 shows the log 10 neutralization indices (NI's) of various pooled antiserums representing known IBV serotypes and the Indiana virus. Indiana antiserum did not significantly neutralize any of the heterologous viruses (NI < 2.00) but did have an NI = 5.38 log ]0 EID 50 with the homologous virus. Heterologous antiserums failed to significantly neutralize the Indiana virus but had high homologous titers. Table 2 demonstrates the ability of M41 IBV vaccination by spray at four weeks of age to protect chickens from challenge by Indiana IBV. M41 vaccination did not produce satisfactory protection as shown by the criterion of virus shed-rate. Similarly, Table 3 indicates the lack of immunity against virus shed from Holland-72 (Massachusetts-type IBV) vaccination at one day of age and subsequent challenge with Indiana IBV. Chemical and Physical Characteristics. This isolate was found to be a filterable agent since the filtrate (0.22 u..) when inoculated into embryos caused typical changes of IBV in the P.I. period. A reduction of 5.16 log ]0 EID 50 was seen after thirty minutes of heat treatment at 56° C. and slO 1 EID 50 was obtained by exposing infective CA fluid to acid pH (3.0) for thirty minutes. The virus was ether sensitive as a reduction in titer of 2.38 logs resulted after treatment. In the presence of chloroform, a 1.96 log 10 EID 50 reduction titer was noted indicating the loss of essential lipids from the virion. These characteristics are compatible with other characterized IBV isolates. Pooled serums from two clinically infected flocks revealed NFs of >4.00 logs and >3.87 logs to the Indiana virus ten weeks after the disease occurred. DISCUSSION The results reveal the isolation of a new serotype of IBV which has not been described previously. This isolate was associated with production problems even though an apparently satisfactory IB vaccination program was used. The immunity from Massachusetts-type IBV vaccination, however, apparently buffered the severity of adverse effects of infection as shown by the immunization trials. Whereas a reduction in respiratory rales was evident from cross-immunization and challenge, virus infection and shed was not significantly prevented. The latter criterion has been shown previously to be a sensitive criterion in evaluating the immune response from vaccination .

v3-fos

2014-10-01T00:00:00.000Z

1976-08-01T00:00:00.000Z

9382931

s2

Association of serum hemolytic complement levels with the major histocompatibility complex in chickens. The total hemolytic complement (C) levels in inbred line 7 chicks and adults were lower than C levels in inbred lines 2 and 3 and in outbred chickens of the same age. In all birds, adult levels of C were obtained in 5- to 6-wk-old chickens. Analysis of F1 and F3 generations clearly showed that the C level in chickens was determined by a dominant gene(s) associated with the major histocompatibility complex. Finding this association in a nonmammal strengthens the importance of the relationship between closely linked genes controlling histocompatibility, immune responsiveness, mixed leukocyte reaction, and C activity. chickens were immunized with sheep erythrocytes. The chicken anti-sheep erythrocyte antisera were inactivated at 56°C for 30 rain. Complement Assay. The method of Gewurz et al. (12) was used with some modifications. Tris buffer, pH 7.4, was used throughout (13). Sheep erythrocytes standardized to 2 × 10 s cells/ml were sensitized with an equal volume of a dilution of hemolysin which gave optimal sensitization. Different volumes of appropriate dilutions of test sera in duplicate were added to 0.2 ml of sensitized sheep erythrocytes and the reaction volumes were adjusted to 1.2 ml. After incubation at 37°C for i h, the reaction was stopped by addition of 2.0 ml of cold physiological saline. After centrifugation at 4°C, the hemoglobin content was determined in a Beckman Spectrophotometer (Beckman Instruments, Inc., Fullerton, Calif.) at 412 nm, and the CH5o units were calculated according to the method given by Kabat and Mayer (14). Results Adult birds from lines 2, 3, and 7 were selected at random without regard to age or sex and their sera were assayed for total hemolytic C. The sera of line 7 birds had 193.7 _+ 10.5 CHs,~ U/ml, whereas the sera of lines 2 and 3 had 249.8 +_ 17.0 and 272.5 +_ 18.3 CHso U/ml, respectively (Table I). There was no relationship between C levels and sex. To determine whether the reduced C level of line 7 birds was related to age, the C levels of outbred cockerels and inbred cockerels and hens from ages 1 day through 7 wk were determined. As shown in Fig. 1, at 3 days of age the C levels of line 7 birds were significantly lower than those of lines 2 and 3; however, the mean level of the outbreds at this time was only slightly higher. By 2 wk, the C levels of the outbred birds were the same as line 2 and 3 and significantly higher than the line 7 levels. By 5 wk, the C levels for all lines leveled off so that by 6 wk the concentrations for all birds were about the same as those found in adult (1-yr-old) birds. Sera from adult F1 hybrids, produced by crossing line 7 with line 2 and with line 3, were analyzed. The C levels were similar to those of lines 2 and 3 (255.7 +_ 13.5 and 259.1 _+ 13.0 CHso U/ml, respectively) ( Fig. 1). Second and third generation birds were obtained from crosses of the three inbred lines, and birds were selected on the basis of their homozygosity and heterozygosity for the B locus, as shown in Fig. 2. All hybrids which were homozygous for the line 7 B allele (B'B 4) had low C levels (174.1 ± 6.8 and 171.0 _+ 8.9) regardless of whether they were derived from crosses between lines 7 and 2 or between lines 7 and 3. All sera from birds with the homozygous B allele of line 3 (B2B 2) and heterozygous at the B allele (B6B 4 and B2B 4) had levels of C comparable to the levels in parental lines 2 and 3. There was no association of C levels with the CS-1 7S immunoglobulin and lipoprotein allotypes (Table II). Discussion All line 7 birds assayed were found to have about 70% of the serum hemolytic C of lines 2 and 3 and outbred production line birds. Line 7 birds also are low Relationship between Hemolytic C Levels and B Blood Group and Allotype Genotypes in Hybrids No. High antibody responders to the copolymer poly(L-Glue%-Alaa°L-Tyr 1°) (GAT), and the response to GAT also is associated with the MHC (7). Lest it be thought that line 7 birds are generally immunologically incompetent, they produce more antibody in response to limiting doses of bovine serum albumin than line 3 birds (18), and they synthesize levels of anti-dinitrophenyl antibodies comparable to outbreds (7). Furthermore, line 7 birds clearly had lower C levels throughout the period of maturation. The Fm hybrids, obtained by mating line 7 birds with either of the high C lines, had C levels comparable to lines 2 and 3. Therefore, the low C level appears to be a recessive trait. The results show that the C level in the chicken is associated with the MHC as it is in mammals. All of the low C birds found in F3 generations had the B genotype of line 7 (B4B4). There was no overlap in the standard deviations of the CH~o values between these birds and the F3 birds with high C levels (B genotypes: B2B 4, B2B 2, and B6B4). Furthermore, there was no correlation between C levels in F3 birds with either the 7S immunoglobulin H-chain allotype linkage groups or a lipoprotein allotype. In the mouse, the gene(s) responsible for the H-2-dependent differences in C levels is associated with the Ss-Slp locus (10,11), and recently C4 was identified as the Ss protein (19,20). In addition, C3 levels were somewhat associated with H-2 (21) and in man C2 deficiency was shown to be HL-A linked (9). At present, we do not know which C component(s) deficiency accounts for the reduced C levels in line 7 birds. Finding the association between C levels and the MHC in the nonmammal strengthens the importance of the relationship between closely linked genes controlling histocompatibility, immune responsiveness, and C activity. It is unlikely that these immunological functions are linked by chance in species as different as chickens, mice, and men; and this linkage favors the view that this region has remained relatively unchanged over millions of years. Perhaps the C gene(s) of the MHC may prove to be the oldest component of the MHC in view of the recently described presence of C3 proactivator in starfish hemolymph (22). We agree with the view of Pazderka et al. (23) that the chicken "is eminently suited" for a species-wide evaluation of the linkage between the MHC-associated functions because of the '~many differently selected populations large and stable enough to permit prolonged and repetitive sampling." For these reasons the chicken also is suited for revealing the evolutionary genetic mechanisms affecting immunoglobulin structural genes as indicated by recent studies on allotypes by E. K. Wakeland in our laboratory. Summary The total hemolytic complement (C) levels in inbred line 7 chicks and adults were lower than C levels in inbred lines 2 and 3 and in outbred chickens of the same age. In all birds, adult levels of C were obtained in 5-to 6-wk-old chickens. Analysis of F1 and F3 generations clearly showed that the C level in chickens was determined by a dominant gene(s) associated with the major histocompatibility complex. Finding this association in a nonmammal strengthens the importance of the relationship between closely linked genes controlling histo-compatibility, immune responsiveness, mixed leukocyte reaction, and C activity.

v3-fos

2014-10-01T00:00:00.000Z

1976-02-01T00:00:00.000Z

1442888

s2

Methods to assess reproductive effects of environmental chemicals: studies of cadmium and boron administered orally. Results of a U.S.S.R.--U.S. cooperative laboratory effort to improve and validate experimental techniques used to assess subtle reproductive effects in male laboratory animals are reported. The present studies attempted to evaluate the reproductive toxicity of cadmium as cadmium chloride and boron as borax (Na2B4O7) and to investigate the mechanism of toxicity in the rat following acute and subchronic oral exposure. In vitro cell separation techniques, in vivo serial mating tests, and plasma assays for hormones were utilized. Effects on the seminal vesicle and prostate were evaluated with chemical and enzyme assays. Clinical chemistry was monitored routinely. Acute oral doses, expressed as boron were 45, 150, and 450 mg/kg while doses for cadmium equivalent were 6.25, 12.5, and 25 mg/kg. Rats were also allowed free access to drinking water containing either boron (0.3, 1.0, and 6.0 mg/l.) or cadmium (0.001, and 0.l mg/l.) for 90 days. Randomly selected animals were studied following 30, 60, and 90 days of treatment. These initial studies, utilizing a variety of methods to assess the reproductive toxicity of environmental substances in male animals, suggest that cadmium and boron at the concentrations and dose regimens tested are without significant reproductive toxicity. Our U.S.S.R.-U.S. cooperative laboratory effort seeks to improve and validate experimental techniques used to assess subtle reproductive effects in laboratory animals. In our laboratory, we routinely use the velocity sedimentation cell separation technique to determine cellular uptake of environmental chemicals and to determine the effect on environmental substances on the incorporation of thymidine, uridine, and L-leucine by specific spermatogenic cell types (1). In vivo serial mating studies assess reproductive function. A variety of other experimental techniques are also to be tested and evaluated jointly. It was agreed that the acute and subchronic oral exposure (in drinking water) of selected environmental chemicals would be studied *National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709. tNational Institute of Child Health and Human Development, Bethesda, Maryland 20014. in the U.S. by in vitro cell separation techniques, in vivo serial mating tests, and plasma assays for hormones. Effects on the seminal vesicle and prostate would be evaluated with chemical and enzyme assays. Soviet scientists agreed to study the male reproductive effects of the same environmental chemicals using techniques common to their laboratories. Cadmium and boron were selected for initial study. Health effects of environmental cadmium are a continuing concern in the United States. Cadmium concentrations in both natural and drinking water are usually less than 1 ppb. Concentrations of cadmium greater than 0.01 mg/l. constitute grounds for rejection of a water supply for drinking. Cadmium is sometimes taken up in grains, such as wheat, depending on the cadmium concentration of the soil. The use of sludge may increase the cadmium uptake in food grains severalfold. Cadmium February 1976 in foodstuffs is generally less than 0.05 ppm. In marine organisms, fish meat contains very small amounts of cadmium, whereas internal organs of fish and shellfish may contain appreciable amounts. Daily uptake of cadmium in nonpolluted areas is estimated to be 50 ug with a possible regional variation from 30 to 70 ,ug (2). Numerous studies of cadmium-induced toxicities have been reported in the past, although most involve parental administration. In the pregnant rat and mouse, cadmium (2.24-4.48 mg/kg) causes rapid placental necroses and fetal death. Cadmium also damages the sensory ganglia in both male and female rats and produces a transient disturbance in liver function in the same dosage range. A single subcutaneous or intramuscular dose of cadmium can induce sarcoma at the injection site and interstitial cell tumors of the testis (3). The toxic effect of cadmium on the testes has been reported to be very selective, and this fact is well documented. Doses as low as 1.12-2.24 mg/kg of cadmium can cause testicular damage without pathological changes of other organs. A single large injection of 10 mg/kg of cadmium leads to the selective destruction of rodent testes. Two theories are generally offered for the mechanism of cadmiuminduced testicular lesions: a circulatory failure due to vascular damage, and a direct action of cadmium on spermatogenic cells which can be reversed by zinc. Doses of cadmium without histopathologic effects have a direct action on germinal epithelium (3). Cadmium metabolism and toxicity have been reviewed recently by Nordberg (4). In the United States, much less concern surrounds the possible health hazards associated with boron compounds. These chemicals are not highly toxic and therefore not considered an industrial problem. Boron is used in medicine as sodium borate, boric acid, or borax, which is also a common cleaner. Accidental poisoning due to boric acid and oral ingestion of borates or boric acid have been reported. The fatal dose of orally ingested boric acid for an adult is somewhat more than 15 or 20 g and for an infant 5-6 g. Weir and Fisher (5) have studied the chronic toxicity of borax and boric acid in laboratory species and confirm the low order of toxicity for these chemicals. Boron can affect the central nervous system. Boron poisoning causes depression of the circulation, persistent vomiting, and diarrhea, followed by shock and coma. Boric acid intoxication can arise from absorbing toxic quantities from ointments applied to burned areas or to open wounds, but it is not absorbed from intact skin. Soviet scientists have been concerned with the long-term use of drinking water with a high boron content. Bokina has reported gastrointestinal tract functional effects of boron in drinking water (6). The United States Public Health Service does not list boron in its 1962 chemical standards for drinking water. In contrast to the boron compounds discussed, decaborane has been used as a rocket propellant and for various industrial purposes and is toxic to exposed workers. Signs and symptoms of human intoxication are in general referable to the central nervous system. The present studies attempt to evaluate the reproductive toxicity of cadmium (cadmium chloride) and boron (borax, Na2B407) and to investigate the mechanism of toxicity in the rat. Both acute and subchronic oral administration were studied. The velocity sedimentation cell separation technique was utilized to identify the spermatogenic cell types which incorporate these substances. Both in vivo and in vitro effects of cadmium and boron on the uptake of tritiated thymidine, uridine, and L-leucine by selected spermatogenic cells were investigated as an indication of alternation of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and/or protein syntheses. In vivo effects on fertility were determined using the serial mating techniques. The effects of subchronic exposure to drinking water containing either cadmium or boron on endocrine function and levels of selected enzymes were also studied. Fructose, zinc, and acid phosphatase levels were assayed in the prostate. Plasma levels of follicle stimulating hormone (FSH) and luteinizing hormone (LH) were also determined. Acute Studies Single oral doses of cadmium chloride and borax were administered to adult male Sprague-Dawley rats to determine reproductive effects during serial mating studies. Acute doses, expressed as boron, were 45, 150, and 450 mg/kg while doses for cadmium equivalent were 6.25, 12.5, and 25 mg/kg. In vivo Assessment of Fertility Serial mating assesses the biological functionality of sperm cells and produces fertility patterns which are inversely related in time to the phase of spermatogenesis damaged by the chemical tested. For instance, effects on spermatozoa appear Environmental Health Perspectives first, and those due to interference with spermatogonia appear last. The successful application of this method depends upon the fact that spermatogenesis proceeds continuously without regard to frequency of mating. Thus, the relationship between a chemical effect on fertility and the type of spermatogenic stage affected can be readily estimated. For these experiments, the serial mating technique of Jackson et al. (7) was utilized. Serial mating studies for control and experimental groups were performed concurrently. Male rats were acclimated to the animal room and breeding conditions for at least 4 weeks before they were used. They were housed in a quiet room maintained at a temperature of 72 ± 1°F with constant air circulation. Animals were treated on day 0 with cadmium chloride or borax. Treatment groups of 10 male rats were used. After treatment, each male rat was housed singly with a virgin female for a period of 7 days. During each 7-day period, female animals were examined daily for vaginal plugs to ensure that the treatment did not interfere with ejaculation and mating capability. After 7 days the female rats were removed from the males and replaced with virgin females. These breeding studies are usually terminated 70 days after chemical treatment. Nine days after the end of the breeding period, when a female could be approximately 9-16 days pregnant, the rats were sacrificed, uteri and fetuses were examined, and the number of reabsorptions and viable fetuses were recorded. The fetuses were examined grossly before fixing representative samples in neutral formalin solutions for future study. Fertility profiles were drawn from these data in which the ordinate expresses the percentage of the males determined to be fertile as indicated by pregnant females. Females that had three or more viable fetuses were considered pregnant. The abscissa indicates the days after drug treatment. The vertical lines indicate the cell type that was present at the time oftreatment. The relative duration of each main type of spermatogenic stage is as follows: spermatogonia, 14 days; spermatocytes, 21 days; spermatids, 21 days; spermatozoa, 14 days. The more mature the cell type at the time of treatment, the earlier it appears in the ejaculate as mature sperm (see Figs. 6 and 7). In addition, morphologic evidence of testicular damage was obtained by sacrificing rats on days 1 and 7 and for six succeeding 7-day intervals; the testes were removed, and after fixation in Bouin's solution and routine histological processing, examined. Velocity Sedimentation Cell Separation Studies Major spermatogenic cell types were separated by velocity sedimentation by using a modified method of Lam et al. as previously described by Lee and Dixon (1). Attempts were made to determine the uptake' of cadmium by individual spermatogenic cell types and to assess the effects of th se chemicals on the incorporation of thymidine, urimine, and L-leucine by selected spermatogenic cell types. The methodology is presented diagrammatically in Figure 1. A is the sedimentation chamber, B is the gradient maker, and C is a small intermediate vessel. The sedimentation chamber is a cylindrical cavity with a conical angle of 300 to the horizontal and made from a block of Lucite. Testicular cells are prepared by removing the tunica albuginea from the testes under a stereomicroscope and cutting the testicular tissue into short segments with an array of stainless steel razor blades spaced 0.2 mm apart. This tissue mince is washed with Spinners solution containing 0.5% bovine serum albumin. Subsequently, the cells are suspended by gently pipetting the suspension up and down. Following this, the heavy tissue segments are allowed time to settle, and the supernatant is filtered through lens paper to remove cell aggregates. Aliquots of cell suspensions containing 2 x 107 cells are loaded into the sample vessel C and 720 ml of a 1-3% linear gradient of bovine serum albumin in Spinners solution is developed through the bottom of the chamber. At the end of 4 hr of sedimentation, the chamber is drained from bottom at the rate of 10 ml min. One hundred thirty-five individual fractions are collected with a fraction collector. Each fraction corresponds to a sedimentation distance of 0.76 mm. The chamber size is such that 1 mm of vertical distance contains 7.2 ml of gradient fluid. Figure 2 presents the relative cell number in each of the fraction collected. The primary cell types indicated were identified by time sequence studies of thymidine incorporation and histologic identification. Methods for preparation of the cell suspensions, preparation of buffers and gradients, cell separation procedures, cell counting and identification, size analysis of cell suspension, and determination of radioactivity have also been described (1). Single-label isotope counting was utilized in these studies with the required consideration for purity of labeled compound, specific activity, quenching and other aspects of liquid scintillation spectrometry. Isotopically labeled chemicals were tritiated thymidine (thymidine-methyl-T, specific activity, 19.3 Ci/mmole), uridine (uridine-G-T, specific activity, 30 Ci/mmole), and L-leucine (Lleucine-4,5-T, specific activity, 22 Ci/mmole). Carrier-free l09Cd was used for cellular uptake and autoradiographic studies. Figure 3 demonstrates the cellular incorporation of radioactive thymidine 1 hr after intraperitoneal injection of the nucleoside. The fraction with greatest activity was identified as spermatogonia which sediments at a rate of 5.7 mm/hr. Figure 4 demonstrates that tritium-labeled uridine appeared in the cell fraction sedimenting at 3.0 mm/hr when cells are isolated 1 hr after injection of the substrate. This peak of activity represents early elongated spermatids. Figure 5 indicates the cell types that incorporate tritium labeled leucine 1 some spermatozoa; the 5.7 mm/hr fraction represented spermatogonia; the 8.7 mm/hr fraction contained early spermatids; the 13.1 mm/hr fractions were secondary spermatocytes, and the 15.6 mm/hr fraction was identified as diplotene and pachytene cells. The effects of environmental chemicals on testicular function are usually assessed both in vitro and in vivo. Rats were sacrificed by cervical dislocation, and the testes were removed, washed in Spinner's salt solution and placed in a Petri dish on ice. The tunica albuginea was removed under a stereomicroscope, and the bundle of seminiferous tubules was carefully teased apart for in vitro studies. Incubation studies, with the test substance, are carried out in polyethylene screw cap vials using a metabolic shaker. In vivo studies usually involve the administration of the chemical directly to the test species. The in vivo effects of cadmium and boron on the uptake of tritiated thymidine, uridine and L,leucine into spermatogonia, early elongated spermatids, and late spermatids, respectively, were determined. Results Figures 6 and 7 present fertility profiles for cadmium-and boron-treated rats. Three oral acute doses were tested. No significant effects on male fertility were apparent at any of the doses tested for either chemical. portion of cadmium was incorporated into spermatogenic cells sedimenting at the rate of 2.2 mm/hr which are the late elongated spermatids. Radioactive cadmium was also incorporated into early elongated spermatids sedimenting at the rate of 3.0 mm/hr and into spermatogonia and early spermatids. No significant effect of either cadmium or boron on the incorporation of thymidine by spermatogonia, uridine by early elongated spermatids, or L-leucine by late elongated spermatids was demonstrated at the test doses. Subchronic Studies Rats were allowed free access to drinking water containing either borax or cadmium chloride. Borax is 11.3% boron and cadmium chloride is 61% cadmium. Treatment groups were exposed to the concentrations of boron of 0. it is assumed that rats drink an average of 35 ml of water daily, the maximum dose for boron can be estimated as 840 and that for cadmium as 14 /Ag/kg-day. However, only about 1% of cadmium is absorbed. Animals were exposed for 90 days, and randomly selected groups were studied following 30, 60, and 90 days of treatment. These test doses and experimental design were selected during conferences with our Soviet co-workers. Animals were evaluated for toxicity by using various techniques. Body weights were recorded as well as the weight of the testis, prostate, and seminal vesicles. Clinical -chemistry included serum determinations for sodium, potassium, chloride, carbon dioxide, total proteins, albumin, calcium, alkaline phosphatase, total bilirubin, blood urea nitrogen (BUN), glucose and serum glutamic-oxalic transaminase (SGOT) and serum glutamicpyruvate transaminase (SGPT). Fructose levels in the seminal vesicles were also determined, as were zinc and acid phosphatase levels in the prostate. Testes were fixed in Bouins solution, washed, dehydrated, and embedded in paraffin for histological studies. FSH and LH plasma levels following 30, 60, and 90 days of cadmium or boron treatment are presented in Figure 9. Neither cadmium nor boron treatment affected FSH or LH in plasma. Although LH levels tended to increase with exposure time in both treatment groups, these effects were not statistically significant. Subchronic tests failed to reveal any reproductive effects or biologically significant change in clinical serum chemistry or weight of the body, testis, prostate, or seminal vesicles. Fructose, zinc, and acid phosphatase levels in the prostate were unaltered. These data are presented in Tables 1-7. Forced breeding studies failed to reveal any effects on male fertility following any of these treatment periods. Animals refuse to drink hormone (LH) plasma levels are presented for individual rats following 30, 60, and 90 days of treatinent with either cadmium or boron in the drinking water. FSH and LH ranges for normal and castrated rats are also presented. Normal FSH and LH plasma levels are 6(18 ± 110 ng/ml of plasma and 60 ± 10 ng/ml of plasma, respectivelv. water containing concentrations of cadmium or boron significantly greater than those tested. Our initial results utilizing a variety of methods to assess the reproductive toxicity of environmental substances in male animals suggest that cadmium and boron at the concentrations tested are without significant reproductive toxicity in the male when administered as a single oral dose or subchronically in the drinking water. Weir and Fisher (5) have demonstrated that doses of borax or boric acid as high as 350 ppm boron in the diet produced no adverse effects on reproductive function. However, these same investigators reported that doses of boron of 1170 ppm produced sterility in test animals. Hopefully, conferences during these meetings will provide time to compare the results of our joint experiments and select other environmental chemicals for future study.

v3-fos

2018-04-03T02:12:04.258Z

1976-01-01T00:00:00.000Z

20004666

s2

Effect of individual amino acid supplements on the toxicity of excess tyrosine in rats. The object of this study was to determine the effects of individual amino acid supplements on the development of tyrosine toxicity in growing rats fed 10% casein containing 5% tyrosine. Each amino acid was added at levels equivalent to its content in 20% casein. Supplement of methionine to the high tyrosine diet partially alleviated both growth depression and pathological lesions. Threonine and cystine had a somewhat beneficial effect, but the single addition of other amino acids was not effective. Besides, some amino acids enhanced the severity of the toxicity even more. The effects of methionine supplementation were highest at 0.66 to 1.32% levels (equivalent to the methionine content in 20 to 40% casein). By the supplement of both 0.66% methionine and 0.90% threonine to the high tyrosine diet, growth was significantly improved and toxic lesions were completely prevented. It was confirmed that the counteracting effects to the toxicity, caused by the extra addition of protein (casein) to rats fed a high tyrosine-low protein diet, were mainly attributed to the effectiveness of the methionine and threonine, i.e., first-and second-limiting amino acids, respectively, contained in it. Summary The object of this study was to determine the effects of individual amino acid supplements on the development of tyrosine toxicity in growing rats fed 10% casein containing 5% tyrosine. Each amino acid was added at levels equivalent to its content in 20% casein. Supplement of methionine to the high tyrosine diet partially alleviated both growth depression and pathological lesions. Threonine and cystine had a somewhat beneficial effect, but the single addition of other amino acids was not effective. Besides, some amino acids enhanced the severity of the toxicity even more. The effects of methionine supplementation were highest at 0.66 to 1.32 levels (equivalent to the methionine content in 20 to 40% casein). By the supplement of both 0.66 % methionine and 0.90% threonine to the high tyrosine diet, growth was significantly improved and toxic lesions were completely prevented. It was confirmed that the counteracting effects to the toxicity, caused by the extra addition of protein (casein) to rats fed a high tyrosine-low protein diet, were mainly attributed to the effectiveness of the methionine and threonine, i.e., first-and second-limiting amino acids, respectively, contained in it. It is well recognized (1, 2) that the growth rate and food intake of rats fed a low protein diet containing high tyrosine are depressed, and the animals develop a specific toxicity syndrome which is characterized by eye and paw lesions. These adverse effects can be overcome by an addition of dietary protein (3)(4)(5)(6)(7). In pre vious work, we have observed (7) that the tyrosine toxicity which occurs in rats fed 10% casein containing 5% tyrosine is entirely counteracted by the supple mentation of 15%, or more, of casein, wheat gluten or corn gluten. The bene ficial effect of extra addition of the proteins on tyrosine toxicity can probably be attributed to the contained amino acids. Actually, the effects of some amino acid supplements on tyrosine toxicity have been reported (8)(9)(10)(11)(12)(13). SULLIVAN et al. (8) observed that a high level of cystine alleviated the toxicity. Glycine and cystine (9), threonine (10), and glycine, methio nine, tryptophan or a mixture of branched-chain amino acids (13) were also found to diminish the toxic signs. ALAM et al. (11)(12)(13) reported that the addition of threo nine to a high tyrosine diet alleviated growth retardation and the severity of symp toms, and lowered the tyrosine level in plasma, liver and muscle. However, more detailed studies on the individual amino acids have not as yet been done. The present study was undertaken mainly, therefore, to examine the effect of supplementation of individual amino acids, which correspond to their contents in 20% casein, on tyrosine toxicity. Since methionine was most effective in all amino acids examined the influence of graded levels of the amino acid was also investigated. acid distribution in 20% casein, respectively. Experiment 2 was done to determine the effect of graded levels of L-methionine, which was simulated to the amino acid content in 10 to 60% casein, on tyrosine toxicity. Experiment 3 was done to test the effects of supplementation of extra casein, essential-, non-essential-, and complete-amino acid mixtures, L-methionine, and L-methionine plus L-threonine on tyrosine toxicity. Amino acids were simulated to their contents in 20% casein and the complete amino acid mixture consisted of all amino acids listed in Experiment 1. Experiment 1 The effects of supplementation of single amino acids on the growth and sus ceptibility of rats fed the 10% casein containing 5% tyrosine (10C5T) diet are sum marized in Table 1. Amino acids were ranked in decreasing order of the mor tality. Rats consuming the 10C5T diet showed a progressive loss of body weight, which was accompanied by the development of eye lesions and high mortality. All animals in this group lost their eye sight due to cataract formation within one week, and half the animals died within 9 to 11 days. These results were consistent with a previous report (7). The addition of phenylalanine, arginine, or lysine to the high tyrosine diet enhanced the adverse effects even more. Phenylalanine and arginine groups caused more a pronounced loss of body weight than that of the 10C5T group. Lysine supplement also caused death of all the animals in this group although the body weight was not substantially decreased. All animals of the phenylalanine, arginine and lysine groups developed severe eye lesions within one week and failed to survive the experimental period. The addition of glycine, aspartic acid, alanine or isoleucine did not improve the growth and failed to decrease the mortality of animals fed the high tyrosine diet. Proline, histidine, leucine, and valine also were not able to promote a weight gain, but the mortality in these groups was decreased, only one out of 4 rats died, respec tively. Serine, tryptophan and glutamic acid were ineffective in increasing the body weight, yet all of the animals survived. The single addition of glycine, aspartic acid, isoleucine, Proline, leucine, valine, serine and glutamic acid were ineffective in alleviating the pathological lesions, and one out of 4 rats of the alanine and histidine groups developed eye lesions. The only single amino acids that had a growth promoting effect were cystine, threonine and methionine. The highest weight gain was obtained with methionine, followed by cystine and threonine. In these groups all rats survived except one animal which died with a cystine supplement. In addition, methionine supplement delayed the onset of eye lesions, and 3 out of 6 rats of the methionine group and one Table 1. Effects of supplementation of individual amino acids on survival, eye lesions (cataracts) and growth of rats fed 10% casein containing 5% L-tyrosine. a Individual amino acids were added at levels equivalent to their contents in a 20% casein. out of 4 animals of the cystine and threonine groups did not develop the patho logical lesions. Experiment 2 The effects of graded levels of methionine supplement on the growth of rats fed the 10CST diet are presented in Fig. 1. As the supplemental level of methionine was elevated, the weight gain was greatly increased. The highest weight gains were obtained with rats fed the high tyrosine diet containing 0.66 to 1.32% methionine (equivalent to the amino acid content in a 20 to 40% casein diet), and these gains were comparable to that of animals fed the 10% casein diet, whereas with the supplement of 1.98% methionine (equivalent to the amino acid content in a 60% casein diet) growth fell to about half of the maximal gains. All of the rats supplemented 0.33 to 1.98% methionine were able to survive for the 2-week experimental period, and half the animals of the 0.33 and 0.66% methionine groups and one-fourth of the 0.99% methionine group developed eye lesions, but the 1.32 and 1.98% methionine groups did not develop either eye or paw lesions. Experiment 3 The effects of supplementation of extra protein (20% casein), essential-, non essential-, or complete-amino acid mixtures, 0.66% methionine, 0.66% methionine plus 0.90% threonine on the growth and pathological lesions of rats consuming the 10CST diet are shown in Table 2. The weight gains of the extra protein (20% Table 2. Effects of supplementation of amino acid mixtures, L-methionine, and L-methionine plus L-threonine on the toxicity of rats fed high tyrosine diet. casein) and the complete-amino acid mixture supplement groups were essentially the same as that of the 30% casein group. The supplementation of the nones sential amino acid mixture caused only a slight increase in weight gain, and eye lesions developed. The effectiveness of the essential amino acid mixture sup plement for growth was comparable to that of the 0.66% methionine supplement, and the growth of the 0.66% methionine plus 0.90% threonine supplement group was approximately 84% of that of the 30% casein group. All of the animals of these supplemental groups survived and did not develop eye lesions except the non-essential amino acid mixture group in which the eye lesions occurred in 4 out of 6 animals. DISCUSSION The beneficial effects of some amino acid supplements against toxicity in rats fed a high tyrosine-low protein diet have been demonstrated extensively. Previ ously, SULLIVAN et al. (8) had reported that the toxic symptoms in rats fed a 5 or 10% tyrosine containing diet could be counteracted to a considerable degree by the addition of 5% cystine. MARTIN (9) showed that the toxicity of tyrosine was reduced by addition of 1% cystine or 5% glycine. BENTON et al. (10) observed that a small supplement of threonine (0.3% DL-Thr) to a 9% casein diet containing 3% tyrosine stimulated growth without alleviating the severity of the pathological lesions. Subsequently, ALAM et al. (11) found that the addition of large amounts of threonine (1.25% L-Thr) to a 3% tyrosine containing diet improved growth and prevented the development of external lesions. In addition, the supplements of glycine, methionine, tryptophan or a mixture of leucine, isoleucine and valine prevented the development of eye and paw lesions, but were not associated with increased growth (13). They concluded (13) that threonine was the only amino acid which alleviated both the growth depression and the pathological lesions. In the present study, we confirmed that the supplementation of methionine, threonine or cystine, in amounts corresponding to their content in 20% casein, to the 10% casein diet containing 5% tyrosine (10CST) improved growth and sur vival, and partially prevented the appearance of external lesions, but the single addition of other amino acids did not appreciably alleviate the tyrosine toxicity, in fact, some amino acids even enhanced the severity. Methionine was the most effec tive amino acid, and both supplements of methionine and threonine significantly promoted growth while completely preventing the development of the pathological lesions. The major part of the counteracting effect caused by the extra casein supplement could be attributed to the action of the two amino acids. In the basal diet (10% casein) used in this study, methionine is the first limiting amino acid, and threonine is the second limiting one (14). Hence it is suggested that the beneficial effects of methionine or methionine plus threonine supplements to a high tyrosine diet are probably due to the improvements of the amino acid balance and the nutritional quality of the protein, thus stimulating growth which AND TYROSINE TOXICITY 403 in turn enhances the tolerance to excess tyrosine. In this connection, it has been demonstrated (6) that the extent of the adverse effects caused by ingestion of excess amino acid was usually dependent upon the protein quality of the diet. It should be noticed, however, that larger amounts of methionine (0.66 to 1.32%) and threonine (0.9%) than the requirements for growth, i.e., methionine 0.5 or 0.6% and threonine 0.5% (15,16), were needed to improve both the growth and the external lesions caused by the ingestion of high tyrosine. Similar observa tions for threonine have been made by ALAM et al. (11), who reported that large amounts of threonine (0.8 to 1.25%), 3 to 4 times the requirement, were more effective than small amounts (0.2%) in improving both growth and the pathological lesions. Therefore, it is expected that both methionine and threonine might also be related to a particular action other than the role of limiting amino acid. Fur ther study is necessary to elucidate the relationship between the limiting amino acids and amino acid toxicity.

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2019-03-20T13:08:08.482Z

1976-02-01T00:00:00.000Z

84126132

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Equivalence of the A genome of bread wheat and that of Triticum urartu Lines of Triticum aestivum Chinese Spring (2 n = 6 x = 42) which were ditelocentric or doubly ditelocentric, in turn, for the 14 chromosomes of the A and B genomes were pollinated by Triticum urartu (2 n = 14). The behaviour of the marked telocentric chromosomes was scored in the 14 distinct hybrids obtained from these pollinations. In 6 of the hybrids in which different A genome chromosomes were marked by telocentrics there were from 50 to 80% of the pollen mother cells in which the telocentrics were paired. In the seven hybrids in which different B genome chromosomes were marked the telocentrics were never paired. It was concluded that the genome of T. urartu matched very closely the A genome of hexaploid wheat and that it did not correspond, as had been proposed by Johnson, to the B genome. The pairing behaviour of the 14 T. aestivum × T. urartu hybrids was compared with earlier results obtained from hybrids between T. aestivum and T. boeoticum . It was proposed that the higher trivalent frequencies seen in the T. boeoticum hybrids could be due to homoeologous pairing and that the genotype of T. boeoticum has the capacity partly to suppress the activity of the Ph locus of chromosome 5B of wheat, as a result of which homoeologous pairing is normally prevented. INTRODUCTION Knowledge of the course of the evolution of the bread wheat of commerce (Triticum aestivum In -6x = 42) to its present cytogenetic structure is not only of intrinsic interest, it is also necessary for the determination of the kinds of genetic manipulation that may be employed in practical breeding programmes. An important part of the evolutionary advance in Triticum has been the change first from diploidy to tetraploidy and subsequently to hexaploidy. The genome structures at the three levels of polyploidy are indicated by the genome symbols: diploid (2n = 2x = 14) AA tetraploid (2n = 4x = 28) AABB hexaploid (2% = 6x = 42) AABBDD In Triticum a genome is a set of seven chromosomes either representing the haploid complement of distinct diploid species or acquired, in duplicate, at each advance in polyploidy. The origins of the genomes of hexaploid wheat have recently been discussed extensively (see Riley, 1965;Kimber, 1973Kimber, , 1974Johnson, 1975) and a review is 70 V. CHAPMAN, T. E. MILLER AND R. RILEY unnecessary in this paper. It is sufficient to point out that the evidence is strong that the A genome was contributed by a diploid form of Triticum such as T. boeoticum, and that the D genome was derived from Aegilops squarrosa. However uncertainty still surrounds the source of the B genome. Certain evidence has been adduced in favour of the notion that the B genome was contributed by Aegilops speltoides (for summary see Riley, 1965). Subsequent evidence was collected which was interpreted as implying that the B set of chromosomes could not have been derived from Ae. speltoides but no alternative source was suggested (see Kimber, 1974). However, more recently Johnson (1975) has claimed that the B genome was contributed to tetraploid and hexaploid wheat by Triticum urartu (2n = 14), a diploid form said to be widely distributed in several Middle East countries. This claim was based on the comparisons of the banding patterns obtained following the electrophoretic separation of albumin proteins extracted from seeds. Johnson pointed out that there were apparently several protein bands in extracted albumins of the two tetraploid species T. dicoccoides and T. araraticum which were also present in the albumin ofT. urartu but not in that ofT. boeoticum the presumptive A genome donor. He concluded that the distinctive proteins present in the tetraploids, but not in T. boeoticum, must be produced by the activities of genes contributed by the B genome donor and thus that T. urartu could be the source of the B genome. While electrophoretic evidence can be very effective at demonstrating the absence of a phylogenetic relationship it is less dependable at showing phylogenetic proximity unequivocally. Consequently it is necessary to test the validity of Johnson's proposal by other methods. This paper reports work showing that the chromosomes of T. urartu correspond to those of the A genome of T. aestivum and not to the B genome. PLANT MATERIALS The aim of this work was to observe the relative frequencies with which A or B genome chromosomes pair at meiosis in T. aestivum x T. urartu hybrids, using hybrids in which specific chromosomes were marked by being telocentric. The form of Triticum urartu Turn, used throughout was obtained from the N. I. Vavilov Institute for Plant Industry, Leningrad, U.S.S.R., under the Leningrad designation K33870. This form has all the distinguishing characters ascribed to T. urartu by Johnson (1975) except that it has white and not red coloured grains. Disc electrophoresis of glutenin proteins carried out by our colleague Mr J. B. Smith, revealed T. urartu to have the band pattern described by Johnson (1975). The bread wheat lines used were all in the species Triticum aestivum ssp. vulgare variety Chinese Spring. Lines of Chinese Spring were used in which, in turn, all of the chromosomes of the A and B genomes were marked by being doubly ditelocentric. These lines were produced by Dr E. R. Sears, University of Missouri, U.S.A., and were kindly supplied by him. The cytogenetic structure of these lines is that they possess 44 chromosomes of which 20 pairs are in the normal condition with median or sub-median centromeres while one chromosome pair is represented by four Equivalence of A genome of bread wheat and T. urartu 71 telocentrics. Each arm of this chromosome is represented by two telocentrics. Thus, except for the duplication of the centromere region the structurally marked chromosome was present in normal dosage. Such doubly telocentric lines were used where the structurally modified chromosome was either 2A, 3A, 4A, 5A, 1A, IB, 2B, 3B, 4JS, 5JB, 6jBor7B. The lines in which chromosomes IA and 6^4 were marked were ditelocentric for only one arm of the chromosome, the other arm being entirely deficient. For IA the telocentric for the long arm was present, for QA the telocentric for the short arm was present. Provision was made for some comparisons in the current experiments between the behaviour of marked telocentric chromosomes in T. aestivum x T. urartu hybrids and in T. aestivum x T. boeoticum hybrids. Lines of Chinese Spring doubly ditelocentric for chromosomes 2A, 3 A and 5 A were pollinated with T. boeoticumHoiss. (2n = 14). The products of these pollinations were handled in a way similar to that used with the hybrids derived from T. urartu pollinations. METHODS The Chinese Spring telocentric lines were pollinated with T. urartu. The grains that developed as a result of these pollinations were removed 16 days after the pollination. The embryos were excised from the grains and cultured on standard nutrient medium, and when the resulting juvenile hybrid plants were large enough to handle they were transferred to soil in pots and grown until maturity in a controlled environment cabinet at 20 °C with continuous light. Anthers from the 29-or 28-chromosome hybrids were fixed in 3:1 absolute alcohol:glacial acetic acid and stained by the Feulgen procedure. Permanent slides were prepared from anthers with pollen mother cells at first metaphase of meiosis from every hybrid. The slides were examined for the participation of the telocentric chromosomes in pairing associations: that is in bivalents or trivalents. So that the results could be expressed quantitatively, 30 first metaphase cells from a hybrid of each type were scored for the number of bivalents and trivalents present and for the involvement of the telocentric chromosomes (Tables 1 and 3). RESULTS Hybridization between T. aestivum and T. urartu was achieved without difficulty. Altogether 25 ears of the Chinese Spring lines were pollinated with the pollen of T. urartu. From these 216 embryos were taken into culture and 176 hybrid plants established. Hybrids were established in which, in turn, every chromosome of the A and B genomes of T. aestivum was marked by the telocentric condition. However, in the case of hybrids in which chromosomes IA and 6A were marked there was only one telocentric present. By contrast in the other 12 combinations both arms of the marked chromosome were simultaneously telocentric. Examination of first metaphase of meiosis in the hybrids showed that telocentrics were never present in a bivalent or trivalent when a chromosome in the B genome 72 V. CHAPMAN, T. E. MILLER AND R. RILEY was marked by the telocentric condition (Table 1). By contrast telocentrics which marked A genome chromosomes were very frequently in bivalents and trivalents (Plate l(i)). This is overwhelmingly strong evidence that the chromosomes of T. urartu correspond closely with the chromosomes of the A genome of T. aestivum and that they have no close affinity with the chromosomes of the B genome. Chapman & Riley (1966) had earlier carried out a test analogous to this on the relationships of the chromosomes of the form of T. boeoticum known as T. thaoudar (Reut.) Scbiem. to those of the A and B genomes of Chinese Spring. In this test however the marked chromosome was represented by a single telocentric, so that one arm was entirely deficient. These earlier results are shown in Table 2. From this it will be seen that there was an equally clear contrast in the frequency of involvement in pairing of A genome and B genome chromosomes. This implies that the pairing affinities of the chromosomes of T. urartu or T. boeoticum with the chromosomes of T. aestivum are essentially similar. This is further confirmed by the pattern of pairing at first metaphase in T. aestivum x T. boeoticum hybrids with either chromosome 2A, 3 A or 5A marked by being telocentric for both arms (Table 3, Plate 1 (ii)). Pairing in these hybrids was comparable with that in the corresponding T. aestivum x T. urartu hybrids (Table 1). There is one anomalous result among those recorded in Table 1. This relates to the T. aestivum x T. urartu hybrid in which supposedly both arms of chromosome 4.4 were present in the telocentric condition. Three hybrids of this combination were available. All had 29 chromosomes which included two telocentrics but the telocentrics were never involved either in a bivalent or a trivalent. It seems likely that this behaviour arose as a result of an error in the maintenance of stocks and that the parent used, which should have been doubly ditelocentric for chromosome 4.4, was in reality telocentric for a chromosome of either the B or the D genome. (2-6) (0-1) Genomes It is clear that T. urartu is not the source of the B genome of the polyploid wheats. It may, however, more nearly match the A genome of the polyploids than any other diploid Triticum. This would account for the possession of apparently similar species of albumin molecules in T. urartu and in T. dicoccoides and T. araraticum as displayed by Johnson (1975). Indeed on the basis of this evidence it appears that the B genome donor should be sought among diploid species with albumin proteins that migrate under Johnson's conditions to the region between 6-0 and 8-5 cm from the origin, since neither T. boeoticum nor T. urartu display bands in this area according to figure 4 of Johnson's (1975) paper but they are present in the tetraploid species. It is a considerable time since Sarkar & Stebbins (1956) and Riley, Unrau & Chapman (1958) suggested that the evidence then available could be interpreted to mean that the B genome of wheat was derived from Ae. speltoides. Subsequently evidence has been adduced in support of, Rees & Walters (1965), or against, Kimber & Athwal (1972), the conclusion that Ae. speltoides could have been involved. This issue is still unresolved but it may be noted that while occasional items of positive evidence support the notion of the participation oiAe. speltoides there is no positive evidence of an alternative B genome parent. Indeed recent evidence shows that the large subunit of fraction I protein, which is determined by chloroplast DNA and therefore inherited maternally, is similar in T. aestivum to that in Ae. speltoides but not to that in any other related diploid species thus far examined (Chen, Gray & Wildman, 1975). Homoeologous chromosome pairing induced by T. boeoticum In T. aestivum x T. urartu hybrids the only trivalents formed were those in which two telocentrics participated (Table 1). They therefore resulted from pairing between fully homologous A genome chromosomes one of which was represented by two telocentric fractions. By contrast in T. aestivum x T. boeoticum hybrids there were trivalents which did not include the telocentric chromosomes (Table 3). Moreover there were trivalents in T. aestivum x T. boeoticum hybrids in which the marked A genome chromosomes were represented by only one telocentric ( Table 2). Some of these trivalents also did not include the marked chromosome. When these trivalents were first observed, Chapman & Riley (1966) concluded that they were indicative of interchange heterozygosity. Moreover since trivalents, rather than quadrivalents, were formed it was also concluded that the interchanges must have been between A and B genome chromosomes of Chinese Spring rather than within the A genome. Separate evidence showed the D genome of Chinese Spring to be unmodified structurally (Riley & Chapman, 1960). So the trivalents were thought to involve an A genome chromosome from T. boeoticum and an A-B and a B-A chromosome from Chinese Spring. However, the interchange hypothesis is shown to be incorrect by the absence of non-telocentric containing trivalents in T. aestivum x T. urartu hybrids. Such hybrids would be heterozygous for the A-B interchanges just like the T. aestivum x T. boeoticum hybrids if the interchanges were present in Chinese Spring. In addition to the differences in trivalent formation between the T. boeoticum and the T. urartu hybrids, two other differences must also be explained. First, in the T. boeoticum hybrids telocentrics marking B genome chromosomes were occasionally observed in bivalents, but this did not occur in T. urartu hybrids. Secondly in the Chinese Spring x T. boeoticum hybrid with chromosome QA telocentric a quadrivalent was observed (Table 2). It seems possible from these differences that the genotype of T. boeoticum may be able, in part, to over-ride the normal suppression of homoeologous meiotic chromosome pairing that is caused in T. aestivum principally by the activity of the PA locus on chromosome 5B (see Riley, 1974). If the genotype of T. urartu lacked this capacity, the contrast in meiotic pairing between the two sets of hybrids would be explained. According to this hypothesis the extra trivalents in the T. boeoticum hybrids would be the result of homoeologous association. Moreover, since there were four representatives of every homoeologous group in the hybrids, occasional quadrivalents could be expected. Additionally, all chromosomes would be expected to display some homoeologous pairing, so it could be anticipated that B genome telocentrics might pair with either A or D genome chromosomes. PLATE 1 First metaphase of meiosis in 29-chromosome hybrids, (i) T. aestivum x T. urartu with 2 telocentrics marking chromosome 5A. There are 7 bivalents, one of which (arrowed) incorporates chromosome 5A, and 15 univalents; (ii) T. aestivum x T. boeoticum with 2 telocentrics marking chromosome 2A. There are 3 trivalents, one of which (arrowed) incorporates both 1A telocentrics, 3 bivalents and 14 univalents. 76 V. CHAPMAN, T. E. MILLER AND R. RILEY If this explanation is correct, genetic variation affecting homoeologous pairing has been recognized in diploid Triticum that is analogous to differences reported within some Aegilops species (Mello-Sampayo, 1971;Dover & Riley, 1972;Dvorak, 1972;Larsen & Kimber, 1973). Of course the present work does not permit the assertion that the different effects on chromosome pairing of one form of T. urartu and one of T. boeoticum will be constant for all genotypes of these taxa. There is need to broaden the evidence by the inspection of more T. aestivum x diploid Triticum hybrids. Moreover the further investigation that is now indicated will attempt to assess whether the A genome incorporated in polyploid Triticum exercises an effect on the control of meiotic pairing which is comparable with that of the T. urartu or T. boeoticum forms used in the present work.

v3-fos

2018-04-03T04:40:48.304Z

1976-09-01T00:00:00.000Z

52867057

s2

Studies on the posterior silk gland of the silkworm Bombyx mori. V. Electron microscope localization of fibroin in the posterior silk gland at the later stage of the fifth instar. Electron microscope observations of thin sections of epoxy resin- embeded posterior silk gland cells at the later stage of the fifth instar revealed that the Golgi vacuoles and the secretory granules (fibroin globules) in the cytoplasm and the glandular lumen contain fine fibrous materials. In frozen thin sections these structures appear as electron-dense granules and electron-dense blocks, or a column, respectively. Immunoelectron microscopy has shown that ferritin particles or products of the peroxidase reaction are localized on these structures. It was concluded that the fine fibrous materials most probably represent native fibroin molecules or their aggregates. The posterior silk gland cells of the silkworm Bombyx mori synthesize exclusively a single exportable protein, fibroin, in the later stage of the fifth instar (26,27). Electron microscope studies (3,26) suggested that fibroin is synthesized on the ribosomes attached to the endoplasmic reticulum membrane, accumulates in the intracisternal space, and is transported via Golgi vesicles to the Golgi vacuoles. Mature Golgi vacuoles leave the Golgi region as secretory granules of fibroin or fibroin globules (3), and these granules accumulate in the apical cytoplasm and finally are secreted into the glandular lumen. The intracisternal space of the rough endoplasmic reticulum (ER), the Golgi vacuoles, the fibroin globules, and the luminal space, where fibroin presumably accumulates, are electron-transparent except for a small amount of fibrous materials (26). This led to the suggestion either that fibroin cannot be fixed with conventional fixatives (glutaraldehyde and osmium tetroxide) and there-fore is lost during the subsequent procedures for electron microscopy, or that fibroin is preserved by fixation but cannot be visualized because of the lack of affinity of fibroin for heavy metals such as osmium, uranium~ and lead (26). We first attempted to visualize fibroin in conventional ultrathin sections of epoxy resin-embedded materials as well as in frozen thin sections of the fresh posterior silk gland. Electron microscope observations of conventional ultrathin sections revealed that under proper conditions the Golgi vacuoles and fibroin globules in the cytoplasm and the newly discharged materials and the central fibroin column in the glandular lumen are exclusively composed of fine fibrous materials, whereas in frozen thin sections the Golgi vacuoles and fibroin globules contain electron-dense granules, and the newly discharged materials and the central fibroin column appear as an electron-dense substance. In order to determine whether or not these 648 THE JOURNAL OF CELL BIOLOGY. VOLUME 70, 1976' pages 648-659 materials do contain fibroin, ferritin-or peroxidase-labeled antibodies against fibroin were applied to the frozen ultrathin sections. Ferritin particles or the products of the peroxidase reaction are localized on the newly discharged materials, on the central column in the glandular lumen, and on the fibroin globules in the cytoplasm. Silkworm The strain of silkworms used is a hybrid of Shunrei and ShOgetsu. The fifth instar larvae were cultivated as described previously (26). Fibroin, Ferritin, Peroxidase, Immunoglobulins (IgG), and Iodination of Fibroin and IgG Fibroin was prepared according to the method described previously (27). Horse spleen ferritin was prepared by Granick's method (7). Horseradish peroxidase type II was purchased from Sigma Chemical Co., St. Louis, Mo. Rabbit Fab fragment was prepared by the method of Porter (23). Rabbit antiserum to fibroin, rabbit antiserum to ferritin, and sheep antiserum to rabbit Fab were produced by four weekly injections of 5 mg, 3 mg, and 100 mg of each antigen in Freund's complete adjuvant (Difco Laboratories Inc., Detroit, Mich.), respectively. Rabbit and sheep IgG were prepared by precipitation with 33% saturation of ammonium sulfate. Antibody activity of each IgG solution was checked by the double immunodiffusion technique of Ouchterlony (20). Rabbit IgG to fibroin, sheep IgG to rabbit Fab, and rabbit IgG to ferritin each formed a single precipitation band against the corresponding antigen. Rabbit specific antibody to fibroin was isolated according to the following sequence of steps: (a) preparation of a water-insoluble fibroin immunoabsorbent by using ethanol and acetic acid as the insolubilizing agents; (b) adsorption from whole rabbit antiserum of the rabbit antibody to fibroin; (c) elution of the adsorbed antibody with 3 M NaSCN. Recovery of the antibody from 20 ml of the antiserum was -30 mg. The activity of the isolated antibody was also determined by the double immunodiffusion technique. ~3q_ or ~2'~I-labeled fibroin and IgG were prepared by the Chloramine T method (8) and fractionated by gel filtration on a Sephadex G25 column. One fibroin molecule has -250 tyrosine residues (14). The iodinated fibroin was calculated to contain ~33 atoms of iodine per molecule and was immunologically identical with the unlabeled fibroin. Preparation of Conjugates Ferritin-conjugated rabbit IgG to fibroin and sheep IgG to rabbit Fab fragment were prepared by the method of Schick and Singer (24) and fractionated by gel chromatography on Bio-Gel A, 1.5 M (Bio-Rad Laboratories, Richmond, Calif.) as described previously (18). The antibody activity of the conjugates was determined by sucrose density gradient centrifugation with radioactive iodine-labeled fibroin and rabbit IgG as the antigen (18). Peroxidase was conjugated to the rabbit Fab fragment to fibroin with sodium periodate as reported by Nakane and Kawaoi (19), and the peroxidase Fab conjugates were fractionated by gel chromatography on Sephadex G100 (Pharmacia, Uppsala, Sweden). Fluorescein isothiocyanate (Sigma) was conjugated to sheep IgG to rabbit Fab fragment by the method of Marshall et al. (15), and the conjugates were purified by gel filtration on a Sephadex G25 column. Immuno fluorescence Microscopy The posterior silk gland was fixed for 1 h with either 0.5% glutaraldehyde or 4% paraformaldehyde in 0.2 M cacodylate buffer, pH 7.3, containing 0.25 M sucrose, dehydrated with alcohol, and then embedded in paraffin. An indirect technique was applied to the paraffin sections previously de-embedded with xylene. Conventional Electron Microscopy An attempt was made to visualize fibroin filaments in thin sections of conventional epoxy resin-embedded specimens. For this purpose the glands were fixed for 1 h in the cold with various fixatives: 6.25% glutaraldehyde in 0.1 M Na cacodylate buffer, pH 7.3, followed by postfixation with 1% OsO4 in the same buffer; 2. After fixation, the tissue blocks were dehydrated with ethanol and embedded in Epon 812 in the usual manner. After ultrathin sectioning with a Porter-Blum MT-2 ultramicrotome, the specimens were usually stained with uranyl acetate and lead citrate and observed under a Hitachi HU-12 electron microscope at an accelerating voltage of 100 kV. Effect of Fixative on the Antigenicity of Fibroin In order that frozen ultrathin sections be made suitable for electron microscopy, tissue should be fixed beforehand. The effect of glutaraldehyde solution on the antigenicity of fibroin was therefore examined by the procedures of Solmon et al. (25). As the antigenicity of fibroin decreased by 18% and 37% after fixation for 1 h with 0.5% and 1.0% cold glutaraldehyde solution, re-SASAKI AND TASHIRO Localization of Fibroin in the Posterior Silk Gland 649 spectively, fixation with 0.5 % glutaraldehyde for 1 h was used for all frozen ultrathin sectioning and immunoelectron microscopy. Frozen Ultrathin Sectioning and Immunoelectron Microscopy on the Sections Posterior silk glands of the silkworm Bombyx mori in the fifth larval instar were fixed for 1 h at 4~ with 0.5% glutaraldehyde in 0.2 M Na cacodylate buffer, pH 7.4, containing 0.25 M sucrose. After washing in the same buffer, the frozen ultrathin sectioning was carried out according to the method of Christensen (6). Some of the sections were dried and observed without any counterstaining, while other sections were transferred onto a drop of 0.3 M K phosphate buffer, pH 7.3, for immunocytochemical staining. Recently, Painter et al. (21) have devised a method for intracellular localization of antigens on frozen thin sections. This method was used for localization of fibroin in the posterior silk gland cells. For direct and indirect ferritin antibody techniques, ferritin-conjugated rabbit IgG to fibroin and ferritin-conjugated sheep IgG to rabbit Fab fragments were used, respectively. In order to avoid loss of antibody activity by the coupling reaction with ferritin, a bridge method which is similar to the IgGenzyme bridge method reported by Mason et al. (16) was also used. Leduc et al. (13) have reported an immunoperoxidase method for intracellular localization of antigens on frozen thin sections. This method was used herein for localization of fibroin on frozen thin sections of the posterior silk gland. Quantitative Analysis of lgG Bound to the Sections on the Grids The grids with frozen ultrathin sections were floated on a drop of 4% bovine serum albumin (Sigma Chemical Co., St. Louis, Mo.) in 0.3 M K phosphate buffer, pH 7.3, for 15 min at -20~ After washing with the same phosphate buffer, the grids were transferred onto a drop of a mixture containing a 0.3% solution of l'~lI-labeled rabbit IgG to fibroin and a 0.3% solution of ~Z'~l-labeled normal rabbit IgG in 0.05 M K phosphate buffer, pH 7.5. After incubating for 30 min, the grids were washed well with 0.3 M K phosphate buffer, pH 7.3, and the radioactivity was counted with a two-channel well-type scintillation counter. The ratio of the amount of IgG specifically bound to the thin section to the amount of the IgG nonspecifically bound to the same section was calculated. Electron Microscopy of the Gland in Conventional Ultrathin Sections We first attempted to examine the effects of various fixatives on the appearance of the intracellular and intraluminal fibroin. It was revealed that these structures appeared as either homogeneous materials with various densities, fine fibrous materials, or coarse fibrous materials, depending on the fixative solutions used, and that under proper conditions fibroin was clearly visible. Briefly, the images of the luminal fibroin were coarser when fixed with 2.5% glutaraldehyde solution in phosphate buffer than in cacodylate or Veronal buffer. In the same buffer series, fixation in the more concentrated buffer solution or with the addition of sucrose resulted in a coarse image of the luminal fibroin. For visualization of the contents of the fibroin globules, fixation with 2.5 % glutaraldehyde in 0.1 M Na cacodylate buffer containing 0.25 M sucrose and postfixation with 1% OsO4 in the same buffer proved to be best (Fig. 1). The same procedure, however, was not effective for visualization of the luminal fibroin, since the glandular lumen appeared clear and homogeneous except for the occasional observation of fine fibrous materials -70 A in diameter (see Fig. 2 and inset), For visualization of the luminal fibroin, fixation with 2.5% glutaraldehyde in 0.2 M Na cacodylate buffer in the presence of 0.25 M sucrose gave better results, as shown in Fig. 3, and the luminal fibroin appeared as fine fibrous materials as in the previous case. The fibroin globules were similar in appearance. The contrast of the cytoplasm in general was, however, lower in this case than in the previous one. Fibroin apparently cannot be extensively extracted once fixed with 2.5% glutaraldehyde and 1% osmium tetroxide, because no change in the electron microscope images of the fibroin in the glandular lumen and in the fibroin globules was observed when the fixed specimens were kept for up to 10 days in cold 0.1 M K phosphate buffer or 0.1-0.2 M Na cacodylate buffer containing 0.25 M sucrose. Fig. 2 shows that there is a large number of moderately dense granules, -0.3 /,tm in average diameter, which occasionally accumulate in great numbers in the apical cytoplasm. It is certain that these are the fibroin globules. Similar granules also exist deep in the perinuclear and basal cytoplasm (Fig. 1). When the cells arc properly sectioned, the granules are aligned in a radial direction together with microtubules and mitochondria. Such an arrangement of the fibroin globules will be described in detail in a following paper? Fig. 1 also shows that Golgi bodies are composed of minute vesicles, vacuoles, and granules. The vacuoles are empty except for some fibrous materials, while the granules contain moderately dense materials and are very similar in appearance and size to the fibroin globules. There seem to be all the intermediate appearances (11) between the fully loaded granules and empty vacuoles. Fig. 3 shows that at the peripheral part of the lumen there are several sheets of lamina, 0.1-0.2 /~m in width, which are not always continuous and are composed of felt-like materials with moderate electron density (Fig. 3 and inset). The most peripheral lamina (LL 1 ) corresponds to the tunica intima, The sheets of lamina usually run parallel to the long axis of the gland, thus dividing the peripheral area of the glandular lumen into several thin compartments. In the central lumen of the gland, fibroin exists Sasaki, S., and Y. Tashiro. Submitted for publication. as a long homogeneous column and is usually termed '~columnar fibroin." Next come thin layers of fibroin (TLF). At the most peripheral part of the lumen, fibroin exists as large blocks, irregular in shape and adapting to the complicated contour of the glandular lumen surface. It is suggested that such blocks are formed by fusion of newly discharged fibroin (NDF). The newly discharged fibroin and the thin layers of fibroin at the peripheral part of the luminal space may correspond to the silk layer described by Akai (2, 3). Fig. 3 also shows that a series of cytoplasmic processes are arranged regularly on the cell surface which contain bundles of fine filaments. These filamentous structures are "a circular microtubule system," as described in a paper which will follow.I Fig. 6 shows that ferritin particles are exclusively localized on the newly discharged materials. Fig. 7 shows that ferritin particles are localized on the denser Golgi vacuoles (arrow) which probably correspond to the condensing vacuoles. The localization of ferritin is, however, not so specifc in this case, as the ferritin particles were also found diffusely in the cytoplasm. Only a few particles are, however, evident in the nuclear region (N). Electron Microscopy o f t he Gland in Frozen Thin Sections Location of nuclear membrane is shown by a dotted line. No, Nucleolus. Fig. 6, x 50.000. Fig. 7, • 60,000. Fig. 3, we can easily identify the newly discharged fibroin blocks and thin layers of fibroin as well as the luminal lamina. Apical cytoplasm characterized by the existence of microvilli and a number of vacuoles loaded with electron-dense content can be seen adjacent to the newly discharged fibroin blocks. It is likely that the electron-dense vacuolar content corresponds to the fibrous materials found in the fibroin globules. Comparing this material with the fibroin in the glandular lumen, we could safely assume that globule and luminal contents are concentrated to the same extent, no heavy metal staining being applied to the frozen thin section. Immunofluorescence Microscopy The previous sections have been written assuming that both the Golgi vacuoles and fibroin globules in the cytoplasm, and the newly discharged fibroin blocks, the thin layers of fibroin, and the central columnar fibroin in the lumen are composed of fibroin. To prove this assumption, immunofluorescence microscopy was carried out. Fig. 5 is a fluorescence micrograph of the posterior silk gland, which shows that the columnar fibroin and the silk layer are fluorescent, indicat-ing that they do contain fibroin. The cytoplasm of the cells, particularly the apical cytoplasm, was also fluorescent, suggesting accumulation of fibroin in the apical cytoplasm. There was a large gap between the central fibroin column and the peripheral silk layer which probably occurred artificially during the sampling procedures for microscopy. Immuno ferritin Electron Microscopy o f the Posterior Silk Gland Cells For the same purpose, we applied immunoferritin electron microscopy to the frozen thin sections of the posterior silk gland cells. As a preliminary experiment, the grids mounted with the frozen thin sections were incubated with an equimolar mixture of ~3q-labeled rabbit IgG to fibroin and ~z'~I-labeled rabbit normal IgG. It was found that 6.8 times more ~alI-labeled IgG was bound to the grid than the control 12"I-labeled IgG, and thus it seems certain that a specific immunoreaction is possible between the antigen molecules (fibroin) on the thin sections and antibody molecules in the incubation mixture. In the real experiment, the specific antibody for fibroin was SASAKI AND TASHIRO Localization of Fibro& in the Posterior Silk Gland used instead of IgG. It is expected, therefore, that the specificity of the antigen-antibody reaction on the grids is better than in the preliminary experiment. Fig. 6 shows newly discharged fibroin blocks in the luminal space, on which a number of ferritin particles are seen exclusively localized. Fig. 7 shows the perinuclear region of the cells where the nuclear envelope runs from the upper left to the lower right corner. The ferritin particles are predominant in the cytoplasm and much less numerous in the nucleus. In the cytoplasm, the ferritin particles were found to be numerous on the Golgi vacuoles with moderate electron density (arrow). They were also found rather uniformly on the electron-dense part of the cytoplasm, and there was no selective staining of the cisternal space of the rough ER. Control frozen thin sections which were incubated with normal rabbit IgG instead of rabbit antibody to fibroin showed that the cytoplasm is, in marked contrast to the luminal content, extensively extracted during incubation for immunoelectron microscopy (not shown). This fact explains why ferritin particles are found exclusively localized on the newly discharged fibroin blocks but not in the intracisternal space of the rough ER in the cytoplasm. Preservation of the cytoplasm during the incubation procedure would be much improved if a more concentrated glutaraldehyde solution was used. Antigenicity of fibroin is, however, rapidly lost by such treatment as described previously (see Materials and Methods). Therefore, the immunoperoxidase method which requires a shorter incubation and less extensive washing was used. Immunoperoxidase Electron Microscopy When the peroxidase-labeled antibodies were applied to tissue blocks, only the surface of the newly discharged fibroin blocks and columnar fibroin and very apical cytoplasm were stained, presumably because the cells are too large to be stained by the conventional diffusion methods. We, therefore, used frozen thin sections for the peroxidase-labeled antibody methods. Fig. 8 shows that the. cytoplasm is preserved quite well after incubation for 30 rain at 20~ Comparing this picture with a control (Fig. 9), it is evident that the contents of the fibroin globules are positively stained (arrows). The intracisternal space of the rough ER, however, showed no deposition of the peroxidase reaction product. DISCUSSION In the present work it was revealed that homogeneous materials with slight electron density, or fine fibrous materials appear in the glandular lumen, in fibroin globules, and in some of the Golgi vacuoles if the silk gland is properly fixed with 2.5% glutaraldehyde in slightly hypertonic salt and sucrose solution. It is suggested that fibroin molecules exist in these spaces probably in a molecularly dispersed state and are not visible if they are fixed in this state. They can be observed, however, as fine or coarse fibrous materials if they aggregate during the sampling procedures for electron microscopy. Further, the existence of buffer salts such as Na cacodylate or K phosphate seems to intensify the staining with the heavy metals. This interpretation provides an explanation for the inconsistent and diverse images of the glandular and intracellular fibroin reported by various authors (3,4,5,10,26). As pointed out by Christensen (6), it is indeed rather surprising that there is sufficient contrast in the frozen sections of unstained material for the structure to be perceived, and the morphology of the posterior silk gland cells that can be seen in these frozen sections was generally similar to that seen in conventional sections, except that the content of the fibroin globules and the fibroin in the glandular lumen were electron dense (Fig. 4). Since the specimens were neither embedded in plastic nor stained with heavy metals, the electron density of the micrographs should reflect the intrinsic density of the specimens. It has been reported that the concentration of fibroin in the luminal space is -30% (wt/vol) (14). Therefore, it is quite natural that the luminal fibroin is observed as an electron-dense mass in the frozen ultrathin sections. The fibroin globules contained similar electron-dense granules in the frozen thin sections; thus, it would be assumed that the concentration of fibroin in the fibroin globules is also high. On the contrary, the intracisternal space of the rough ER was always electrontransparent, and so fibroin was probably concentrated mainly in the Golgi vacuoles, as in the case of the condensing vacuoles in exocrine pancreatic cells (1 l, 22). This is supported by the existence of the Golgi vacuoles with intermediate density in conventional ultrathin sections (Fig, l). These vacuoles are probably condensing vacuoles in which fibroin is concentrated for intracellular transport. The present work demonstrated that it is possi-656 THE JOURNAL OF CELL BIOLOGY" VOLUME 70, 1976

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Integrated pest management in the U.S.: progress and promise. In the U.S., where heavy use of insecticides has been commonplace for years, the development of proper integrated insect pest control cannot get underway unless there is a changed use pattern for such chemicals. A changed use pattern, however, cannot be accomplished without much study to establish the requirements for integrated control for each major crop situation. In this paper recent developments in a number of crop areas in the U.S. in which the necessary study has been begun are reviewed. Important phases in the development of integrated control programs include: the single tactics phase, the multitactic phase, phase, the biological monitoring phase, the modeling phase, the management and optimization phase, and the implementation phase. Several crops are discussed in relation to how far along we are in the development of practical programs of insect pest control. These are cotton, apples, alfalfa, soybeans, citrus, corn, cereal grains, tobacco and pine forests. Several of these programs have already made substantial headway, e.g., those for cotton, alfalfa, apples, tobacco, and soybeans, although the accomplishments have not been even or parellel with respect to the phases of development where progress has been good. The review of developments in these crops suggests that programs of control for individual crops and perhaps for complexes of associated crops will be developed according to specific needs of the crop, the geographic area and the pests, the technologies available and the socioeconomic and political factors of relevance. The tendency will be toward greater use of science in pest control decision-making, with extensive use of biological monitoring to establish realistic levels of threatened damage to the crop, and greater concern given to possible profit reductions and environmental disturbances of applying an insecticide, as well as the possible gain from doing so. Introduction In the last decade there has been much reappraisal of where we stand in pest control, particularly in insect pest control. The problem in this country is rather different from that of developing countries. Here, there already has been an extensive use of broad-spectrum organosynthetic insecticides which certainly gave striking results for many pests. However, detrimental side effects soon developed, including resistant types of pests and *This publication was supported in part by a NSF-EPA grant GB-34718 to the University of California. The resurgence of target species, destruction of natural enemies and release of previously innocuous ones to pest status, residue and public health problems, and other more general environmental effects. These problems have been elaborated many times (1-3) and will not be dealt with here. In the developing countries, pests continue to take a significant part of the harvest, e.g., in South America, 33%; in Africa, 42%; in Asia, 43% (4), and not much is done to prevent it. Insecticides are not used extensively. Consequently pest control measures in these countries can be integrated to begin with; whereas in the U.S. we can only accomplish this by reducing and modifying the existing intensive and automatic use of insecticides so that alternative measures can have their potential effects. Here we must rethink and research our whole program of pest control in order to determine if a multifactorial approach (i.e., use of a combination of chemical, cultural, and biological strategies or tactics) April 1976 can be used to reduce the heavy costs and detrimental effects of unilateral reliance on chemicals. In the past decade, considerable effort has been expended in this country to do this. For example, in North Carolina, a significantly changed approach to control of insects and diseases of tobacco has been developed; the same is true in Washington for spider mites on apples. The concept of integrated control is not really new; it had an early proponent in Woodworth in the early 1900's (5), and later advocates were Pickett (6) in Nova Scotia and Michelbacher (7) in California. In the past five years, an additional dimension has been added to the way we approach pest control problems. We are not yet sure what its ultimate impact will be, but it appears that the technology being developed will result in a greater integration of pest control with other aspects of agricultural management as the focus is properly placed on crop production. We are referring especially to the introduction of systems analysis and the computer science technology which is developing in relation to pest control research and making recommendations to pest managers. In developing this new technology, several efforts were simultaneously initiated. One has been concerned with cereal leaf beetles in Michigan (8); another with pests of cotton in Mississippi, Arizona, and Texas (9,10). Still another was the broader effort embracing not only cotton but five other crops, including alfalfa, stone and pome fruits, soybeans, citrus, and pine forests (bark beetles), initiated by the U.S./International Biological Program (IBP), funded by the National Science Foundation (NSF), the Environmental Protection Agency (EPA), and 18 participating universities, with some support by the USDA. The goals of this program have been expressed in different ways, but in general they are: to develop ecologically based and structured systems of management of pest populations at noneconomic densities so as to optimize economic returns on a continuing basis consistent with minimal environmental damage and to demonstrate that agricultural research can be done in a more productive way than in the past through unified, interdisciplinary approaches utilizing systems analysis. These projects noted above and other related ones have provided a major thrust to the use of modeling as an important tool in structuring pest management research. The first goal expressed above for the US/IBP project would generally apply to the nationwide efforts underway to develop integrated pest management. In the remaining portions of this document the success achieved in attaining these goals and its probable impact in the future are discussed. Progress in Integrated Pest Management Entomologists have learned that an excellent way to estimate future trends in insect populations is to study the historical records of their population dynamics in the past. Perhaps also the future promise of integrated pest control in the U.S. can be estimated from reviewing its recent advances. In this paper, developments primarily in insect control for 9 crops are dealt with. Emphasis is placed on crops carrying heavy pesticide loads or those of great importance to food or fibre production. Each is considered in terms of three evaluation criteria. First, has the program resulted in increased economic return to the grower (and/or to the consumer) as contrasted to non-integrated control programs Second, has the program resulted in a reduction in the adverse influences of the pesticide program to the environment or public health and to what extent, contrasted to nonintegrated programs And last, has the program resulted in scientific and methodological advances by which its success can be measured. In addition, several phases of development of an integrated control program, beyond those given by Smith (11), may guide us in judging advancement of a given program. These are: a single-tactic phase, a multitactic phase, the biological monitoring phase, the modeling phase, the management land optimization phase, and the systems implementation phase. The single-tactics phase is somewhat of a "straw man" which is often associated with strictly calendar date spray programs, i.e., with nonintegrated pest control measures; it is not an actual phase in the development of an integrated control program. The multitactics phase embraces the search for new tactics or strategies for controlling or manipulating insect populations, including cultural, mechanical, physical, biological, and regulatory measures (1,2,12). These tactics have received a great deal of attention following the recent emphasis of the integrated control concept. In recent years, considerable attention has focused on developing more refined sampling and/or biological monitoring methods which allow pest control advisers and managers to determine more precisely the changes in the state of an insect, natural enemy or crop plant population, in relation to the need for applying a control measure and/or the best time to do so (i.e., economic damage levels, use of pheromone traps, and/or parasite-host or Environmental Health Perspectives predator-prey ratios). Progress and interest in this phase is exemplified by the efforts of the Federal Extension Branch of the USDA to establish pest management scouting and advising systems for a variety of agricultural crops. The modeling phase, as broadly defined, includes the conceptualization of a process by mental, pictorial, flowchart or mathematical means and has as its objective to understand, manage, or predict some feature or totality of the process in question. For prediction, there is of course a tradeoff between monitoring the development of an insect, natural enemy, or crop population and forecasting its expected development based on simulation or mathematical models. The more one can predict, the less he needs to monitor and vice versa. Models, through sensitivity testing and validation, can also play an important role in elucidating control elements and critical parameters in a pest management system to which research can be directed. As many individual components of a pest management system or beyond this, of a crop production system, are developed as subunits or submodels, they must be integrated or coupled into a total crop management system. This is the management and optimization phase. The difficulties of integrating such a complex of variables so that each can be managed in a way to give optimal benefits are apparent. For many years this type of integration has been carried out by intuitive experience gained from trial-and-error experimentation, but more recently, mathematically-based pest management models for entire complexes and techniques for economic analysis and optimization are also being used on a more quantitative basis. In theory, the systems implementation phase is the culmination effort which concentrates or simplifies the best system of control methodologies and integrates monitoring, modeling and management tools into a system of delivery to the pest manager. Although effective systems can be based on traditional methods (i.e., off-line mode, without mathematical models) use of real-time weather acquisition systems which interface with biological monitoring systems (8,13,14) provide for rapid delivery of decision-making information to the pest manager, including feed-back data which provide up-dates on the state of the entire crop-pest system. Beyond the initial development, research in this phase is undertaken only when refinement or further improvements are needed in response to pest adaptations or changing crop technology. As indicated in the following discussion, for no crop have we reached this level of sophistication. Cotton Cotton is grown in the U.S. from California to the Carolinas and from the Gulf of Mexico northward as far as Illinois. Losses to insects exceed $500 million annually and $150 million is spent on insecticides, which is about 45% of the total used in agriculture. About 50% of the cotton acreage is not treated in most years. Thus the potential benefits to be derived from advanced pest management on this crop are substantial. In general, three major cotton agroecosystems exist, and the insect pests for each case be characterized as follows (15): (1) the irrigated deserts of the Far West where the major pests are the pink bollworm, lygus, bollworm, and spider mites; (2) the semiarid regions of the Southwest where the bollweevil, fleahopper, bollworm, and tobacco budworm are the major pests; (3) the humid regions of the mid-South and Southeastern U.S., where the bollweevil, plant bug, bollworm, and tobacco budworm are the major pests. In each of these cotton areas there is a major pest which must be controlled; there are also ones which appear to be largely pesticide-induced, and these can be avoided by properly chosen insecticide regimes or by use of other alternative methods. In the 1950's and 60's, and even today, cotton insect control on substantial acreages has been and still is almost exclusively a single-tactic situation, i.e., a systematic application of insecticide every 5-7 days, according to Casey, Lacewell, and Sterling (16). These control programs were and are plagued with problems, including the development of resistance to the point of ineffectiveness for some species, environmental problems, and high costs for treatments in the face of a darkening economic picture for cotton production. This situation, however, is rapidly changing and was changing even before the establishment of the NSF/IPM project or the USDA Extension Service Action programs which are also helping to alter this situation. Practical entomologists, extension people, consultants and supervised control specialists or "applied insect ecologists" had earlier developed practical programs, the latter by selling advice to growers. They made practical evaluations of the pest-crop system, pest occurrence, natural enemy presence, and probable impact on yield before advising use of chemicals; systems science in its limited technical usage had no part, but at the broader conceptual level it was involved, especially at the level of integration. Recently there has been interest in developing new tactics for cotton insect control. Most of these April 1976 (17). bR = resistant; N = no effect; S = increased susceptibility. fall within the strategy of integrated control or pest containment. However, programs for eradication by initial, intensive use of insecticides, followed by massive release of sterile insects or use of other genetic eradication tactics, fall in the strategy of eradication and are outside the scope of this paper. Work to develop varieties of cotton resistant to or tolerant of insect pests has been going on for some years; the NSF/IPM project has assisted to speed up and better coordinate some of this activity (Table 1). Different degrees of resistance to various cotton insects is afforded by lines possessing such characters as frego bract, glabrous, nectariless, pilose, high square gossypol, okra-leaf, and X-factor, and these traits are being incorporated into productive agronomic backgrounds. At least one nectariless variety has been released commercially (18). The possible benefits from combined use of resistance, built into the new, smaller, short-season cottons, combined with crop residue destruction, are considerable. Various insect pests (and diseases) may thus be better managed or avoided and, moreover, costs for pesticides, labor, and fossil Environmental Health Perspectives fuels would be much reduced (19,20). Nevertheless, the work has shown that certain problems will be difficult to overcome by breeding alone. For example, frego bract types are more resistant for weevil but more susceptible for plant bugs; glabrous types are more resistant for Heliothis but more susceptible for leafhoppers (21,22). Natural enemies are important in cotton ecosystems, and the role of predators and parasites is being evaluated, as is the utility of microbial insecticides. There have been experimental efforts to suppress bollworms by mass release of Trichogramma and Chrysopa (23) and to increase the complement of natural enemies by introductions from Latin America (none yet successful). Research has been done on both descriptive and predictive modeling for cotton. This started with models for the growth and development of a single cotton plant (24,25); they are now developed for a population of field plants and have been verified for three seasons in California (26). Workers in Mississippi, Texas, Arkansas, Arizona (15) and several other states (9, Colwick and H. D. Bowen, in press) have also reached high levels of attainment in plant and pest modeling. Submodels have been developed in the IPM project or are being developed for the following: leaf photosynthetic production (Texas) (27), drying of abscissed cotton squares (relative to mortality of boll weevils) (Texas), light penetration of row crops, such as cotton (Texas), drift of an insect pheromone (Texas), modified SIMCOT plant growth models (California, Texas, Mississippi), insect movement (North Carolina, Texas), bollworm ecosystem(s) (including one for natural enemies) for evaluation of alternative control tactics (Mississippi, Arkansas), natural enemies of bollworm (Arkansas), fleahopper dynamics (Texas), boll weevil (physiology and population dynamics) (Texas), pink bollworm and lygus bug dynamics (California), single-treatment, two-variable model for optimizing pesticide treatment (California) (28), addition of a nonlinear kill efficiency to the Hall and Norgaard model (Texas), economic thresholds and interfacing of plant growth and insect models (Mississippi) (29), an overview model of a cotton pest management system (Texas) (30). In addition, various models have been developed or are being developed to deal with all aspects of cotton production from planting to harvest. For cotton pest management, however, all the submodels above have not been interfaced into a management decision model and, indeed, all of them probably will never be used in such a system. It will be necessary in developing practical (implementable) guidelines to simplify submodel ele-ments to essential components, but this can best be judged after the necessary insight has been gained by simulation and validation studies. With respect to implementation, use of a new integrated control strategy by the Texas Department of Corrections in cooperation with Texas A & M University, as a result of increasing pesticide resistance of the tobacco budworm, further substantiates that economic and environmental benefits may be gained by IPM approaches if adopted for cotton. The new program was: bollweevil control with a fall diapause program, fleahopper control with low dosages of insecticides applied early in the season, termination of fleahopper treatments quickly to allow natural enemies to build up and corntrol bollworm and tobacco budworm populations, careful sampling, to initiate control techniques only after pest populations are determined to exceed economic thresholds, and harvesting of the crop and destruction of residuals as early as possible. Following use of this program in the Brazo River area, insecticide use declined from 12 to 6.4 lb/acre while lint yield increased from 229 to 345 lb, of which 50% was attributed to improved pest control. In the Trinity River region, insecticide use was reduced from 10.8 to 5.6 lb/acre and yields were increased by 80 lb/acre. Extrapolation of these effects to the 215,000 acres of cotton in this area suggests that annual insecticide use could be reduced by 1.4 million lb and cost benefits alone would increase by $5.4 million (16). Impressive statistics for the diapause bollweevil suppression program in the Texas High Plains (31) and the Mississippi scouting programs have also been attained, including a 50% reduction in insecticide usage. In many areas, the introduction of real biological monitoring (as opposed to quasisampling provided by insecticide salesmen) in relation to evaluating economic thresholds and crop compensation capacities have provided for significant benefits (e.g., the state-supported "scouting" programs developed in Arkansas and the private advice of applied insect ecologists in California). These above programs have been improved by the IPM modeling efforts and when coupled with the biological monitoring programs described, they begin to approach the systems implementation stage. Apples The value of stone and pome fruits grown in the U.S. is well over $700 million annually. Losses from insects and mites average about 25% of this 171 April 1976 total and costs for pesticides are about 12% Among the pome and stone fruits, apples receive the greatest amounts of pesticides; they are only behind cotton and corn in total use and on a per acre basis they rate even higher. Concerning methodological advances associated with apple pest control, development of alternative tactics for control of the direct fruit pests of this relatively stable crop are limited because cosmetic appeal is a significant feature and little damage is tolerated. By and large, insecticides are the principal measure used for direct pests (those attacking the fruits) of apple, although substantial reductions in pesticide usage have been achieved by improved pest monitoring techniques, and therefore better timing of applications and more precise appraisal of the need to spray. This has been greatly facilitated by the identification of sex pheromones and/or their synthetic mimics which have been made commercially available for such species as the codling moth, oriental fruit moth, red-banded leafroller, tufted apple budmoth and others. Pheromones of several apple pests (e.g., codling moth, red-banded leafroller) are also being experimentally evaluated as direct control measures in mass trapping and pheromone confusion studies. Considerable success has been achieved in reducing the pesticide load and costs for control of several indirect pests which feed on the foliage or woody tree parts, including mites, scales and aphids. In Washington, integrated programs have achieved an approximate 50% reduction in the use of chemical pesticides (32) and in the midwestern and eastern U.S. where the pest complex is more varied a 20-30% reduction has been realized (33). The greatest success has involved the integration of biological and selective chemical control of plantfeeding mites (e.g., European red mite and McDaniel spider mite). Use of the coccinellid beetle, Stethorus punctum, and two phytoseiid mites, Amblyseius fallacis and Typhlodromus occidentalis, has been the basis of these programs. Their successful exploitation is largely due to the fact that each predator is either tolerant or has acquired high levels of resistance to organophosphate insecticides which are commonly applied for fruit pest control (34). With respect to modeling, three prototype efforts are being conducted and coordinated throughout the major fruit producing states through the NSF/IPM program (35). A system of forecasting codling moth phenology based on pheromone trap monitoring and use of physiological-time modeling has been developed and validated, and is currently delivered to apple growers as a component of an on-line Extension Service delivery system in Michigan (36). Another direct fruit pest which is similarly being modeled is the tufted apple budmoth. Also, additional efforts to develop models for timing spraying for the codling moth (37) and predicting its population dynamics following sterile male release programs (38) in the western U.S. are almost completed. A second important prototype modeling effort (for a disease) is in progress in New York, Michigan, and Pennsylvania for the key apple disease, apple scab. Submodels for this fungus which have been completed or are in progress include: a forecasting system for primary scab infection, an ascospore maturity submodel, a secondary lesion submodel, fungicide spray components, and lastly a total simulation for apple scab development-VISIM (39). The third prototype modeling effort, in this case for a secondary arthropod pest of apple, is centered on plant-feeding mites and incorporates their key predators. Three institutions, Pennsylvania State University, Washington State University, and Michigan State University, are cooperatively involved. In Pennsylvania, a simulation model for the interaction involving the European red mite, its predator S. punctum and the acaricide-fungicide Dikar, has been developed, validated and is currently being used in a simplified form by fruit growers (40). In Washington, model development emphasizes the spider mite, T. mcdanieli and the phytoseiid predator T. occidentalis. In Michigan, European red mite and the predator A. fallacis are the prinicpal prey and predator species. In each of these studies, coupling of the respective pest and natural enemy submodels has been accomplished and model simulations provide outputs which predict whether biological control will be successful or if selective acaricides should be applied to establish more favorable predator-prey ratios. As noted previously, employment of models in actual pest management programs for apples has been developed in both "off-line" and "on-line" modes. In Washington, Michigan, New York, and Pennsylvania, states that grow more than 70% of the U.S. apple crop, Action programs sponsored by growers and receiving Federal support are providing for rapid establishment of integrated control programs. In Michigan, models for control of codling moth, apple scab, and plant-feeding mites are being used presently as the focus or essential core of a pest management system for the whole complex of apple pests. Output recommendations from these models are delivered daily to Extension personnel via telecommunications lines to teletype terminals. This prototype pest management delivery system for apple pests utilizes on-line weather from 26 Environmental Health Perspectives 172 NOAA sites located throughout the Michigan fruit belt (14). It is part of a larger network embracing pest management for many crops (see discussion of cereal crops). Alfalfa Alfalfa in the U.S. is a major crop in its own right and is used primarily as a feed for domestic livestock. It is not a crop which receives large amounts of pesticide, but it does support a wide variety of insects including destructive species, pollinators and particularly beneficial natural enemies which overwinter or build up in alfalfa before migrating into neighboring crops. The point is that insects initially associated with alfalfa often affect associated crops (e.g., cotton, soybeans), and gives alfalfa pest management particular significance beyond that commonly ascribed to a single crop system. Development of integrated pest control systems for alfalfa is among the most advanced in the U.S. This progress is due in part because this crop is grown in virtually every state and it has a single principal insect pest, the alfalfa weevil (or Egyptian alfalfa weevil) which is similarly distributed; it is also because the philosophy of integrated control has long been accepted by alfalfa entomologists (41) and extensive use of chemicals has simply been prohibitive from the point of view of cost. Researchers readily pooled their talents to develop integrated pest management programs and modeling expertise was incorporated at a very early stage (35,42). To date, the effective tactics for control of the alfalfa weevil have included cultural, chemical, biological, and host plant resistance means. Integration of chemical, biological, and cultural methods for alfalfa weevil control is greatly dependent upon the biology and ecology of the respective weevil, its parasites, and the phenology of the plant's development. These features differ seasonally and from place to place. The tactics used range from spraying or cutting the alfalfa during early summer and thereby destroying the larvae or by doing the same in late fall so as to reduce the number of overwintering eggs laid in the southern states, to precisely timing the cutting of the first crop in the spring so that many of the weevil larvae and eggs are killed, and applying a stubble spray if needed after first harvest to clean up the remaining larvae in northern states (42). Inclusion of host plant resistance in integrated control programs for alfalfa insects, including the alfalfa weevil and especially for the spotted alfalfa aphid, has been substantial, but as yet multiple pest resistance has not been achieved to the extent that resistant varieties adapted to a wide range of cultural and climatic conditions are available. In recent years, biological control of the alfalfa weevil in most of the U.S. has been greatly increased by the establishment of several natural enemies, the most important being the larval endoparasite, Bathypletes curculionis, and the braconid, Microctonus aethiops, which attacks the adult weevil. In many areas, these natural enemies provide for significant control of the weevil, but in California the practical situation has drastically changed with the appearance and spread of the Egyption alfalfa weevil which is ineffectively parasitized by the former species. Since rather refined manipulations of control measures (e.g., insecticides or cutting) in relation to pest, parasite, and predator numbers, damage potential, and crop development are critical to achieving optimal pest control,the application of system science technology was a natural research development that was begun about 4 years ago. Models for the growth dynamics of alfalfa under California (Gutierrez et al. in press) and midwestern-eastern nonirrigated conditions (43, Ruesink, in press) and for the population dynamics of the alfalfa weevil (44, Ruesink, in press), its parasitoid, B. curculionis (Ruesink, in press) and the Egyptian alfalfa weevil (Gutierrez et al., in press) have been developed and tentatively validated in the field. Coupling of plant, insect, parasite and economic models is at an early stage of development, but results from these efforts have been very promising (44,45,Gutierrez et al., in press). Development of optimization techniques for decision-making, embracing cost/benefit analysis is also the most advanced of any of the NSF/IPM projects. Such methods as dynamic programming and projectory decomposition have been used to select optimal single-season tactics for a single grower, such as cutting or spraying the crop (45)(46)(47)(48). In addition, efforts to develop solutions optimal for a group of growers located within a range of common effect have been investigated (48). To date, implementation of control programs using management models for alfalfa production and pest control has been carried out in two ways. A system of on-line or real-time alfalfa pest management based on survey data taken on weevils and/or crop development in early season and almost current weather from 21 Agricultural Meterological stations in Indiana and 10 first order National Weather Service stations has been developed at Purdue University (13). Outputs from this system include plant growth and pest development simulation summaries which provide extension personnel with decision-making information. In an off-line mode, general control recommendations which were model generated and based on past and projected environmental conditions have been used in simplified form by Illinois growers in making pest management decisions (Ruesink, in press). Soybeans Soybeans are grown mainly in the midwest and southern U.S. The global importance of this crop is widely recognized. The worldwide shortages of proteins and oils for human consumption became acute in 1974; in the U.S., soybean exports became a major means by which this country was able to restore a more favorable balance of world trade. In several ways an appraisal of where we stand in management of soybean pests is confronted with difficulty. Unlike cotton, relatively little insecticide is used, but as the increased acreage in the last decades has come mainly in the South where a complex of pest insects is capable of causing significant damage, there has been an intensive campaign by industry to develop extensive insecticide treatment programs for soybean. If this campaign were successful, the situation could follow the same disastrous path which developed for cotton. The research program underway is thus designed to prevent this. Soybean is unlike cotton also in that there is no single key pest in any region but a complex of threatening pests, and the backlog of biological and ecological knowledge concerning these pests is much less understood. There is evidence that the existence of a complex of predators, parasites, and diseases that attack soybean insects in the South where they are the most threatening is the main reason why no one species has become a key pest. There is also evidence that strains of some pest species are becoming better adapted to feed on soybean, and this applies even in the Midwest where insects have caused little trouble in the past. Consequently, there is at times a real need to use insecticides, and any usage may increase the need for further usage, for it will tend to lessen the effectiveness of natural enemies. Little is known about the effects of soybean insects or diseases on soybean yields or the capacity of the plant to compensate for damage at different stages of the plant's development. Moreover, since most soybean pests are 174 polyphagous feeders on other crops and noncrop vegetation, the relative densities, patterns of distribution, and phenology of these plants affect both pest dynamics (sources, distribution, densities, movement, phenology) and natural enemy effectiveness. Consequently, this complexity makes doubly necessary a systems approach to the problem. This is especially so as the economic threshold for damage by a given species, or a complex of species, will vary during the season with the growth of the plant and with economic developments. Pest management for soybean has been confronted with both the burden and blessing of being able to mount a coordinated multiple-tactics approach from almost point zero, and in this sense it is much like various crops in developing countries. Only a few years ago soybean insect control in the U.S. essentially was at the single-tactic phase, i.e., chemicals alone were advised if a problem arose, although, depending upon the particular adviser, possible effects on natural enemies were given some consideration. At present, commercial implementation has reached at least the multitactic phase wherein many tactics are used in some regions or states, incorporating also significant biological monitoring, certainly in those areas where "action" or "scouting" programs are used to ascertain need for pesticides. Trends indicate that, even in preliminary programs not significantly utilizing modeling, insecticide usage can be held to half what it would otherwise be, with corresponding increases in profits and improved environmental quality. Costs of scouting can also be reduced by half by using information from recent studies on the phenologies of the pests and the soybean plant and the lack of damage potential except at critical times. Basic research on several tactics necessary in a systems approach is being conducted. In Illinois a mathematical expression of economic injury levels and data correlating yield decrease with defoliation have confirmed that soybean can (at times) tolerate substantial foliage injury without adverse effects on yield (Ruesink and Carlson, in press). As noted above, resident natural enemies appear to be very important in economical soybean production. A surprising finding is that predators and diseases of soybean insects are much more important than insect parasites. Ways are being studied to make them more useful; a special problem exists with the diseases, as epizootics are triggered only by high humidity and/or heavy dews. Efforts to introduce new natural enemies are being made, especially for the Southern green stinkbug. Varieties exhibiting multiple resistance to several insect pests and diseases are being sought. Starting with lines initially Environmental Health Perspectives resistant to the Mexican bean beetle, resistance to a number of defoliating insects has been added, e.g., for bean leaf beetle, soybean looper, velvetbean caterpillar and Heliothis spp., and lines should soon be commercially available (S. Turnipseed, W. Campbell, and M. Kogan, personal communication). As information on these tactics is gained, modeling and management and optimization are being explored simultaneously. A broad cooperative effort supported by CSRS-USDA to develop a plant growth model for soybean has been launched. While this is being fully developed scientists at Louisiana State University have formulated a simple model based on records of the progress of soybean development at different dates from planting time. Coupling of insect damage with this rather gross plant model predicts yield to ± 10% . A model framework for corn earworm, velvetbean caterpillar, sou-thern green stinkbug and soybean looper has been developed and it adequately reproduces their development through the season. Lastly, a prototype management model is being developed. It is too soon to appraise, generally, either the economic or environmental benefits developing from such new programs. One of the most striking benefits is in scientific methodology-the multidisciplinary cooperation that it has engendered. Moreover, as noted above, costs of scouting where scouts are used can be much reduced. Where an insecticide must be used, the amount used has in many areas been reduced by half. It is more difficult to assess just how many acres have not been treated that would have been, except for these new developments. It does seem hopeful that soybean pest control can be prevented from going the way of cotton, whether or not sophisticated modeling has a key role. Corn Corn is second to cotton with respect to the total use of pesticides and is one of the most valuable crops (exceeding $12 billion annually) grown in the U.S. Development of integrated control measures for grain corn in the Midwest corn belt of the U.S. has been difficult due to the wide variations in climate and inadequacy of natural control factors in regulating pests of this crop which frequently and sporadically achieve outbreak status. In addition to insecticides, integrated control programs have relied heavily on such measures as host plant resistance, rotation and other cultural measures and intensive monitoring or sampling of pests. Use of resistant varieties for first generation European corn borers (ECB) and corn leaf aphids contribute significantly to control of these major pests throughout the corn belt (49,50). Although the light trap is still considered the best tool for detecting ECB activity, pheromone research has a potential for improved monitoring. Other measures used in integrated control programs are early planting to reduce the potential for development of 15 to 20 insect pests of corn (this treatment increases the damage potential of the ECB), manipulation of irrigation to reduce ECB larval survival, early harvesting to reduce ECB and corn rootworms in succeeding years, and crop rotation which is the best measure for managing corn rootworms (50). With respect to sampling and economic threshold determinations, methods are available for foliage and stalk inhabiting species, but techniques for assessing populations and economic thresholds of the contagiously distributed soil-inhabiting insects, including rootworms, wireworms and cutworms, have been more difficult to develop. The emphasis given to monitoring of corn insect pests is reflected by the six USDA/CES sponsored "pilot" pest management projects which are currently under development in Illinois, Indiana, Iowa, Missouri, Nebraska and Ohio. Research on modeling for corn pests is at an early stage. Work has been done in forecasting population levels of rootworms based on physical parameters taken from the fields and rootworm samples taken during the previous season, and preliminary models for the black cutworm and ECB are being developed and validated (51). Pine Forests* Bark beetles are so important in pine forests (our most important coniferous timber type) that all of the NSF/IPM effort has been put on these insects. They are generally the most destructive insect pest on pines, and nearly all major U.S. pine types or regions have a major bark beetle problem-western pine beetle (WPB) in the Pacific states, mountain pine beetle (MPB) in the Intermountain and Rocky Mountain states, and southern pine beetle (SPB) in the South. For the first two bark beetles, extensive blocks of research data have been accumulated for more than 50 *The data on pine forest are taken mostly from Waters (52). April 1976 17.5 years, yet at the initiation of the NSF/IPM project it was felt that the causes of outbreaks and the proper recommendations for their management were still unknown or at least highly controversial. It was necessary that these data be collated and analyzed to elucidate the ecology of bark beetles, the nature and extent of losses, the role of stand age-class and other characteristics, tree species diversity, certain conditions predisposing outbreaks (e.g., disease, fire, wind-throw), and the role of natural enemies and various management tactics. In these studies researchers do not view their objective as the handing to forest managers of a flat recommendation of what to do about bark beetles. There are many aspects of managing a crop which will not be harvested for 15 to 100 yr that may be affected by a given measure for bark beetle control. Hence, bark beetle specialists expect to develop planning-management advice relative to bark beetles which forest managers would then use in their overall management of the forest. They view as their main objective to obtain an understanding of the role of destructive bark beetles in forest ecosystems and to develop strategies for minimizing the adverse effects of these pests with minimal disruption of the ecosystem and minimal environmental degradation. Sub-objectives are to develop descriptive and predictive models of the dynamics of bark beetle populations as bases for parameter inputs to stand dynamics and treatment strategies; to develop forest stand growth and development models to include the effects of beetle-caused tree mortality in the context of all destructive agents affecting stand parameters; to develop criteria and analytical models which will permit evaluation of the socioeconomic impacts of bark beetles on forest uses and values; to develop treatment strategies and tactics and models for predicting and evaluating their outcomes, with pertinent information on the costs and environmental safety of these strategies; and to better define the benefits and costs of forest pest management and develop sound methodology for benefit/cost evaluation of the management alternatives for pine bark beetles. The pine bark beetle ecosystem is complex; it encompasses a wider range of ecological conditions and the space-time dimensions are greater than in most agricultural systems. A pest management system for this ecosystem is even more complex since there is a diversity of social and economic values involved. Over the past few years the participating institutions have developed and refined a model structure of the bark beetle management system (Fig. 1). The primary information flows in the research and development section are shown by the heavy arrows and feedbacks by light arrows. In terms of modeling, both sets of arrows indicate which components provide inputs in some form that are parameters for another. The four major modeling components are beetle population dynamics, forest stand dynamics, pest impact, and treatment strategies; each is a complex subsystem. The insect population and forest stand dynamics components require basic ecological and biological information to develop the explicit models needed in the pest management system; the impact components require basic ecological and biological information to develop the explicit models needed in the pest management system; the impact component requires the application of economic, social and mathematical theory in order to provide criteria for assessment of potential benefits; and the development of treatment strategies requires a thorough knowledge of ecological and environmental effects of the treatments used. Definition of the system permits refinement of the problem and focuses the research effort. Essential to orderly progression of the work is a thorough knowledge of what is known, and efficient data management systems. A stand growth model has been developed by the Forest Service, USDA, and this or some modification of it will be coupled with bark beetle population dynamics models. Within-tree population dynamics and stand population dynamics are receiving attention. A study in California on use of the western pine beetle pheromone in assessing beetle attack potential and "confusion" and/or "trapping out" potential is under way. As an example of what has been accomplished thus far, the following important conclusions have been drawn relative to mountain pine beetle (MPB): beetle epidemics occurred in stands with a high proportion of thick-phloem trees (not all stands with this characteristic support epidemics); qualitative visual classification of tree response 3 weeks after inoculation with Europhium clavigerum showed promise for identifying resistant trees (no trees with heavy reactions were killed and trees with light or medium responses were killed at the greatest rate); and important root rots (e.g., Verticicladiella wagenerii, Coniophora putaena and Pythium sp.) were associated with dead trees, lending support to the hypothesis that root diseases play a role in predisposing lodgepole pine to MPB attack (this relationship has been demonstrated by the western pine beetle group for other species and hosts). These studies and prior knowledge led to conceptual model of the dynamics of MPB populations. The concepts are summarized as follows: endemic populations are maintained at low levels either by scarcity of thick-phloem trees (in which large numbers of beetles survive and emerge) or by tree resistancethe major regulatory process is competition for food; release to epidemic levels results when resistance in thick-phloem trees is lowered by stresses (e.g. drought, over-stocking, old age, root diseases). The increase in available food results in lower attack densities (per attacked tree), reduced competition, and higher survival and emergence densities. Outbreak momentum is generated by high beetle populations (which apparently can overcome and kill even relatively resistant trees by sheer numbers of attacks. Collapse of epidemics occurs when the larger, thick-phloem trees have been removed from the stand or overall stand resistance increases (predation by insects and woodpeckers may aid population decline.) A large project along the lines of the NSF/IPM project was initiated this year on the SPB. Economic impact, population dynamics and other work started under the IPM project is being expanded. One of the toughest problems for the systems analyst in the area of biology is how he can reduce the enormous complexity of biological systems to be able to describe them analytically and yet meaningfully. When the system is reduced to manageable terms, it often no longer represents the real world. Bland Ewing and associates in California have confronted this problem. The space-time dimensions for a bark beetle ecosystem is of the order 1014 for area and 1010 for time. They have developed a flexible computer language, groups of submodels, and equations and algorithms for computer use by which they can create a simulation which mimics the structure and dynamics of a population of organisms and the behavior of its individuals. By looking at a stand of trees as a system of interlocking clusters, any given cluster can be described quantitatively. The method is being used to model the western pine beetle-root disease relationship. This disease (caused by the fungus Verticicladiella wagneri) has been found associated with a high percentage of bark beetle attacked trees. The model will make it possible to test, by simulation experiments, whether the bark beetles in fact may be an instrument in stopping a root disease epiphytotic, and thus beneficial! Asked what they would be able to recommend for bark beetle management by the end of the project, Waters replied (52), "...possibly, (1) use of stand manipulation (cutting treatments, beetle management by pheromones or insecticides, (2) capability of predicting stand dynamics over space and time, (3) capability of predicting outbreaks and (4) liberation of the forester from some erroneous ideas that have cQnstrained his management options." Cereals Cereal grains do not receive heavy amounts of pesticide due to the limited number of pests which attack these crops; also the moderate value of the crop on a per acre basis precludes the extensive use of costly pesticides. Cereals, however, are crops of great importance in terms of U.S. food production. For example, wheat, the most valuable cereal is grown on ca. 65 million acres and is valued at approximately $6 billion, annually. Considerable efforts to develop integrated pest control programs for cereal pests have been made. The principal species to which these programs have been directed include the hessian fly, the wheat stem sawfly, the greenbug and the cereal leaf beetle. Host plant resistance has played a significant role in development of integrated control tactics against the above species. For the hessian fly and wheat stem sawfly, an estimated 8.5 and 1.5 million acres of wheat, respectively, were planted to resistant varieties in the U.S. during 1969 (49). Varieties of wheat resistant to the greenbug are being developed but have not yet been released, whereas several resistant barley varieties are available (49). Wheat germ plasm resistant to the cereal leaf beetle, based on leaf pubescence factors, has been identified, and some barley lines show considerable promise, whereas there has been little success with oat varieties (53). The total value of research on development of resistant varieties for the hessian fly, wheat stem sawfly and the two noncereal pests, the European corn borer and spotted alfalfa aphid, has been estimated at $3 billion dollars over 10-yr period (49). Considerable success in biological control has been achieved with parasites of the cereal leaf beetle (CLB), of which four or five imported from Europe have been established (54). An integrated control system for managing the CLB, including manipulations of their parasites, has been developed (55)(56)(57). It is based on planting tolerant varieties of oats or wheat, encouraging native natural enemies, such as the spotted ladybird beetle, Coleomegilla maculata, establishing the introduced eulophid parasite Tetrastichus julus, and the mymarid, Anaphes flavipes, treating cereal seeds with propoxur or carbofuran at planting to control the adult beetles so as not to affect the parasites which emerge later, and lastly, if populations of the pest exceed an economic threshold, spraying with carbaryl or malathion. Considerable efforts have been made to model the population dynamics of the CLB. Gutierrez et al. (58) modeled its within-field dynamics on wheat and oats in Indiana, and in Michigan extensive efforts have been made to model its dynamics in relation to its parasites and various host plants, using both a discrete component approach (59) and a continuous time model (Lee et al., in press). In addition, the basic elements for an on-line environmental monitoring system for managing this pest have been developed (8), and extensive real-time implementation programs are in the early stages of development. Citrus* Citrus comprises about 3% of all food consumed in the U.S., and U.S. production of oranges is 36% of world production; that of grapefruit 75%. The national acreage is ca. 1,325,000. This production has been threatened many times by newly invading insect pests and some of them pose continuing problems, in spite of phenomenal success with biological control of a number of species. Insecticides have continued to be widely used, some 11,000,000 lb in 1966. Such use of broad-spectrum insecticides has led to trouble from a number of formerly minor species. The IPM project for citrus was established largely on the assumption that insecticide use could be reduced and sound insect control achieved by using natural enemies and selective, reduced use of insecticides. From the system analysis viewpoint, the citrus project has perhaps suffered from its blessings; the biological control potential for managing citrus insects seemed so promising that the design of the program was conceived in anticipation of being able to rely on natural enemies, with only minor use of insecticides or acaricides or use only of highly selective types. An experience of ca. 70 years in *Data on citrus were taken mostly from Riehl (60). Environmental Health Perspectives California had suggested this possibility for all citrus areas. Biological control certainly offers real potential, sufficient that a number of major pests in citrus growing states may not require treatments or else reduced treatments in major areas. The parasite Aphytis melinus appears to be that effective in much of southern California in control of California red scale, and other parasites of purple scale, whereas, for example, A. melinus has so far proved incapable of controlling red scale in the San Joaquin Valley; A. lingnanensis has been widely colonized in Florida for control of snow scale and is showing much promise. Complete control of purple scale has been achieved in Texas by A. lepidosaphes (61). It has also been shown for citrus red mite that the cost of controlling it can be substantially reduced in southern California by low volume application of an acaricide chosen selectively; that generally now in the San Joaquin Valley it is inherently not as damaging as formerly thought and that usually treatments for it can be much reduced because climate, virus disease and predatory mites adequately control it. One of the main insects for which treatments are made, even at times or in areas where insecticides would not otherwise be needed, is the citrus thrips. Damage from this insect (external quality and yield) is presently distinctly important to growers and citrus marketing associations, yet the problem is controversial and subject to assessment. Where A. melinus is established, ryania (control efficiency of ca 50% ) is the only selective thrips material currently available. Much of the damage is minor and affects the surface of the peel only. Yet, appearance is certainly a marketable quality. The packing industry uses a sliding scale in culling thrips-damaged fruits for the fresh fruit market, depending upon the supply/demand situation, a feature that dismays the systems ecologist trying to develop a managementdecision model for citrus insect control. All this means that management-decision modeling has not gotten under way. The modeling effort in California has been devoted to the population dynamics of California red scale, the key pest, and of its parasites, and presently relating the model to San Joaquin Valley conditions. A model for citrus tree phenology and management of rust mites is being developed in Florida. In Texas, Florida and California, practical systems of integrated control are in operation in some areas. These programs have been initiated without modeling input. While one might deplore the lack of an earlier effort in management-decision modeling, it can be argued that if the snow scale is brought under control in Florida by the parasites the program introduced, the benefits from this alone would dwarf in a few years' time the cost of the entire NSF/IPM project. Tobacco Six major classes of tobacco are grown in the United States, largely along the east coast. Fluecured tobacco makes up 60% of the crop, and North Carolina is the leading grower of this class. In North Carolina a major effort has been made to develop a multidisciplinary integrated approach to control of the insects and diseases (including nematodes) that attack the crop. Rabb et al. (62) noted that the use of such teams of scientists well grounded in the relevant basic and applied sciences is the key to progress and necessitates a study of the cropping system as a whole, with attention given to pest populations as system components. Most of the progress made to date has occurred through a very elementary application of systems science and through trial and error. However, the value of the systems approach is realized and advanced techniques of systems analysis are now being researched and offer the potential to further improve the existing system. The first subsystem the researchers sought to understand was that of the central feature-the tobacco plant and its culture-then the pests, insects and diseases, their economic effects, and the possibilities for reducing those effects. Historically, preventive or purely intuitive treatments with insecticide have been used on fluecured tobacco in North Carolina. The growers gradually developed an overestimation of their problems, and insecticides came to be extensively overused and poorly timed. This overuse of such chemicals added unnecessarily to costs, left unnecessary residues on the product, increased personal hazards, contributed to some of the problems and had other adverse environmental consequences. The goal of the team (63) became that of integrating pest control measures with those of crop production to effect economically sound crop protection with a minimum disruption of natural controls and environmental values. The program had two basic objectives: to lower the mean level of pest abundance over a wide geographic area and thus reduce the frequency of outbreaks and to suppress pest outbreaks when they do occur, according to sound economic and ecological principles. The initial effort (1971) was aimed at the tobacco hornworm and tobacco budworm, but in 1972 tobacco flea beetle and green peach aphid were included, and various tobacco diseases have now been brought in (Rabb et al., in press), with three additional measures added. The methods for insect control consisted of sucker control, early stalk destruction, and applications of selected insecticides only when infestations exceed economic thresholds (using scouting and advising programs). Ellis et. al., (63) stated: "The first two practices comprise a diapause control program and serve to lower the mean level of abundance of the key pests. The latter practice allows suppression of pest outbreaks on a field to field basis, but requires frequent field inspection to determine the presence and infestation level of various pests." The insect monitoring or scouting program, done weekly in 1973, involved 813 farms and 11,350 acres of tobacco. Data on the phase of growth of the crop, the above pest species, and their principal parasites and predators were recorded, with recommendations being made to each grower on the basis of whether densities exceeded economic thresholds: 5 or more fourth or fifth instar hornworms per 50 plants; "heavy infestation" of fleabeetles or aphids. The sucker control and early stalk destruction (even of nematode-resistant types prone to suckering), combined with heavy mortality from parasites and predators, resulted in satisfactory control at reduced cost and reduced natural disturbance and residue problems (always a very sensitive feature in the market). Excessive use of insecticides in the management area in 1973 decreased by 52.9% compared to 1971, the first year of the program. However, there is still room for improvements in the timing and methodology of treatments and the acceptance by growers of the concept of the economic threshold and the need for scouting (Rabb et al, in press). Changing tobacco markets and cropping practices strongly affect grower acceptance of suggested IPM practices, however. Two important developments have been the elimination of chlorinated hydrocarbons and the wide area adoption of chemical sucker control. Toward the end of the chlorinated hydrocarbon period, the chief motivating interest in IPM by growers was to eliminate undesirable residues from tobacco products-not to reduce costs, not to improve effectiveness of control, and not to protect the environment. They wanted to get top dollar for their tobacco. Now that chlorinated hydrocarbons have been replaced with effective insecticides (including B. thuringiensis formulations but chiefly organic phosphates and carbamates) which leave no significant residues, the original motivation for moving to IPM has been reduced. However, there is now less reason for using frequent applications of insecticides for hornworms than prior to the mid-1960's because of uniform adoption by growers of chemical sucker control. This practice was adopted to suppress sucker growth during the preharvest period, but since the material used (maleic hydrazide) acts systemically, it inhibits sucker growth during the postharvest period also. This post-harvest effect is most important to removing food resources for hornworms. Entomologists had tried to reduce overwintering hornworms, but with little success. Ironically, they can take no credit for the success achieved serendipitously by the insertion of chemical sucker control. The increased emphasis on more effective sucker control and earlier crop residue destruction a,s promoted by "Action" IPM programs has resulted in demonstrated benefits with respect to both insect pests and plant pathogens. Rabb et al. (in press) warn that while further improvements will surely come from use of systems science, an examination of costs and benefits from this advanced approach show that costs increase with accuracy, and that the benefits also increase but then level off at some point due to diminishing returns. Thus, there is the problem of seeking the practical level of sophistication in methodology which is financially rewarding. Promise of Integrated Pest Management Many meritorious programs of integrated pest management in other crops are not dealt with here, and the summaries presented in this review are of necessity rather sketchy. This overview should, however, indicate the current status of integrated pest management in the U.S. and provide a basis for speculating on its future promise. We feel that those innovations which are currently being developed (the new), when integrated with essential traditional integrated control elements (the old) (e.g., natural enemies, minimal insecticide use), will provide in the future for significant economic, environmental and social benefits in this country. One might even say that this transition is already in progress and many benefits are now becoming apparent. Of course there is a lag time between research and implementation and by the very nature of research, all of its products will not be used. Yet the evidence suggests that we presently Environmental Health Perspectives are only beginning to see the outlines of the potential benefits that may be reaped from these developments during the next two decades. The old and the new contain elements of value, and we will do a disservice to society if we do not try to put them together harmoniously. What integrated pest management programs might look like 10 years hence is a speculative question. It is likely that individual programs for single crops and probably for complexes of associated crops-e.g., cotton, soybeans, and alfalfa-will be developed in response to specific needs, available technologies, and socioeconomic and political factors in the various areas of the country. Such programs will be implemented in a variety of ways and they will probably replace conventional single component systems of pest control. The speed of their development will vary, for example, with factors such as the uniqueness of the crop and the production system, the economic resources available, and the intensity of the pest problems. However, a degree of commonality in these programs appears to be developing. The tendency is towards interdisciplinary, multiple component approaches where improved pest monitoring and prediction systems are essential elements and in many cases relatively sophisticated systems science and computer technology will be used; the final delivery message to the producer, however, must be clear and plain, unencumbered with excessive details. As noted previously, the value of models and the system science approaches which were previously developed with great success in relation to physical systems, remains to be fully tested and evaluated for pest management applications. However, certain benefits from these methods have already been proven. For example, the logic and methodological approach associated with system science has greatly facilitated research, especially in identifying priorities through modeling, sensitivity analysis and validation. With respect to its use in practical management or prediction, the answer is less clear. Much depends upon the weather and our ability to predict it. Many workers, however, who have had experience with these methods are optimistic. Ruesink (in press), in speculating on the progress of integrated pest management programs for alfalfa, has stated: ". . .we have models describing the population dynamics of the alfalfa weevil and B. curculionis and a growth model of the alfalfa crop. During the next 2 years, these models will be compared with field data from a wide range of conditions, and refinements will be made as needed. Simultaneously, the technology associated with online and off-line utilization of the models will con-tinue to develop. It is our expectation that by 1977 these results will be used to manage the alfalfa weevil on many acres, nationwide. With continued support and encouragement we hope to add models for aphids and leafhoppers towards the end of this decade. By 1980 perhaps we will have the capacity to manage the majority of insect pest problems in alfalfa, based on the use of our model." If this optimism, which is shared by many others, is correct, there will have to be an intensive period over the next few years of development, validation, simplification, and integration, and then an economic and environmental comparison with traditional methods of development and implementation of pest control. We must remember that trial-and-error experimentation in response to use: need is an essential feedback mechanism which is necessary for improving these systems in response to adapting pests and changing technologies. Furthermore, acceptance and utilization by farmers is really the ultimate test as to the utility of any integrated pest managemenrt program. It is certain that many unforseen problems in the area of delivering this type of decision-making information to the grower or pest manager will be encountered. However, we do not see these as insurmountable problems, but more as an exciting challenge which will require the best efforts of our profession.

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2017-06-18T10:43:27.317Z

1976-07-15T00:00:00.000Z

5213335

s2

Some genetic aspects in two strains of chicken and their crosses SUMMARY An experiment was carried out to study the ratios of female to male body weight means and variances in Rhode Island Red (RIR) and Dandvawa (Dand.) strains as well as in their two possible crossbred combinations (RIR-Dand. and Dand-RIt2). Heterosis percentages for female and male body weight at different ages up to 20 weeks of age were also calculated. Four successive hatches comprising 1 439 chicks were obtained within one season at intervals of one week. Individuals of the two strains and their reciprocal crosses were given identical managemental conditions. Results could be summarized as follows. Differences between the mean body weight of the two sexes in each RIR-Dand. and Dand.-RIR crosses was larger than that of any of the two parental strains, and this indicates the probability of the existence of homogametic heterosis (S TONAKER , i 9 6 3). Ratios of female to male body weight variance in chicken were not in accordance with the corresponding ratios of body weight means. These ratios varied for the four breeding groups of the study and this may be due to specific biological causes which are affected by heredity. Heterotic effects on body weight of RIR-Dand. and Dand.-RIR crosses up to 20 weeks of age were clearly observed but with greater degree in Dand.-RIR crossbred group. Heterosis percentages of body weight of the crosses obtained were always higher in males (the homogametic sex) than in females (the heterogametic sex). INTRODUCTION During the last twenty years, the subject of using linecrossing and crossbreeding attracted the attention of poultry breeders. S TONAKER (i 9 62 et r 9 6 3 ) reported greater ratios of hybrid to inbred weights in the homogametic sex than in the heterogametic sex and suggested the existence of a phenomenon known as « homogametic hete-rosis ». Result of some other investigators showed different degrees of heterosis in males and females, but did not consistently support the previous hypothesis (KlDWEi.i<, 19 6 3 and KiDWEM< and N ASH , tg6 4 ). The latter investigators recommended additional studies to determine the nature and extent of homogametic heterosis. S TONAK E R ( 19 6 3 ) indicated that the greater the heterosis in the homogametic sex, the greater the sex difference is expected in turkeys and broiler chickens. M ARAT and A YOUB ( 1973 ) stated that the previous phenomenon might go further than the ratio of mean body weight of the two sexes to the ratio of body weight variance of females to that of males. The present study was carried out to test the hypothesis of the existence of homogametic heterosis in body weight of Rhode Island Red and Danda y awi single crosses at different ages up to 20 weeks. The ratio of body weight variance of females to that of males and heterosis percentage were also studied with the aim to obtain further information to be used in a breeding program for improving broiler meat production. Materials Rhode Island Red (RIR) and Dandarawi (Dand.) strains were bred and mated to each other to produce two purebred groups and their two reciprocal crosses (RIR-Dand and Dand-RIR). The Dand. strain originated in Upper Egypt and has been handled for improvement, 25 years before the start of.this experiment, in the Poultry Farm of the Ministry of Agriculture at Dokki. Data included were collected on 1 439 chickens of four successive hatches taken at intervals of one week during one breeding season. There are four sires per experimental group. Statistical analysis The ratio of body weight variance of females to that of males for the purebred and crossbred groups was tested according to the method which was mentioned by M fRAT and A YOUB ( 1973 ) for comparing groups two by two Heterosis percentage was calculated as the percentage increase of each of the crossbred groups over the mean of its two parental purebred groups using the following equation. where : H % = heterosis percent e = mean of the crossbred group and p = mean of its 2 parental purebred groups RESULTS Mean and variance of male and female body weight for Rhode Island Red (RIR) and Dandarazvi (Dand.) strains as well as those of their two reciprocal crosses at different ages studied, are presented in tables 1 , 2 , 3 et 4 . The ratios of female to male body weight mean and variance of the same breeding groups at the same ages are also given in the same tables. Table 5 presents the significance of the group-sex interaction variance for each age. Heterosis percentages for each cross and sex are given in table 6. DISCUSSION Ratios of female to male body weight mean and variance Data in tables I to 4 suggest that the ratio of female to male mean body weight of any of the two reciprocal crosses was of lower value than that of each of the two purebred groups at all ages studied except that of Dand.-RIR crosses at 8 weeks of age when compared with RIR purebred group, since their ratios were nearly the same. Table 5 confirms that the sex X group interaction is significant at all ages. Generally, these observations also reveal that the difference between the means of the two sexes in each of the crossbred groups of this study (RIR-Dand. and Dand.-RIR groups) was larger than that of any of the purebred groups. This may be due to the existence of homogametic heterosis. In this concern, S TONAK E R ( 19 6 3 ) reported that the greater the heterosis in the homogametic sex the greater the sex differences is expected in turkeys and broiler chickens. As shown in tables 1 , Z , 3 et 4 , the ratios of female to male body weight variance of the four breeding groups were not in accordance with their corresponding ratios of body weight means. Ratios of female to male body weight variance in the four groups did not differ significantly at four weeks of age (table i). At eight weeks of age, the two purebred groups showed homogeneity in their ratios of female to male variance, but the crossbred groups differed significantly in this respect (table 2 ), and the RIR-Dand. crossbred group differed significantly from the RIR purebred group (P < 0 . 025 ) and from the Dand. group (P < 0 . 05 ) at the same age. Significant difference was also observed between the ratio of female to male variance in Dand. group and that in RIR-Dand. crossbred group at twelve weeks of age (table 3 ), as well as between the same ratio in Dand.-RIR crossbred group and that of each of RIR and RIR-Dand. groups at 1 6 weeks (table 4 ). Although these results correspond to two by two comparisons, amoy which the probability to obtain at least one significant comparison by chance is higher than in a unique test, it is unlikely that all can be explained in this way, so that they may have some further interpretations. The heterogeneity of the ratio of female to male variance in chicken body weight could not have been caused by natural or artificial eliminations done more in one sex than in the other as can be observed from the numbers of individuals of the two sexes involved in the analysis (tables i, 2 , 3 et 4 ). On the other hand, the average body weight may be expected to have some influence on its variance due to the expected correlation between them. The result obtdined in this experiment indicated that there was no relationship between the ratio of variances in the two sexes and either the magnitude of variance or mean of any sex. The differences in the ratio of female to male variance may have specific biological causes. Bearing in mind that the purebred and crossbred groups of this study were brooded and reared under the same conditions, then the following interpretations may be offerred : I . Different manifestation of genes in both sexes due to different internal environment such as internal physiology and different external environment such as physical activity and social characteristics (E ISUN and LEGATES, ig66). 2 . Different variation between strains (or breeding groups) relative to sexlinked genes. 3 . Differences in the degree of homozygosity between strains concerning the sex-linked genes (L ERN ER, 1954 Heterosis percentage for 4 -, 8-, i2-, 1 6-and 20 -week body weight of each of the two crossbred groups were higher for males, the homogametic sex, than those for females, the heterogametic sex (table 6). These observations coupled with findings on the sex difference in chicken body weight, previously discussed in the present report, may confirm the hypothesis of the existence of homogametic heterosis suggested by ST ONAK E R ( 19 62 et 1963). Comparisons of heterosis percentages calculated for crosses resulting from mating Dand. cocks to RIR hens (Dand.-RIR crosses) with those of their reciprocals (RIR-Dand. crosses) illustrate that the amount of heterosis for either the female or male body weight of the former crosses at all ages studied were greater than their correspondings of the latter crosses (table 6). This observation may be due partly to the fact that RIR females are heavier and produce larger eggs and chicks than Dand. females. However this does not seem to be the only explanation, as the difference does not decrease with advancing age.

v3-fos

2014-10-01T00:00:00.000Z

1976-12-01T00:00:00.000Z

18967249

s2

The unreliability of direct gas chromatographic analysis of polychlorinated biphenyls. detected in these blank determinations that we were not certain whether it came from the Soluene or other sources. You may note that the blood of control rats which was analyzed with the use of Soluene did not contain detectable amounts of asbestos and zero fibers per gram was reported. We have since repeated this work (as yet unpublished) in an experiment in which asbestos was fed to rats and the tissues analyzed by a low temperature ashing technique similar to that reported for fecal asbestos (2). Essentially similar results were obtained as in the original work with significantly higher levels of asbestos fibers found in treated than in control animals. Polychlorinated biphenyls (PCBs), ubiquitous environmental pollutants, are usually assayed by gas chromatography of cleaned-up extracts with electron capture detection. Quantitation is relative to some arbitrary standard, usually one of the commercial PCB mixtures (Aroclors, Clo-phens, etc.). The mixtures recovered from environmental samples (soil, feces, various animal tissues, milk and the like) almost never match the PCB standard mixture in composition, but the commercial mixture best approximating the unknown in general distribution of peaks on the chromatograms is necessarily selected as quan-titation reference. Although it is well known (1,2) that electron capture detectors respond differently to different PCBs, the assumption is usually made that the errors in comparing an unknown mixture with a more-or-less similar standard mixture will cancel out. Accordingly, the literature is replete with data on "PCB content" of all sorts of environmental samples (3). In a few cases (4), the numbers reported were roughly confirmed by perchlorina-tion to the single compound decachlorobeiphenyl (which can be accurately quantitated with an electron capture detector), but in the vast majority of cases, there has been no confirmation. To illustrate the unreliability of the more common direct gas chromatography of PCBs versus an Aroclor standard, we performed the following simple experiment. A 1-g portion of Aroclor 1260 was chromatographed on 50 g of Florisil PR, eluting with ligh petroleum ether. Under these conditions, the PCBs tend to "tail". The portion eluting between 200 and 300 ml of petroleum ether weighed about 4 mg and showed all of the same peaks as the original Aroclor 1260 during gas chromatography. However, the relative proportions of the peaks differed from those of stock 1260. Three preparations were supplied for gas chro-matographic analysis; the original Aroclor 1260, for use as reference standard, cottonseed oil spiked … Dear Sir: Polychlorinated biphenyls (PCBs), ubiquitous environmental pollutants, are usually assayed by gas chromatography of cleaned-up extracts with electron capture detection. Quantitation is relative to some arbitrary standard, usually one of the commercial PCB mixtures (Aroclors, Clophens, etc.). The mixtures recovered from environmental samples (soil, feces, various animal tissues, milk and the like) almost never match the PCB standard mixture in composition, but the commercial mixture best approximating the unknown in general distribution of peaks on the chromatograms is necessarily selected as quantitation reference. Although it is well known (1,2) that electron capture detectors respond differently to different PCBs, the assumption is usually made that the er-rors in comparing an unknown mixture with a more-or-less similar standard mixture will cancel out. Accordingly, the literature is replete with data on "PCB content" of all sorts of environmental samples (3). In a few cases (4), the numbers reported were roughly confirmed by perchlorination to the single compound decachlorobeiphenyl (which can be accurately quantitated with an electron capture detector), but in the vast majority of cases, there has been no confirmation. To illustrate the unreliability of the more common direct gas chromatography of PCBs versus an Aroclor standard, we performed the following simple experiment. A 1-g portion of Aroclor 1260 was chromatographed on 50 g of Florisil PR, eluting with ligh petroleum ether. Under these conditions, the PCBs tend to "tail". The portion eluting between 200 and 300 ml of petroleum ether weighed about 4 mg and showed all of the same peaks as the original Aroclor 1260 during gas chromatography. However, the relative proportions of the peaks differed from those of stock 1260. Three preparations were supplied for gas chromatographic analysis; the original Aroclor 1260, for use as reference standard, cottonseed oil spiked with 11.0 ,ug Aroclor 1260/g oil, and cottonseed oil spiked with 10.0,g Aroclor 1260 "tail" (simulated environmental sample)/g oil. The analyst was simply instructed to analyze the two cottonseed oil samples for "total PCB content", using the Aroclor 1260 as reference standard. He did not know what PCB mixture or what concentration range to expect to find in the oil (i.e., a single-blind experiment). Work-up of the samples was typical of procedures used for fatty materials, involving extraction into hexane:benzene, 5:1 (v/v), partitioning with sulfuric acid to remove lipids, drying with sodium sulfate-sodium carbonate mixture, and chromatography on Florisil. In every case, elution (with hexane:diethyl ether, 94:6) was shown to be sufficient to remove all of the PCBs from the column (no PCBs were seen in a subsequent elution with hexane:diethyl ether, 85:15). Gas chromatography was routine, with a Varian 2100 gas chromatograph, Sc3H electron capture detector, and a glass column (2 mm ID x 6 ft) of 1.5% OV-17 + 1.95% QF-1 on 80/100 Gas Chrom Q. Each sample was analyzed five times. Quantitation was done in two ways: by summing all of the peak areas attributable to PCBs, and by summing the areas of four conspicuous peaks selected from chromatograms of the Aroclor 1260 standard. Both methods are commonly used in different laboratories. The analyst reported that "Sample B" (the oil spiked with Aroclor 1260) contained 10.4 ± 1.7 .Ag PCB/g oil. Thus, his procedure gave a recovery of 94.5% of the PCB spike (the cottonseed oil had previously been foung to be free of PCBs at the 10 ppb level). For the 1260 "tail" sample, he found a PCB content of 3.53 ±1 0.30 ,ug/g oil using total peak areas, and 3.06 ± 0.66 using four major peaks. Thus, the analytical results were low by about a factor of three. We used the high molecular weight 1260 to avoid the risk of evaporative losses, and, since the variation of electron capture response among isomers is least for the PCBs having more than four chlorine atoms (1), to bias the analytical results in favor of as little error as possible. Thus, we believe that this experiment casts doubts upon the reliability of all literature data on "PCB content" of environmental samples, whenever the sample analyzed did not exactly match the reference standard in distribution of components and an electron capture detector was used for quantitation.

v3-fos

2014-10-01T00:00:00.000Z

1976-10-15T00:00:00.000Z

12076508

s2

Arthrogryposis in charolais cattle a study on gene penetrance SUMMARY SAP (Syndrome of Arthrogryposis and Palatoschisis) is a congenital abnormality affecting Charolais cattle and is conditioned by an autosomal recessive gene. Among pure Charolais animals in France the gene penetrance seems low while among Charolais crosses in Canada penetrance is almost complete. In a study of 3 case histories in Canada at least five Charolais bulls that may be homozygous and recessive for SAP have been identified. Such bulls show zero penetrance which is probably brought about by the selection and accumulation of modifier genes through an evolutionary process. Charolais crossbreds however have nearly complete penetrance probably due to an incomplete complement of the necessary modifiers which are able to supress or alter the gene penetrance. The presence of homozygous recessives normal overlap which had not been individually demonstrated in French herds does result in the low penetrance of the SAP gene, according the INTRODUCTION A Syndrome of Arthrogryposis and Palatoschisis affecting Charolais cattle (SAP) has been reported in several countries and an annotated bibliography has been prepared by I,A UV E R G IV E and FA U C ON (ig 7 6). In general the defect is characterised by crippling of the fore and/or hind limbs and is often associated with a cleft palate. In studies conducted on pure Charolais populations in France, the penetrance of the gene appears to be around o.io and 0 . 14 for males and females respectively (I, AUV >r RGN E, 1972 ). Among Charolais crossbreds in Canadian herds the gene penetrance seems to be almost complete (N AWRO T, i 973 ; BERG and G OON E W A RI I EN E, I974) ! The purpose of this paper was to study the differences in gene penetrance among pure and crossbred Charolais animals by studying three case histories from the beef industry in Canada. MATERIALS AND METHODS The data for the study came from two sources namely, the Experimental Beef Breeding Station at Kinsella, Alberta maintained by the University of Alberta and from the records of the Canadian Charolais Association. At the Experimental Station up to 1975 33 calves have been identified as having SAP and 2 8 carrier females recognized. The dams which are proven carriers together with those suspected of carrying the recessive gene, based on their pedigrees are being maintained in a herd referred to as the crippled herd. The dams in the crippled herd were derived from a hybrid line established in 19 6 0 using three foundation breeds namely, Angus, Galloway and Charolais, the latter being introduced by bulls both by A.I. and natural service. Two cases from the beef industry that will be discussed in this report come from the breeding records maintained by the Canadian Charolais Association. OBSERVED CASES AND THEIR INTERPRETATION Case (I). In 19 6 5 at the Experimental Beef Breeding Station at Kinsella, one sire, a purebred Cha y olais bull was mated in the same year to four of his half blood Charolais daughters the sire himself being proven a carrier of SAP. Of the four daughters one did not settle but the three that did conceive produced crippled progeny (fig. i). As the defective gene is not of a dominant type the case could be explained in one of two ways. Assuming that the original sire was a carrier of the defective gene, i.e. heterozygous, when bred to the non Cha y olais dams only 50 % of the progeny would be heterozygous carriers. If the daughters were bred back to him in a second generation, assuming that half were heterozygous, I2 . 5 % of all calves would be expected to be defective. Although numbers are small the probability of the observed occurrence under the hypothesis is only 0 . 002 and a second and more probable hypothesis could be considered. If the original sire was a homozygous recessive carrier of the defective gene then all his progeny would be carriers, i.e. heterozygous. Crossing back to his daughters will then result in expectation of 50 % of the progeny being defective, and a probability of 0 . 125 of three daughters from three matings being defective. Thus, the hypothesis that the sire was a homozygous carrier with zero penetrance is more compatible with the observations of an above normal frequency of defectives in the second generation. Phenotypically the sire appeared normal; on the other hand his grand progeny which were 75 % Charolais showed complete penetrance. In 19 68 a pure Charolais bull (A) was bred to a group of non-Charolais females, mainly Shorthorns, the resulting progeny being half blood Charolais ( fig. 2 ). In 1971 a second Charolais bull (B) was bred to fifteen of the daughters of bull (A) along with other yearling heifers. In the spring of 1972 a total of thirty two calves were obtained,through the use of bull (B) and fifteen of them were from the daughters of bull (A). Of the fifteen calves, eight were normal and seven showed SAP. Analysing the records it seems unlikely that sires (A) and (B) were merely heterozygous carriers of the defective gene and if this be the case the expectation in the second generation would be about 2 -3 defective calves. A second possibility that could account for the higher number of defective calves observed would be that both sires were homozygous carriers of SAP and if this were the case a near i: i ratio for crippled and normal progeny would be expected in the second generation which is very close to the observed ratio. The third example comes from a case encountered by a cattleman in Saskatchewan. Thirty heifers were purchased by him, the heifers being from three pure Charolais bulls referenced (X), (Y) and (Z) (fig. 3 ). All heifers were either Charolais X Hereford crosses or three quarter Cha-tolak* ose quarter If!!M crosses. Of the thirty heifers eighteen were from (X), ten fr o ta I t And one fidi! (1).! the pedigree information on the last heifer was incomplete it was left Of!,tJi6 atudy. In 10 6 0 the twenty nine heifers were exposed to bteediMg to a single Cho&dquo;s bull (C) and eleven defective calves were observed, ten ()î!e$'l'èi!£tOtn:då!ters of sire (X) and one from the daughter of sire (Z). No de!<!v< prog,!ny were produced from the daughters of (Y). The case shows that sire l'X) &dquo; § w# g 'd o 6h ' th#' d£kttive gene to at least ten out, of eighteen of his daughters. K ! sbo ev!d!tt that the gene was introduced into the heifers through sire (X) as all his matm were Horefords. The most probable hypothesis is that sires (X) aøå..1C)w!höm.! re&oelig;ssivecatriers of SAP. .. From the three cases studied it seems evident that homozygous sires do exist and are able to live and reproduce u<)rlman -y. The defective gene in the homozygous. state shows no apparent penetrance although bull (B) referred to in ease (II) has shown signs of crippling with age. The ctossbred progeny having the recessive genes in the homozygous state do show almost complete penetrance. DISCUSSION The sires falling into this classification of homozygous normal overlaps were pure Clsarvlais while those showing fall penetrance af the defective gene were Charolais ' crossbreds. The expressivity of the gene varies considerably and some SAP calves have been observed with mild symptoms such as slight flexion and/or over extension of the fore and/or hind limbs (Gr!.oun-and GusGui4x, 1973 ), while others show extreme rigidity in the joints with severely reduced muscle tissue and abnormal knee caps and could be classified as being grossly affected. A third category of abnormalities occurs where limb defects associated with a cleft palate have also been observed but there seems to be no apparent association between the severity of the limb defect and the observance of a cleft palate (t A tJ V ERG 1I &OElig;, r975)! .. Modifier genes play an important role in determining phenotypic expressions of characters. The ultimate phenotypic expression of any character depends on the actions of primary or major genes, interactions among major and modifier genes and the environment. Thus the phenotypic expression depends on the entire genome and environment rather than just a single pair of recessive genes as has been often visualized. Genes code for amino acid sequences or polypeptides and the products of gene action are proteins and enzymes. Each phenotypic character expressed ' arises due to a series of biochemical processes mediated by enzymes with specific properties conferred by the sequence of base pairs in the DNA. Modifier genes are translated and transcribed in the same way as major genes and in general play a very significant role in altering gene expression by fitting into certain biochemical pathways. Modifier genes may produce an end product that inhibits or augments the production of a particula r protein or enzyme or that may provide a more or less ' efficient alternate pathway for an intermediate reaction in a basic biochemical pathway thereby modifying the phenotype. The normal overlap homozygous recessive carriers of SAP probably have the required complement of modifier genes to correct the abnormality by fitting into specific biochemical pathways or providing alternate pathways, eventually modu lating the phenotype. In the supression of the recessive genotype seen to occur n the pure Chaxoda%s, the entire genetic system would have evolved the ability to overcome the defect as a direct result of natural selection of modifier genes and this could account for the very low penetrance. Among Chas!ola%s crossbreds penetrance seems to be almost complete as they apparently do not have the correct complement of modifier genes to alter or completely supress the defect. The evolution of decreasing penetrance by natural selection is similar to the phenomenon of « Evolution of Dominance theorized by FISHER (igz8 a, 192 8 b) and reviewed by S HEPPARD and FORD (ig66). It carries the concept one stage further as the recessive homozygote is moved toward a normal phenotype, whereas F ISHER ' S phenomenon deals with the gradual modification of the heterozygote to a dominant phenotype. The study clearly establishes that pure Charodais sires that are homozygous recessive do exist. The reason for the low penetrance that LAU V E RG NE ( 1975 ) observed with respect to SAP in French Charolais herds may well be due to an overlap of homozygous and heterozygous carriers in his herds, the homozygotes being recognized as carriers rather than showing the defect. Individual bulls that are homozygous and recessive for SAP have as yet not been identified in France but there existence was anticipated (see I,ALIV!RGNE'S, fig. I, 1974). In French Charolais herds modifier genes that supress the SAP syndrome may be fixed to such an extent that the penetrance is kept at a very low level. CONCLUSIONS Evidence for the existence of homozygous recessive carrier sires are presented and at least three cases have been observed. The study confirms that the penetrance of the SAP gene type appears to differ in relation to the genotype, where full penetrance is observed among Charola%s crossbreds and almost no penetrance is observed among certain pure Charolais cattle.

v3-fos

2016-05-13T20:27:53.313Z

1976-04-15T00:00:00.000Z

3085894

s2

A cytogenetic survey of 1101 australian cattle of 25 different breeds SUMMARY The karyotypes of i 101 cattle in Australia were examined in a population screening survey. XX : XY chimerism was found in the 5 animals of the Hereford and Friesian breeds. The incidence for Friesian cattle was 2 p. xoo. A Herefo y d bull with 6o,XY : 6 1 ,XXY mosaicism was identified. Autosomal trisomy was found in two bulls and one cow of the Hereford breed. Poly-ploidy was observed at a low level in samples from 577 animals. No polyploid cells were found in three muscular hypertrophic Angus animals. Chromosome associations were found in samples from 2 6 animals. Secondary constrictions were found in a proportion of the best quality cells in samples from 686 animals. No case of Robertsonian translocation, tandem fusion or of any of the other documented chromosomal aberrations was found. INTRODUCTION Population cytogenetic studies are directed at the estimation of the incidence of chromosomal aberrations. In cattle G USTAVSSON (ig6g) found a high 14 p. 10 0 for a Robertsonian translocation in a select group of the Swedish R and W breed, HA RV E Y ( 1974 ) reviewed several reports to arrive at an estimated incidence of i.5 3 p. 100 for the same defect in normal cattle of both British and European breeds and FEC HH E IM F R ( 1973 ) investigating only British breeds found no case of translocation carriers but did find a significant incidence of sex cell chimerism. No attempts have been made to arrive at estimates of the incidence of a further nine classes of chromosomal aberration mentioned in a review by H A I, NAN (z9!5). This paper reports on the collected data from the study of I 101 cattle in Australia. MATERIALS AND METHODS The survey comprised accumulated data during the period 19 6 7 to 1975 . Acctzss to the animals studied was opportune and not by planned random selection. The greater part of the sample comprised 8 00 animals examined at the request of stud owners and the remainder were in herds belonging to public institutions. The details are presented in table i. The results were obtained by a peripheral blood lymphocyte method (H ALNAK ,197 6). A small number of cases were further studied by fibroblast cultures from other somatic tissues. The chromosomes were stained by orthodox methods using orcein or Giemsa's stain and the work on banded chromosomes is not included. The results were compiled from records of microscopic examination supported by photo-micrography and karyotyping. In about 100 animals as few as 15 cells were studied ; for the remainder a minimum of 30 cells and as many as 300 cells were analysed. A list of recognised chromosome aberrations, as described by H ALNAN ( 1975 ), depicted the types of aberration looked for in the material. RESULTS I . -General presentation The chromosomal aberrations observed and their incidence are summarised in table 2 . 2 . -XX : XY chimerism Five freemartins or cotwins were studied. A summary of the results is presented in table 3 . None of the animals was available for dissection or histological study. The bull 3 was an AI donor and had a conception rate of 65 p. ioo which was 10 p. 100 below the average for other bulls in the same center. The heifer 4 had an enlarged clitoris, a small uterus and larger ovaries than normal, which did not manifest signs of cyclical activity although she did show a degree of oestrous behaviour. The bull was used for natural service and found to have a greater return to service of his cows than normal, he was culled. -Polyploidy Polyploidy was recorded as present in an animal on the finding of only one cell. In a select sample of 50 animals the level of incidence was determined, it varied from less than o.i p. ioo to 1 . 0 p. ioo of the total lymphocytes. The higher figures for the incidence of polyploidy were always found where the mitotic index was low. In three double muscled Angus animals no polyploid cells were found. The possibility of correlation with disease was investigated and no significant evidence was found. However, in 33 progeny of one bull 3 half sibs were found to carry the same triad configuration as the sire. . -Secondary Constrictions The study of secondary constrictions is to be the subject of a later paper. Two examples only are presented here of seven types recognised so far (H A r, NAN , unpublished data) ( fig. 3 b and d). The incidence in Friesian cattle at 2 . 2 p. ioo agrees closely with the 2 p. 100 derived from the F ECHHE iMER ( 1973 ) data. The subnormal fertility in the three bulls studied here is reason enough for reconsidering the practice of using cotwins in breeding programmes. . -Mosaicism 60 XV 61 XXY The very small percentage of aneuploid cells suggests that no major departure from normal genotype had occurred in this animal. The fertility problem is regarded as a fortuitous event and the conditions are noted without presumption. This case differs from the mosaic described by R WC x et al. ( 19 6 9 ) OHNSON , 1972 ) if the three cases recorded here are included this brings the total to 9 . The total number of cattle examined cyto-genetically may be estimated from the literature mentioned by Hnr,rrArr ( 1975 ) together with the results reported here and the figure arrived at after allowing for 400 unreported normals was about 5 . 200 . Thus the incidence of autosomal trisomy is . 17 p. 100 which compares closely with human studies at . 15 p. 100 of live births (H AM E RTON , 1971 ). In both human and animal studies skeletal deformities are recognised as one of the phenotypic concomitants of autosomal trisomies. -Polyploidy The incidence of polyploidy in the population was high, in keeping with the earlier suggestion that it is an artifact of culture (H ALNAN , zg 7 6). The incidence of polyploid cells within individual normal animals agreed with the 2 -6 p. 100 found by DA RR f et al. ( 1970 ). Several correlations have been suggested for abnormally high levels of polyploid cells such as muscular hypertrophy (P OPESCU , 19 68 and DARRf et al., ig 7 o). in-breeding (Z ARTMAN and F'!CHH!IM!R, zg6 7 ) and neurological deformities (H!xzoG and HÖ HN , 1971 ). The failure to find polyploid cells in 3 muscular hypertrophic animals may indicate breed differences rather than contrary evidence of correlation. FORD and MA DAN ( 1972 ) reported studies of chromosome associations describing them as branched chromosomes in preference to the term « endoreduplication selective » (LEJEUNU el al., 19 66). The chromosome associations described here bear a close resemblance to those described for man. Moreover, as in man, they were found here to be heritable. -Secondary Constrictions The work on secondary constrictions, achromatic regions or gaps in human studies has been summarised by H AMERTON ( 1971 ). Secondary constrictions seem to be involved in the formation of satellited chromosomes of the D and G groups where they are accepted as nucleolar organisers. In cattle the secondary constrictions in small autosomes ( fig. 2 b), bear a resemblance to the human satellited condition. Therefore, it will be interesting to see if these cattle chromosomes are also the site of nucleolar organisers. The other types of secondary constriction in cattle, figure 2 d, resemble the type found in the Nos. 2 , 3 , 9 and 13 -15 chromosomes of man. No mention was made by HnM!xTOrr ( 1971 ) of the heritability of these secondary constrictions in man, however, in cattle they have been seen in the same position on the same chromosomes in the progeny of the propositus and in animals from related family lines (Har,NAN, unpublished data). The possible occurrence in relation to phenotypic defects (H ALNAN , 1972 ) is being further studied. That minor aberrations of this type should not be disregarded because they have been seen only in cells of the highest quality is encouraged by the remarks of B OBROW ( 1974 ) in relation to small differences observable in human banded preparations. . -Robertsonian Translocation and Other Aberrations Robertsonian translocations and several other chromosomal aberrations noted previously (H A I, NAN , 1975 ) were not encountered. This study, like that of F ECH -H E IM E R ( 1973 ), was mainly concerned with cattle breeds collectively described as British. The results suggest a lower incidence of chromosome aberrations than among the continental European breeds, with the exception of chimerism and autosomal trisomy. CONCLUSION The results have shown a low incidence of major chromosomal aberrations in the cattle studied, as the mosaics and trisomies were found only in a minority of the cells. Chimerism was found at levels similar to those expected from previous publications. Polyploidy is a condition which requires closer study as present evidence does not correlate polyploidy with phenotypic variation in mammals. The same conclusion applies to associations and secondary constrictions. However, if it is remembered that polyploidy is rarely observed in vivo, whereas, associations and secondary constrictions are, then the latter may have a definite role to play in the identification of phenotype variation.

v3-fos

2018-04-03T04:31:41.824Z

1976-01-01T00:00:00.000Z

20550500

s2

Effects of penicillamine on the contents of B6 vitamers of the mouse brain. The contents of B6 vitamers were measured in the brains of mice treated with DL- and D-PeA. When a single convulsant dose of DL-PeA was injected, PLP content was decreased, being accompanied with production of PLP-thiazolidine. The effect of DL-PeA on PLP content was evident far before the occurrence of the convulsions. The administration of PN together with DL-PeA prevented the onset of seizures and lessened the effect of DL-PeA on PLP content. The same amount of D-PeA did not invoke seizures, but caused a small but significant decrease in PLP content. L-PeA was shown to form a thiazolidine compound with PLP (12, 13). Sub sequent studies, however, indicated that D-PeA also formed a thiazolidine with PLP (14); hence it too might be expected to have antivitamin B6 effect. In fact, an antagonism of D-PeA to vitamin B6 has been demonstrated, using the urinary excretion of xanthurenic acid as a parameter of vitamin B6 deficiency (15). The present studies were undertaken to test the effect of DL and D-PeA on the distri bution of B6 vitamers in mouse brain which have previously been shown to reflect the deficiency of vitamin B6 (16). METHODS Animals. DDY mice (weighing approx. 20g) were used in this experiment. Administration of drugs. Solutions of PeA and PN (Nakarai Chemicals) were prepared daily in 0.9% saline, the pH being adjusted to 7.0 immediately before use. The final concentration of the drugs was adjusted so that the required dosage was administered in a volume equivalent to 1% of the body weight of the animals. Injections of PeA and PN were intraperitoneal and intramuscular, respectively, and the injected animals were kept in a laboratory with minimal background noise. Determination of B6 vitamers. Brain extracts to be assayed were prepared by homogenizing about 400mg of brain with 9 vol. of cold 1N perchloric acid, centrifuging, reextracting the residue twice with 3 vol. of cold 0.2N perchloric acid, and neutralizing the combined extracts with 5N KOH to pH about 6.0. The assay of each B6 vitamer with Saccharomyces carlsbergensis 4228 was per formed by the procedure previously reported (18). Since PL-thiazolidine was also active for the yeast, the fraction containing both PLP and PLP-thiazolidine was hydrolyzed by acid phosphatase to PL and PL-thiazolidine (18), respectively, and the hydrolyzed products were separated again with the same column, and then each one was assayed with the yeast. AND DISCUSSION Effect of DL-PeA on the contents of B6 vitamers in brain The effect of convulsant dose of DL-PeA on the contents of B6 vitamers was determined at the onset of seizures (Table 1). DL-PeA administration induced a significant decrease of PLP concentration and tended to decrease that of PL. In contrast, the DL-PeA administration did not influence the PMP concentration and other B6 vitamers. The fate of the PLP concentration, following the DL-PeA administration, is illustrated in Fig. 1. The diminution of the content of PLP which was caused by DL-PeA was already apparent immediately after the admin istration although the convulsions only occurred between 110 and 130min later. This latency suggests that this fall in the PLP level in the brain may be causally related to the convulsant phenomenon. Since the DL-PeA-induced seizures are known to be prevented by the simul taneous administration of PN (11), experiments with this B6 vitamer were included in the present investigation. The administration of PN along with DL-PeA pre vented convulsions induced in mice by DL-PeA and lessened the concomitant decrease of PLP concentration without altering PMP concentration. The PN administration gave also remarkable increases in the other two non-phosphorylated vitamers (Table 1). These results seem to suggest that there are rapid inter conversion reactions going on in the brain and that the recovery of PLP content is possibly the result of PLP formation through PL or PNP. In spite of the marked diminution of PLP content by the DL-PeA administra tion, there was little amount of thiazolidine derivative of PLP in the mouse brain tested (Table 1). This may be due to elimination of the formed thiazolidine com pound from the brain tissue. An administration of PN alone gave remarkable increases in non-phosphoryl ated B6 vitamers but did not significant changes in PLP and PMP ( result may represent a complete regulation of the phosphorylating mechanism, although there is no direct evidence to support this supposition. Effect of D-PeA on the contents of B6 vitamers in brain The next studies were undertaken to test the effect of D-PeA on the distribution of B6 vitamers in mouse brain. Since an injection of D-PeA could not cause at tacks of convulsions, levels of B6 vitamers after the D-PeA administration were determined at the time corresponding to a mean convulsion time when the same amount of DL-PeA was administered. The effects of the D-PeA administration on the levels of B6 vitamers in brain are reported in Table 2. The PLP content of brain was reduced to between 80 and 90% of the control level, and PLP-and PL-thiazolidine derivative were also produced in trace amounts. These results show that as compared with L-PeA, D-PeA causes a far slight diminution of PLP content which is accompanied by the production of PLP-and PL-thiazolidine compound. Previous fact that L-PeA but not D-PeA exerts an antivitamin B6 effect in animals may be interpreted by the difference in the potency reducing PLP content betwee n L-PeA and D-PeA.

v3-fos

2019-04-03T13:06:12.929Z

1976-03-01T00:00:00.000Z

91556620

s2

Composition and volume of the rumen microbiota of sheep fed on grass silage with different sucrose, starch and cellulose supplements A comparative study was made of the effect of different sucrose, starch and cellulose supplements and the effect of different silage preservatives on the quality and quantity of the rumen microbiota of sheep fed on grass silage. The levels of the carbohydrate supplements were 15 % and 30 % of the dry matter of the daily rations, representing 2 1/ a and 5 g/kg animal live weight per day. The silages were prepared with three different preservatives: 1) AIV I solution (25 % formic acid and 20 % hydrocloric acid), 2) formic acid and 3) Viher solution (26 % formic acid and 70 % formalin). The total number of ciliates was highest in the animals receiving sucrose with the silage, lower in those given starch and lowest in those given cellulose. On the pure silage diet, it was between those for the starch and cellulose diets. The total number of bacteria decreased in the opposite direction on the different diets. The sucrose supplements increased the numbers of small ciliates especially and the cellulose supplements those of the bigger ciliates. The total volume of the ciliate fauna was thus highest in the animals on cellulose diets, lower in those on starch diets and lowest in those on sucrose diets. The total microbe mass constituted the following percentages of the rumen content on the different diets: only silage 4.1, 15 % sucrose 3.5, 30 % sucrose 3.5, 15 % starch 4.0, 30 % starch 4.5, 15 % cellulose 4.6 and 30 % cellulose 5,2. Bacteria constituted 77 —B6 % of the total microbe mass on the different diets, the precentage being highest on the sucrose diets and lowest on the cellulose diets. Only small differences were found between the different silage preservatives in the effect on the rumen microbiota. In the fermentation processes occurring during ensiling of grass, the sugars and other soluble carbohydrates disappear from the grass more or less completely, depending on the preservation methods used, producing organic acids and gases. At the same time the solubility of the nitrogenous components increases. As readily fermentable carbohydrates are important as an energy source for rumen microbes, their scarcity or absence might be expected to affect the quality and quantity of the rumen microbiota, and also the utilization of the feed, in animals on grass silage-based diets. However, little information is available on these questions. Another subject that needs study is the effect and quantity of the rumen microbiota of sheep fed on grass silage. The levels of the carbohydrate supplements were 15 % and 30 % of the dry matter of the daily rations, representing 2 1 / a and 5 g/kg animal live weight per day. The silages were prepared with three different preservatives: 1) AIV I solution (25 % formic acid and 20 % hydrocloric acid), 2) formic acid and 3) Viher solution (26 % formic acid and 70 % formalin). The total number of ciliates was highest in the animals receiving sucrose with the silage, lower in those given starch and lowest in those given cellulose. On the pure silage diet, it was between those for the starch and cellulose diets. The total number of bacteria decreased in the opposite direction on the different diets. The sucrose supplements increased the numbers of small ciliates especially and the cellulose supplements those of the bigger ciliates. The total volume of the ciliate fauna was thus highest in the animals on cellulose diets, lower in those on starch diets and lowest in those on sucrose diets. The total microbe mass constituted the following percentages of the rumen content on the different diets: only silage 4.1, 15 % sucrose 3.5, 30 % sucrose 3.5, 15 % starch 4.0, 30 % starch 4.5, 15 % cellulose 4.6 and 30 % cellulose 5,2. Bacteria constituted 77 -B6 % of the total microbe mass on the different diets, the precentage being highest on the sucrose diets and lowest on the cellulose diets. Only small differences were found between the different silage preservatives in the effect on the rumen microbiota. In the fermentation processes occurring during ensiling of grass, the sugars and other soluble carbohydrates disappear from the grass more or less completely, depending on the preservation methods used, producing organic acids and gases. At the same time the solubility of the nitrogenous components increases. As readily fermentable carbohydrates are important as an energy source for rumen microbes, their scarcity or absence might be expected to affect the quality and quantity of the rumen microbiota, and also the utilization of the feed, in animals on grass silage-based diets. However, little information is available on these questions. Another subject that needs study is the effect of the organic acids formed during silage fermentation and the pH of silage on the rumen microbes. Although the feeding value of lactic acid is almost as great as that of glucose (Barnett 1954) and the value of volatile fatty acids is considerable, since they are absorbed both directly from the rumen and through the intestinal wall, these fermentation products are not an energy source for so large an amount of the rumen microbes as the soluble carbohydrates. The use of different carbohydrate supplements with hay and similar forage has been investigated in many experiments (Mitchell andHamilton 1940, Hamilton 1942, Barnett and Reid 1961, Komkris et al. 1965, Sutton 1968, Kellogg 1969, Syrjälä 1971, Salo et ai. 1973, with widely varying results. Less attention has been paid to the effect of adding sugars or other readily available carbohydrates as an energy source for rumen microorganisms in silage diets. The digestibility coefficients of silage were found to decrease with increasing amounts of sucrose or starch supplements, whereas with cellulose supplements they tended to increase in some cases. Sucrose supplements seemed to promote the utilization of the nitrogenous components of silage better than starch or cellulose supplements at the same levels, animals on the sucrose diets showing a somewhat higher N balance and lower rumen NH 3 -N contents than those on the other diets (Syrjälä 1972). Salo et ai. (1973 a) found that varying the amounts of sucrose and starch supplements had little or no effect on the digestibility of silage by sheep. The capacity of sucrose and starch supplements to increase the live-weight gain, silage intake and feed utilization of growing lambs seemed to depend on the fermentation level of the silage (Syrjälä 1975). The purpose of this experiment is to examine the quantity and quality of the rumen microbiota of sheep on pure silage diets and the effect on the microbiota of different sucrose, starch and cellulose supplements. Attention will also be paid to the effect of different silage preservatives. This study is closely connected with the earlier investigation on carbohydrate supplements (Syrjälä 1972), because it provided the rumen samples for the present experiment. Experimental design and rations The experiment was performed with nine rumen-fistulated adult Finnsheep rams according to a Latin-square design. The design of the experiment, the composition of the rations and the feeding of the animals are explained in detail in the earlier study (Syrjälä 1972). The silages were prepared with three different preservatives: AIV I (25 % formic acid and 20 % hydrochloric acid), formic acid (86 %) and Viher solution (26 % formic acid and 70 % formalin). The carbohydrate supplements given with the silages were pure sucrose, potato starch and sulphite cellulose from the wood industry. Each carbohydrate supplement was given at two levels, constituting 15 % and 30 % of the dry matter of the daily rations, which averaged 928 g. The supplements thus represented about 2 1 / 2 g and 5 g per kg animal live weight. Pure silage diets were also given. Sampling The samples of rumen liquid were collected as described by Syrjälä (1972). Only the samples taken in the morning before feeding were used in this microbial work. Five millilitres of rumen liquid was transferred to a glass bottle containing 44 ml of 4 % formol (10 % formalin). Before the counting, Iml of methyl green (1 g methyl green and 2 ml glacial acetic acid and 100 ml aqua destillata) was added to each sample. In this way the original rumen sample was diluted to 1: 10, and the nuclei of the ciliates were stained deep bluish green. The staining was completed within 30 minutes. Counting the ciliates The ciliate cells were counted and identified by a modification of the method of Westerling (1970 a). The modified Fuchs-Rosenthal chamber B. S. 748 Chawksley & Sons Ltd, London), which is divided into 12 rows of 12 square fields, was used. The side of a square is 0.25 mm and the depth of the chamber is 0.2 mm. The total capacity of the chamber is thus 1.8 mm 3 . Ten counts were performed on each rumen sample. In this way, the ciliates of 18 mm 3 of diluted rumen contents were counted. Identification of the ciliates The species were identified on the basis of the descriptions and figures of Dogiel (1927), and Kofoid and MacLennan (1930, 1932, 1933 and according to the taxonomy of Dogiel, account also being taken of the principles of Noirot-Timothee (1960). Counting the bacteria The bacterial cells were counted as described by Syrjälä et ai. (1973). Three preparations were made of each rumen sample for the counts. Determination of the volume of the fauna and flora The mean cell volume of each ciliate species was calculated according to the geometrical method introduced by Schumacher (1962). The mean dimensions used and the cell volumes for each ciliate species are shown in Table 1. The cell volumes are rounded up to two digits on account of computer processing. For the bacterial cells the mean volume of 1 ju 3 was used (Warner 1962). Results and discussion Effect of sucrose, starch and cellulose supplements The results are combined for all the silages and are the averages of 54 samples for the 0 % carbohydrate diet and of 18 samples for each of the diets containing carbohydrate supplements. Altogether 162 samples were analysed. Dogiel (1927). 3. Kofoid and MacLennan (1930, 1932, 1933. 4. Noirot-Timothee (1960). 5. Westerling (1970 b). Number and kinds of ciliate and bacteria cells Giliate species. The ciliates found in the rumen fluid of the experimental animals represented 14 different species (Table 2). Not all the species were found in every sample. For instance, Entodinium vorax and Eudiplodinium negleclum were found in only one third of the samples and Entodinium triacum, E. loboso-spinosum , Diplodinium dentatum and Eudiplodinium maggii in two thirds of the samples. Only Entodinium nanellum and E. duhardi were found in all the samples. Charon was the only representative of the holotrichs. It was found in large number and occurred in all the animals except one in one period. On average, 50 % of the total ciliates were holotrichs (Charon) and 50 % entodiniomorphs, about 45 % belonging to the genus Entodinium and 5 % to the genus Diplodinium. Descriptions of the ruminal fauna in the literature reveal large variations in the quality and quantity, depending on nutritional and many other factors (Hungate 1966). In domestic ruminants on normal feeding, the fauna sometimes comprises more than 30 different species and sometimes less than 10. On some special diets, such as high urea diets, all the ciliates may be lacking (Virtanen 1967). Charon has been found to occur only occasionally in the rumen (Hungate 1966), whereas the holotrichs Isotricha prostoma, I. inleslinalis and Dasytricha ruminantium occur frequently. However, these Isotricha and Dasytricha holotrichs were completely lacking in the animals in this experiment. Holotrichs require soluble sugars as a source of energy (Hungate 1966). For this reason they are present in especially large number in grazing ruminants and those on a diet containing a considerable fraction of good quality hay. Their absence from the animals on the pure silage diet is thus understandable, as its sugar (Syrjälä 1972). But it is more difficult to explain why they did not occur when the sucrose supplements were given. One reason may be that before the experiment began the animals were kept for a long time on silage alone, during which period these ciliates probably disappeared. Moreover, during the experiment they were separated from other ruminants and there was no possibility of contamination. Total numbers of cells. Some differences were found between the total numbers of ciliates in the animals on different carbohydrate diets, but these were not significant (P > 0.05). The sucrose and starch supplements increased the total numbers of rumen ciliates and cellulose supplements decreased them, as compared with the pure silage diet (Table 2, Fig. 1). If the number of ciliates on the pure silage diet is taken as 100, the relative values are 107 for the 15 % and 30 % sucrose diets, 103 for both the starch diets, and 92 and 99 for the cellulosa diets. The numbers of bacteria were lower on the sucrose (P < 0.05) and starch diets than on the cellulose diets. When the number of bacteria on the pure silage diet is taken as 100, the relative values are 83 and 86 on the 15% and 30 % sucrose diets, respectively. 92 and 101 on the starch diets and 104 and 112 on the cellulose diets. The finding that the sucrose and starch supplements tended to increase the quantity of ciliate cells and decrease that of the bacteria cells, and that cellulose had the opposite effect can be at least partly explained by the different solubility of these carbohydrates. The rumen ciliates ingest sugars and starch very rapidly and thus remove them from bacterial attack (Hungate 1966). Cellulose is ingested more slowly, so that the bacteria are probably better able to compete for it. Besides being superior in the competition for food, protozoa can also use bacteria as their food, which explains the decreased bacterial population in faunated sheep as compared to defaunated (Eadie and Hobson 1962). The pH of the rumen fluid on the carbohydrate diets was somewhat lower than on the pure silage diets. Even when fermentation was strongest, however, it kept within the limits of 6.1 and 6.9 (Syrjälä 1972). This range is optimal for ciliates, which are rather sensitive to acidity (Purser and Moir 1959). Entodinium species are somewhat more resistant to acid than are the holotrichs (Abou Akkada et al. 1959), but all the ciliates die rapidly at very high rumen acidities (Hungate et al. 1952). The pH range outside which they do not survive has been found to be 5.5 -8.0 (Krogh 1959, Quinn et al. 1962). Some variations in the numbers of the different ciliate species were found between the diets having different carbohydrate sources. The cellulose supplements decreased the numbers of Charon, compared with the pure silage diet, whereas the sucrose and starch supplements had little effect on them (P > 0.05). The total numbers of entodinia were highest on the sucrose diets and lowest on the starch diets, especially the 15 % starch diets. However, not all the entodinia species were affected in this way by the starch supplements; on the contrary, only Entodinium loboso-spinosum, E. nanellum and E. dubardi were decreased and the numbers of the other entodinia, especially E. vorax, E. longinucleatum and E. caudatum, were higher on the starch than on the other diets, sometimes significantly so (P < 0.05). The total numbers of diplodinia decreased with the sucrose supplements, especially the 30 % supplement, but increased with the starch and cellulose supplements. On the 30 % cellulose diet the number was 2.6 times as high as on the 30 % sucrose diet. In grass silage-based diets, the effect of the different carbohydrate supplements on the ruminal fauna can be explained by the nutritional requirements of the different genera (Hungate 1966). All the entodiniomorphs, except some small species of Entodinium, utilize starch, whereas holotrichs assimilate soluble sugars. Entodiniomorphs are thus usually abundant in the rumen of animals given a high-concentrate ration and holotrichs appear in greatest quantity in animals given hay or other forages rich in soluble sugars (Warner 1965, Abe et al. 1973. The cultural, enzymatic, and microscopic evidence suggests that cellulose is also digested by many entodiniomorphs (Hungate 1966). Oxford (1955) showed that rumen ciliates are capable of digesting both starch and cellulose and may utilize a number of soluble carbohydrates. The differences in the numbers of the various ciliate species between the different levels of the same carbohydrate source were not significant (P > 0.05), except in the case of Eudiplodinium neglectum, which was more numerous (P < 0.01) on the 15 % starch than on the 30 % starch diet. This shows that neither the level of the carbohydrate nor the level of the protein in the diet had any clear effect on the rumen microbiota in this experiment. As the silage was the only source of protein in the diet, the protein content decreased when the carbohydrate supplement increased. The crude protein content averaged 18.7 % of dry matter in the pure silage diet, 15.9 % in the 15 % carbohydrate diets and 13.1 % in the 30 % carbohydrate diets (Syrjälä 1972). However, even at the lowest levels in this experiment, the protein content was too high to inhibit the growth of the rumen microbes. In experiments where a high positive correlation has been found between the level of protein in the diet and the total amount of microorganisms in the rumen, the protein content of the diet has been lower, the maximum being 12 % of dry matter (Moir and Williams 1950). Percentage composition of the fauna. The carbohydrate supplements caused some changes in the composition of the ciliate fauna as compared with that of the pure silage diet (Fig. 3). The representation of the genera and subgenera in the total fauna generally varied less than that of the single species. Charon constituted more than half of the total ciliate numbers on the pure silage diet and on the 15 % sucrose and starch diets. The lowest percentages for Charon were obtained on the cellulose diets. Its representation on the cellulose diets was about 10 % units lower than on the pure silage diet. The proportion of the genus Entodinium was lowest on the 15 % starch diet, being sligtly more than one third of the total number of the fauna, and highest on the 30 % sucrose diet and both cellulose diets, where it constituted about half of the fauna. The genus Diplodinium composed 3-B % of the fauna on the different diets, its contribution being lowest on the sucrose and highest on the cellulose diets. Volume of the microbe mass Significant differences (P < 0.05) were found in the total volume of the ciliates between the different diets. The total volumes were highest on the cellulose diets, somewhat lower on the starch diets and lowest on the sucrose and pure silage diets (Table 3, Fig. 2). The changes in total volume thus went in the opposite direction to those in the total numbers of ciliate cells, which decreased from the sucrose diets to the starch diets to the cellulose diets. This is due to the size of the ciliates cells affected by the carbohydrate supplements. Cellulose increased the numbers of the bigger entodiniomorphs especially, whereas sucrose raised the numbers of the small holotrichs. The large ciliate volumes on the cellulose and starch diets, as compared with the sucrose diets, are attributable in particular to the massive Polyplastron (Fig. 4). The total volume of the microbe mass varied from 3.5 % of the rumen content on the sucrose diets to 5.2 % on the 30 % cellulose diet (Fig. 5). The values were significantly higher (P < 0.05) on the two cellulose diets than on all the other diets, and significantly lower (P < 0.05) on the sucrose diets than on all the others except the 15 % starch diet. The ciliates constituted a rather small proportion of the rumen content, between 0.5 and 1.2 %. One reason is that some large ciliate species, for instance Isotricha and Dasytricha, were completely lacking. Ciliate volumes of about this magnitude have found in cows (Schumacher 1962), but the values in domestic ruminants have generally been higher (Mowry and Becker 1930, Warner 1962, Harmeyer 1963. The contribution of the bacteria to the total microbe volume was fairly high, the average for all the diets being 4.5 times as high as that of the ciliates. The ratio of bacteria to ciliate volumes was highest, 6.0, on the 30 % sucrose diet and lowest, 3,3, on the 30 % cellulose diet. In earlier studies, the ciliate mass has been found to be roughly equal to that of the bacteria in domestic ruminants (Abou Akkada 1965) and 4,6 times as high in semidomestic reindeer (Syrjälä et ai. 1973). In other experiments (Oxford 1964, Warner 1965, the bacteria have been found to compose the largest part of the microbial mass, as in the present study. Effect of silage preservatives The values for the diet consisting of silage alone are the averages of 18 rumen samples and those for the diets containing carbohydrate supplements are the averages of 54 samples. Only slight and non-significant differences (P > 0.05) were found in the total numbers of ciliates and bacteria between the silages prepared with different preservatives (Table 4, Fig. 6). The differences in the numbers of the various ciliate species were significant (P < 0.05 or P < 0.01) in only a few cases. The total numbers of both dilates and bacteria tended to be higher on the Viher solution silage diets than on the other diets. The numbers of bacteria were higher on the formic acid silage diets than on the AIV I silage diets. The number of ciliates was higher on the AIV I silage diet than on the formic acid diet when only silage was given, but the situation was reversed with the diets containing the carbohydrate supplements. Statistical analysis performed separately for diets consisting only of silage and for diets also containing carbohydrate supplements. Meaning of index letters same as in Table 2. The volumes of the different ciliates were about the same on the different silage diets, especially on the pure silage diet (P > 0.05, Fig. 7). The total volumes of ciliates on the pure AIV I, formic acid and Viher solution silages were 6094, 5975 and 6078 /u? X 10 8 per ml rumen content, respectively. The corresponding values on the diets containing carbohydrate supplements were 7284, 7881 and 6818. The genus Entodinium constituted the highest proportion of the ciliate volume on the Viher solution silage diets and a higher proportion on the formic acid silage diets than on the AIV I silage diets. The contribution of the genus Diplodium decreased in the opposition direction: AIV I > formic acid > Viher solution. The contribution of Charon was about the same on all the silage diets. The differences in the total microbe mass between the different silage diets were also non-significant (P > 0.05, Fig. 8). General considerations The investigation reported in this paper concern the effect of sucrose, starch and cellulose supplements, at levels of 2 x / 2 g and 5 g/kg live weight per day, on the composition and volume of the rumen microbiota of sheep fed on grass silage prepared with different preservatives. The carbohydrate source affected the rumen microbiota more than the level at which it was used. The total number of ciliates was highest in the animals receiving sucrose with the silage, lower in those on starch diets and lowest in those given cellulose. On the pure silage diet, the ciliate number was between those for the starch and cellulose diets. The total number of bacteria was highest on the cellulose diets, lower on the pure silage and starch diets and lowest on the sucrose diets. These variations in the numbers of ciliate and bacteria cells can mainly be attributed to differences in the solubility of the carbohydrates. The sucrose supplements increased the numbers of small ciliates especially and the cellulose supplements raised those of the bigger ciliates. The total volume of the ciliate fauna was thus highest in the animals on cellulose diets, lower in those on starch diets and lowest in those on sucrose diets. The contribution of the total microbe mass to the rumen content decreased in the same direction: cellulose > starch > sucrose. In the earlier study (Syrjälä 1972) in which the same rumen samples were used as in this experiment, the average ammonia content of the rumen fluid was lower on the sucrose diets than on the starch and cellulose diets with the same levels of carbohydrate supplements. When this finding is considered together with present results, it appears that the decrease of the rumen ammonia content depends more on the numbers of the ciliate cells than on the volume of the ciliate fauna. Perhaps the bigger ciliates contain relatively more polysaccharides than the smaller ciliates, so that the protein content is relatively higher in a ciliate mass formed from smaller cells than in one formed from larger cells. The average amounts of total volatile fatty acids behaved in the same way in relation to the different carbohydrate diets as the volume of the ciliate fauna, decreasing from cellulose to starch to sucrose. However, these results cannot be considered to reflect the fermentation activity of the ciliates alone or its dependence on, for instance, the volume of the ciliates, because the amount of bacteria was not the same on the different diets. The bacteria counts were highest on the cellulose diets, lower on the starch diets and lowest on the sucrose diets. The importance of the bacteria flora in this experiment is evident from its high contribution to the total rumen microbe mass compared with that of the ciliate fauna. It constituted 77 -B6 % of the mass on the different diets, the percentage being highest on the sucrose diet and lowest on the cellulose one. The ciliates and bacteria were counted on samples taken only once a day, in the morning before feeding. In contrast, the pH, ammonia and VFA content were determined on samples taken three times a day (Syrjälä 1972). This investigation of the microbiota thus describes only the situation at the begin-ning of feeding and does not give any information on qualitative and quantitative changes occurring during the day (Purser andMoir 1959, Warner 1962). Consequently, the results cannot be used as a measure of the protein utilization of the host. They do, however, permit a comparison of the effects of the different diets on the rumen microbiota. The other indices of the utilization of the nitrogenous compounds of silage, such as the nitrogen balance and the biological value of protein, were also somewhat better on the sucrose diets than on the corresponding starch and cellulose diets. In contrast, the apparent digestibility coefficients were generally higher on the cellulose diets than on the starch or especially on the sucrose diets. It must, however, be remembered that the metabolic nitrogen substances of the faeces are not separated when apparent digestibility is determined and that soluble carbohydrates have been found to increase its proportion in the faeces (Hamilton 1942, Fontenot et al. 1955, Paloheimo et ai. 1968, Syrjälä 1971). The different silage preservatives were found to have little effect on the composition and volume of the ruminal microbiota. Similarly, little variation was found in the digestibility values of the silages, except that of crude protein, which was lower for the Viher solution silage than the other silages (Syrjälä 1972). Some significant differences were found in the rumen fermentation products between the different silage diets. The ammonia concentration in the rumen fluid was, on average, lower on the Viher solution silage diets than on the other silage diets, which was attributed to the formaldehyde contained by the Viher solution (Syrjälä 1972). But according to the results of the present experiment, the formaldehyde did not have a notable effect on the rumen microbiota.

v3-fos

2018-04-03T04:16:28.596Z

1976-08-01T00:00:00.000Z

38483539

s2

Reactivation mechanisms of thiamine with thermostable factors. It was observed, in vitro, that the water extract of the fermented-tea customarily chewed by Thai people has a similar thermostable thiamine-inactivating factor to that found in the water extract of fern. It was also observed that the percentage of thiamine disulfide formed from thiamine with some flavones, catechol, pyrogallol, caffeic acid, dihydroxyphenylalanine, and hemin is greater at pH 7.5 than at pH 7.0. With some flavonoids, such as quercetin, rutin, and 6,7,4'-trihydroxyisoflavone, and pyrogallol, hemin, catechol and caffeic acid at pH 7.5, around 30-100% of thiamine is changed into thiamine disulfide. Water extract of shiitake, okra, coffee, black tea and fukinoto have only weak activities of thermostable thiamine-inactivating factors as a large percentage of thiamine disulfide is formed from thiamine even at pH 7.0. 2-Methyl-4-amino-5-aminomethylpyrimidine was isolated from the reaction mixture of 1 g thiamine with 20 mg catechol (1:0.5 mole) at pH 7.0, 45 degrees C, and identified with the synthesized pyrimidine. Summary It was observed, in vitro, that the Nakabayashi (5) isolated flavonoid pigments, astragalin and isoquercitrin from bracken and also demonstrated the presence of rutin, which they considered the thermostable antithiamine factor. On the other hand Fujita and his group (6-8) studied the mechanisms of thia mine inactivation with the thermostable factors, and reported that isolated crystals from the reaction mixtures of thiamine with some fl avonoids were identical with thiamine disul fi de, which is an active form of thiamine. The above results on studies of thermostable thiamine-inactivating factors led to the study of factors in fern and bracken etc. and the reaction mechanisms of thiamine with fla vonoids. In the meantime Berutter and Somogyi (9) reported isolation of 3,4-di hydroxycinnamic acid (caffeic acid) from the fern as one of the thermostable antithiamine factors. Davis and Somogyi (10) attempted to clarify the mechanism of the inactivation of thiamine by caffeic acid and observed that the reaction was made partially reversible by the addition of cysteine to the assay mixture, indi cating that some thiamine disulfide might be formed. However, the remaining thiamine inactivating mechanism with caffeic acid had not been clarified. Since there are many o-dihydroxy compounds beside caffeic acid in food, reaction mecha nisms of thiamine with caffeic acid, catechol, and other dihydroxy compounds should be studied not only for the basic science involved but also for practical dietary considerations. Vimokesant et al. (11) observed that people in Thailand chew fermented-tea leaves showed signs of thiamine deficiency even though they ingested the normal daily requirements of thiamine. We attempted to determine the reaction mechanisms of thiamine with some diphenol compounds including caffeic acid and fla vonoids. Part of the results had already been reported (12) and data from further experi ments is presented herein. RESULTS 1. Thermostable thiamine-inactivating activi ties (TIA) of fermented-fish and fermented-tea Fermented-fish and -tea were purchased from a market in Bangkok and Chaingmai respectively, and brought to our laboratory and stored in a freezer until use. Extraction of the heatstable antithiamine factors in these samples and determination of the activities were carried out as described in EXPERIMENTAL. The activities were compared with those of fern and shellfish. The results were shown in Fig. 1. Optimum pH of TIA of the fer mented-tea and a fern obtained in Japan was almost the same at pH 7.0. Activities in the fermented-fish and shellfish were rather weak compared with those of the fermented-tea and fern as seen in the figure. 2. Thiamine-decomposing activities with some thermostable factors Thiochrome negative percentages of thia mine and formation of thiamine disulfide from thiamine with quercetin, rutin, catechol, pyrogallol, caffeic acid, hemin and dihydroxy phenylalanine (DOPA) were estimated at intervals in the condition of both pH 7 and 7.5 3. Percentages in thiamine disulfide formation to the thiochrome negative form in the reac tion mixture of thiamine with plant extracts As mentioned in our previous paper (12), some of the plant extracts and diluted tea extract produced large percentages in thiamine disulfide to the thiochrome negative form of thiamine. The experiment was repeated care fully and obtained the results as shown in Table 2. Only small percentages of thiamine were inactivated with these extracts as indicated in the last column of the table. 5. Isolation of 2-methyl-4-amino-5-amino methylpyrimidine from the reaction mixture of thiamine with catechol As shown in Fig. 6, pyrimidine was detected in the eluate of fraction number 720-860. (about 23% of thiochrome negative form of thiamine) was formed from thiamine (data not included in Fig. 1). Factors in the fermented tea leaves may thus be similar to the caffeic acid isolated from fern or other diphenol compounds such as catechol, some isoflavones, which have been studied as model experiments. In samples of shiitake, coffee, black tea, okra and fukinoto, high activity of thermostable thiamine-inactivating factor could not be dem onstrated ( Table 2). Fujita et al. (13) sug gested that thiamine disulfide may be occurred from thiamine with the shiitake extract. Factors, which accelerate formation of thia mine disulfide from thiamine, have not been elucidated. Hemin reported has antithiamine activity as determined by the thiochrome pro cedure of Kundig et al. (14,15). ®n the other hand, Kuo and Hilker (16) observed that heroin reacts with thiamine to form a fluo rescent thiamine derivative, and used the term "thiamine -modifying activity" of heroin. As the derivative can be utilized as thiamine or changed to such in the body. The relation ship between such fluorescent thiamine deri vatives and the thiochrome negative form of thiamine reacted with hemin is the subject of future studies.

v3-fos

2018-04-03T03:45:35.471Z

1976-04-01T00:00:00.000Z

7920361

s2

Integrated pest management. Integrated pest management, in which the conventional pesticides are augmented by one or more nonchemical control practices, has been receiving renewed interest. What is new in this revitalization of an old technique is the careful and more knowledgeable application of a variety of control techniques. 4/15 (26%) grade 1, 5/18 (28%) grade 2 and 3/5 (60%) grade 3 tumours. There was no clear relationship to tumour stage, lymphocytic infiltration or stromal content of the tumours. Clinical review one year after the 2 year period of tumour collection showed that 6/9 (66%) of patients with tumours with reduced levels of transcript had died or had disease which was not controllable by local resection and 3/9 (33%) had developed tumour re-occurrences. In comparison, in the group with normal levels of expression of TGFPi1, 3/18 (17%) had disease which was not controllable by local means, 9/18 (50%) had tumour re-occurrence and 6/18 (33%) had no evidence of disease. The association of reduced expression of TGF%1 and advanced disease was statistically significant P<0.02 (Fisher's test). Although the sample size is small, we suggest that the loss of expression of TGFP1 may be a potential marker of progressive disease or prognosis in transitional cell carcinoma and warrants further study. The TGFPI group of peptide regulatory factors is a large expanding family of multi-functional genes displaying marked homology and evolutionary conservation (Roberts & Sporn, 1990). TGFP is secreted in a latent form which is unable to bind to its receptor (Wakefield et al., 1987). Activation of this latent TGFP may be achieved in several ways including transient acidification, alkalinisation or chaotropic agents (Kryceve-Martinerie et al., 1985). The action of TGFP is mediated through binding to specific cell surface receptors. Almost all cells, regardless of origin, bind TGFP (Roberts & Sporn, 1990). Three distinct classes of receptor with various affinities for TGFP1 and TGFPI2 have been described (Cheifetz et al., 1987) and it has been suggested that the cell specific effects of the individual forms of TGFP may be regulated by differences in the levels of receptors of different affinities present on those cells (Cheifetz et al., 1990). The pleiotropic effects of the TGFPi family have been extensively documented (for recent reviews see Moses et al., 1990; Roberts & Sporn, 1990). The role of TGFPI in cell transformation is unclear. Most normal epithelial cells in tissue culture are growth inhibited by TGFP (Moses et al., 1985;Masui et al., 1986;Jetten et al., 1986;Kurokowa et al., 1987). In contrast many carcinoma cells show reduced inhibition by TGFPI (Wakefield et al., 1987;Lechner et al., 1983;McMahon et al., 1986) and many transformed cell lines secrete increased amounts of TGFPI (Derynck et al., 1987;Jakowlew et al., 1988;Niitsu et al., 1988) which is reflected in an increase in the steady-state levels of TGFPI mRNA in these cell lines and in tumours. Such increased TGFPI production could contribute to tumour development and progression in multiple ways via paracrine effects on neovascularisa-tion, extracellular matrix formation, chemotaxis and immunosuppression . However, changes in TGFP production and responsiveness are not demonstrated in all transformed cells. Some remain growth inhibited by TGF,B and not all transformed cells secrete increased amounts of TGFPi (Derynck et al., 1987;Wakefield et al., 1987). There have been few studies of TGFP production by human tumours. Raised levels of TGFP mRNA were reported in breast and renal tumours (Coombes et al., 1990;Gomella et al., 1989). Similarly, TGFP1 RNA was detected in all glial tumour cells in a spectrum of cerebral malignancies (Mapstone et al., 1991). TGFP secretion and growth response of urothelial cells has not been studied in detail. The only report on bladder epithelial cells to date showed that foetal urothelial cells but not transformed urothelial cells responded to exogenous TGFP1 by a decrease in plasminogen activator activity secondary to increased transcription of PAI-I activity (Hiti et al., 1990). Transitional cell carcinoma is the fourth most common cancer in males in the United Kingdom and the incidence is rising in both men and women (31% between 1971(31% between and 1984(31% between , OPCS, 1971(31% between -1984. Studies of the natural history of transitional cell carcinoma have identified an aggressive subset of tumours (Pryor, 1973). Identification of the molecular events involved in the genesis of transitional cell carcinoma may offer potential markers of disease progression and prognosis. As part of a study aimed at identifying some of these lesions in transitional cell carcinoma, we have examined the structure and expression of the genes encoding TGFPi1 and TGFPi2 in human urothelial cancer cell lines and transitional cell carcinomas. We show that in bladder tumours, marked reduction or loss of expression of the gene encoding TGFPI is associated with advanced disease. No TGFP2 transcript could be detected in these tumours. In urothelial cancer cell lines, variable levels of TGFPi1 and TGFP2 mRNA were expressed with no apparent relationship between the relative amounts of these transcripts. 'cold' cup biopsy forceps and was removed from the bladder as soon as possible, trimmed of debris and a representative sample excised (including the base and attached normal tissue) for histological assessment. Tumour size ranged from 60 mg to many grams but the majority (>80%) were small and were processed as a single sample. Where biopsies of normal urothelium or carcinoma in situ were taken, the epithelial layer was dissected free of submucosa and muscle. In these cases, urothelium from at least four biopsies from the same patient was pooled and processed together. Tissues were placed immediately at -70°C. The tissues used are shown in Table I. Tumours were graded according to W.H.O. recommendations (1973) and staged using the TNM system (UICC, 1978). Cells and cell culture The cell lines used were EJ (Evans et al., 1977), VM-CUB-2 (Williams, 1980), SCaBER (O'Toole et al., 1976), SD (Paulie et al., 1983) and SW1710 (Kyriazis et al., 1984) Isolation of DNA and RNA DNA and RNA were isolated from the same tumour sample by the guanidine isothiocyanate method (Maniatis et al., 1982). DNA was extracted twice with phenol, twice with phenol:chloroform and once with chloroform, ethanol precipitated and dissolved in 1 x TE prior to quantitation and use. The RNA pellet was washed in 70% alcohol, air dried and dissolved in 0.3 M sodium acetate pH 6.0 prior to precipitation with two volumes of absolute ethanol and storage at -70'C. Southern blotting DNA samples were digested with EcoRI (Gibco BRL, Paisley, Scotland) according to the manufacturer's instructions and the fragments separated in 0.8% agarose gels. Gels were stained with ethidium bromide and photographed prior to capillary transfer (Southern, 1975) onto Hybond-N membranes (Amersham UK). Lambda DNA digested with Hind III was used as size markers and lymphocyte DNA from normal volunteers as a normal DNA control on each gel. Blots were baked at 80°C for two hours and pre-hybridised and hybridised following the manufacturer's instructions. Following washing to high stringency (0.1% SSPE and 0.1 % SDS), blots were exposed to Hyperfilm-MP (Amersham UK) at -70°C with intensifying screens. Northern blotting Total cellular RNA was electrophoresed in 1 % agarose/ formaldehyde gels (modified from Thomas, 1980) and transferred by capillary blotting to Hybond-N membranes. These were pre-hybridised and probed according to the manufacturer's instructions. Some gels were stained with ethidium bromide to compare loading and RNA inetegrity with the results obtained from control probes. Blots were hyrbidised sequentially to probes for TGFP1, TGFP2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The 1.4 kb GAPDH transcript migrated suitably close to the 2.5 kb TGFI31 message to act as a good control for RNA loading and degradation. Non-specific binding of the GAPDH probe to the 28S ribosomal band was used as a control for TGFP2 (transcripts of 4.1 kb, 5.1 kb and 6.5 kb). In addition, selected blots were hybridised to c-erbB-2 (transcript size 4.5 kb, not illustrated) to confirm the presence of intact RNA in the size range of TGFP2. Levels of transcript in tumours were compared with levels in control issues and scored as normal (+), raised (+ +) or reduced or undetectable (0). Blots were stripped by washing for two hours in 5 mM Tris-HCl pH 8.0, 2 mM Na2 EDTA and 0.1 x Denhardt's solution at 650C. Slot blotting Slot blots were made on Hybond-N membranes using a Schleicher and Schuell vacuum slot blotting apparatus. Each slot received 2.5 jig denatured total RNA. RNA was denatured in 50% formamide, 5% formaldehyde, 1 x SSC at 680C for 15 min. Following application of the sample, the wells were flushed with 100 gl 20 x SSPE and the membranes baked at 800C. Hybridisation was as for Northern blots. Slot blots were assessed by comparing the ratio of signals obtained with GAPDH and the gene of interest for a reference slot containing control tissue RNA with those obtained for tumour tissues. Probes The probes used were the 2.1 kb EcoRI fragment of phTGFb-2 (TGFP1) cloned by Dr G. Bell and kindly supplied with his permission by Dr J. Scott, the 2.3 kb EcoRI fragment of pPC-21 (TGFi2, Madisen et al., 1988) supplied by Oncogen Science (Manhasset, NY., USA) and the 1.3 kb Pst-I fragment of pRGAPDH-13 (glyceraldehyde-3-phosphate dehydrogenase, Fort et al., 1985). Probes were labelled by random priming (Feinberg & Vogelstein, 1983) and used at 106 c.p.m. ml-' of hybridisation fluid. (Figure la). SCaBER, a cell line derived from a squamous cell carcinoma of the bladder, showed the highest levels of expression and VM-CUB-2 and JO'N the lowest levels. After allowance was made for variations in loading and RNA degradation, 5637, SW1710, EJ and SD were judged to express similar levels of TGFP1I. Results Levels of TGFPI2 expression were more varied. The level of expression of the three expected transcripts of 4.1 kb, 5.1 kb and 6.5 kb differed within the same bladder tumour cell line (Figure lb). Expression of the 5.1 kb and 6.5 kb transcripts was greater than that of the 4.1 kb transcript. Total expression of TGFP2 RNA also varied markedly between cell lines. All cell lines expressed two of the three expected transcripts of 6.5 kb and 5.1 kb respectively. VM-CUB-2 appeared to express higher levels of the 6.5 kb than the 5.1 kb transcript (Figure lb) but all other lines expressed more of the 5.1 kb than the 6.5 kb transcript. This can be seen clearly for SWI710 in Figure Expression of TGF I3 and TGFl32 in primary transitional cell carcinoma Northern and slot blots of total RNA extracted from transitional cell carcinomas were hybridised sequentially to TGFP1, TGFP2 and GAPDH probes. RNA from 38 tumours, two cases of CIS, ten matched and three unmatched field biopsies, 11 normal urothelial biopsies, two biopsies of follicular cystitis and one biopsy from a bladder with cystitis following BCG treatment were analysed by Northern and slot blots. Since large samples of normal urothelium from individuals with no history of urological symptoms are not available, samples of macroscopically normal urothelium from several sources were assessed to provide a measure of 'normal' levels of expression. These included samples from bladder tumour patients with no tumour at check cystoscopy and from prostatectomy patients. Expression of TGFI in all but one of these samples (see below) was similar and was taken as the baseline level of expression. No aberrant transcripts of TGFP1 were detected and the levels of transcript in 25 tumours were similar to those in normal urothelial controls. In 12 tumours, reduced levels of transcript were observed and in three tumours raised levels of transcript were detected. Examples are shown in Figures 2 and 3. The characteristics of these tumours are outlined and compared with grade in Table II. Although the number of G3 tumours analysed is small (five), the marked difference in the incidence of tumours with reduced expression 3/5 (60%) suggests that there is an association between high tumour grade and reduced expression of TGFPI. No relationship between the reduced levels of transcript of TGFPI and tumour stage or the amount of stroma in the tumour biopsies was detected. Three tumour re-occurrences (we have used this term for subsequent tumours or 'recurrences' in the same patient, since the relationship between initial and subsequent tumours is unclear) were subsequently analysed. In two of these, the level of expression of TGFPI was the same as that detected in the initial sample. In the third, expression was reduced in the initial biopsy and normal in the second. Of the ten matched field biopsies, four showed similar levels of expression of TGFP to the tumour from the same bladder. These were comparable to levels detected in normal urothelium. In three cases, transcript levels were reduced in the tumour and not the field biopsy. In one patient, levels were reduced in the field biopsy but not the tumour, in one patient levels were reduced in both field and tumour biopsies and in one patient transcript levels were raised in the tumour but normal in the field biopsy. Of the three unmatched field biopsies, one showed raised levels of transcript. In addition, two biopsies of follicular cystitis (both from patients in whom transitional cell carcinomas had been resected in the past) were assessed. One of these showed reduced levels of transcript. Only one of the samples of macroscopically 'normal' urothelium showed altered levels of transcript. This sample was obtained from a patient in whom no re-occurrences were detected at check cystoscopy. Two biopsies from this patient were assessed. Table III. These exclude the two patients with CIS, one of whom was treated with BCG and one with cystectomy, and one patient with a transitional cell tumour who was too ill for any therapy (all of whom expressed normal levels of TGFI). Correlation between reduced expression of TGFJIl and disease progression was found P = 0.02 (Fisher's test). Discussion The members of the TGFPi family of peptide regulatory factors have been shown to play important roles in the control of growth and differentiation of normal cells. A number of observations suggest that differences in production of, or response to TGFPi may play a role in transformation. Here we have shown that expression of TGF,B1 and TGFP2 vary considerably in both urothelial carcinoma cell lines and tumours. Nevertheless, results indicate a correlation between decreased expression in tumours and clinical behaviour. Northern analyses of total RNA from human bladder tumour cell lines showed the expected transcripts for TGFPI and TGFPi2. The three TGFP2 transcripts may result from differential splicing and/or polyadenylation events. The 4.1 kb and the 6.5 kb messages are considered the major transcripts of TGFP2 and have been described most commonly in other cell lines (Madisen et al., 1988;Derynck et al., 1988). However, we found that the 6.5 kb and 5.1 kb transcripts were the most abundant in urothelial carcinoma cell lines and the 4.1 kb transcript was only clearly demonstrated in one cell line. Since this did not reflect mRNA degradation, it is likely that the various transcripts identified in the urothelial tumour cell lines are differentially expressed messages. TGF,1I mRNA was expressed at higher levels than TGFP2 and although those cell lines with the highest levels of TGFPI transcript tended to have lower levels of TGFP2 transcript, there was no clear inverse relationship. Tissue and species specific differential expression of TGFPI1 and TGFP2 has been reported (Seyedin et al., 1985;Cheifetz et al., 1987;Assoian et al., 1983;Derynck et al., 1988;Wrann et al., 1987;Ikeda et al., 1987). However, results obtained using cultured cells must be interpreted with caution. In this study, the cells were harvested at semi-confluence from media containing serum. Cell density in culture has been shown to affect response to exogenous growth factors including TGFP (Ke et al., 1990) Increased levels of RNA were also found in one unmatched field control sample and 1 sample of macroscopically normal urothelium and in one case of follicular cystitis reduced expression was found. This supported the impression that a steady state of expression of TGFP1 occurs during relatively 'controlled' growth in urothelium. The role of TGFPI1 in transformation remains unclear although the ability of transformed cells to respond to TGF,B1 is frequently altered (Lechner et al., 1983;McMahon et al., 1986;Shipley et al., 1986;Wakefield et al., 1987). A number of studies suggest that altered expression of TGFP1 may play a part in transformation (Derynck et al., 1987;Jokowlew et al., 1988;Niitsu et al., 1988). Increased levels of TGFPIB mRNA in tumours compared to adjacent tissues has been reported in a number of tumours (Derynck et al., 1987), including breast and renal cell carcinoma (Coombes et al., 1990;Gomella et al., 1989). It has been suggested that in-creased expression of TGFP1 by non-responsive tumour cells may stimulate tumour growth indirectly via paracrine effects and may also confer an additional advantage on the tumour by suppressing the hosts immunological surveillance. We detected raised levels of transcript in only three tumours and in these there was no obvious correlation with any clinical parameter. Reduced levels of TGFPI1 transcript have been reported in some tumour cell lines (Jakowlew et al., 1988). Undetectable or reduced levels of TGFPi1 transcript were seen in 12 bladder tumours. The significance, if any, of the association between reduced levels of transcript of TGFPI1 and high tumour grade is not clear. A larger sample size is required to clarify this point. However, this was not as striking as the apparent association of reduced transcript levels with tumours which in the relatively short period of follow up became uncontrollable by local means (P<0.02). A similar reduction in TGFP expression has been reported in a series of breast tumours analysed by immunohistochemistry, where expression of the TGFP1 gene product was detected in only 38% (31/82) of tumours and was unrelated to stage and grade (Mizukami et al., 1990). In these breast tumours, it was observed that tumours expressing TGFPI1 were associated with a better prognosis over 2 years. Thus, a reduction in TGFP expression may be common to both aggressive breast and bladder tumours. A number of other molecular changes have been described in both tumour types. These include amplification and overexpression of ERBB2 (Coombs et al., 1989Slamon et al., 1987Slamon et al., , 1989, amplification at 1 1q13 involving INT2, HST and BCL1 Adnane et al., 1989), and loss of heterozygosity of the RB gene Varley et al., 1989). The association of reduced expression of TGFPi1 mRNA and poor prognosis is in keeping with the known growth inhibitory activity of TGFP1 on normal epithelial cells. Loss of expression of TGFP1 might result in reduction of extracellular matrix formation, increased pericellular proteolysis and removal of the negative effect on proliferation (Sporn & Roberts, 1990). Further studies are now required to investigate the relationship of TGFPi1 transcript levels to levels of the mature active gene product in bladder tumours. Although only a relatively small number of tumours have been examined, there appears to be a relationship between reduced levels of expression of TGFPI1 transcript and disease progression. From the information obtained in this study, the presumed loss of the inhibitory activity of TGFP1 in transitional cell carcinoma may be a late event in bladder carcinogenesis. However, this does not preclude its utility as a clinical marker of progression or prognosis.

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2016-05-04T20:20:58.661Z

1976-07-15T00:00:00.000Z

11263393

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Sex-linkage as a factor in the inheritance of sex differences for body weight in two strains of chickens The interactions « sex X Sire families » and a sex x dam within Sire families » in two strains of chickens, Dokki4 and White Plymouth Rock, were studied for body weight at 6 and 8 weeks of age. The results showed that Sire x sex interactions were not significant (P > 0.5 ) in all cases, but dam families within Sire x sex interactions were significant (P 0.5 ) at 8 weeks of age. The effect of sex-linked genes and the low magnitude of heritability of sex-differences were discussed. INTRODUCTION Many investigators showed evidence of the importance of sex-linkage in the inheritance of quantitative characters and some concluded that its effects are too great to be ignored for maximum efficiency in breeding, B EILHARZ (i 9 62). With respect to the phenomenon of sexual dimorphism in poultry, only researches proved the superiority of selection based on dam families over that based on sire families, S HAKL E E et al. ( 1952 ), A YOUB and MA R AT (1972). B EMHARZ ( 19 6 0 ) explained the results of Sxngr.E! et al. ( 1952 ), by the fact that cocks generally distribute identical sex chromosomes as well as autosomal chromosomes to offsprings, while dams (being X-) distribute their sex chromosomes in different manner. This reason may be the cause of the low estimates of heritabilities for body weight differences found by A y ouB and M AGRABY (i9!g), as the method suggested by KI NNEY and Sxo>·>rr!R ( 19 6 5 ) for the estimates of h 2 q_,)) gives only estimates based on sire components of variance. The present work therefore was designed to test the interaction « sex X sire families » and « sex x dams within sire families » in two strains of chicken in order to detect the effect of sex-linkage in the inheritance of sex differences. MATERIAL AND METHODS Two strains of chickens, namely Dokki 4 and White Plymouth Rock, were used in this study. Data on body weight at 6 and 8 weeks of age were analysed using the unequal number model described by S NEDECOR and C OCHRAN ( 19 6 7 ). Differences between Siye families For Dokki 4 strain (table i) highly significant differences in body weight at 6 and 8 weeks of age were observed between both sexes, and sires families, However the sire X sex interaction was insignificant at the same ages. Similar results were obtained for white Plymouth Rock strain with respect to the effect sex, at 6 and 8 weeks of age. But the variance between sires was highly significant at 6 weeks and not significant at 8 weeks of age. The interaction between sire families and sex, was found to be insignificant, F value being i.i¢ and o. 95 (table 2 ) at 6 and 8 weeks respectively. Differences between dam families In the Dokki 4 strain (table 3 ), highly significant differences were observed in both weights at 6 and 8 weeks of age between sexes, dams families within sires and for the dam families within sires X sex interaction. In White Plymouth Rock (table 4 ) a highly significant difference between sexes was observed at the two ages studied. The analysis of variance showed significant differences between dams within sires (P < 0 . 05 ) in both ages. The interaction proved to be significant at 6 weeks of age (i.¢o) at 5 p. 100 level of significance but not at 8 weeks of age (F value being 1 .05). GENERAL DISCUSSION AND CONCLUSION In general the results showed that sire X sex interactions were insignificant in all cases, but dam families within sire X sex interactions showed significance in three cases out of four, the interaction between dam families and sex for Plymouth Rock failing to reach significance at 8 weeks of age (table q.). The inheritance of sexlinkage in the two strains may perhaps be different, although this cannot be unequivocally proven by the present data. On the other hand, A YOUB and M AGRABY ( 1975 ) obtained low heritability estimates of sex differences for the same two strains, which were explained by the mathematical derivation showed by W s!rr and LEGATES ( 19 66). Since the genetic variance of the difference between males and females was reported to be twice the genotype X sex interaction, the heritability in the narrow sense of the difference between sexes for the trait can be written as follows : ( 1 -2 ) -20 ' os la p (¡-2 ) where 0' 2 os is the additive genotype X sex interaction variance component, and a $ P( 1 _ % ) is the phenotypic variance of the differences between sexes for the trait. This derivation assumes that the trait is controlled primarily by additive autosomal genes, and the effect of sex-linkage, dominance and epistasis are not included. Thus the low estimates obtained for h 2 ( ¡ -2 ) in these two strains may be largely due to the small magnitude of Sire X Sex interaction as found in the present work. Moreover, the expected genetic progress as shown also by E IS E N and LEGATES ( 19 66) is 3G(1 -2) = .J 2 ih '(I.-2 ) J!! ,. (Prime notation denotes a Parameter free of scaling effect). One may then conclude according to the results obtained herein of Sire X Sex interaction, that no great change is expected if selection is based on sire families. The present study may suggest the need of the development of a method or system of mating to estimate heritability of the difference between sexes for the trait based on the dams component of variance.

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Extended part-lactations on the basis of last testday milk yield 0. 36 (s h2 = 0.172 ) for three year old trotters and h 2= 0.25 (Sh E = 0.4 8) over all age groups. The expected selection response after selection on racing performance of three year old stallions and mares has been calculated. It amounts to 2.03 seconds per generation if selection is only applied on the male path, and 2.57 seconds per generation after selection on the male and the female path. Assuming a generation interval of five years on the male side and of 10 years on the female side, the expected selection response per year amounts to 0.3! seconds or o. f p. 100 . The record times of trotters have increased during a period of 120 years by i minute which makes an average increase of 0.50 seconds per year. RECOMBINATION OF BLOOD GROUP FACTORS IN THE B SYSTEM IN CATTLE Z. DORYNEK, A. KACZMAREK. Agricultural Academy, Posnan, Poland. Bei i9!9 Kälbern wurden die Blutgruppen in 10 Systemen unter Anwendung von 5 8 Sera bestimmt, wobei 20 Sera zur Identifizierung der zum B-System gehörenden Antigene dienten. In zwei Fällen wurde eine irregulare Blutgruppenvererbung im B-System festgestellt, was als Folge von Crossing-over betrachtet werden kann. Department of Animal Genetics and Breeding, Agricultural University of Norway, !I s-NLH, Normay. A study concerning extention of part-lactation records is done on about 6 200 first and -4 I50 secondto fifth lactations records from the milk recording in Norway. The monthly and cumulative montly milk yield records were pre-adjusted for the effect of age, month of calving, herd, length of first period and calving interval. Studies of the genetic and phenotypic correlations between different information from the lactation showed that the last test was the single information with the highest correlations with the unknown part of the lactation. Five different extention equations were compared : three ratio and two regression equations. The ratio equations were : i) a ratio extention of part-lactation to total lactation directly and 2 ) two ratio equations to estimate the rest-lactation from the last test. The regression equations were a linear regression of the rest-lacation on the last test and a multiple regression of partlactation, last-test and test before last test on total total lactation. The last method was found to be the best in terms of the precision of the extention, but the three methods using last test to estimate rest-lactation were very close to the multiple regression. It is concluded, with reference to the applicability, that the ratio equations to estimate the rest-lactation from the last test should be prefered in practice. The relationships between the resistance to mastitis, measured by the C.NIT, on the one hand and ease of milking and various characteristics of udder and teats, on the other, were studied on 8 farms with Swedish Red and White (.SRB) cows and 9 farms with Swedish Friesian (SLB) cows. RELATIONSHIPS BETWEEN THE RESISTANCE TO The correlations between rate of mastitis and ease of milking were very low and generally non-significant. The relationships with the proportion of the milk from the fore udder were significant in most cases. Large proportions are correlated to higher rates of mastitis in the hind quarters and small proportions to higher rates of mastitis in the fore quarters. Significant correlations were also found with udder height, while other relationships were weaker. It has been observed from the literature that hormone activity of the fetal placenta stimulate mammary developements during the pregnancy. The milk production is also indicated to be limited by the number of milk-synthesizing cells. Observing these two findings together may suggest a working hypothesis that the genotype of the fetus affects the milk production of the dam. EVIDENCE FOR In order to test this hypothesis, first lactation cows calved in the fall were used. altogether four production years were analysed. The data contained j8 8 52 lactations and 1 2 j 7 sires of the calves. The characters considered were lactation yield and maximum daily yield. The lactation yield was corrected for age and expressed as deviation from the herd-average .The maximum daily yield was corrected for the herd-average by using linear regression. The components of variance were obtained by nested half-sib analysis. The components of variance of the sire of the calves were estimated by nesting the sires of the cows within the sires of the calves. The fetal sire effect on the milk production of the dam was expressed as the coefficient of correlation between the phenotype of milk production of the dam and the genotype of the sire of the calf. The estimates of these correlations range from 0 . 0 8 to 0 . 13 for lactation yield and from 0 . 07 to 0 . 09 for maximum daily yield. These coefficients were all significantly different from zero. The genetic correlation estimated between the proof of the sire of the dam and the proof of the same bull as calf sire was essentially zero and non-significant.

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Serum enzymes and metabolites related with high production and longevity Mat in two laboratories. In each year there appeared significant differencies in protein-p. 100 between laboratories but these grew continuously smaller. These differencies like the effects of days after calving, of average herd milk yield and of month of test day were eliminated with statistical corrections prior to estimating the heritabilities and progeny testing, Daughter's fat-p. ioo, milk yield of test day, protein/fat-ratio and fat and protein yields of test day were computed in the same way. The heritabilities of different traits on the basis of one test day were as follows : milk yield 25 p. 100 , fat-p. 100 2 8 p. ioo, protein-p. 100 25 p. 100 , protein/fat-ratio i 7 P. 100 , fat yield 21 p. zoo, protein yield 19 p. 100 and fat + protein yield 21 p. 100. The estimates varied slightly in each year ; it seems that the most reliable estimates were from the year 1973. On the basis of the mentioned heritabilities the number of daughters required for a repeatability of 70 p. 100 in progeny testing were 35, 3T, 35! 53, 4 2, 47 and 42 respectively. In the number of samples these mean zo-3 o p. ioo of what is needed to reach the same accuracy with a monthly sample of i 6 daughters during lactation. Progeny tests based on at least ten daughters were obtained from 13 6, 134 and 15 6 bulls each year respectively. It seems as though with a relatively small number of samples the progeny testing of bulls can be managed. In the progeny testing results the correlation of milk yield with fat-p. 100 was-0. 42 , with protein-p. 100-0. 29 , with fat yield o.8 1 , and with protein yield o. 91. The correlation between fat-p. 100 and protein-p. 100 was 0. 44 , and between fat yield and protein yield o.8 4. The genetic correlations calculated from single observations were similar to these both for signs and values. Metabolites and enzymes in the blood which are normally used for clinical diagnoses (total bilirubin, glucose, cholesterol, GOT, CK LDH and LDH-isoenzymes), were regularly determined in a high yielding dairy herd, during all stages of lactation. The correlations between serum levels and the dairy traits were analysed. What we are really looking for by analysing these correlations is to find indicators for the individual physiological reactions of dairy cows on the stress … of the mentioned heritabilities the number of daughters required for a repeatability of 70 p. 100 in progeny testing were 35, 3T, 35! 53, 4 2, 47 and 42 respectively. In the number of samples these mean zo-3 o p. ioo of what is needed to reach the same accuracy with a monthly sample of i 6 daughters during lactation. Progeny tests based on at least ten daughters were obtained from 13 6, 134 and 15 6 bulls each year respectively. It seems as though with a relatively small number of samples the progeny testing of bulls can be managed. In the progeny testing results the correlation of milk yield with fat-p. 100 was -0 . 42 , with protein-p. 100 -0 . 29 , with fat yield o.8 1 , and with protein yield o. 91 . The correlation between fat-p. 100 and protein-p. 100 was 0 . 44 , and between fat yield and protein yield o. Metabolites and enzymes in the blood which are normally used for clinical diagnoses (total bilirubin, glucose, cholesterol, GOT, CK LDH and LDH-isoenzymes), were regularly determined in a high yielding dairy herd, during all stages of lactation. The correlations between serum levels and the dairy traits were analysed. What we are really looking for by analysing these correlations is to find indicators for the individual physiological reactions of dairy cows on the stress caused by a very high milk production. We think that physiological differences between high yielding cows do exist. There might be cows with a very stabile organism which easily give a high yield, and others which are suffering much more by the stress caused by the production of the same amount of milk. The investigations were carried out during a period of two years in the dairy herd of the University of Munich consisting of about 100 Holstein-Friesian cows. The average milk yield amounted to 7 ooo kg milk per year with 3 .8 7 p. 100 butterfat. Each cow was bled 8 weeks before parturition, i week before parturition, 6-24 hours after parturition, 1 , 2 , 3 , 5 weeks after parturition and afterwards in intervals of 8 weeks. The serum levels of all examined blood constituents were significantly influenced by diseases, age, season and stage of lactation. The influence of diseases was eliminated by only analysing data taken from samples of cows which showed no signs of disease. The influence of season was accounted for by adjustment factors. To avoid biases caused by differences in age and stage of lactation intraclass correlations for different stages of lactation were calculated within age classes. Numerous significant correlations between the serum levels and the milk yield (milk kg, FCM, fat-p. 100 , fat kg) at different stages of lactation were found (r = 0 . 23 -o.61). For all parameters, rank correlations were calculated. The ranking was determined by the deviations of the measured serum levels from the regression line relating milk yield and serum level. Significant rank correlations between glucose and GOT, cholesterol and glucose and between GOT and LDH-isoenzymes i !-2 were detected. The correlations between the serum levels of two successive lactations were calculated as measurements of the repeatability of the different parameters. Some parameters showed close correlations. In all stages of lactation glucose had the closest correlations of all the parameters examined (up to 0 . 997 ). The serum levels of glucose and cholesterol samples, taken 8 weeks before parturition when they are nearly uninfluenced by the milk yield, were about 10 p. 100 higher in animals selected than in animals culled during second lactation. However, the difference is not significant. The other parameters showed no differences between selected and culled animals.

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Some considerations on selection criteria and optimization for terminal sire breeds SUMMARY The efficiency of selection of beef breeds for terminal crossing has been analysed in terms of selection criteria for growth and methods for selection of sires, notably with reference to the situation and research in France. With respect to selection of specialized beef lines, the advantage of control of the individual feed intake and of carcass performances as compared to control of growth only is shortly discussed. For this purpose, not only the favourable indirect response but also the resulting increment of feed intake and changes in body composition according to the reference system used (weight, duration, age) should be taken into account. Because of the increase in birth weight due to the intensification of selection for muscle growth within the beef breeds and the strong genetic correlation between this weight and the paternal component of calving difficulties, it seems advisable to use restricted indices for selection on birth weight, at least for terminal sire lines used in heifers. In the French situation of terminal crossing practised on suckling cows of dual purpose or hardy breeds with the aim of producing veal calves, a special attention has been paid to the genetic and economic advantage of two stage selection for choosing bull sires : selection on a sta ( 1 ) Report presented at the first European Hconomic Community (E.E.C.) Genetic Seminar on a Optimization of Cattle Breeding Schemes ,, Dublin, Ireland, November 2 6-2 8th, 1975 . France is much concerned by terminal crossing on account of the great number of AI performed for this purpose (about 2 millions), the availability of beef breeds and the efforts made to set np an integrated selection scheme. As pointed out by HILL (I9!I), two separate aspects are to be considered : i) the choice of breeds, ii) the choice of selection programmes maximizing the efficiency of selection. The first important point of comparaison of the relative merit of different terminal sire beef breeds or lines will not be discussed here. Furthermore, the optimization methods of breeding schemes will be divided into two parts : genetic and economic. For genetic considerations, it is not the purpose of the author to present an exhaustive review of objectives and criteria for selection of a terminal beef breed after the very comprehensive previous papers, especially those of C ARTWRIGHT ( 1970 ), P RES T ON and W ILLIS ( 1970 ) and M É N ISSIER et al. (I9!5). Therefore only two topics related to selection on growth will be considered as growth for a terminal sire line appears to be the most important factor to be controlled genetically. After trying to elucidate selection criteria for fattening efficiency and improvement of calving g ability, some results will be supplied about theoretical optimization of selection procedures for a terminal sire line. GROWTH, CONSUMPTION, FEED EFFICIENCY AND BODY COMPOSITION Many studies have been reported on these topics (Koc H et al., I9 6 3 ; GREGORY, I9 6 5 ; T AYLOR , I9' jI ;F ITZHUGH and T AYLOR , 1971 ). Only some general results will be supplied here and only not entirely elucidated questions for which there is no general agreement will be treated. -Rather high genetic variability exists for growth rate and appetite and these two traits are very highly correlated genetically : PRESTON and WILLIS (1 970 ), PETTY and CART-WRIGHT ( 19 66) indicate heritability mean values of 0 . 52 and 0 . 54 for postweaning growth rate, 0 . 70 and o.62 for final weight after performance test or in feedlot, respectively. The estimated heritability values for feed intake range between 0 . 35 and 0 . 7 6 according to P RESTON and W ILL I S ( I97 o). KOC H et al. ( I9 y 3 ) found a genetic correlation estimate of -!-0 .6 4 between daily gain for a postweaning period of 1 6 0 days and daily feed intake (adjusted for mid-weight). -In these conditions, a very close genetic correlation generally appears between growth rate and the economic character (feed efficiency) measured by the ratio of gain to feed (or inversely) consumed in the same period, as shown by numerous experimental data (rg = &mdash; 0 . 32 to -O. !9 for PRESTO N and WILLIS, 1970 ;-0.8!, -o.g6 for N EIMANN -S ORENSEN and A NDERSEX cited by K RAU SS I C H , 1974 in Danish cattle) and demonstrated theoretically as well by S UTHERLAND ( I g65). -Therefore, as clearly proved in long term selection experiments in laboratory animals (S UTHERLA :V D , 1974 ) and illustrated in table I for beef cattle, selection on growth rate is very efficient to improve feed efficiency : at least about 8 0 p. 100 of the improvement expected by direct selection. As a matter of fact, efficiency of selection on growth for feed efficiency appears, to a great extent, to be due to an indirect selection for appetite. KocH et al. ( 1973 ) evaluated to 4 o p. 100 the amount of genetic differences in feed consumption which explains genetic increase of gain. For other authors : T AYLOR and F ITZHUGH , ( 19 68) in cattle; T IMON andE ISEN ( 1970 ) andS UTHERLAND ( 1974 ) in mice, increased feed efficiency by selection on growth should essentially result in increase of appetite, but in no change of net intrinsic feed efficiency of tissue growth in opposition to the figures of Iiocx and table i. -Another important question is the influence on body composition of selection on growth or feed efficiency. Since selection for growth will alter the shape of the growth curve increasing particulary the mature weight and modifing the degree of maturity, change in the body composition can be expected. T IMON and EisErr ( 1970 ), SurxERtnNn et al. 1974 ) report that fastly growing lines of mice show higher fatness percentage at constant age ( 7 and i 5 p. ioo fatter than the controls, for males and females at 49 days of age for generation 42 in S UTHERLAND ' S experiment) and even, but to a less extent, at constant weight. The same tendency could be expected in beef cattle from the results of genetic analysis of maturing patterns made by F IT2HU <;H and T AYL O R ( 1971 ) ; SMITH et al. (i 975 ). Since there are a relatively large amount of genetic variablity of the degree of maturity until 1 8 months and positive genetic correlations between, on the one hand the degree of maturity and, on the other the weight at the same age and growth rate during this period, selection for increased growth rate or final weight at constant age will increase average degree of maturity at this age and therefore fatness. On a within breed basis, PRESTO N and WILLIS ( 1970 ) give experimental evidence of this fact that, on a constant age basis, faster growing animals exhibit fatter carcasses. But, on a fixed end-weight basis, there is either no change or the animals will be leaner. Selection on feed efficiency would lead to the same tendency for leaner carcasses as reported by these authors. Thus, recording of feed or/and carcass assessment can be justified in order to limit the unfavorable effect of increased appetite on fatness at a given age by selecting on growth only. However, as the final objective must be measured on an economic basis (value of carcass less feed and time variable costs) usefulness of the different criteria (growth, feed-intake, body composition) appears to be not so easy to elucidate and will also depend on reference or/and production systems (constant weight, constant age or constant degree of finish). Thus, DICKERsox et al. ( 1974 ) studying selection indices to predict the economic efficiency during postweaning period, found that more emphasis should be laid on reduction of back-fat and feed intake on a weight constant basis than on an age constant basis ; these two criteria add very little as compared to accuracy obtainable from weaning weight and postweaning gain (R = 0 . 5 6 vs 0 . 59 respectively). These theoretical considerations are very important for defining test procedures as argued by IixAi''ssticx et al. ( 1974 ) in a cattle commission of the E.A.A.P. It is sure that in the light of present knowledge on these topics, especially genotype-environment interactions which would be interesting to consider in connection with so different types of production and management systems as those existing in France, it is often difficult to take decisions without compromises. SELECTION FOR GROWTH WITH LIMITATION OF BIRTH WEIGHT As well established by M ÉNISSIER ( 1975 ) the choice of the optimum combination of parental breeds allowing production of the heaviest veal or yearling calves without exceeding the critical threshold of calving difficulties can certainly be planned more objectively. This procedure offers the possibility of using a large range of sire lines, on account of the diversity in size and calving ability of indigeneous dam populations, the effect of age of cows and the replacement rate combined with maximum possible rate of crossing (C UNNINGHAM and M C C LINTOCK , i 974 ; E L S EN and : B 10C QUOT , 197 6 a). In crossbreeding schemes, which take into account the age of the cows by use of different kinds of sires for young and mature cows, selection policy, particularly for growth, might be very different for these two situations. A. -Genetic increase in birth weight due to present section for growth Since the main economic traits such as weaning and yearling weight are strongly correlated genetically with birth weight, selection for growth practised at present will indirectly increase birth weight and also the direct paternal component of calving difficulties (table 2 ; B ELIC and . M!NISSIER, I9 68 ;FOULLEY et al., 1975). This theoretically expected increase of birth weight due to selection is generally well confirmed by field and experimental data. Under French conditions of field progeny-testing of t1I bulls for veal production on 75 -day-weight and muscling score, the genetic superiority in birth weight of selected bulls over contemporary tested bulls is estimated to be -!-0 . 4 kg for the Limousin breed (F OULLEY and G AILLARD , 1975 unpublished). P OIVEY ( 1973 ) found similar values of -! 0 . 54 and !-o.61 for Charolais and Blonde d'Aquitaine bulls, respectively. As a matter of fact, genetic change will increase with improved efficiency of the different selection stages involved in the selection scheme of AI terminal beef bulls (G AILLARD et al., 1974 ). Thus, for instance, in an individual selection experiment carried out on 3 Hereford lines for weaning weight, yearling weight and index of yearling weight and muscling score, KocH at al. ( 1974 ) observed over a 10 year-period, substantial genetic responses on birth weight in al three lines ; mean change was estimated to be o.zz, o.z8 and 0 . 2 8 (expressed in standard deviation units : about I kg) per generation in weaning weight, yearling weight and index lines, respectively. From a theoretical point of view, with individual and progeny selection rates of 1/4 and 1/3 , respectively, a genetic change of about + 1 . 5 to + 2 p. 100 per generation can be expected on birth weight by selecting bulls on yearling weight. So, it seems very important to control genetic change in birth weight. In France, the set up of a national sample of reference bulls in each beef breed since 1971 will be very useful for this purpose (CoLL$au et al., 1974 ;F OULLEY and G AILLARD , 1975 ). Thereby, it might be relevant to consider for the case of a terminal sire line, selection for growth with some limitation with respect to birth weight since direct selection for rate of birth difficulties would not be as efficient unless a very high number of progeny are recorded ( 250 to 300 progeny according to VI AHON and CUNNINGHAM, 1975). B. -Efficiency of restricted indices Using the theory of restricted indices and calculation procedures developed by MALLARD ( 1972 ), F OULLEY and R OUVIER ( 1971 ) primarily studied genetic consequences of imposing the restriction of no genetic improvement in birth weight for AI terminal beef sires. This restriction leads to a 5 8 and 74 p. 100 reduction of expected genetic improvement in 75 dayweight in Charolais and Limousin calves and to 22 and 4 6 p. I oo lowering of the economic value. The same restriction has been applied by M OLINUEVO ( 1971 ) for individual and progeny selection of Chavolais and Limousin bulls used in purebreeding ; his results imply similar conclusions as far as relative genetic cost of restriction in the two breeds is concerned (Four. L t:v and !'IOLINUEVO, I97I ). In the same situation of purebreeding, F ITZHUGH ( 1975 ) compared convientional growth criteria ( 12 month-weight, absolute growth rate) with relative growth rate and restricted selection indices with no change either in mature weight or in mature and birth weight (table 3). Relative growth rate seems to be a very powerful mean (much more than absolute growth rate) for reducing especially the genetic increase in birth weight. But it implies higher reduction in yearling weight than restricted index selection for mature weight or both mature and birth weight (&mdash; 113 vs -27 P . I oo ; table 3 ). Thus, for the particular purpose discussed here, the use of restricted selection indices seems to be more advantageous. As a matter of fact, such absolute restriction on birth weight is an extreme goal. Perhaps, some intermediate restrictions might be more realistic since the relation between the restriction on birth weight and the corresponding decrease of weaning or yearling weight is not linear (F OULLEY and M ANISSIER , 1975 ; fig. I ) For instance, in the case of a progeny selection for yearling weight with 20 calves per sire, genetic increase of birth weight can be theoretically reduced by 50 p. 100 when selecting on a yearling weight minus 2 . 4 times birth weight with a corresponding decrease of improvement for yearling weight of only 7 . 5 p. 100 vs 25 p. 100 with the absolute restriction of no change in birth weight (F OULLEY , 1975 , unpublished). These results are in good agreement with those of D ICKERSON el al. ( 1974 ) established by somewheat different reasoning and genetic parameters. Selecting on « yearling weight minus 3 . 2 times birth weight » to improve the economic efficiency of beef production from weaning to slaughter on a constant age basis including expected changes in cow-herd costs from associated increases in mature and birth weight, they found reductions of expected genetic improvement in birth and yearling weight of 5 6 and 9 p. ioo respectively. For this purpose it can also be suggested to apply the independent culling levels, although they are theoretically less efficient (loss of 1 8 to 3 8 p.ioo for total selection rates on 75 day-veight varying between 5 and 50 p. ioo respectively, according to FouLr.EY and M>: N isstEx, 1975 ). C. -Discussion Even if it seems theoretically feasible to improve weaning or/and yearling weight by selection with little genetic change in birth weight, it may however be asked whether such kind of selection is really efficient. In spite of a large variability in observed responses between replicates, the selection methods based on restricted indices or independent culling levels prove generally to be effective as attested especially by results of S HERIDAN and BARKER ( 1974 ) in Drosophila, OKADA and HARDIN ( 19 67) in Tribolium castaneum and M CCARTHY ( 1971 ) in mice. However, in such antagonistic selection « genetic correlation may be more powerful in impeding component responses than predicted from presently available theory » as concluded by RuTLEDGr: et al. ( 1973 ) from data of a selection experiment in mice on tail length and body weight. Which is the effect of this selection on the change of genetic correlation ? It will be theoretically increased when selecting on a restricted index according to simulation studies of ScxLO2r·: ( 1970 ). Finally, these preliminary studies suggest some methods of selection in order to improve calving ability. Other predictor traits such as calf morphology and gestation length can be considered. The efficiency of these selection criteria have also to be discussed, especially concerning the consequences of direct selection for reduced gestation lengt on calf mortality and calving difficulties. ECONOMIC OPTIMIZATION OF THE SELECTION SCHEME FOR TERMINAL SIRE LINES The development of AI in cattle and the wide diffusion of genetic progress owing to frozen semen techniques as well as the national organization of breeding by the livestock act of 19 66 have allowed the setting up of large and powerful selection units. This is the case now in France for selection of terminal beef sire lines. In spite of the obvious advantages of these large structures, the decisions which have to be taken in order to develop profitability of AI industry and breeders become rather complicated. Under these conditions and taking into account the importance of the national outlay for this purpose, it appears necessary to develop synthetic studies for comparing the different selection alternatives. These studies must take into account not only animal genetic improvement, but also costs implied by selection operations, returns expected by breeders through better performance of calves sired by selected bulls and different time flows of costs and returns according to selection programmes. Since the early work of PouTous and V l ssac ( 19 6 2 ), many studies have been made in this area essentially for dairy or dual purpose populations. Primarily these studies have clearly demonstrated : . A. -The interest of use and selection of terminal sire beef breed especially in dual purpose populations .!NDERSON and L.INDH! ( 1973 ), C UNNINGHAM ( 1974 ) have shown the favourable effect of increasing the rate of terminal crossing on dual purpose or dairy breeds for increasing the total beef production provided that the beef breeds used show some higher (but relatively modest) beef progeny performance than straight-dairy progeny. Furthermore, an increased beef crossing improves the return for investment of dairy selection since the dairy merit is transmitted to the population by fewer inseminations (CU NN I NG HAM and MCCLINTOCK, 1970). In these conditions where terminal crossing is practised on a dual purpose population, C UN -N I N GHAM ( 1974 ) suggested that a selection within the terminal crossing breed would be economically effective : I p. I oo of improvement in a beef crossing breed used in 40 p. 100 of a cow population of I million would give an annual benefit of 1 . 17 million UA ( 1 ) which would justify the setting-up of the selection programme. HILL ( 1971 ) concluded in the same way for a population of the same size with 25 p. I oo of terminal crossing. Furthermore, he showed that selecting ( 1 ) Unit of Account. in the beef breed for beef characteristics leads to a better profitability than selecting within the dual purpose breed : on a 20 year-evaluation period the rate of return was estimated to 27 and 1 6 p. 100 , respectively. But practically no attempt was made to study the optimum design of the selection programme. B. -Optimization of selection procedures within terminal beef breeds Considering now the French suckling cow population of dual purpose and hardy breeds crossed with beef breeds mainly for veal production. M OCQUOT and FouLLr;Y ( 1973 ) compared the following three selection methods : --selection on a station performance test ( 12 month-weight) ; -selection on a field progeny test ( 3 month-weight) with 50 progeny per sire ; .--selection in two stages : at first, on individual performance and then on the breeding value for 3 month-weight estimated from the performance at the two stages. Two stage selection appears to be the best way for selecting bull sires since it does not only provide the best profitability for the most probable values of genetic parameters, but it is also less sensitive to their variation. Two stage selection is always more efficient than progeny selection alone. It is also better than performance test selection except in the case of a low heritability coefficient for 3 monthweight (h 2 = 0 . 10 ) and a high correlation (R = 0-4 ) between individual and progeny performance. When this correlation is low (R = 0 . 2 ), the selection after performance test becomes rather inefficient. Although generally this does not seem to occur, especially with the same recorded traits, we can imagine that for very specialized terminal strains (such as double muscled) needing a very protected environment, individual performance under current conditions might be negatively related with the economic value of their crossbred progeny (V ISSAC , personal communication). On the other hand, as reported by M OCQUOT ( 1972 ), comparison of performance and progeny test will also depend on the relative importance of veal and yearling calves. When the relative number of these latter calves exceeded r / 4 , he concluded that, on a constant cost comparison basis, performance test on yearling weight becomes more interesting than progeny test on 3 month-weight. The commercial use of no selected bulls except they are sons of the best proven bulls justifies by itself the investment made. But, as reported also by HILL ( 1971 ), a choice of these bulls after performance test enhances the profitability of selection. Therefore, performance and progeny test will have quite different goals. Even if the latter remains of high interest for detecting elite bulls, it will not necessarily be applied to bulls before a commercial use, provided they are sons of proven sires. Besides, the choice of bulls for this purpose and also at all stages, will be improved if cumulative information procedures such as the one proposed by C OLLEAU and PO UTOUS ( 1973 ) are applied. Finally, different ways using not only AI can be imagined for disseminating genetic improvement realized through the best proven sires. CONCLUSION An attempt has been made to gather all elements available in order to define optimum selection schemes of bulls for terminal crossing. As far as the application is concerned, it clearly appears that goals and schemes have to take into account : -the differences in age and genetic types of females to be crossed ; -the reproduction conditions : AI vs natural service in various environments. Should we select several lines adapted to each goal ? Or should we take advantage of the efficient integrated schemes devoted to increasing as much as possible muscle development in order to produce for other situations (mating of heifers, natural service), the most fitted bulls by crossing those from integrated schemes with various females adapted to each particular case ? Truth lies probably between these two possibilities. Selecting several terminal sire lines cannot be avoided since this aim will be pursued in several populations and countries in order to realize particular production objectives or to promote breeding stocks. Moreover, the prospect of developing techniques for ova transplantation and possible control of sex ratio in the future will unavoidably lead to a better efficiency of selection through the dam path (LAND and HILL, 1975 ;Er.sEN and M OCQUOT , 197 6 b ;C UNN I NG HAM, 1975 ). Integrated selection units for terminal sire lines are favourable structures for this purpose. ACKNOWLEDGEMENTS The author is indebted to Messrs. B. V ISSAC , F. MA NISSIER and B. B ONAITI for their helpful suggestions and comments during the preparation of this paper and to Mrs. Kirsten RA RAT for her valuable assistance in translation.

v3-fos

2018-04-03T06:11:38.213Z

1977-01-01T00:00:00.000Z

21638392

s2

Role of intestinal microbes in body composition in germ-free, gnotobiotic and conventional mice. To study the role of intestinal microbes in body composition and the effect of Staphylococcus epidermidis (Staph.) on body nitrogen (N) accumulation in host mice, ICR strain male, germ-free (GF) mice, gnotobiotic (GB) mice, obtained from GF mice monocontaminated with Staph. at three weeks of age, and conventional (CV) mice were used. All mice were separated into two groups, one group was killed at five weeks of age (B5) and the other group was killed at eight weeks of age (B8) after being fed an irradiated purified whole-egg protein diet for three weeks. The body weight gains in the three-week period were higher in CV mice than in GF and GB mice. The moisture contents of a carcass per 100 g of body weight were lower in CV mice than in GF and GB mice in both the B5 and B8 groups. The lipid and energy of the carcasses of CV mice in B8 group were higher than in the other mice. Crude protein of the carcasses of CV mice in the B5 group was higher than in the other mice. Accumulation of dry matter, lipid and energy per mouse for three weeks was higher in CV mice than in GF and GB mice. Crude protein accumulation per unit body weight gains in GB mice was higher than that of GF or CV mice. With respect to N and energy retention per unit food intake by the slaughter method, CV mice tended to show high values. The results of N balance by the balance method showed the similar tendencies as the results of the slaughter method. GF mice showed higher values for dry matter, N and energy of gut contents per 100 g of body weight than those of the other mice. Gross energy of crude protein and lipid in carcass showed no differences among the three groups of mice. Summary To study the role of intestinal microbes in body composition and the effect of Staphylococcus epidermidis (Staph.) on body nitrogen (N) accumulation in host mice, ICR strain male, germ-free (GF) mice, gnotobiotic (GB) mice, obtained from GF mice monocontaminated with Staph. at three weeks of age, and conventional (CV) mice were used. All mice were separated into two groups, one group was killed at five weeks of age (B,) and the other group was killed at eight weeks of age (B8) after being fed an irradiated purified whole-egg protein diet for three weeks. The body weight gains in the three-week period were higher in CV mice than in GF and GB mice. The moisture contents of a carcass per 100 g of body weight were lower in CV mice than in GF and GB mice in both the Bb and B8 groups. The lipid and energy of the carcasses of CV mice in B$ group were higher than in the other mice. Crude protein of the car casses of CV mice in the B5 group was higher than in the other mice. Ac cumulation of dry matter, lipid and energy per mouse for three weeks was higher in CV mice than in GF and GB mice. Crude protein accumulation per unit body weight gains in GB mice was higher than that of GF or CV mice. With respect to N and energy retention per unit food intake by the slaughter method, CV mice tended to show high values. The results of N balance by the balance method showed the similar tendencies as the results of the slaughter method. GF mice showed higher values for dry matter, N and energy of gut contents per 100g of body weight than those of the other mice. Gross energy of crude protein and lipid in carcass showed no differences among the three groups of mice. The purpose of this investigation is to explain the role of intestinal microbes especially Staph. in body composition in GF, Staph. monocontaminated gnoto biotic (GB) and conventional (CV) mice by comparative slaughter methods. Animals. ICR strain OF mice, GB mice produced by gastric administration of 0.2ml of Staph. incubated culture to three-week old GF mice and CV mice were reared in plastic rearing cages (three mice per cage) until five weeks of age. At this time, six mice were chosen from the 12 mice in each group and were sacrificed (at 10 AM) by etherization (B5 group). Among the mice chosen, the body weights of the 12 mice were measured and these mice were separated into two groups with the same body weight levels as much as possible. The other six mice were reared individually in metabolism cages with the diet shown in Table 1 to those based on 100g of body weight. Accumulation of body components per mouse or per 10g of body weight in crease in the period of purified whole-egg protein diet are shown in Fig. 2. A mounts of dry matter and energy accumulation per mouse for three weeks in CV mice were higher (P<0.01-0.02) than those of GF or GB mice and lipid in GB was lower (P<0.01-0.05) than that of GF or CV mice. Crude protein showed no significant differences but there were higher tendencies in CV mice than in GF or GB mice. Ash accumulation in CV mice tended to be lower than that in GF and GB mice. Dry matter, lipid and energy accumulation per 10 g of body weight gain showed no significant differences among the three groups of mice . Crude protein accumulation showed the highest value in GB mice (vs GF: P<0.02, vs CV: P<0.001). Ash accumulation in CV mice was lower than those in GF (P<0 .02) and GB (P<0.001) mice. The ratios of N, ash and energy retention per food intake and N balance per mouse for three weeks are shown in Table 4. N and ash retention observed by the comparative slaughter method showed no differences among the three groups of mice. However, ash retention of CV mice tended to be lower than that of GF and GB mice. On the other hand, energy retention of CV mice was higher than that of GF (P<0.05) and GB (P<0.01) mice. In the results of the balance method , N retention of CV mice was higher (P<0.02) than that of GF mice. Nitrogen balance per mouse for three weeks=I-E I: nitrogen intake. E: nitrogen excretion in feces and urine. Dry matter, N and energy of gut contents from stomach to rectum per 100g of body weight are shown in Table 5. The data for CV mice of the B5 group were significantly lower (P<0.001) than those for the GF and GB mice, and GB mice were also lower (P<0.01-0.02) than GF animals. In the B8 groups, values in CV mice were lower (P<0.001) than those of GF and GB mice. Between GF and GB mice, no significant differences were shown except in the dry matter results. Gross energy of protein or lipid of the carcass and dry matter of defatted carcass are shown in Table 6. The gross energy of dry matter of defatted carcasses and protein of the carcass did not differ in any of the mice. Gross energy of lipid in GB mice was higher than those in GF (P<0.05) and CV (P<0.001) mice in the B5 group and also GF (P<0.02) mice in the B8 group. DISCUSSION Previous experiments (1-6) indicated that intestinal microbes affected body N accumulation and they had different effects according to the sort of microbes. Staph. increased N accumulation of the host mice. In these experiments, how ever, only an autoclaved CL-2 diet was used. Thus, to examine whether the pre vious results were caused by the use of the CL-2 diet, the irradiated purified diet containing whole-egg protein was used in the present study . The reason for using this protein is that it is considered to be a good quality protein and such a diet can easily be reproduced. Crude protein measurements of the purified whole egg protein diet (10.0%) showed lower values than those of the CL-2 diet (23 .9%), while crude fat measurements of the purified whole-egg protein diet (7 .9%) showed higher values than those of the CL-2 diet (4.6%). In the results of gross energy measurements, purified whole-egg protein diet showed 4 .60 kcal per g of dry matter and autoclaved CL-2 diet 4.37 kcal per g of dry matter . One percent vitamin mixtures are usually used, but the reasons for using a 2% vitamin mixture in the purified whole-egg protein diet were that vitamins might be lost by sterilization and the short experimental period of three weeks. Body crude protein of CV mice was higher than that of GF mice and GB mice also showed higher value than that of GF mice (Tables 2 and 3) . Moisture and ash contents of CV mice were lower than those of GF or GB mice , but lipid and energy contents of CV mice were higher than those of GF or GB mice . The increase in lipid contents in the B8 group of CV mice was especially remarkable . This might be due to the influence of intestinal microbes , but the details are un known. Lipid contents in the B8 group of GB mice were lower than those of the GF mice, and moisture showed the opposite results . The GF and GB mice were not offsprings from the same parents, and therefore these results might be caused by the difference in parents or times of experiments . Moreover, Staph. might have some activities which prevent body lipid accumulation in host mice . For example, gross energy of lipids in GB mice had a tendency to be higher than that of GF mice in both the B5 and B8 groups. STANIER and MOUNT (11) showed a body composition of CV mice as follows: moisture: 65-70g, lipid: 5-12g, N: 2.5-2.9g and ash: 3.1-4.0g per 100g of body weight; our experimental results agreed with the above report . ROBINSON and LAMBOURNE (12) reported that the body lipid content of mice was the reverse of the moisture content, but body protein content was stabilized. In the experiments by TANAKA et al. (13) where rats were fed diets with various ratios of protein to energy for three weeks, moisture contents of the carcasses were decreased by aging and body lipids gave opposite results. In the body protein or ash contents showed similar tendencies in the results of our experiments and also in those of CZAJKA-NARINS and HIRSCH (14). In body component accumulation (Fig. 2), dry matter in CV mice was signi ficantly higher than those in GF and GB mice. In a case not illustrated , lipid accumulation per unit dry matter accumulation in GF or CV mice was higher than that of GB mice and for protein and ash, GF and CV mice gave lower values than GB mice. In the accumulation of body N per unit ash accumulation , CV mice showed higher values than GB and GF mice. The rank was CV, GB and GF mice. In the previous report (6), body N accumulation per 100g of body weight showed increases in the order of GF, E, coil GB, Staph. GB and CV mice. Retention ratios of body components per food intake were generally high in CV mice and low values were shown in GB and GF mice in that order. These results supported the previous results obtained by balance method. Intestinal microbes affect the gut contents more directly than the body com ponents. Dry matter, N and energy of gut contents (Table 5) in GF mice were higher than those of GB and CV mice. The rank was GF, GB and CV mice with significant differences among three groups of mice. These results showed reverse tendencies to body components and agreed with the results of total N of gut con tents in the previous report (6). It is well known that the cecum of GF mice is much larger than that of CV mice and the GF cecum has more contents than the CV mice cecum, as seen in Table 5. No differences in gross energy of protein and lipids of carcasses were observed among three groups of mice. Several researchers have reported the gross energy per g protein in animal bodies as follows: 5.74 kcal for rats (13), 5.348 kcal for pigs, 5.411 kcal for sheep and 5.447 kcal for cows, and also reported on the gross energy per g lipids, i.e. 9.608 kcal for pigs, 9.414 kcal for sheep and 9.499 kcal for cows (15). The value of gross energy of mouse lipid ranged from 8.80 to 9.09 kcal per g of lipid (Table 6). These values were lower than the results of REID et al. (15) for pigs, sheep and cows and the above gross energy of mouse lipid resembles the results of 8.95 kcal per g of rat lipid obtained by TANAKA et al. (13). The value of 5.7 kcal per g of animal protein and 9.5 kcal per g of animal lipid are already known as the values of gross energy. From the results of other researchers and these experiments, it appears that there are species differences in gross energy of protein and lipids.

v3-fos

2017-07-29T04:25:04.276Z

1977-01-15T00:00:00.000Z

40714180

s2

Reproductive efficiency of iceland sheep II. Prolificacy and reproductive performance of adult ewes cross and native breed crosses widened as the animals became older. A study is underway at one Ministry of Agriculture, Fisheries and Food, Experimental Husbandry Farm to determine whether improved nutritional levels, from 6 months of age, will maintain the advantage in prolificacy thoughout life and arrest the widening of the differences in ewe body weights as the animals get older. ! La vitesse de croissance des agneaux Finnois X Aragonais élevés en Aragon (région N.-E.) et Romanov x Mérinos élevés en Extremadure (région S.-O.) effectuée dans des fermes privées a été comparée à celle des races pures respectives. De même on a fait des comparaisons dans une ferme expérimentale de croisements Romavrov X Mévinos, Romanov x Manchega, Romanov X Avagonais avec des races pures en étudiant la croissance et les qualites bouchères des carcasses. Dans tous les cas traités, les agneaux issus de croisement de races prolifiques et de races locales espagnoles ont montré une vitesse de croissance plus élevée que celle qui correspond aux races locales pures, ce qui doit être attribué à la très petite taille des brebis de races locales. La croissance de ces animaux croisés a été presque égale à celle des agneaux issus du croisement avec Ile de France. La conformation des agneaux croisés Romanov a été la même ou légèrement supérieure à celle des animaux de race locale. L'état d'engraissement a été aussi supérieur avec les agneaux croisés. A brief description is given on the fertility of the Iceland sheep. The aim of the breeding the last 20 years has been to increase the fertility. The average number of lambs weaned pr. ewe for the whole population has increased from 1 , 07 in 1950-54 to 1 , 42 in 1 975. There are given examples of differences between districts. From the Sheep Recording Associations there is taken an example of change in fertility through 20 years, from 1 , 04 lambs born in 1950-54 to 1 , 7 6 lambs born in 1975. Results from 10 Sheep Recording Associations for the years 1974 and 1975 are given, showing the average of 0 ,9 up to 2 , 3 per cent of all ewes being barren and 1 , 1 og 3 , 9 per cent of all ewes having triplets. Results of investigations on losses of lambs are referred, showing average losses of lambs from birth (including stillborn) to weaning of 5 … REPRODUCTIVE EFFICIENCY OF ICELAND SHEEP II. PROLIFICACY AND REPRODUCTIVE PERFORMANCE OF ADULT EWES S. HALLGRIMSSON. The Agricultural Society of Iceland, Reykjavik, Iceland. A brief description is given on the fertility of the Iceland sheep. The aim of the breeding the last 20 years has been to increase the fertility. The average number of lambs weaned pr. ewe for the whole population has increased from 1 , 07 in 1950 -54 to 1 , 42 in 1 975. There are given examples of differences between districts. From the Sheep Recording Associations there is taken an example of change in fertility through 20 years, from 1 , 04 lambs born in 1950 -54 to 1 , 7 6 lambs born in 1975 . Results from 10 Sheep Recording Associations for the years 1974 and 1975 are given, showing the average of 0 ,9 up to 2 , 3 per cent of all ewes being barren and 1 , 1 og 3 , 9 per cent of all ewes having triplets. Results of investigations on losses of lambs are referred, showing average losses of lambs from birth (including still-born) to weaning of 5 , 7 per cent. Investigations on fertility of white and non-white ewes are showing higher fertility of the Data on the ovulation rate of mature ewes of the following breeds were analysed: Finnish La,ndrace (F), Galway (G), Fingalway (FG) (Galway ewes X Finn rams) and High Fertility (HF). Average ovulation rates were 3 .8, 1 .6, 2 . 3 and 2 .6, respectively. The repeatability of ovulation rate was o.66, 0 . 15 , 0 . 15 and 0 . 7 8 for the F, G, FG and HF ewes. The corresponding values for the repeatability of litter size were 0 . 07 , o. 2 o, 0 . 0 6 and 0 . 13 . The correlation between ovulation rate and average litter size at the three previous lambings was o.oo, 0 . 0 8, 0 .11 and 0 . 35 for F, G, FG and HF ewes, respectively. Evidence was obtained for a quadratic relationship between ovulation rate and litter size and the data suggested the existence of an optimum ovulation rate. The heritability of ovulation rate was 0 . 3' . fnt Finn ewes and hence selection should be affective.

v3-fos

2014-10-01T00:00:00.000Z

1977-05-01T00:00:00.000Z

11207859

s2

Why the healing gods are twins. The association of twins with health-giving powers is widespread in mythology, folklore, and religion. The Ashvins of the Rig-Veda, the classical Dioscuri, and the early Christian saints Cosmos and Damian are among the many examples of twins divinely empowered in the area of health and fertility. A characteristic set of attributes of twins recurs in different mythologies of wide distribution. In addition to healing, divine twins are often empowered with the ability to revive the dead, increase the fertility of man, animals, and crops, influence the weather, predict the future, and insure victory in battle. In some traditional societies these special attributes are thought to extend to all of the twins and their parents in the tribe. Ancient and primitive societies supposed that the birth of twins was associated with divine influence, the mother having been visited or otherwise affected by supernatural powers. A frequent explanation was that twins were the result of superfetation, a divine impregnation occurring along with that by the lawful husband. The specific powers of divine twins appear to be a reflection of the particular form of origin of twins through divine interference with the fertilization process. The twins thus share some of the powers of the divine parent, particularly those pertaining to fertility. Their dual paternity and its inherent competition is related to their martial interests as well as their ability to resolve ambivalent or ambiguous situations and predict outcomes. Ye gave to Kali, when he had grown old in years, To him, the singer, all his youthful strength again; And Vandana ye rescued from the deep abyss, And quickly Vicpala the maimed ye made to walk [1]. It has long been noted in folklore, mythology, and religion, that a pair of divine or saintly twins were often associated with the healing arts. This relationship has occurred widely in time and place and has posed an intriguing folkloristic pattern. Sigerist states, for example, in observing the widely dispersed occurrences of supernatural twins with healing powers, that no valid explanation for this relationship has been offered [2]. The interesting question as to the meaning and origin of this association brings together the viewpoints of the separate disciplines of the study of mythology and the history of medicine. The healing powers of twins are to be found in the context of a widespread folkloristic pattern of attributes and powers associated with twins [3]. It will be of value to survey briefly the themes of myth and folklore associated with twins in order to focus on the specific question of the healing powers of twins. The birth of twins as a natural variation occurs infrequently enough (one in 80 to 100 live births) to be a source of curiosity and to provoke theories regarding their origin. Perhaps most basic is the fact that twins are a natural and available symbolic representation of the tensions and forces in man and nature. Twins serve the imagination as a symbol for philosophic dualities and formulations on the theme of opposites and as a metaphor for balances and competing forces. The mythologies of every continent contain twin 307 gods, twin offspring of gods, twin heroes, and ordinary mortal twins in various story cycles. In addition to folklore accounts, every society has its special practices and attitudes toward the birth of twins. In view of the recurrent findings in folklore and religion, we might ask if we are dealing with a universal human phenomenon. Direct cultural transmission can account only in part for the worldwide distribution of similar twin folklore features. Every continent and every ancient society appears to have taken note of twins and developed folklore, mythology, or legend in relation to twins. On the other hand, direct cultural transmission is a significant factor in some aspects of twin culture. In particular, a cult of divine twin healers appears in a wide belt stretching from ancient India to the Mediterranean. The various features which are similar at many points in this vast geographical swathe point to a common religious development in the ancient world in relation to twins as a worshipful object. By far the most well known twins of western mythology are the Dioscuri, Castor and Pollux (Polydeuces), whose supernatural powers included those of healing. Other twins figure prominently in Greek mythology. The twin of the mighty Heracles was Iphicles. The offspring of the incestuous union of Oedipus and Iocaste are twins, Eteocles and Polyneices. Of particular interest in the present context is the mythical healer Asclepius who had a twin Ericthonius. Asclepius was fathered by the god Apollo while his brother was the result of a second liaison by his mother with a mortal. Asclepius in turn was father to twins, Macaon and Podaleirius, both highly skilled in medical and surgical arts, who served as physicians to the Greeks in the Trojan War [4]. Twins are important in native American mythology and folklore [5]. North American Indians as well as South American, including Peruvian and Amazonian tribes, all have twins in significant sequences in their mythologies. In Iroquois and Huron Indian mythology a pair of twin heroes are responsible for opposing good and bad forms of nature [6]. Radin in extensive studies of North American Indian mythology has placed the twin myths as the last phase of a sequence in heroic mythology [7]. Utilizing the details of the Winnebago cycle of myths Radin delineates a progression through four evolving phases: (1) the trickster, an undifferentiated primordial figure;8 (2) a partially but imperfectly differentiated individual, usually the hare; (3) a more differentiated Olympian figure, called Red Horn; and finally (4) the twins, a Promethean pair who wander widely attempting to master the world and to change the cosmos. They subsequently meet their limits and end in tragedy. Radin feels that this grouping of myths in a progressive sequence is to be found widely, e.g., Polynesian, Japanese, Greek, and Hindu mythologies, and is reflective of the evolution of primitive man's thought. A highly detailed twin sequence has been preserved in the record of the creation myths of the Quiche nation of the pre-Columbian Mayan culture of southern Mexico [8a]. In this intricate and convoluted account, two pairs of mythical twins are involved. The first twins, Hun-Hunahpu (or One-Hunahpu) and Vucub-Hunahpu (or Seven-Hunahpu), are summoned to the Underworld and are eventually killed by the death gods. The head of One-Hunahpu is hung up in a calabash tree where it spits into the hand of a daughter of one of the death gods [8b]. She becomes pregnant giving birth to the Hero twins, Hunahpu and Xbalanque. They grow up, defeat various opponents including a rival pair of cousins, and eventually are summoned to the Underworld. They overcome the Lords of Xibalba by a series of tricks, ending in the destruction of the two gods, One-Death and Seven-Death [8c]. The Hero twins then rise up to become the Sun and the Moon. [8d]. As will be discussed below, this latter element in particular bears an interesting similarity to Egyptian theology. Christianity has its twin themes as well. The two sons of Zebedee, James and John, are called Boanerges by Christ, a name meaning "sons of thunder" (Mark 3.17). This epithet is equivalent to that used for Castor and Pollux, the Dioscuri ("Zeus' boys"), and it has been suggested that this is a remnant of the worship of twin divinities in the ancient Near East [9]. Christ is, of course, a divine healer; and it is noteworthy that he is referred to several times in an apochryphal work as the identical twin of the apostle Judas Thomas [10]. The work entitled the Acts ofthe Holy Apostle Thomas, which is believed to have been composed in Edessa in the 3rd century [11], makes explicit mention of the twinship and describes how Judas Thomas and Christ are mistaken for each other. Thus Christ provides another example of a twin with healing powers. Also famed as divinely guided healers are the early Christian saints Cosmos and Damian. The folklore of twins often presents two contrasting themes, one a constructive role of twins as builders or founders and the other, a destructive role as warriors or competitors. Mythology, folklore, and religion often cast twins in a key role in beginnings or origins. Thus twins often are associated with the establishment of the ethnic group, the founding of a city, the beginning of civilization, or the creation of the world. There is often a etiological motif in the twin narratives, i.e., the circumstances pertaining to the twins account for some enduring feature of the descendents. In the Bible the first natural born of mankind are the twins Cain and Abel; and the offspring of Isaac, the twins Jacob and Esau, are the sources of the two great contesting Semitic strains, the Hebrews and the Arabs. The founders of Rome are Romulus and Remus. The Spartan kings are descended from the famed Dioscuri [12]. Amphion and Zethus are the mythological twin builders of Thebes in Greece [13]. The constructive aspect of twin mythology includes the roles of civilizers or culture-bearers. As mentioned above, Radin notes twins at the phase in the cycle of myths in which mankind is given gifts for his control of nature after the earlier less settling phases of creation. In the mythologies of North American Indians, e.g., Navajo, Zuni, divine twins fathered by the Sun undertake prodigious tasks to reduce the dangerous forces opposing mankind. The twins instruct men so that survival of the human race is assured in the face of great dangers and forces of destruction [14]. South and Central American Indian mythologies similarly portray a pair of divine twins who bring the gift of fire and the arts of civilization to man [15]. The murderer twin of the Bible, Cain, is also connected with man's technical advancement. He founds or builds the first city (Genesis 4.17) and according to tradition, contributes the associated arts of boundary-fixing, measuring, weighing, and fortifying. As befits a murderer who becomes a culture-bearer Cain also taught vice, luxury, and indulgence and put an end to man's simplicity [16]. In contrast to the builder theme, the mythology of twins often features destructive elements in the form of competition, rivalry, and aggression. The rivalry of the twins may have a dynastic significance as with Jacob and Esau. Furthermore, the rivalry is foreshadowed by a struggle which starts while the twins are still together in their mother's womb (Genesis 25.22). Other examples of intra-uterine twin antagonists are Pharez and Zarah (Genesis 37.28-30), Proteus and Acrisius [17], the Iroquois twins, Flint and Sapling [18], and the Huron Creator-twins [19]. The competition in mythical twins often culminates in fratricide. Romulus and Remus have a fatal quarrel as do Biblical Cain and Abel and Theban Amphion and Zethus. A variation of the fratricide theme is the simultaneous mutual slaughter of twins in combat with each other. Such is the case of Eteocles and Polyneices [20] as well as the gigantic twin sons of Poseidon, Ephialtes and Otus, who had besieged the gods on Olympus by piling Pelion on Ossa [21]. A variation on the twin's sibling rivalry is found in classical mythology where Castor and Pollux have a protracted feud not with each other but with another pair, their twin cousins Idas and Lynceus. This rivalry culminates in a titanic struggle and the deaths of Idas, Lynceus, and Castor [22]. Still another variation is in the myth of Aeolus and Boeotus, also twin sons of Poseidon, who are the object of a plot by their foster mother Theano. She instructs her own twin sons to kill Poseidon's but the tables are turned and Theano's twins are killed by Aeolus and Boeotus [23]. In the case of the Quiche Mayan myths, a fatal competition of an intergenerational nature occurs several times involving sets of twins and/or pairs of brothers [23a]. An interesting feature in the mythology and folklore of twins is the multiplying of the participants. Twins are found to be reduplicated and born as quadruplets, other twins appear as rivals, and twins give birth to twins. Poseidon has at least five sets of twin offspring [24]; Jacob has twelve. Why the replication of twins in mythology? Levi-Strauss explains this tendency of mythic and oral literature to multiply the same sequence as a thematic repetition serving to make the structural aspect of myth more apparent [25]. Paul Radin has taken note of a related feature in his studies of American Indian mythology and regards it as a reflection of a particular mentality in primitive man. Radin has characterized the majority of primitive men as actionoriented, seeking a repetitive rhythm of events, and unconcerned with progression or evolution of concepts [26]. The man of action, as Radin has labeled him, in contrast to the thinker philosopher, is not concerned with the monotony which comes with repetition but finds in it the preoccupying and diverting quality of ever-changing external reality as he perceives it. Kluckhohn in a wide-ranging formulation of the phenomenon of myth has pointed up the satisfactions served by myths in primitive society where they occupy a position comparable to that of literature in literate society [27]. The myth serves the need for emotional discharge and expression. It edifies and entertains. The repetitive character of myth and its use of the familiar word and form is basic to its efficacy and charm since it utilizes well established channels of communication which enhance and color the myth. In the world of the supernatural, the special personal gifts of twins form a highly characteristic pattern. Both mythical and human twins are ascribed extra-ordinary powers and abilities. The broad super-natural powers of Castor and Pollux are well depicted in classical writings and remains. They are the saviours of shipwrecked sailors, senders of favorable winds, guardians of hospitality, inventors of martial music and the war dance, patrons of bards who sing of ancient battles, and the particular protectors of the Spartans in battle. In addition, they have healing powers and aid women in childbirth [28]. Their many adventures in classical mythology relate to their prowess in war and athletics. On their white chargers they might swoop down to answer the prayer of a nearly doomed individual. The feats of the Dioscuri as rescuers or saviours provide a particularly interesting theme [29]. As will be discussed, the role of miraculous rescuer was a central theme in the mythology of the Ashvins and one of the clear indications of their connection with the Dioscuri. Euripides casts the Dioscuri in the role of rescuers of their sister Helen, the chorus singing: May you riding down through the bright air, swift on your horses, sons of Tyndareus, come down the storming courses of your stars' flaring, oh, dwellers in the sky, saviors of Helen . . [30] Just as the Dioscuri lead the Spartans in battle, the human twin was also regarded as particularly potent in warfare and designated to go at the head of an African tribe into battle [31], or required to engage in particular ritual performances prior to battle in order to ensure victory. A particular focus of twins' powers lies in relation to the weather [32]. Among many African tribes the mortal twin was believed to have special abilities to bring about good weather, abundant rainfall, and consequently good crops. The twin was considered able to manipulate the forces which made for good weather or act as an intermediary for the tribe in obtaining good weather. The Kwakiutl and Nootka Indians of British Columbia regard the birth of twins as a good omen for salmon fishing [33]. Another quality prominently associated with twins is the ability to divine the future or carry out various clairvoyant or prescient activities. Thus, Iroquois twins are associated with the prediction of the future; the Golah of Liberia, the interpreting of dreams [34]; and the Peruvian Indians and African Zulus, the foretelling of weather [35]. Among mythological twins their clairvoyance is translated into a role as a teacher of navigation and a patron of travelers, wayfarers, and sailors, features common to both the Dioscuri and the Vedic Ashvins [36]. It is natural that beliefs in the healing powers of divine twins should be closely tied to ancient religious practices. The mythology of supernatural twins takes its most coherent form in what has been termed the cult of the divine twins or dioscurism. From India to the Mediterranean there is ample evidence of the ancient worship of markedly similar twin gods. Many of the sites of worship throughout this broad cultural belt show a variety of etymological links [37]. From the Dioscuri of the classical world, the tradition of healing twins was passed on to Christianity. The Christian saints Cosmas and Damian were revered for their miraculous cures such as the transplantation of a limb from a cadaver to a maimed man and the removal of a snake from a boy who had swallowed one. Following the martyrdom of Cosmas and Damian in a Diocletian persecution in 303 A.D. [38], the sick sought faith cures by sleeping overnight at their tomb at Cyrus in northern Syria. Two centuries later Justinian I moved their remains to Constantinople where the pagan shrine of the Dioscuri was rededicated to Cosmas and Damian. The practice there of healing by incubation was thus passed from the one religion to the next [39]. By far the most ancient and detailed writings referable to dioscurism are those of Vedic mythology which speak of the twin Ashvins. They are often mentioned in the most ancient of the Vedic writings, the Rig Veda, and occupy a place of honor in the Vedic pantheon as well as in later Hindu mythology. The Rig Veda in which the accounts and references to the Ashvins are to be found is dated at somewhere around 1500 to 2000 B.c. The Rig Veda is written in ancient Sanskrit and is the surviving work of the Vedic or Aryan people who preceded the Indian and Iranian civilization [40]. It is the most ancient of the varied complex of Vedic writings, and the most ancient written source associated with the cult of the divine twins. The Vedic references pre-date the earliest classical Homeric writings by as much as 1,000 years. Attesting to the ancient status of the Ashvins is the famous Mitanni inscription in northern Mesopotamia dated about 1400 B.C. which lists one of the names of the Ashvins, Nasatyas [41]. The Ashvins, often pictured as divine horsemen, are referred to 400 times in 50 out of the 1,028 hymns of the Rig Veda. The parents of the twins are variously mentioned as the sun, heavens, ocean, earth, or two great kings [42]. They ride in a golden shining chariot, herald daybreak, overcome darkness, and appear as the respective stars of morning and evening. They are the physicians of the gods and perform extraordinary medical feats including the revival of the dead, the restoration of sight to the blind, helping the lame to walk again, replacing a head which has been cut off, and providing prostheses for amputated limbs. They also assist in childbirth and help to cement happy marriages. The aged and impotent are restored to full vigor by them. They are the inventors of medicines and bring healing arts to mankind. Like Prometheus their philanthropic acts earn them the disfavor and suspicion of the gods. They are inseparable companions. Their harmonious ability to coordinate themselves in good works is a model for all happy dualities. They are compared, for example, to a happy married couple, the two horns or two hoofs of an animal, the two seeing eyes, the two lips speaking sweetly, or the two confluent rivers [43]. Like two hands be investing us with vigour, like heaven and earth subduing the atmosphere, Our songs, Asvins, that proceed towards you, sharpen them well, like an axe upon the whetstone [44]. The Ashvins are ever ready to correct wrongs or outrages; and they magically snatch from danger those who call on them for help. They seek out the man who has been captured by bandits and free him, save one from drowning, and pull another from a burning chasm. The quail in the mouth of the wolf is rescued. Throughout the lore of the Ashvins is the,theme of their marvelous ability to reverse or negate nature or some nearly final catastrophic state. They are thus mediators between an outcome and its reversal, mirroring their existence as harmonious twins and bearers of the light of dawn into darkness. They are the great reconcilers. Levi-Strauss in his structural analysis of mythical thought formulates a conceptual framework of polarized terms which are mediated to form a third term. The third term is polarized against another in a process of continual structured contrasts [45]. The Ashvins are symbolic of the process of accommodation of opposing forces. Various contrasting or competing forces (light and darkness, sickness and health, violence and tranquility, etc.) are brought together to produce a harmonious synthesis. The Ashvins are the personification of coordinated action by a duality. The myth of their birth may be interpreted as an allegory on the theme of reconciliation. In one version, the goddess Sanjna ("Conscience" or "Understanding") being unable to stand the brilliance of her husband, the sun, Surya or Vivasvat ("Righteousness" or "Eternal Law"), separated from him to a shaded spot and assumed the form of a mare. He accommodated himself to her transmutation by becoming a stallion and followed her to her forest retreat. The offspring of their union were the Ashvins [46]. Thus the benevolent healers are the result of godlike brilliance being united with the retiring form of understanding needed to relate to mankind's limitations. Folklore offers several different theories on the origin of twins. The birth of twins was regarded with particular awe and concern by ancient man and primitive and traditional societies; and throughout the world recognition of the birth of twins is noted in folklore and custom in the attempt to resolve the tension created by their birth. It is apparent that the birth of twins raised questions regarding the conduct of the parents. The mother of twins in some societies was required to undergo special rites of purification and separation from the tribe for a specified period, as for example among the African Ibibios tribe [47]; she may be exposed to physical punishment or even killed [48]. The offspring at birth might also receive special treatment, in certain cultures one twin being sacrificed. As an alternative to severe punitive measures refuges were set up where the mother could go in safety with her newborn to live out her life or the period of the taboo. Whereas the twins and their mother were regarded with fear and rejection in some cultures, in others they were objects of particular favor. Thus, the Bantu mother of twins receives special recognition by the tribe [49]. The twins themselves might be highly regarded and entitled to special privileges and honors. The treatment accorded the mother or twins was related to the particular theory of twin origins entertained by the culture. Paternity appears to be the central question for most folklore theories explaining twin births. One theory among traditional societies is that twins are the result of superfetation, i.e., they are the result of two fathers and are therefore evidence of infidelity on the part of the mother. This is so among those tribal cultures where the basic belief is that a human father can only beget one child at a time. It is natural in such societies that the birth of twins will be regarded in a negative way. Another theory is that twins are the result of divine intervention or even impregnation of the mother by a divinity or divine force. The theme of divine intervention appears in the case of Jacob and Esau where their father Isaac had asked for God's help in relieving the barrenness of his wife Rebecca (Genesis 25.21). The more primitive belief is that the god is actually the impregnating force. In Greek mythology the sea god Poseidon appeared to be particularly endowed with the ability to produce twin offspring in his liaisons with both mortal and divine partners [50]. A variation on the theme of divine intervention is that the birth of twins is related to an act of reincarnation, a claim made by shamans at times as support for their access to nonrational realms. The theory of divine impregnation as the source of twins is frequently combined with that of superfetation in a belief that the twins represent one mortal and one divine father. This is a common feature in Greek mythology where an account may describe a god visiting the wife already impregnated by her legitimate spouse or conversely where a woman impregnated by a god takes a mortal partner. In either case there may be hurt feelings and vengeful consequences. The Dioscuri were born to Leda the wife of Tyndareus who was pregnant by him as well as by Zeus. Their miraculous generation is variously described. In one version they form quadruplets born along with a pair of sisters; in another version the four are born from two eggs laid by Leda as a result of a pregnancy by Zeus in his form as a swan [51]. The accompanying female pair are the famous Helen and Clytaemnestra. An interesting parallel is a midrashic tale of the first offspring of Adam and Eve. According to the account, Cain and Abel each had a female twin born along with him [52]. Another midrashic tale tells of a female twin born with each of Jacob's twelve sons [53]. Besides the Dioscuri and their sisters, other cases of god-mortal superfetation in Greek mythology include: Lynceus and Idas, Heracles and Iphicles, Dardanus and Iasion, Acrisius and Proteus, Asclepius and Erichthonius, Eurytus and Cteatus, and Amphion and Zethus [54]. The competitiveness of twins and their struggle with each other while still in the womb might be seen as a natural extension of the two paternal rivals for the mother's sexual favors. In life a more material issue might develop in terms of inheritance or legitimacy of one twin over the other. This is clearly portrayed in the Biblical accounts of Cain and Abel and Jacob and Esau, one twin being very much offended by loss of a blessing. Another primitive theory is that twins are the result of animal impregnation, animal pregnancies being more readily explainable as twins due to their more frequent occurrence in various species. This belief is a part of the folklore of some North American Indians in reference to the births of human twins. The animal origin theory is reflected in the myths in which the twins are hatched from an egg, e.g., the Dioscuri, the Moliones of Greek mythology. Finally, certain foods, particularly fruits which are paired or double in form, are given as the basis for twin pregnancies. The folklore connected with foods is found widely [55]. In Europe, for example in Bavaria, the eating of two apples grown together was believed to be a cause of twins; a Wendish belief attributed twins to eating two plums; in Porton, a fruit with two kernels. In the Western Hemisphere, some Northwest Coast Indians believed that eating from both sides of an animal caused twins; Paraguay natives, a double ear of maize [56]. Running through all of these various theories is the idea that the mother of the twins is implicated in a supernatural or unusual process. She appears singled out for an aberration of nature or a special favor due to a force external to her conjugal arrangement. The reproductive process has been tampered or interfered with or varied from the normal due to one of the several possibilities. As contrasted with the single birth, twins suggested to ancient or primitive man that some awesome supernatural force was at work. The living twins remained as evidence of the intervention of a procreative force other than that accounting for one child. Primitive societies note the special relationship of twins to the divine or supernatural by designating them with animal names, e.g., twins are called birds by the Nuer [57], salmon by the Kwakiutl [58], children of apes in Togo [59]. Levi-Strauss theorizes that using an animal name for a human provides a symbol for a relationship between nature (spiritual) forces and man. The twin is perceived as not entirely natural, occupying a position intermediate between man and spirit. The designation of the twin as an animal structures this position of the twin and provides a metaphor for grasping this relationship. Both Levi-Strauss's structuralist theory and the primitive and ancient theories of twin paternity place twins in a position of bridging the natural and supernatural worlds. Twins are the living evidence of exuberant or supernatural procreative powers. Following from this fact, extraordinary powers fall to the twin as the inheritance from his unique parentage. In particular it might be expected that twins will have great powers in relation to fertility, both in human and non-human species. The ability to influence fertility will be reflected in powers of fructifying the barren woman as well as rejuvenating the effete male. Might not these powers over fertility be extended to restoration of health and of healing generally? Closely akin to powers over nature's generosity is the ability to predict the outcome of crops, the weather upon which crops depend, and the offspring of domesticated animals. Thus, the divine twin has broad prognosticator powers which in turn are related to other more general abilities at foretelling, such as the outcome of battle and the navigation of voyages. Where twins do not exist, man's imagination has sometimes created them. It is a widely held primitive belief that the products of conception along with the fetus are vital parts with a continuing life of their own after birth. They are dealt with in various ceremonial ways in primitive societies. In some tribes the placenta is regarded as the individual's twin. The Hamitic tribes of Africa treat the placenta with particular reverence and have specific rituals pertaining to the placenta [60]. The Kooboos of southeastern Sumatra regarded the uterine products as siblings and guardian spirits of the newborn [61]. In ancient Egypt, in the earliest dynasties, the pharaoh's umbilical cord was preserved and carried in a specific kind of a case during his lifetime [62]. The famous Narmer palette from the archaic period of Egyptian history contains a drawing of the carrying case of the cord and a beardless servant bearing the placenta on a standard [63]. Later, in the New Kingdom a model was substituted for the placenta [64]. The placenta was believed to be the pharaoh's twin [65]. It has been suggested that the paired pyramids of the pharaohs are the respective tombs of a pharaoh and his placenta [66]. Among the Bagandan and other North East African tribes the placenta and cord of the king are preserved throughout his life as sacred ritual objects [67]. This is of particular significance since the tribes of North East Africa are believed to share a common Hamitic origin with ancient Egypt and show other resemblances in their religious practices to those of ancient Egypt [68]. The ancient drawings of the carrying case for the pharaoh's cord resemble a similar article used by the Bagandan and Ugandan natives [69]. The Bagandan employ the preserved placenta of the king in a monthly ritual display of the afterbirth to the new moon [70]. Upon the king's death the placenta and king are given separate burials just as two tombs were provided for the Egyptian king. The ancient Egyptian viewed the afterlife existence with absolute conviction and maintained a highly elaborate formulation of the emanations or spiritual aspects of man. Their belief system included the non-corporeal entities called the Ka, Ba, and Akh which existed for each individual. Each of these entities has specific roles or functions both during life and after death. It is the Ka which is of particular interest to us here since it was believed to be the pharaoh's double. In a simultaneous act of creation, the pharaoh and his double were fashioned by the creator god Khnum. Khnum or Khnemu is portrayed in temple art as a ram-headed potter who models the pharaoh and his double on his potter's wheel [71]. This double, called the Ka, was an invisible guiding genius throughout life which assumed critical importance in the continuity of the kingship upon the death of the pharaoh [72]. In the afterlife, the Ka was the significant force maintaining the "health" and vitality of the dead pharaoh. Tomb offerings were made and prayers directed to the Ka so that it might exercise its benign influence over the dead king [73]. In addition the living successor to the dead pharaoh depended on the Ka to transmit the vital power of the kingship to him [74]. Thus the placenta-double of the pharaoh which becomes the Ka is a highly important element in the king's well-being, both living and dead. The pharaoh is dependent on his placenta in effect for his rebirth. The placenta of the pharaoh appears also to have represented the moon. The hieroglyphics for "pharaoh's placenta" is also the spelling for Khons or Khonsu, the moon-god, son of Amon and Mut. [75]. Amon and Mut are the male and female creator gods who with Khons formed the Theban triad or holy family of Egyptian religion. The moon-god was hawk-headed, held sway over reproduction and growth, and was labeled "the traveler" as befits his lunar wanderings [76]. In the Egyptian cosmology of Hermopolis the moon god Thoth and sun god Re are addressed as twins [77]. The pharaoh and his placenta provided a parallel twinship to the sun and moon. The sky in Egyptian mythology was conceived as a great face with the sun and moon as its two eyes. The Egyptians designated several gods as the sun and moon, different gods personifying the great orbs in the course of evolving Egyptian religious history [78]. Several of the moon gods were associated with healing. Thoth was a god of wisdom and patron of the sciences. His most sensational cure was in relation to the wounds inflicted in the conflict between the gods Horus and Seth. He separated the combatants, healed their wounds, and then replaced Horus' eye which had been plucked out in the struggle [79]. Thoth also assists Isis in restoring to life the infant Horus [80]. Khons, the placenta-moon god, also demonstrates magic healing abilities [81]. Khnum, the potter-creator, who made the pharaoh's Ka, is also a moon god. It is apparent that the Egyptian was untroubled by conflicting representations and meanings for the same object. It was this type of mental process, called by Frankfort a "6multiplicity of approaches," which permitted the Egyptian thinker to link together three different and baffling dualities of nature into a single system. The newborn and his placenta, the sun and the moon, and the corporeal and non-corporeal aspects of man were woven into a system of dualistic symbolic forms. These various "twins" were then pictured, copied, and symbolized for the pleasure, satisfaction, and solace of the Egyptian in the course of his daily worship and ritual. An interesting variation on the twin theme occurs in Egyptian mythology in the account of the birth of Horus. This account is a reversal of twin birth by having one individual born twice. Osiris impregnates his two sisters who each give birth to Horus [82]. Horus completes the non-logical cycle by having two burial places [83]. In summary the religion of ancient Egypt made use of the twin in important ways as a bridge to the spiritual world. The ancient Egyptian simultaneously regarded the pharaoh's placenta as (a) his twin, (b) a spiritual emanation of supreme importance, the Ka, and (c) an equivalent of the moon as the god Khons. The placenta is the lunar twin to the pharaoh in his role as a god incarnate and the earthly emanation of the sun. Thus, pharaoh and placenta are the material reflections of sun and moon and a basis for an Egyptian religious dualistic formulation. Throughout the dualistic formulation, powers of healing, health-granting, or rejuvenation are regularly attributed to the divine participants. EPICRISIS The question of the basis for the mythological association of twins and healing may be related back to a linking theme of procreation. Twins are the living embodiment and natural symbol of exuberant and unique reproduction. As an extension of twin symbolism, the other products of conception are sometimes considered as siblings. Twins in folklore have been universally viewed as a living indication of increased or altered fertility. For both primitive and ancient man twins were interpreted as the outcome of superhuman generativity. Following from this twins were thought to have special powers of their own inherited from their exceptional procreators. When the placenta has been viewed as a twin, it has been venerated and closely associated with the well-being of its original owner. In the case of the ancient Egyptians, the placenta was equated with a non-corporeal entity which was responsible for the pharaoh's vitality and transmission of his power in death to his successor. The powers of mythological and legendary twins in relation to reproduction have been widely elaborated into a recurring folkloristic pattern, including the ability (a) to make fertile the barren wife, (b) to facilitate childbirth, (c) to restore youth and potency to the aged, (d) to increase harvests from both plant and animal sources, and (e) to influence the weather upon which harvests depend. Closely related to this group of generative attributes is the lore involving the special ability of twins to predict the future or to prognosticate: crops, offspring, weather, warfare. The supernatural powers of twins are readily relatable to the areas of health and healing. The twin empowered with the ability to ease birth pangs or reverse sterility will naturally be sought for broader health care assignments. Thus, we may view the broad association of twins and healing as stemming from their own exceptional procreation as elaborated in folklore.

v3-fos

2017-06-18T09:36:24.010Z

1977-07-15T00:00:00.000Z

5628281

s2

Polymorphism of egg white proteins egg weight and components weight in the Fayoumi hen Centre national de Recherches zootechniques, LN.R.A., ., 78 Jouy-en-Josas Summary The genetic variations in egg-white proteins were detected in Fayoumi eggs. G locus proved to be the only polymorphic among four egg-white protein loci investigated. Two types were determined, Gand G,BB. The heterozygote proved to have heavier eggs and accordingly heavier egg components. The phenotypic correlation, with respect to the homozygote type, between albumen weight and yolk weight proved to be insignificant. Introduction Egg-white contains a number of proteins, which have been investigated by many workers for existence of genetic variants detected by electrophoresis. Polymorphism of some egg-white proteins has been investigated in relation to embryonic mortality (M ORTON et al.,ig65). Other works have been done, recently, concerning possible association with the economical traits (B UVANENDRAN , V., 19 6 7 ; T IT OK, I. G., 1970 ;KOVALENKO et C lt., 1972 ;LAZUK, MAKAROVA, 197 6;STRATIL, VO NDREJEC , 1973 andMAR TINKEVITCH et al., 1974 ). The first investigator (B UV A NENDR A N , 19 6 7 ) found an association between body weight, egg weight and ovalbumin; the AA genotypes were heavier and laid heavier eggs than the two other genotypes. On the other hand, for G, globulin, the heterozygotes laid more egg than either homozygote, but G 2 seemed to have no effect on the characters studied. He added, that selection on gene basis is not practical. MARTIN-( * ) This work had been done in collaboration between " I,aboratoire de G6n6tique Factorielle CNRZ, jovy-eii-josas, France and the Animal Breeding Department, Faculty of Agriculture, Cairo University, Egypt. K EVI T C H B l al. ( 1974 ) found a significant effect of genotypes on egg production for G 2 locus. This study had the purpose to determine the genetic variations in the eggwhite proteins of Fayoumi eggs. Moreover, if polymorphisms in egg-white proteins would exist, it was thought of interest to know if this has some bearing on egg weight and weights of its components. Material and methods The eggs tested in this study were transported to France from Egypt; they came from the Fayoumi breeding stock of the Experimental Poultry Breeding Farm, Faculty of Agriculture, Cairo University. The flock was hatched in winter 197 6. All birds were kept on floor under the same environmental conditions and fed the same rations at lib. The total number of individual hens used is !z2; they are the progeny of 20 sires. One egg per hen was collected in january 1977 . Each egg corresponding to each hen was broken and yolk was separated from albumen. Yolk and shell (with membrane) were weighed to the nearest mg. Meanwhile, a sample from egg-white had been taken from each egg. Albumen weight was obtained by difference between egg weight and both yolk and shell weight. Electrophoretic analysis, using starch gel (LusH, ig6i; C ROIZIER , ig6g). was practiced, firstly, to detect ovalbumin variants, but all eggs tested proved to be homozygote to this locus (AA type). The same holds true for conalbumin and G 3 , which were of BB and AA types. The only polymorphism detected in this investigation was for G,. The weights of eggs and egg components were compared on within sire and dam pairs according to the genotypes. An analysis of variance was made on this material with genotype (at G 2 locus) and sire as sources of variation. Results and discussion Two types were determined at locus G 2 as shown in Figure I , BB and AB types. The number of BB hens was 58 0 vs 132 AB hens. No AA individuals were found. Gene frequency computed for A is 9 . 3 % and for B is go. 7 °,!°. Comparing observed and expected numbers in the random mating hypothesis gives a significant X2 of 7 . 39 for 2 d.f. (P < . 025 ). The absence of AA birds may suggest some disadvantage attached to this genotype. The mean values, for egg weight, yolk weight, shell weight and albumen weight obtained for the homozygote (G 2 BB) are in grams 37 .6 9 , x 2 .55, 4.28 and 20 .8 9 respectively and 40 . 27 , 13 . 40 , 4 .6 3 and 22 . 24 respectively for the heterozygote (G 2 AB). The differences between the two genotypes are all in favor of AB: 2 .55 g. for egg weight, 0 .85 g. for yolk weight, 0 . 35 g. for shell weight and 1 . 35 g. for albumen weight. As egg weight is determined by its components weights, relatively, the largest difference is due to albumen weight. The differences between the two genotypes within each sire for the four characters confirm the overall genotype effect (Table i). They are in the same direction with only two exceptions (slight difference in the reverse direction) for egg, yolk and albumen weight and three for shell weight. The percentage of the four differences from the corresponding homozygotes weights, are: 6.8 per cent for egg weight, 6.8 per cent for yolk weight, !8.2 per cent for shell weight and 6. 4 per cent for albumen weight; This may indicate the superiority of the heterozygote type. O BEIDAH and M OSTAGEER (unpublished data) obtained a genetic correlation of o.8 between shell weight and shell thickness. The higher percentage for shell weight may be in favor of a higher shell thickness for the heterozygote. Such results on egg components do not seem to have been reported previously in the literature: M ARTINKEVITCH et al. ( 1974 ) reported a significant effect of G 2 locus for egg production in white Leghorn. T ITOK ( 1970 ) found that the homozygotes of G, and G 2 types were the best ones for both productivity and fertility in comparison with the two respective heterozygotes. The analysis of variance for the four traits is shown in Table 2 . There are no interactions between sires and genotypes for the four traits. The variation between the two genotypes proves to be highly significant in all cases. The variation between sires for shell weight is the only case which proved to be insignificant. Comparing the variances between sires and between genotypes, the first one is about one fifth of the second for egg weight, more than one fourth for yolk weight and albumen weight and about one tenth for shell weight. This indicates, that the variations in egg weight and components weights are due in large proportion to the genotypes, while the sire effect is relatively of less contribution. This is quite apparent in the case of shell weight. On the other hand, the variance attributable to this locus, approximately, is 1 . 032 for egg weight; as a fraction of the additive genetic variance, which is 2 . 32 , it is 44 . 4 per cent. Thus selection for egg weight on basis of this gene is convenient or /and may be used in deciding the choice of lines and the mating systems.

v3-fos

2018-04-03T04:39:06.593Z

1919-02-01T00:00:00.000Z

39811783

s2

A METHOD OF EXPRESSING NUMERICALLY THE GROWTH-PROMOTING VALUE OF PROTEINS.* A young animal continuously increases its food intake during growth, hence two which grow at, different rates eat unlike qua.ntities of food. We have already pointed out’ that comparisons of the value of different proteins for growth can be made when t,he animals eat the same amount of food in the same number of days andgain the same amount of weight, the protein factor being the only variable. As it is exceedingly difficult to conduct experiments which fulfil these requirements we have sought to develop a method whereby the relat,ive values of prot,eins for growth can be expressed numerically. Since food intake is quite closely regulated by the calorific requirements of the animal approximately as much food is eaten under otherwise similar conditions whether this contains a high or low percentage of protein. If a single series of experiments is made with diets containing a given percentage of protein it is obviously impossible to demonstrate the maximzLm power of any protein to promote growth. When the proportion of protein in the food is so restricted that the protein factor alone determines the rate of growth, it should be possible to find the concentration which promotes the greatest gain of body weight relative to the protein ingested by supplying foods containing d$erent percentages of protein. However, this is possible only within A young animal continuously increases its food intake during growth, hence two which grow at, different rates eat unlike qua.ntities of food. We have already pointed out' that comparisons of the value of different proteins for growth can be made when t,he animals eat the same amount of food in the same number of days and-gain the same amount of weight, the protein factor being the only variable. As it is exceedingly difficult to conduct experiments which fulfil these requirements we have sought to develop a method whereby the relat,ive values of prot,eins for growth can be expressed numerically. Since food intake is quite closely regulated by the calorific requirements of the animal approximately as much food is eaten under otherwise similar conditions whether this contains a high or low percentage of protein. If a single series of experiments is made with diets containing a given percentage of protein it is obviously impossible to demonstrate the maximzLm power of any protein to promote growth. When the proportion of protein in the food is so restricted that the protein factor alone determines the rate of growth, it should be possible to find the concentration which promotes the greatest gain of body weight relative to the protein ingested by supplying foods containing d$erent percentages of protein. However, this is possible only within 224 Growth-Promoting Value of Proteins limits, for differences in physical activities and the inherent capacity of the animal to grow may affect the amount of growth made within a fixed time. Consequently we cannot expect that, on a given diet, each animal will make quite the same gain of weight during equal intervals of time. How great these individual variations may be is illustrated by the data given in Tables I and II. From these we see that while the absolute gains of body weight may differ by even 75 per cent, the differences between the gains in body weight per gm. of protein eaten are very much less, though by no means inconsiderable. It is consequently necessav to employ a larger number of animals than were used for these experiments in order properly to minimize errors caused by such individual variations. Table I shows that when the food contained 7.9 per cent of lactalbumin the maximum gain of body weight per gm. of protein eaten was 3.0 gm., which probably represents approximately the Growth-Promoting Value of Proteins maximum growth-promoting capacity of lact,albumin when fed to albino rats under the conditions of these experiments. Table II gives similar data obtained with casein as the sole protein of the diet. Here the greatest gain per gm. of protein eaten was 2.25 gm., the food containing 12 per cent, of casein. To make this maximum gain the rats ate on the average 24.4 gm. of cascin and 203 gm. of food and gained 56 gm. When the food contained 7.9 per cent of lactalbumin the rats ate on the average 19.7 gm. of lactalbumin and 252 gm. of food and gained 59 gm. Thus to make the same gain in t,he same time under the conditions of maximum efficiency 24 per cent more protein and 20 per cent more food were needed when the protein was casein than when it was lnctalhumin. When an animal is rest,ricted to such a quantity of protein that a maximum gain of body weight is made per unit of protein eaten, it grows at less than the normal rate. A longer time thercforc is required to make a given gain of weight, and conscquently more food is needed for maintenance than if growth had not thus been delayed. In these experiments nearly t,hc same amount of food was cntcn whether this contained 6.2 or 16.2 per cent of lactalbumin (see Table I), but the gain in weight was almost twice as great on the high as on the low protein food; namely, 61 and 35 gm. respect,ively, or 0.32 and 0.17 gm. of gain per gm. of food eaten. Although growth on the 6.2 per cent ration was made with the least expenditure of protein, the. consumption of food was almost twice as great per gm. of gain as that on the 16.2 per cent diet. Consequently, although an economy in the consumption of protein may be effected by reducing its concentration in the diet, this is necessarily accompanied by a larger consumption of food. The data given in Tables I and II may be compared with those previously obtained in attempting to establish by other methods the relative value for growth of lactalbumin and casein. Series A of the earlier experiments differed from those described in the present paper in that the food intake was restricted by supplying the diet in weighed quantities, estimated to be about 10 per cent less than was needed for full normal growth, and also in being continued for 11 weeks. From the data. given in Table II of the earlier paper we have calculated the gains in body weight per gm. of protein eaten for the entire 11 weeks, and also, for comparison with the experiments just described, for periods of 4 weeks following the time at which these rabs had reached a body weight of about 70 gm. In recalculat(ing these results we have estimated the protein from the nitrogen content of the diet whereby the nitrogen of the "protein-free milk" was included, .as was done for the experiments described in this paper. The gains of weight per gm. of protein eaten during the 4 week periods were slightly higher than during the 11 week periods, presumably because the former coincide with the time of most rapid growth. The.maximum gains were decidedly less in these 4 week periods than those obtained in our new experiments, probably because in the earlier trials grow'th also was restricted by the amount of food supplied, and not solely by the protein eaten. When growth is limited by food intake protein can be used as a source of energy and consequently a smaller part may be available for growth than when sufficient energy is supplied in other forms and growth is determined solely by protein. In our former paper we called attention to criticisms, based on other grounds, which might be made to Series A of the earlier experiments, which was designed to show the relative value of proteins for growth. The comparison just made gives additional reasons for rejecting such methods of experimentation. In Chart II of our earlier paper we showed that rats made equal gains of weight in equal periods of time and ate practically Growth-Promoting Value of Proteins the same amount of food when diets of essentially the same composition were fed, but containing 8 per cent of lactalbumin, 12 per cent of casein, or 15 per cent of edestin. While the method employed for these experiments shows the relative growthpromoting power of these proteins it does not show their maximum efficiency. Thus the gain of weight per gm. of protein eaten during the 8 weeks of these experiments was for lactalbumin 2.34 gm., for casein 1.70, and for edestin 1.35 gm. These figures for lactalbumin and casein are decidedly lower than the maxima found by the method described in this paper, and the difference in the relative value of these proteins appears greater than it actually is. For the investigator we believe that this new method, which shows with some degree of accuracy the maximum efficiency of individual proteins, or mixtures of them, for growth, will be of use. By its aid we can determine and express numerically the efficiency of combinations of proteins and compare this with that of either one alone. Differences in food intake and rate of growth are largely eliminated and experimental data can be used which cannot be compared in any ot,her way. This method has its limitations, for obviously when the protein of the diet is capable of promoting growth only at a very slow rate the amount of protein eaten .per gm. of gain made will approach infinity as the gain approaches zero. The error thus introduced of course affects all comparisons made between figures obtained by the simple method of dividing the gain of weight by the gm. of protein eaten, but when the rate of growth on the two proteins thus compared is fairly rapid the magnitude of the error is small. The practical feeder does not wish to know what quantity of a given protein is the smallest which he can use to secure a given amount, of gain, if this quantity can only be used under unprofitable conditions. Rather does he want to know the least proportion which will give him the greatest gain in the shortest time, for although he may thereby waste some protein he may save food. The method described in our earlier paper (Series B) is better adapted to the use of the dietitian or the agriculturist in determining the relative value of proteins for growth than the method now described in this paper. Comparing the gains made on diets containing similar percentages of each protein when the food intake was restricted, with those made when it was unrestricted, it is seen that with only three exceptions these were greater under the latter conditions of feeding. Economy of food can be effected only by supplying the young animal with as much as it will eat; economy of protein only by reducing the nutritive ratio below that at which the normal rate of growth can be maintained.

v3-fos

2019-03-21T13:05:21.089Z

1977-06-01T00:00:00.000Z

84483644

s2

Physico-chemical properties of calf-diarrhea coronavirus Replication of calf diarrhea coronavirus was not inhibited by 5-iodo-2′-deoxyuridine, indicating that the virus is an RNA virus. Sensitivity to ether and chloroform indicated that the virus is enveloped, and this was confirmed by electron microscopic observation of the virion. The virus was readily inactivated by trypsin and sodium deoxycholate. The virus was labile at 50°C in diluted medium, but readily stabilized in the presence of MgCl2. It was stable at pH 5 and 7, while a slight loss of infectivity was observed at pH 3. The virus was readily filtered through membrane filters of 200 and 100-nm pore sizes, but not through 50-nm filters. The buoyant density of the virion in CsCl was estimated to be 1.25 g/ml. INTRODUCTION Calf diarrhea coronavirus was recently recovered from the feces of calves with neonatal diarrhea and proved to be a causative agent of the disease (Stair et ah, 1972;Mebus et al., 1972, 1973 a andb;Sharpee et al., 1976). The virus multiplied in bovine embryonic kidney cell cultures, but failed to induce readily recognizable cytopathic effect . Recently we have obserced that the virus readily replicates and induces a marked I cytopathic effect in cultures of a continuous cell line, BEK-1, derived frovll bovine embryonic kidney. This observation has provided a sensitive, practical assay method (Inaba et al., 1976), whereby the virus and its disease can now be studied more systematically. The present study was undertaken to learn some of the physico-chemica] properties of the virus. Viruses. --The calf diarrhea coronavirus , passaged in cultures of bovine embryonic kidney cells, was kindly supplied by Dr C.A. Mebus, University of Nebraska. The virus was passaged twice in calf kidney cell cultures and five times in BEK-1 cell cultures in our laboratory (Inaba et al., 1976) before use in the present study. Strain BF!I of bovine enterovirus (BE virus) (Inaba et al., 1962) and st, rain Los Angeles of infectious bovine rhinotracheitis (IBR) virus were also used for comparison. All these viruses were grown in BEK-1 cell cultures and the supematant fluid o~ infected cul. tures was used as seed virus in the present study. Infectivity assay. --Serial dilutions of the viral mate]Sal were made with maintenance medium and each dilution was inoculated in 0.1-ml amounts into three tubes of BEK-1 cell culture. After virus adsorption at 37 ° C for I h, the inoculated cultures had 0.5-ml amounts of malntec~ance medium added and were then incubated at 34*C in a roller drum for ~i days. The titer was expressed in 50% tissue culture infectious doses (TCIDs0) per 0.1 ml. Hemagglutination (HA). --HA titer was measured by the method described previously (Sato et al., 1977) and was expressed as the reciprocal of the highest antigen dilution which showed complete hem~,~glutination. RESULTS Effect of 5-iodo-2'.deoxy ~. ri~:.?#ze (IUDR) Calf diarrhea coronav~r~ls was tested along with IBR and BE viruses. A group of tube cultures of ~;EK-1 cells were inoculated with virus at an input multiplicity of 0.1 TCID~ ~}ceU. After virus adsorption at 37 ° C for I h, ~me half of the inoculated cultures received 0.5-ml amounts of maintenance medium and the remaining half received maintenance medium containing 50 ~g/ml of IUDR. The cultures were incubated in a roller drum at; 34 ° C, and, at intervals, four cultures were taken f~t a each group. The fluid phase of the cultures was pooled and stored at -SjOC after clarification by tow-speed centrifugation. All the specimens were assayed for infectivity ~imultaneously. The results are illustrated in Fig. 1. The replication of calf diarrhea coronavirus was not affected by IUDR, indicating that the virus is an RNA virus. The control viruses employed behaved as expected; BE viras was not affected by IUDR, but IBR virus did not multiply in the presence of IUDR. Sensitivity to ether and chloroform In a test tube fitted with a robber stopper 1.6 ml of infectious culture fluid and OA m! of anesthetic ether were mixed, kept at room temperature for I h, and centrifuged to separate the water phase, which was used for infectivity assay after evaporation of the residual ether under reduced pressure. ~e treatment with chloroform was carried out by mixing 1.9 ml of infectious culture fluid and 0.1 ml of chloroform. The mixture was kept at room temperature for 1 h, centrifuged~ and the resulting wate~ phase was used for infectivity assay. As shown in Table I, calf diarrhea coronavirus was sensitive to ether and chloroform. Of the viruses used as controls, IBR virus was completely inactivated but BE virus was resistant to these treatments. Sensitivity to deoxycholate (DOC) One milliliter of infectious culture fluid and I ml of 0.2% DOC in PBS were mixed and the mixtures were kept at room temperature for 1 h and assayed for infectivity. As shown in Table I, calf diarrhea coronavims was completely inactivated by DOC. IBR virus was also inactivated by DOC, but BE virus was not. Effect of molar magnesium chloride One milliliter of infectious culture fluid, diluted ten-fold with distilled water, was mixed with I ml of 2.0 M MgC12 solution, the maintenance medium or distilled water, and the mixtures were incubated in a water bath at 50°C for I h and assayed for infectiviW. As shown in Table II, calf diarrhea coronavirus, like BE virus, was stabilized by M MgCI2, but readily inactivated in diluted medium at 50°C within 1 h, while IBR virus in diluted medium was as stable as in the maintenance medium but was rapidly (within 1 h) inactivated in M MgCI2 solution. Effect of pH Two tenths of a milliliter of infectious culture fluid and 1.8 ml of McIlvain's buffer solutions at pH 3.0, 5.0 or 7.0, or PBS (pH 7.2) were mixed in test tubes fitted with rubber stoppers. The mixtures were incubated at room temperature (22°C) for I h and assayed for infectivity. At pH 3 a slight loss of infectivity was shown, while at pH 5 and 7 no loss in infectivity was shown (Table III). Effect of trypsin One milliliter of infectious culture fluid and I ml c~ 2, I, 0.5 or 0.25% trypsin ~olution (Difco, 1 : 250) in PBS (0.8% NaCI, M/150 phosphate buffer, pH 7.4) were mixed. The mixtures were incubated at 37°C for 1 h and assayed for infectivity after addition of 2 ml of trypsin inhibitor solution (1 mg/ml). As shown in Table IV, calf diarrhea coronavirus was sensitive to trypsin but somewhat less sensitive than IBR virus, whereas BE virus was res~stan~ to trypsin. Filtration The virus was readily filtered through Sartorius membrane filters uf 200 and 100-nm pore sizes, but not through 50-nm filters (Table V). Caesium chloride equilibrium density gradient centrifugation Infectious culture fluid was centrifuged at 3 000 Ilam for 30 rain to remove coarse debris and virus was sedimented by centrifugation at 100 000 × g for 2 h, and resuspended in 0.01 volume of PBS. The resulting virus suspension was mixed with a CsCI solution to a density, of 1.25 g/ml and centrifuged in a Spinco SW 50.1 rotor at 300 000 X g for 20 h, Fractions were obtained by puncturing the tube bottom, and assayed for infectivity and hemagglutinin (Fig. 2). Infectivity showed a peak which coincided in position with the peak of hemagglutinin. The density of the peak fractions was estimated to be 1.24 g/ml. Electron microscopic examination of these peak fractions by the phosphotungstic negative staining technique revealed numerous spherical virions. Although most particlcs were more or less damaged, they were shown to have an envelope covered with widely spaced club-shaped projections about 20 nm long (Fig.3). The size of the virions ranged from 110 to 160 nm, the average diameter including surface projections was estimated to be about 130 nm. DISCUSSION The present knowledge concerning the basic properties of calf diarrhea coronavixus is limited. The results presented in this ~port contribute some information on the physical and chemical properties of ",he virus, and support 79 . ). This finding should be further confirmed and extended by direct extraction and analysis of the nucleic acid from highly purified virions, including determination of whether it is single or double stranded. Sensitivity to lipid solvents, ether and chloroform, indicates that the virus is enveloped, confirming the electron microscopic observation of the virion reported in this paper and by previous workers Sharpee et al., 1976). The virus was readily inactivated by trypsin and sodium deoxycholate. The virus was labile at 50°C in diluted medium, but readily stabilized in the presence of 1 M MgCI2, agreeing with previous results (Sharpee et al., 1976). Similarly, stabilization to heat by divalent cations has been reported for avian infectious bronchitis virus (Hopkins, 1967). The virus was stable at pH 5 and 7, while a slight loss of infectivity was observed at pH 3. Previous reports disagree regarding the stability of various coronaviruses at acid pH (McIntosh, 1974). Recently, calf diarrhea coronavirus was reported to be as stable as transmissible gastro-enteritis vh~s at acid pH, while avian infectious bronchitis virus was acid labile (Sharpee et al., 1976). The virus was readily filtered through membrane filters with 200 or 100nm pore size, but not through 50-nm filters. On the other hand, the size o:~ the virion including surface projections, as determined by electron microscopy, ranged from 110 to 160 nm with an average of 130 nm, substantit~ting the reports of Stair et al. (1972) and Sharpee et al. (1976). ~l~nese measurements seem to agree with the present results of filtration, because virions t~re pleomorphic and may be collapsed and flattened when negatively stained. The buoyant density of the virion in CsC1 was estimated to be 1.24 g/ml, confirming the previous report by Stair et al. (1972), while that in sucrose has been reported to be 1.18 g/ml (Sharpee et al., 1976).

v3-fos

2017-07-29T04:25:02.040Z

1977-01-15T00:00:00.000Z

597861

s2

Ergebnisse der zerlegung von lammerschlachtkorpern mit verschiedenen anteilen Finnischer Landrasse The report discusses the investigations upon some valuable properties of the declining old Polish sheep breed Wvz6sowka concerning fecundity and sheepskin utility. Before 50 years the yzos6wka breed composed r /q of the sheep population of the NorthEast region of Poland and was characterized by the following useful parameters: live weight of single born lambs amounted to 1,8 kg, of twin born lambs to 0,9 at weaning to 14 ,4 kg and in ripe age to 34 ,6 kg. The fecundity amounted to 150 -84per cent at 22 per cent of single born lambs, 72 per cent of twin born lambs, 52 per cent of triplets and o,8 per cent of quadruplets. The yield of greasy wool of grey colour in the tint from brown to fai, in a yearly regrowth amounted to 1,3 kg at a staple length of 9,5 cm after a regrowth of Y2 year. The anatomic composition of the fibres in the coat amounted to about 70 per cent down fibres and 30 per cent medullated fibres, shorter than the down fibres. At present observations in the breeding methods are carried out on 477 sheep in the Expe1’ Ïnental Station of the Institute of Zootechnics at Czechnica. The data obtained for a period of 5 years (1971 -5 )in comparison with those obtained before 50 years show an increase of live weights of about 15 -2 per cent, however a distinct fecundity decrease of about 25 to 30 per cent took place at a smaller intensity of about 50 per cent twin-born lambs and triplets. Nevertheless the results obtained till now indicate the possibility of fecundity restitution, improvement of the quality of coat assigned to artistic tissues and of the pelt to the production of sheepskins, at a simultaneous maintenance of a high vitality and immunity of this breed from new diseases. A further adequate mating of these animals at a comperatively high percentage of rams in relation to the ewes will permit to maintain in the breed examined its different properties without parallel not only in other breeds and breeding varieties in Poland but also as may be supposed lambs, 72 per cent of twin born lambs, 52 per cent of triplets and o,8 per cent of quadruplets. The yield of greasy wool of grey colour in the tint from brown to fai, in a yearly regrowth amounted to 1 , 3 kg at a staple length of 9 , 5 cm after a regrowth of Y 2 year. The anatomic composition of the fibres in the coat amounted to about 70 per cent down fibres and 30 per cent medullated fibres, shorter than the down fibres. At present observations in the breeding methods are carried out on 477 sheep in the Expe-1' Ï 1 nental Station of the Institute of Zootechnics at Czechnica. The data obtained for a period of 5 years ( 1971 -1975 ) in comparison with those obtained before 50 years show an increase of live weights of about 15 -25 per cent, however a distinct fecundity decrease of about 25 to 30 per cent took place at a smaller intensity of about 50 per cent twin-born lambs and triplets. Nevertheless the results obtained till now indicate the possibility of fecundity restitution, improvement of the quality of coat assigned to artistic tissues and of the pelt to the production of sheepskins, at a simultaneous maintenance of a high vitality and immunity of this breed from new diseases. A further adequate mating of these animals at a comperatively high percentage of rams in relation to the ewes will permit to maintain in the breed examined its different properties without parallel not only in other breeds and breeding varieties in Poland but alsoas may be supposed in the majority of old European breeds. School of Agviculluve, Old Aberdeen AB 9 I UD, Scotland. THE DEVELOPMENT OF The paper describes the development, over a ten year period, of a new breed of sheepthe Cambridgedesigned to combine high prolificacy with other attributes of British breeds. The development was based on a initial screening of 12 British sheep breeds on the basis of individual ewe prolificacy and the incorporation of a small proportion of genes from the Finnsheep breed. Following the initial screening the foundation population has been improved by within population selection largely based on the selection of ram lambs on their dam's prolificacy and the culling of ewes on their own early performance. Generation length has been minimised by the use of rams only as ram lambs and of severe culling of ewes at three years of age. The breed is being further tested and multiplied by the cooperation of several commercial breeders and trials are in progress to assess the value of the breed as sires of crossbred ewes for commercial slaughter lamb production. Results so far indicate that a mature pure bred ewe performance target of almost 3 lambs born per ewe lambing is being achieved in practice and that this should lead to a substantial increase in lambs sold per ewe and overall profit in crossbred ewes sired by rams of this breed. Results are presented for the breeding performance of Finn x Dorset Hovn ewe lambs or ewes in a once per year breeding system. Conception rate at a mean age of 23 8 -t-9 . 4 days was gi. 7 per cent and mean litter size 1 . 52 . Ccriesponding values for 294 ::!: 11 . 4 days were 94 . 3 per cent and 1 . 70 . An interaction between time of year of birth and onset of puberty was observed, with ewe lambs bom as late as November showing behavioural oestrus before the end of the breeding season. Ewes bred once per year had a mean litter size of 2 . 34 . Results are also given for a frequent breeding experiment in which two flocks each of 4 8 ewes were repeatedly subjected to artificial daylength regimes, weaned after one month of lactation and synchionised in cestrus by using a synthetic progestagen. Total annual production was 3 . 5 lambs per ewe. The amounts and pattem of food intake during the reproductive cycle required to achieve this level of production are presented. The results of experiments designed to show whether the increased frequency of breeding and high level of production achieved under conditions of artificial daylength control could be achieved under natural conditions are presented. In these studies a conception rate of 92 .8 per cent was obtained in ewes bred during the normal period of anoestrous using 400 LU. of pregnant mares serum gonadotrophin at pessary withdrawal. La saison sexuelle bien que de durée à peu près similaire pour les trois génotypes, semble pourtant, commencer et finir plus tard chez les croisées que chez les Rasa. Lambs of the Iceland breed of sheep are found to be early maturing. The level of reproductive efficiency in the flock is raised by breeding from ewe lambs and by using ram lambs for mating. The reproductive potential of the lamb is increasingly being realized by farmers in Iceland.

v3-fos

2017-07-29T04:25:02.878Z

1977-10-15T00:00:00.000Z

52805519

s2

Cytogenetic Studies on Inherited Neurofibromatosis in Calves Between the many types of chromosomal aberrations, the translocations of centric fusion type are very interesting, because they result in change of the form and number of chromosomes of one given species. These aberrations were found in several domesticated animals. In domes- tic cattle (B. taurus) there are at least four different types of centric fusions distributed among different breeds and in various countries. The most frequent centric fusion is the 11 / 29. This aberration is found with greater frequency between the best animals used for reproduction. Wether the different types of centric fusion have a positive or negative effect on zootechnical trails is yet unclear, and so also its origin. In the present work a centric fusion of the i /z9 type in a German Brown Cattle from Bavaria is described. The centric fusion was found in a very good A.I. bull, and before it was known that it was a 1 /29 translocation carrier his semen was used in round 30 00o inseminations. The material studied consists in more than 400 cows who finished at least their first lactation. The parameters studied does not show any important devia-tions as expected for a normal good reproducer. The behaviour of chromosomes in crossbreds of the Gayal or Mithan (Bibos Irontalis) with German Blackpied cattle (Bos taurus typicus) has been investigated in two successive crossbred generations. Bibos Jvontalis has a karyotype of zn -- 58 (2 submetacentric and 54 acrocentric autosoms, XX and XI’ submetacentric), Bos taurus typicus karyotype is zn --- 60 (58 acrocentric autosoms, XX and XY submetacentric). Fl crossbreds have 2n = 59, XX or 59, XY. 56 autosoms are acrocentric, one unpaired autosom is submetacentric and gonosoms are submetacentric. Two Fl females produced a calf each from German Blackpied bulls. F2 crossbred females (25 p. cent Gayal 75 p. cent Blackpied) exhibit karyotypes of 2n = 59, XX or 2n = 60, XX. Various banding methods are used to compare banding patterns of the Fi and Fz crossbreds with those of the P generation. A Fl bull from a Gayal cow and a Blackpied bull is being reared and tested andrologically. This report is concerned with the mass incidence of congenital neurofibromatosis of the skin in calves born in one area during a three-year period. The clinical picture consisted of unilateral progressive tumours on the face, and its incidence was limited to the progeny of one bull. Exten- sive laboratory examinations including virological examination and bioassay yielded negative results. Cytogenetic examination could be made on only 8 affected calves and 4 of the dams, since the remaining calves and the sire had been slaughtered before our examination began. Of the 8 calves examined four showed 11 / 29 translocation in the heterozygous state, one animal showed this translocation in the homozygous state and the remaining three calves were free of this aberration. None of the dams showed translocation. It is of interest to note that some quantitative cytogenetic data (counts of cells with breaks or gaps) obtained for the calves corres- ponded to those obtained for their dams. The cytogenetic findings are interpreted as indicating random association of two inherited ealth disorders. The chromosomes of Bos taurus and Ovis aries have been studied using the R-banding tech- nique (DUTRILLAUX and LEJEUNE, 1971). The banding pattern obtained allows an accurate identification of chromosome pairs, including those of Ovis aries involved in the centromeric fusion. But the most striking observation concerns the centromeres. All centromeres appear darkly stained, just as if C- banding had been used. As a control, human chromosomes were stained in identical conditions, in the same containers than the Bovoidea chromosomes: the usual R-banding pattern of Man was observed. This shows first that the centromeric staining of Bos taurus and Ovis aries is not an artefact, second that very likely it is not constitutive hete- rochomatin that has been detected. Should R and C-banding be linked with the nature of the chromosomal DNA (eu - or heterochromatin, repetitive short or long DNA sequences, etc.), the present observation is sug-gestive of the presence of more than one kind of DvTA at the centromeric region of the Bovoidea. ratios in progeny groups have been reported for bulls born as dizygotic twins. Experience from the last 20 years’ of A.I. work within the Swe-dish Red and White cattle breed is reported. A total of 33 bulls born as dizygotic twins and divi- ded into three groups according to sampling method, were investigated with respect to semen quality and quantity, conception rate at first service, non-return (NR) rates of 28 and 56 days, and sex ratio of progeny groups. The information available neither point to deviating semen characteristics and reduced fertility of the bulls nor to deviating sex ratios of the progeny groups. Therefore the diagnosis « born as a twin » has hitherto been omitted from consideration in breed-ing work of cattle in Sweden. Between the many types of chromosomal aberrations, the translocations of centric fusion type are very interesting, because they result in change of the form and number of chromosomes of one given species. These aberrations were found in several domesticated animals. In domestic cattle (B. taurus) there are at least four different types of centric fusions distributed among different breeds and in various countries. The most frequent centric fusion is the 11 / 29 . This aberration is found with greater frequency between the best animals used for reproduction. Wether the different types of centric fusion have a positive or negative effect on zootechnical trails is yet unclear, and so also its origin. In the present work a centric fusion of the i /z 9 type in a German Brown Cattle from Bavaria is described. The centric fusion was found in a very good A.I. bull, and before it was known that it was a 1 / 29 translocation carrier his semen was used in round 30 00 o inseminations. The material studied consists in more than 400 cows who finished at least their first lactation. The parameters studied does not show any important deviations as expected for a normal good reproducer. The behaviour of chromosomes in crossbreds of the Gayal or Mithan (Bibos Irontalis) with German Blackpied cattle (Bos taurus typicus) has been investigated in two successive crossbred generations. Bibos Jvontalis has a karyotype of zn --5 8 ( 2 submetacentric and 54 acrocentric autosoms, XX and XI' submetacentric), Bos taurus typicus karyotype is zn ---6 0 ( 5 8 acrocentric autosoms, XX and XY submetacentric). F l crossbreds have 2 n = 59 , XX or 59 , XY. 5 6 autosoms are acrocentric, one unpaired autosom is submetacentric and gonosoms are submetacentric. Two F l females produced a calf each from German Blackpied bulls. F 2 crossbred females ( 25 p. cent Gayal 75 p. cent Blackpied) exhibit karyotypes of 2 n = 59 , XX or 2 n = 6 0 , XX. Various banding methods are used to compare banding patterns of the F i and F z crossbreds with those of the P generation. A F l bull from a Gayal cow and a Blackpied bull is being reared and tested andrologically. This report is concerned with the mass incidence of congenital neurofibromatosis of the skin in calves born in one area during a three-year period. The clinical picture consisted of unilateral progressive tumours on the face, and its incidence was limited to the progeny of one bull. Extensive laboratory examinations including virological examination and bioassay yielded negative results. Cytogenetic examination could be made on only 8 affected calves and 4 of the dams, since the remaining calves and the sire had been slaughtered before our examination began. Of the 8 calves examined four showed 11 / 29 translocation in the heterozygous state, one animal showed this translocation in the homozygous state and the remaining three calves were free of this aberration. None of the dams showed translocation. It is of interest to note that some quantitative cytogenetic data (counts of cells with breaks or gaps) obtained for the calves corresponded to those obtained for their dams. The cytogenetic findings are interpreted as indicating random association of two inherited ealth disorders. The chromosomes of Bos taurus and Ovis aries have been studied using the R-banding technique (DUTRI LLAUX and LEJEU N E, 1971 ). The banding pattern obtained allows an accurate identification of chromosome pairs, including those of Ovis aries involved in the centromeric fusion. R-Banding studies in But the most striking observation concerns the centromeres. All centromeres appear darkly stained, just as if Cbanding had been used. As a control, human chromosomes were stained in identical conditions, in the same containers than the Bovoidea chromosomes: the usual R-banding pattern of Man was observed. This shows first that the centromeric staining of Bos taurus and Ovis aries is not an artefact, second that very likely it is not constitutive heterochomatin that has been detected. Should R and C-banding be linked with the nature of the chromosomal DNA (eu -or heterochromatin, repetitive short or long DNA sequences, etc.), the present observation is suggestive of the presence of more than one kind of DvTA at the centromeric region of the Bovoidea. Often reduced fertility and deviating sex ratios in progeny groups have been reported for bulls born as dizygotic twins. Experience from the last 20 years' of A.I. work within the Swedish Red and White cattle breed is reported. A total of 33 bulls born as dizygotic twins and divided into three groups according to sampling method, were investigated with respect to semen quality and quantity, conception rate at first service, non-return (NR) rates of 2 8 and 5 6 days, and sex ratio of progeny groups. The information available neither point to deviating semen characteristics and reduced fertility of the bulls nor to deviating sex ratios of the progeny groups. Therefore the diagnosis « born as a twin » has hitherto been omitted from consideration in breeding work of cattle in Sweden.

v3-fos

2014-10-01T00:00:00.000Z

1977-08-01T00:00:00.000Z

13394341

s2

Levels of arsenic in the United States food supply. At the present time, the Food and Drug Administration (FDA) accords the highest priority to mercury, lead, cadmium, arsenic, selenium, and zinc in its program on toxic elements in foods. The only regulatory levels for arsenic in foods in the U. S. are the tolerances which have been established for its residues in specified foods, resulting from the application of arsenical pesticides on food and feed crops and from animal feed additives. FDA has monitored for arsenic in its Total Diet Survey since the inception of this program. The data from this program indicate that the average daily intake for arsenic (as As(2)O(3)) has decreased from about 130 mug/day in 1968 to about 20 mug/day in 1974. Most of the arsenic is found in the meat-fish-poultry food class of the total diet. In individual foods, the highest levels were found in fish, with a mean level of about 1.5 ppm (as As(2)O(3)) in the edible portion of finfish. Much lower levels were found in all the other food types analyzed; of these, the highest levels found were a mean level of 0.08 ppm in chicken and 0.16 ppm in rice. FDA toxicologists do not believe that the average daily intake of arsenic, or its levels in the different food commodities, pose a hazard to the consumer. The Food and Drug Administration has conducted various studies on toxic elements in foods for many years. At the present time, the Agency accords highest priority to mercury, lead, cadmium, arsenic, selenium and zinc. This report summarizes the results FDA has obtained in recent years in monitoring for arsenic in foods. Neither the toxicological aspects of arsenic nor details of analytical methodology will be discussed in detail, but will be referred to only as they are related to our monitoring activities. The only regulatory levels for arsenic in foods in the U. S. are the tolerances that have been established for its residues in specified foods, resulting from the application of arsenical pesticides on food and feed crops and from animal feed additives. Generally, the tolerance for arsenic residues (as As,O3) on various fruits and vegetables resulting from pesticidal use of copper, magnesium and sodium arsenates is 3.5 ppm As203 (Table 1). Tolerances for residues resulting from the use of lead arsenate, sodium arsenite, and the sodium salts of methane arsonic acid and of cacodylic acid are also given. A number of arsenic compounds are also permitted for use in food producing animals either as *Bureau of Foods, Food and Drug Administration, 200 C St., S.W., Washington, D. C. 20204. growth stimulants or for prevention or treatment of diseases. Among these are arsanilic acid and its sodium salt, 4-nitrophenylarsonic acid, 3-nitro-4-hydrophenylarsonic acid, and 4-nitrophenylarsonic acid. Tolerances for total combined arsenic residues (calculated as As) resulting from these uses are given in the Code of Federal Regulations, Title 21, part 556.60. Essentially, the tolerances are as follows: 0.5 ppm arsenic in eggs; 0.5 ppm in the muscle of chickens, turkeys, and swine; and 2 ppm in edible by-products of chickens and turkey and the livers and kidneys of swine. The tolerance for arsenic in other edible by-products in swine is 0.5 ppm. A 5-day withdrawal period is required when arsenicals are used in these species to allow for excretion, so that residue levels in the tissues will not exceed these tolerances. The Food and Drug Administration has monitored for arsenic in its Total Diet ("market basket") Survey since the inception of this program, to determine the levels of this element in the average diet. The composition of the "market basket" used in FDA's Total Diet Program (1) is based in large part on the survey of U. S. household food consumption conducted by the U. S. Department of Agriculture (USDA) in 1965. Diet guides are provided for each of four geographical regions of the U. S. as defined by USDA: South, Northeast, North Central, and West. Each market basket rep- Table 2 are prepared by the FDA Kansas City laboratory, and each of the composites is analyzed for the residues specified. All arsenic findings are calculated as As203 because residue tolerances for most arsenic-containing pesticides are expressed in terms of As203. As203 can be converted to As by multiplying by 0.76. The average levels of arsenic (as As203) found in the different food composites from 1967 to 1974 are shown in Table 2. It can be seen that the levels in all food categories have decreased significantly since 1967-1970, to the extent that the meat-fish-poultry composites now are the only major source of arsenic in the total diet. Table 3 shows the estimated average daily intake of arsenic (as As203) represented by the different food composite categories. Again, the levels of arsenic intake from all the food categories have significantly decreased, with the meat-fish-poultry category contributing most to the daily intake. It is believed that much of this drop may be due to the decreased use of arsenic-containing pesticides on food crops since the late 1960s. To explain the apparent increase in arsenic levels in the meat-fishpoultry and fruit categories from 1973 to 1974, it will be necessary to evaluate the data from the Total Diet Studies for several more years to determine whether this represents a trend, or if it is merely the random variation one may expect from year to year. The estimated average daily intake of As203 from the total diet has varied from about 100 Ag/day in 1967-1969 to about 10-20 ag/day in 1971-1974. FDA toxicologists believe that these levels do not represent a hazard to the consumer, but that they do warrant continued surveillance. On a priority basis, mercury, lead, and cadmium have been of more concern to FDA in recent years than has arsenic. To obtain a better idea of the levels of arsenic in individual food items, during the past three years (Fiscal Years 1974-1976, FDA has carried out exploratory surveys for arsenic in various food commodities in interstate commerce, which are obtained primarily at the retail level. The data obtained on arsenic levels in meats, eggs, and milk are shown in Table 4. The mean levels in meats were all less than 0. 1 ppm As20. The highest levels were found in chicken, with a mean value of 0.08 ppm and a maximum level of 0.5 ppm. Generally the levels were about the same in the samples of muscle and liver examined. 'N D = not detected; T = <0.00 1 ppm As203 average for the given food class composite in the given year (30 composites of each of the 12 food classes each year). The limit of quantitation for a single analysis was approximately 0.1 ppm (As203). Detections below that level are estimations only. The U. S. Department of Agriculture (USDA) conducts a continuing monitoring program for various contaminants in red meat and poultry obtained in slaughterhouses. USDA has the responsibility for assuring the safety of meat products in the U. S. and has, therefore, examined many more meat samples than FDA has. The mean levels of arsenic that USDA found in chicken, swine, and cattle meat products in 1975 agreed fairly well with the FDA findings presented in Table 4. One difference was that USDA found somewhat higher levels in liver than in muscle; the highest was an average of about 0.8 ppm arsenic as As203 in chicken livers. Actually, we would expect to find somewhat higher values in the liver, since it is the target organ for arsenic. As in the FDA survey, levels in chickens were higher than those found in swine or cattle. The higher levels in chickens may result partially from the greater use of arsenical additives in poultry feed. Table 5 presents the results that FDA obtained for arsenic levels (as As203) in vegetable and fruit products in the 1974-1976 period. The mean levels in all types examined were well below 0.1 ppm. The highest value in any single sample was 0.3 ppm As203 in a potato product. The levels obtained for arsenic (as As203) in cereal, nut, and sugar products are shown in Table 6. The mean values are again all well below 0.1 ppm, with the exception of rice, where the mean level was 0.16 ppm. The highest individual level in the foods in Table 6 was 0.4 ppm in a sample of rice. Environmental Health Perspectives During Fiscal Years 1973 and 1974, the Food and Drug Administration carried out a comprehensive fish survey to determine the levels of pesticides, PCBs, and heavy metals in fish. The species were selected either on the basis of their commercial significance, or on the fact that bottom feeders are indicators of pollution. The samples were generally obtained at the wholesale level in order to determine the quality of fish available to the consumer. All analyses were carried out on the edible portion of the fish. Arsenic was added to this surveillance program in 1974. However, this survey was cut short in order to divert resources to the botulinumin-mushrooms crisis at that time. As a result, only 105 samples were analyzed, all of which were finfish, except for 10 samples of shrimp. FDA also conducts a continuing surveillance program for residues of heavy metals in samples of oysters and clams obtained from approved commercial harvesting areas. Table 7 shows the results obtained by FDA in its Fiscal Year 1974 Comprehensive Fish Survey and in its Fiscal Years 1974 and 1975 Heavy Metals in Shellfish Program. Since it was possible to analyze only 105 samples in the comprehensive fish survey, we do not feel that we can draw conclusions about species likely to have higher arsenic contents, and thus we have classified the species from this survey only as "finfish" and "shrimp." "Limit of quantitation approximately 0. 1 ppm As2O3 in an individual analysis. Detections below that level are estimates only. As can be seen, there is very wide variation between the levels found for individual samples. The highest finding in an individual sample of finfish (19 ppm) was more than 10 times the mean level (1.47 ppm). Similarly, some individual samples of molluscs contained more than 10 times as much arsenic as the mean level for the species. With respect to the results for arsenic in clams and oysters, we should point out again that all samples were obtained from approved commercial harvesting areas. We do not know whether the same general levels would be found in molluscs in all areas where they may be caught. The nature of the sampling in FDA's comprehensive fish survey precludes the ability to determine the precise origin of catch, since entire lots of fish are sampled primarily at the wholesale level. Even though analysis of a lot is an appropriate method of determining the average intake of arsenic for the general public, it will be beneficial to learn more about the levels of specific species caught in specific locations. In this regard, surveys being conducted by the National Marine Fisheries Service will provide valuable additional information, since their sampling population is much larger, and they obtain their samples at specified locations. In summary, the total diet monitoring program carried out by FDA since 1967 shows that the aver-age daily intake of arsenic (as As2O3) has decreased drastically from about 130 gg/day in 1968 to about 20 ,ug/day in 1974. Most of the arsenic is found in the meat-fish-poultry composite of the total diet survey. In analyses carried out on individual foods, the highest levels were found in fish, with a mean level of 1.47 ppm in finfish. Much lower levels were found in all other food types; of the latter, the highest levels found were a mean level of 0.08 ppm in chicken and 0.16 in rice. We do not believe that this average daily intake, or the levels in the various foods, pose a hazard to the consumer. However, we do believe that continued surveillance for arsenic in our food supply is necessary. For the future, FDA plans (1) to continue to measure arsenic in its total diet studies to determine 'vhether there are any trends in arsenic levels in the average U. S. diet, and to follow up on any unusually high total diet findings; and (2) to carry out special surveillance programs on individual food commodities when unexpected levels warrant.

v3-fos

2018-04-03T04:09:01.702Z

1977-01-01T00:00:00.000Z

37934134

s2

Effect of pyridoxine deficiency on cholesterogenesis in rats fed different levels of protein. Hepatic cholesterol contents in rats fed a 70% or 20% casein diet with or without pyridoxine was determined. In the case of the 70% casein group, pyridoxine-deficient rats showed a higher content than the control. The increment was mainly due to the accumulation of an ester form of the cholesterol. On the other hand, pyridoxine-deficient rats in the 20% casein group showed a slightly lower content. The cholesterol content in liver microsomal fractions was lower in the 20% casein pyridoxine-deficient group and serum cholesterol level was lower in the 70%-casein pyridoxine-deficient group than those in respective control groups. Incorporation of [14C]acetate into cholesterol was studied using liver slices, and significant stimulation was observed in pyridoxine-deficient rat fed a 20% or 70% casein diet. There was no difference in intestinal cholesterogenesis between the control and the deficient groups. Summary Hepatic cholesterol contents in rats fed a 70 % or 20% casein diet with or without pyridoxine was determined. In the case of the 70% casein group, pyridoxine-deficient rats showed a higher content than the control. The increment was mainly due to the accumulation of an ester form of the cholesterol. On the other hand, pyridoxine-deficient rats in the 20% casein group showed a slightly lower content. The cholesterol content in liver microsomal fractions was lower in the 20% -casein pyridoxine-deficient group and serum cholesterol level was lower in the 70%-casein pyridoxine-deficient group than those in respective control groups. Incorporation of [14C]acetate into cholesterol was studied using liver slices, and significant stimulation was observed in pyridoxine-deficient rat fed a 20% or 70% casein diet. There was no difference in intestinal cholesterogenesis between the control and the deficient groups. Since RINEHART and GREENBERG found artherosclerosis in Rhesus monkey fed a pyridoxine-deficient diet for almost one year (1), divergent observations have been reported in the literature with regard to cholesterol metabolism in pyridoxine deficiency. There are findings of hypercholesterolemia in pyridoxine-deficient rats (2), chicks (3) and rabbits (4) as well as no change (5) or even a decrease (6) in plasma sterol level of depleted rats. The liver cholesterol ester was reported to be reduced in pyridoxine-deficient rat (6), while both LUPZEN et al. (7)(8)(9) and SHAH et al. (10) reported no change in cholesterol contents and an increase of cholesterogenesis in the pyridoxine-deficient rat liver. These previous studies have not paid any special attention to the feeding condition of animals, although cholesterogenesis in liver is known to show a diurnal rhythm (11)(12)(13). Recently, we found that dietary protein levels influence lipid metabolism in pyridoxine deficient rats (14,15). On being fed a high (70%) protein diet, the pyridoxine deficient rats accumulated triglyceride and cholesterol ester in the liver. We have also obtained evidence of increased cholesterogenesis on rats fed a 70%-casein pyridoxine-deficient diet (16). In the present paper, we describe the results of a more detailed study on cholesterol metabolism in pyridoxine-deficient rats fed a 20% or 70% protein diet. Determination of the reaction products. Radioactivity of CO2, absorbed in 0.1ml Hyamine solution, was determined in 10ml dioxane scintillator (18) with an Aloka liquid scintillation counter. After incubation, liver slices were taken up and homogenized with a mixture of 4ml 1N H25O4 and 15ml CHCl3-methanol (1:2). The homogenate was centrifuged to obtain the supernatant and the resultant precipitate was washed with a mixture of 2ml 1N H25O4 and 7.5ml CHCl3-methanol. The residue was used for the determination of protein content. The washing was combined with the original supernatant, then 6ml water and 7.5ml CHCl3 were added to the mixture. After mixing, the two phases were separated and the lower phase was used for the following lipid analyses. The solvent was removed by evaporation, the residue was dissolved in petroleum ether and washed several times with 0.1M sodium acetate to remove the radio active substrate completely. After concentrating this solution to a small volume, the lipid fraction was separated by thin-layer chromatography on Kiesel Gel with a solvent mixture of petroleum ether-ethyl ether-acetic acid (80:30:1). The lipid bands on the plate were detected by iodine vapor, scrapped off, extracted with CHCl3-methanol (1:1), and the radioactivities were determined in toluene based scintillator. Analyses. Protein was determined by the biuret reaction (19). Alanine aminotransferase [E. C. 2 .6. 1. 2] activity was determined by measuring pyruvate produced from L-alanine and 2-oxoglutarate as phenyl hydrazone (20). Total lipid in liver tissue was gravimetrically determined after extraction by the method of FOLCH et al. (21). Cholesterol was measured with an FeCl3 reagent as de scribed by ZAK (22), and free and ester cholesterol was separated by a thin-layer chromatography prior to the chemical determination. with and without supplemented pyridoxine, were used for this experiment. The pyridoxine supplemented group was divided into paired-control and ad libitum control groups to observe effects of feeding patterns. As shown in Table 2, the acetate incorporation into free cholesterol fraction at midnight gave similar high values in all groups. The midday activities were, however, different among three groups; ad libitum control group showed almost the same value with that at mid night, while the midday activities were decreased to 20% and 40% of the midnight activities in pair-fed control and the deficient groups, respectively. The incorpora tion into cholesterol ester in various groups showed a similar trend with that into free cholesterol. Liberation of 14CO2 and triglyceride synthesis would serve as internal standards of the experiment. Triglyceride synthesis was active at mid night in all groups and decreased at midday in the order of ad libitum control, pair-fed control and deficient group. Liberation of 14CO2 was almost constant at any time tested in all groups. Cholesterol synthesis in the livers of fasted rats The results so far obtained indicate that the change in feeding conditions profoundly influences the rate of hepatic cholesterol synthesis. Therefore, rats fed a 20% or 70% casein diet were fasted overnight to set a constant condition in all groups and the cholesterol synthesis was measured in the liver slice system ( Table 3). The deficient groups on both 20% and 70% casein diets showed significantly higher activities than the control groups. We thus conclude that hepatic cholesterol synthesis is enhanced in pyridoxine deficiency, not reflecting the change in the levels of cholesterol in the liver. Triglyceride synthesis and 14CO 2 liberation were not significantly different between the control and the de ficient groups. Intestinal cholesterogenesis in pyridoxine-deficient rat Cholesterol synthesis was also measured in rat intestine which is considered to be the most important site of extrahepatic cholesterogenesis. Since cholesterol synthesis has been shown to be different in the central and distal portions of intestine (23), the incorporation of acetate was studied in both portions. As shown in Table 4, the distal portion displayed a higher synthetic activity in the 20% casein group. But, in both levels of protein intake, pyridoxine deficiency exerted no effect on intestinal cholesterogenesis. DISCUSSION Pyridoxine deficiency causes disturbances in lipid metabolism in the liver. We previously reported that the total liver lipid was elevated twice in the pyridoxine -deficient rats fed a 70% casein diet, although no such elevation was observed in the case of a below 30% casein diet (14,15). The present study has shown that the content of cholesterol, particularly the ester form, is increased in the livers of pyridoxine-deficient rats fed a 70% casein diet and is decreased in pyridoxine -deficient rats fed a 20% casein diet. Thus the discrepancy in the literature with respect to the effect of pyridoxine deficiency on the hepatic cholesterol level can be attributed to the different protein contents in the diets employed by various investigators. The present study has shown that hepatic cholesterogenesis is increased in rats fed pyridoxine-deficient diet containing either a 20% or 70% casein. The rate of hepatic cholesterogenesis is known to be influenced by the level of cholesterol content in the liver. JAKOI and QUARFORDT (24) have proposed that microsomal cholesterol content may regulate hepatic cholesterogenesis. In the present study, however, no direct relationship was found between the microsomal cholesterol content and the rate of cholesterol synthesis. Serum lipoprotein fraction has also been implicated to play a regulatory role in hepatic cholesterogenesis (25). We found that serum cholesterol level was somewhat decreased in the pyridoxine-deficient groups, but it is questionable if the increased cholesterogenesis by pyridoxine deficiency can be explained by this finding alone. LUPIEN et al. (7)(8)(9) and SHAH et al. (10) reported that cholesterol synthesis was stimulated by pyridoxine deficiency in livers of rats fed a 18-22% casein diet, although the hepatic cholesterol content was unchanged. LUPIEN et al. attributed the stimulation of cholesterogenesis by pyridoxine deficiency to the activation of taurocholate conjugation in the liver since the conjugation reaction in the liver homogenate was inhibited by pyridoxal phosphate. This is not likely since cysteine metabolism in general and taurine formation in particular have been found to be decreased in pyridoxine deficiency (26). We have in fact observed a marked decrease in taurocholate conjugate in the bile of pyridoxine-deficient rat (27) . Obviously further work is necessary to clearify cholesterol metabolism in pyridoxine deficiency . We are now investi gating the level of 3-hydroxy-3-methylglutaryl CoA reductase activity and the rate of cholesterol turnover in pyridoxine-deficient rats .

v3-fos

2018-04-03T01:56:01.069Z

1977-01-01T00:00:00.000Z

31607520

s2

Effects of thiamine administration on blood lactic and concentration and mineral metabolism in sheep. The object of the present experiment is to determine the effect of thiamine administration on lactic acid concentration in blood plasma and the mineral metabolism in sheep given low and high concentrate rations. A high concentrate ration containing 90% concentrate and 10% roughage was fed to one group of sheep and a low concentrate ration containing 60% concentrate was given to the other group. Thiamine was intramuscularly injected into every sheep at a level of 50 mg of thiamine tetrahydrofurfuryl disulfide per day. No great difference was found in the plasma lactic acid concentration between the animals fed the low and high concentrate rations, and those between the control and thiamine injection periods. Thiamine injection appeared to increase urine and fecal magnesium excretion and significantly decreased magnesium balance (p less than 0.05). It may be conceivable that endogenous magnesium excretion is enhanced by thiamine administration in sheep. Summary The object of the present experiment is to determine the effect of thiamine administration on lactic acid concentration in blood plasma and the mineral metabolism in sheep given low and high con centrate rations. A high concentrate ration containing 90% concentrate and 10% roughage was fed to one group of sheep and a low concentrate ration containing 60% concentrate was given to the other group . Thiamine was intra muscularly injected into every sheep at a level of 50mg of thiamine tetrahydrofurfuryl disulfide per day. No great difference was found in the plasma lactic acid concentration between the animals fed the low and high concentrate rations , and those between the control and thiamine injection periods . Thiamine injection appeared to increase urine and fecal magnesium excretion and significantly decreased magnesium balance (p<0 .05). It may be conceivable that endogenous magnesium excretion is enhanced by thiamine administra tion in sheep. Ruminants given a high concentrate ration frequently showed symptoms of acidosis (1)(2)(3)(4). It was indicated that rapid recovery from acidosis caused by acute overeating was accomplished with injection of thiamine (5) . However, HUBER (6) reported that an intravenous injection of thiamine was not effective in increasing blood lactate clearance. On the other hand, a change in mineral metabolism was found in ruminants in which acidosis occurred (4), especially in phosphorus excretion . Urinal phos phorus excretion increased with the lowering of blood pH. Because microbial synthesis occurs in the rumen , ruminants are unlikely to show thiamine deficiency (7)(8)(9). Therefore, little information is available about the action of thiamine in cattle and sheep . The present experiment was conducted to determine the effects of thiamine administration on lactic acid concentrations in blood plasma and mineral meta bolism in sheep given low and high concentrate rations. MATERIALS AND METHODS Six sheep averaging about 38kg weight were divided into two groups. One group was fed with a high concentrate ration containing 90% concentrate and 10% roughage, and the other group was fed with a low concentrate ration containing 60% concentrate and 40% roughage. The concentrate ration consisted of 90% flaked corn and 10% soybean meal. Rice straw was used as a roughage source. Calcium carbonate and sodium chloride was added to the concentrate ration at a level of 0.5% to obtain adequate amounts of calcium and sodium. After feeding with experimental rations for a thirty-day control period, all sheep were injected intramuscularly at the level of 50mg/head/day with thiamine tetrahydrofurfuryl disulfide for a fourteen-day injection period. Urine and fecal samples were taken for the last seven days in both the control and injection periods. Blood samples were taken from the jugular vein puncture three hours after the morning feeding on the fourth day of both sampling periods. Fecal and dietary samples were ashed with nitric acid and perchloric acid. Lactic acid in blood plasma was analyzed by the method of BARKER and SUMMERSON (10). Total thiamine in blood was assayed by the thiochrome method (11). Calcium and magnesium in urine, serum, feces and ration were determined by an atomic absorption spectrophotometer. Inorganic phosphorus of these samples were analyzed by the method of FISKE and SUBBAROW (12). RESULTS The total thiamine concentration of blood is shown in Table 1. Although little difference was found in blood thiamine between the two groups-low and high concentrate groups-the blood thiamine evidently became higher in the in jection period in both groups. Thiamine concentration in the injection period was two times over than that in the control period. The results of lactic acid concentration in blood plasma are presented in Table 2. There was no great difference in the plasma lactic acid concentration between the groups given the low and high concentrate rations, and between the control and thiamine periods. Thiamine injection did not affect calcium and phosphorus retentions in this ex periment. The rations of low and high concentrate groups contained, respec tively, 0.12% and 0.11% magnesium. As shown in the mineral balance data, dietary magnesium concentrations seemed to be adequate for animal requirements. ZIEVE et al. (14,15) and ITOKAWA et al. (16)(17)(18)(19) have studied the relationship between magnesium and thiamine in rats. ITOKAWA et al, (16)(17)(18) observed that magnesium concentrations in serum and bone were decreased by oral administra tion of thiamine. Although serum magnesium concentrations were not apparently changed in this experiment, magnesium excretion via urine and feces tended to become higher as thiamine was given to sheep, From the result that a negative magnesium balance was induced by thiamine injection, it is conceivable that serum magnesium could decrease if thiamine is administered for a prolonged period. It may finally be concluded that, when a large amount of thiamine is given, there is the similar association between thiamine and magnesium metabolism in sheep as that found in rats.

v3-fos

2019-04-02T13:11:51.984Z

1977-01-01T00:00:00.000Z

90293094

s2

The Control of Growth and Development in Bombyx mori. XXXVI: Larval-Larval and Larval-Pupal Apolysis, Especially on a Hormonal Antagonistic Balance It was shown an antagonistic action between juvenile hormone (JH) and molting hormone (MH) (Morohoshi 1972, XV) . A similar view was expressed by Masner and Hangortner (1973) in vivo experiments with the German cockroach. This concept has been asserted by Morohoshi since 1957 and supported by several investigators in vitro experiments (Congote et al. 1969, Lezzi et al. 1969, Laufer et al. 1970). This study deals with antagonism between JH and MH. Materials and methods. Eggs resulting from bivoltine hybrids between J. 106 and Daizo were incubated at 17°C in the dark condition (L-series) and at 25°C in the light condition (H-series). The application of JH into the larval skin was performed in the 5thinstar-larvae at the level of 15 pg/1 pl per gr. Results. 1. Appearance o f extra molted larvae in early (Lme), intermediate (+ Lm) and late (Lm) maturing strains following the JH-application. Recently Komori (1976) showed extra molting which occurred earlier in the female larvae having the sex-linked recessive early maturing gene (Lme) than in the female ones carrying the dominant late maturing gene (Lm) (Table I (1)). The frequency of extra molted larvae in the 96-hr-old 5th-instar-female larvae following the JHapplication was most in the Lme-larvae, medium in the Lm-larvae and least in the Lm-larvae. Proc. Japan Acad., 53 (1977) [ MAKINO, M. J. A., Jan. 12, 1977) It was shown an antagonistic action between juvenile hormone (JH) and molting hormone (MH) (Morohoshi 1972, XV) . A similar view was expressed by Masner and Hangortner (1973) in vivo experiments with the German cockroach. This concept has been asserted by Morohoshi since 1957 and supported by several investigators in vitro experiments (Congote et al. 1969, Lezzi et al. 1969, Laufer et al. 1970. This study deals with antagonism between JH and MH. Materials and methods. Eggs resulting from bivoltine hybrids between J. 106 and Daizo were incubated at 17°C in the dark condition (L-series) and at 25°C in the light condition (H-series). The application of JH into the larval skin was performed in the 5thinstar-larvae at the level of 15 pg/1 pl per gr. Results. Recently Komori (1976) showed extra molting which occurred earlier in the female larvae having the sex-linked recessive early maturing gene (Lme) than in the female ones carrying the dominant late maturing gene (Lm) ( Table I (1)). The frequency of extra molted larvae in the 96-hr-old 5th-instar-female larvae following the JH-application was most in the Lme-larvae, medium in the Lm -larvae and least in the Lm-larvae. 2. Appearance o f extra molted larvae in the L-and H-series following the JH-application. Extra molted larvae were produced earlier in the L-series-larvae than in the H-series-larvae after the JH-application (Table I (2)). Discussion. In the last 5th larval instar the secretion of JH ceases at the early stage, while the secretion of MH rises with age after the 3rd day. A similar hormonal sequence has been induced in younger larvae either by decapitation or by allatectomy. Under such hormonal environmental conditions, larvae naturally induce larvalpupal (L-P) apolysis. The L-P apolysis was changed into L-L apolysis by applicating JH, Mil or BH (brain hormone). The L-L apolysis as well as L-P apolysis are controlled by the presence or absence of JH in haemolymph, while the timing of apolysis is determined by the titer of JH or MH. The time (hour) needed to prepare for pupal ecdysis usually exceeds that of larval ones. Fig. 1(1) illustrates L-L apolysis following the application of JH (Morohoshi et al. 1975: XXV), and Fig. 1(2) shows the difference (L-L) apolysis by application by strains on the induction of L-L apolysis with the application of JH (Komori 1976). Fig. 1(3) shows L-L apolysis by the application of MH (Morohoshi et al. 1969) or BH (Kobayashi et al. 1973). The L-L apolysis after the implantation of the brain-corpora allata (Br-CA) complex into the 4th-instar-larvae 24 hours after decapitation is illustrated in Fig. 1(4) . The amount of BH in haemolymph seems to determine the period of L-L or L-P apolysis in the final larval instar (Fig. 2, Rep. XXVII, XXIX and XXXV). The higher titer of BH induced the higher frequency of L-L apolysis and the quicker period of apolysis. These results were quitely in accord with Kobayashi's results (1973) in which extracted BH was injected into decapitated 4th-instar-larvae. These results indicate that the L-L apolysis in the last larval instar occurs under high dosages of JH, MH and BH, respectively. By applying JH the 5th-instar-larvae carrying the Lme gene were higher in percentage of L-L apolysis than the 5th-instar-larvae with the Lm gene. This may be caused by higher titer of MH (or BH) in the former. The secretion of JH in the 5th-instar-larvae ceased on the 3rd day. When much JH would be applied to larvae, they may induce extra larval ecdysis. Even if insects would be young, they could be pupated following the cease of JH-secretion. Thus, two hormones (JH and MH) show an antagonistic action upon protein-metabolic tissues or organs such as body wall, silkglands or ovaries. The JH acts as a larva-maintaining (growth) hormone, whereas the MH serves as a pupation-promoting (development) hormone. However, the function of JH on ecdysed types is always superior to that of MR. When the MR-titer in haemolymph is constant, the length of each instar is controlled by the amount of JH : under a sufficient amount of JH, the length of each instar becomes long. When the JH-titer is constant, the length of each instar is controlled by the amount of MH : a sufficient amount of MH induces a short larval instar. Under a poor amount of JH and rich amount of Mil, the length of each instar becomes shortened to a considerable extent. The larvae carrying the sex-linked early maturing gene (Lme) release more BH than those carrying the sex-linked late maturing gene (Lm). The CA activity of the larvae of the former type is strongly inhibited by more BH introduced or stored in the CA while the PG (prothoracic gland) activity is strongly promoted by the BH released into haemolymph from the CA. The JH induces the delay of larval development (Rep. XVI) with the inhibition of protein synthesis (Rep. Vu-TX). The MH induces the acceleration of larval development (Rep. XIII) with the promotion of protein synthesis (Kobayashi et al. 1971). The JH and MH are clearly antagonistic hormones. The change of molt-numbers at earlier larval instars is well explained by this antagonistic action between JH and MH. Tetramolting groups segregating some trimolters in the L-series accelerate molting cycles more than tetramolting groups segregating no trimolters in the H-series. Some larvae out of accelerated normal tetramolters in the L-series delay molting cycles at earlier larval instars and they become trimolters. When much BH is produced in the L-series-larvae, a lower titer of JH and a higher titer of MH result in haemolymph. This feature induces the acceleration of molting cycles. The delay of the molting cycle in appeared trimolters may be controlled by a higher titer of JH and a lower titer of MH due to the acceleration of larval development.

v3-fos

2016-05-14T03:58:30.200Z

1977-10-15T00:00:00.000Z

18535293

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Sensitivity of inbred fayoumi chicks to seasonal variations The aim of this experiment was to study the sensitivity of inbred lines of Fayoumi chicks to seasonal variations. The results indicate that the inbred lines were more sensitive to seasonal variations than the control ones and L25 showed the maximum responses in fertility and hatchability. The results also show that the local breed has been well adapted and often resists the harmful effect of inbreeding. Slight inbreeding had maximum response in viability and this may be due to early homozygosity occurring in alleles responsible for viability. For body weight, it was noticed that sensitivity of inbred lines decreased as chicks advanced in age. Therefore, these findings are important to stabilize the production of the local breed, all the year around. Introduction In 1970 an inbreeding trial had been carried out to get different inbred lines of Fa.youmi chicks. The aim of this experiment was to study the sensitivity of inbred lines to seasonal variation (i e. -winter and summer) for some economic traits of Fayoumi chicks. Material and Methods Full-sib, half-sib and first cousin matings were used to get different inbreeding intensities. The inbreeding coefficient percentages were 6. 25 , 12 .5, 25 .o and 37 .5. The control chicks were resulted randomly from a flock mating system. The pedigree chicks with control ones were brooded and reared during Winter 1 973 and Summer ig 74 . All the experimental conditions, except seasonal variation, were the same as far as possible. The available numbers of chicks were 95 6 for control and 3 o68 for the lines during the two years. Three pedigree pens were used for eachline. Each pen contained 1 5 females and one sire. The data are illustrated in Table z.. Traits studied were: fertility, hatchability, viability and body weight. All the percentage data were converted to angles with the arcsin transformation prior to statistical analysis using tables given by S N E D EC OR , 1959 . D UNCAN ' S new multiple range test was also used for statistical comparisons (STEEL and T ORRI E, ig6o). Corrections according to variation in control means in winter and summer were made to show the sensitivity of inbred lines to seasonal variation. Results and Discussion Fertility Data presented in Table 2 show the sensitivity of inbred lines to seasonal variation for fertility. The inbred lines were more sensitive to seasonal variation than the control chicks and L 25 showed the maximum response which reached 8. 0 6 p. cent. The range of fertility percentage was 2 .6 in winter, while it was 6.8 in summer. However, there were no significant differences among all the means during the two seasons. This may be due to the good adaptation of the inbred Fayoumi to our circumstances and to the harmful effect of inbreeding. This adaptation may be due to the custom of the Egyptian Farmers to raise flocks with limited numbers of cocks. Hatcha bility Inbred strain L 25 showed the greatest response to seasonal variation and its effect amounted to 8. 7 8 p. cent in relation to winter season (Table 2 ). However, it was noticed that most of the inbred lines were less affected by seasonal variation and this may be due to the fact that proper conditions of the air draft machine were almost the same during the two seasons. Moreover, the hatching eggs in summer were not stored and set at the next day of laying. A BDOU and M OUKH -D UNCAN ' S test of significance on transformed dams hatching percentages presented in Table 2 shows that there is no significant difference between control and slightly inbred line during both seasons. The marked difference was observed between I,12.5 and L!5, the latter being more affected by seasonal variation. In winter season there was no significant difference between them, while it was significant in summer. It was of interest to notice that intensive inbreeding (L 37 . 5 ) showed less variation and this may be due to the type of gene action in which homozygosity occured. Moreover, these results reveal again good adaptation of the local breed and its homeostasis. Viability Data presented in Tables 3 and 4 indicate that seasonal variation of the inbred lines for viability increased as chicks advanced in age. The seasonal differences in control chicks were -0 . 33 p. cent and &mdash; 4 . 49 p. cent at 4 and 8 weeks of age, while these figures in the inbred lines ranged from &mdash; 0 . 59 to -5.8 2 and &mdash; 2. 95 to -8. 40 , in the same order. The seasonal differences at 'i2 and 1 6 weeks were -4.15 and &mdash; 4 . 25 in the control and ranged from &mdash; 6.5 3 to -1 6. 14 and from &mdash; 8.m to -1 8. 5 8 in inbred lines, respectively. After substracting seasonal effect from total differences to get net difference due to inbreeding, it was noticed that L 625 gave the maximum response. This corresponds to the great differences between its viabilities in winter and summer. In winter the difference between L 6 . 25 and control chicks was larger than its corresponding in summer. Moreover, the viability means of L 6.25 was more neaily related to the control means in summer than in winter. D UN C AN ' S test showed the same trend where L 6.25 in winter was more related to intensive inbreeding lines, while in summer it was more related to control. These results indicate that slight inbreeding had maximum effect and this may be due to early homozygosity occured in alleles responsible for viability. Low total seasonal variation of viability at earlier ages may be due to heating system in brooding period which was almost the same during the two seasons. Therefore, after 8 weeks of age seasonal effect was more clear as chicks grew older and heating stopped. Duncan results reveal these findings where the differences among the means at earlier ages were somewhat the same during the two seasons. At older ages, these differences largely differed from winter to summer. These results indicated the sensitivity of the inbred line viability to seasonal variation, while the control chicks were less affected. Body weight The demand for poultry meat all the year around calls special consideration to seasonal variation in chicken raising. Therefore, seasonal effect and its interaction with the type of gene action in Fayoumi chickens are important from the economical viewpoint. Moreover, most of the local flocks are almost closed and having a limited number of cocks and inbreeding often occurs. Seasonal differences in the control chicks were 4LI2 ,29 .86,20 .88 and 8.8 4 ,at 4 ,8,12 and 1 6 weeks (Tables 5 and 6). It was clear that seasonal differences decreased as chicks advanced in age, and this may be due to genetic homeostasis in local breed. A BDOU ( 19 6 4 ) reported that seasonal variation in control chicks was 23 p. cent at 1 6 weeks of age. Tables 5 and 6 show that sensitivity of inbred chicks decreased as chicks advanced in age. It may indicate that alleles responsible for early growth rate were more sensitive to seasonal variations. This sensitivity in early age corresponded to great differences between the means of the two seasons. D UNCAN ' S tests revealed these findings where the differences among the means, at earlier ages were nearly the same during the two seasons. At older ages, these differences differed largely from winter to summer. Therefore, these findings are important to stabilize the production of local breed all the year around. Data presented in Re!u pour publication en decembve 19 7 7 .

v3-fos

2018-04-03T03:24:21.010Z

1977-01-01T00:00:00.000Z

3101688

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Nutritive efficiencies of lactalbumin and wheat gluten at very low levels of intake in adult rats. The nutritive values of proteins in relation to their intake levels were evaluated by feeding adult male rats weighing 250 g diets containing 0%, 0.39%, 0.78%, 1.56%, 2.34%, 3.90%, 7.79% and 15.58% lactalbumin or wheat gluten for three weeks. The biological values (BV) of both proteins were high at low levels of protein intake but decreased with increase in protein intake. The BV of wheat gluten was estimated to be about 100 at a level of intake of 1.56% but only 25 at a level of 15.58%. Similarly, the BV of lactalbumin decreased with increase in the protein level, being 67 at a level of 7.79%. The BVs of both proteins at low levels of dietary protein (below 2.34% of lactalbumin or 0.78% of wheat gluten) were apparently more than 100 because urinary N excretion was less than endogenous N. The BVs also decreased with time during the three-week test period. It is concluded that BV of a protein is not a fixed value but varies with the experimental conditions especially with changes in the amount of intake, and that differences in the qualities of various proteins cannot be compared quantitatively at a single level of protein. The results were briefly discussed in relation to protein requirements. in itself and is important in monitoring the qualities of proteins during food proces sing and in determining the quality of a new protein. The other aspect is concern ed with the physiological conditions of the man or animal eating the protein. Thus, in assessing protein requirements accurately, it is important to clarify the uti lization of proteins under various experimental conditions. The utilization of a protein is influenced by many factors, such as the age of the test animal, its nutritional state, its energy intake, the amount of protein con sumed, the other components of the diet and the length of experimental period. It has long been recognized that the efficiency of protein utilization is low when the protein supply is abundant, because excess protein cannot be stored in the body and the surplus is used as a source of energy. Therefore, protein quality has usually been measured at a single, low or moderate, test level of protein, which is assumed to be adequate for obtaining constant efficiency. However, several workers re ported that the nutritional values of proteins in men and rats change with protein intake, even when supplied at near or below the maintenance levels (1)(2)(3)(4)(5)(6). Fur thermore, MCLAUGHLAN and NOEL (7) reported that lysine-deficient amino acid mixture gave an erroneously high biological value (BY) when estimated at relatively low nitrogen levels. The utilizations of proteins at very low levels of intake have not been studied extensively in relation to protein requirement. Therefore, we examined the nutritive values of lactalbumin and wheat gluten to adult rats in terms of protein level and the length of the feeding period. MATERIALS AND METHODS Adult male rats of the Sprague-Dawley strain were fed a control diet containing 11.69% lactalbumin3 for 7 to 10 days until they weighed about 250g (Table 1). Table 2 shows that the food intake of rats on a protein-free diet was least. The intakes of lactalbumin and wheat gluten diets increased with increase in the protein content of the diet and there was no significant difference in food intakes of rats on lactalbumin and wheat gluten diets of equal protein contents. For some unknown reason, rats on 3.90% wheat gluten diet ate significantly less food than rats on a 2.34% or 7.79% wheat gluten diet. As shown in Fig. 1 lactalbumin, but it became curved with above 3.90% protein. Rectilinear regres sion lines relating N balance to N intake were calculated from results with between zero and 3.90% lactalbumin and between 2.34% and 15.58% wheat gluten in week 3. The slopes of these lines, representing the approximate efficiencies of utiliza tion of these proteins at around their maintenance levels, were 0.827 and 0.186 for lactalbumin and wheat gluten, respectively. The maintenance requirement of N, estimated from the regression lines in week 3, were 0.24g/kg/day for lactalbumin and 0.55 g/kg/day for wheat gluten. Changes in food intake and body weight Urinary N excretion at low levels of protein intake Although urinary N excretion generally rises with increase in protein intake, it remained essentially constant in rats on 0.39, 0.78, 1.56 and 2.34% lactalbumin diets (Table 3-A). Furthermore, rats on these low levels of lactalbumin or on 0.78% wheat gluten diet excreted significantly less nitrogen than rats on a protein free diet. Apparently the BVs of more than 100 in the limited ranges of protein intake shown in Fig. 4 and Table 4 were due to the nitrogen-sparing effect of feeding small amounts of proteins. The urinary N of rats on 3.90% lactalbumin diet or 1.56% wheat gluten diet was as high as that of the protein-free diet group, but above these levels of protein it significantly exceeded the endogenous output (p< 0.01). Relation of By to N intake The BVs of lactalbumin and wheat gluten calculated from the N balance data BVs calculated for limited intakes of wheat gluten and lactalbumin were apparently more than 100 because animals receiving small amounts of these proteins excreted less than the endogenous level of nitrogen in the urine ( Table 3). The difference in quality of the two proteins was obvious with above 2.34% protein, where the BYs of wheat gluten were distinctly lower than those of lactalbumin. At a level of 7.79 protein or more, lactalbumin could not be utilized with 100% efficiency. Effect of the length of the experimental period on the BV The BVs of the two proteins varied not only with the level of protein but also with the experimental period (Table 4). At higher levels of lactalbumin and wheat gluten, BVs in week 1 were higher than those in week 3, while no definite changes were observed at lower levels of intake. For instance, the BY of lactalbumin at a concentration of 7.79% protein was 91 in week 1 but only 67 in week 3, and simi larly the BV of wheat gluten at a concentration of 15.58% protein fell from 31 to 25. DISCUSSION SAID and HEGSTED (4) demonstrated that the regression line relating to change in body water with protein intake in adult rats fed wheat gluten diet at concentra tions of 3.20% to 9.20% protein was rectilinear but that on extrapolation this line did not intersect the dosage level at zero. To explain this they suggested that the relation was curvilinear at low protein concentration, but did not test this. In the present study, we paid special attention to utilization of wheat gluten at low levels of intake. Confirming the results and suggestion of Said and Hegsted, we found that the dose-response curve for wheat gluten between zero and 15.58 protein intake could not be represented in terms of body weight or N balance by a single straight line as shown in Figs. l and 3-B. The curves had points of inflec tion. The point of inflection was around 2.34% with wheat gluten, whereas it was above 3.90% with lactalbumin. With neither protein was utilization constant with up to 9% protein, the level at which protein quality is usually assayed (8). Similar ly, BERDANIER et al. (6) observed a decreased utilization of casein and soy protein with increased protein intake in the Thomas-Mitchell method (9) of determining the BV. However, recalculating the BV using non-urea nitrogen value for the endo genous nitrogen, they showed an independence of BV from nitrogen intake. YANEZ and MCLAUGHLAN (5) evaluated the quality of lysine-deficient proteins at two levels of intake and showed that NPR values were much higher when deter mined at the 5% level than at the 10% level. They also found that addition of lysine to wheat gluten diet gave higher NPR values at the 10% level of protein than at the 5% level. From these results they concluded that the wheat gluten diet was satisfactory for maintenance purposes but inadequate for growth and they suggest ed that amino acid requirements for maintenance and for growth were different in rats. However, INOUE et al. (3) reported that even in adult men whose body weight was in a steady state wheat gluten was utilized as well as egg protein at levels of below 0.2g/kg of protein intake. Therefore, the efficient utilization of wheat gluten at low levels of intake observed in the present study did not seem to be due to a difference in the lysine requirements for maintenance and growth. SAID and HEGSTED (10) demonstrated that rats conserved lysine and leucine more efficiently than other essential amino acids. The metabolism of lysine is very specific and its degradation is rather slow, particularly in lysine-deficient animals (11); the amount of lysine in the body relative to the amounts of other amino acids may be sufficient for protein synthesis when protein intake is very low. At protein intakes of below 1.56%, lactalbumin and wheat gluten were both almost fully utilized and no difference could be detected in their qualities. Thus the lysine content of wheat gluten did not seem to be limiting at this low level of protein although lysine was the first limiting amino acid when higher levels of pro tein were fed. At very low levels of dietary protein, the BVs of both wheat gluten and lactalbumin were more than 100, apparently because when the dietary level of proteins was low urinary N excretion exceeded the endogenous level. Nitrogen sparing effects were also observed with 3.5% egg protein diet (12) and methionine supplemented protein-free diet (13). YOSHIDA and MORITOKI (14) demonstrated that addition of methionine and threonine to a protein-free diet reduced urinary N excretion more than addition of methionine alone. YOKOGOSHI and YOSHIDA (15) further reported that the nitrogen-sparing action of methionine plus threonine was observed even when as little as 0.0188% of each amino acid was added to protein free diet. However, 1.30% zein diet, which is deficient in lysine but contains more methionine and threonine than wheat gluten, did not have nitrogen-sparing action (unpublished data). The nitrogen-sparing effect observed in this study requires further investigation. The difference in quality between lactalbumin and wheat gluten could be seen at protein levels of above 1.56 %: utilization of wheat gluten decreased markedly to a BV of 25 at 15.58% protein determined in week 3. In contrast, lactalbumin was utilized with 100% efficiency a level of up to 3.90% protein under the present experimental conditions, but its BV was only 67 at a concentration of 7.79% pro tein, estimated in week 3. These results show that even good quality protein, such as lactalbumin, is only utilized with 100% efficiency under specific conditions, such as during recovery from depletion, at low levels of protein intake or in rapidly growing animals. The results also show that no protein can be used at a safe level of intake for adult man as a reference of 100% efficiency of usage. The BV was also affected by the length of the experimental period: the BVs of both lactalbumin and wheat gluten decreased with time during the feeding period (Table 4). This tendency can be seen from the data on N balance in Figs. 3-A and 3-B. Comparison of the relations of N balance and N intake in weeks 1 and 3 showed that N balance in week 3 tended to approach zero from both positive and negative balances. For instance, with 7.79% lactalbumin, N intake did not change but the urinary and fecal N outputs increased with time and so the extent of N retention was less in week 3 than in week 1 (Table 3-A). In contrast, at mainte nance levels of protein or less, urinary N excretion decreased with time more signifi cantly than fecal N or N intake. Thus, the N balance improved with time and the improvement was greatest at zero protein concentration. Consequently, the BVs tended to decrease both in and below the maintenance range of N intake during the 3-week feeding period. These changes may be regarded as adaptations of the animals; but if the test period is long, the factor of aging of animals should also be taken into consideration. As described above, the BV was not a fixed value but varied with the experi mental conditions. Moreover, the changes in the BVs of wheat gluten and lactal bumin with the dietary protein levels were different (Fig. 4), and so the relative nutritive values of wheat gluten and lactalbumin varied at different protein levels. This means that differences in the qualities of the various proteins cannot be repre sented by a single correction factor determined at a given level, such as 9 % pro tein (8). One of the most important problems in estimating protein requirements is how to represent protein qualities. For practical purposes, HEGSTED (16) proposed measuring protein quality by the slope-ratio assay and suggested defining the ef ficiency of utilization of a protein as the slope of the dose-response line around the requirement level relative to that of a standard protein such as lactalbumin. The nutritive value of wheat gluten relative to that of lactalbumin based on the slope ratio assay in this study was 22.5% in week 3 (Fig. 3-B), and this value is very similar to that of 21,8% for growing rats reported by HEGSTED and CHANG (17). The ef ficiency of utilization of lactalbumin, estimated as the slope of the regression line between N intake and N balance between zero and 3.90% protein, was 83% ( Fig. 3-B). This figure is the same as that given by Block and Mitchell in their review (18) for growing rats. In human subjects, CALLOWAY and MARGEN (1) reported that the NPU of egg protein was about 65 rather than the expected value of 100. A similar value was obtained by YOUNG et al. (2) and a still lower value was observ ed by INOUE and his co-workers (19). These values show the great variability in utilization of dietary protein and the importance of estimating the nutritive value relative to that of a standard protein. However, the problem of how to evaluate the efficiency of utilization of a standard protein remains to be solved. We are grateful to Professor G. Inoue for his advice in conducting the present investigation and in preparing the manuscript.

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2014-10-01T00:00:00.000Z

1977-08-01T00:00:00.000Z

18964875

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Epidemiology and toxicology of arsenic poisoning in domestic animals. Arsenic poisoning is one of the more important causes of heavy metal poisoning in domestic animals. Two species--dogs and cattle--are intoxicated more frequently than other animals; yet sporadic instances of poisoning have been observed in cats, horses, and pigs. Cases observed by veterinary clinicians are either peracute, acute, or chronic intoxications. Frequently the initial and only indication that a severe problem exists with peracute poisoning in a cattle herd is dead animals. Chronic intoxications are also observed in cattle. Acute intoxication is the most common form of arsenic poisoning observed and documented in the dog. Also intoxicated dogs were younger, i.e., 2-6 months of age. Arsenic is a severe alimentary tract irritant in domestic animals, and treatment in most instances consists mainly of symptomatic and supportive treatment. The source of intoxication, when it can be determined, is usually dips, sprays, powders, or vegetation contaminated by pesticides containing arsenic. Introduction Arsenic is second only to lead as a cause of heavy metal intoxication in domestic animals. Clinically, arsenic intoxication occurs as an acute or peracute intoxication, although chronic forms of the disease have been observed, especially in cattle. Intoxication results from consumption of the trivalent inorganic or organic forms of arsenic. Trivalent arsenic is the more toxic form of the element for domestic animals (1). Arsenic occurs in small amounts naturally in soil and native vegetation (2,3). However, most instances of intoxication result from the consumption of soil, vegetation, or discarded materials that contain or are contaminated with high levels of arsenic. When the source of the intoxication can be found, it is usually herbicides or insecticides containing arsenic. Described in this report is a review of the basic toxicology of arsenic intoxication in animals and an evaluation of the medical records from 13 North American veterinary coliegest for the 11-year period of [1964][1965][1966][1967][1968][1969][1970][1971][1972][1973][1974]. We also reviewed cases from the Missouri State Diagnostic Laboratory at Columbia, Missouri, as well as selected data collected by the authors in their field investigations of suspected intoxication outbreaks in cattle, later documented as arsenic intoxication. Background Man and lower animals are highly susceptible to inorganic arsenic. But in the diagnostic laboratory, arsenic poisoning is most frequently encountered in the bovine and feline species resulting from contamination of their food supply. Occurrence of arsenical poisoning in these two species is closely followed in other forage-eating animals, such as the sheep and horse. Arsenical poisoning in most animals is usually manifested by an acute or subacute syndrome. Chronic poisoning, although it has been reported in animals, is seldom seen and has not been clearly documented. Experience with field cases of arsenic poisoning indicates that animals which are weak, debilitated and dehydrated are much more susceptible to arsenic poisoning than normal animals. This may be because of reduced excretion rate via the kidneys (4). August 1977 183 Inorganic arsenic is found in nature and is synthesized in many complex and varied forms, having many uses from medicinal to forensic. In practice, the most dangerous arsenical preparations are dips, herbicides, and defoliants in which the arsenical is in a highly soluble trivalent form, usually trioxide or arsenite. Unfortunately, animals (such as dogs and calves) will frequently seek out and eat materials such as insulation, rodent baits, dirt, and foliage that have been contaminated with an inorganic arsenical. Soluble forms of arsenic, such as sodium arsenite, are readily absorbed from all body surfaces. Arsenic trioxide and other less soluble arsenicals are poorly absorbed from the digestive tract and are largely excreted unchanged in the feces. Once absorbed, pentavalent arsenic is readily excreted by the kidneys, whereas trivalent arsenic is more readily excreted into the intestine via the bile. It is generally considered that regardless of whether an arsenical is introduced into the body as trivalent or pentavalent arsenic, all the major actions can be attributed to the trivalent form. Arsenicals are suspected of having a metabolic function (5) and ultimately, they exert their effects by reacting with sulfhydryl groups in cells (6). While most textbooks report that arsenic is accumulated in the tissues and slowly excreted, this phenomenon appears to be true only in rats. Most species of livestock and pet animals apparently rapidly excrete arsenic (7). This phenomenon is very important when one considers arsenic levels in tissues as a means of confirming suspected poisoning. Peracute and acute episodes of poisoning by inorganic arsenic are usually explosive with high morbidity and moderate mortality over a 2to 3-day period. Symptoms are manifested by intense abdominal pain, staggering gait, extreme weakness, trembling, salivation, vomiting (in dogs, cats, pigs, and perhaps even cattle), diarrhea, fast, feeble pulse, prostration, rumen atony, normal to subnormal temperature, collapse, and death. In subacute arsenic poisoning, animals may live for several days, exhibiting depression, anorexia, watery diarrhea, increased urination at first followed by anuria, dehydration, thirst, partial paralysis of the rear limbs, trembling, stupor, cold extremities, subnormal temperature, and death. The watery diarrhea may contain shreds of intestinal mucosa and blood. Convulsive seizures have been reported but are not an expected manifestation. Poisoning from arsenical dips usually results in some of the signs noted previously, in addition to blistering and edema of the skin followed by cracking and bleeding with associated secondary infection (4). Characteristic gross lesions associated with inorganic arsenic poisoning include reddening of the gastric mucosa (abomasum in ruminants) which may be localized or general, reddening of the small intestinal mucosa (often limited to the first few feet of the duodenum), fluid gastrointestinal contents which are sometimes foul smelling, soft yellow liver and red, edematous lungs. In peracute cases of poisoni.ng, occasionally no gross postmortem changes are noted. The inflammation is usually followed by edema, rupture of the blood vessels and necrosis of the mucosa and submucosa. Sometimes the necrosis progresses to perforation of either the stomach or intestine. The fluid gastrointestinal contents may contain blood and shreds of mucosa. Hemorrhages on all surfaces of the heart and on the peritoneum may occasionally be observed (4,8). Histopathologic changes include gastric and intestinal edema of the mucosa and submucosa, necrosis and sloughing of mucosal epithelium, renal tubular degeneration, hepatic fatty change and necrosis, and capillary degeneration in vascular beds of the gastrointestinal tract, skin, and other organs. In cases involving cutaneous exposure, a dry, cracked, leathery, peeling skin may be a prominent feature. The urine of poisoned animals may contain protein, red blood cells and casts. The arsenic level in the urine varies with the form of arsenic, route of exposure and species but usually ranges from 2-10 ppm (4). Materials and Methods Medical records in the Veterinary Medical Data Program (VMDP) for the years 1964-1974 were reviewed for cases with a diagnosis of arsenic intoxication. The VMDP is a data collection and storage registry sponsored by the National Cancer Institute, to which veterinary university hospitals/clinics submit standardized abstracts about each medical episode occurring at their facility. All diagnoses of arsenic poisoning were considered regardless of the diagnostic techniques used to arrive at a diagnosis. In a majority of instances the diagnosis of arsenic intoxication was based only on clinical observations. Also, because a majority of the intoxications occurred in two species-dogs and cattle-major considerations of the epidemiology and toxicology were confined to these two species. Furthermore, comparison with controls, i.e., cases presented to one of the clinics without arsenic intoxication, will be considered only for dogs as approximately 70% of the arsenic intoxicated animals observed in the 13 veterinary hospitals/clinics occurred in this species. The medical records in the VMDP registry were either compiled directly from a disk file or Environmental Health Perspectives they were abstracted onto computer cards and analyzed using the Statistical Package for the Social Sciences (SPSS) or the Statistical Analysis System (SAS) computer programs (9,10). The effects of the independent variables of age, breed and sex upon a diagnosis of arsenic poisoning were evaluated by estimating the relative risk of occurrence by Gart's method (11). Comparisons were made with a reference population drawn from the same medical facilities and study period as the case series. The reference population was tabulated by the patient-years-at-risk method. In addition to reviewing the veterinary clinic records, the records of one of the diagnostic clinics in Missouri was reviewed for possible instances of arsenic intoxication. With these records, a majority of the instances of arsenic intoxication occurred in cattle. In some of these outbreaks one of the authors conducted field investigations and/or had analyzed tissues or feed for arsenic. "Typical" examples of these investigations will be presented to facilitate our discussion of the toxicology and epidemiology of arsenic intoxication in domestic animals. Results Between June 1964 and July 1974 a total of 93 animals were diagnosed as having arsenic intoxication in the VMDP registry. Of these 93 animals shown in Table 1, 64 (69%) were dogs, 18 (l9o) cattle, 5 (5%) cats, 4 (4%) pigs, and 2 (2%) were horses. Thirty-three (35%) of the cases were treated as outpatients. Of those animals hospitalized the median length of hospitalization was less than 2 days. Sixty-eight (73%) of the animals survived the initial intoxication experience. Approximately 10 patients with arsenic intoxication were diagnosed annually from 1968 to 1974, except 1970, when 21 animals were seen with arsenic intoxication. A majority of these animals were intoxicated cattle. This represents 44% of the total of cattle diagnosed with arsenic intoxication during our survey period. Table 2 presents a comparison of selected variables for dogs (N = 64) and Cattle (N = 18). Approximately twice as many dogs as cattle were treated as outpatients, and for those animals hospitalized the median number of days that they were hospitalized was half as long for dogs as it was for cattle. Another contrasting variable was that 83% of the dogs were discharged alive, where only 50% of the cattle were discharged alive from the clinicS. Concerning clinical procedures, 53% of the dogs were diagnosed strictly on clinical findings, whereas only 22% of the cattle were diagnosed with the same criteria. Figure 1 gives the percentage distribution by month of arsenic poisoning diagnosed in dogs and cattle for the complete study period. The month which was the mode in most species was June. The fluctuation in this particular chart for cattle percentages may be due in part to the small number of animals observed. August 1977 Taking the analysis a step further, Table 3 presents the crude attack rate per 100,000 patientyears-at-risk for the 64 dogs observed with arsenic poisoning. This distribution appears bimodal with two peaks, one in 1970, one in 1972 respectively. However, in pursuing this type of analysis further, ity of those that were diagnosed in this laboratory were cattle. Taking this analysis a step further, the records that were available between [1970][1971][1972][1973][1974][1975] were then examined to try to determine the number of animals in the herd, number of animals affected, -' / , _< _ > _ ,,i.e., primarily those that died from the intoxication, possible case fatality rate, as well as the source of the arsenic intoxication. These results for some 12 outbreaks are summarized in Table 6. As shown in JAN FEB FR APR M/AY JUN JLY AUG SPT OCT NOV DEC ing examples are presented. (p -0.05) between arsenic poisoning and these patient characteristics. We then turned our attention to an evaluation of the Veterinary Medical Diagnostic Laboratory rec-Outbreak A. Weeds were sprayed with a herbicide containing sodium arsenite as part of the maintenance program on a defense establishment in west central Missouri. The person applying the Environmental Health Perspectives Fi, weedkiller sprayed through the fence, covering weeds for a yard or so outside the defense station fence, and 24 of 26 young cattle in this adjacent pasture died of arsenic poisoning. Field observations and a history suggested that the cattle appeared to be attracted to foliage which had been sprayed with arsenical herbicide. Outbreak B. An owner uncovered a metal drum in a corner of a combination machinery shed and hay barn. The container was so old that any original labels were lost or had become illegible. He thought the drum contained a molasses supplement, so he mixed the contents with feed which was then fed to feeder calves. The cattle became seriously ill within a few hours and many died. Necropsy of the dead cattle revealed evidence of arsenical poisoning. Tests for arsenic on tissue and contents of the digestive tract were positive for arsenic. The remains of the material in the drum which the owner thought was molasses proved to contain 40% sodium arsenite, undoubtedly a weed killer and not a molasses supplement! The owner lost 18 of 25 calves. Outbreak C. A rancher stated that his cattle were "eating dirt," and it was poisoning them. He submitted a dead animal to necropsy after his local veterinarian had made a tentative diagnosis of arsenic poisoning. The soil sample, as well as tissues from the dead animal, contained high levels of arse-nic. Search of the site where the cattle were seen to be "eating dirt" revealed that the cattle had unearthed an old metal drum, buried many years ago along with other trash. Time and erosion had exposed the arsenic container, and the cattle finished uncovering it. As in outbreak A, cattle seemed to be attracted to discarded material or soil containing arsenical compounds. Discussion The clinical and toxicological signs of arsenic intoxication in domestic animals have been the same since the disease was first reported. Arsenic is not highly corrosive; it does, nevertheless, cause severe inflammation, eventual edema, and subsequent necrosis of the gastrointestinal mucosa and submucosa, if the animal survives the peracute phase. Thus, the major clinical sign seen is a severe gastroenteritis, primarily a diarrhea. If one counts specimens sent to the diagnostic laboratory with a request that the specimen be analyzed for arsenic, one would include a majority of the diarrheas of unknown etiology. What is presented herein are the confirmed cases of arsenic intoxication in domestic animals; as such, they represent only a small portion of the true incidence. The incidence of arsenic intoxication reported to our diagnostic laboratory agrees with the observa-August 1977 tions of Hatch and Funnell, who reported 21 positive diagnoses of arsenic poisoning in cattle during an 8-year period in Ontario, Canada (12). Common sources of arsenic, when the source could be determined (Table 6), included sprays, dips, and powders used as insecticides or herbicides. In a majority of the intoxicated cattle this material was consumed voluntarily from such sources as feed, contaminated soil, materials left on trash piles, in vegetation along fence rows, or around buildings that had been sprayed with a weed killer. In approximately 80% of the dogs intoxicated with arsenic, the source was found to be ant or roach bait containing arsenic, e.g., a sodiumarsenate compound. In these instances, the dogs were presented to the clinic with a history of vomiting and some muscular weakness and muscular trembling. Subsequent interview with the owner suggested that in a majority of cases an insecticide containing arsenic had been used in the area where the dog was housed or allowed to roam free. Many times owners of poisoned animals are not aware that an arsenical compound was available; that is, they do not realize that ant bait may contain arsenic. In many cases of arsenic intoxication in large animals, no source of arsenic was detected. Often considerable effort is required on the part of the clinician or diagnostician to assist owners in finding the source. It is characteristic that the owner will "look harder" when a positive diagnosis of arsenic intoxication has been confirmed or suggested. A positive clinical diagnosis of arsenic intoxication stimulates owners to help detect the source of the compound. In the instance of intoxication in large animals, often a field trip is an invaluable aid to the clinician and toxicologist in defining the location of the source of the compound, since the owner may not be aware of what to look for (e.g., signs of eating treated foliage, remains of containers or powders) as a source. Cattle owners used the compound frequently as a weed killer, and it was only on subsequent investigation that it was documented that such compounds were the cause of the intoxication. One statement we have made to owners in the past on field investigations is: "You and I have been searching this pasture for an hour looking for the source of the intoxication. These calves have been present in this pasture for approximately 3 to 10 days, and they have been more successful in locating the source for consumption and intoxication." It sometimes appears that animals, especially cattle, develop an increased desire to consume weeds sprayed with an arsenic weed killer, not because of a change in palatability of the plant but possibly because arsenic compounds tend to taste salty, and thus attractive, to the animals. 188 In diagnosis of arsenic intoxication, wet chemistry tests, such as the Reinsch test (13), are used as a screening tool; and, if positive, then more definitive chemical analysis must be used to quantitate the levels of arsenic; e.g., arsine evolution (14). A positive test does not conclusively confirm arsenic intoxication, for this test is sensitive to as little as 2 ppm of arsenic. In Missouri, for example, the geometric mean of arsenic found in soil varied from 13 ppm in the glaciated prairies portion of the state to approximately 7 ppm in the oak-hickory-pine area. In every sample of soil that was analyzed for arsenic, the inorganic form of the element was detected in all of the major vegetation-type areas (3). If we contrast the epidemiology of arsenic intoxication in the major species that were presented to our diagnostic laboratory and 13 veterinary clinics, we see quite a different view. A majority of the small animals, dogs and cats, are admitted to the veterinary clinic; whereas a greater majority of cattle cases are submitted to the diagnostic laboratory or seen on ambulatory service. The picture of arsenic intoxication by species is in part related to the type of animal population that these two facilities observe during the year. Dogs are more apt to be treated as outpatients than cattle. Also dogs were hospitalized a shorter period of time and more of them survive the intoxication, possibly not because of greater resistance to the arsenic, but rather because of a lower degree of exposure. In addition, most intoxications in dogs were diagnosed solely on a clinical examination and history. Younger animals are more likely to be intoxicated with arsenic. For example, in calculating the rate of arsenic poisoning for 100,000 dog-years-at-risk, adjusting for sex and age (see Fig. 2), there was a threefold increase for dogs 2-6 months of age compared to dogs under 2 months of age. Our initial impression before adjusting for age and sex was that possibly younger animals were being exposed to arsenic in the dam's milk; this is not the case. Further evaluation of the clinical records, however, did not support this hypothesis. It has also been suggested that Thiacetarsamide, a trivalent organic arsenical and the only drug that has consistently been found to kill adult canine heartworms that infect dogs (15), might cause arsenic intoxication. Recent work by a number of clinicians shows that an animal treated for heartworms with the arsenical treatment schedule does have a temporary rise in some liver function tests and in some instances animals will die shortly after treatment, suggesting a reaction to the arsenical drug (16,17). Yet, overt arsenic intoxication has not been confirmed as the cause of death. Finally, in an earlier report we were concerned with the potential public health aspects of arsenic Environmental Health Perspectives intoxication, primarily in cattle and other foodproducing animals (18). Depending on the form of the compound, arsenic may be excreted in the feces without absorption, with minimal absorption, or, if absorbed, it is primarily excreted through urine and the half-life is found to be fairly rapid, being a number of hours rather than days or weeks, compared to many other chemical compounds. The potential public health risks in cattle were felt to be minimal, if nonexistent. Preslaughter withdrawal after a single, acute exposure was recommended at 14 days; with multiple exposure 6 weeks withdrawal. In contrast, in the dog, the second species in which greater numbers of cases occurred, arsenic generally is consumed by a younger dog who is active, playful and inquisitive. The potential public health risk relative to dogs is not from a foodproducing standpoint, but rather for children who are exposed to the same area as the pet. There is also a potential public health risk that has been associated with another domestic animal; i.e., sheep. It has been recognized that arsenic can cause skin and lung cancer, especially in sheep dip workers (19). Monitoring domestic animals with a history of arsenic intoxication may offer clues to environmental hazards yet unknown to humans. Addendum According to information received from Bencko (20), excessive contamination of the environment by arsenic has resulted in the extinction of bees' colonies up to 30 km in the direction of the prevailing winds from the Novaky power plant in Czechoslovakia. Examination of soil and vegetation in the exposed area showed a relationship between arsenic in the soil and vegetation. The quantity of nitrogen in the soil was diminished with increasing quantity of arsenic. Also, soil samples containing approximately 165 ppm of arsenic showed diminished quantities of bacteria as well as protozoa, and no worms were found in samples containing arsenic in values about 150 ppm. Influence of arsenic on the reproductive functions of domestic animals was encountered in this area. In a village near the power plant, there was a pig-breeding farm specializing in large-scale production of piglets. After the power plant went into operation, the incidence of abortions among sows increased with time to the point where it was necessary to close the farm and move it away from the power plant. Arsenic is known to be a capillarytoxic poison. Due to environmental pollution, the pregnant sows received in their forage sufficient doses of arsenic to damage the placental blood capillaries to the extent that abortions began to occur. Abortion rates also increased among cattle in this area but did not reach epidemic proportions.

v3-fos

2018-04-03T00:00:37.100Z

1977-02-01T00:00:00.000Z

8613175

s2

Proposed Calfhood Immunization Program for the Commercial Dairy Herd Immunization programs never will usurp the central role of sound management practices and good nutrition in the disease prevention program of the commercial dairy operation. However, certain immunizations against disease such as brucellosis, leptospirosis, and clostridial infections should be routine. Other disease such as infectious bovine rhinotracheitis, bovine virus diarrhea, parainfluenza-3, colibacillosis, and pasteurellosis should be considered if it can be determined that the herd is infected chronically. The present knowledge of other disease conditions, vaccine effectiveness and safety makes the use of vaccines for other diseases of questionable value. INTRODUCTION The task of rearing healthy" calves as milking-line replacements always has been a major problem of the dairyman. Little thought is required to realize that the death loss of potentially great producers due to scours and pneumonia in the baby calf is a major difference between a financially rewarding and a marginal or bankrupt operation. Management and nutrition are the keys to the door of successful disease prevention. Prophylaxis and treatment can be considered only, at best, a second line of defense against decimating diseases of the dairy calf. The prevention of disease by enhancing the immune mechanisms of the baby calf to increase its resistance to disease never should become more important in the dairyman's mind than the strict adherence to good management practices and sound nutritional programs. Such sound principles as the assurance by the dairyman that the neonatal calf receives adequate amounts of antibodyladen colostrum within the first few hours of life cannot be overemphasized. Even though this practice has a sound immunologic basis, it will not be considered in this discussion except as it applies to specific vaccination programs. The purpose of this report is to make an unbiased evaluation of the many vaccines available and try to develop recommendations 1 for a reasonable vaccination program based upon scientific knowledge of the immunogenicity of the product, the resistance developed in the animal vaccinated, and the reasonable risk involved in not vaccinating the animal. The report is organized around bacterial and viral disease agents that may be expected in neonatal, young, and growing calves. Diseases of the Neonatal Dairy Calf The most prominent diseases in the neonatal dairy calf may be divided for convenience of discussion into septicemias and diarrhea-respiratory disease complexes. Septicemias. Septicemic diseases usually occur in the first few hours of life. The syndrome is due to the growth and circulation of pathogenic bacteria and their toxigenic products within the blood stream. The clinical signs of fever, hemorrhages in the mucosal surface, lethargy, prostration, and hypoxia are indications of a grave prognosis. The disease can be caused by bacteria in virtually every genera of bacteria, but the most common ones, Streptococci, Escbericbia, Salmonellae, Corynebacteria, Klebsiella, and Proteus, usually enter the blood stream through superficial wounds in the mucosaI surfaces or severed naval cords. The value of colostrum to prevent such serious infections is unknown. Maternal antibody could play an important role as a protective mechanism in light infections, but gross contamination always would overwhelm such protective mechanisms. No known vaccines to be used in the neonate have been effective nor would be expected tO be developed against these bacterial agents. Viremias are in the same category as the above, but both conditions seem to be reasonably rare. Diarrhea-respiratory disease complex. Bacterial and vital pneumoenteritis of the neonatal calf is perhaps the most prevalent cause of calf losses. A review of the literature indicates that many different agents are involved in the pathogenesis of diarrheal disease of the neonatal calf. These agents may be singularly causal or may serve as concert pathogens in combined infections. They may be divided conveniently into bacteria and viral agents. Bacterial causes include such well known agents as Escbericbia coli (colibacillosis), Salmonella typbimurium, S. Dublin, and other sp. (Salmonellosis) and, occasionally, other genera of bacteria. The viral agents associated with pneumoenteritis include bovine virus diarrhea-mucosal disease virus (BVD), reovirus, coronavirus, adenoviruses, infectious bovine rhinotracheitis (IBR), bovine parvoviruses, and probably enteroviruses. Bacterial Diseases of the Neonatal Calf Colibacillosis. It has been estimated that E. coil is responsible for 90% of the calf losses in early neonatal life. Colibacillosis may account for as many as 50% of the calf losses in a herd, and death rates of 10 to 20% are not uncommon. Because the disease strikes so early in life, it probably is not reasonable to believe that a vaccine could be developed that would immunize the individual effectively soon enough to prevent disease. Recent experimental data shows promise, however, in immunization of the dam with bacterins prepared from known pathogenic strains of the organism (31). Myers and his colleagues (30) have shown that effective resistance to experimentally induced cotibacillosis is induced in calves nursing dams immunized with bacterins injected subcutaneously 1 yr previously and 1 wk before the expected beginning of calving within the herd. The practice of preparing autogenous bacterins from E. coli strains isolated from problem herds to vaccinate dams has been recommended by veterinarians for years, but these reports are sound scientific evidence that the procedure is effective. Though commercial vaccines are not now available, Myers (30) reports that "a commercial firm has prepared the vaccine for sale and will release it for marketing as soon as a license becomes available." He recommends that the primary vaccination be administered 6 mo before and a secondary booster be given 1 mo before the expected calving date. The effectiveness of such a vaccination program on experimental infections with the homologous strain of E. coil provides hope for the dairyman, but the results of this and other investigations (8, 11) must be verified carefully against the efficacy of the procedure on a routine herd basis. The impact of strain differences in vaccine development and immunospecificity cannot be predicted with certainty. Salmonellosis. Acute enteritis in neonatal dairy calves caused by salmonella infection are not rare, but the incidence is much lower than E. coli infections. The same type of general conditions apply as in the colibacillosis situation. Vaccination programs designed to immunize the dam and thereby provide antibodies via the colostrum offer hope. Since salmonella infections are more prone to be a serious disease in later life, it would be more feasible to suggest the possibility of developing a bacterin to be used in early calfhood to protect from Salmonellosis. The availability of effective vaccines for commercial use is unknown to the author. The procedures for development and potency testing of Salmonella vaccines have been reported (2). Viral Diseases of the Neonatal Calf Bovine virus diarrhea (BVD) causes serious disease in calves (35). Experiments by Lambert et al. (23) indicated that deaths due to acute enteritis may occur within 18 to 96 h of birth and that chronic diarrhea could exist for weeks. Widespread use of attenuated live viral diarrhea (BVD) virus vaccines has been reported (22). Their use in dairy calves has been recommended in conjunction with concentrated anti-BVD bovine serum (20). However, the seriousness of side reactions and the chronic nature of BVD infections has prompted caution and has promoted the investigation of killed virus vaccine development (21). Experimental evidence indi-SMITH cated that such inactivated vaccines can be prepared (9, 10) but currently are not available commercially. Infectious bovine rhinotracheitis (IBR)virus has caused intestinal disease in young and neonatal calves (12,34). The wide range of symptoms recorded (12,16) exemplifies the complex nature of the disease in chronically infected herds. The agent is apparently widespread in dairy herds (16), but each of these reports indicates that the widespread use of modified live virus vaccines may be responsible for some of the evidence for the existence of chronic infections. The intranasal type vaccine to prevent disease is recommended by workers actively pursuing research in this area (17). The primary vaccination should be given at 4 to 6 days of age, a secondary booster vaccination should be given at 6 mo of age and again at one yr and annually thereafter. The dangers of a modified live virus vaccine causing overt disease in immunodeficient calves or of reverting to the virulent form at present cannot be evaluated properly. Our own research indicates that few passages in bovine tissue culture cells and an increase in the viral titer of the inoculum will cause similar lesions to the field strain when inoculated by the same routes and that recrudescence of the vaccine virus by chemical immunosuppression causes overt disease. The use and effectiveness of an attenuated nonabortigenic temperature sensitive mutant of IBR has been reported (41). The theory behind such an effort is intriguing, but the vaccine must still be considered experimental. Adenovirus infections have caused diarrheal disease in newborn calves in Europe (3) and North America (5,24). Bovine adenovirus type 5 has been implicated as being one of the causal agents in the so-called "weak-calf syndrome" and has produced mild diarrhea when inoculated into neonatal calves (25). Adenovirus vaccines have been used widely in Europe (6), but their effectiveness is questionable. A limited field trial is underway in this country, but recommendations for their use in a herd health program must be withheld until such time as a proven potent, safe vaccine is available and the extent and importance of the disease condition in this country is known. Bovine reoviruses have caused diarrheal disease in young calves (26,39), and it has caused high incidence of herd epizootics in beef herds (40). The use of a modified live virus has been reported and, its use recommended to control outbreaks of the disease when a definite diagnosis has been made in a herd (27). Since adequate nonvaccinated controls were not used, it has been difficult to evaluate the effectiveness of the vaccine in controlling natural outbreaks of diarrhea. Expanded studies were reported later (28), but the evidence for the necessity for routine vaccination programs and efficacy of the vaccine in preventing diarrhea is not convincing. Coronaviruses have caused diarrheal disease in neonatal calves. Their morphology and pathogenicity in neonatal gnotobiotic calves were described (36). The infections were in 12 of 19 beef herds examined. Though commercial vaccines are available, the scientific data are insufficient to determine the efficacy, potency, safety, and necessity of vaccination as a routine procedure. Enteroviruses and paroviruses have been associated with diarrheal disease of the young calf (1,37). They are apparently widespread infections, and diagnosticians and other investigators often isolate one or both from calves with diarrhea. These viruses frequently are isolated from calves that are apparently healthy. The knowledge of the pathogenicity of these two groups of viruses is not complete. Our studies indicate that massive doses of bovine enterovirus inoculated orally in neonatal colostrum-deprived calves even when immunosuppressed with corticosteroids did not cause serious enteric disease. Diseases of the Juvenile and Young Adult Cattle Consideration of routine immunization of calves against several well known diseases should be incorporated into recommendations for a vaccination program for herd replacements. Brucellosis. The vaccination of dairy calves with Brucella abortus strain 19 is a well accepted and scientifically sound principle (18). The vaccine is recommended for calves, except bulls (19), at 4 mo of age. However, the establishment of persistent infections with shedding of live organisms in the milk following standard vaccination procedures (29) (14). The outlook is promising, but commercial vaccines are not available. Vibriosis. General infections with Campylobacter (Vibrio) fetus virtually has been eliminated in the modern dairy industry principally through artificial insemination procedures (4). However, in infected herds, vaccination of adult cows (15) and bulls (7) can prevent and eliminate the problem. Recommended Vaccination Program The following recommendations are based upon the author's personal judgment and predicated upon personal ownership of an imaginary average commercial herd operation. Several routine immunization practices can be recommended as outlined in Table 1. Diagnosis of a specific disease condition within a herd would indicate that other vaccination programs should become routine, and in those conditions listed in Table 2 should be continued in a routine. In each situation I believe the determination of the herd status by serologic or other accepted diagnostic techniques should receive high priority to enable one to make specific recommendations for the herd immunization program. Immunization against bacterial diseases listed in Table 3 should become routine when the disease condition has been diagnosed or vaccines have been proven efficacious and are available for commercial use.

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2018-04-03T05:44:11.701Z

1977-01-01T00:00:00.000Z

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Determination of free monosaccharides and detection of sugar alcohols in mature soybean seeds. Although the oligosaccharide contents of soybeans are well documented, the exact values of monosaccharide contents have not been reported. Elaborate methods of preparative paper chromatography together with gas chromatography established the following data for one variety, Kyushu No. 12. The air-dried cotyledon part (admixed with hypocotyls) contained 0.030% glucose and 0.039% fructose. The hull part contained 0.018% galactose, 0.028% glucose, 0.023% fructose, 0.005% arabinose, and 0.002% xylose. Gas chromatograms of trimethylsilated monosaccharide fractions revealed the existence of minute amounts of sorbitol, arabitol, xylitol, and mannitol in decreasing order (about 0.03% to 0.001% of whole seeds). Summary. Although the oligosaccharide contents of soybeans are well documented, the exact values of monosaccharide contents have not been reported. Elaborate methods of preparative paper chromatography together with gas chromatography established the following data for one variety, Kyushu No. 12. The air-dried cotyledon part (admixed with hypocotyls) contained 0.030% glucose and 0.039% fructose. The hull part contained 0.018% galactose, 0.028% glucose, 0.023% fructose, 0.005% arabinose, and 0.002% xylose. Gas chromatograms of trimethyl silated monosaccharide fractions revealed the existence of minute amounts of sorbitol, arabitol, xylitol, and mannitol in decreasing order (about 0.03% to 0.001% of whole seeds). Simultaneous determination of sugars is very difficult when various sugars are present in extremely different amounts. When soybeans were analyzed for mono and oligosaccharides, their hulls gave measurable data for both, whereas their cotyledons and hypocotyls did not, since the monosaccharides were far less than the oligosaccharides. Our previous data (1) were calculated without regard for the presence of monosaccharides in cotyledons and hypocotyls. Therefore, the values for monosaccharides were too low since they were calculated only from the data of monosaccharide contents of hulls, consisting of only about 7% of total seeds. The data (average of all the nine varieties of soybeans) are shown in Table 1. In this paper, we report the monosaccharide contents of the hulls and cotyle dons (mixed with small amounts of hypocotyls) of mature soybean seeds deter mined by careful methods using paper partition and gas liquid chromatography. Soybeans. Mature seeds used were obtained from soybeans, Glycine max cultivar. Kyushu No. 12, which were planted in July 1969, at the University Farm of our faculty. They were separated into the two parts, cotyledons (hypocotyls present in about 2% of the total seeds were not removed) and hulls. They were pulverized to pass through a 32-mesh sieve. Extraction of sugars. (a) For the cotyledon part. Eleven parts of 80% ethanol were added to one part of cotyledons and refluxed 1 hr in a boiling-water bath. About four parts of ether were added to the ethanolic extracts and the upper layer discarded after settling. This defatting process was repeated several times. Lead acetate and then sodium carbonate were added to remove proteins. The concentrated filtrate was passed through a column consisting of Amberlite IRC-50 and Amberlite IR-45 (1:2) to remove remaining salts, and the eluate was concen trated to about a 10% sugar solution. (b) For the hull part. The procedure is the same except the omission of the defatting process. Separation of monosaccharides from oligosaccharides. Preliminary experi ments showed that the carbon column chromatography according to WHISTLER and DURSO (2) was the most suitable, when compared with ion exchange chro matography with Dowex-50 (K+type), cellulose column chromatography, and gel filtration with Sephadex G-15. Chromatography on a column of Darco G-60 and Celite (1:1) (2) gave monosaccharide fractions which were concentrated to 1 or 2ml. Qualitative paper chromatography. Since 5 monosaccharides were detected in soybean seeds by preliminary gas chromatography, a search was made for the most convenient solvent system to clearly separate the five sugars, i.e., glucose, fructose, galactose, arabinose, and xylose. A solvent system consisting of ethyl acetate, pyridine, acetic acid, and water (30:11:1:6) was selected since the time necessary for chromatography was relatively short and five repetitions were satis factory to separate all five sugars. matography after they were identified from the retention time of gas chromatog raphy. The absorbance at 530nm of the authentic sugar was as shown in Table 2 . The result of determination calculated (average of three measurements) on air dry basis is shown in Table 3. Thus, soybean cotyledons do contain 0.030% glucose and 0 .039% fructose. Though both are very small in amount, it is noteworthy that they contain more free fructose than glucose. It is interesting that soybean hulls contain five free monosaccharides . They are glucose, fructose, galactose, arabinose, and xylose in decreasing order (0 .028% to 0.002%). When calculated for whole seeds by putting the yield of hulls to be 7 .23% (1), the sugar content of cotyledons (plus hypocotyls) have far larger effect . Air-dried soybeans (cultivar. Kyushu No. 12) contained 0 .038% fructose, 0.030% glucose, 0.001% galactose, 0.0004% arabinose, and 0.0001% xylose . Previous data on monosaccharides (Table 1) (1) are extremely incorrect . (The previous arabinose content of hulls, 0.02%, was erroneously high .) Approximate estimation of sugar alcohols from gas chromatograms Sugar alcohols were identified by retention time, but they were not determined quantitatively. However, their approximate amounts may be estimated from the corresponding peak areas. The values are shown in Table 4 . As shown in Fig. 7 the xylose fraction of hulls contain a considerable amount of an unidentified compound (possibly a sugar alcohol) with a low retention time . Anyway the air dried whole soybeans contain about the same order of free sugar alcohols with free monosaccharides: about 0.03% sorbitol, 0.01% arabitol, 0.002% xylitol, and 0.001% mannitol. In conclusion we obtained reliable data for free monosaccharides present in mature soybean seeds. At the same time we discovered that soybeans contained sugar alcohols, though in very small amounts. We thank Dr. Hiroshi Suzuki and Mr. Sumizo Tanusi of our Laboratory for their helpful discussions.

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2018-04-03T00:10:57.317Z

1977-02-01T00:00:00.000Z

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Probable Role of Viruses in Calfhood Diseases Bovine enteroviruses, bovine viral diarrhea virus, rotavirus (formerly called reovirus-like agent), coronavirus-like agent, bovine adenovirus, and bovine parainfluenza-3 virus have been isolated from calves suffering from neonatal disease. The experimental disease produced by these viruses is not necessarily severe or fatal, but under farm and ranch conditions, each probably serves as an added stress factor that contributes significantly to mortality from neonatal disease. After initial losses following the introduction of a virus into a herd, the subsequent losses will be limited because the cow will produce antibodies to protect the fetus during gestation. Antibodies will also be concentrated in colostrum to protect the calf at birth. However, colostrum must be fed immediately after birth, before the calf becomes infected. nutrition. The value of colostrum for protecting the calf from many infectious agents common to a normal herd is well known (1,2,25). In experimental work calves may be fed colostrum before exposure, deprived of colostrum until after exposure or deprived of colostrum for the entire experiment. When exposure to an infectious agent isolated from a sick calf does not produce disease in colostrum-fed calves on experiment, it may suggest the agent crossed the placental barrier, infection taking place before birth causing the calf to be born weak, prolonging the time between birth and colostrum feeding, or that the agent may be in the environment, infection taking place immediately at birth before colostrum feeding. Colibacillosis, caused by particular strains of E. coli, is an important factor in neonatal disease today, as it was when Theobald Smith elucidated certain aspects of the disease (24). However, not all neonatal disease can be attributed to E. coll. In this paper, we will consider some of the viruses associated with neonatal disease and suggest possible methods of control. Bovine Enteroviruses A virus of the size and characteristics of a bovine enterovirus, with the same resistance to heat and certain chemicals, was found in the lungs and other tissues of 2-and 3-day-old dairy calves affected with acute fatal scours (4,17). The acute fatal disease could be reproduced by exposing colostrum-deprived calves to aerosol inhalation of a filtrate of tissues containing the virus. These calves also carried a natural intestinal flora of E. coli. However, only those colostrum-deprived calves experimentally exposed to a filtrate containing virus developed the acute fatal disease. This virus, and the lesion produced, were also in the lungs of apparently healthy dairy cattle in the same area which were sent to slaughter. Colostrum-deprived calves exposed to a filtrate of such lung tissue also developed the acute fatal disease. The natural protective value of colostrum was shown readily in the experimental disease. However, the prevalence of the disease on the farms was difficult to explain because all calves born under natural conditions and kept for replacement either were allowed colostrum from the dam or were fed colostrum by nursing bottle. A study of time in feeding colostrum in relation to experimental exposure revealed that colostrum had to be fed before experimental exposure if it were to protect the calf. Feeding colostrum within 30 min after exposure failed to protect the calf, and the acute fatal disease developed. Assistance in getting colostrum to the calf immediately after birth resulted in favorable reduction in the incidence of scours on the farms (17). Inasmuch as the reservoir for the vires was in the lungs of apparently normal cattle, there was a probable natural aerosol of the virus in the atmosphere into which the calf was born. The conclusion, therefore, was that exposure began as soon as the calf began to breath, and infection was fought by feeding colostrum as soon as the calf was delivered. Eight serotypes of bovine enteroviruses can cross the placental barrier and infect the fetus. Stillborn calves or aborted fetuses infected with these viruses responded to the infection with the production of antibodies (10). The significance of bovine enteroviruses in the role of stillbirths and abortions has not been investigated adequately. Recently, an enterovirus was isolated from a calf that died within 1 day of birth. Antibodies to this virus were shown in the precolostral serum samples from three other calves in this herd. These antibodies indicated that this virus also can cross the placental barrier and infect the calf before birth. Bovine Viral Diarrhea Virus Bovine viral diarrhea virus (BVD) is a pathogen that can cross the placental barrier of susceptible cattle and cause infection at any stage of gestation (7,14,23). The disease in calves which develops after an intrauterine infection depends on the stage of gestation when the fetus is infected. If the fetus is infected during the first trimester, it may die or brain damage may result in underdevelopment or hypoptasia of the cerebellum. Calves that develop an intrauterine infection during the second half of gestation also may be born weak and scouring (14). The immune system of the fetus is functional at 6 mo gestation (7). Consequently, if the fetus is infected with BVD during the last trimester, it will produce antibodies and be resistant to disease produced by BVD. Some evidence shows that infection with certain strains of BVD during the last trimester results in BVD-immune healthy calves that stay healthy (3,19). However, there are many strains of BVD; more susceptible pregnant cows should be infected during the last trimester with different strains of BVD before this practice can be recommended. Bovine viral diarrhea virus also can infect newborn calves (14,15). When susceptible neonatal calves are exposed to BVD, they develop pyrexia, leucopenia, and varying degrees of scours. Nasal and lacrimal exudate also may develop. Under conditions of isolation in experimental exposures, neonatal calves usually recover. However, BVD is an immunosuppressant, and infected calves exposed to microbiological flora of the herd may develop a more severe disease that results in death (12,22). Rotavirus Mebus found a virus-like agent by electron microscopy in the fluid fecal material from scouring calves (20). This agent was first called reovirusqike but now is called rotavirus because of similarity in appearance to a gear wheel (5,11). The virus does not grow readily in cell culture and, therefore, has not been isolated and propagated for characterization extensively. Serological surveys to determine the incidence of the disease have not been undertaken, but the virus is believed to be widespread in the United States. It has been isolated in Canada (21) and England (27). Filtrates of feces containing the virus will reproduce the disease in hysterectomy-derived, colostrum-deprived calves when introduced into the duodenum by means of a cannula or in natural-born, colostrum-deprived calves when fed orally. The incubation period of the experimental McCLURKIN disease appears to be about 24 h. The epithelial cells of the intestine slough to some degree, and infected cells can be shown by staining the feces with fluorescein-stained specific antibody. Disease caused by this virus appears to be associated with calves less than 1 wk of age. If the virus has been in the herd, colostrum feeding before exposure appears to prevent appearance of disease. There is no proof of an intrauterine infection; the agent causing the infection probably is introduced orally before or concurrent with colostrum feeding. Coronavirus-like Agent By electron microscopy, Stair found a coronavirus-like agent in the feces of scouring calves (26). This agent is called coronavirus-like because, like the viruses of infectious bronchitis of chickens and transmissible gastroenteritis of swine, there is what appears to be a corona around the virion. Calves from 1 to 3 wk of age are more likely to be infected with the coronavirus-like agent, but unlike the reovirus-like agent, colostrum feeding does not protect the calf completely. Workers believe that the virus and colostral antibody become disassociated as digestion takes place and the material moves through the intestine. The cornavirus-like agent can be identified by staining frozen sections of the spiral colon with fluoresceinconjugated specific antibody. The coronavirus-like agent does not grow readily in cell culture; therefore, virus has not been isolated extensively from different herds nor have epizootiological studies been undertaken to determine by serological means the extent and incidence of the virus in the cattle population. Bovine Adenovirus A disease of neonatal calves in Idaho and Montana, "weak-calf syndrome," has been described (6). This is a complex disease in which the stresses of cold wet climate and marginal nutrition have a bearing on the severity of the disease. However, two viruses, BVD and a bovine adenovirus subgroup II, have been isolated from the tissues of calves affected with this disease (8,16,18). The results from experimental inoculation of calves with the bovine adenovirus indicate that the virus is a pathogen that produces subcutaneous hemorrhages over the hock, polyarthritis, and arteritis (9,18). These lesions also are in some calves affected with "weak-calf syndrome," but the virus is not fatal for experimentally infected calves nor does it produce diarrhea. Parainfluenza-3 Virus Parainfluenza-3 virus (PI-3) is widespread in the cattle population; practically no herd has escaped infection from this virus. Generally, it is associated with respiratory disease and particularly is associated with feedlot problems. However, examination of the serum from stillborn calves has shown that some animals have antibodies for PI-3 ; these antibodies indicate an intrauterine infection (10). We have not proved yet that PI-3 infection can cause stillbirths, but any viral infection of the fetus is undesirable. DI SC USSI ON Vaccines are available for BVD, rotavirus, the coronavirus-like agent, and PI-3. All are live-virus vaccines. Recommended use and effectiveness of any of the vaccines for cow-calf operation depend on an accurate diagnosis in each particular herd and the severity and nature of the disease problem. Most mature cattle are immune to PI-3; their antibodies should protect the fetus from an intrauterine infection, and the colostrum should protect the newborn calf. However, intrauterine PI-3 infections apparently do occur, and fetal death may result (10). Because of the danger of intrauterine infections in the first half of gestation, commercially available BVD vaccines usually are not recommended in cow-calf operations; but if BVD is a problem, animals should be immunized before they are bred. Vaccination of the replacement animals at weaning and again before breeding should produce sufficient antibodies to protect the calf from an intrauterine infection and provide antibodies in the colostrum. Killed-BVD vaccines have been prepared and are effective (19), but they have not been produced commercially. They are being used for cow-calf operation and appear to be effective against neonatal disease, but the studies will have to be continued before definite trends can be determined. The immune status of the animal should be determined before it is bred; this is the time to use BVD and PI-3 vaccines to help boost antibody titers. Viruses do not produce necessarily a frank clinical disease or an acute fatal disease in newborn calves used in experimental infection experiments. Why then do so many newborn calves from which these viruses have been isolated die? I believe that virus infections cause added stress, mild debilitation, and suppression of part or all of the cellular system that provides immunity to the calf against the infectious agents that are always in the environment of the cattle population. As virological techniques improve, better cell culturing systems evolve, and more investigators take up the problem, other viruses likely will be isolated from neonatal disease of calves. But this isolation does not imply new and complex management procedures. From our present understanding of the nature of viral infections within a herd, we cannot assume that only the sick calves have been exposed. The question arises as to what factors have tipped the balance in favor of the healthy calf. Probably no single factor alone can shift the balance in favor of the healthy calf. When a virus gains entry into a herd, results of good management practices such as good nutrition for the dam and calf and clean dry calving and calf-rearing areas may be disappointing. However, many viral infections are self-limiting in that mature animals do not die but have either overt disease or inapparent infection and recover and begin to produce antibodies against the agent in question. These antibodies will prevent an intrauterine infection and concentrated in the colostrum will protect the calf at birth if the colostrum is fed before the calf is infected heavily. Good management and sanitation will keep exposure of the calf to a minimum, and proper feeding of the calf will cause a minimum of stress for the gastrointestinal tract. Proper nutrition for the dam will allow her to produce the optimum quantity and quality of colostrum. The most important factor of management which tips the balance in favor of the healthy calf is the herdsman with the cow at time of parturition and his assistance and aid to the calf in feeding colostrum immediately. Colostrum should be fed at least 2 to 3 days to give the calf benefit of antibodies and nutrients.

v3-fos

2016-05-12T22:15:10.714Z

1977-10-15T00:00:00.000Z

271385

s2

Polydipsia and polyuria at high environmental temperature in association with the productive traits in the fowl This work had the purpose to investigate the difference between normal birds and birds with high water consumption and water in feces, in the experimental flock, Jouy-en-Josas, in association with productive traits, body measurements, body temperatures and egg components at two different environmental temperatures. The results can be summarized as follows: i. The difference between the two genotypes (polydipsic and normal) for water intake proved to be highly significant at the normal temperature (20 °C) and at the high environmental temperature (27-34 °C cycle), but it was more important in the first environment. z. The difference between the two genotypes for water output proved to be significant in the normal condition. The same holds true with rectal temperature. 3. Body temperature measurements, rectal, comb and shank, were increased significantly for both genotypes at the high environmental temperature. Introduction A significant number of researches has been done to study the effect of environmental temperature on chickens and laying hens (see review by SMITH and OLIVER, 1971 ). Most of these researches discuss the response, to cold and hot conditions, of physiological parameters, mainly rectal temperature and respiration rate. The effect of environmental temperature on egg production and water consumption in laying White Leghorns has been studied by ToS H lo et al ( 1970 ). Few works conversely relate to possible genetic influences on heat tolerance of laying hens. On the other hand, it is known that one of the ways for the hen to maintain its body temperature during heat stress is increasing its water intake and water evaporation by respiration. Water intake and output by laying hens has been, also, investigated by many workers (L EESON et al., ig 7 6). The evidence for genetical influence on excessive water intake had been, firstly, reported by Buss and MURPHY ( 19 6 5 ). That diabetes insipidus is an inherited character has been reported by WILLIAM and Buss ( 19 68). They added that the kidneys of affected birds are capable of an antidiuretic response when lysine vasopressin or arginin vasotocin is injected. Meanwhile, the association between the productive traits and diabetes insipidus has been investigated to determine the effect of excessive and normal drinking on the economical traits (WILLIAM and Buss, 19 68, S U E I, INT!RNMOORE, 1972 , $WA!,D et al., 19 67). Water output had been also studied in the same trend by Buss and MURPHY (ig6 5 ), and R OB E RT et al.. ( 1974 ). The principle of this study was, therefore, to investigate the effect of polydipsia and polyuria on the productive traits and egg components weights under normal and hot climatic conditions. The experiment was performed in order to determine which of the genotypes under study is more tolerant to heat. Material and Methods Since several years, two strains of the experimental flock of the Laboratory at Jouy-en-Josas were found to contain a certain proportion of individuals with abnormally high water consumption. A summing up of the corresponding data showed that these individuals were very likely to be homozygous for a major recessive gene (BoRnns et al., in press). This gene seems analogous and possibly identical to that described by WILLIAMS and Buss (ig68); in particular, it is associated with a large increase in the water /feed ratio. In view of the present experiment, breeding birds of both sexes issued from the two strains mentioned aboBe e were chosen in families containing individuals with high water intake. These breeding birds ( 5 males, each with 5 females) were mated without any discrimination according to their strain of origin. In april 197 6, they gave rise to two hatches separated by a two week-interval. The female progeny were kept, raised on floor and put at 1 6 weeks of age in individual cages in a climatized room at 20 °C, with 10 hours darkness and 14 hours light per 24 hours. The feed was in the form of pellets. Age at first egg, egg number till mid-December (average duration from lst egg about 3 months), body weight and two body measurements (wattle length and shank length) in this same period were noted. The age of birds at this date was about 9 months. Their total number was then 94 . They were tested during 11 successive days in the second half of December for their water intake, so as to identify their genotype for the « polydipsia » factor. In this same testing period, feces were collected over 4 8 hours (Starting from 10 a.m.), weighed, then dried in an oven at 6 0 °C during 4 8 hours to evaluate their dry matter. This test allowed to identify two groups of hens, one with « normal » water intake opposed to another with obviously « excessive » intake. In fihis second group for the experiment we kept only birds with a value for water intake appreciably higher than the average of « normals » plus twice their standard deviation, as the previous result (Boxn:!s et al., in press) showed that such individuals could be considered, with a very high likelihood, as homozygotes for the postulated recessive gene. Individuals with a value close to the limit between the two groups were discarded. _ Using this criterion, 24 pairs of full-sisters, one « normal », the other « polydipsic », both laying at the time of the test, were kept. During this first testperiod, beside the measurement of water intake, weight of feces and water in the feces, four consecutive eggs were weighed per hen, the albumen height and shell thickness (including membrane) were determined for each; then, on one egg per hen, yolk weight, shell weight and albumen weight (the last by difference from total egg weight) were measured. Finally the hematocrit value was determined for each hen and one measurement was made (always around 2 p.m.) of its rectal temperature, along with superficial temperatures for comb (central zone) and shank (at mid-length). These temperatures were read to the nearest. i °C with a thermocouple connected with two different probes, one for rectal and another for superficial temperatures. The 4 8 hens forming these 24 pairs, remaining in individual cages in the same cell, were then submitted, from 6th to 14 th January 1977 inclusive (after a progressive rise of temperature from January 3 rd to 6th), to a temperature cycle per day consisting of 10 hours at 27 °C during the dark period followed by 14 hours at 34 °C during the lighted period. Percentage humidity was kept around 50 p. cent as far as possible. During this « heated » period, with this temperature cycle, again individual water consumption was recorded, and feces were collected over 4 8 hours, weighed and put to the oven as previously for determinat!p'n of dry weight. The same egg traits and components as in the first test period were measured (albumen height and shell thickness on 2 eggs per hen, yolk, shell and albumen weight on one) as well as rectal, comb and shank temperature, each being taken once for each individual at the end of the period (around 2 p.m. in the day). Moreover, the respiration rate was noted per hen on each of the last three days of the « heated » period, also around 2 p.m. for half a minute and doubled to get values per minute. Each of the 4 8 studied females can be considered as having been submitted to two successive environments :The first one includes the whole period where ambient temperature was 20 °C (also called « normal » environment) and more specifically the test period in December. Some measurements were taken only in this phase (age at first egg, egg number till December, body weight and measurements, hematocrit). The second environment comprises the period from January 6 to 1 4 , 1977 with the daily cycle of temperature ( 27 -34 O C). We will refer to it also as « heated environment » or by the abbreviation « at 34 °C » (although this temperature concerns only the lighted period). A number of traits are measured in both environments as already mentioned (water intake, weight and percent water in feces, body temperatures, egg traits and components). Conversely, the egg number registered in the « heated » period is not comparable to that noted in the first environment (later stage in the laying year, short duration) and respiration rate is not measured at 20 °C. In the following tables, as concerns traits measured several times for each individual (egg traits, respiration rate), only the mean value per bird is used. The traits measured in one or both of the environments are symbolized in an abbreviated way in the text and tables, as shown in the following list: At 3 ¢ °C the differences, for water intake, between the two genotypes decreased about one third. The same trend, almost, can be observed for water intake as a percentage to body weight. The differences between the two genotypes for water output and percentage of water in feces proved to be significant in normal conditions (z! °C). In this. respect, R OB E R T et al. ( 1974 ) reported significant differences between 12 stocks. of white Leghorns. The value of percent moisture for the 12 stocks were within the normal range, which averages about 70 p. cent, but can reach at maximum 8z p. cent in the normal birds and as high as g8 p. cent for polydipsic (Su!-LiN-. TERNMOORE, 1972). In the hot environment, water output in the feces becomes little different for the two compared genotypes. Possibly the difference between the two genotypes in total water output may have been somewhat underestimated because of occasional projection of droppings of (di) hens into the trays collecting feces of neighbouring normal hens. At 34 °C, the increase in water intake for the (di) birds is 107 . 0 (g) and the increase in water output is 6 9 . 7 (g). The increase for the (Di) birds, in water intake, is 193 .8 (g) and 10 6. 0 (g) in water output. These differences due to temperature proved to be highly significant (Table I b). According to heat stress the birds consume more water for both genotypes to maintdin body temperature as is well known (e.g. see Tosxio et al., 1970 ; S N >;TS IN GE R , ig75); but although the interaction term is not significant in the variance analysis the increase in water intake for the normal birds is about double the increase for the polydipsic. This may be due to the fact, that as the normal birds consume, in normal condition, less water than the polydipsic the latter need less increase in water consumption. Our data seem in agreement with the results obtained by SU E I, INTERN-MOOR! ( 1971 ), that wet droppings are result of primary polydipsia rather than obligatory loss of body water followed by an increase in water intake. & d q u o ; It must be noted, finally, that there is no significant difference between families, except for percentage of water in feces, and no significant interactions as already mentioned. 2 . -Body temperature and res!iration rate The difference between the mean values for rectal temperature, for the two genotypes (Table 2 ) proved to be significant only at normal temperature. On the other hand, the differences between the measurements at 20 °C and 34 °C, which proved to be highly significant (Table zb), for comb, shank and rectal temperatures are for (di) birds 4 . I2 °C, 6. 75 °C and 0 . 17 °C respectively, and for (Di) they are 4.19 °C, 7. 41 °C and 0 .2 0 °C respectively. Although, again, there is no significant interaction, one can remark that the differences on the (Di) birds are slightly higher than those of the (di) birds. In fact, body temperature rises above normal depend on the relative humidity of the atmosphere and the degree of acclimatization of the birds. W ILSON ( 194 8) found that rectal temperature increased at environmental temperature above 2 6. 5 °C although the increases were small until the environmental temperature was 32 . 3 °C. I,E! et al. (1945) noted that up to an environmental temperature of 29 . 5 °C rectal temperature was unaffected but an environmental temperature of 32 . 3 °C increased body temperature by 0 .2 0 °C to 0 .83 °C. The difference between the mean values of the two genotypes for respiration rate at 34 °C. proved to be the only significant one. The (di) birds are less panting than the normal birds ( I ); the increase in respiration rate, here, for the (Di) birds has the effect of lowering body temperature by evaporation. I,orrGxous! et al. ( 19 6 0 ) found that pullets kept at 32 . 3 °C lost 6 0 p. cent of their body heat by evaporation. Total heat loss from laying hens due to respiration is relatively constant below 2 6. 7 °C ambient. Between 2 6. 7 °C and 32 °C it increases rapidly. (i) In a further experiment on other hens kept in cages with the same temperature cycle ( 27 °C during io hrs darkness, 34 °C during 14 hrs light) from april to jtine 77 , 9 additional pairs of sisters were obtained, one « normal the other « polydipsic ». Average respiration rate /mn was 12 6. 2 for the normals and 10 6. 9 for the polydipsic (t = 4 . 37 , P < o.oi) which is in concordance with the present results. Above 32 °C the rate of increase declines, indicating a breakdown in the body temperature control mechanism (B ARO TT and P RINGL E, 1941 ). The amount of increase of water loss by hens can be from 5 (g) per hour at normal temperatures up to 30 (g) per hour when the bird is panting (L EE et al., ig 45 ). From the above mentioned results, it can be concluded that the polydipsic birds are more tolerant to high environmental temperature since they show lesser increase in respiration rate, water intake and water output at high environmental conditions. . -The productive traits Only mean values for egg weight proved to be statistically different in normal environmental temperature in favour of the (di) genotype. At 34°C the differences for egg number between the two genotypes proved, also, to be statistically significant in the same trend in spite of the short period of recording (only eight days). The same holds true with egg weight; moreover, there are no much changes in the mean values, with respect to the two genotypes, between the two different environmental temperatures. . -Egg weight and weights of egg components Egg weight and weights of egg components corresponding to the two genotypes at the two environmental temperatures are shown in Table 4 . The mean values of egg weight, yolk weight, shell weight proved to be statistically different in favour of the (di) hens in normal conditions. At 34 °C, no character showed significant difference between genotypes. This investigation for egg weight and components, weights for the two different environmental temperatures does not seem to have been done before. The differences between the mean values at the two environmental temperatures, which proved to be statistically significant as concerns egg components, are 3 . 02 (g), -0 ,66 (g), + 0 , 7 8 (g), -f-2 . 1 2 (g), and 3 .88 (mm) for egg weight, yolk weight, shell weight, albumen weight and shell thickness with respect of the (di) genotype. With respect to the (Di) genotype, the differences are -0.02 ( g ), -1,49 ( g ), + 0.19 (g), -!-0.55 (g) and -!-3.7 4 mm for egg weight, yolk weight, shell weight, albumen weight and shell thickness respectively. The decline in mean values for shell thickness has about the same magnitude for both genotypes. Shell weight and albumen weight are more depressed at heat stress for (di) genotype. Most of the decline in mean egg weight is due to the decline in albumen weight for this genotype. On the other hand, both means of the two genotypes, for yolk weight, are increased, the magnitude of increase for the (Di) mean being higher than for the (di) mean. One must mention that there are no genotype X treatment interactions except for shell weight. The modifications occurring for egg formation at heat stress may be due among other things to water metabolism. Additional experiments would be required to confirm this hypothesis and know more precisely the way of action of the « polydipsia » genotype on egg components in hot climate. . -Correlation between water intake and output, and physiological parameters (shank, comb, rectal temperatures, packed cell volume and respiration rate) for di and Di genotypes at 20°C and 3¢ °C Owing to the great number of traits recorded, the correlations are presented in four tables. Due to the limited numbers available, some of these correlations may need to be further checked on additional data. In normal condition ( 20°C), for both genotypes, water intake is correlated significantly with water in feces and percentage of water in feces (Table 5 ). Buss and MURPHY ( 19 6 5 ) found statistically significant correlation fcr visual score of excreta and water (feed ratio of immature and mature birds. At 34 °C water intake, for the (Di) genotype, is significantly correlated with water output but not with percentage of water in feces. Water output is significantly correlated, for (di) genotype, with percentage of water intake to body weight at 34 °C. In normal condition, for (Di) genotype, only water output showed significant correlations with comb, shank and rectal temperatures. The same holds true with the correlation between percentage of water in feces and comb temperature. With respect to the (di) birds, the correlation between the percentage of water intake to body weight and shank temperature is negative, and the correlation between percentage of water in feces and packed cell volume is the only significant one. At heat stress, shank temperature is correlated with water intake and output, comb temperature and respiration rate; also, comb temperature and respiration rate show a correlation of about the same magnitude for the normal birds. The (di) birds show another distinct result, respiration rate is negatively correlated with percentage of water in feces and positively with rectal temperature. It may be added to the suggested tolerance of the (di) birds to heat stress, that the increase in ambient temperature causing an increase in body and accordingly in rectal temperature, both respiration rate and percentage of water in feces regulate body temperature: see reviews of SMITH and OLIVER ( 1971 ) and L!ESOr! et al. ( 197 6) on this subject. But the absence of correlation between RR and RT in (Di) birds suggests a lesser regulation potential of the former by the latter. 6. -Correlations between water intake and output, comb, shank and rectal temperatures and the productive characters and two body measurements The correlations between water intake and output, comb, shank and rectal temperatures and the productive characters and two body measurements for the two genotypes at the two environmental temperatures are shown in table 6. In temperate condition ( 20 °C), the correlation between egg number and water intake, for the (Di) birds, proved to be significant. Wattle length showed, also, significant correlation but with a negative sign. The (di) birds, in the same conditions, showed another trend. Water in feces and percentage of water in feces are correlated positively with sexual maturity; the two figures are of about the same magnitude. Wattle length is correlated negatively with water in feces. Egg number is correlated negatively with percentage of water in feces. So a higher percentage of water in feces decreases egg number for (di) birds, meanwhile, water intake increases egg number for the (Di) birds. On the other hand, for the (di) birds, shank temperature is correlated positively with body weight and negatively with wattle length, and rectal temperature is correlated negatively with average egg weight. R OBERT et al. (ig 7 q.) found, for three lines of White Leghorns, that body weight was consistently positively correlated with dropping weight. Egg production was negatively correlated with percentage of fecal moisture and fecal wet weight which seems to agree with our results, especially those concerning the di birds. E WALD et al. ( 19 6 7 ) noted that it is twice more effective to predict egg production through water ingestion rather than body weight. On the other hand, Buss and MURPHY (rg6 5 ) noted that no statistically significant correlations were found for the water /feed ratio and egg number, egg weight, albumen quality, shell thickness and body weight. At 34 °C, the (Di) birds showed a significant positive correlation between water in feces and wattle length, and a negative one (&mdash; 0 . 42 ) between wattle length and respiration rate. For the (di) birds, the correlation between body weight and water consumption in percentage to body weight and water in feces with body weight are negative (which anyway is expected for the first one). The same holds true with the correlation between shank length and percentage of water in feces and shank temperature with sexual maturity. 7 . -Correlations between water intake and output and egg weight and components weights and egg characteristics The correlations between water intake and output and egg weight and components weight for both genotypes at the two environmental temperatures are represented in Table 7 . Shell thickness is correlated positively with water intake, percentage of water intake to body weight and percentage of water in feces for the (Di) birds at 20 °C. The same holds true with correlation between shank temperature and Haugh units. Meanwhile, the (di) birds, in the same temperature condition, show a positive significant correlation between Haugh units and water output. At heat stress only the Di birds showed positive significant correlations between shank temperature and egg weight, albumen weight, the albumen /yolk ratio and Haugh units. 8. -Correlations between egg weight and egg components' weight with respect to the Di and di genotypes in the two environmental conditions The correlations between egg weight and egg components weights and egg characteristics for the two genotypes under study at 20 °C and 34 °C are shown in Table 8. As egg weight is determined by it's component weights, it is normal that the correlation between egg weight and components weights for the two genotypes in the two environmental conditions are positive and significant. The correlations between the components of the egg for the two genotypes in normal conditions are as follows: for (Di) birds yolk weight is not significantly correlated with both shell and albumen but shell weight is correlated with albumen weight, while, for (di) birds only yolk weight is correlated significantly with shell weight. Meanwhile, both genotypes show positive significant correlations, of about the same magnitude, between shell weight and shell thickness. O BW n 9H et al, ( 1977 ) showed in a study for G 2 locus of egg white protein that the correlation between yolk weight and albumen weight existed for G 2 AB while there was no correlation for the G 2 BB genotype between the two characters, which may have some analogy with the present situation. At 34 °C the correlations changed as follows: for (Di) birds yolk weight is significantly correlated with albumen weight and for (di) birds shell weight is correlated with albumen weight. Meanwhile, the albumen /yolk ratio is correlated with both egg weight and albumen weight for the (Di) birds, while it is negatively correlated with yolk weight for the (di) birds. Another result in favour of (di) genotype is that in hot environment, still there is a significant positive correlation between shell weight and shell thickness which does not exist for the (Di) genotype. This may add to the efficiency of indirect selection for shell thickness for eggs produced by the (di) birds. Anyway the correlations seem to confirm that the gene for polydipsia has an appreciable effect on the components of the egg.

v3-fos

2018-04-03T03:52:46.186Z

1977-01-01T00:00:00.000Z

25277734

s2

Effect of supplementation of limiting amino acids on tyrosine toxicity in rats fed wheat gluten diets. The effect of supplementation of limiting amino acids on rats fed a 10 or 20% wheat gluten diet containing 5% tyrosine has been investigated. Growth-limiting amino acids in a 20% wheat gluten diet were lysine and threonine. When rats were fed a 20% wheat gluten diet containing excess tyrosine, the addition of 1.0% lysine-HCl or 1.0% lysine-HCl and 0.4% threonine completely prevented the development of tyrosine toxicity, and was accompanied by a lowering of free tyrosine concentrations in plasma, liver and muscle. In rats receiving a 10% wheat gluten diet, lysine, threonine and methionine were limiting for growth. The addition of 1.0% lysine-HCl to the 10% wheat gluten plus 5% tyrosine diet failed to alleviate the tyrosine toxicity, but the supplement of 1.0% lysine-HCl and 0.4% threonine could partially alleviate the toxicity. The combined addition of 1.0% lysine-HCl, 0.4% threonine and 0.5% methionine was most effective. The free tyrosine concentrations in plasma, liver and muscle were lowered greatly by the supplementation of three limiting amino acids, but amounts of free tyrosine and phenolic metabolites excreted in the urine did not decrease with addition of these limiting amino acids. first limiting amino acid, lysine. The amounts of some amino acids such as threonine and methionine are also insufficient in the low wheat gluten diet (10,11), but the effect of these limiting amino acids in wheat gluten on tyrosine toxicity had not been investigated. Therefore, the present study was performed in an attempt to elucidate the effect of those limiting amino acids in wheat gluten on tyrosine toxicity. Growth depression, free tyrosine concentration in plasma, liver and muscle, together with urinary excretion of tyrosine and its phenolic metabolites were determined. EXPERIMENTAL Animals and diets. Male weanling rats of the wistar strain were fed a stock diet (25% casein) for 3 to 4 days prior to starting the experiments. Rats weighing 60 to 65g were divided into two series and separated into several experimental groups of five animals each (Table 1). In experiment I, 20% wheat gluten2 was used in each group, and 5% L-tyrosine 1.0% lysine• HCl alone to the 20% wheat gluten diet (20G Lys) produced a weight gain of 95% of that found in the 20G Lys Thr group (Table 2). When rats were fed the 20% wheat gluten diet containing 5% tyrosine (20G5T) a growth depression was produced and two out of five animals suffered from pathological lesions of the eye and paw after three weeks. In rats receiving the 20G Lys diet including 5% tyrosine (20G5T Lys), pathological lesions were not observed and the growth depression was partially alleviated. When 0.4% threonine and 0.5% methionine were supplemented in the 20% wheat gluten diet without additional lysine (20G Thr Met), growth retardation was produced, and the inclusion of 5% tyrosine in the diet (20G5T Thr Met) caused eye and paw lesions in two out of five animals. In rats given wheat gluten at the 10% level, the combined addition of 1.0% lysine • HCl, 0.4% threonine and 0.5% methionine (10G Lys Thr Met) was most effective in growth stimulation (Table 3). When excess tyrosine was included in the 10% gluten diet (10G5T), severe growth depression was caused and all animals suffered from eye and paw lesions. Addition of lysine• HCl alone to the diet (10G5T Lys) could alleviate neither growth retardation nor eye and paw lesions. When 1.0% lysine. HCl and 0.4% threonine were supplemented to 1OGST diet (10G5T Lys Thr), the growth depression due to excess tyrosine was partially alleviated and the pathological lesions diminished. When 0.5% methio nine was added to the diet (10G5T Lys Thr Met), the growth was stimulated and the weight gain was about 92% of that found in the 10G Lys Thr Met group. Ratio of weight gain to food intake correlated well with changes in body weight (Experiments I and II). Tissue tyrosine concentrations Table 4 shows the concentration of total phenols in plasma, and free tyrosine Y. YAMAMOTO, Y. ORITA, and K. MURAMATSU concentrations in plasma, liver and muscle. When rats were fed the 20G5T diet, tyrosine concentrations were elevated very markedly in plasma, liver and muscle. Addition of lysine, the first limiting amino acid, to the diet effectively lowered free tyrosine levels of plasma, liver and muscle, although their values exceeded slightly that for the 20G group. The plasma tyrosine levels in animals fed the 20G5T Thr Met diet were not lowered; in fact the levels in liver and muscle exceeded even those in the 20G5T group. DISCUSSION In an early study (9) we observed that the toxic effect of tyrosine, which deve GLUTEN AND TYROSINE TOXICITY 121 Table 5. Effect of supplementation of limiting amino acids on urinary excretion of total phenols, tyrosine metabolites and free tyrosine in rats fed the 10% wheat gluten diets containing excess tyrosine. loped in rats fed a low wheat gluten diet containing excess tyrosine was not alle viated by addition of lysine, the first limiting amino acid. The present study confirmed that the growth retardation and the pathological lesions which developed in animals fed the 10% wheat gluten-high tyrosine diet were alleviated by the concomitant supplement of three limiting amino acids, lysine, threonine and methionine, but not by the addition of lysine alone. Furthermore, when 20 wheat gluten was used as the source of dietary protein, the tyrosine toxicity could be overcome by adding lysine alone or both lysine and threonine. The first and second limiting amino acids in this diet were lysine and threonine, respectively. Another report (7) indicated that, when 10% casein was used as the protein source, the tyrosine toxicity was partially alleviated by the addition of methionine alone. In addition the growth retardation could be markedly reversed and the pathological lesions completely prevented by the supplement of both methionine and threonine which are limiting in this diet. Thus, the findings in the present and previous studies (7,8) demonstrate clearly that the addition of the limiting amino acids to a low protein-high tyrosine diet has a beneficial effect in alleviating the toxicity. The adverse effects resulting from amino acid additions can be classified by ELVEHJEM (16) and HARPER (17) into three categories: "toxicity," "antagonism" and "imbalance." Detrimental effects due to ingestion of diets containing excess amino acids have in general been included in amino acid "toxicity." However, it is suggested that in the case of tyrosine toxicity, the "toxicity" somewhat re sembles a phenomenon of "imbalance," which is prevented by a small supplement of the limiting amino acid(s). The present investigation has also demonstrated that the beneficial effect of the limiting amino acid supplements in reducing the toxicity in animals fed wheat gluten-high tyrosine diets was accompanied by a lowering of free tyrosine concen trations in plasma and tissues. This is in agreement with the results reported previously (8), in which the high levels of free tyrosine in plasma and tissues of animals fed the 10% casein diet containing excess tyrosine were lowered signifi cantly by the addition of the limiting amino acids, methionine and threonine. ALAM et al. (2) have also shown that the supplement of threonine to a 6% casein diet containing excess tyrosine lowered the tyrosine concentration in plasma, liver, muscle and eye (3). O n the other hand, amounts of phenolic metabolites of tyrosine excreted in the urine of rats fed the low wheat gluten-high tyrosine diet supplemented with limiting amino acids, lysine, threonine and methionine, did not decrease signifi cantly, as was the case in the results of a previous report (8) in which low casein high tyrosine diet was supplemented by methionine and threonine. It would seem likely that the phenolic metabolite level in urine dose not reflect the tyrosine toxicity.

v3-fos

2017-09-10T16:14:40.175Z

1977-01-01T00:00:00.000Z

15887775

s2

Observations on squamous cell carcinomas of sheep in Queensland, Australia. Observations were made over a 4-year period on squamous cell carcinomas on the ear and other areas poorly covered by hair or wool, sheep pastured in the hot, dry environment of north-western Queensland. Overall incidence in the flock was higher than in flocks kept at greater latitudes. Increased incidence with advancing age was demonstrated, and ewes appeared to be more susceptible than wethers. Metastases were observed in 4 of 33 affected ewes (12%) submitted to detailed necropsy examination. Measurements of tumour growth in 4 ewes revealed an increase in size of about 3-5 mm per month. Ovine aural squamous cell carcinoma was considered to be a good model for studies on skin cancer in man. ImagesFig. IN MAN the relationship between high levels of solar radiation and an increased occurrence of skin cancer is well recognized (Silverstone and Searle, 1970). Lloyd (1961) has reviewed the literature in other animal neoplasms considered to be associated with solar radiation. Recent publications describing such neoplasms include those of Vandegraaff (1976) on vulval carcinoma in sheep, Burdin (1964) and Wettimuny (1974) on vulval carcinoma in Aryshire cattle, Naik, Balakrishnan and Randelia (1969) on horn cancer in Indian Zebu cattle, and Ramadan (1975), on squamous cell carcinomas (SCC) in the perineum of 4 goats. In New South Wales, SCC in sheep, on the ear or other areas poorly covered by hair or wool, has been described in detail by Dodd (1923) and Lloyd (1961). The present observations were made to compare the occurrence and biological behaviour of this neoplasm in a tropical environment with earlier observations on sheep pastured some 90 further from the equator, and therefore exposed to considerably lower levels of u.v. radiation. MATERIALS AND METHODS Animals and their environment.-Observations were made over a 4-year period on an experimental flock of approximately 8000 medium-wool Peppin Merinos, maintained at the Toorak Sheep Field Research Station, Julia Creek, Queensland (Lat. 21°S; Long. 141°E). All the sheep had been born on the research station, and were themselves the progeny of animals which had been in the area for at least 3 generations. Mean structure of the flock during the period of the study was 59% ewes, 20% wethers and 4 % rams, the remaining 18% being unsexed lambs and weaners. A specific age analysis of the flock was not possible except for the ewe portion during the first 2 years of the study. As the flock was an experimental one, however, ewes were retained longer than in commercial flocks in the area, in which the ewes are normally culled at 5-7 years of age. Wethers were culled from the experimental flock at 5-6 years of age. The topography of the research station is characteiristic of the open treeless Mitchell grass plains of north-western Queensland, where summer maximum temperatures may reach 46°C, and where mean monthly temperatures exceed 350C for 5-6 months of the year (Farmer, Everist and Moule, 1947). High levels of solar radiation occur throughout the year and may reach 2-93 MJ/m2/h during the summer months (McFarlane, Morris and Howard, 1958). Rainfall is mainly restricted to the period between December and March, and averages 42 cm, with a range of 19-60 cm per annum. All animals were shorn in June-July of each year, and management procedures required that any animal showing obvious " cancer " lesions be removed from the flock prior to shearing. Because of this, lesions studied were assumed to have developed de novo, or from precursor lesions, within a period of not more than 1 year. Examination of cases.-On one occasion during each year of the study, sheep were individually examined, and those with detectable clinical lesions (tumours more than 5 mm in diameter) on the aural or facial regions were restrained. Some or all of the following observations were then made in regard to each affected animal: sex, age, location of lesion or lesions (right or left ear or elsewhere), whether the tumour was associated with an identifying earmark or ear tag " punch " hole (involving no more than 1 cm2 of pinna), type of lesion (cutaneous horn, ulcerating lesions or " other "), size and if unusual, comments concerning shape, consistency, or mode of growth. Sheep with hyperkeratosis of the outer surface of the pinna of the ear (scaly, rough appearance) were frequently observed, but these lesions were not recorded. When clinical lesions were on the ear, the whole tumour, together with some normal tissue, was removed by cutting through the entire pinna. Sheep with lesions on the muzzle or face were killed, and necropsy examination was made on some of them. Five sheep found on initial examination to have comparatively small ear lesions (up to 1 cm2), and one with a small ulcerating lesion on the muzzle, were kept in an unshaded paddock, and were examined approximately monthly for 8 months (July to March). At each examination the type and size of lesion was recorded. After the final observation, sheep with lesions that continued to enlarge were necropsied. A total of 33 sheep with SCC, including 4 of the 6 observed for rate of tumour growth, were killed and subjected to detailed necropsy examination for detection of metastases. Portions of the primary lesion, and any lesions 8 considered as possible inetastases, were fixed in 10% buffered neutral formalin (BNF). For histopathological study, paraffinembedded sections cut at 6 ,um were stained with haematoxylin and eosin (H.E.). A total of 48 aural and facial lesions, sampled by biopsy or at necropsy, were studied. Freslh tissue from tumours in 6 sheep was autocultured and co-cultured with normal sheep kidney monolayers, and cells wvere observed over a period of 28 days. Occurrence of lesions During the period of study, records were made of clinical lesions in 132 sheep. Occurrence in relation to sex was: 120 (950o) in ewes, 5 in wethers, and one in a ram. The other 6 lesions were in sheep whose sex was not recorded. Histopathological examination of lesions in wethers revealed that only 2 were SCC; the remaining 3 were inflammatory lesions, in 2 cases associated with pseudoepitheliomatous hyperplasia. Table I summarizes data on the occurrence of tumours in relation to age for the first 2 years of the study. In both years, all cases confirmed histologically as SCC were observed in ewes. Differences in occurrence between age groups in both years were significant (P < 0.01). The mean occurrence in ewes in the 2 years was 0.95%. When all ages and sexes were included, the occurrences in the flock in Years 1 and 2 were 0.86% (54/6307) and 0.49%o (36/7237) respectively. In the second year, clinical lesions were found in 9 of 75 12-year-old ewes examined (12%). Of the 132 affected sheep in the study, 6 (40o) were 0-3 years, 28 (21 %) were 4-7 years, and 83 (63%) were 8-12 years old; the ages of 15 sheep were not recorded. The youngest sheep with a SCC confirmed by histopathology was 3 years. Location and type of lesions A total of 146 clinical lesions were recorded in the 132 affected sheep. Of these, 110 (76%) were on the ears, 32 illl (22%) were on the muzzle, and 4 were on the lower lip or adjacent skin. In 9 sheep, lesions were present on one or both ears and the muzzle. In one 12-year-old ewe, an SCC was situated on the inside of the pinna of the ear, near its caudal margin; another ewe had multiple fine papillomatous proliferations in this location. Of the ear lesions, 42 of the 110 observed (39%0) were associated with ear marks or ear-tag punch holes, located respectively on the anterior margin and centrally on the pinna. Most tumours (60%) were present as cutaneous horns. Ulceration was present in the remaining cases, probably in many instances as a result of removal of the cutaneous horn by trauma. Tumours of the ear tended to ulcerate less frequently (25% of cases) than those in other sites (86%); large ear lesions, however, were usually ulcerated, suppurative and often infested with blow-fly larvae. No relationship was evident between size of lesions and age of the sheep. Mean size of ear and other lesions, respec-tively, were 3-5 x 2-5 cm and 4 0 X 3-3 cm. The largest one found was approximately 12 x 8 x 6 cm in size. Necropsy findings Metastases were found in 4/33 (12%) of the ewes necropsied. Details of these 4 ewes are shown in Table II. The lymph node metastases were detected grossly as firm, discrete, pale areas. Metastasis to the parotid salivary gland in Ewe 3 probably resulted from lymph node lesions in that area; such lesions were not demonstrated, however, as the ulcerated lesion was suppurative, oedematous and infected with blow-fly larvae. Microscopic findings Microscopic examination of 48 lesions, considered on clinical study to be SCC, confirmed that 41 (86%) were typical squamous cell carcinomas or epitheliomas. Acanthosis and pseudoepitheliomatous hyperplasia were apparent in 3 and 4 other sheep respectively, and associated swelling Year 2 in these lesions was largely attributable to trauma and inflammation. Lymph node metastases all exhibited pronounced infiltration, with necrosis and capsular penetration. Keratinization was evident in each case. Similar infiltration of the parotid salivary gland was apparent ( Fig.) but ulceration had resulted in a more severe inflammatory response. Tumour growth obseruatiomu Lesions in 2 sheep, both ulcerating, located on the muzzle and on an ear mark on the right ear respectively, regressed over a 3-month period. However, no histology was done on the ear lesion, and an initial biopsy of the muzzle lesion revealed only pseudoepitheliomatous hyperplasia. Ear lesions in 4 other sheep increased in size over the 8-month period of observation. Mean initial and final sizes (height x diameter) of the lesions in these sheep were 1-0 x 0-9 cm and 3-1 x 2-2 cm respectively, so that mean increases in height and diameter were approximately 0-4 and 0-3 cm/month respectivelv. No metastases were found in these sheep at necropsy. Virology No evidence of a cvtopathic agent or of transformation of marker cells was detected in material from any of the 6 sheep examined in titro over a 4-week period. DISCUSSION As the present study was made in a tropical environment, we expected a greater occurrence of SCC than that found in sheep in temperate areas. Certainly the occurence we found was higher than the 0.2% occurrence reported by Lloyd (1961) as normal in one flock in New South Wales. Comparison is difficult, however, because the maximum of age of sheep studied bv Llovd was onlv 6 vears. With regard to the relationship between sex and the occurrence of SCC, our finding of only 2 cases in wethers compared with more than 100 in ewes, strongly suggests susceptibilitv of the latter, and indicates an aspect requiring further studv. In cattle also, the occurrence of ocular carcinoma is greater in the female, but the significance of this observation is complicated by the later disposal of cows for slaughter (Moulton, 1961). Our finding of an increased occurrence of SCC with increasing age, to a maximum of 120% in 12-year-old ewes, extended the observations of Lloyd in a flock of younger sheep. In the present study, details were not recorded of the frequently observed hyperkeratotic, and presumably precursor lesions on the ears. Such lesions appear to be comparable to the solar keratoses observed in man, and their conversion to SCC would seem to occur most readily in sheep over 7 years of age. The single SCC we observed on the inside of the pinna of the ear of one ewe is of interest. Lloyd (1961) found no tumours in this location, and interpreted this finding as being consistent with solar radiation being an aetiological factor in pathogenesis of these tumours. Although Lloyd, in his cases, found no association between site of the tumour and identifying ear marks, our observations that 3900 of SCC were associated either with ear marks or " punch " holes, tends to support the original opinion of Dodd (1923) that inflammation associated with ear marks may sometimes precede neoplasia. Our findings of metastasis in 4/33 sheep (120%) necropsied was comparable to that of Lloyd (1961) who observed them in 3/28 sheep (11%). Extension of metastatic tumour through the lymph node capsule into adjacent salivary gland, with ultimate ulceration through the skin, demonstrates the infiltrative nature of the neoplasm. The only report we found of metastasis other than to lymph nodes was a pulmonary metastasis observed by Lloyd in one sheep. Observations in the present study suggest that factors other than solar radiation per se are involved in the genesis of SCC in grazing sheep. Because of the importance of skin cancer in man in tropical areas, research using sheep as a model seems to be indicated. Affected sheep are readily available, and the peripheral location of ear lesions would be advantageous for studies involving such aspects as pathogenesis, iso-enzyme patterns, chemotherapy and immunotherapy.

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2018-12-05T17:53:16.551Z

1977-01-01T00:00:00.000Z

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Residual Herbicides for Use in the Wheat-Fallow System 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 1977 Kansas State University Agricultural Experiment Station and Cooperative Extension Service. Residual Herbicides for Use in the Wheat-Fallow System Charles A. Norwood, Research Agronomist Several residual herbicides are suitable for controlling weeds during fallow in the wheat-fallow system. The most widely used herbicide for this purpose is atrazine. Atrazine persists in the soil for over a year, and should be applied as soon as possible after wheat harvest to avoid injury to the following wheat crop. Cyanazine (Bladex), metribuzin (Sencor or Lexone), and terbutryn (lgran) do not persist as long in the soil as does atrazine, and are usually applied in the spring. Cyanazine and terbutryn, however, can be tank mixed with atrazine and applied following wheat harvest. Chlorsulfuron (Glean) is a new herbicide, which obtained full labeling for wheat and wheat-fallow in 1983. Chlorsulfuron is different from the other herbicides currently used in wheat-fallow in that it was developed specifically for use in wheat and barley. Thus, it does not injure wheat and can be applied either to growing wheat (between the 2-3 leaf and boot stages) or anytime during fallow. Its persistence in the soil for weed control purposes is similar to that of atrazine. However, small quantities can remain in the soil for long periods of time and cause injury to following crops. For this reason, chlorsulfuron is currently labeled only for weed control in barley, wheat, and wheat-fallow on soils having a pH of 7 .5 or lower. All of these herbicides have been tested extensively at the Garden City Experiment Station to determine their suitability for the wheat-fallow system. The results of the experiments reported here are intended This publication from Kansas State University Agricultural Experiment Station and Cooperative Extension Service has been archived. Current information: http://www.ksre.ksu.edu. Results Postharvest, Spring, and Sequential Applications. Results of postharvest, spring, and sequential (postharvest + spring} applications are presented in Tables 1 and 2. Results from 1982, contained in Table 1, indicate that lib of atrazine gave good broadleaf weed control for 10 months after application, with less control from lower rates. Spring applications of cyanazine and metribuzin controlled witchgrass, in addition to the broadleaf weeds. All rates of atrazine controlled weeds from application through winter freeze-up . Therefore, the best season-long control was obtained from a postharvest application of atrazine, followed by cyanazine or metribuzin in the spring. Data from 1983 are presented in Table 2. Only Russian thistle and kochia were rated, since pigweed and witchgrass were not present in any quantity. Weed control with atrazine, cyanazine, and metribuzin was similar to that obtained in 1982. The control resulting from these herbicides, however, lasted longer into the summer of 1983, because a cool spring delayed weed growth. The plots were rated twice in 1983, and several of the treatments required tillage on July 14, prior to the second rating, including all of the non-sequential atrazine treatments. Chlorsulfuron was included in 1983, and the 0.375 oz rate, applied postharvest, resulted in longer lasting weed control than did 1 lb of atrazine. Conversely, plots receiving the 0.25 02 rate of chlorsulfuron , postharvest, required tillage on July 14, along with the plots receiving atra2ine. The sequential application of 0.25 oz chlorsulfuron following atra2ine was still giving nearly perfect control on July 21, 1983. Spring (non-sequential) applications of chlorsulfuron resulted in better weed control than t~at obtained with cyanazine or metribuzin . A comparison of tank mixes containing chlorsulfuron, cyanazine, and metribuzin is given in Table 3. Tank mixes of 0.125 02 chlorsulfuron with either cyanazine or metribu2in resulted in weed control as This publication from Kansas State University Agricultural Experiment Station and Cooperative Extension Service has been archived. Current information: http://www.ksre.ksu.edu. good as that obtained with higher rates of chlorsulfuron applied alone, and better control than with higher rates of cyanazine or metribuzin applied alone. Although not evaluated in this test, such tank mixes can be expected to provide control of certain grassy weeds, in addition to broadleaf weeds. Extende d Control from In-Crop Applications. Of the herbicides suitable for reduced tillage, chlorsulfuron is unique in that it can be applied to growing wheat. It will control most broadleaf weeds in This publication from Kansas State University Agricultural Experiment Station and Cooperative Extension Service has been archived. Current information: http://www.ksre.ksu.edu. the growing wheat, and if a high enough rate is used this control can be extended into the fallow period. In Table 4 it can be seen that satisfactory control of Russian thistle and kochia was obtained in September 1982, from an application of chlorsulfuron made to jointing winter wheat on April 3, 1982. Kochia was controlled with only 0.125 oz, while the control of Russian thistle required 0.375 oz. When used in this manner, the maximum labeled rate (0.375 oz) is recommended if control l:mtil winter freeze-up is needed. A lower rate can be used if a shorter period of weed control is desired. Control.of.-Volunteer Wheat. The LO lb -rateof atrazine applied prior to emergence has given adequate control of volunteer wheat. Lower rates of atrazine, when used on high pH soils have also given satisfactory control. Occasionally, control of volunteer with atrazine has been less than satisfactory. In such instances, controJ·has been improved when 1.6 lb of cyanazine was tank mixed with 0.81b of atrazine. The use of chlorsulfuron alone will result in essen· tially no volunteer control; tank mixing chlorsulfuron with atrazine will aid in controlling volunteer. In the event that an excessive amount of volunteer escapes the residual herbicide, tillage or postemergence herbicides should be used. Delaying the application of the residual herbicides until the volunteer has emerged, and tank mixing the residual herbicide with a postemergence herbicide also can be effective. Conclusions Herbicides for wheat-fallow can be applied postharvest, in the spring, or sequentially. In addition, chlorsulfuron can be applied to growing wheat. All of these methods result in good weed control for varying periods of time. The length of time of weed control depends on the persistence of the particular herbicide in the soil, the application rate, and climatic conditions. Atrazine and higher rates of chlorsulfuron can be applied after harvest for control that will last into the spring, while short-term residual herbicides such as metribuzin, cyanazine, or a low rate of chlorsulfuron are best suited for application in the spring, or for seuential treatments following a postharvest application. Ideally, herbicides should be selected to control weeds during the entire fallow period. Unfortunately, there is no single herbicide that will control weeds for the entire time. The best control, therefore, will result from an in-crop or postharvest application followed by a spring application. Trade names are used to identify products. No endorsement of these products or criticism of similar products not mentioned is intended. This publication from Kansas State University Agricultural Experiment Station and Cooperative Extension Service has been archived. Current information: http://www.ksre.ksu.edu.

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2014-10-01T00:00:00.000Z

1977-11-01T00:00:00.000Z

5555675

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Mammary tumors and mammary tumor virus expression in hybrid mice of strains C57BL and GR. Mammary tumorigenesis in genetic crosses between the high mammary tumor incidence GR and the low incidence C57BL mouse strains is highly correlated with murine mammary tumor virus expression in milk. Although the F1 and first backcross females had a mammary tumor incidence which was consistent with a single dominant gene segregation, the tumor incidence in the critical second backcross segregants disproved the single gene hypothesis. Genetic factors were clearly involved in regulation of virus expression which in turn correlated with both tumor incidence and tumor latency; these complex phenotypes are however best explained as threshold or quasicontinuous characters. As predicted from this model, the age specific incidence of mammary tumors showed a broad peak at 14-19 mo of age with no evidence of an early or late phase. Hematopoietic tumors showed no correlation with virus expression or mammary tumorigenesis suggesting different etiologies for these tumors. Geneticists in 1933 and 1934 made the original discovery that led to the identification of the murine mammary tumor virus (MMTV) (1,2). ~ By using reciprocal hybridization of high and low mammary tumor strains of mice, they discovered an extrachromosomal factor or maternal influence in mammary tumorigenesis. Bittner (3) later showed through foster nursing experiments that this factor could be transmitted through the milk and hence it was called the milk agent. Through logical steps this agent was eventually purified and characterized as an RNA-containing virus and shown in its mature form to be a characteristic particle now known as the type B particle or finally the MMTV. Bittner and Huseby (4) never considered the issue of mammary tumorigenesis as solely viral and persisted with the concept of three sets of interacting factors, the genetic constitution of the host, the hormonal influence, and the milk agent. He carried genetic crosses beyond the F1, and in the maternal line, F~, and in first backcrosses observed tumor and nontumor segregation ratios that were in accord with single dominant gene segregation (5). Subsequently, genetic crosses and foster nursing studies of strains C3H and C57BL carried out by Heston and co-workers in 1945 (6) showed that replication and transmission of virus was under genetic control. Females of the backcross to the susceptible C3H male could transmit the virus more effectively than those of the backcross to the resistant C57BL male. Again the C57BL backcross females showed a 50-50 tumor nontumor segregation ratio but nontumor females could also transmit virus although not as effectively as those that later developed tumors. Successive backcrossing of these C57BL backcross females to C57BL males completely eliminated virus by the third backcross suggesting that the number of genes controlling virus was few and possibly only one (7). However, a subsequent more detailed study of the second backcross populations did not support single gene control (8). In the 50's and 60's Mtihlbock (9) developed strain GR that was unique in that female mice had a very high incidence of mammary tumors which was transmitted by the male as readily as by the female. It was of special interest to us when Bentvelzen in 1968 and1972 (10, 11) suggested from his data that the GR virus was genetically transmitted as a structural gene, the provirus, controlled by a regulator gene. From tumor nontumor segregation ratios, Bentvelzen concluded that single gene segregation accounted for mammary tumorigenesis. In GR and C57BL crosses carried out by van Nie and co-workers (12) the single gene hypothesis seemed to be supported. However, data from the second backcross appeared to be inadequate particularly in the light of the past observations already discussed. As a further complication in crosses between GR and BALB/c, Nandi 1 Abbreviation used in this paper: MMTV, murine mammary tumor virus. 1206 THE JOURNAL OF EXPERIMENTAL MEDICINE • VOLUME 146,1977 and Helmich (13) observed MMTV segregation ratios in the F2 and backcross generations that fit a two gene model. We therefore initiated a study of the genetic transmission of MMTV in which the presence and segregation of MMTV expression in F~, F2, and backcross hybrids of the low mammary tumor strain C57BL and the high mammary tumor strain GR was analyzed (14). To test the single gene hypothesis special emphasis was given to the second backcross. In crosses in which passage of virus was only through the male, MMTV expression in milk samples from early lactations segregated in first backcross females in a 60:40 ratio not significantly different from the expectancy for either single or two gene control as postulated by either Bentvelzen or Nandi and Helmich, respectively (10,13). Among the second backcross progeny of MMTV-positive first backcross females there were more virus-positive females than among the offspring of virus-negative first backcross females indicating significant segregation of genetic factors influencing virus expression. However, all second backcross families had some virus-positive females and further the families from virus-positive first backcross females had an incidence of positive females above the 50:50 ratio expected with single gene control. Similarly, of 25 second backcross families of first backcross males all but one had MMTV-positive females with no evidence of grouping of families. These results suggested strongly that virus expression in crosses between GR and C57BL mice was regulated by more than a single locus. In addition to the measure of MMTV expression, females from these crosses have now been observed for appearance of mammary tumors. In the present report an analysis of the genetic segregation of the tumors in these hybrids and the correlation of tumor frequency with the early measure of expression of virus in milk is reported. These results indicate that MMTV expression and mammary tumorigenesis are highly correlated. Also, analysis of the segregation ratios reveals that genetic control is more complex than can be explained by the single gene hypothesis. Materials and Methods A detailed outline of the breeding experiment (see below), the care of the animals, and the measure of MMTV expression was given in the previous publication (14). The C57BL parent strain was the line from our laboratory in which less than 1% of females normally develop mammary gland tumors by 2 yr of age (15). The strain GR parents were derived from the GR strain developed by Mfihlbock (9) and received from his laboratories in 1960. This strain has a mammary tumor incidence of approximately 100% males transmit MMTV as readily as do females. It was in strain GR that Bentvelzen et al. (16) described male transmission of MMTV and proposed single dominant gene control for genetically transmitted virus. The various hybrids produced from these strains are listed in Table I and their designation is given. Our intent was to study only male-transmitted genetic influences to eliminate putative milk-transmitted virus. The first cross between a C57BL mother and a GR father produced the F1 population which was 100%; positive for virus expression (14); brother sister matings of this F1 population produced F~ populations. The first backcross to C57BL populations were done as reciprocal crosses to evaluate the putative milk-transmitted virus influence on virus expression in mammary tumorigenesis. Thus, either an FI mother and/or a C57BL mother were employed in matings to produce the two reciprocal groups of first backcross animals designated BC~. Second backcross populations were derived from 25 male and 25 female BC1 animals by using C57BL mates. The BC~ females had been further segregated based on early lactation virus-positive and early lactation virus-negative groups. After MMTV expression was measured during the first or second lactation, females were retired to holding cages. However the 25 first backcross females were allowed to produce as many second backcross litters as possibl~ before being retired. All females were examined twice weekly (17). Similarly, viral polypeptide expression levels in milk correlate highly with the likelihood of any given female within an inbred strain of developing a mammary tumor (18)(19)(20). Until the recent small study by van Nie et al. (21), no study had evaluated the correlation between virus expression and tumorigenesis in various hybrid generations by using high and low mammary tumor incidence mice strains. As shown in Table II, in a very large number of animals the percentage of animals with tumor virus expression parallels the percentage of the various hybrid groups which developed tumors. In subsequent hybrid generations, as increasing genetic information from C57BL is introduced there is a marked decline in both virus expression and tumor frequency, and further, virus expression always exceeds tumor incidence. For example, 88% of the F2 generation females express virus, but only 73% develop tumors. The incidence of virus-positive BC-1 females with a C57BL mother was 60%, intermediate between the 50% expectancy for a single gene and the 75% expectancy if either of two genes resulted in virus transmission (14), The incidence of virus-positive first backcross females with an F1 mother was 88%, considerably higher than in the reciprocal cross. Interestingly, the incidence of tumors in both groups was 41 and 42%. These data and similar results from reciprocal second backcross offspring suggest that the gentically-transmitted virus and/or genetically-transmitted factors are more important in mammary tumorigenesis than is milk-transmitted virus. A similar lack of maternal influence on virus expression was noted in the second backcross populations. The frequency of virus expression regardless of the first backcross parent was approximately 50%, and the tumor incidence in both groups was approximately 15%. Thus, in both the first and second backcross generations there was no evidence of a maternal influence on tumorigenesis and only in the first backcross generation was there an apparent influence on the frequency of virus expression. Although the incidence of virus-positive females is significantly greater than would be predicted with a single gene in all crosses, the incidence of mammary tumors in the F2 and the first backcross groups shown in Table II do not differ significantly from ratios expected with a single dominant gene hypothesis; 73% observed, 75% expected (X 2 = 0.104; 0.80 > P > 0.70); 41 and 42% observed, 50% expected (X ~ = 1.7418; 0.20 > P > 0.10) (×2 = 1.1486; 0.30 > P > 0.20). However, such an analysis of tumor incidence breaks down upon the analysis of the various second backcross generations as will be shown subsequently. Correlation between Virus Expression and Tumors in GR and C57BL Strains and Their Hybrids. The quantitative relationship between mammary tumor virus expression and mammary tumorigenesis is not clear from the above data. Thus, a comparison of tumor-positive and virus-positive mice in the hybrid groups is shown in Table III. With but few exceptions, all mammary tumors occurred in females that had been mammary tumor virus positive at either the first or second lactation. The exceptions were three C57BL females which were virus negative and developed mammary tumors. It is possible that the three females listed in Table III may have become infected venerally at later matings with their MTV-positive GR and F1 cage mates. Other exceptions to the high correlation between virus expression and mammary tumors were one fir §t backcross and six second backcross females that had been classified as virus negative and later developed mammary tumors. These tumors could not be attributed to venereal infection since these females had been mated only to C57BL males, but might be explained by testing errors or tabular mistakes. Additionally, there was a mammary tumor in a virus-negative second backcross female that was probably caused by an active chromophobe adenoma of the pituitary gland that occurred in this animal. In subsequent backcross generations, the proportion of virus-positive females that developed mammary tumors was much lower. As will be shown subsequently, lower tumor incidence was in part due to lower levels of virus expression. Therefore, the simple presence of virus expression in the milk did not necessarily equal tumor formation. As is also shown in Table III, decreasing virus expression in backcross groups prolonged the latency to tumor formation from 9 mo in the GR strain to approximately 16 mo in the second backcross generations. Thus, the correlation between the proportion of any parental strain or hybrid group which expresses MMTV virus and the percentage of that group that develops tumors is very high and further correlates with tumor latency. Correlation between Virus Expressor Status and Mammary Tumors in Second Backcross Females. With the high degree of correlation between virus expression and the propensity for mammary tumorigenesis, it was possible to evaluate critically various second backcross generations for tumor and nontumor segregation ratios. Specifically, this analysis is to test the single gene hypothesis for mammary tumorigenesis to test the role of milk-transmitted virus and finally to extend the correlation between virus expression and tumor formation. As shown in sections A and B of Table IV, by selecting families from virus-positive and virus-negative first backcross mothers the frequency of mammary tumorigenesis differed significantly, 35 and 1%, respectively. Female progeny from virus-positive first backcross mothers (Table IV, Section B of Table IV shows the correlation between virus expression and tumorigenesis in the female offspring of 13 first backcross females classified at early lactations as virus negative. Only one mammary tumor was noted in 170 offspring and only 32% were virus positive. The ability to separate first backcross mothers so strikingly into two groups clearly indicates that genetic factors are important in the regulation of virus expression and tumorigenesis. More importantly, factors regulating viral expression, regardless of their complexity are also important determinants of mammary tumorigenesis. As indicated earlier, the larger number of animals available from the progeny of these first backcross females shown in both sections of Table IV enables a more precise statistical analysis of the single gene hypothesis for mammary tumorigenesis. This hypothesis would predict a 50% mammary tumor incidence among the BC2 female offspring of virus-positive BC1 mothers. Of 113 BC2 female offspring of the early positive backcross females, 35 of those with virus developed tumors. However, this incidence does not support the single gene hypothesis since the observed 31% tumor incidence of tumors associated with virus is significantly lower than 50% expectancy; X 2 = 8.01 P < 0.01. Such an analysis of these second backcross females (section A) is complicated by possible milk transmission of virus in addition to the genetic influences transmitted by these first backcross mothers. However, if milk virus transmission was operative one might expect an enhancement of genetically-transmitted virus and actually expect a figure greater than 50% tumor incidence. Given that the observed values were significantly lower than would be predicted by a single gene, antibody influences in milk (22) or negative influences from the C57BL genotype must be postulated to be consistent with a single GR gene hypothesis. Analysis of the Reciprocal Second Backcross Population with a Male First Backcross Parent. The breeding test of the 25 first backcross males to produce second backcross females has the disadvantage that it was not possible to classify the fathers as virus positive or negative. However, this cross has the advantage that large numbers of female offspring are produced allowing analysis of individual second backcross families. As previously reported 24 of the 25 first backcross males produced female progeny that were virus positive (14). As shown in Table V, 15 males produced female offspring that developed mammary tumors. If a single gene were involved we would assume that these 15 BC1 males had that gene and the incidences of mammary tumors in their families would be distributed about a 50% incidence. In contrast, the tumor incidences range from 5-58% with no evidence of grouping (Table VI). There was a total of 347 offspring in these 15 second backcross families of which 95 animals or 27% of the total had tumors. This incidence is significantly below the 50% expected with a single gene segregation: ×2 = 37.43; P < 0.001. Thus, although the incidence of mammary tumors in the F2 and first backcross generations do not deviate significantly from that expected for a single gene hypothesis, such an hypothesis is not supported with the data from this second backcross generation. These data strongly support a more complex genetic situation than can be explained as a single mendelian trait. 1854 2 3 9 1866 3 5 1867 3 2 1 1888 3 11 1940 3 1978 11 9 1979 4 18 1980 2 8 2114 2 13 2115 4 14 2159 3 5 7 2160 1 1 3 6 2257 2 12 Total -1 * All first backcross mothers were virus positive in their first or second lactation and are identified in the previous publication. 10 of these 12 mothers developed mammary tumors, the exceptions being 1853 and 2060. $ Number in parenthesis represents percent of total. § Of 13 virus-negative mothers tested in early lactations, 12 were tumor free at the time of death, One mother, 1979, died and was cannibalized, thus no final record is available. Comparison of Agouti Gene Segregation and Mammary Tumor Segregation. Evidence that typical mendelian segregation occurred in the GR-C57BL crosses came from studies of the agouti locus through the second backcross generation (Table VI). In the second backcross females there was single gene 9 6 3 8 2207 8 2 1 2 12 1931 7 3 1 14 2358 11 2 12 2110 11 3 4 5 2357 3 1 2 2809 10 1 1 4 3 Total 8-7 Distribution of Age and Tumor Death. It has been considered by some authors that there are two kinds of mammary tumors, those that arise relatively early in the life of the animal and are induced by MMTV and those that arise relatively late and are caused by other factors and/or genetically transmitted MMTV (21,23,24). The many mammary tumors occurring in these hybrids were tabulated according to tumor age to see if there was evidence for such separation. The distribution presented in the histogram in text Fig. 1 spreads from 8 to 25 mo but fails to indicate any grouping according to age at which the tumors appeared. We conclude that early and late are more likely the result of levels of virus expression interacting with a particular genotype. Further, it should be noted that by 2 yr the age-specific incidence of mammary tumors appears to be declining markedly suggesting that most mammary tumors occurred in this population by 20 mo of age. Reticular Cell Neoplasms in Second Backcross Females. Earlier studies have shown that type C expression is correlated with reticular cell neoplasms but not with mammary tumorigenesis although no measure of MMTV expression was reported (25). We were interested to determine if mammary tumor virus expression could be directly demonstrated to be independent or dependent of reticular cell tumors in various hybrid groups. An analysis of the second backcross groups for reticular cell neoplasms indicated no correlation between virus expressor status and reticulum cell tumors (Table VII). Thus, the high association between type B virus expression and mammary adenocarcinomas appears to be specific to that class of tumors and not general for all tumor types. Other Tumors in C57BL, GR, and Their Hybrids. All neoplasms observed in the parent strains and their various hybrids in this study are listed in Table VIII. Most of the mammary tumors were Dunn's types A and B (26). In all cases they were classified depending on whether the cell arrangement was predominantly an adenoid, type A, or predominantly sheets and cords, type B, because most tumors had areas that were of the alternate type. The pale cell carcinoma has been described by van Nie and Dux (27) in the GR strain. The pale cell carcinoma reveals cells that appear to lie in compact sheets but take a very pale stain with hematoxylin and eosin. Thus far we have noted this type of mammary tumor only in the GR strain and hybrids derived therefrom. It was of interest that adenocanthomas observed in these groups occurred in females whose milk was virus positive. In other studies (28,29) this type of neoplasm had been noted in strains such as the C3HfB, thought to be free of milk-transmitted virus and especially among mammary tumors induced by chemical carcinogens. Thus adenocanthomas may represent an aberrant or unusual result of the activation of type B virus expression (30). Further studies will be necessary to validate this hypothesis. Aider mammary tumors, the next most frequently occurring neoplasm in this study were the reticulum cell neoplasms probably because of the influence of the C57BL strain. This group included both reticulum cell types A and B described by Dunn (30). Other neoplasms in the GR strain included lymphocytic leukemias and lung tumors, but were limited to these two groups probably because of the early death of the GR females from mammary tumors. The list includes in the other groups a number of other tumors, none of which occurred in significant numbers in the population. Discussion The initial reports of mammary tumor virus segregation as a single dominant gene in crosses between the GR and C57BL used mammary tumor development as the measure of virus expression (10)(11)(12). Previous studies had clearly shown a high degree of correlation between virus expression and mammary tumorigene- (14). The present study was designed to investigate this point specifically for mammary tumors through the second backcross generation. The analysis of mammary tumorigenesis in the Fl's, F2's, and first backcross generations yielded results that were consistent with a dominant single gene determinant for mammary tumorigenesis in agreement with the earlier observations of Bentvelzen and van Nie et al. (10)(11)(12). However, our concern in initiating these studies, was that statistically, too few second backcross segregants had been analyzed. In our analysis of the second backcross generation the hypothesis for a single gene is not proven. Although we cannot exclude a negative genetic influence contribution from the C57BL genotype, we favor the view that mammary tumorigenesis in these crosses is not a single gene influence but are a threshold expression of multifactorial genetic inheritance from the parental phenotypes. Such inheritance patterns can be expected with threshold or quasi-continuous characters. Several examples of such phenomena in mammalian genetics are known (32)(33)(34)(35). The evidence from this and other studies suggests that the genesis of mammary tumors in such hybrids is an interplay between multiple positive and negative influences. Many of the positive influences appear to affect the expression of mammary tumor virus in early lactations and promote expression of high virus titers. Animals that display this particular phenotype for whatever reasons appear to have a greater potential for the development of mammary tumors. Negative genetic influences that may influence virus expression are largely undefined at the present time and may be exemplified by the regulatory genes present in the C57BL mouse that prevent expression, or the spontaneous 10-fold drop in constitutive virus expression noted in mammary cells in culture (36). Since both the GR and C57BL strains have comparable although not necessarily identical amounts of mammary tumor viral genes in their DNA (37), it would appear that the regulation of the expression of these genes is an important determinant in natural mammary tumorigenesis. The definition of genetic influences that regulate mammary tumor virus expression is an important future concern in the understanding of the regulation of murine carcinomas. This rather genetic interpretation of mammary tumorigenesis does not adequately deal with what is commonly presumed to be a largely milk-transmitted disease. The milk influence, presumably a milk-transmitted mammary tumor virus into susceptible recipient newborn animals may also play a role in natural disease (38). In this context, the ability to lower or alter tumor incidence by foster nursing (39) suggests that the milk-transmitted virus is relevant although such experiments have not been repeated in the past 20 yr. Certainly the view that cancer is a delicately balanced interplay between positive and negative influences has been altered to a significant degree by geneticist's selection for particular tumor phenotypes. For example, high mammary tumor incidence strains where milk transmission is an important factor might not commonly exist in situations where natural selection is operative. The results of reciprocal cross analysis (Tables I, II, IV and Fig. 1 There is no natural situation where mammary tumor virus is expressed at high levels and the population is not at risk for some mammary tumors. Nor is there a mouse population that has a mammary tumor incidence of greater than 20% that can be shown to be free of detectable mammary tumor virus expression. Thus, as shown herein there is a high degree of association on the level of individual animals, within inbred strains and at the level of the whole population between virus expression and propensity for the development of mammary tumors. It would appear however that environmental influences may play important roles in determining whether or not an animal expresses high levels of mammary tumor virus. For example, glucocorticoids have been shown to be an important determinant of mammary tumor virus expression in tissue culture (40) and similarly this and other hormones are likely to play important, although as yet largely undefined roles, in natural mammary tumorigenesis. In view of the clearcut association between virus expression and mammary tumorigenesis it is perhaps surprising that we consider the possibility that mammary tumorigenesis is not the direct effect of a mammary tumor virus gene or genes. However this possibility should be considered in light of recent results from type C virus studies in AKR thymic leukemias (41). The virus currently regarded as the cause of this well-studied disease appears not to be the classic ecotropic AKR virus but the result of recombinational events between the AKR virus and endogenous xenotropic viruses in the AKR mice (41). Similarly in rats, another murine system, recombinational events have been clearly demonstrated which result in altered viral properties and the ability to transform cells in cell culture (42). As yet there is no direct evidence of a viral-encoded transformation gene nor evidence for recombinational events in mammary tumorigenesis. Nevertheless these possibilities are being pursued (24). Summary Mammary tumorigenesis in genetic crosses between the high mammary tumor incidence GR and the low incidence C57BL mouse strains is highly correlated with murine mammary tumor virus expression in milk. Although the F1 and first backcross females had a mammary tumor incidence which was consistent with a single dominant gene segregation, the tumor incidence in the critical second backcross segregants disproved the single gene hypothesis. Genetic factors were clearly involved in regulation of virus expression which in turn correlated with both tumor incidence and tumor latency; these complex phenotypes are however best explained as threshold or quasicontinuous characters. As predicted from this model, the age specific incidence of mammary tumors showed a broad peak at 14-19 mo of age with no evidence of an early or late phase. Hematopoietic tumors showed no correlation with virus expression or mammary tumorigenesis suggesting different etiologies for these tumors. Received for publication 6 June 1977.

v3-fos

2016-05-04T20:20:58.661Z

1977-01-15T00:00:00.000Z

655499

s2

Reproductive efficiency of iceland sheep I. Puberty and early reproductive performance Results are presented for the breeding performance of Finn x Dorset Hovn ewe lambs or ewes in a once per year breeding system. Conception rate at a mean age of 23 8-t-9. 4 days was gi. 7 per cent and mean litter size 1. 52. Ccriesponding values for 294 ::!: 11. 4 days were 94. 3 per cent and 1. 70. An interaction between time of year of birth and onset of puberty was observed, with ewe lambs bom as late as November showing behavioural oestrus before the end of the breeding season. Ewes bred once per year had a mean litter size of 2. 34. Results are also given for a frequent breeding experiment in which two flocks each of 4 8 ewes were repeatedly subjected to artificial daylength regimes, weaned after one month of lactation and synchionised in cestrus by using a synthetic progestagen. Total annual production was 3. 5 lambs per ewe. The amounts and pattem of food intake during the reproductive cycle required to achieve this level of production are presented. The results of experiments designed to show whether the increased frequency of breeding and high level of production achieved under conditions of artificial daylength control could be achieved under natural conditions are presented. In these studies a conception rate of 92 .8 per cent was obtained in ewes bred during the normal period of anoestrous using 400 LU. of pregnant mares serum gonadotrophin at pessary withdrawal. Mean daily lamb growth rate and estimated milk yield in twin bearing ewes given ad libitum a diet containing 10 MJ metabolizable energy per kg was o.2g + 0. 05 and 2.93 ! 0. 51 kg respectively. Les performances des F, obtenues avec les races Romanov et Finnoise sur un troupeau de 100 brebis Avagonaise Rasa (p. v. 45 kg), sont étudiées depuis 1974 , dans le cadte des essais d'utili-sation de ces races prolifiques en milieu Méditerranéen. D'après les résultats constatés, il semblerait que l'utilisation du Romanov ou de Finnois sur une race rustique de format moyen permet une amélioration de la croissance par rapport à la race l ustique. Dans le cas présent, l'avantage a été de l'ordre de 15-20 p. 100 pour le RO x RA et de 10-15 p. 100 pour le FI X RA. Le croisement dans ces conditions, bien que sans améliorer notablement l'aspect des carcasses, permettrait l'obtention de carcasses plus lourdes pour … The amounts and pattem of food intake during the reproductive cycle required to achieve this level of production are presented. The results of experiments designed to show whether the increased frequency of breeding and high level of production achieved under conditions of artificial daylength control could be achieved under natural conditions are presented. In these studies a conception rate of 92 .8 per cent was obtained in ewes bred during the normal period of anoestrous using 400 LU. of pregnant mares serum gonadotrophin at pessary withdrawal. Mean daily lamb growth rate and estimated milk yield in twin bearing ewes given ad libitum a diet containing 10 MJ metabolizable energy per kg was o.2g + 0 . 05 and 2.93 ! 0 . 51 kg respectively. A!avtado 202 , Zaiago7a, Espana. Che les femelles, la précocité sexuelle (un mois d'avance au 1 er oestrus) et surtout le taux d'ovulation, sont nettement améliorés par effet du croisement, avec un léger avantage des croisées RO sur les croisées FI. Le taux d'ovulation moyen au cours d'une saison à l'âge de 2 ans, a été Lambs of the Iceland breed of sheep are found to be early maturing. The level of reproductive efficiency in the flock is raised by breeding from ewe lambs and by using ram lambs for mating. The reproductive potential of the lamb is increasingly being realized by farmers in Iceland. Icelandic lambs are normally born in May. Most of the ewe lambs experience their first cestrus from late November to late December aged 6, 5 -7 , 5 months and weighing 30 -45 kg. They may exhibit cestrus 4 -5 times on the average during the breeding season, the mean oestrous cycle length being i6 days. It has now become a common practice to breed from ewe lambs weighing 3 j kg or more in December so that they will lamb in May at zz months of age. Normally some 70 per cent of all ewe lambs exposed to rams will conceive. They have an average gestation period of 141 days. The growth rate of their lambs from birth to weaning at q months exceeds that of twins reared by adult ewes. Early breeding does not have any detrimental effects on the overall lifetime productivity of the ewe provided well grown and adequately nourished ewe lambs are selected for breeding. Icelandic ram lambs attain puberty, judged by their anatomical development, at an early age of !-5 months and they are used successfully for breeding in December when 7 months of age. Kaposvár (Hungary). The authors examined the possibility of developing such a population of great prolificacy and requiring intensive breeding, the genetic parameters cf which are the following: first lambing about the age of one year, at least 3 lambs per lambing, lambing interval between 6 ans 8 months. Newertheless, it could be achieved, that 13 of 17 aboundantly foraged tegs should lamb at the average age of 2 8 2 days, their lambs were not viable enough. Data were collected concerning the reproductive quality of 37 Finnish tegs imported from Finnland, 30 Homanov tegs imported from the Soviet Union and their progeny, originating from own breeding. The lambing rate of tegs, having lambed for the first time about at the age of one year was smaller than those of having lambed for the first time at an older age, but the earlier taking into breeding did not influence disadvantageously their later productivity and prolificacy. In the average of m6 lambings the Finnish ewes had 2 , 00 lambs and the Romanov ones in the average of 159 lambings 2 , 42 lambs. Their lactation milk yield proved to be insufficient for the lambs, therefore the growth of them was not satisfying. Especially the F i lambs of Finnish mothers, originating from I?oiiiaiiov rams, were raising more poorly during the lactation period and could less compensate the lag after the weaning, too. Therefore it is not worth leaving more than 2 lambs with the mother and the lambs above this number must be raised artificially with reconstituted milk. Both the Finnish and the Romanov breed, as a selection basis, are suited for the development of such an ewe population, which can fulfill the requested genetic parameters. But the development and application of breeding and feeding technologies is of fundamental importance. The special acclimatization ability of the Romanov breed must be emphasised. It can well tolerate the rearing in great -100 -200 headsgroups. MODEL EXPERIMENTS Not more than two lambs should be left together with the ewe. The feeding of lambs with milk or milk substitute, whether they are raised artificially or suckled by the mother, is not reasonable over the age of 35 -45 days. At this age they must be weaned. Concerning the period of re-lambing with respect to both breeds, so great individual differences were obtained, that it can be advised for the future to put the examination of heredity and repeatability of this trait on the agenda. The h 2 values obtained for the prolificacy of sheep, must be revised. The change of both, market requirement and husbandry techniques in the sheep industry of the FRG call for investigation of genetic alternatives to the most widespread breed in the country, the 3lerinolandschaf (l!Vuvttemberg Mevino). In a first experiment from 19 6 9 to 1973 ,

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2017-07-29T04:25:02.114Z

1977-04-15T00:00:00.000Z

2753391

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Characteristics of some less-common breeds of sheep in Southern Europe: a preliminary survey Preliminary findings of a field study of Mediterranean sheep are presented for twelve of the fifty-three breeds surveyed during the period September 1974 -August 1975 : Spanish Mevino, Levant Red (Spain); Peone (France); Carapelle (Italy); Dubvovnik (Yugoslavia); Xatafigion, Skopelos, Chalkidiki, Dvama native, (Greece); Sakiz, 7!!o!, Odemis (Turkey). For disappearing breeds, factors which account for decline are identified. Each breed is briefly discussed as to its location, present population and trend, significant morphological and performance characteristics, management, and adaptive conditions. Breeds are identified whose fleece and skin samples showed affinity to fine-wooled types. Introduction This paper is a preliminary and partial report of a survey to seek out and identify the less-common breeds of sheep in Mediterranean Europe and western Anatolia, to map their distribution, and to ascertain their populations and rates of change. The object was also to identify breeds in danger of extinction, and to determine the factors contributing to their decline. Where published details were lacking or inadequate, it was proposed to record breed characteristics, performance data, and adaptive qualities. Unimproved sheep likely to throw light on the affinities and evolution of modern breeds were also sought, with particular reference to the spread of the fine-wooled type around the Mediterranean, and its emergence in Spain as the Me y ino breed. The field investigation for the study, supported by a grant from the National Science Foundation (U.S.A.), was carried out from September 1974 to mid-August Division, FAO, Rome. The FAO has a long-standing interest in animal genetic resources, and, in this regard, our study provides continuity to J.-J. L AUVERNE ' S survey of disappearing breeds of cattle in Europe and the Mediterranean basin (LAUVERGNE , 1975 ;C O LLINS, 1975) Methods The scope of the survey was similar to that of MASON ( 19 6 7 ), except that it was limited to countries north of the Mediterranean: Portugal, Spain, France, Italy, Yugoslavia, Greece, and Turkey. Fifty breeds were investigated, and these are listed Table i. be allowed to die out as a result of the crossing that is now taking place with improved Merino. 2 . Levant Red (also, Guirra, or Sudat) is a breed found near the coast in the Spanish provinces of Alicante, Valencia, and Castell6n de la Plana. During the past decade the breed has steadily declined to the extent that its survival is uncertain. In 1974 , the total number of pure-bred Levant Red was reported to be 1 . 33 6 (SAN CHEZ B ELD A, 1974 ). Of these 3 g 7 were in the province of Alicante, all in six localities within a few kilometers of the shore (from Villajoyosa to Javea). This northern section of the Costa Blanca is rapidly being transformed into a multistoried resort area. The open, uncultivated coastal areas, in which the sheep have traditionally grazed, are disappearing as more land is developed to accommodate the hundreds of thousands of tourists that come to this part of Spain each year. Almost all of the 8 44 Levant Red in the province of Valencia were in six places near the town of jativa, about 25 km inland from the Costa del Azahar. In this area, sheep raising has declined as production and profits from horticulture (vine, citrus, fresh vegetables) have substantially increased. All of the g 5 Levant Red sheep in Castell6n de la Plana are kept by one owner near the outskirts of the provincial capital. In the course of our survey, Levant Red flocks were seen in all three of the provinces. The early history of the breed is not known, but according to S A :vcHEZ B ELDA ( 197 6), the Levant Red developed from crossings of the Manchega with thin-tailed North African breeds. Except for about 5 per cent which were black, the color of the fleece of the animals seen during the survey varied from reddish-brown to yellow-white. The color of new-born lambs is dark reddish-brown. With age the coat lightens, becoming at full maturity a dirty cream color. Sheep with white coats are not considered to be pure Levant Red, nor are those that do not have reddish-brown legs. (« Guirra » is the word for « reddish » in the Valencian dialect; « sudat » means « greasy)), in reference to the oily condition of the uncleaned wool.) Both sexes lack horns. The tail is thin and of medium length. The breed is notably frugal in its feeding requirements and appears to thrive on the poor, rough grazing of the region. Veterinarians reported that the breed is remarkably healthy. The Levant Red is considered to be a good milking breed, but today production is almost entirely for lambs (slaughtered three to four months after birth). In the recent past, milk was of primary value. Ewes have two oestrous periods annually, but usually lamb once a year with a twinning rate reported to range from 5 0 per cent to 8 0 per cent. Of the five fleece samples taken, one animal had wool of almost Merino fineness, ranging is diameter from 1 6 to 3 6 ¡ 1 .m with a mean of 24 .6 and a mode 27 ¡1.m, but its S /P ratio was only 5 . 7 , lower than expected even for a Merino cross. The remaining four fleece samples had hairy fibres (mean percentage of medullated fibre 14 . 25 per cent) in addition to the bulk of the coat which ranged up to about 5 0 ¡1.m in diameter. The mode or each was 24 ¡ 1 .m, but the means ranged from 27 . 3 to 34 .8, with an overall mean of 31 ¡1.m. The mean S /P ratio in the skin was 5 .4 /i. By the the classification of R YDER (ig6g) these are hairy medium wools. The color and the suggestion of a tendency to moult indicate a primitive type akin to that of the Shetland in northern Europe, which also has fine as well as hairy fleeces, and a similar S /P ratio (R YDER , 19 68). L YDEKKER ( 1912 ), p. 7 6, referred to a primitive colored breed of sheep in Spain called Ovejas marinas, and it is of interest that PLINY (Natural Histo y y VIII, igi) stated that Spain was famous for black and brown sheep in Roman times. B. -France The Peone (also, Mauvevousse) is an old migrant breed of sheep found in the southeastern corner of France. The breed has been decreasing during the past twenty years as a result of crossings with Al!ine, Avles Merino, and Pvealpes du Sud. In Alpes-Maritime Department Peone are kept in the upper valleys of the rivers Vésubie and Var, with most of them in scattered holdings in the area of Guillaumes. The population of pure-bred Peone was reported to be approximately I . 250 , with about an equal number cross-bred. This is half the number reported in an earlier survey (G ILBERT , 1975 a). Production is for meat (lamb), with wool accounting for only three per cent of the total value. The breed is not milked. In general appearance, the Peone resembles the Rouge du Roussillon (fig. 3 , c). In both breeds the head and legs are brown or reddish-brown, the fleece white, and the ears are medium long, nearly horizontal or drooping forward slight In this survey, significant differences were noted between the two breeds. Peone ewes are smaller animals: for mature ewes the mean wither height is 6 1 cm., compared to 73 cm. for the Rouge du Roussillon; for mature rams: Peone 75 cm., and Rouge du Roussillon 8o-go cm (G ILBERT , i 975 b). In proportion to the trunk, the legs of the Peone are shorter. Unlike the Rouge du Roussillon, which has bare head and belly, the Peone has a top knot, and its fleece extends below the sides to cover the belly. The bridge of the nose is commonly white in both breeds, but the muzzle of the Peone is broader than that of the Rouge du Roussillon. Both sexes of the Rouge du Roussillon are polled; in the Peone, ewes are polled, and about half of the rams are horned. From October to June flocks of Peone are kept in open pastures on the lower valleys where they are exposed to the cold and violence of the mistral. It was to produce cross-breeds immune to the effects of these bitterly cold winds that the Alpine (Commune des Alpes) and the Arles Merino were brought to the region. During the winter only ewes with lamb (and the few flocks that are in the high valleys) are stall fed. Individual holdings range from 200 to 300 head of pure and cross-bred sheep. In late June several holdings are combined into flocks of 8 00 to 1 200 sheep and walked from the valleys to the high grazing places in the moutains where they remain until October. Ewes are in oestrous twice a year but usually lamb once per year, either during October-November or February-March. The lambing rate was reported to be between 130 and 150 with about ten per cent mortality before market age (four to six months). Local breeders reported that although the Peone is less hardy and somewhat less frugal than the Alpine and Arles Merino, it is superior in these characteristics to the P y jalpes du Sud. The four fleece samples ranged from 20 to 40 mm in length, with a mean of 30 mm. They were all clearly fine, with a quality of 5 8s, but lacked crimp. The wool therefore resembled that of a British Down type rather than Merino. The overall fibre diameter range was from 14 to 6 2 >m the modes ranged from 2 6 to 40 >m with a mean of 32 . 5 !tm and the mean diameters ranged from 27 . 9 to 35 . 3 to give an overall mean diameter of 32 . 7 !.m. The similarity of the modes and means reflects the symmetrical diameter distribution, all being true medium fleece types, with one tending towards the fine type. The three skin samples had the relatively high mean S /P follicle ratio of 6. 2 y , which is, however, in keeping with the relatively fine mean fibre diameter (above). One animal had a small amount of non-latticed medullation in the pri-mary fibres making the overall mean 2 per cent; z 3 per cent of the primary follicles, and 5 per cent of the secondaries showed evidence of regrowth after shedding (at the beginning of July). C. -Italy The Carapelle is a triple-purpose sheep formerly kept in flocks of mixed breeds in the Carapelle valley southwest of Foggia, Italy. A local name is « Black Merino », although FEDERCO!rsoRZr (ig6i) identifies Carapelle as a variety of Moscia Leccese, a carpet-wool breed popular in Apulia. During the past twenty years the large agricultural estates in the area have been broken up into small holdings for irrigated farming, and the changes in the land-use and tenure system have brought about a precipitous decline in numbers of sheep there. The spread of hy!ericum sp!. concurrent with the introduction and extension of irrigation, also is a factor in the decline of sheep. F EDERCONSORZI in ig6i recorded only 300 Ca y apelle. Today the breed appears to be very near extinction. In our survey, only one Carapelle was found, a two-year old ewe, 6 1 cm. high at the withers. The coat was a very dark brown, and the face and legs were black and bare of wool ( fig. 3 , d). This sheep was horned according to F EDERCONSORZI (ig6i) and MASON ( 19 6 7 ) both sexes are polled. The mean wool fibre diameter of 31 . 2 >m was higher than that of the local Mevino, and S /P follicle ratio of 5. 3 lower. Three samples taken from Gentile di Yuglia sheep for comparison had a mean fibre diameter of 24 .8 >m and an S /P follicle ratio of 8. 9 / 1 . The Carapelle therefore appears to be of primitive, rather than 'llerino type. D. -Yugoslavia Dubvovnik. To the description of this white-faced, polled breed given by MASON, can be added the following points: the rams are sometimes horned, the nose is bare and slightly convex, but there is a wooly itop knot (fig. 3 e). The wool from the 10 per cent of sheep with colored fleeces was desired in the past to make naturally-colored garments. The fleece weight is from one to two kg., and the tail is long. In the recent past each peasant family had from fifteen to twenty sheep, but now only about one to six are held. The five flocks we saw numbered three to five ewes. The breeds is declining rapidly. Locally, tourism offers greater income than sheep husbandry and many young people today leave home to work in this and other industries. Barrenness is common because there is only one ram in each district, and no attempt is made to detect heat. But among ewes lambing there is 145 per cent lambing, and there is a long breeding season; ewes often lamb thrice in two years. Both sexes will mate in their first year. The sheep are grazed in olive groves in the summer, and kept indoors during the winter, despite the mild climate. A common lambing time is late autumn, and lambs are killed at 35 -40 days, crossbreds dressing out at about m kg. The ewe is then milked for cheese from February until July, the yield declining from a maximum of two litres at the beginning of lactation, to about one-half litre at the end. As to the fleece, most fibres had a diameter within the range 15 to 55 fL m, but of the nine samples taken, six had a few hairs ranging up to 88 t L m in diameter, and thus were hairy medium wools on the classification of R YDER (ig6g). These had a skewed diameter distribution, with the bulk of the fibres being fine, while the remaining three samples had a symmetrical distribution, being fine medium wools. The overall mean diameter was 34 !m, and the average of the most frequent diameters was 31 !m. This is somewhat coarser than the figure of 2 8-30 !m given by MASON. But even this is coarser than the quality of 5 8s-6os he quotes. The wool lacks crimp and is no more than about 5 os quality. The mean percentage of medullated fibres was six per cent. There was virtually no medullation in the skin, which is in keeping with the time of sampling in winter when medullation is lost. There was evidence of inactivity in 2 -3 per cent of the follicles. The secondary /primary follicle ratio ranged from 3 . 5 to 6.o with a mean of 4 .7 y . The accepted view quoted by MASON is that the Dub y ovnik breed derives from a recent cross between the Merino and the Pramenka, and introductions of the Merino into the area are well documented. But there are various features such as the lack of crimp, and the high lambing percentage, which suggest that this breed cannot derive solely from this cross. The S !P ratio values obtained in the present study also oppose this origin. Assuming that the Merinos introduced would have had an S /P ratio as least as high as the Spanish Merino ( 10/1 , see above), the Dubyov!cik would be expected to have a value intermediate (6.6) between this and that of the Pramertha ( 3 . 3 ); yet, the figure was only 4 . 7 . It therefore seems likely that the Dubrovnik is a relic of an ancient fine wool. This accords with historical evidence that this area had contacts with peoples who could have introduced such a sheep, and in the Middle Ages the city of Dubrovnik had a well-developed wool-textile industry. E. -Greece z. The Drama native breed, discovered in the village of Volax north of Drama, is not listed by MASON, but is probably a variety of the Vlachos (Mou!2tain Zccchet). Some animals were closely similar to the colored Vlachos sheep illustrated in his Plate I IO. The black sheep we saw (fig. z, f ) had a relatively finer fleece than the remainder, and were also similar to the Chalkidihi sheep, (see below). On the whole, however, as with other Greek breeds, the appearance was very variable: horned and polled animals, black, white and grey fleeces, speckled faces and legs, and a moderately long tail. Black around the eyes, and a woolly « top knot », were common, as in other Greek breeds. The sheep mate first at 20 months, and lamb only once a year, but the ewes are kept for eight years. Owing to barrenness and a 10 per cent lamb mortality, the weaning percentage was only 8 0 per cent. Lamb provides 6 4 per cent of the income, milk 32 per cent and wool 4 per cent (fleece weight one kg). In contrast, in the Se y rai breed of the plain, 6 0 per cent of the income comes from milk, 35 per cent from meat and per cent from wool. In three of the five fleece samples taken the bulk of the wool was between 1 6 and 6 0 !.m in diameter, with a few hairs up to 70 pm in diameter, making them hairy medium wools. Two of the sheep had a more continuous distribution with hairs up to 112 >m in diameter, being therefore true hairy types, although they had no more than 10 per cent medullated fibres. The overall mean percentage of medullated fibres was 5 per cent. The mean diameter was 3 g.i fL m (range 33 . 3 to 47!5 tim) and the average of the modes 31 .6 fL m (range 22 to 3 8 !tm). There was no medullation at all in the skin, and 13 per cent of the primary, and 4 per cent of the secondary follicles were inactive. The S !P ratio of 3 . 2 was low (cf. Chatkidiki breed below). 2 . The Kata fegion is a migratory breed of the Pieria Mountains in southeastern Macedonia. It has almost disappeared as a result of cross-breeding and socioeconomic changes in the hearth of the breed. From a population of several thousand in 19 6 0 (G EORGIOU , ig6o), the number of pure-bred animals has declined to less than 100 today. Very few pure Kata figion sheep remain, as they did formerly, on the western slopes of the Pieria. That area was toured extensively in the course of the survey but no sheep were found that could with certainty be identified as pure Katafigion. However, some pure-bred Kata fcgion were found at the town of Katerini among small flocks owned by former residents of Katafigion village. The village, set high in Pieria Mountains, had a population of several hundred families in the mid-195 o's. At that time, each family maintained a flock of ioo-r 5 o sheep. Today only a few elderly persons remain there. During the past twenty years the others left the village to establish new homes in Velvendos, in Katerini, and in other towns, where most of them have adopted an urban way of life. However, each year about thirty families from Katerini return to Katafigion village and remain there from May to October. Some families bring their sheep, transporting them by motor trucks. Owners reported that the breed is unusually hardy and is eminently able to thrive on graze of poor quality. Production is for milk and lambs. For many years breeders have crossed the Kata fcgion with Greek Zackel (especially Karaganiko). In the course of our survey, it was reported that in 19 6 4 an artificial insemination programme was introduced in the Velvendos area to cross the Katafigion with the Chios and East Friesian breeds. The informant stated that the program was dropped after several years because the cross-bred sheep, unlike the pure-bred Kata fcgion, did not thrive unless given supplemental feed. White is the prevailing color of the Katafigion breed ( fig. 4 a). Speckled or brown sheep are not regarded as pure-bred. The face, legs, and (commonly) the belly are bare of wool. Most have a top-knot. The tail is thin and of medium length. Rams are horned; ewes polled. The nose is slightly convex, and the ears horizontal and of medium length. The wither heights of eight mature ewes ranged from 5 8 cm to 6 5 cm, with a mean of 6 2 cm. For the one ram that could be accepted as cc probably pure-bred » the wither height was 70 cm. Ewes are first mated at two-years of age, and it is common practice to breed for two lambings annually, in August and in February. The twinning rate is reported to be 50 per cent. MASON ( 19 6 7 ) described the Katafigion as the only migratory breed among the uniform wooled sheep. He gave the fleece weight as I to 1 . 5 kg, and the sheep illustrated (Plate m8) has a « tippy fleece like a hairy Shetland. The mean fibre diameter of 3 6 ¡ L m he quotes is coarser than the wool quality of 50 s to 5 8s he also quotes. Nor does the staple length of 15 cm warrant the description short. The ten fleece samples obtained in the present study were variable. Half were hairy, i. e. comparable to the Scottish Blacktace type of carpet wool. There was further variation among the remaining finer ones, one being crisp like the British Down type, and one being wavy ( 2 .5 waves per cm). The staple length ranged from 5 0 to 1 8 0 mm with a mean of 100 . The overall diameter range was from 12 to II4 ¡ L m. The mean mode was 32 .6 ¡ L m (range 30 to 3 8 !cm). The average of the mean diameters was 39 . 9 ¡ L m (range 31 .6 to 4 8. 3 !.m). The diameter distributions were either symmetrical (four medium fleece types) or skew fine /continuous (three hairy medium and three true hairy fleeces). One animal was black and had 100 per cent pigmented fibres, and another had z2 per cent pigmented fibres. Seven animals had medullated fibres, the proportion ranged from I to 17 per cent with a mean of 5 . 7 per cent. The sheep with the hairiest coat, and the greatest mean diameter, was the only one with any medullation in the skin, which amounted to 20 per cent. This animal also had the highest proportion ( 33 %) of inactive primaries. The overall mean of inactive primaries was i 5 per cent, and of the secondary follicles 6 per cent. The S /P follicle ratio ranged from 2 . 0 to 4 . 1 with a mean of 2 .g y. 3 . The finer fleeced Chalkidiki is native to the peninsula of the same name southern Macedonia. The numbers are declining, and it was possible to locate only individuals in flocks that are being crossed. The most primitive animal was an all-black yearling ram had horns and a convex nose, and a withers height of 6 4 cm; 10 per cent of the sheep were black (cf. the Drama native breed, above)! The general appearance, and the fleece, were like the Shetland breed, but the tai. was longer (medium length). The ewes were polled. The face, legs, and sometines the belly, were black, altough others were white, speckled, or had black around the eyes ( fig. 4 , b). Some of the white animals had black hairs, which, as in the Shetland breed (R YDER , 19 68), give rise to grey fleeces, and some dark grey animals were seen. Four mature ewes ranged in height from 6 5 to 6g cm, with a mean of 6 7 . 25 cm. The sheep were said to be hardy. They do not mate until their second year, the litter size is 1 . 2 , but the ewes are fertile until the age of seven or eight years (cf. Drama native breed, above). Among the brief details of the Chalkidiki breed given by MASON are a ewe fleece weight of r. 4 kg, a staple length of i5 cm, and mean fibre diameter of 3 6 ¡ L m. Two fleece samples indicated hairy animals with a mean of 4 6. 4 ¡ L m, a mode of 34 ¡ L m, and z 5 per cent medullated fibres. The mean diameter of the remaining four was 33 ¡ L m and the average mode 25 ¡ L m. Three of these samples had a few hairs (mean proportion of medullated fibres, 12 %) and were therefore hairy medium wools (cf. hairy Shetland); the remaining animal was a generalized medium wool (cf. woolly Shetland). The skin samples had a mean of only 5 per cent non-latticed medullation. The smaller amount than in the fleece accords with the time of sampling in winter. There were 31 per cent of the primaries, and m per cent of the secondary follicles inactive, which indicates a tendency to moult the fleece as in the Shetland. The S !P ratio of 3 . 3 was low (cf. Drama native breed). The general appearance, and fleece measurements, indicate similarities between the Chalkidiki and Drama native breeds. Both appear to be broadly of generalized medium fleece type, the ancient fine wool that from textile remains can be traced back to about 5 oo BC in the Near East, and which spread through Europe during Roman times (R YDER , ig6g). It persists in northern Europe in such breeds as the Shetland. 4 . The Sko!elos (or Glossa), a dairy breed of the Greek Zackel type, was developed on the island of Skopelos in the Northern Sporadhes. The ancestry of the breed is obscure. According to MASON ( 19 6 7 ) there is no record of sheep on Skopelos before 1 8 00 , and he considers that a fertile breed with a coarse fleece could have been introduced from St. Eustratios island, and the fleece selected to give the modern Skopelos. His alternative, possible origin from the Chalkidiki breed, seems more likely and is supported by N. P. Z ERVAS at the A y istotelian University of Thessaloniki (personal communication). At the time of our survey (February, 1975 ) the number of mature Skopelos sheep on the island was g 7 (85 ewes and 12 rams); the total in ig6o was 470 (GEOR-G iou, ig6o). About goo Sko!elos sheep are on the mainland, most of them in or near several small towns on the coast south of the city of Volos. Small flocks of the breed are maintained also at Thessaloniki University Farm and at Yannitsa Fa y m Breeding Station. During the past fifteen years, land-use on Skopelos has changed to give greater emphasis to horticulture, and increasingly, young persons leave the island to find employment in the cities. Sheep husbandry, which was similar to that described for the Dub y ovvcik breed has disappeared. Formerly, from one to three ewes were taken out to graze each day and returned to stalls at the house in the evening. Currently, there are only six owners of Skopelos sheep on the island, and their holdings range from eight to twenty animals. Goats have replaced sheep as the source of milk for home consumption. On the mainland, Sko!elos sheep are kept in flocks of from 20 to 25 head, which are stall-fed throughout the year. Milk from the ewes is sold to buyers from local cheese factories. Attempts during the 1950 's to establish the breed on the mainland failed, with high mortality of sheep. The present flocks of Sko!elos in the area south of Volos are descended from breeding stock brought from the island during 19 6 4 -65 to replace Chios ewes that had become diseased with « parmara o, an udder infection that seriously reduced lactation. With careful husbandry, and especially the provision of supplemental feed, the Sko!elos breed now appears to be successfully established on the mainland. In the course of our survey, the description of the breed by MASON was confirmed, the chief characteristics being precocity and prolificacy ( fig. 4 , c). Sexual maturity is achieved seven months after birth, with ewes lambing at age 13 to 15 months. Our findings support the statement by G EORGIOU ( 19 6 1 ) that with good management « two lambings a year are easily obtained with parallel multiparity». His data (for 94 ewes lambing) shows a lambing percentage of 1 8 4 , with singles accounting for 37 per cent, twins 45 per cent, triplets 15 per cent, and quadruplets 3 per cent. During our survey, the owner of one flock of Skopelos (at Nea Ankhialos) reported 70 lambs per lambing from the 3 o ewes he managed. The fleece was noticeably finer than that of other Greek breeds, and the wool more crimped. The mean fibre diameter of 2 6 fL m quoted by MASON accords with the quality number of 5 0S to 56s he quotes. Three groups of five fleece samples were obtained : from the island, and 1BTea Ankhialos and Yannitsa on the mainland, and skin samples, too, were taken from the latter. The fleece was in general comparable with that of a British « Down o type, but with a tendency towards a coarser staple tip, which was also greasy, and waviness. The best defined of these had two waves per cm. The sheep on the island had the shortest and finest fleeces, the staple length ranging from 6 0 to 70 mm with a mean of 6 3 mm. The staple length of the Nea Ankhialos samples ranged from 70 mm to 140 mm in one uncharacteristically hairy individual with a mean of 105 mm. That of the Yannitsa animals ranged from 8 0 to I IO mm with a mean of 93 mm. Except for the hairy animals, and one with a few hairy fibres, the overall diameter range was from 1 6 >m to 6 0 pm and most animals had a symmetrical diameter distribution, although two had an unusual skewed-to-medium distribution, but nearly all were true medium fleece types on the classification of Ryder (ig6g). The skewness was so great in one of the Yannitsa samples as to make the mode 50 >m which increased the mean mode to 3 g.8 fL m compared with 33 . 3 and 33 . 4 >m in the other two groups (ranges 3 0 to 40 !m and 30 to 37 i t m). The overall mean fibre diameter of the Island group was 34 .8 fL m (range 32 .6 to 3Z .5 !tm) while that of the Nea Ankhialos group was 40 . 7 !m, which was raised by the mean of 5 8. 7 >m in the hairy animal already discussed. If this animal is excluded the mean is reduced to 3 6. 2 !.m (range 33 .6 to 3 g.5 !tm). The mean fibre diameter of the Yannitsa group was 37 .8 >m (range 35 . 2 to 42 . 1 !.m). Only one of the Yannitsa sheep had any medullated fibres, the proportion being 2 per cent and the overall mean was 0 . 4 per cent. Four of the Nea Ankhialos animals (including the two hairy animals) had medullated fibres ranging up to 20 per cent of the total, the overall mean being g.8 per cent. Despite the relatively fineness of the island group, three of the animals had medullated fibres, the proportion ranging up to 2 8 per cent, making the overall mean 7 .6 per cent. Follicle counts in the skin samples indicated a secondary /primary follicle ratio of 3 . 7 (range 3 . 2 to 4 . 4 ). There were no medullated fibres within the skin which is in keeping with the loss of medullation in winter. A mean of 4 per cent of the primaries and 0 . 5 per cent of the secondaries were inactive. Scottish Cheviot than the Down type, although one had wave like that in the Border Leicester breed, and another was more hairy, although lacking in kemp. The staple length ranged from 100 to 1 8 0 mm with a mean of 132 mm. The mean diameter was 42 . 7 ¡ L m (range 3 6.6 to 4 6. 7 !tm). The mean mode was 3 g.6 ¡ L m (range 34 to 44 !.m). The overall diameter range was from 20 to no J im, which was found in one hairy individual. The diameter distribution was either symmetrical in the three sheep with medium . fleece types, or skew-fine /continuous in the hairy sheep and the one with a hairy medium fleece. Three individuals had medullated fibres, the proportion ranging up to 19 per cent in the hairy animal, with an overall mean of 5. 2 per cent. The skin samples indicated a secondary-primary follicle ratio of 3 . 5 , with 3 per cent of the primary follicles and 2 per cent of the secondaries inactive (in December). F. Turkey I . In Turkey the Sakiz breed is localized to a narrow zone towards the end of the Erythraen peninsula opposite Chios, and its numbers have declined from about 5 00 o in recent years to goo. In one small flock visited, the litter size at lambing was 2 .8 and at weaning, 2 . 3 . The main product is milk, the daily yield being quoted as 3 kg. The Sakiz samples were much coarser than the Chios wool; four of the six samples were very kempy, one had hair instead of kemp, and only one had a denser more woolly fleece. The staple lengths ranged from 55 to 105 mm with a mean of 79 mm. The overall fibre diameter range was from 14 to 1 8 0 ¡ L m, the modes ranged from 22 to 2 6 ¡ L m with a mean mode of 24 . 3 ¡ L m. The mean diameters ranged from 31 .8 Ji m to 4 8. 5 5 >m giving an overall mean for the breed of q.o.g !.m. Each animal had a skew-fine /continuous fibre diameter distribution, and the fleece was of true hairy type. Every animal had medullated fibres the incidence ranging from 5 per cent to 23 per cent, giving a mean of 1 6. 3 per cent. One animal had 10 per cent of pigmented fibres, giving an overall mean of 1 . 7 per cent pigmented fibres for the group of sheep. 2 . The local Odemis breed, not listed by MASON ( 19 6 7 ) but described by S 6NMEZ ( 19 66), was seen at Ege University farm and near Odemis in the Kiiçiik Menderes valley. It is a mostly polled, fat-tailed breed with lop ears; one flock had brown, and the other, black faces ( fig. 4 , e). The tail is short, broad and twisted. There are no clear indications of the origin of this breed, although a cross between the Dagliç and a fine wooled breed is a possibility. The fleece appeared variable, some animals had a double coat reminiscent of that of « hair » sheep with a coarse, brittle, outer coat, and fine underwool. Others apparently had a generalized medium type of fleece comparable with the Chalkidiki of Greece. The five animals sampled at Ege had staple lengths ranging from 30 to 50 mm with a mean of 37 mm. The two at Odemis had obviously not yet been shorn and had fleeces g5 and 145 mm in length. The overall diameter range was from 12 ¡ L m to r 3 6 !.m, the modes from 20 to 30 ¡ L m with a mean of 2 2.8 !.m and the means from 32 .6 ¡ L m to 42 . 2 ¡ L m with an overall mean of 3 6.8 fi m. One animal had a skew-fine diameter distribution and was of hairy medium type; the remainder had a continuous distribution, and so were true hairy fleeces. Every animal had medullated fibres, the incidence ranging from 2 per cent to 2 6 per cent with a mean of 17 per cent. Three animals had pigmented fibres, the maximum proportion being 20 per cent and the overall mean 4 per cent. Skin samples were obtained from the five animals at Ege, and in these a mean of 6 2 per cent of the primaries, and 23 per cent of the secondaries were inactive. Two animals had some primary fibres with a non-latticed medulla, the overall mean being 3 . 4 per cent. The S /P follicle ratio ranged from 3 .6 to 5 . 2 , giving a mean of 4 . 5 /i. This is a relatively high figure for a hairy breed, and is the same as that of the Sakiz. 3 . The Im y oz breed takes its name from the Turkish island z 5 km west of the Gallipoli Peninsula. The breed is also found on the mainland of Turkey in the area between the towns of Canakkale and Ezine. The total number of Imroz is about 30 , 000 (Sandikcioglu, personal communication). Production is for milk (by estimated value 6 0 per cent), lamb ( 35 per cent), and wool (5 per cent). Imroz are reported to be hardy, resistant to cold weather, and relatively free of disease. The breed was seen at Kumkale Animal Breeding Station, where the Turkish Government maintains 979 pure-bred Imroz. The fleece is white and covers the top of the head and the trunk to the belly, which is bare ( fig. 4 , f). Commonly blackor reddish-brownpatches occur around the eyes, the nose, and the tips of the ears. The head is narrow; the bridge of the nose straight in profile. Legs are white and bare of wool. The tail is of medium width, reaching to just below the hocks. About two-thirds of the ewes seen at Kumkale were polled; the remainder had small scurs. Rams develop horns which spread widely in open spiral (very much like those of So!ravissana rams seen in central Italy). Wither heights were recorded at the station for six ewes and one ram. The values for the ewes ranged from 6 0 cm to 6 7 cm, with 6 2 .8 cm the mean; the ram was 71 cm. Live weight is reported to be from 35 to q.o kg (Ozcarr, 1972, p. 19). Lambing occurs once a year, and at the rate of 129 per cent at Kumkale Station; elsewhere (according to OzcA!r) from m2 per cent to 120 per cent. Annual milk production per ewe varies widely&mdash;from 50 kg to 15 o kg. The breed is shorn twice a year; annual wool production per head is about 1 .5 kg to 2 kg. The fleece, which hangs in curled locks 25 cm. to 30 cm. long, is not uniform in quality. « Although the average fibre diameter is 35 -40 !.m, coarse wiry examples 3 6s to 40 s in quality are found, and the value of the wool is correspondingly low » (OzcnN, 1972 , p. 1 8). Twelve Imroz fleece samples ranged in length from 6 0 to 1 6 0 mm with a mean of 123 . 3 mm. Most samples appeared hairy, with a hairy tip, and varying proportions of kemp and underwool, being typical of carpet wool type; four were black. The overall fibre diameter range was from 12 to 194 fL m, and the modes ranged from 20 to 34 pm with a mean of 25 . 1 fL m. The mean diameters ranged from 31 . 0 to 4 6.8 !m with an average of 3 6. 5 !.m. Most of the diameter distributions were skew-fine to continuous, and so typical of a hairy type, but two with hairs no greater than 70 pm in diameter had skewfine distributions, and so were hairy medium wools. Every sample had some medullated fibres which ranged in incidence from 3 to 24 per cent with a mean of 12 per cent. Only two of the six skin samples had any medullated fibres, possibly because of the season of sampling, the overall mean being 2 per cent. An overall mean of 31 per cent of the primary follicles, and 8 per cent of the secondaries were inactive. The S /P follicle ratio ranged from 4 . 1 to 5.8 with a mean of 4.5 !i, which is a relatively high value for a hairyfleeced type. Conclusions Breeds are usually thought to decline because they have become uneconomic, and this is true of such breeds as the Chalkidiki and even the Spanish Merino. The present survey has shown that other factors, including demographic change and the impact of new socio-economic mobility in rural communities, contribute markedly to the decline and extinction of breeds. And, if a husbandy system is becoming disused, the breed associated with it, e.g., Dub y ovnik, Skopelos, will decline. But clearly, decisions as to whether or not a breed should be preserved must be based on the need to maintain genetic diversity, and not on the vagaries of the economic value of the breed. The question of declining breeds with the consequent loss of genetic resources is complicated by the lack of basic information, such as breed numbers and location, genetic characteristics, management, and the nature of the natural and cultural milieu in which the breed is found. For this reason, the problem is best approached through interdisciplinary cooperative effort, including field investigation by a team of research workers from the biological, physical, and social sciences. Within the biological sphere, specialist attention to such aspects as genes with visible effects, blood type, and milk protein is necessary, in addition to fleece type which has been emphasized in the present paper. Re ç u pour publication en mai 1 977 .

v3-fos

2016-05-12T22:15:10.714Z

1977-06-01T00:00:00.000Z

2229697

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Influence of milk source on transplantability of histocompatible mammary tumours in mice. It is confirmed that C3H mammary tumours are much more easily transplantable in histocompatible recipients when these have been reared on C3H milk, than when they have been reared on milk from the inbred Swiss/B strain. By contrast, A.CA mammary tumours transplanted in histocompatible hosts reared on A.CA or Swiss/B milk, grow almost equally well in both sorts of recipient. Thus, rearing on Swiss/B milk has different effects on the transplantability of mammary tumours of C3H and A.CA. On the other hand, recipients which were reared on C3H or A.CA milks accept grafts of C3H mammary tumours about equally, suggesting that milks from A.CA and C3H have the same effect on the transplantability of C3H mammary tumours. The different action of Swiss/B milk on tumours of C3H and A.CA seems best attributed to differences between C3H and A.CA tumours or between mouse strain genotypes. By contrast, the transplantability of C3H mammary tumours is significantly changed when the recipients were reared on milk from the RIII strain instead of C3H. These facts suggest that the milk from RIII has an action which differs from that of both C3H and A.CA in this respect. The data are discussed on the basis of a differential tollerance-inducing action of mammary tumour viruses (MTVs) which infect C3H, A.CA and RIII, and have an important role in tumour induction. important role in tumour induction. WE have previously shown that several 03H spontaneous mammary tumours, when transplanted in histocompatible hosts, are strongly influenced by the milk on which the graft recipients had been fed in the neonatal period. In particular, if they had been fed with milk from a Swiss/B mother, a strong inhibition of growth was often observed, relative to recipients fed with C3H milk (Oth et al., 1968;0th, Robert and Dumont, 1972). This effect of milk on transplantability, which has been observed for a long time (Barrett and Morgan, 1949), has been explained as the neonatal induction of tolerance by the mammary tumour virus (MTV) antigens contained both in the milk of some murine strains and in the MTV-induced tumours themselves (Oth et al., 1968;Morton, 1969). Thus, mice exposed to MTV-containing milk develop less resistance to the MTV antigens, and are therefore better acceptors of a tumour graft than are unexposed mice. In a study of several C3H mammary tumours, we previously found some variability between tumours in this effect of milk growth inhibition (Oth et al., 1972). The aims of the present studies were: (a) To estimate, in a more significant number of cases, the proportion of C3H mammary tumours sensitive to this effect; (b) To test if mammary tumours from another strain, known to be also infected with the MTV, are also sensitive to this effect: mammary tumours of the A.CA strain, congenic with the A/Sn strain, were used; (c) To test the action of the milks from other MTV-infected murine strains on the transplantability of C3H mammary tumours: milks of C3H, A.CA and R III strains were compared. MATERIAL (Guggiari and Rudali, 1977). The Swiss/B inbred strain was created 20 years ago in the Faculty of Medicine of Strasbourg, France. It has been kept inbred in our laboratory for 16 years, by strict brother x sister matings. No mammary tumours are observed in them, and suckling their milk contents strong resistance to the transplantation of MTV-induced tumours (Oth et al., 1968;0th et al., 1972). They are therefore considered as free of MTV, or of MTV variant with an activity comparable with those of strains such as C3H, A or RIII. The different Fl hybrids have been created in our laboratory, In the following notations, the maternal strain always comes first in the Fl hybrid symbols. Tumour grafts.-Tumours are transplanted as solid fragments of 1 mm3, s.c. on the dorsum of the recipients. Since the effect of milk on transplantability may be lost after several passages (Oth et al., 1972) only the early passages were used. Some tumours. both from C3H and A.CA. have been examined histologically and classified as mammary carcinomas. Animals of both sexes were used, since sex apparently does not influence the sensitivity to the action studied (Oth et al., 1968(Oth et al., , 1972. Their ages at the moment of graft varied from 3 to 6 months. For a given experiment, ages and sexes were either the same or equally partitioned among the groups. Plaque-forming cell assay.-The plaqueforming cell (PFC) assay was performed according to the method of Jerne and Nordin (1963). For the indirect PFC, goat antimouse-gammaglobulin serum (Cappel, Downing Town, Pennsylvania, lot 70694) was added to complement at a final dilution of 1:200. Skin grafting.-A piece of skin was removed aseptically and grafted on to the recipient's neck. The graft bed was slightly smaller than the grafted piece, which was fitted by inserting the edges beneath the skin of the host and pulverization of surgical plastic film. Syngeneic grafts are permanently accepted. The grafted mice were housed one per cage. Changes in the colour of the graft and mechanical rejections were observed daily. RESULTS (1) Comparison of the influence of milk on transplantability of mammary tumours from C3H and A.CA mice Seventeen C3H tumours were transplanted by the standard graft technique to (Swiss/B x C3H) Fl hybrids (raised on Swiss/B milk), and to C3H mice (raised on C3H milk). Syngeneic C3H animals were used instead of (C3H x Swiss/B) Fl hybrids for convenience: it had been previously checked that the heterozygosity status of the hybrids does not, in the present case, interfere with studies designated for studying only the influence of the milk. Actually (C3H x Swiss/B) Fl hybrids raised on C3H milk are as good acceptors of C3H mammary tumours as are the C3H themselves, in contrast to (SwisslB x C3H) Fl hybrids raised on Swiss/B milk (Table I). In other genetic combinations (C3H x A.CA) Fl and (C3H x RIII) Fl hybrids raised on C3H milk accept the tumour graft at a high rate, near to 100%, like the C3H (data in Table IV). Thus, in the case of these tumours, no " hybrid effect" comparable to that found with other tumours is observed, and the data obatained by Morton (1969) agree. When the tumour incidence (number of takes divided by number of grafted mice expressed as a percentage) was reduced by 20% or more in the mice raised on Swiss/B milk as compared with those raised on the milk of the original strain, the tumour was classified as sensitive to inhibition. Table II, it appears that 12/17 C3H tumours are inhibited in the hosts which had been nursed by a Swiss/B mother. Eight A.CA tumours were transplanted in (Swiss/B x A.CA) Fl hybrids (Swiss/B milk) and in A.CA controls, and it may be seen that none of them are inhibited in the Swiss/B-nursed recipients. The different degree of inhibition between the C3H tumours (12/17) and the A.CA tumours (0/8) is highly significant (P < 0-01 using the x2 test, after Yates' correction for continuity). This difference could be attributed to the murine strains, to the tumours, or to the milks. However, it seems that A.CA milk is nevertheless able to influence the growth of A.CA mammary tumours. Tumour TMA-10, spontaneous in an A.CA female, has been grafted to A.CA-nursed A.CA and (A.CA x Swiss/B) Fl, and to Swiss/Bnursed (Swiss/B x A.CA) Fl hybrids. shows that all animals accept the grafts, but that mice of the last group die later than the other two, suggesting an action of the A.CA milk on A.CA tumours. In the next section, we shall see that the A.CA milk is fully capable of influencing the transplantability of C3H tumours. This suggests that the difference in inhibition between C3H and A.CA tumours is not due to a difference between the milks of these strains. (2) Comparison of the influences of A.CA and C3H milks on C3H tumour transplantability Neonatal exposure to C3H milk abolishes the natural resistance of (Swiss/B x C3H) Fl hybrids to C3H tumours (Oth et al., 1968). To test whether A.CA milk has a similar effect, we transplanted tumour TM8 into (Swiss/B x C3H) Fl hybrids, some of them having been fosternursed by an A.CA female. The results in Fig. 2 show that A.CA milk abolishes the resistance of (Swiss/B x C3H) Fl, just as C3H milk did. If milk from A.CA and C3H have similar capacities of enhancing C3H tumour grafts, it is expected that reciprocal (C3H x A.CA) Fl and (A.CA x C3H) Fl hybrids will be equally good acceptors of such grafts. Seven tumours, sensitive to growth inhibition in (Swiss/B x C3H) Fl, were tested. Table IV shows that the tumour incidences are equally high in the reciprocal hybrids, also suggesting that A.CA and C3H milks have very comparable activities. A slight difference may be observed, however, in some cases: TM8, TM14 and TM26growfasterinthe(C3H x A.CA)F1 than in the (A.CA x C3H) Fl hybrids. Also, Sanford and Soo (1971) reported that an A mammary tumour grew better in (A x C3H) Fl than in (03H x A) Fl. But these observed small differences of behaviour between A and C3H milks do not affect our criterion: tumour incidence. (3) Comparison of the influences of RIII and C3H milks on C3H tumour transplantability Four C3H tumours, tested for their sensitivity to the inhibition effect in (Swiss/B x C3H) Fl hybrids, were grafted in (C3H x RIII) Fl and (RIII x C3H) F1 hybrids. Fig. 3 shows that 3 of the tumours which are well inhibited in (Swiss/B x C3H) Fl animals, are also inhibited, to a lesser extent, in (RIII x C3H) Fl hybrids. On the contrary, the (C3H x RIII) Fl hybrids were approximately as susceptible as C3H hosts. Tumour TM20 was found to grow equally well in (C3H x RIII) Fl and (RIII x C3H) Fl hosts (not shown) but this tumour was not inhibited in (Swiss/B x C3H) Fl, denoting the absence of sensitivity to inhibition in this case (Table II). The overall differences between (RIII x C3H) Fl and (C3H x RIII) Fl, in the case of the 3 inhibition-sensitive tumours, is significant for tumour incidence (50/55 vs 48/64, P < 0-05), and for the mean survival time of tumour-bearing mice (P < 0 05 in each case). It is therefore concluded, that for inhibition-sensitive tumours, RIII milk does not enhance tumour growth to the same extent as C3H milk. (4) Comparison of some immunodepressive effects of C3H and RIII milks That hybrids which have been fed with RIII milk present some spontaneous been observed (Blair et al., 1971), to variable extents (Griswold, Heppner and Calabresi, 1973), in several mammarytumour-susceptible mouse strains. We tested the possibility of a difference in the non-specific immunodepressive action of RIII and C3H milks. With that aim, we used (Swiss/B x C3H) Fl litter mates nursed by Swiss/B, RIII and C3H mothers. Plaque-forming cells (PFC), both direct and indirect, and rejection times of allogeneic skin grafts have been used as criteria for the immune status. The PFC results (Table V) show that RIII milk had no immunodepressive effect in comparison with Swiss/B milk, while C3H milk had some depressive effect, particularly with the indirect PFC. On the whole, RIII milk seems to confer a higher immune reactivity than C3H milk on these animals, in the PFC test. If this reflects the actual immune capacity, this could represent an alternative, non-specific explanation for the lowest tumour incidence observed with animals nursed with RIII milk. Using the other test, namely the rejection of an allogeneic skin graft, we did not observe any immunodepression due either to RIII or C3H milk. On the contrary, in both cases a slight potentiation of the capacity to reject grafts is suggested (Table VI). DISCUSSION Although we have not, in these experiments, verified the presence of MTV in the C3H, A.CA and RIII strains, these are known to be infected by this or have not, been nursed with milk from an MTVpositive mother. That is, feeding the recipients with either C3H or A.CA milk considerably abolishes the spontaneous resistance which is observed in other recipients. The previously proposed interpretation of this fact (Morton, 1969;0th et al., 1968), based on the induction of tolerance to MTV antigens by the milk, seems the most probable. In this connection, the actions of C3H and A.CA milks on transplantation of C3H tumours are similar. One can interpret this as a closely related antigenicity between the MTVs contained in C3H and A.CA milks, inducing tolerance to MTV-related antigens in C3H tumours. This finding is not too surprising, because C3H and A.CA (congenic with the A strain) are known to be distantly related (Vlahakis, 1973), and the sharing of cross-reactive antigens by their MTVs has been shown with other tests (Blair, Weiss and Smith, 1970 of C3H and A.CA tumours must have another basis. It must first be noticed that spontaneous mammary tumours are much less common in A.CA than in C3H. Thus, considering that antigenically comparable MTVs are responsible of tumour induction, there must exist other differences between both strains. Some regulatory mechanisms could be stronger in A.CA, permitting less tumours to appear, and also resulting in the appearance of less antigenic tumours. Those less antigenic tumours would therefore be much less sensitive to transplantation inhibition in MTV-negative hosts, as observed with C3H tumours, and consequently suppression of inhibition not demonstrable in MTV-positive hosts. Such differences between C3H and A.CA strains could result from differential infection capacity (Vlahakis, 1973) or immunosuppressive effects of their respective MTVs (Stutman, personal communication) or from differences in sensitivity to the induction of immunological tolerance in general, as suggested by some results (Yunis et al., 1974). A strong effect of hormonal status might also explain the different rates of appearance of mammary tumours in C3H and A.CA mice. On the other hand, RIII and C3H milks have clearly different actions on the transplantability of C3H tumours. As the former is of European origin and the latter American, it may be expected that they are more different from each other than C3H-MTV and A.CA-MTV. Some antigenic specificities common to RIJI-. MTV and C3H-MTV have been observed, however, using neutralizing antibodies raised in the rabbit (Blair et al., 1970). Nevertheless, a difference in toleranceinducing antigenic parts remains possible and would explain the results presented. On the other hand, the differential actions observed between RIII-MTV and C3H-MTV might also explain on a non-specific basis, the difference we observed. The immunosuppressive and/or immunostimulating action (Griswold et at., 1973) of MTV seems to be a complex problem, and the effect could depend on both the mouse strain (Griswold et al., 1973) and the MTV strain. Certainly, additional experiments are necessary to clarify this point.

v3-fos

2016-05-04T20:20:58.661Z

1977-10-15T00:00:00.000Z

8479054

s2

Cytogenetic analysis of cattle chromosomes; current utilization and speculation of future applications a correct diagnosis of the phenotypic condition concerned. On the contrary chromosomal polymorphisms, due to their small phenotypic effects, mean great risks for distribution of chromosomal aberrations in the population. Too little is still known about aberrations such as chromosome breaks and small deletions but most often they appear to be parts of the phenotypic effects. Different trends of developing cattle cytogenetics are considered and future applications of cytogenetics to chromosome mapping, genetic engineering and embryo transfer are briefly discussed. The development and application of banding techniques will, no doubt, reveal in the near future a host of hitherto undescribed chromosome markers. It is stressed that new ideas must be introduced to get cytogenetics further established as an important research branch of practical cattle breeding. It is concluded that chromosomal abnormalities with grave effects on conformation and fertility are rapidly eliminated by artificial selection and mean no great economic losses for agriculture. Nevertheless the cytogenetical investigations of those anomalies are of importance for a correct diagnosis of the phenotypic condition concerned. On the contrary chromosomal polymorphisms, due to their small phenotypic effects, mean great risks for distribution of chromosomal aberrations in the population. Too little is still known about aberrations such as chromosome breaks and small deletions but most often they appear to be parts of the phenotypic effects. Different trends of developing cattle cytogenetics are considered and future applications of cytogenetics to chromosome mapping, genetic engineering and embryo transfer are briefly discussed. The development and application of banding techniques will, no doubt, reveal in the near future a host of hitherto undescribed chromosome markers. It is stressed that new ideas must be introduced to get cytogenetics further established as an important research branch of practical cattle breeding. When we start the present section containing many, what I hope, very interesting communications in cattle cytogenetics it is quite natural to ask: What is the current use of cattle cytogenetics and What will future purposes of the discipline be? Particularly the last question I have asked myself several times. Of course I am not the right person to answer, but I can at least briefly draw the principal lines of cattle cytogenetics, give some personal thoughts on the future and in that way hope to provoke you to discussions. I suppose our following communications of to-day and to-morrow will deal with the details of each special field of cattle cytogenetics. The first observations of cattle chromosomes were carried out 50 years ago (K R A LL I N GE R , i 9 27), but chromosome pictures as we are used to see them occurred much later. I would like to remind you that it was just twenty years ago that the first cattle chromosomes from tissue-cultured cells were obtained (M ELANDER , ig5!). We had to wait until the next decennium, however, to know more about chromosomes of cattle, but, during the late sixties and early seventies a number of phenotypic malformations were associated with chromosome aberrations (for a review see R IFCK , ig!q.). At the turn of ig6g-i 97 0 the first new staining techniques were introduced which for cattle cytogenetics culminated in 197 6 with the description in Reading, England, of an international standard arrangement of the chromosomes into a karyotype (Proceedings of the First International Conference for the Standardisation of Banded Karyotypes of Domestic Animals, to be published) . If we consider the work done during the last 2 o years we might divide the chromosome aberrations into three groups according to phenotypic manifestations which are given within parenthesis. The first group, here labelled chromosomal anomalies, include spontaneous or familiarly occurring cases of cattle more or less malformed and /or with greatly reduced fertility. Common for this group of animals is that they mean little risks for extensive distribution of the anomaly concerned. Summarizing the information hitherto available we must concede that very few chromosomal anomalies (with the exception of XX /XY chimerism) have been associated with considerable phenotypic abnormalities in cattle, and for each type of anomaly only a few cases have been described. Then the question arises: Why are the incidences so low compared to corresponding incidences in man? Even though we don't know if mutation rates are similar in the two species one reasonable explanation would be that the phenotypic abnormalities in cattle seldom come to investigation. Anyway I think the conclusion is correct if I say that chrosomome anomalies giving considerable phenotypic effects don't represent important economical losses for agriculture because such anomalies become eliminatcd very quickly from the cattle population due to their pathological effects. Nevertheless and it should be stressed very hard the knowledge and the study of the cytogenetical background is often of utmost importance for the outcome of a correct diagnosis and is also of great importance for the understanding of similar conditions in man. The second group of chromosome deviations, chromosomal polymorphisms, presents a quite different problem. Evidently those types of aberrations constituting polymorphic systems give very small phenotypic manifestations such as a small reduction of fertility. The last fact concerns at least the i /2 Q translocation (GusTavssoa, ig6g; R EFSDAL , 197 6) and I am bold enough to say that we had better presume the same also for other centric fusion translocations occurring in A. I. populations. The small deviations in phenotypic effects mean that there are great risks for extensive distribution of such polymorphic systems and in cattle populations making use of A. I. it is therefore absolutely necessary to keep the cytogenetic situation under control with continuous investigations of breeding animals. Although direct cytogenetical proofs of reduced fertility are still lacking, I think the fertility figures for the i /S Q translocation are so convincing that we should discuss if it is not highly urgent to introduce recommendations about cyto-genetic investigation before transfer of breeding materials between countries. There is also a third group of chromosome aberrations, here labelled secondary chromosome anomalies. We know very little of those types of anomalies but the chromosome anomaly appears to be a part of the phenotypic effects. Due to the need of diagnosis and the control of cattle intended for breeding work to-day's cattle cytogenetics has been founded on the first two groups of chromosome aberrations. Those reasons have been and still are good motives for the establishment of cytogenetic laboratories in different parts of the world. What will be the future of cattle cytogenetics ? Of course we have to expect that further work involving application of new techniques will increase our knowledge of different chromosome aberrations and their phenotypical manifestations. Since there are still large cattle populations hitherto quite unknown from the chromosomal point of view it may be possible to find new polymorphic systems which are geographically widespread. With the increasing utilization of A.L, routine cytogenetic investigations will get increased importance. Therefore I think the initiative, at present meeting, of Paul Popescu to continue the work, started by David Pollock, in Reading 197 6, compiling present knowledge about number of cattle analysed in each breed, breed incidences and types of aberrations is worth encouragement. As Mike Harvey expressed some years ago (H ARV E Y , zg7.!) it is urgent to form an international central reference library where the results of all chromosomal analyses carried out could be deposited and published. However, when we are talking of future trends in cattle cytogenetics we must not only look to the near future but be as far-seeing as possible. In a strained financial world it is necessary, as you know, to have a long term plan for your projects. At least in my country we are to-day confronted with the problem that developing cytogenetics must include projects in which the outcome means predictable return of money. Although our works have given what we think important results for diagnosing different abnormal conditions and increasing fertility of cattle in Sweden, cytogenetics is still considered by many people to be, a very exclusive science of genetics, of small economical importance, and with little possibilities of development. I suppose several collegues here also have met the same experience and I am quite sure that everyone who wants to increase his resources will be confronted with the arguments I just mentioned. It is of course not easy to predict future development of cattle cytogenetics, but I think it is evident that we must present new ideas on how to use cytogenetics in future animal breeding. It is to-day not easy to predict future use of chromosome maps, genetic engineering techniques and so on, but personally I am quite sure that manipulations of the hereditary material will gain increased interest in the near future because of the rapid development of egg transplantation techniques. I would also like to point out the practical utilization of so-called chromosome markers which I think will be very important in research as well as in routine work. The sex chromosomes have been important chromosome markers since they indicate the sex of the animal. The type of work carried out by Doug Hare and coworkers (HARE et al.,197 6) by sexing embryos before cryopreservation and lor transplantation will be one very important task for future cytogenetics. The new banding techniques have given us very detailed information of individual chromosomes and it is my and my co-worl!ers impression that there is a very extensive variability in chromosome banding patterns within several domestic animals. Some years ago Paul P OPESCU ( 1974 ) demonstrated centromere polymorphism in the cattle chromosomes by using C-banding, and we have observed the same phenomenon using the T technique. Extensive chromosome variability, similar to that observed between different inbred strains of the laboratory mouse (l!It!-LER et al., ig 7 6), thus make possible identification of the single individuals. Such a system can be used for several in vivo as well as in vitro experiments. Finishing my brief introduction it is my hope that the present meeting will give some new ideas and also provoke the participants to take up discussions of future developments.

v3-fos

2018-04-03T02:15:27.875Z

1977-01-01T00:00:00.000Z

32874414

s2

Niacin and pantothenic acid excretions of humans fed a low-methionine, plant-based diet. The addition of a vitamin to a diet of humans has been shown to increase the excretion of that vitamin. The effects of an increase of one vitamin on another have not been investigated. The objective of the current project was to compare the effects of two supplementation patterns on the niacin and pantothenic acid excretion values of humans consuming a peanut butter-based diet. Two groups each received one of two supplementation regimens. One group received niacin, a multi-vitamin, or no supplement. One group received methionine alone, pantothenic acid alone or methionine plus pantothenic acid. The addition of either vitamin resulted in increased excretion of that vitamin. Urinary niacin excretion of the group that received pantothenic acid and/or methionine was greater than that observed with a multi-vitamin or no supplement. Urinary pantothenic acid excretion was suppressed when niacin was a supplement. Urinary pantothenic acid excretion of the methionine supplement group was greater than the excretion of the groups which received either niacin or multi-vitamin supplements. These data suggest some possible dangers in indiscriminate supplementation of food products. Summary The addition of a vitamin to a diet of humans has been shown to increase the excretion of that vitamin. The effects of an increase of one vitamin on another have not been investigated. The objective of the current project was to compare the effects of two supplementation patterns on the niacin and pantothenic acid excretion values of humans consuming a peanut butter-based diet. Two groups each received one of two supplementation regimens. One group received niacin, a multi vitamin, or no supplement. One group received methionine alone, pantothenic acid alone or methionine plus pantothenic acid. The addition of either vitamin resulted in increased excretion of that vitamin. Urinary niacin excretion of the group that received pantothenic acid and/or methionine was greater than that observed with a multi-vitamin or no supplement. Urinary pantothenic acid excretion was suppressed when niacin was a supplement. Urinary pantothenic acid excretion of the methionine supplement group was greater than the excretion of the groups which received either niacin or multi-vitamin supplements. These data suggest some possible dangers in indiscriminate supplemen tation of food products. Pantothenic acid and niacin are both known to be essential to metabolic processes, as a part of NAD and NADP and acetyl Coenzyme A (1-10). While these two vitamins operate independently of one another, an indirect relationship between niacin and pantothenic acid can be observed biochemically (11). However, little information exists on the practical significance of a niacin-pantothenic acid interrelationship as it occurs against a background food. An earlier study from this laboratory6 observed a three-way interrelationship between niacin, pantothenic acid and nitrogen balance. Supplemental panto thenic acid resulted in a depressed niacin excretion and an improved nitrogen balance. The addition of either niacin or pantothenic acid depressed excretion of the other vitamin. Low-protein diets can be classified into either of two groups: those limited in the amino acid lysine and those limited in the amino acid methionine. Since the earlier study7 investigated pantothenic acid and niacin excretion against a lysine-limited background food (wheat), the current study investigated pantothenic acid and niacin excretion against a methionine-limited background food (peanut butter). The objective of the current study was to compare the effects of methi onine and/or pantothenic acid supplementation or niacin/multi-vitamin supple mentation to a peanut butter based diet on the excretion of niacin and pantothenic acid by normal human subjects. EXPERIMENTAL Two 18-day human bioassay studies were carried out simultaneously. Each study was composed of a two-day nitrogen depletion period, a four-day nitrogen adjustment period and three experimental periods of four days each. The experi mental plans for Studies 1 and 2 are given in Tables 1 and 2, respectively. In Study 1, methionine and pantothenic acid were the experimental variables. Subjects received plant-based diets supplemented with 0.5g methionine, 25.0mg pantothenic acid or 0.5g methionine plus 25.0mg pantothenic acid. Nitrogen intake during the depletion period was 0.7g nitrogen/subject/day, as shown in Table 1. During the adjustment and experimental periods, subjects received 4.7g of nitrogen/subject/day. In Study 2, niacin and a commercial multi-vitamin8 were the experimental variables, as shown in Table 2. Subjects received plant-based diets supplemented with 25mg niacin, 1 multi-vitamin or no supplement. Nitrogen intake during the depletion period was 0.7g of nitrogen/subject/day. During the adjustment and experimental periods, subjects received 4.7g of nitrogen/subject/day. Experimental periods were randomly arranged in order to minimize the influence of the order of presentation. The menu pattern was the same for Studies 1 and 2 and in Tables 1 and 2. Supplements were consumed at the midday meal. Subjects consumed a constant amount of coffee or tea each day throughout the study. Caloric intake was adjusted with soft drinks, hard candy, sucrose and jelly, honey or butteroil for subject weight maintenance. Sugar-free soft drinks and water were allowed ad libitum. Subject data is included in Table 3. All subjects were normal, healthy young women who were students at the University of Nebraska and continued normal daily living patterns except for the collection of urine and feces and the eating of meals at the diet laboratory. This project was reviewed and approved by the Committee on Human Investigation, University of Nebraska Medical Center. Subjects were required to collect all urine and feces. Urine was collected in 24-hr lots and preserved under toluene. The 24-hr urine collections for the final three days of each 4-day experimental period were composited and frozen until analysis. Niacin and pantothenic acid were measured by microbiological assay (12) using Lactobacillus plantarum ATCC 8014 as the test microorganism. Daily creatinine determinations were done on 24-hr urine samples to assure completeness of collections (13). Urinary and fecal nitrogen were determined by the boric acid modification of the Kjeldahl method (14) for another part of this project. Resulting data were subjected to statistical analysis, including analysis of variance and Student-Neumann-Keuls tests. These computations were done by the Statistical Laboratory of the Agricultural Experiment Station, University of Nebraska, Lincoln. AND DISCUSSION Individual urinary niacin excretions and urinary pantothenic acid excretions for Studies 1 and 2 are shown in Tables 4 and 5, respectively. A comparison of urinary niacin, pantothenic acid excretion and nitrogen balance is given in Table 6. Details on nitrogen balance data were given in an earlier paper. A statistically significant change in urinary niacin excretion was observed with the addition of a niacin supplement. No differences among the mean urinary niacin excretion values were observed when subjects received methionine and/or pantothenic acid. However, subjects receiving methionine and/or pantothenic acid had slightly higher mean urinary niacin excretions than those of subjects receiving no supplement or a multi-vitamin supplement. This could be a demon stration of an indirect relationship between Coenzyme A and NAD-NADP, which would ultimately involve pantothenic acid and niacin. Two possibilities exist when increased vitamin excretion occurs. The obvious is that vitamin needs have been met and the excess is being excreted. The other possibility is that the vitamin is "rejected" because of the inability of the body to use that substance. In view of the known indirect relationships between Coenzyme A and NAD NADP (1-3), it is possible to assume that the increased excretion observed with pantothenic acid and/or methionine supplementation is an observation of an indirect relationship between pantothenic acid and niacin. Moreover, it can be assumed that body needs are being met. Total mean urinary pantothenic acid losses, as shown in Table 5, show statistically significant differences between the means. Methionine and/or panto thenic acid supplementation significantly increased pantothenic acid excretion over that observed with a vitamin or a niacin supplement. Pantothenic acid excretion was significantly greater when a mufti-vitamin supplement was included, as com pared to periods in which niacin or no supplement was given. This may be par tially due to the effect of the pantothenic acid in the mufti-vitamin supplement, but it may also reflect a more favorable total nutritional environment. These data also support the position of the U. S. Food and Drug Administration that increases in vitamin levels above minimum requirements in mufti-vitamin supple ments have no beneficial effect. A factor (see footnote to Table 5) was used to correct differences in panto thenic acid excretion data due to previous supplementation. The need for use of a factor with the pantothenic acid excretion data gives evidence that pantothenic acid may be retained in appreciable amounts for periods of four days. Niacin excretion data did not show a carry over from one period to the next, and therefore were not corrected. NIACIN AND PANTOTHENIC ACID EXCRETIONS OF HUMANS 487 Comparison of the current study and the results of Unver10 reveal that while niacin excretion was seemingly suppressed by pantothenic acid supplementation in Unver's study, no such effect was observed in the current work. However, the conditions under which niacin excretion was suppressed in Unver's experiment were not the same as in the current study. In Unver's11 experiment, which was carried out with human subjects, a wheat -based diet was used. Comparison between a lysine-limited diet in Unver's study and methionine-limited diet in the current work creates a different nutritional environment. Unver's study was conducted on three levels of protein intake, while the current study used one level of protein intake. Observations from both studies on similar protein intake levels (4.7 and 4.5g of nitrogen per day) were nearly alike. However, in Unver's experiment niacin excretion was suppressed by pantothenic acid intake at the higher protein level (6.5g nitrogen/day). There was no comparable data in the data from the current study. Larger pantothenic acid excretion in the current study as a result of methi onine supplementation could be due to a positive relationship between methionine and pantothenic acid. Evidence exists suggesting that methionine improves the condition of pantothenic acid deficient animals (16,17), but there is no evidence on the effects of methionine on pantothenic acid excretion. Mean nitrogen balance data shown in Table 6 indicates that niacin, "full" vitamin, methionine and pantothenic acid all have a positive influence on nitrogen balance; however, methionine pantothenic acid supplementation combination resulted in a negative effect. In an earlier report of nitrogen balance data, KIES and FOX12 proposed that the more negative nitrogen balance that resulted from methionine plus pantothenic acid supplementation was due to methionine toxicity induced by pantothenic acid supplementation. This could be an explanation for the increased pantothenic acid excretion as a result of methionine supplementation, either alone or combi nation with pantothenic acid. Examination of pantothenic acid excretions of subjects receiving niacin seemingly shows the effects of an indirect niacin-pantothenic acid relationship. Examination of the niacin excretions of subjects receiving pantothenic acid does not show such a clear-cut relationship. In comparison to periods in which subjects received pantothenic acid as a supplement, pantothenic acid excretions of subjects receiving niacin were suppressed. Increases in pantothenic acid resulted in a greater need for niacin, thus a lower excretion. Pantothenic acid excretion as a result of multi-vitamin supplementation was significantly different from that observed with either niacin or no supplement. The expectation is that the difference between the excretions would not be so great. In this case, the effect of the addition of other vitamins may result in higher excre tions of pantothenic acid. It would seem that if the above is true, then niacin excretions of subjects receiving pantothenic acid would also be suppressed. However, this is not so clear from the data. One explanation for these differences may be the differences between the supplement groups themselves. The relationships between panto thenic acid and methionine and the multi-vitamin and niacin were not the same. Niacin was a part of the multi-vitamin, while pantothenic acid and methionine were linked by a common coenzyme, Coenzyme A. However, this still does not fully account for the differences observed.

v3-fos

2018-12-27T00:41:45.587Z

1977-01-01T00:00:00.000Z

87824120

s2

Field Evaluation of Insecticides for Control of Alfalfa Weevil Larvae The alfalfa weevil, Hypera postica (Gyllenhal), is a serious pest of alfalfa in Kansas. The large numbers of larvae generally found in alfalfa fields during early spring can completely destroy the first crop and may sometimes retard early growth on the second crop. A native of Europe, the weevil was first discovered in the United States in 1904 in Utah. For nearly 50 years, it remained confined to 12 western states. In 1952, the weevil was discovered in Maryland and from there it spread rapidly through the East, South, and Midwest. In Kansas, the weevil (western strain) was first discovered in Hamilton and Cheyenne Counties in 1960. By 1969, it had spread into 40 western Kansas counties. In eastern Kansas, the weevil (eastern strain) was detected in Cherokee County in 1967. It spread westward and northward, causing severe damage by 1972. Both larvae and adults are injurious, but larval feeding causes the greatest damage. The larvae feed on the buds, growing tips, and leaves of alfalfa. The leaves may become skeletonized and dry. A field of alfalfa damaged by heavy populations of alfalfa weevil will take on a grayish-white appearance, similar to CORE Metadata, citation and similar papers at core.ac.uk January 1987 Field Evaluation of Insecticides for Control of Alfalfa Weevil Larvae L.J. DePew, Research Entomologist Southwest Kansas Branch Station and Department of Entomology The alfalfa weevil, Hypera postica (Gyllenhal), is a serious pest of alfalfa in Kansas. The large numbers of larvae generally found in alfalfa fields during early spring can completely destroy the first crop and may sometimes retard early growth on the second crop. A native of Europe, the weevil was first discovered in the United States in 1904 in Utah. For nearly 50 years, it remained confined to 12 western states. In 1952, the weevil was discovered in Maryland and from there it spread rapidly through the East, South, and Midwest. In Kansas, the weevil (western strain) was first discovered in Hamilton and Cheyenne Counties in 1960. By 1969, it had spread into 40 western Kansas counties. In eastern Kansas, the weevil (eastern strain) was detected in Cherokee County in 1967. It spread westward and northward, causing severe damage by 1972. Both larvae and adults are injurious, but larval feeding causes the greatest damage. The larvae feed on the buds, growing tips, and leaves of alfalfa. The leaves may become skeletonized and dry. A field of alfalfa damaged by heavy populations of alfalfa weevil will take on a grayish-white appearance, similar to severe freeze damage. Larval feeding not only lowers yield, but also reduces quality and nutritional value of the hay. Alfalfa weevils pass through egg, larval, pupal, and adult stages in their development. Usually, there is only one generation a year. Full grown larvae are legless, 3/8" long, and green in color with a shiny black head. A conspicuous white stripe can be seen down the middle of the back. Young weevils are light brown in color, with a dark stripe extending half-way down the middle of the back. As weevils age, they darken in color and become uniformly dark brown to almost black. The adult weevils are approximately 3/16," long and have a short, distinct snout. Alfalfa growers often must rely on chemical control measures to suppress weevil outbreaks and prevent economic damage to the alfalfa crop. This report presents the results of field tests conducted in 1985 and 1986 to determine the comparative effectiveness of various insecticides for controlling alfalfa weevil larvae. Procedure Tests were conducted in fields of Riley alfalfa located at the Southwest Kansas Branch Experiment Station near Garden City. Insecticides were applied with a C0 2 -activated backpack sprayer calibrated to deliver 10.5 gallons of water per acre at 30 psi through 4 nozzles (6x) mounted on a 6-foot boom. Plots were sampled for larval densities by counting all living larvae collected from 20 stems randomly selected from each plot. Stems were shaken against the sides of a white bucket to dislodge the larvae from the plants. Data were subjected to analysis of variance for significance, and Duncan's multiple range test was applied to separate pair comparisons. This publication from the Kansas State University Agricultural Experiment Station and Cooperative Extension Service has been archived. Current information is available from http://www.ksre.ksu.edu. Plots were 18 x 50 feet, arranged in a randomized complete block design. A single application of 7 insecticides (Table 1) was made on April 20. Alfalfa was 14 inches tall at the time of application. Posttreatment assessments were taken at 3, 7, 14, and 21 days after application. Test Plots were 12 x 30 feet, arranged in a randomized complete block design. A single application of 7 insecticides (Table 1) was made on April 11. Alfalfa was 10 inches tall at the time of application. Posttreatment assessments were taken at 3. 7, 12, and 19 days after application. Test At 3 days posttreatment, all insecticides significantly reduced larval numbers below the untreated check (Table 1). Furadan gave the best control, but it was not significantly better than the other materials, except Lorsban. At 7 days posttreatment, all treated plots continued to have significantly fewer larvae than the untreated check. Furadan and Supracide gave the best control, but they did not differ significantly from the malathion treatment. At 21 days posttreatment, all treated plots had significantly fewer larvae than the untreated check. Furadan and Supracide again were the most effective insecticides, significantly better than the other materials tested. Leaf spotting was noted in plots treated with methyl parathion, Guthion, and Sevin XLR, Some insecticides may injure tender foliage when they are applied to plants that are wet from either heavy dew or rain. At 3 days posttreatment, all insecticides significantly reduced larval numbers below the untreated check (Table 1). Furadan gave the best control, but it was not significantly better than Lorsban, Supracide, Capture, or Bathroid. Pydrin and malathion were less effective. Two hard freezes (25°F) occurred between the 3-and 7-day evaluations and, as a result, larval densities declined in the untreated check plots. At 7 days posttreatment, all insecticides continued to give significant reductions in larval numbers compared to the untreated check. Furadan was the best treatment, significantly better than either Pydrin or malathion. All insecticidal treatments gave significant reductions in larval numbers at 14 days posttreatment. Except for Lorsban and malathion, all insecticides provided at least 96% control. At 21 days posttreatment, all treated plots had significantly fewer larvae than the untreated check. Except for malathion, no significant differences existed among any of the insecticides. Plots were not sampled after 21 days because many of the larvae in the untreated plots were beginning to spin pupation cocoons, and further sampling would not have been meaningful. Conclusions In these control trials, the best overall results were obtained with Furadan. Supracide provided excellent control through 21 days at the 0.5 lb. rate and appears to be comparable ratewise to Furadan. The synthetic pyrethroids (Capture, Bathroid, and Pydrin) look promising for controlling alfalfa weevil larvae and could be alternative insecticides in the future, if they are registered for use on alfalfa. Brand names are used to identify products. No endorsement is intended, nor is any criticism implied of similar products not mentioned. Contribution 87-216-S from the KAES This publication from the Kansas State University Agricultural Experiment Station and Cooperative Extension Service has been archived. Current information is available from http://www.ksre.ksu.edu. Iarvae (1985Iarvae ( -1986 Mean no. larvae /20 stems after ( This publication from the Kansas State University Agricultural Experiment Station and Cooperative Extension Service has been archived. Current information is available from http://www.ksre.ksu.edu.

v3-fos

2017-07-29T04:25:02.991Z

1977-01-15T00:00:00.000Z

52804261

s2

The productivity and efficiency of border leicester × cheviot, finn × blacface and east friesland × blackface prolific cross-bred ewes for lamb and carcass meat production in England Experiments have been carried out over 8 years to investigate systems of lamb production designed to make the fullest use of grassland, based on high levels of prolificacy of the ewes and high annual stocking rates per hectare. The results show that for suckled fat lamb production, both the Border Leicester /Cheviot and East Fviesland /Blackface crosses possess the necessary attributes of prolificacy and high milk yield, but the smaller size of the latter enables higher output per hectare to be obtained. The Finn IBlacklace cross, despite higher prolificacy and smaller size, are less efficient for suckled fat lamb production, but have merit for producing « store u lambs for subsequent winter fattening The results show that for suckled fat lamb production, both the Border Leicester /Cheviot and East Fviesland /Blackface crosses possess the necessary attributes of prolificacy and high milk yield, but the smaller size of the latter enables higher output per hectare to be obtained. The Finn IBlacklace cross, despite higher prolificacy and smaller size, are less efficient for suckled fat lamb production, but have merit for producing « store u lambs for subsequent winter fattening- During the years 1969-1975 the prolificity and adaptability of 42 sheep, 14 rams and their offspring of .Es!/ytMtaM breed were followed. The sheep and rams were from GDR imported where they were kept in small numbres of 1 -5 animals. In our country this animals were kept together with other sheep breeds in a herd of i8o-45 o animals. The individual tupping occured at an age of 1 6-19 months from i August to 31 October of each year. No synchronisation checks took place. The lambs were kept till to the age of 4 months with their mothers. The daily feed rations for the sheep: 2 , 470 kg dry matter, o,i 55 kg digestible protein and 1 , 10 kg Ste; for the lambs: 1 ,8 24 kg dry matter, o,i 5 8 kg digestible protein and i,o 2 q kg Ste. Imported sheep: Average recurrence oi oestrus till to the conception (I-V lambing) occured 2 , 39 times. 6 1 , 7 per cent of sheep became pregnant. The prolificity of the tupped sheep amounted to 12 8, 1 per cent. Exit of lambs 35 , 3 per cent. Sheep born in our country: Average occurence of oestrus till to the conception (I-V lambing) occured i,o6 times. 93 , 75 per cent of sheep became pregnant. The prolificity amounted to. 1 43 ,3 per cent and the exit of lambs 2 , 2 per cent. The adaptability of the imported Eastfviesian sheep to the herd breeding is fairly low, especially in the worse conditions in which case the efficiency and disease-resistance and the state of health are reduced. If the Eastfviesian sheep and lambs were born and kept in a herd their adaptation and reproduction abilities and the state of health were considerably better. The observation of the Easli p iesian sheep will continue. The use of Finnish Landvace &verbar;Border Leicestev rams to produce crossbred ewes, in place of the more traditional ram breeds used for crossbreeding, has resulted in lambing percentages being increased by about 14 per cent and rearing percentages increased by about 1 6 per cent over four lamb crops. Although individual lamb weights have been reduced by 6 per cent at ten weeks, the weight of lamb produced per ewe mated was 6 per cent greater from the Finnish Landvace lborder Leicestev crosses. These figures for increased lambing percentages and decreased individual lamb weights are about one half of the values found when purebred Finnish Landrace rams were used as alternative crossbreeding sires, and hence are not unexpected. The advantagein using Finnish Landrace &verbar;Border Leicestev rams instead of pure Finnish Landvace rams lies in the easier management of the crossbred ewes and their lambs, because of the more easily handled lambing percentages and relatively heavier lambs. The improvement in prolificacy from the Finnish Landvace lborder Leicester crosses occured in the I year old and 2 year old dams particularly but was less marked in the older ewes. Furthermore, the differences in ewe weights, prior to mating, between the Finnish Landvace &verbar;Border

v3-fos

2016-05-12T22:15:10.714Z

1977-01-15T00:00:00.000Z

9835540

s2

Reproduction and adaptation abilities of eastfriesian sheep in the ČSR Experiments have been carried out over 8 years to investigate systems of lamb production designed to make the fullest use of grassland, based on high levels of prolificacy of the ewes and high annual stocking rates per hectare. The results show that for suckled fat lamb production, both the Border Leicester /Cheviot and East Fviesland /Blackface crosses possess the necessary attributes of prolificacy and high milk yield, but the smaller size of the latter enables higher output per hectare to be obtained. The Finn IBlacklace cross, despite higher prolificacy and smaller size, are less efficient for suckled fat lamb production, but have merit for producing « store u lambs for subsequent winter fattening- The results show that for suckled fat lamb production, both the Border Leicester /Cheviot and East Fviesland /Blackface crosses possess the necessary attributes of prolificacy and high milk yield, but the smaller size of the latter enables higher output per hectare to be obtained. The Finn IBlacklace cross, despite higher prolificacy and smaller size, are less efficient for suckled fat lamb production, but have merit for producing « store u lambs for subsequent winter fattening- During the years 1969-1975 the prolificity and adaptability of 42 sheep, 14 rams and their offspring of .Es!/ytMtaM breed were followed. The sheep and rams were from GDR imported where they were kept in small numbres of 1 -5 animals. In our country this animals were kept together with other sheep breeds in a herd of i8o-45 o animals. The individual tupping occured at an age of 1 6-19 months from i August to 31 October of each year. No synchronisation checks took place. The lambs were kept till to the age of 4 months with their mothers. The daily feed rations for the sheep: 2 , 470 kg dry matter, o,i 55 kg digestible protein and 1 , 10 kg Ste; for the lambs: 1 ,8 24 kg dry matter, o,i 5 8 kg digestible protein and i,o 2 q kg Ste. Imported sheep: Average recurrence oi oestrus till to the conception (I-V lambing) occured 2 , 39 times. 6 1 , 7 per cent of sheep became pregnant. The prolificity of the tupped sheep amounted to 12 8, 1 per cent. Exit of lambs 35 , 3 per cent. Sheep born in our country: Average occurence of oestrus till to the conception (I-V lambing) occured i,o6 times. 93 , 75 per cent of sheep became pregnant. The prolificity amounted to. 1 43 ,3 per cent and the exit of lambs 2 , 2 per cent. The adaptability of the imported Eastfviesian sheep to the herd breeding is fairly low, especially in the worse conditions in which case the efficiency and disease-resistance and the state of health are reduced. If the Eastfviesian sheep and lambs were born and kept in a herd their adaptation and reproduction abilities and the state of health were considerably better. The observation of the Easli p iesian sheep will continue. The use of Finnish Landvace &verbar;Border Leicestev rams to produce crossbred ewes, in place of the more traditional ram breeds used for crossbreeding, has resulted in lambing percentages being increased by about 14 per cent and rearing percentages increased by about 1 6 per cent over four lamb crops. Although individual lamb weights have been reduced by 6 per cent at ten weeks, the weight of lamb produced per ewe mated was 6 per cent greater from the Finnish Landvace lborder Leicestev crosses. These figures for increased lambing percentages and decreased individual lamb weights are about one half of the values found when purebred Finnish Landrace rams were used as alternative crossbreeding sires, and hence are not unexpected. The advantagein using Finnish Landrace &verbar;Border Leicestev rams instead of pure Finnish Landvace rams lies in the easier management of the crossbred ewes and their lambs, because of the more easily handled lambing percentages and relatively heavier lambs. The improvement in prolificacy from the Finnish Landvace lborder Leicester crosses occured in the I year old and 2 year old dams particularly but was less marked in the older ewes. Furthermore, the differences in ewe weights, prior to mating, between the Finnish Landvace &verbar;Border

v3-fos

2017-06-20T19:35:18.189Z

1977-04-15T00:00:00.000Z

8653154

s2

Heterosis in Fayoumi strain incrossing Summary Many experiments have proved the superiority of hybrids resulting from incrossing of mbred lines, which have been utilized commercially in poultry production. The aim oi this experiment was to study the effect of incrossing inbred lines of Fayoumi chickens upon their combinability. The results can be summarized as follows: The fertility percentage 96.7 in comparing with 92.9 (L2j while reached in The heavier incross Lz5 X L3!.j had body weight equal to 118, II2 and io3 % in relation to the control at 4, 8 and i6 weeks of age, respectively. Heterosis in body weight became less obvious as chicks advanced in age and alleles responsible for early growth rate showed a great response to incrossbreeding. Incrossbreeding improved feed utilization of Fayoumi chicks. These results show the possibility for improving the native breed by incrossing and detecting the most efficient incrossbreds. Introduction Superior vigor of hybrids resulting from inbred line crosses has been utilized commercially in the production of chickens for both meat and eggs. Most of the studies dealing with in crossing showed that heterosis produced considerable positive effect on ecor!o.mical characters in chickens. R IZK ( 19 6 7 ) working on Fayoumi incrosses reported that incrossing increased hatchability. Similar findings on feed efficiency, body weight and viability in other breeds were reported for instance by MARSHALL and QulsErrsERRY (1959) and COLE and H UTT ( 19 6 2 ). The main objective of the present investigation was to study the effect of incrossing on some economic traits in Fayoumi chicks. Full-sib, halfsib and first cousin matings were used to get different inbreding intensities. Fayoumi sires and dams were mated in family pens supplied with trap nests, and pedigree chicks were used to consist different inbred lines during the experimental period (i.e. 1971-1 975). The data were collected by using z 5 females mated to 2 sires for each type of cross. Thus 1 8 sires and 135 dams were used in this experiment, giving 7 6 9 offspring. For the « control n group 205 individuals were obtained. The experiment was carried out during february 1975 and 4 hatches were available. All the chicks were reared in batteries in the same conditions using a diet of a standard type. The eontrol chicks (outbreds) were resulted randomly from a flock mating system. Three inbred lines (having inbreeding coefficients 25 , 37 ,5 and 5 0 °,o) had been incrossed in all possible combinations. Results were subjected to statistical analysis using the method of analysis of variance (Srr EDECOR , 1959 ). Results and discussion Effect of f incrossing on f ertility Data in Table I summarize the effect of incrossing on fertility of Fayoumi chicks. The fertility percentage in the control chicks was c!2,9, while it was improved in L 25 X L 50 incross and reached 9 6. 7 . The reciprocal incross (L5o x L 25 ) had the lowest fertility percentage (6 9 . 4 %). Moreover, all the incrosses having L 25 as a male had the highest fertility percentages, while their reciprocal incrosses had the poorest percentages. These results suggest a possible effets of the inbreeding coefficient of the male line on fertility. Hatchability of Fayoumi chicks resulting from incrosses showed considerable hybrid vigor. Most of the incrosses had higher hatchability percentage than the control chicks ( Table 2 ). The incross L 25 x L 37 . 5 gave the most positive effect on hatchability, which reached 143 . 7 % in relation to the control. COLE and H UTT ( 19 6 2 ), and R IZK ( 19 6 7 ) proved this positive effect on hatchability. Ettect of incrossing on viability Table 3 presents hybrid vigor in viability of the Fayoumi chicks at different ages. Early viability (i.e. from o-4 weeks) was improved in some incrosses, where it reached 93 .8 % in L 25 X L 37 . 5 compared to 88. 0 % in the control. No significant differences were secured among all the means of this early period. At 8 weeks of age, the viability percentage of the control chicks was 74 . 4 , while it was g2 .3 and 86 .4 in L2 5 X L 37.5 and L 50 X L37.5, respectively. Although there was a marked difference between the incrosses and the control, yet it was statistically not significant. This may be due to the small number of replicates. At 1 6 weeks of age, the viability percentage of the control chicks was 6 7 . 5 and this figure is low comparing with the figures given in standard breeds. This revealed its lack of genetic potentiality concerning high viability. However, some incrosses gave desirable results and viability percentage reached 8 4 .6 at 1 6 weeks of age (L 25 X L 37 . 5 ). This figure was equal to 125 , 3 % in relation to the control and such increasing in some incrossbreds could not be ignored. Similar results were obtained by B RILES and K RUEGER ( 1955 ). These results gave the possibility for improving the native breed from the viewpoint of the viability by detecting the most efficient incrossbreds. Meat production of chickens is considered as a short way to solve the meat shortage in some countries. Hybrid vigor in body weight is widely used in broiler industry and almost all the broilers produced now are hybrids. Table 4 presents body weight of incrossbred chicks in different systems of mating. At hatching day, almost all the incrossbred chicks were lighter than the control ones. The unweighted mean of both sexes of the control chicks was 27 . 3 grams and it was 25 . 2 -27 . 1 grams in the incrossbreds. This may be due to maternal effect, where chick weight is largely affected by egg weight and all the inbred dams were lighter than their corresponding controls. At 4 weeks of age, hybrid vigor became obvious and some incrosses were heavier than the control. It was noticed that the body weight mean of both sexes of control chicks was 102 grams while it was 121 grams in L 25 x L 37 .5 incross. In other words, this incross had body weight equal to no % in relation to the control one. At 8 weeks of age, the mean of body weight was 2 6 0 grams in the control while it varied from 252 to 29 o grams in the incrossbreds. The heavier incross (i.e. L25 X L37.5) had body weight equal to m2 % in relation to the control chicks. The same trend was also observed in body weight at 12 weeks of age. At 1 6 weeks of age, the body weight mean was 6 57 grams in the control and varied from 6 45 to 6 79 grams in the incrossbreds. Although most of the incrossbreds were heavier than the control chicks, yet the differences among these means were not significant. To summarize the effect of incrossing on body weight, it was noticed that heterosis in crossbred Fayoumi became less obvious as chicks advanced in age and it was more pronounced during the early period when the growth rate was high. On other words, alleles responsible for early growth rate (during broiler age) showed a great response to incrossbreeding. Modern trends in poultry production have clearly emphasized the need for research on the efficiency of feed utilization in chickens where feed cost is the largest single item of total cost. At 8 weeks of age, feed efficiency of the incross L 25 x L 37 .5 was 5. 000 , while it was 6. 15 in the control chicks ( Table 5 ). Most of the incrossbreds (except L 37 . 5 X L 50 -0 ) showed better feed efficiency than controls. Similar results were reported by MARSHALL and Q UI SENBERRY (1959). At 1 6 weeks of age, feed utilization was less efficient and this way be due to low growth rate of the chicks at this older age ( * ). Units of feed required to produce one unit of gain were 9 . 3 z in the control and varied from 8. 07 to 10 . II in the incrossbreds. L 25 X L 37 . 5 was the most efficient incross and its feed efficiency was 8. 07 . These results showed that it was possible to reduce the amount of feed required per unit of gain by incrossing and detecting the most efficient incrosses.

v3-fos

2014-10-01T00:00:00.000Z

1977-07-15T00:00:00.000Z

10084225

s2

Estimation of general and specific combining abilities from a diallel cross of three inbred lines of Fayoumi chicks Three inbred lines of Fayoacmi chicks were incrossed in diallel mating system to estimate general and specific combining ability. The inbreeding coefficients in three inbred lines were 25 , 37. 5 and 5 o per cent. L 25 showed the highest g.c.a. in fertility and body weight at 8 weeks of age. L 37 , 5 exhibited the best g.c.a. in viability, while L 50 gave the best g.c.a. in hatchability. The inbreeding coefficient of sire had a highly significant effect on both fertility and hat-chability while its effect on viability and body weight at 8 weeks of age was not significant. L z 5 X L 37 , 5 incrossbred chicks gave the best s.c.a. in viability and body weight, fertility and hatchability. Introduction The model of inbreeding and incrossing has been applied successfully in chicken production and has played a major role in many broiler and egg production investigation programs. Y AO (ig6i) using diallel crosses of inbred lines of chickens reported that line effect was highly significant for 10 -week body weight. C OLE and HuTT ( 19 6 2 ) found that hybrids resulted from crossing 2 strains of white Leghorns showed a high degree of heterosis in egg production, hatchability and body weight. ErsErr et al. ( 19 6 4 ) reported that cross effects were highly significant for 8 week body weight and adult mortality. H AZEL and L AN to REU x (zg 47 ), and GoTO and NoRDSKOG (rg5g), using Leghorn breed of Chicken suggested that nicking was not of major importance. HALE and C LAYTON ( 19 65) found the same concept in Leghorn breed and crosses, showing that genetic interactions were unimportant. H ILL and N ORDSKOG ( 195 8) stressed the importance of general combing ability over specific combining ability. W EARDEN et al. ( 19 6 4 ) in a full-diallel crossing experiment using 3 white Leghorn and 3 Rhode Island Red &dquo; closed flock &dquo; strains suggested that general combining ability was much more important than specific combining ability for five and ten-month body weights. Contrarily, specific combining ability was estimated to be of more importance for sexual maturity, hen day and hen-housed egg production to 2 6 0 and 470 days. HowEVER E RASMUS et al. (rg68), illustrated the importance of specific combining ability as compared with the general combining ability. Material and methods The main objective of the present study was to estimate general and specific combining ability (g.c.a. and s.c.a.) of Fayoumi chicks resulting from incrossing of three inbred lines. Full -and half-sib matings were used through 4 years (i.e. 1971 -1974 ) to get different inbreeding intensities. Inbreeding coefficients were 25 , 37 .5 and 5 0 per cent in three different inbred lines. All possible combinations along with the three inbred lines were represented using diallelic mating system. The traits studied were fertility, hatchability, viability and body weight. The birds used in this study are the same as described in the paper by S OLTAN et al. (in press). General and specific combining ability were calculated as means (Fnr.co-NER , 19 6 4 , p. 2 8 4 ) and also tested by the analysis of variance (G ALAL et al., ig 7 2). ( * ) Maternal influence for each inbred line was estimated as the average of differences between the reciprocal incrosses which include that inbred line. Heterosis percentages were also estimated as the deviation of the incross mean from the average of the two parental inbreds. All the percentages data were converted to angles with the arcsin transformation using tables given by S NEDECO R, 1959 . Results and discussion Data presented in Table i show the average of general and specific combining ability on some traits of Fayoumi incrossbreds. It was noticed that L 25 showed the highest general combining ability in both fertility and body weight at 8 weeks of age, while it was moderate in hatchability and viability percentage at the same age. L 37 .5 gave the highest general combining ability in viability, while L 50 showed the best general combining ability in hatchability. The interaction between sires and dams, present in all the traits except body weight (see table 2 ) could be due to nicking between the genes of the sires and dams. Incrossbred chicks produced from L 25 X L 37 ,5 mating showed the best effect on viability and body weight. These improvements in viability and body weight may be due ( * ) General combining ability is defined as the average performance of a breed, strain or line in a cross combination, while specific combined ability (« nicking ») is defined as the deviation in performance of a cross from the expectation on the basis of the average performance of the parental breeds, strains or lines involved. to better general combining ability in L 25 and L 37 .5 and also to a favourable specific combining ability between them. However, this incrossbred was moderate in fertility. Table 2 shows the effect of sire and dam on different traits. It was noticed that the coefficient of inbreeding of sire hadhighly significant effect on both fertility and hatchability, while its effect on viability and body weight at 8 weeks of age was not significant. It was also noticed that the dam's inbred effect was highly significant on hatchability, while non-significant for all the other traits. Table 3 shows maternal influence of different inbred lines on traits studied. It was noticed that L 25 exhibited the best maternal ability in fertility, viability and body weight, while L 50 gave the highest maternal ability in hatchability. The same trend, except viability, was observed in g.c.a. (Table i). Therefore, it seems resonable to take both g.c.a. and maternal ability into consideration when evaluating an inbred line performance in incrosses. Heterosis percentages for different incrosses are given in Table 4 . It was observed that L 25 X L 5 0 incross gave the highest heterosis in fertility and hatchability, while L 25 X L 37 .5 gave the best heterosis in viability and body weight. It was also clear that both s.c.a. and heterosis percentages gave almost the same trend and it is quite resonable to consider any one of them in explaining differences among incrosses.

v3-fos

2017-07-29T04:25:02.318Z

1977-10-15T00:00:00.000Z

5570487

s2

Survey of recent situation of chromosome pathology in different breeds of german cattle 2. Considering the impossibility in karyotyping all calves with congenital anomalies extensively, most of them being stillborn, and in regard of the impractibility of a cytogenetic exploration of whole the population, it seems to be impossible to derive more than rough estimations of the real frequencies of chromosome anomalies within the observed population. 3. The first rank of different types of chromosome anomalies is occupied by the z / 29 translocation, followed by the trisomy 18. (In this connection the XX /XY chimerism in freemartins Introduction The following report presents the results of systematic cytogenetic studies in newborn calves with congenital malformations, in adult individuals with disturbances of sex differentiation, and those which have been fallen sick with maladies caused by (or combined with) chromosomal aberrations. Those results have been obtained in the regional population of cattle in Hessen (Fed. Republ. of Germany), composed of the four breeds Black Pied I,owland (partially Holstein-Friesian), Red Pied Lowlands, German Simmental (Fleckvieh), and German Red Cattle, during 19 68 till May 1977 . The report offers a review of prior papers of the authors emphazising recent advances in etiological and nosological research in Bovine Hereditary Parakeratosis, and the Trisomy 1 8 Syndrome. The results of cytogenetic observations reported in this paper are not derived from systematic investigation of whole the population, but the sample of karyotyped animals collected from the population is preselected regarding pathological traits of the animals only. Therefore, it seems to be inconvenient to derive out of those figures presented in Table I statistically established informations about the frequencies of chromosomal defects within the population, and differences of chromosomal defects within the population, and differences of frequencies between the breeds. But, if we compare the percentages of chromosomal anomalies with the numbers of examined animals belonging to the different breeds, extending from 3 .8 p. cent in Black Pied Lowlands (partially Holstein-Friesian), 2 . 7 p. cent in Red Pied Lowlands, to 14 . 1 p. cent in the German Simmental Cattle (Table 2 ) we may establish the number of chromosomal aberrations being biased in disfavour of the Simmental breed. The real relations of percentages of chromosomal anomalies to the percentages of breeds composing the population you may evaluate by a short glance at the left column of Table 2 . Table 1 exhibit that regularly only three types of f structural and numerical autosomal aberrations are found in the observed population associated with different pathological traits of newborn calves or of adult patients during the last five years. Incidentally or seldom gonosomal numerical aberrations are observed, except the XX /XY chimerism, which is not from interest in this connection, mainly four types of them. The roughly estimated frequencies of the different types of autosomal anomalies are differing in wide range; none of them gained a remarkable expansion with corresponding economical importance. pancies between the phenotypical effects of translocations in Swedish and Norwegian breeds (R!FSDAI&dquo;197 6) probably are attributed to the comparatively very small sample of karyotyped animals corresponding to the low distribution of translocations in our breeds. b) -Autosomal aberrations in Bovine hereditary parakeratosis (BHP) Since ig6o it has been observed in several regions of the world a metabolic disturbance in Holstein-Friesian breed, which very soon had been recognized to be caused by a nutrient zinc deficiency (MILLER and MILLER, 19 6 0 ), which is connected with more or less severe lesions of the skin in the sense of parakeratotic dermatosis and other pathological traits. The hereditary etiology of this syndrome, originally named &dquo; parakeratosis &dquo; (L EGG and SEARS, ig6o), had been first stated in Denmark by A NDRESEN et al. ( 1970 ) Originally, there aroused some doubts wether the chromosome anomalies observed in patients with BHP are acting a role in the etiology of this lethal trait, but, rather only being one symptom of this syndrome together with several others. But, the demonstration of the chromosome anomalies described above in all patients with BHP (Table 3 ), and, moreover the observation of breakages in metaphases of white blood cells in the parents of BHP calves with few exceptions seem to elucidate a more important role of those autosomal structural defects in pathogenesis of this metabolic disturbance. Table 4 is demonstrating a frequency of breaks in autosomes of fathers of HP calves of m.r p. cent, and in those of mothers of 9 . 5 p. cent on the average of all karyotyped parents. In comparison with the frequencies of breaks in the normal Black Pied Lowlands population of Germany from 0 -0 . 02 8 p. cent (E L -N AHASS et al., 197 6) to 4 . 1 p. cent in an own control sample (Table 5 ) the difference between the values of m.r p. cent resp. <!.5 p. cent and of i. 5 p. cent are significant. Therefore, it seems to be justified to declare relatively high frequencies of autosomal breaks to be characteristic for the heterozygous condition of the gene &dquo; Hereditary Parakeratosis &dquo;. In bovine adults suspicious to be heterozygous for BHP, autosomal breaks exceeding a limit of about 5 p. cent may assigned to detect heterozygosity. Self-evident, this assertion must not neglect the fact, that some other causes may produce structural defects in chromosomes than BHP. c. -Autosomal breaks in cases of Hereditary Nanism Autosomal breaks too had been observed in 197 6 in three calves belonging to the sibship of a Simmental bull bred in Baden-Wurttemberg. This bull brought about 25 p. cent dwarf calves within his offspring, obviously being heterozygous for an autosomal quasi dominant Mendel factor &dquo; Hereditary Nanism & d q u o ; , a viable non chondrodysplastic, proportionate dwarfism. The breaks occurring in the first blood sample in frequencies of about 30 -43 p. cent of metaphases of white blood cells diminished in the course of one month about 50 p. cent ( Table 6). The father of the dwarf calves didn't exhibit any chromosome anomaly. This spontaneous elimination of cells with chromosomal defects seems to affirm the doubts about the etiologic resp. pathogenetic role of autosomal breaks, because the possibility the breaks are caused for instance by an intervention in mitotic processes by virus particles in contaminated culture medium or by latent infections of the probands by IBR-IPV virus or others is not to be ruled out. -Autosomal aneuploidies In contrast to the tremendous role of numerous trisomies causing in human beings nea.rly 10 typical malformation syndromes, and a high percentage of spontaneous abortions, in cattle this group is represented only by one sing,1'2 trisomy, the trisomy 1 8. (LBTS) (Trisomy 1 8 syndrome) and its cytogenetic background first had been published by H!xzoG and H6 HN in 19 68. Afterwards this syndrome combined with a trisomy of an autosome belonging to the group C had been established by MoRr et al. (ig6g) in Japan, and, furthermore by DurrN et al. ( 1972 ) in U.S.A. In the meantime, we collected 1 8 cases of LBTS in all breeds of our region, i. e. in In present, we are not able to present a statistically proved assertion of the real frequencies of this syndrome, and the differences of frequencies between breeds, because in most cases the probands with the pathologic traits of LBTS are stillborn. Therefore, it is not possible to gain viable tissue cultures from them beyond the limit of q.8 to 72 hours post mortem. Indeed, we get the impression, that this autosomal aneuploidy gained a broader distribution, preferably in the German Simmental, than primarily supposed. In this connection, we are allowed to discuss the identification of those autosome of the C group involved in the trisomic process, and, which had been identified by the Standardization Conference of Reading 197 6 to be the autosome No. y . In regard to the statistical impossibility to separate the autosome No. 17 from No. 1 8 by biometrical methods (arm length) it seems to be without any significance to destine the position of both autosomes in the karyogram more or less arbitrary ( fig. 3 ). Therefore, we prefer to insist in the original designation of &dquo; trisomy 1 8 &dquo; preliminary ascertained in our first publication of 19 68 (H ERZOG and H6 HN ) before usage of banding techniques. Pathology of the Lethal Brachygnathia T y isomy Syndrome The association of several different types of congenital malformations composing the LBT syndrome had been analyzed by means of autopsies of the 1 8 probands; the results are summarized in the following Table 7 : . Other incidentally defects observed in LBTS probands are: -lVIaxillo-facial dysplasia, mostly connected with -Palatoschisis (cleft palate), -Aplasia of cerebellum, -Ascites and Hydrothorax, all with a high variability of manifestation and combination of traits. Apparently the structure of this syndrome on no account is homogeneous: several probands with trisomy 1 8, partially with trisomy 1 8 mosaics, deviate from the scheme presented in Table 7 . We like to quote as illustrations of those irregular types, the case with a trisomy mosaic (6 0 , XX /6i, XX, 1 8 -!-) exhibiting extremely severe dysplasias of the face, hydrocephalus, cleft palate, and multiple arthrogrypotic anomalies of all limbs, H ERZOG and H6 HN published in 1974 . During 197 6 a similar case with dysplasia of mandible, cleft palate, arthrogryposis of front limbs, and gigantic development had been registered ( fig. 5 ). The karyotype of this stillborn calf (Reg. No. 4431 / 7 6) revealed 100 p. cent of trisomy 1 8 metaphase in cultured kidney cells. Etiology of LBT synd y ome The empiric impression, the I,BT-syndrome being genetically influenced, is derived from several observations of its familiar incidence in the breed German Red Pied Lowlands as well as in German Simmentals. Especially in the descent of two bulls belonging to the last mentioned breed used in A.I. we found three different types of meiotic disturbances, which are establishing the initial supposition of &dquo; an inherent genetical disposition to meiotic disturbances &dquo; in these families, we uttered in 1970 . In this paper we demonstrated the occurrance of one case of XXY-syndrome, and another with X-trisomy in one half sib group. 1975 and 1 97 6 these observations had been supplemented by three cases of trisomy 1 8 ( fig. 6). Moreover, one of the involved bulls, the Dmo 1744 brought during the past 10 years on the whole 7 descendents with the Lethal Brachygnathia Fetal Hypoplasia Syndrome (Table !8), all stillborn. Out of this reason we succeeded only in two cases ( 41 6 2 and q. 34 8) in gaining viable tissue cultures post mortem, mostly from kidneys, and to demonstrate the trisomy 1 8. Those relative seldom observations, self-evident, are not suitable for calculating a mode of heredity involved with the manifestation of the LBT syndrome, because in both systems of heredity, in the monogenic and in the polygenic, it depends on the frequencies of the corresponding genes in the population, which are absolutely unknown. But, the unique accumulation of meiotic resp. mitotic disturbances in two families among some hundreds of unsuspicious sibships steadily controlled in the breeds of our region rules out an accidental coincidence of such rare events. In Considering the detection of a trisomy 1 8 and a trisomy 21 in half-siblings, both from different fathers, DAVID and J ONES conclude 1975 that it may be possible that in some woman is a predisposition to nondisjunction. Their opinion, certain families apparently being &dquo; more prone to the occurrence of chromosomal aneuploidy &dquo;, we estimate -together with all observations mentioned above&mdash; as confirmation of our hypothesis of a &dquo; familial disposition to meiotic disturbances &dquo; in cattle, we postulated in 1970 (R I P CK et al.). Furthermore, we appreciate the appearance of two cases of LBTS (with proved trisomy I 8), and several more cases of a from pathological point of view identical malformation syndrome (without evidence of a trisomy because the probands have been stillborn) in the half-siblings group of the bull D IVO (Teratogram (Table 8) as a further proof for the same genetic background of the autosomal and gonosomal aneuploidies and their pathological effects. -Polyploidies Polyploidies in general are incompatible with vitality of mammals. Nevertheless, some cases in human infants with triploidies (6 9 , XXX) have been published in the past five years. Some of them survived to birth, others even nine days post partum ( DE Gxoucx y et al., I97 q). In cattle, too, tetrato dekaploidies have been found in individuals of Charolais cattle with double muscled condition (culard) in percentages from 17 to 24 p. cent of examined cells (P OPESCU , 19 68). One of us (R I E CK ) demonstrated 1973 a case of diploidy/triploidy mosaicism in a German Simmental individual, quite a similar case D UNN et al. published in 1970 . The XXY gonosomal complement of this mosaic obviously was the cause of masculinization of the gonads, and of disturbances of differentiation of the sinus urogenitalis. This type of polyploidy seems to remain a unique case. Some doubts about the etiological role, resp. pathogenetic significance of certain chromosome anomalies have been mentioned above. Apparently this turns out to be true in a high degree concerning the polyploidies observed in white blood cell cultures. This problem grew acute in human genetics considering the observations, that up to 100 p. cent polyploidy has been found in cultures of amnioticfluid cells gained by amniocentesis from pregnant women with normal diploid embryos. It has been suggested that prolonged cultivation of cells increases the frequencies of polyploid cells. This tendency to disturbances of mitosis in cells in culture could be avoided by introduction of a special technique (N AKAKOM E et al.,I(!72). In regard to our experience with a &dquo; control group &dquo; of nearly one thousand routine charges of tissue cultures we believe to possess sufficient estimations of frequencies of polyploid cells cultured from normal animals. The observations on chorionic cells of human beings, therefore, may not be verified in bovine cells of streaming blood, and of other organs. In comparison with our results obtained in the &dquo; control group &dquo; the significance of polyploidy findings statistically may be proved, self-evident on the assumption of constant conditions of culture techniques. Out of this reason we are convinced the polyploidies not to be artificial products of culture techniques, but representing a symptom of lability of cells in performing the mitotic processes in culture. This seem to be the case especially in endomitotic polyploidies, in most cases tetraploidies, observed in nearly 25 probands with congenital defects of central nervous system and eyes of newborn calves (H ERZOG and H6 HN , 1971 ). These findings recently have been confirmed by further observations in the same category of defects by S TIX ( 197 8). The same functional lability of mitosis in culture W EINHOLD demonstrated conclusively 1970 in aneuploid and polyploid white blood cells and tumor cells of cows with lymphatic leukaemia, estimating this phenomenon to be a fundamental characteristic of mitotic disturbances of tumor cells. Cells which reveal the tendency to polyploidization in culture are estimated in human oncology to be a &dquo; characteristic feature of malignant change &dquo; of a blastoma (K NO E RR -GX R T N E R , H. and W., 1977). This interesting phenomenon of abnormal cellular functional behaviour in realization of mitosis in culture is till now very poorly understood. It requires further investigation, because its explanation with disturbing influences by culture medium, mentioned above, apparently not at all is its only cause. In teratological aspects it is to evaluate whether cell types exhibiting polyploidization in culture may have similar or homologous effects on developmental processes of certain blastema (neuroblastema f. i.) in embryos as in malignant blastomas. Hence, we may infer, that the problem of polyploidy in mammals include two items: (i) Polyploid cells in culture are they a symptom of cellular functional lability, resp. insufficiency, to realize mitotic processes in artificial medium, or, (z) are they originally present in blood cells or cells of other organs, and, assertive, how many percent of polyploid cells in mosaics are sufficient to disturb the embryological development or to individual mortality? 4. -Gonosomal numeric anomalies including gonosomal chimaeras The most comprehensive contingent of chromosomal aberrations presented in Table i is represented by the gonosomal numeric anomalies, first of all the XX /XV chimerism of freemartins, and other gonosomal mosaics. Notwithstanding the high share of this type of blood chimerism, the XX /XV chimerism possess little significance in connection with the present discussion, because the frequency of freemartins only depends from the frequencies of dizygotic twins in the different breeds. High interest, indeed, is to be claimed to the problem of the XX /XY mosaicism in singleborn animals and in isosexual twins. Although the reports on this extraordinary type of gonosomal mosaicisms seem to increase recently, the frequency of the so called &dquo; Autonomous XX /XY syndrome &dquo; in the breeds of Germany is very low. The last case we observed in Black Pied Lowlands in ig 7 ¢ in the environment of Hanover (R IECK , 1975 ). The pathogenic activity of those second X-chromosome in genetic male individuals, obviously originating from a &dquo; whole body mosaicism &dquo; (B FNIRSCHKE , 1970 ) seem to be rather obscure in the present. Partially those XX /XY individuals are absolutely normal, another part of them exhibit more or less severe disturbances of differentiation of sexual organs within wide limits of variation. Likewise very low population frequencies in German breeds exhibit the gonosomal trisomies, i. e. the bovine XXY syndrome, a homologue to the human K LINEFELTER ' S syndrome, and the trisomy X. Since 19 6 7 (R I EC K , 197 0), and ig 7 o no further gonosomal trisomy had been observed. A symptom of their scarcity in other European cattle breeds, too, is the finding of only one more case of an X trisomy in a heifer of the Norwegian Red cattle in 197 6 (Nous!RG et al., zg 7 6), not at all in all other breeds in the world. Most of the cases of intersexuality or of malformations of sexual organs without intersexuality in cattle are not combined with (or caused by (?)) gonosomal aberrations. Conclusions In spite of a large amount of karyograms which had been collected in the course of etiological research of developmental disturbances in cattle during the last ten years it is impossible in the present moment to gain an impression of true frequencies of chromosomal anomalies, considering the lack of comprehensive investigations of whole of the population. Moreover, the fact, relatively seldom to obtain surviving cells from stillborn calves being fit to divide in tissue culture make it impossible to get real figures of frequencies of chromosomal defects. Nevertheless the findings of chromosomal anomalies associated with congenital malformations in newborn calves, or with clinical symptoms in adolescent animals allow some rough estimations of their frequencies and their economic importance. So, it is evident that the incidence of trisomy 1 8 in all breeds just follow that of z /2g translocation; the frequencies of all other chromosome aberrations are ranging widely below those just mentioned. Considering the chromosome aberrations specialized in groups of anomaly types it had been found within the group of structural autosomal defects cases of hereditary nanism combined with breakages in the sibship of a bull of German Simmextal breed; breakages and reunion figures in calves suffering from Hereditary parakeratosis ( & d q u o ; Zinc deficiency syndrome & d q u o ; ) in Black Pied Lowlands, furthermore breakages too in a solitary case of hydrocephalus, rachimyeloschisis etc. The group of nume y ical autosomal aberrations (aneuploidy) is represented by the trisomy 1 8 alone, causing the Lethal Brachygnathia Trisomy Syndrome ' ; whereas the group of polyploidies mainly consisted in tetraploidies observed in primary cultures of blood cells originating from several cases of different defects of the central nervous system. The etiological role of structural anomalies of autosomes in disturbances of embryological development is not quite clear yet. It is not known whether the chromosome defect is merely a symptom in the range of others within a syndrome, or, whether it reveals pathological effects autonomously in sense of an effective etiological factor. But, there is strong evidence autosomal breaks being markers for heterozygosity of healthy parents of calves with Hereditary parakeratosis. The group of gonosomal numerical anomalies is represented particularly by the XXY syndrome, the &dquo; Bovine hypogonadism & d q u o ; , and the X trisomy, both being very seldom in the observed populations. The different mosaicisms, i. e. the diploidy /triploidy mosaic, include of course a multiplication of gonosomes in triploid cells resulting in disturbances of genital development such as the different types of intersexuality. This group comprises also the &dquo; Autonomous XX /XI' syndrome &dquo; in singletons and isosexual twins. Certain observations exhibit a hereditary disposition of families to disturbances of meiotic and /or mitotic processes, based on the presence of autosomal together with gonosomal aneuploidies in two families of the German Simmental breed. Reçu pouv publication en de c em b ve 1 977.

v3-fos

2018-12-07T08:04:05.397Z

1977-01-01T00:00:00.000Z

55452958

s2

Field Evaluations of Selected Insecticides to Control Russian Wheat Aphid on Winter Wheat 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 1977 Kansas State University Agricultural Experiment Station and Cooperative Extension Service. AES The Russian wheat aphid (RWA), Diuraphis noxia Mordvilko, was recently introduced into Kansas and has the potential to be a serious pest of small grains. It was first detected in the United States in the Texas Panhandle in March 1986. The first report of this aphid in Kansas was from Stanton County in April 1986. The RWA is indigenous to Russia and has been regarded as a pest of wheat and barley in southern Russia and surrounding Mediterranean countries since 1900. It does not usually damage oats or rye. The winged aphids probably ~ached the United States from Mexico (where they were discovered in 1980) by making use of strong, prevailing air currents. The RWA can be easily confused with other common cereal aphids (greenbug, English grain aphid, corn leaf aphid, bird cherry-oat aphid), but it can be distinguished by some unusual physical characteristics (Figure 1). Unlike other aphids, the RWA has no visible cornicles or 'tail pipes' protruding from the rear do!sal portion of the body. The aphid has a prominent supracaudal appendage at the end of the abdomen, which gives it a 'double-tail' appearance when viewed frQm the side. The RWA has short antennae extending from the head and is lime-green in color, with an elongated, spindle-shaped body. Experience in controlling this pest with chemicals is still very limited in the United States. Information gleaned from the literature suggests that the RWA may be difficult to control because it is protected within the leaf sheaths and tightly rolled leaves of plants. In order to obtain more information on the effectiveness of insecticidal treatments, field tests were conducted in Kansas during 1987. Procedure Field-plot tests were conducted at two locations to determine the efficacy of selected insecticides against the RWA, using a randomized complete block design with three replications. Sharon Springs (Wallace Co.) Test. Six insecticide treatments {Table 1} were evaluated for RWA control in a farmer-owned field of dryland wheat. Experimental plots measured 18ft X 25ft. A single broadcast application of each insecticide was made on May 8 using a COz-pressurized, backpack sprayer calibrated to deliver 12.8 gal/acre through four hollow-cone 6X nozzles, mounted on a hand-held boom, at 20 psi. The crop was in Feekes growth stage 10 (boot) at time of application. Population densities of the RWA were assessed prior to treatment and at 4-, 10-, and 21-days ( (Table 2) were evaluated for RWA control in a field of dryland wheat at the Fort Hays Branch Experiment Station. Experimental plots measured 15 ft x 30 ft. Plots were artificially infested with RWA by taping two infested wheat culms (placed in vials of water) per plot to the plants so that infested flag leaves were at the same height as those of the planted wheat. A single broadcast application of each insecticide was made on May 12 using a self-propelled, high-clearance, ground sprayer that delivered 20 gal/acre. Treatments were assessed for initial mortality 1 day after application by removing the infested culms and counting the number of live RWA. Residual effectiveness was determined by caging 30 RWA on the flap leaf of each of two randomly selected, treated plants per plot at 7 and 21 days posttreatment. The number of surviving aphids in the cages was recorded 1 day after each posttreatment date. Results and Discussion Sharon Springs (Wallace Co.) Test. All insecticide treatments, when compared to the untreated check, significantly reduced RWA 4 days after application (Table 1). The Di-Syston treatment had the fewest RWAs, but it did not differ significantly from the other insecticides. Ten days after application, all treated plots continued to have significantly fewer RWAs than the untreated check. Except for malathion, there were no significant differences between insecticides. At 21 days posttreatment, all treated plots still had significantly fewer RWAs than the untreated check. Lorsban was the most effective insecticide at that time and was significantly better than either parathion or malathion. Grain yields did not differ statistically, but all treated plots yielded more grain than the untreated check (Table 1). The late application of insecticides, extremely high RWA densities at the time of application, and plot variation were probably responsible for the low yields and nonsignificant data. Hays (Ellis Co.) Test. All insecticide treatments significantly reduced the number of RWAs, when compared to the untreated check, at 1 day posttreatment (Table 2). Except for Capture, there were no significant differences between insecticides. Seven days after application, Di-Syston, Lorsban, Karate, and Capture provided significant residual control. The other insecticides did not differ significantly from the untreated check. Karate and Capture gave 100% residual control at 21 days posttreatment, but they did not differ significantly from Di-Syston, which gave 89% control. Except for Furadan, there were no significant population differences among treatments compared with the untreated check. Conclusions Results of these trials indicate that among the insecticides broadly labeled for aphids on small grains, Di-Syston, Cygon, and parathion gave excellent initial control of RWA, but residual effectiveness varied. Among the insecticides not currently labeled for application to wheat, Lorsban, Karate, and Capture gave excellent initial control and good residual activity at the rates tested. · Producers are urged to consult with their local county agricultural extension agent and/ or the current issue of the Kansas 'Wheat Insect Management Guide' for assistance in selecting the right insecticide (s) for RWA control on winter wheat. Brand names are used to identify products. No endorsement is intended, nor is any criticism implied of similar products not mentioned. l.orsban, Karate, and Capture insecticides do not have current label registration for use on wheat and CANNOT be utilized for that purpose until they re· ceive proper labeling. Fur a dan is not currently labeled specifically for aphid control on wheat and, therefore, _ CANNOT be used for RWA control on that crop unless future labe l changes are made. 5-88-3M 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

2014-10-01T00:00:00.000Z

1977-08-01T00:00:00.000Z

15002159

s2

Occurrence and distribution of arsenic in soils and plants. Inorganic arsenicals have been used in agriculture as pesticides or defoliants for many years and, in localized areas, oxides of arsenic have contaminated soils as a result of fallout from ore-smelting operations and coal-fired power plants. Use of inorganic arsenicals is no longer permitted in most agricultural operations, and recent air pollution controls have markedly reduced contamination from smelters. Thus, this paper will concentrate on the effect of past applications on arsenic accumulation in soil, phytotoxicity to and uptake by plants as influenced by soil properties, and alleviation of the deleterious effects of arsenic. Once incorporated into the soil, inorganic arsenical pesticides and arsenic oxides revert to arsenates, except where the soil is under reducing conditions. The arsenate ion has properties similar to that of orthophosphate, and is readily sorbed by iron and aluminum components. This reaction greatly restricts the downward movement (leaching) of arsenic in soils and the availability of arsenic to plants. Several methods of estimating plant available arsenic in soils have been developed. They involve extraction of the soil with reagents used to estimate phosphorus availability. This extractable arsenic is reasonably well correlated with reduced plant growth by, and plant uptake of arsenic. For most plants, levels of arsenic in the edible portion of the plant are well below the critical concentration for animal or human consumption, even when severe phytotoxicity occurs. Alleviation of arsenic phytotoxicity has been attempted by increasing the soil pH, by use of iron or aluminum sulfate, by desorbing arsenate with phosphate and subsequent leaching, and by cultural practices such as deep plowing. Only limited benefits have accrued from these procedures the cost of which is often prohibitively high. Since attempts to reduce arsenic toxicity have not been very successful, its excessive accumulation in soils should be avoided. Introduction Inorganic arsenicals were originally used in agriculture for the control of various insect pests such as the Colorado potato beetle, codling moth in apples, horn worm in tobacco, and boll weevil in cotton. Control often required the application of considerable quantities of lead or calcium arsenate or cuprous arsenite (I). More recently, arsenic trioxide has been widely used as a soil sterilant and sodium arsenite for aquatic weed control and as a defoliant to kill potato vines prior to harvest (2)(3)(4)(5). *Department of Soil Science, University of Wisconsin, Madison, Wisconsin, 53706. tVisiting Professor, Department of Soil Science, University of Wisconsin, Madison, Wisconsin, 53706; on sabbatical leave from the Department of Soil Science and Agrometeorology, University of Natal, Pietermaritzburg, South Africa. Arsenical sprays have also been used to control the ripening of grapefruit and Valencia oranges (6). Arsenic is one of the few metal elements for which recent developments indicate a decreasing rather than increasing concern over concentrations in the food chain. The primary reason for this stems from the banning of sodium arsenite as a defoliant and the replacement in fruit orchards of substantial quantities of lead arsenate by carbamates and organic phosphates (7). Organic arsenicals are now used where required, and are applied at much lower rates than the inorganic arsenic compounds. The net result has been a drastic lowering of the amount of arsenic reaching the soil. Poultry are often fed 3-nitro-4-hydroxyphenylarsonic acid, but the arsenic from their manure does not accumulate in corn grain grown on soils receiving up to 600 tons manure/ha and does not seem to pose a hazard (8). With this in mind the objectives of this paper will August 1977 be to review arsenic chemistry in the soil in relation to its immobilization and methods for its determination which have prognostic value in terms of plant uptake and phytotoxicity; to define as far as possible the conditions under which arsenic becomes phytotoxic and potentially hazardous to humans and animals ingesting plant material from treated soils; and finally, to evaluate methods of decreasing the arsenic hazard in contaminated soils. Arsenic Chemistry in Soils Arsenic is ubiquitous in nature, occurring in most soils and rocks in detectable quantities. Concentrations of arsenic in uncontaminated soils range from 0.2 to 40 ppm. No clearly defined relationship exists between the arsenic content of soils and the parent material or climatic conditions under which the soils were formed. In uncontaminated soils the level of arsenic is not sufficiently high to cause phytotoxicity and does not therefore represent a health hazard. Soils from land repeatedly treated with inorganic arsenicals contain levels of arsenic often 10-to 100-fold those of untreated areas ( Table 1). Use of arsenicals as insecticides usually results in higher concentrations of arsenic in the soil than when they are used as defoliants. Arsenic chemistry is in many ways very similar to that of phosphorus, especially in aerated systems, and it is generally held that the arsenate ion closely resembles orthophosphate. However, arsenic is more labile than phosphorus, and unlike phosphorus it can undergo valence state changes over the range of redox conditions likely to be found in soils and biological systems (10). Also, it apparently does not accumulate in organic forms in soils. Under strongly reducing conditions elemental arsenic (As,) and arsine (AsH3) (-III) are the stable forms. In less reduced environments, such as those in flooded soils, the relatively toxic arsenite (MAsO2) (+111) can be formed. However, in aerated soils, arsenate (MAsO,) (+V) predominates. The above valence states can form compounds containing the C-As bond and are readily interconverted by soil microorganisms which do not seem to utilize the energy produced by oxidation for growth (11,12). Arsenite added to a well-drained soil having significant biological activity is oxidized in a matter of a few days (13). In reduced environments such as sediments, arsenate is reduced through arsenite to dimethylarsinic acid, which is extremely toxic (12). Evidence exists that this compound may be more ubiquitous than previously realized due to lack of methods for its detection and estimation (14). Soluble arsenic has been observed to increase in flooded rice soils (15). This was attributed largely to the reduction of ferric to ferrous iron, but the possibility of arsenite formation was not ruled out. The level of soluble arsenic in soils is determined by the relative arsenic sorptivity of soil components, chiefly iron and aluminum compounds. For example, all of the arsenic added to soils is removed by treatment with oxalate, which also removes iron and aluminum bound in allophanic materials and hydrous oxides (16). These are now termed shortrange order iron and aluminum components. Hydrous ferric oxides have been shown to be effective in sorption of arsenate (17). The importance of adsorption is further supported by the decrease in extractable arsenic from arsenic-amended soils with time (9,16,18) and the apparent ability of phosphate to replace arsenate from soils (19,20). The high proportion of sorbed arsenate extractable by neutral salts (16) also indicates that it is weakly sorbed by soils relative to phosphate. Although arsenic can form moderately insoluble salts with ferric iron and aluminum, the previous observation on the mobility of arsenate in soils would indicate that discrete arsenate compounds do not form in soils, and arsenate is retained in soils by sorption mechanisms. As a first approximation, the sorptive capacity of a soil for an ion such as arsenate is a function of its surface area, and hence of its clay content (3,16,18,(21)(22)(23). Thus, arsenic would be expected to be more labile in low clay (sandy) soils, explaining ob-Environmental Health Perspectives servations that arsenic phytotoxicity is more likely on sandy than on fine-textured soils (24)(25)(26) and that arsenate can leach through the profile of sandy soils (27). Soil Arsenic and Plant Growth Plant growth stimulation has often been observed as a result of low levels of arsenic additions to soils, despite the fact that it is not considered as an essential element for plant growth. Two mechanisms have been proposed to explain this stimulation, the one being presumed to be similar to that obtained by other pesticides (e.g., 2, 4-D) at sublethal dose levels (18) and the other being due to the displacement of phosphate by arsenate from soil surfaces, thereby increasing phosphate availability (27). Depression of plant growth usually occurs at higher levels of arsenic application and is particularly severe when treated orchards are removed and the land replanted to an agronomic or horticultural crop (3,28). Plants vary considerably in their tolerance to high levels of soil arsenic. Potatoes, cabbage, tomatoes, carrots, tobacco, rye, Sudan grass, and grapes are highly tolerant; strawberries, corn, beets, and squash are moderately tolerant; and onions, cucumbers, and legumes have low tolerance (24,26,29). Plants take up arsenic from the solution phase of soils and therefore measures of labile forms should be better related to plant uptake than the total arsenic content when soils with widely varying properties are compared (21,26). The labile forms are usually measured by extracting the soil with hot water, dilute acids, salt solutions or complexing agents. These remove a certain reproducible proportion of the total arsenic which happens to be soluble in the particular reagent. A summary of a few recent studies (Table 2) shows the "critical" level of arsenic at which a yield depression might be expected is influenced by plant species and varies with differing soils. Arsenic extracted by many of these extractants, such as Bray P-1 (0.025N HC1 + 0.3N NH4F), O.5N NaHCO3, or O.05N HCI plus 0.025N H2SO4 are satisfactorily related to the amount of arsenic available to the plant (18,24,25). Examples of correlation coefficients between yields of a number of crops and arsenic extracted by various methods are presented in Tables 3 and 4. Even though water-soluble and total arsenic can satisfactorily predict phytotoxicity in certain cases, soil testing laboratories can more conveniently use the "available arsenic" extractants because they are routinely used to estimate available P. Bioaccumulation of arsenic would be hazardous bAll of the correlation coefficients are significant at the 0.05 probability level. to humans and animals because of its possible relationship to cancer, arteriosclerosis and chronic liver disease (30). (Table 5). Toxicity to animals or humans usually is due to the ingestion of surface residues of arsenic on plant material. The U. S. Public Health Service tolerance level for arsenic in edible plant material is 2.6 ppm and most products grown on arsenic-treated soils would comply with this requirement (32,33). Even though the arsenic additions reported in Table 5 caused yields to decrease to approximately half of those obtained on unamended soil, the tolerance level was not exceeded in the above-ground portions of the plants (31). Tobacco seems to be an exception to this general rule. Concentrations as high as 14 ppm arsenic in flue-cured leaves of tobacco grown on a soil which received 54 kg of arsenic/ha (as lead arsenate) have been obtained (23). This poses an additional health hazard for smokers. There is evidence to suggest a relationship between the arsenic content of cigarettes and lung cancer (34). One might expect root crops (e.g., potatoes) to contain high levels of arsenic when grown in treated soils. However, this is not the case, as most of the arsenic (2 to 3 ppm) is confined to the peelings, and much of this arsenic probably is due to minute quantities of contaminated soil adhering to the tuber surface (27,35). Little arsenic accumulates in the reproductive tissues, and hence seed crops would not exceed the arsenic tolerance level even where phytotoxicity reduced growth by 50% (24). In general, available soil arsenic is well correlated with arsenic concentration in the whole plant, but because plants tend to exclude arsenic from seeds and fruits, soil tests for available arsenic would not be reliable predictors of the concentration likely to be found in edible plant tissue (24). The contamination of the aerial portions of plants with arsenic-containing dust from treated soil can occur (26). This possibility casts some doubt on published values for arsenic uptake in cases where steps were not taken to remove adsorbed soil particles from plant organs (26). 70 Arsenic Toxicity in the Future Because the use of inorganic arsenicals on nearly all vegetable and agronomic crops has been banned since 1968 in the U. S. (7), the major problem to be addressed in the future is the reclamation and restoration of arsenic-contaminated soils to their optimal production level. A number of possibilities exist in this regard. As pointed out earlier, the available arsenic level in many soils slowly decreases with time, although no information is available to predict how long would be required for the available arsenic to decrease to background concentrations. Another approach which has been proposed is to add sufficient phosphate to the system to depress arsenate uptake by the plant. This has been shown to be true in solution cultures (36,37), but in soil systems the results are less clear because phosphate and arsenate compete for the same sorption sites in soil. Results have been reported where phosphate additions have either had no effect (38) or increased arsenic toxicity as a result of displacement of arsenate from sorbed sites by phosphate into solution increasing its availability (19,20). Thus phosphate additions to decrease arsenic phytotoxicity appear to be of little value. Since arsenate is sorbed by iron and aluminum compounds, another obvious approach is to add iron or aluminum salts, along with sufficient lime to neutralize the acidity produced, to increase the surfaces potentially available for arsenate sorption. While this is theoretically feasible, such large amounts of iron and aluminum need to be added that this approach is uneconomical. Deep plowing to dilute the arsenic concentration in the surface soil and expose the arsenate to more sites for fixation has been suggested as one of the most economical methods of decreasing toxicity (17,39). Arsenic toxicity has sometimes been lessened by growing and plowing under tolerant cover crops such as rye or Sudan grass (22). Because excessive arsenic in some fruit trees induces zinc deficiency, foliar sprays of zinc sulfate or zinc chelates have in some cases helped to overcome arsenic toxicity. Concurrent high nitrogen applications to the soil also were beneficial (40)(41)(42). In soils in which added phosphate desorbs arsenate, deliberate leaching of the soil after phosphate addition may be a viable approach to removing arsenic from the root zone (20). This would be particularly true of sandy soils which have been shown to desorb arsenate and are easy to leach (19,20,27). Work in the Netherlands has indicated that in humid regions leaching of arsenic can result in considerable attenuation of phytotoxicity. A half-life value for arsenic in soil of 6.5 + 0.4 yr was calculated from this study (42). Environmental Health Perspectives Support of M.E.S. from the Council for Scientific and Industrial Research, Pretoria, South Africa, is gratefully acknowledged.

v3-fos

2016-05-04T20:20:58.661Z

1978-01-01T00:00:00.000Z

23872286

s2

Simultaneous typing of alfa s1, beta, kappacaseins, betalactoglobulin, alfa-lactalbumin and serum albumin in cow milk by polyacrylamide gel electrophoresis The possibility for genetic change in production traits of salmon looks good when considering the magnitude of genetic variation together with the selection differential which can be applied. At the Fish Breeding Expevim.ental Stations Sunndalsora and Averoy a selection programme is being carried out. Selection for body weight is based on strain, fulland half sibs and individuals information. Strongest selection was applied on weight after two years in the sea. The first generation respons of salmon fingerlings to selection for high weight at igo-day old was as high as 30 p. ioo. This response is higher than expected and can not be explained as a direct selection response only. It is thought that part of this response is a domestication effect. The possibility for genetic change in production traits of salmon looks good when considering the magnitude of genetic variation together with the selection differential which can be applied. At the Fish Breeding Expevim.ental Stations Sunndalsora and Averoy a selection programme is being carried out. Selection for body weight is based on strain, full-and half sibs and individuals information. Strongest selection was applied on weight after two years in the sea. The first generation respons of salmon fingerlings to selection for high weight at igo-day old was as high as 30 p. ioo. This response is higher than expected and can not be explained as a direct selection response only. It is thought that part of this response is a domestication effect. Cholesterol concentration and alkaline phosphatase activity in blood plasma were analysed in zqq Swedish calves of both sexes and 249 young Danish bulls. Thyroxine degradation rate was in addition determined in the Danish bulls. In both animal materials, blood samples were taken one to three times per animal at different ages. The cholesterol level was higher in the heifers than in the bulls at all ages, while the alkaline phosphatase activity was higher in the heifers at 10 ans 1 6 months. Both cholesterol concentration and thyroxine degradation rate rose with increasing age, while the age influence on alkaline phosphatase activity was more complicated. The repeatabilities of cholesterol concentration between ages were in the range 0 . 2 to 0 . 4 . The repeatability estimates of alkaline phosphatase activity were between 0 . 3 and 0 . 5 For thyroxine degradation rate, the estimate for bulls was o. 4 . When average data from the three ages were used, the following estimates of heritabilities and genetic correlations were calculated. The heritability of cholesterol concentration was o. 4 and o.8 for the two animal materials. The heritability estimates of alkaline phosphatase activity were 0 . 3 and 0 . 7 respectively, and for thyroxine degradation rate, the estimate was o.8. The genetic correlations between growth rate and the blood parameters were 0 . 7 and o.8 for cholesterol concentration, 0 . 2 and T . 0 for alkaline phosphatase activity and o. 9 for thyroxine degradation rate. Due to the positive genetic correlations in bulls between growth rate and the blood parameters studied, it may be possible to include them in an indirect selection for growth rate. S-75o 0 7 U pp sala, Sweden A simple method of horizontal polyacrylamide gel electrophoresis in a discontinuous buffer system (Tris-citrate-borate, pH 9 . 0 ) was described for the simultaneous phenotyping of as , -, o -, x-caseins and !-lactoglobulin, x-lactalbumm and serum albumin in cow milk. A step gra-dient gel was used. Polymorphism was observed for a si -, 3-, x-caseins and 3-lactoglobulin. The pretreatment of milk samples was found to be an important step for the improved separation observed. As serum albumin fraction in mastitic cows was considerably stronger than in milk from normal cows, this method also may be suitable for an indirect diagnosis of mastitis. Dep. of Animal Bveeding and Genetics, The Swedish Univevsity of Agricultural Sciences, 7$0 0! Upfisala, Sweden Horizontal polyacrylamide gel electrophoresis with a discontinuous buffer system (Triscitrate-borate, pH 9 . 0 ) and io p. ioo separation gel was used to analyse plasma samples of Zlotnicka breed cf pigs from Poland. The material consisted of samples from 3 sires, 29 dams and their 374 offsprings. Six different postalbumin (Po) phenotypes were observed. The analysis of family data showed that the Po types were controlled by three codominant autosomal alleles PoA, PoB and PoC. Each of the homozygote type showed a major band and 3 weak bands moving cathodic to the major band. In the heterozygote types, all fractions of the two homozygotes types were represented with half the staining intensity. It was, however, found difficult to distinguish between the PoA and PoAB types. Prealbumin (Pa), transferrin (Tf) and hemopexin (Hpx) could also be typed on the same gel. TfB and TfC could be differentiated only when the pH of the gel and electrode buffer was reduced to !.5. The use of pH 9 . 0 buffers was however found to be a more convenient and rapid method for the simultaneous typing of samples for Pa, Po, Hpx and Tf on the same gel. Further studies are needed to identify the polymorphic Po protein described in the present report. In order to estimate breeding values for dairy bulls, the individual test day results in a lactation were combined, using selection index theory. The method can in theory allow lactations of varying length (varying numbers of tests) to be combined and also the economic weighting of individual test days to differ. POST ALBUMIN VARIANTS Some of the results of general interest from the aspect of selection index theory are presented here. These concern the influence of genetic correlations among individual test days and their heritability on accuracy of index, variation in selection index weights, and expected response. Accuracy of index was not affected when the genetic correlations among the test days were altered or when the phenotypic variances differed. Individual b-values showed a wide variation and were especially affected by changes in the pattern of heritabilities for the individual test days. The percentage distribution of expected genetic response on individual test days was not affected by the wide variation in b-values in some alternatives. Altering the phenotypic variation of single traits had the greatest effect on the percentage distribution. Finally, it was concluded that as selection index theory assumes error-free parameters, the choice of parameters in any given situation is of prime importance. The commonly used YTI -value is not efficient for measuring the influence of variations in parameters.

v3-fos

2016-05-04T20:20:58.661Z

1978-06-01T00:00:00.000Z

8583409

s2

Test procedures to evaluate effects of chemical exposure on fertility and reproduction. Various methods have been developed to assess the effects of chemical substances on reproduction. In some instances, the tests have been developed to define the effects of treatment on specific segments of the reproductive cycle. In other cases, studies are conducted to determine the cumulative effects of treatment during one or more generations. The structure, advantages, and disadvantages of three types of conventional reproduction studies are reviewed. An outline of the procedural sequences, observations, and record evaluation required in the three-period reproduction study, the three-generation reproduction study, and the multigeneration reproduction study are presented. Introduction Experiments are conducted to assess the effects of chemical substances on reproduction for a variety of reasons. These may include: screening programs to discern therapeutic materials to preserve pregnancy; test systems to identify materials that can be used for contraceptive uses; and finally, tests to identify unwanted chemical alterations on reproduction. It is evident that the induction of a similar result from several different substances could lead to different interpretations regarding the potential hazard. An example might involve a series of experiments in which the absolute prevention of implantation was produced if the substances were administered within 48 hr after copulation. This result would be viewed as highly favorable if the test system were designed to discover a postcoital contraceptive. The result would be extremely unfavorable if we were evaluating an over-the-counter analgesic; this observation would prevent further consideration of the substance as an over-thecounter drug. A similar result from an industrial chemical used in a completely closed system would not weigh heavily in the hazard evaluation of the material. Many factors must be considered when performing a hazard assessment for use in a risk-benefit analysis. Reproduction is a cyclic process ( Fig. 1) that can be modified at any one of several points. Classically, changes occurring during differentiation and development are considered teratogenic responses. Embryonic exposure to appropriate doses of mercury or thalidomide can result in abnormal morphological development. Changes produced during embryonic exposure might include somatic abnormalities that would subsequently prevent gametogenesis. Postnatal exposure to estrogenic or progestational compounds, ionizing irradiation, and nutritionally inadequate diets can also impair gametogenesis. During the preimplantation stage, blastocyst survival can be reduced by exposure to metallic ions, particularly copper or mercury. Tubular or uterine infections can also reduce the blastocyst survival. An impairment in libido, reducing the incidence of copulation, could result in a direct reduction in the number of viable blastocytes. Materials producing a sedating effect will reduce mating behavior in experimental animals. Certain nutritional deficiencies and nonreproductive-system systemic toxic effects can reduce mating performance. Impairment in reproductive efficiency can result from adverse effects on neonate survival at parturition or during lactation. Parturition initiation may be initiated prematurely or may be delayed. Uterine motility might be influenced in a manner that would impair parturition; maternal behavior changes might be induced that would cause rejection of the newborn. Maternal behavior effects commonly observed in rodent animals involve hyperactivity which may lead to cannibalism, or hypoactivity which may result in neglect of the young. Lactation may be adversely affected by delaying initiation or reducing the total volume or nutrient content of milk. It is also possible for chemical agents to be transferred into the milk and be subsequently consumed by the nursing young. Test procedures used to evaluate the effects of chemical exposure on fertility and reproduction must be capable of detecting functional impairments in each of the sequential events shown in Figure 1. Test Procedures Studies designed to evaluate the effects of chemicals on the reproductive processes usually utilize rodent animals. These animals are selected because of their early sexual maturity, short gestation and lactation periods, and their comparative ease of maintenance (Table 1). Evaluation should not be limited to rodents if other organisms will provide information more directly applicable to human exposure or response. Historically, two different types of tests have been recommended for evaluation of overall reproductive affects. One has been recommended by the Food and Drug Administration, Bureau of Drugs; the other by the Food and Drug Administration, Bureau of Foods and by the Environmental Protection Agency in their administration of the Federal Insecticide, Fungicide and Rodenticide Act for pesticide regulation. A third type of protocol has been proposed (7). This protocol retains the advantages of the three-generation reproduction study but requires only approximately one-half the calendar time to complete. These test procedures reflect the anticipated differences in exposure of the consumer to drugs, food additives, foods, and other materials. The types of observations taken in each type of study are similar. Differences exist in length of exposure to the test material and frequency of observations. Routinely, separate studies are conducted to evaluate potential teratogenic or mutagenic responses. Three-Period Reproduction Study The three-period reproduction study is usually specified for assessing the undesired reproductive effects of drug agents. Drugs are normally taken intentionally; therefore, it is assumed that exposure during various periods of the reproductive cycle can be controlled. Environmental Health Perspectives The three periods of exposure are outlined in Table 2. Period II restricts chemical exposure to the period of organogenesis and is, thus, a conventional theratology study. Procedures for the evaluation of a teratogenic response have been presented (7). In period I, the males and females are dosed with the test material on a daily basis for a period equal to the gametogenic period for that sex prior to mating. Treatment of the males and females continues during the mating period. Females are treated continuously during gestation and lactation. The parameters that are evaluated include fertility in both the male and female, lactation and behavioral effects in the female, and neonatal survival and development in the offspring. The experiment is terminated when the litters are weaned. Period III permits treatment only during the last fourth of gestation and during the subsequent lactation period. The male receives no treatment. Lactation and behavioral responses are evaluated in the female; survival and development are evaluated in the neonates. An advantage of this test sequence is the partial separation of exposure during specific segments of the reproduction period. Another advantage is that only four months of calendar time are required to complete this test sequence. A serious disadvantage is that there are no subsequent studies conducted to evaluate the reproductive performance of the F1 offspring. Three-Generation Reproduction Study The conventional three-generation reproduction study is usually used to evaluate direct or indirect food additives and pesticide residues on food crops. June 1978 The Fo parental animals are exposed to treatment for approximately 60 days prior to mating, (Fig. 2). Treatment then continues, uninterrupted, until the final F3b litters have been weaned. Each parental generation produces two litters. The Fib litters provide the F1 parental generation producing the F2,, and F2b litters. The F2b litters provide the F2 parental generation. This test procedure offers the advantage of providing subsequent reproductive performance evaluations on animals in both the F1 and F2 parental animals following in utero exposure. Two disadvantages are associated with the three-generation reproduction study; there is no sequence separation and the study requires 20 months to complete. Multigeneration Reproduction Study A multigeneration reproduction study protocol has been developed (7); treatment of the Fo parental females is initiated prior to the implantation of the Fia litters (Fig. 3). taken in this protocol are similar to those taken in the conventional three-generation study. This study does not provide sequence separation. It has the advantages of providing in utero exposure and subsequent reproductive performance evaluation of the F1 parental generation and a required calendar time of the ten months to complete. This reduction in required calendar time, when compared to a threegeneration study, offers the opportunity of increasing the number of materials evaluated and defining the affected sequence if effects are observed. Materials and Methods Similar animal models and methods are utilized in each of the general reproduction studies described. Normally an experiment consists of a control group and three treatment groups. Fo parental animals are obtained from a stock colony at an age to provide young sexually mature animals after the required pre-or post-mating treatment period. The procedures that are customarily used require approximately 10 male and 20 female animals in each treatment group. Mating Procedures and Records When the parental animals reach sexual maturity (Table 1), each male is paired with two females from the same group. Successful mating is determined by the presence of a copulation plug, sperm, or blood in the vagina. If a female does not exhibit additional evidence of copulation at the end of a subsequent estrous cycle, she is returned to her original cage. At the end of two estrous cycles, all males within the same group are rotated and exposed to different females in the same group. No more than three males should be paired with any female during a given breeding cycle. The number of observed copulations, the number of estrous cycles required to obtain a mating, and the number of resulting pregnancies are recorded. These data are used to calculate the mating and fertility indices ( Table 3). The offspring are weaned 21 days post partum. If a second litter is to be produced, the females are mated approximately 28 days after the first litter is weaned. All pups are examined for physical abnormalities at birth. The numbers of viable, stillborn and cannibalized members of each littler are recorded. Observations for clinical signs are made daily. The numbers of survivors on days 1, 4, 12, and 21 post parturition are recorded. On the fourth day of lactation, litters with more than ten pups may be reduced to that number by sacrificing randomly selected individuals. A final examination for physical abnormalities is made. Individual body weights are recorded at weaning on lactation day 21. These data are used to calculate the progeny survival indices shown in Table 4. Body Weight Evaluations Body weights and weight gains should be recorded to determine if the treatment is having an adverse effect on food consumption or general animal well being. The following sequence will provide adequate information: parental females, weekly from selection and days 1, 4, 12, 21, and 28 following parturition; parental males, weekly from selection and at time of paring for mating; progeny, as litters on days 1, 4, and 12 following birth and individually at weaning on day 21. Results Evaluation Data obtained from treated groups should be compared by appropriate statistical methods to the concurrent control results. Applicable methods should be used for parameters which yield discon-tinuous or nonparametric results. Parental body weight gains and weight of progeny may be compared by F-test and students' t-test (8). Anomalies may be compared by either the chi-square or binominal expansion method (8). Reproductive and survival indices may be compared by a nonparametric rank order method (9). Other statistical methods may be substituted where appropriate.

v3-fos

2017-07-29T02:08:52.610Z

2005-01-01T00:00:00.000Z

21931969

s2

A new coronavirus-like particle associated with diarrhea in swine Summary Coronavirus-like particles were detected by electron microscopy in the intestinal contents of pigs during a diarrheal outbreak on 4 swine breeding farms. Diarrhea was reproduced in experimental pigs with one of the isolates, designated CV777, which was found to be distinct from the 2 known porcine coronaviruses, transmissible gastroenteritis virus and hemagglutinating encephalomyelitis virus. Summary Coronavirus-like particles were detected by electron microscopy in the intestinal contents of pigs during a diarrheal outbreak on 4 swine breeding farms. Diarrhea was reproduced in experimental pigs with one of the isolates, designated CV777, which was found to be distinct from the 2 known porcine coronaviruses, transmissible gastroenteritis virus and hemagglutinating encephalomyelitis virus. In 1946, Do Yr.E and HUTC~I~GS (2) described a viral diarrhea, in swine and called it transmissible gastroenteritis. Until recently, transmissible gastroenteritis virus was the only virus known to be specifically associated with diarrhea in swine of all ages. In 1976, following the discovery of rotaviruses in different, animal species, a porcine rotavirus was detected in the feces of pigs with diarrhea (14). Diarrhea could be reproduced experimentally in piglets with this virus. In a search for rotaviruses on Belgian swine breeding farms with diarrheal problems, a new coronavirus-like particle was detected b y electron microscopic examination of intestinal or fecal samples from sick pigs. The present report describes the morphology of this coronavirus-like particle, and shows that it is distinct from the known porcine coronaviruses and causes diarrhea. Up to now, the only coronaviruses isolated from swine have been transmissible gastroenteritis virus {TGEV) and hemagglutinating encephalomyelitis virus (HEV). TGEV has been described as a cause of diarrhea in swine in countries all over the world (13). Numerous studies have been performed on the v i r u s --a n i m a l interactions of TGEV, which is usually detected either by its isolation from fecal These studies were supported by the Institute for the Encouragement of l~esearch in Industry and Agriculture (IWONL), Brussels, Belgium. material in cell cultures or by immunoflu orescence in the smM1 intestinal epithelium of infected pigs (7,12). TGEV infections can also be diagnosed serologically. J:IEV was first described in Canada in 1962 as a cause of centrM nervous disorders in pigs (4). The same virus was later associated with a disease syndrome called vomiting and wasting disease in several European countries (1,8). The virus can easily be detected by cultivation in several porcine cell cultures (11). Both TGEV and HEV have been classified as coronaviruses mainly on the basis of their specific morphology (10). In 1977, a sudden outbreak of diarrhea was observed in swine of all ages on 4 Belgian swine breeding farms. The morbidity in sows was very variable and the animals recovered after a diarrhea which lasted 3 to 4 days. All the pigs showed a watery diarrhea. Death occurred up to the age of 7 days and the overalI mortality rate in these piglets was approximately 50 per cent (9). It decreased with increasing age. TGEV was suspected as the cause of this diarrhea. However, the direct immunofluorescence test for the diagnosis of TGEV, which is routinely applied on cryostat sections of the small intestine of sick pigs, was negative for these pigs. The absence of seroneutralizing antibodies to TGEV in the blood of sows collected 6 to 12 weeks after the outbreak confirmed that TGEV was not involved. In an attempt to arrive at an etiologie diagnosis, fecal material and intestinal contents from pigs of each farm were subsequently processed for examination in an electron microscope by negative staining. They were diluted t to 5 (v/v) in phosphate-buffered saline, pH 7.3 and clarified at 3000 × g, at 4 ° C, for 30 minutes. The supernatant was layered on top of a 20 per cent sucrose solution and centrifuged at 150,000 x g, at 4 ° C, for 40 minutes. The resulting pellet was resuspended in a few drops of distilled water, placed on 200 mesh formvar coated grids, and stained with 2 per cent K-phosphotungstate, pH 6.1. Grids were examined using a Zeiss EM 9 S-2 electron microscope at an acceleration voltage of 60 KV. Micrographs used for particle size measurement were taken at an instrumental magnification of 28,000 X, which were then photographically-enlarged to 84,000 X or 168,000 ×. gotavirus particles were not detected. However, eoronavirus-like particles were observed in specimens of pigs from each of the 4 breeding farms. One of the fecal samples containing these coronavirus-like particles was designated CV777 and was used for further studies. The etiologic relationship between the corona virus-like particles, CV777, and the occurrence of diarrhea was established by oral inoculation of a 20 per cent suspension of the fecal material contMning CV777 into a one day old colostrumdeprived pig. The experimental pig was killed 30 hours later, at the height of diarrhea, and a virus stock was prepared from an homogenate of its small intestine and contents. A bacteria free filtrate of the supernatant of a 20 per cent suspension of this material was used for inoculation of 12 colostrum-deprived-hysterectomy-derived piglets, kept i n isolation. Seven control pigs were used. The pigs were inoculated at the age of 3 to 15 days. All the inoculated pigs developed a watery diarrhea within 24 to 36 hours after inoculation whereas the control animals remained normal. Coronavirus-like particles were detected by electron microscopic examination in the watery feces or intestinal contents of each of the experimentally inoculated pigs. Such particles were not found in the feces of the same pigs prior to inoculation or in the fecal samples of the control animals. The particles, shown in Figure 1, had typicM coronavirus morphology. They were pleomorphic with a range in diameter of 95 to 190 nm, including the projections, which were approximately 18 nm in length. Most particles were between t30 and t70 nm in diameter. The projections formed a single fringe radiating from the core. They appeared to be club-shaped. Only the dilated distal ends of the projections were seen on the micrographs. The negative stain also appeared to settle on the surface of some particles and an electron opaque central area covered by surface projections was often seen (Fig. 1 a --a r r o w s and lc). No internal structure was observed. It was impossible to distinguish these coronavirus-like particles morphologically from TGEV or H E V particles from similar preparations. Other particles, different from these coronavirus-like particles, were also observed in the majority of the fecal samples. As seen in Figure 2, they were pleomorphic and very variable in size, ranging in diameter from 95 to 650 nm with an average diameter of 190 to 225 rim. They carried numerous short projections, of approximate length 9 nm, on their surfaces. Similar particles of unkno~m identity have been described in human and animal fecal samples (3,5,6). In the present studies such particles have also been found in the solid fecal samples of the control pigs. They appeared, therefore, not to be associated with diarrhea. Rotaviruses and other recognizable virus particles were not seen in control or experimentally inoculated pigs. As already mentioned, TGEV was eliminated as the cause of the diarrhea on the origina 1 farms. AdditionMly 9 out of the 12 experimentally inoculated pigs, killed at, the heigth of diarrhea, were negative for TGE viral antigens in their small intestinal epithelium by the direct immunofluorescenee test. Furthermore, the remaining 3 pigs, inoculated with CV777 at the age of 15 days, were allowed to recover after a diarrhea which lasted 4--5 days. A serum sample, collected from these pigs 3 weeks later, did not contain neutralizing antibodies against the cell culture adapted Purdue strain of TGEV. Fig. 2. One eoronavirus-like particle CV 777 (arrow) together with pleomorphie particles of unknown identity. Bar represents 100 nm The possibility that the CV777 particles consisted of H E V was less likely since the latter virus does not cause diarrhea in pigs. Cryostat sections of the sma~ intestine of experimentMly inoculated pigs were negative for fluorescence by the direct test using a conjugate directed against the VW572 isolate of H E V (8). Furthermore, the pigs that had been allowed to recover did not possess hemagglutination-inhibiting or seroneutralizing antibodies against this t t E V isolate. Preliminary attempts were made to cultivate the coronavirus-like particle, CV777, in primary pig kidney cell cultures and in secondary porcine thyroid cells. Four weekly blind passages were made. The cells were examined for cytopathic effect and hemadsorption with chicken red blood cells, and the cell culture fluids were examined for hemagglutination. No evidence of viral replication in the cell cultures was obtained. It is kno~n that the tIEV can easily be isolated in primary pig kidney cell cultures using the same criteria (8). The present data suggest that, as well as TGEV and HEV, another previously unrecognized coronavirus-like virus is prevalent in swine. The results indicate that diarrhea can be reproduced in experimental pigs with this virus and that it is associated with certain outbreaks of epizootic diarrhea on Belgian swine breeding farms. More details on the clinical disease in the field and on the results of the experim e n t a l infections will be reported later.

v3-fos

2018-04-03T06:24:12.783Z

1978-01-01T00:00:00.000Z

46524004

s2

Effects of various metabolites (sugars, carboxylic acids and alcohols) on riboflavin formation in non-growing cells of Ashbya gossypii. The effects of various sugars and sugar derivatives on flavinogenesis were examined using non-growing cells of a high flavinogenic mold, Ashbya gossypii. Glucose, fructose and galactose were found to be the most stimulative. Glycerol and glucono-delta-lactone were less stimulative; next in order were n-propanol, n-butanol, glycols and butanediols, which were likewise effective; acetate, lactate and pyruvate were slightly stimulative. In contrast, ribose, xylose, arabinose, ribitol, citrate, succinate, oxaloacetate, glyoxylate and malate were rather inhibitory, in additions at 1.0%. Among these compounds, ethanol (1%) greatly stimulated riboflavin formation. Maximum flavinogenesis with the above stimulants was attained by the additions of 1% ethanol, 1.25--3.0% glucose, 1.25% glycerol, 4.0--6.0% propane and butanediols, 1.0% pyruvate and 0.9% acetate after 37 hr incubation, respectively. These compounds inhibited flavinogenesis with increasing concentrations above their optimum concentrations. The stimulation effect of ethanol far exceeded those of other stimulants but ethanol had almost no effect on growth and pH values during incubation. With the addition of ethanol (1%) during incubation, maximum formation (1,776 microgram/g wet mycelia) of riboflavin was achieved when added at the start of incubation and the most effective utilization was observed when added at the logarithmic phase of flavinogenesis, although the maximum formation of riboflavin in the latter case was much lower than in the former case. The relation of sugar metabolism, especially ethanol metabolism, to flavinogenesis was discussed with the flavinogenic activities of these additives. In the present paper, we tried to obtain some knowledge on the sources of the o-xylene ring, the 4-carbon compound at the biosynthetic level of 6, 7-dimethyl -8-ribityllumazine, or the ribityl side chain, using non-growing cells of a high flavinogenic mold, Ashbya gossypii. MATERIALS AND METHODS Materials. Sodium acetate, sodium pyruvate, sodium formate, n-propanol, iso-propanol, n-butanol, ethylether, ethylacetate (95%), formic acid (90%), acetoin dimer and ribitol were purchased from Wako Pure Chemical Industries, Ltd., Osaka. Ribose from Kohjin Co., Ltd., Tokyo. Other tested compounds were from Nakarai Chemicals, Ltd., Kyoto. Peptone from Daigo Nutritive Chemicals, Ltd., Osaka was used for the preculture and the culture in a basal medium and that from Kyokuto Pharmaceutical Industrial Co., Ltd., Tokyo was used for the slant culture. Yeast extract and sucrose were from Nakarai Chemicals, Ltd., Kyoto. Tween 80 was from Wako Pure Chemical Industries, Ltd., Kyoto . Malt extract and agar were from Difco Laboratories, Inc., Detroit, U.S.A. Potato extract was prepared from commercially available potatoes which were cut up and boiled in water (5ml/3g) for about half an hour. Methylglyoxal (40% in water), glyoxal (40% in water) and acetoin (80% in water) were used as the reagents under test. Diacetyl dimer was prepared by self-condensation in an alkaline solution (22). Other chemicals were of the best quality available. Fermentation. Ashbya gossypii (IFO 1355) was obtained through the courtesy of the Institute for Fermentation, Osaka, and maintained on an agar slant con taining 2.0% potato extract, 0.5% yeast extract, 2.0% sucrose and 2.0% agar (pH 5.5). The mycelia were subcultured every seven days on an agar slant con sisting of 1% glucose, 0.5% peptone, 0.3% yeast extract, 0.3% malt extract and 2.0% agar (pH 6.5). A loop of the mycelia was taken from the seven days agar pathway of the pentose phosphate cycle enhanced riboflavin formation to a little less extent than hexoses as above. Monoatomic alcohols, methanol , n-, iso propanol, n-, sec-butanol and benzyl alcohol were not stimulative except for n propanol and n-butanol. Whereas, ethyleneglycol, propyleneglycol and tri methyleneglycol or 1, 3-butanediol, 1, 4-butanediol and 2, 3-butanediol stimulated riboflavin formation by 1.5-2.0-fold compared to the control. On the other hand, the metabolites in a tricarboxylic acid cycle, citrate, malate, fumarate , oxaloacetate, succinate and glyoxylate inhibited riboflavin formation except for citrate without effect and succinate being a week stimulant. Carboxylic acids , formic, propionic, butylic and glyoxylic acids and acetoacetate restricted riboflavin formation as well although acetate, lactate, pyruvate and glucuronate (sodium salts) were slightly stimulative. In addition, among the members and their deriva tives in a 2, 3-butanediol pathway, acetoin and 2, 3-butanediol were stimulative but diacetyl, acetoin dimer and diacetyl dimer were inhibitory. The trapping agents, which are known to conjugate with the diamino-and triamino-type pyrimidine to form the pteridine ring (23)(24)(25), glyoxal, diacetyl, diacetyl dimer and methyl glyoxal, reduced riboflavin formation. Among all these compounds ethanol stimulated riboflavin formation to a great extent. 2. Effects of ethanol at varied amounts on riboflavin formation during non-growing cell incubation Effect of ethanol in a 0-2.0% concentration range on riboflavin formation was examined till 86hr after non-growing cell incubation. Results are given in Seven flasks, differing in concentration of ethanol added, were incubated till 86hr after vacuum infiltration. During incubation an appro priate volume (10ml) of the mycelial mixture was sampled at the indicated incubation times from each flask and used to determine the total flavins. on flavinogenesis were studied by incubation with non-growing cells of A . gossypii for 37hr. Results are shown in Fig. 2. Maximum production appears to be respectively attained at 1.0, 1.25, 1.25, 1.0 and 0.9% in the order of ethanol , glucose, glycerol, pyruvate and acetate. Riboflavin formation was more stimula tive by the additions of ethanol, glucose, glycerol, acetate and pyruvate in this order. Of these compounds, the addition of ethanol at 1% greatly enhanced riboflavin formation under experimental conditions. Moreover, non-growing cell incubation was carried out for 37hr in the presence of various concentrations of ethanol, glucose, pyruvate, diacetyl , acetoin and 2, 3-butanediol and effects of these compounds on flavinogenesis were followed . Results are shown in Fig. 3. Ethanol was most stimulative of these compounds , showing the maximum yields of riboflavin at 1.5% in this case. 2, 3-Butanediol exerted the stimulation effect in higher concentrations, in which maximum forma tion was attained at 5%. While, the other metabolites in a 2, 3-butanediol pathway , diacetyl and acetoin, exhibited only a slight stimulative effect on flavinogenesis in tion under the experimental conditions used. In this section, the relation of ethanol to flavinogenesis is further examined by the addition of ethanol (1%) in the course of non-growing cell incubation. Results are given in Fig. 6, show that the earlier the addition of ethanol, the higher the maximum formation of ribo flavin become. However, the utilization of ethanol after addition was most effective when ethanol was supplemented at the logarithmic phase of flavinogenesis, though the maximum yields of riboflavin were much smaller compared to those with the addition at the start of incubation. Moreover, the addition of ethanol at the stationary phase of flavinogenesis, especially even after 61 hr incubation, enhanced riboflavin formation. Accordingly, these results may demonstrate that ethanol principally serves as the carbon source and the energy source needed for the formation of riboflavin although the supposition may be easily expected also from the non-growing conditions of the mold. Each ten grams of the starved mycelia was placed in nine flasks of a 1 liter Erlenmyer flask containing 0.1M phosphate buffer (200ml). A small volume of ethanol was added to the flasks containing the mycelial suspension to give a final concentration of 1% at the incubation times shown by arrow symbols in the figure, without taking account of the increase of the volume. Incubations were respectively carried out under the same conditions throughout the experiments. Riboflavin formation in the respec tive experiments was followed by the same methods as in Fig. 1. The control experi ments are shown by solid circles. DISCUSSION Many studies have been done to elucidate the origins of the o-xylene ring and the ribityl side chain on the riboflavin molecule. PLAUT showed the specific incorporation of acetate (11,12) and the poor incorporation of pyruvate (26) into the o-xylene ring by tracer experiments, but GOODWIN and TREBLE (13) exhibited the effective incorporation of labelled acetoin, but not acetate, into riboflavin. In addition, ALT and AL-KHALIDI (14) have reported that metabolites in a pentose phosphate cycle are highly incorporated but acetate, pyruvate and acetoin are slightly incorporated into the isoalloxazine ring and the ribityl side chain of riboflavin, and, recently, that a glycolytic pathway is closely related to the bio synthetic pathway of the 4-carbon compound (15). BRYN and STORMER (16) found the possibility of the direct involvement of a 2, 3-butanediol pathway in the originat ing pathway of the 4-carbon unit. Thus, the origin of the 4-carbon compound remains unclear. On the other hand, it is to be noted that other alcohols, glycols, propane and butanediols show a good stimulation in flavinogenesis in higher concentrations. Thus, the mycelia appear to effectively utilize also these alcohols except ethanol as carbon sources and thus supply their metabolites for the formation of riboflavin. Experiments to clarify the origins of the 4-carbon compound and the ribityl group on the riboflavin molecule are under way with these effective alcohols.

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Field concentrations and persistence of polybrominated biphenyls in soils and solubility of PBB in natural waters. Soil samples were collected from 28 fields which had received manure from Michigan's most highly contaminated dairy herds. The number of fields in each concentration range of PBB in soil were: 2, not detectable; 15, 0.0 to 8.0 ppb; 6, 14-102 ppb, and 5, 153 to 371 ppb. Plant tissue sampled from the 10 most highly contaminated fields contained no detectable PBB. No evidence of significant degradation of PBB was noted after 1 year incubation in soil. When 14C hexabromobiphenyl and heptabromobiphenyl isomers were incubated in soil less than 0.2% of the 14C was volatilized. Also gas chromatographic analysis of soil extracts showed no difference in recovery of the six major PBB isomers between sterilized and nonsterilized soil. Analysis of these extracts by thin layer chromatography and autoradiography showed no 14C-PBB intermediates. Photodegradation products of the major hexa- and heptabromobiphenyl isomers showed more but still minor (approximately 3%) biodegradation in soil. Much of the photodegradation products appeared bound to soil, since these products could not be extracted from soil. Photodegradation does not appear to be a significant fate of PBB in manures spread on fields since no change was noted in the relative concentrations of isomers in soil samples from our field survey. Studies with distilled, tap, river, and soil waters showed that PBB solubility was markedly influenced by water composition. Introduction Some unexpected soil pollution occurred in Michigan as a result of the accidental addition of an industrial flame retardant, FireMaster BP-6 (a mixture of polybrominated biphenyls, or PBBs), to livestock feed in place of magnesium oxide (1)(2)(3)(4). This incident led to contamination of many Michigan farm soils through disposal of PBB-tainted manure, milk, feed, and dust cleanings from buildings, etc. This contamination posed questions about the fate of PBBs in soils, including the potential for recycling PBB to farm animals, wildlife and man. In our preliminary greenhouse studies, we found no PBB in the tops of orchard grass and carrots grown in soils amended with high concentrations of PBB (5). Some traces (20-40 ppb) of PBB were found associated with carrot roots. More recently, additional laboratory and greenhouse studies have confirmed that PBB was not transfqrred from contaminated soil to plant tops (6). Therefore, significant contamination of clean animals by recycling PBB through feed crops is unlikely. We have also investigated the degradation (5) and retention (7) of PBBs in soils. Initial degradation studies showed PBBs to be extremely persistent, with only one pentabromobiphenyl isomer showing any significant disappearance after 24 weeks of incubation in soil. Leaching and adsorption experiments with the major hexabromobiphenyl isomer in PBB suggested that PBBs should not leach below the depth of incorporation in surface soils. This study was undertaken to determine the concentration of PBBs in Michigan soils to which PBB contaminated manures had been applied, to further evaluate the degradation of PBBs in soils by using 14C-PBB and to determine how the solubility of PBB was influenced by the composition of different natural waters. Field Samples Thirty Michigan farms were selected from a list provided by the Michigan Department of Agriculture of dairy herds which had >5 ppm PBB in their milk (on a fat basis). One poultry farm and 26 dairy farms were visited in April 1976. Fields which had April 1978 I received the greatest quantity of contaminated manure 2-3 years earlier were sampled. A 2.5-cm diameter soil probe was used to collect soil from the surface 20 cm. A soil core was taken from 20 random locations in the field; the soil from the cores was thoroughly mixed, and two duplicate subsamples of 50 g each were taken for extraction and analysis. Additional samples were also taken on some farms from milk dumping areas, feedlots, holding lots, manure piles not yet spread, and gardens. In August 1976, the 10 manured fields which were found to have the highest PBB concentrations were revisited. Plants were sampled from 15 random locations within each field and a soil core was taken from around the roots of the sampled plants. The plant and soil samples were a composite for each field. Two corn fields which had not been contaminated with PBB were sampled similarly to serve as controls in the event that some indigenous plant compounds produced peaks with retention times similar to PBB. All soil samples were air-dried and passed through a 2-mm sieve before extraction; plant samples were refrigerated until extracted for analysis. Since PBB is easily photodegraded by sunlight, products from ultraviolet photolysis of PBB were also added to soil. The photolyzed '4C-PBB solution was prepared by irradiating a mixture of 50 ppm FireMaster BP-6 and 9.4 ppm of '4C-PBB in hexane for 30 min in a Hanovia photolysis vessel (Ace Glass Incorporated, Vineland, N. J.). The exposed solution turned light yellow and showed some precipitate. The clear fraction was decanted and concentrated to 25 ml. This photolyzed material was diluted in ethanol (14x) to achieve a radioactive concentration similar to the nonphotolyzed sample before addition to soil. Soil Incubation A 25-g portion of Brookston sandy loam soil which had passed through a 2-mm sieve was placed in a 250-ml Erlenmeyer flask. The soil was not allowed to air dry. Half of the flasks were sterilized as follows prior to addition of the two PBB substrates. Each of these flasks received 2 ml water and, 1 hr later, 3 ml of propylene oxide was distributed dropwise on the soil surface. The flasks were covered with foam plugs and placed in a hood for 3 days to allow the sterilant to volatilize. Sterilization was confirmed by inoculating a loopfull of soil on a trypticase agar slant and finding no growth after one week of incubation. Sterilized and nonsterilized flasks received either 1 ml of the filter sterilized 14C-PBB solution or 1 ml of the ultraviolet irradiated '4C-PBB solution distributed dropwise on the soil. All flasks were then sealed with a sterilized rubber stopper. A 2-ml sterilized plastic beaker which contained 1 ml of IN NaOH was suspended above the soil to trap respired '4CO2. All samples were incubated in the dark at 28-+ 1C for 0, 3, 6, 9, or 12 months. The '4CO2 traps were counted periodically as described elsewhere (9). Four replicates of each treatment were extracted for 14C and GLC analysis after each incubation period. Solubility of PBB in Natural Waters Five waters varying in inorganic and organic content were selected for preliminary studies to determine whether composition significantly influenced PBB solubility. These waters were: (a) distilled water, glass distilled from water in which the trace organic matter had been removed by permanganate oxidation; (b) tap water, from cold water tap in laboratory; (c) Red Cedar water, collected from the Red Cedar River which flows through the MSU campus; (d) Spinks water, extracted from a Spinks loamy sand soil (1.10% organic C); and (e) Brookston water, extracted from a Brookston sandy loam soil (3.14% organic C). Water was extracted from the two soils by shaking 100 g soil with 1 liter distilled water, allowing the mixture to settle, and filtering the supernatant through Whatman GF/C glass fiber filters. These waters had the following pH and specific conductance (in ,umhos), respectively: distilled (6.3, 2); tap (8.0, 619); Red Cedar (8.3, 681); Spinks (6.3, 68); and Brookston (7.1, 61). To 1 liter of each water was added 1 ml of an acetone standard containing 1000 ppm of FireMaster BP-6 and 1 ml of acetone containing 13.9 ,ug of Environmental Health Perspectives the 14C-PBB isomers described above. The PBBtreated water was shaken on a rotary shaker at 150 rpm for 24 hr. Following shaking, an aliquot of each water was centrifuged at 10,000g in a stainless steel tube for 3 hr. The remaining portion of each water and the centrifuged water in their tubes were placed in a constant temperature chamber (28 ± 1°C) to stand undisturbed. The centrifuged (10,000g) and uncentrifuged (1g) waters were sampled periodically by inserting a pipet approximately 1 cm below the surface and removing a 1-ml aliquot for PBB analysis by liquid scintillation counting. Two aliquots were taken at each sampling time for duplicate analyses. Analyses PBB was extracted from soil with three 40-ml portions of hexane-acetone (9:1, v/v). This extraction mixture was found to be better than the benzene-isopropanol mixture used previously (5), since it reduced the amount of soil organic matter extracted yet recovered as much of the PBB. Before extraction the soil samples were moistened and vibrated on a minishaker to ensure moisture uniformity. The extraction procedure was the same as used previously except that the soil-solvent mixture was allowed to stand in the flask for 1 hr rather than overnight. Plant samples were macerated, extracted with hexane-acetone, and the extract cleaned-up by passage through Florisil as described by Chou et al. (6). Concentrated soil and plant extracts were analyzed on a Beckman GC-5 gas chromatograph equipped with an electron capture detector (7) and a 2% SE-30 on 100/120 mesh Gas-Chrom Q column operated at 250°C with carrier flow of 40 ml/min. Minimum detectable PBB concentrations were 0.1 ppb (wt/dry wt soil) for soil and 0.3 ppb (wt/plant wet wt) for plants. The "4C was assayed by liquid scintillation counting. A 1-ml aliquot of water or concentrated hexane-acetone extract was counted in 15 ml Bray's solution (10). The "4CO2 trapped in IN NaOH was counted in Bray's solution containing 4% Cab-O-Sil. All counts were corrected for quenching by external standardization and for machine efficiency. For thin-layer chromatographic analysis, the hexane-acetone extracts were concentrated to 0.5 ml; 10 ,ul was spotted on 250 ,u precoated Kieselguhr G plates (Analtech, Inc.) pretreated with paraffin according to the method of de Vos and Peet (11). The precoated plate was soaked in petroleum ether (bp 40-60°C) containing 8% of liquid paraffin until the adsorbent layer was saturated with the sol-vent. The plates were developed in paraffinsaturated acetonitrile-acetone-methanol-water (20:9:20:1, v/v). After development the plates were examined for 14C spots by autoradiography with Kodak No-Screen X-ray film, exposure time 10 days. Field Samples The PBB concentrations found in manured soils and in miscellaneous samples from highly contaminated farms in Michigan are shown in Tables 1 and 2. Concentrations in cropped fields which had re- ceived PBB-contaminated manure ranged from not detectable to almost 200 ppb for the April sampling. The presence of PBBs was confirmed in most samples by the presence of the other isomer peaks in the chromatogram. For samples with PBB concentrations too low to see the other peaks, the presence of PBB was confirmed by the ultraviolet sensitivity method of Erney (12). Since these cropped field samples came from the most highly contaminated April 1978 farms in Michigan, we expect the great majority of farm soils which were contaminated to have <10 ppb of PBB with most having "nondetectable" levels. Several miscellaneous areas had considerably higher PBB levels ( Table 2), particularly where contaminated milk had been dumped and in feedlots and holding lots. Two manure piles, which had never been spread, were sampled and found to contain approximately 1.5 ppm of PBB. These manure levels probably represent some of the highest concentrations which occurred, since these two farms had highly contaminated herds. No PBB was detected in plant tissue sampled from the ten fields which contained the highest PBB concentrations (Table 1). Plant samples included seven corn, two alfalfa, and one sudax. The second soil sampling illustrates the variability one can encounter when sampling field soils for trace quantities of an added chemical. For example, one does not need to encounter much PBB-contaminated manure residue in a random soil sample to significantly change the resulting PBB concentration. Therefore, one could expect to find some variability when resampling PBB contaminated fields. Soil Degradation The recovery of the two major PBB isomer (hexaand heptabromobiphenyls after various periods of incubation in Brookston sterile and nonsterile soil is shown in Table 3. Whether analyzed by '4C or gas chromatography the data clearly show no detectable biodegradation after 1 year. It is also striking that the 14C and GLC analysis of each peak showed virtually identical quantities on each date. GLC data for the recovery of non-'4C-labeled isomers is shown in Table 4. The only possible evidence for degradation is for the 5-Br-I isomer, which does not confirm our previous suggestion which indicated that only the 5-Br-II isomer might have been subject to slight biodegradation (5). Of interest is the significant loss of extractability with time of all isomers (Tables 3 and 4) in the sterile as well as nonsterile treatments. Total 14C collected in IN NaOH is shown in Table 5. Though slightly more label was trapped from nonsterilized soil, the amount of additional label volatilized in the viable treatment was insignificant. Soils incubated with photodegradation products of '4C-PBB showed enhanced though still only minor conversion to 14CO2. Products created by the photodecomposition of PBB by ultraviolet irradiation are apparently more volatile than nonirradiated PBB as shown by the increase in 14C collected from sterilized soils, especially at the first sampling. The microbial activity which occurred in the nonsterilized treatments appeared to increase the amount of 14C volatilized, suggesting that some of the '4C-degradation products of ultraviolet irradiation may be metabolized. PBB irradiated by ultraviolet light forms lower brominated biphenyls (13). The persistence of PBBs and the possible degradation of the ultraviolet products is consistent with the evidence reported for PCBs which shows that the more heavily chlorinated moieties (penta or greater) are resistant to degradation though many lesser chlorinated biphenyls are metabolized (14)(15)(16). The extractability of the 14C photodegraded PBB is shown in Table 6. Much of the added material was not extracted, in marked contrast to PBB (Table 3). Apparently the photodegraded products are more reactive with the soil organic matter thereby preventing their extraction. The early loss of extractability (0 and 3 months) is supportive of this explanation. Since partially degraded PBB would not yield '4CO2, we also examined soil extracts for other '4C products by TLC-autoradiography (Fig. 1). The TLC system used was shown to separate the isomers of FireMaster (detection by ultraviolet). No intermediates of PBB degradation could be found. From the autoradiogram it is apparent that the two '4C-PBB isomers were the only '4C products in the soil. The autoradiogram of ultraviolet-treated '4C-PBB extract from soil (Fig. 2) d The isomer abbreviation used in the tables and text is the same as we defined previously (5), e.g., 5-Br-I = the first major pentabromobiphenyl isomer on the chromatogram. b Each value is the mean of four replicates. difference between the original amendment and the extract from the soil incubation. The marked change in the PBB isomers due to the ultraviolet irradiation is clearly shown by Figure 2. Virtually none of the original '4C-labeled isomers remains. Most of the label is at the solvent front, the position where lesser brominated forms would be expected to run. The label at the origin and the streaking indicates that some of the 14C-PBB products may have com-plexed with the soil organic matter which is consistent with the low extraction efficiency of these photolyzed products. We examined the fate of the photodegraded PBB since the higher brominated forms are readily degraded by ultraviolet light to lesser brominated forms which could be more toxic. It can be reasoned that the PBB in contaminated manures spread on soil surfaces might show some photo-April 1978 degradation. However, this does not appear to have been a significant reaction in the field since the ratios of peaks 5-Br-I:6-Br-I:7-Br found in most of the soils surveyed (2-5:100:40-50) did not vary significantly from the PBB standard (3:100:43). Other types of samples surveyed ( Table 2) showed no striking differences in peak ratios except for the samples from the milk dump areas where the major heptabromo isomer (7-Br) was markedly reduced or absent. The above findings are consistent with metabolism of PBB in cows; most of the PBB is directly excreted in the manure, thus resulting in little change in isomer composition, while the PBB reaching the milk is reduced in the heptabromo isomer content (17). PBB Concentrations in Water The mobility of a chemical like PBB in soils will largely be governed by its solubility in water and its adsorption, or interaction, with soil particles. (1) ultraviolet-irradiated '4C-PBB standard; (2) '4C-PBB standard; (3,4) extracts from sterilized soil after 6 and 12 months incubation, respectively, (5, 6) extracts from nonsterilized soil after 6 and 12 months incubation, respectively. comparing the concentrations of PBB which stayed in solution after the addition of 14 ppb PBB to laboratory and naturally-occurring waters. Following gravitational settling, significant differences were obtained between the resulting PBB concentrations in tap and distilled waters (10-20 ppb) compared to 100-200 ppb, about 200 ppb, and about 400 ppb for the Red Cedar, Spinks, and Brookston waters, respectively. Fine particulates and water-soluble organics present in the latter three waters no doubt contributed to the higher PBB concentrations which remained in solution or in suspension, with time. These materials have been shown to result in higher, gravitationally stable concentrations of DDT in water (18), another highly water-insoluble compound. However, fine particulates in suspension apparently do not dominate PBB concentrations in natural waters. Data for the centrifuged (10,OOOg) portions of these waters indicate that PBB removed by centrifugation will come back into solution, or suspension, to varying degrees depending on the nature of the water. Soluble organics in solution likely have a major effect on the amount of PBB remaining in each water with time. The Brookston water extract could be expected to contain the largest quantities of these organics, since this soil has a high organic matter content. Also, the precipitation of humic acid and other organic compounds were visually observed with time for all three of the natural waters which coincides with the corresponding loss of PBB from solution for these waters. The environmental implications of the different PBB solubilities observed (Fig. 3) should depend on the particular circumstances. For example, with soil concentrations of PBB such as those reported in Table 1, the attraction of PBB by soil organic matter particles would likely be so dominant that little, if any, PBB would reside in and move with the soil water. However, where high quantities of solid PBB are present like in a landfill, the organic content of the water passing through would likely effect PBB mobility. Summary Since PBBs are not degraded, are not leached, are not taken up by plants (5), and are not volatilized (because of their low vapor pressure), we expect PBB to be a rather permanent component of contaminated soils. Because PBB is bound to soil, wherever contaminated soil moves, whether by 1 . .2 zoo wind or water erosion or animal ingestion and migration, traces of PBB (if present) can be expected to follow. However, because of the low PBB concentrations in soil and the low quantities of soils moved by these means, any serious contamination of animals, wildlife or aquatic environments seems unlikely. As a precautionary measure, areas with much higher levels of contamination, such as manure piles, milk disposal areas and feedlots should be managed to minimize runoff, erosion, and animal contacts.

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Comparison of findings among residents on Michigan dairy farms and consumers of produce purchased from these farms. Consumers who had purchased farm products from both quarantined and nonquarantined farms were examined during the cross-sectional clinical survey of 1,029 Michigan residents. Since PBB had inadvertently contaminated cattle and other farm animals, ingestion of meat, milk, eggs and other farm products was thought to have possibly resulted in significant PBB body burdens in some consumers. Findings were considered in comparison with those made among farm residents. Prevalence of symptoms in consumers of farm products from quarantined farms (CQ) was similar to that found in farmers on quarantined farms (FQ); the prevalence was lower in consumers of products from nonquarantined farms (CNQ). Liver function abnormalities were found with similar prevalence in dairy farmers and consumers. Distribution, mean and median values of PBB serum levels in consumers were found to be similar to those of dairy farmers. These results indicate that significant body burdens of PBB had been accumulated by some consumers of farm products in Michigan and that prevalence of symptoms and liver function abnormalities resembled those found among dairy farm residents. Introduction The accidental addition of polybrominated biphenyls (PBBs) as FireMaster BP-6, instead of magnesium oxide, to farm feed in Michigan in 1973 resulted in widespread contamination of farm animals (cattle, hogs, sheep, chickens, etc.) (1)(2)(3). Butter, cheese, dry milk products, eggs had to be removed from the commercial market, and tens of thousands of animals had to be destroyed. Meat, milk, butter, eggs and cheese containing PBBs, entered the human food chain before the nature of the Michigan problem was identified in 1974, i.e., before it was known that the cause of widespread disease in farm animals was the accidental contamination of their feed with PBB. The sampling program for various food products started * Environmental Sciences Laboratory, Mount Sinai School of Medicine, New York, New York 10029. after the identification of the problem. Given the technically difficult and demanding testing procedures and the large number of samples to be tested, PBB analyses of various products (meat, milk, butter, cheese, etc.) was relatively limited and often delayed and, since the decision on the quarantined status of a farm was dependent on PBB levels considered excessive identified in products of that specific farm, various farms were quarantined at different times. Information for PBB absorption and body burden in humans has been meager (4)(5)(6). A Michigan Department of Public Health study analyzed PBB levels in over 100 persons residing on quarantined farms and a comparable number of residents on nonquarantined farms. PBB serum levels up to 0.02 ppm were detected in the group from nonquarantined farms. Levels in fat were, as expected, consistently higher than those in serum. Another study of the Michigan Department of Public Health, conducted in 1976 (7) by using a ran-dom sampling method, found that 96% of 53 women in the lower peninsula excreted PBB in their breast milk; the proportion was lower, but still impressive (43%), in a group of 42 women from the upper peninsula, which many had considered spared from major PBB exposure. The pattern of absorption, distribution, metabolism, and excretion of PBBs, although not yet completely clarified, is nevertheless known to parallel quite closely that of polychlorinated biphenyls. Important characteristics are the marked accumulation of these compounds, mainly in fat tissue, but also in tissues with a relatively high lipid content and the very slow rate of metabolic degradation and very limited excretion. Cumulative body burden with continued ingestion is therefore a major feature of the metabolic model for polybrominated biphenyls. This is of interest, since with such toxic substances adverse health effects may occur either as a result of short-term, high levels of absorption, or as a consequence of long-term, repeated absorption of relatively small amounts. At the time the accidental Michigan contamination occurred, only fragmentary data were available concerning PBB toxicity. Effects in various species of animals indicated, nevertheless, high potential for toxicity; species differences were noted, and no extrapolation as to the relative susceptibility of humans was warranted. Therefore, in the clinical field study conducted by the Environmental Sciences Laboratory (8), farmers and their families from both quarantined and nonquarantined farms were included. Consumers of products from these quarantined and nonquarantined farms were also invited, since it was thought that, given the sequence of events, i.e., the time interval (at least 9 months) until the identification of PBBs as the contaminant, the schedule for testing of various food products, the time constraints associated with deciding whether a farm was quarantined or not and the changes in the FDA action levels for PBB in meat, milk, etc., it was theoretically possible for consumers to have ingested undetermined amounts of PBB. Methods The medical examination protocol used is given in detail by Anderson et al. (8). After the information was collected, for the purpose of this analysis, four groups were considered: farmers residing on quarantined farms (FQ); consumers of dairy farm products from these quarantined farms (CQ); farmers residing on nonquarantined farms (NQF); consumers of dairy farm products from these nonquarantined farms (CNQ). It was believed that a comparison of findings among farmers residing on quarantined farms with those among farmers residing on nonquarantined farms would be of interest in evaluating the dimensions of the potential problem of adverse human health effects due to PBBs. Of similar interest, and of potentially wider significance, would be the comparison between farmers and consumers of farm products. The prevalence of symptoms, liver function tests abnormalities, the distribution of PBB serum levels, the mean and median PBB values in serum, were compared for the four subgroups of the population studied. Results Symptoms were grouped into four major categories, defined as dermatologic, neurologic, musculoskeletal, and gastrointestinal. The neurologic syndrome was the most prominent and was marked by tiredness and fatigue, an important decrement in the individual's capacity for physical or intellectual work and a significantly increased requirement for sleep (14-18 hr/day); other symptoms such as headache, dizziness, and irritability were often associated (9). The gastrointestinal syndrome included loss of appetite, weight loss, abdominal pain (with no characteristic pattern), and diarrhea. These symptoms were most often found in conjunction with the neurologic syndrome, especially with tiredness and hypersomnia. It is noteworthy that hepatomegaly was not a prominent finding, nor was liver tenderness on palpation. The musculoskeletal syndrome consisted of arthritislike changes: swelling of the joints with deformity, pain, and various degrees of limitation of movement. The knees and ankles were generally most affected, but the small joints of fingers and hands were also frequently involved. Tendonitis, with swelling, pain and crepitation, most often affecting the extensor and flexor muscles of the hands, was also found in some cases with joint involvement. The dermatologic changes will be reported in detail elsewhere. Prevalence of symptoms among farmers living on quarantined farms did not differ significantly from that among farmers on nonquarantined farms for any of the four groups of symptoms considered. All were at least as prevalent among farmers on nonquarantined farms as in those on quarantined farms. Furthermore, the prevalence of symptoms among consumer of dairy farm products was similar to that found in farmers; only among consumers of products from nonquarantined farms were neurologic symptoms less frequently reported (Fig. 1). The neurologic symptoms had the highest prevalence in all population groups considered; they were followed by musculoskeletal symptoms. Liver function test abnormalities (alkaline phosphatase, SGOT, SGPT, and LDH) were also compared (Table 1). Liver function abnormalities were found with similar prevalence in farmers from quarantined and nonquarantined farms, and in corresponding consumers. The prevalence of abnormal SGOT and alkaline phosphatase levels was especially high in the subgroup of consumers of products The distribution of PBB serum levels was compared (Table 2). A striking similarity of distribution patterns in farmers from quarantined farms and consumers of products from these farms was found (Fig. 2). When farmers from nonquarantined farms were compared with consumers of products from nonquarantined farms, the distribution patterns were, again, very similar (Fig. 3). The median values for PBBs in serum (Fig. 4) reflected the similarities in distribution pattern. Discussion and Conclusions The results indicate that significant body burdens of PBBs, as reflected in the serum PBB level, have been accumulated by at least some consumers of farm products in Michigan. Although the number of consumers examined was relatively small, the data suggest that this conclusion is warranted. Similarities in prevalence of symptoms and liver function abnormalities in consumers of farm products, with those among dairy farm residents in Michigan, and the fact that these far exceeded those found in Wisconsin dairy farmers (8) indicate that adverse health effects due to PBB toxicity should be considered among segments of the Michigan population, other than dairy farmers.

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1978-10-15T00:00:00.000Z

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A badger-face-like color variant in Texel and in Dutch sheep in the Netherlands A new color variant has been found in Dutch and in Texel sheep from the Netherlands. Phenotypically this variant localy called « blue » is very close from the well known badger-face pattern: black belly, legs and bars on head but one can see in the « blue » Dutch sheep a tendency of greying of the dark zones, specially with age. As for the other « badger-face » patterns which have been genetically studied, the breeding results are in favour of an allele at the Agouti locus. Many cases of badger-faces have been described in breeds which often are not closely related, at least recently but, even, one hesitates to consider each case and specially Dutch Texel on as deriving from an original mutation each time. In 197 6 one of us (P. H.) was called to see a new color variant in a flock of Dutch( 1 ) sheep in Friesland. This color variant was named " blauw " (blue) by the owner. A first note has been given (HooGSC H AGE N ,197 8). The present article brings some additional data and interpretations. Material and methods All the data concerning appraisal of the first case including breeding results were collected in the first flock and the reproducing animal from that flock which were dispersed by sale have been followed in their new flocks. Enquiry was made among the sheep world in the Netherlands in order to find out another cases through an article in « Het Schaap » (issue of august and october 1978). Results A. -Cases history The first detected blue lamb a male was born in Ing. A. O OSTERBAAN ' S flock, Synaede State Tzummarum, Friesland, more than ten years ago, among triplets from a mating between white Dutch parents. A few months after the birth of this blue lamb Mr O OSTERBAAN heard that a similar lamb had been born nearly contemporarily in another flock. He was able to purchase the animal and started to create a blue line. Furthermore we learn that a blue lamb was born at the IVO (Institute for Research in Animal Husbandry at Zeist) some years ago (M. V ISSCHER , personal communication). After the call in « Het Schaap » a few sheep breeders responded that, in their flocks too, blue animals had been born in 197 8 or earlier. The details concerning all the cases up to now detected in the Netherlands have been summarized in table i. B. -Descvi!tion At birth the belly of the blue animal is rather dark, the dark colour extending backwards to the innerside of the hind-legs, up around the anus and the thighs. The dark colour extends forwards along the brisket to the inner side of the forelegs up along the underside and the side of the neck and also on the shoulders. A dark line widens from the neck on to the underjaw. The inside of the ears is dark and there is a dark bar above each eye and another one less wide under each eye plus usually a dark mask on the nose. The pattern is highly symmetrical and the parts of the body which are not dark look white. Some of the dark areas look black at birth and other are already grey by intermixing biack and white fibers. With ageing the greying usually increases. This greying giving some blue glints, is responsible for the name of « blue o sheep given by the breeders. The extent of coloration and its variation is illustrated in fig. I One knows also that at HE NS T RA and B RUINSMA ' S flock the maternal grandfather in 4 cases was Cyrus which has a common ancestor with the father Berend. Discussion The data from tables I and 2 show that the blue variant behaves as a recessive towards the common white coloration in Texel and Dutch sheep. One can think that the gene inducing the blue is allelic with the A ?i h gene in Agouti locus which, according I,AUV!xGN! and HOOGSCHAGE N ( 197 8) is responsible for the white colour of these breeds. This interpretation is in accordance with the phenotypic observations of the blue variant which is very close from the badger-face pattern, a pattern first genetically described by HELLER ( 1917 ) in the sheep and which precisely has been demonstrated to be given by an allele in Agouti locus (B ROOKER andD OLLING , 1969 , ADAI,ST!IN5SON, 1970). There is an alternative hypothesis: blue being given by recessive allele at another locus which were epistatic on Agouti genotypes. A definite proof of the first hypothesis were by crossing white animals heterozygous for the blue factor to homozygous recessive black animals aa and obtaining some « blue ». Some cc blue » lambs have been born in white X black crossings (cf. (.MnsoN, 1977 ). This list is probably incomplete as several archaic sheep breeds in Africa in the Middle East and in Central Asia have not yet been seriously checked for colour patterns. Of course it is impossible to assert that all the badger-face patterns are given by the same gene originating from a given mutation but there are certainly not as many badger-face mutations as there are breeds wearing this colour variant even if several independant mutations with a close expressivity are possible. Comparing badger-face variants in Corsica and Iceland I,auv!xGrrE and Ann!,sT!INSSOrr ( 197 6) have implicitly admitted it was the same gene: All. For the « blue o variant in the Texel and Dutch sheep HooGSCxnGErr ( 197 8) brought another allele A bl . This may be supported by the greying of « blue » Dutch, a usually absent trait in badger-face from other breeds but detailed studies of badger-face expressivity are lacking in most of the cases and the existence of modificator genes may be posponned to explain this greying in the white breeds of the Netherlands.

v3-fos

2014-10-01T00:00:00.000Z

1978-04-01T00:00:00.000Z

12075516

s2

PBB fed to adult female chickens: its effect on egg production, reproduction, viability of offspring, and residues in tissues and eggs. Two experiments with light breed laying chickens fed polybrominated biphenyl (PBB) as FireMaster FF-1. The first involved feeding PBB at dietary levels of 0.2, 1, 5, 25, 125, 625, and 3125 ppm, the second involved levels of 30, 45, 60, 90, and 120. Each group had 24 hens, and each experiment had a control group of 24 hens. PBB diets were fed for 5 weeks. Feed intake, production, reproduction, tissue residues and viability of offspring were monitored during that time and a subsequent 8 weeks. Production, hatchability, and viability of offspring were significantly affected by feeding PBB at 45 ppm. Marked inanition occurred at levels of 625 and 3125 ppm, and there was some loss of feed intake at 125 ppm. There was a return to normal production and hatchability in 3 to 4 weeks after PBB withdrawal of diets with levels of 125 ppm or less. Dose--response lines are presented for PBB in muscle, liver, kidney, adipose tissue, and eggs. Withdrawal curves for PBB from these tissues are also given. Introduction Two laboratories have been involved with elucidating the toxic effects of polybrominated biphenyls (PBB) in chickens since the 1973 accidental contamination of PBB in feeds within Michigan. Two preliminary reports appeared from the USDA, Agricultural Research Service in 1973 on the consequences of 20 ppm PBB given to laying hens (1,2). This was pure serendipity; the cause of the problem in Michigan was not yet known. However, the experience gained with the PBB compound was no doubt responsible for the eventual establishment by Dr. Fries of the USDA that the problem was due to PBB contamination. In the spring of 1974, extensive studies on PBB contamination were initiated at Michigan State University with the first report given in August, 1975 (3). That same month, Cecil and Bitman (4) from the USDA reported on extensive studies they had completed. There were some slight differences in the data from the two laboratories on the minimum level in the diet for an adverse effect on reproduction, the most * Michigan State University, East Lansing, Michigan 48824. sensitive criterion of several evaluated. The data from Michigan State University were developed on levels ranging from 0.2 to 3125 ppm. In these studies no adverse effect on hatchability or chick viability was detected at 25 ppm, in contrast to the studies (1,2) showing a PBB effect at 20 ppm. The details of our data on this study, and a subsequent one involving PBB levels of 30 to 120 ppm are reported in this paper. Some of the data appeared in an earlier report (5) in which PBB and PCB were compared for their toxicity in the chicken and quail. That report included data which had appeared from the USDA laboratory (4,6) providing details on the results reported in 1973 and 1975. There is a very important difference in the type of PBB used by the two laboratories in their investigations. The compound used at Michigan State University was a sample from a bag representative of the material involved in the contamination. This material is FireMaster FF-1, the product of Michigan Chemical Corporation, which contains added anticaking compounds. The compound used at the USDA laboratories, known as FireMaster PB-6, was the product of the company which lacks the small (unknown) amounts of the anticaking compounds, and which had not been milled to obtain a free-flowing compound. Thus, particle size may be another difference between the two compounds tinder evaluation. Methods and Procedures The first experiment was started in June, 1974: the second in January, 1975. Both were conducted at the Michigan State University Poultry Research and Teaching Center. An end room of one of the buildings was used so that the experiments could be isolated from other studies in the building. The adjacent room was kept vacant. Eventually, all wastes, left-over feed, eggs, and chickens in the experiment were buried in a specially selected site to prevent contamination of ground water. The White Leghorn laying chickens in experiment I were 10 months in production (about 60 weeks of age) at the time they were assigned at random to one of seven treatments or to a control group. Each group consisted of 24 hens. The hens were in colony cages, each holding six hens. Feed and water were supplied ad libititun. Treatment levels, as shown in Table I, were selected to start at 0.2 ppm with levels increasing at fivefold intervals. PBB diets were fed for 5 weeks, at which time three hens from each group were selected at random and sacrificed to obtain tissue samples of adipose, liver, and muscle. Then, during the last day of weeks 2, 4, 6, and 8 following withdrawal of PBB diets, three hens from each group were sacrificed for tissues. On the Thursday of every week of the experiment, eggs were saved for measurements of weights of yolk, albumen and shell, as well as examination for any unusual physical appearance. Eggs from this col-lection were set aside and frozen for eventual PBB analysis by the State of Michigan, Department of Agriculture Laboratory. All other eggs collected during the week were saved for incubation. Adult male chickens, kept in a separate room, were fed clean feed (id libitium and used to supply semen for artificial insemination of the hens. Experiment 2 was initiated on January 23, 1975. White Leghorn hens 36 weeks of age were used. Again, 24 hens were randomly assigned to each of five treatments with PBB at 30, 45, 60, 90, and 120 ppm, and 24 hens to a control group. PBB diets were fed for 5 weeks and then withdrawn to follow the resultant effect on reproduction during the next 8 weeks. In addition to measurements on reproduction, which included egg production, fertility of eggs, and embryo mortality during hatch, feed consumption, and mortality of offspring were also monitored. The latter was ascertained by rearing the hatched chicks from treated hens for 21 days on a control chick starter ration. Tissue and egg samples submitted to the laboratory for the Michigan Department of Agriculture for assay of PBB were coded. After gas chromatographic analyses these data were returned to us for collating and data processing. PBB analysis was performed on a homogenized sample weighed accurately to 4.0 to 5.0 g in a tared aluminum moisture dish. The sample was covered with granular anhydrous Na2SO, and mixed in with a small stirring rod. The sample was then dried at 100°C for 30 min and extracted on a Soxhlet apparatus into a tared flask with 6% ethyl ether in petroleum ether for 4-8 hr. The extract was brought to dryness, the flask weighed and the percent fat calculated. Following this, the fat was quantitatively transferred to 10.1(I 11.7)" " PBB was fed for 5 weeks at graded levels of 0.2 to 3125 ppm (mg/kg diet) and 30 to 120 ppm. b From data of Ringer and Polin (5). Environmental Health Perspectives an approximately 40 g of activated Fluorisil column topped with anhydrous sodium sulfate. The sample was then eluted with 200 ml petroleum ether-ether (94:6). The petroleum ether as evaporated on a steam bath, and the residue from the column chromatography was resolved with high-temperature GLC by either one or both of the following procedures after diluting the residue to 5 ml with hexane. One procedure used a 3H-foil electronic detector with a temperature of 220°C in the column and detector, and 250°C in the injector. The other used a 63Ni-detector at a temperature of 270°C in the column and 3 10°C in the detector and 300°C in the injectorport. Nitrogen was the carrier gas in both procedures at a flow rate of 100 ml/min. The column was 182.9 x 0.64 cm in both procedures and was packed with 3% OV-1 on Gaschrome Q, 60-80 mesh. The normal working range for PBB analyses was 25-400 pg. Average recovery of spiked samples was 97.8%. Feed Intake In the first experiment, adult female chickens showed a small but significant (p < 0.05) decline in food intake when fed a diet containing 125 ppm PBB (Table 1). Hens fed diets with PBB levels lower than this consumed normal amounts of feed. The decline in feed intake was drastic for those hens offered the diet with 3125 ppm PBB. These hens were starving themselves to death, so at the end of the fourth week, a week earlier than the schedule had indicated, these hens were switched to clean diets. Cecil and Bitman (4) reported that pair-fed controls in their experiment as well as hens fed PBB at 640 and 2000 ppm in the diet died within 30 days with the effect of PBB producing death because of starvation. Babish et al. (7) noted that Japanese quail refused to eat a semipurified diet containing 500 ppm PBB, but showed no inanition at 10, 20, or 100 ppm. Egg Production Coincident with the dramatic reduction in feed intake by laying hens fed 625 or 3125 ppm PBB in their diets was the complete loss of egg production within two weeks (Fig. 1). Interpretation of these data without pair-fed controls to separate out that effect on egg production caused by PBB vs. that produced by loss of nutrient intake, was not possible. However, investigators (8,9) established that hens cease egg production when without feed for longer than two days. Egg production in the group of hens provided with 125 ppm PBB in their diet declined from 66% to 48%, and returned to normal within 2 weeks after withdrawal of PBB diet. No other significant decline in egg production was detected for birds treated with levels lower than this. The data on the hens fed 0.2 ppm PBB were interpreted to be that their production was characteristic of that group and was not an effect from PBB, particularly because production averaged almost the same at about 65% for the weeks during and after feeding PBB. A clearer picture of the effect of PBB on egg production was obtained in the second experiment in which PBB levels ranged from 30 to 120 ppm (Fig. 2). Egg production declined significantly (p < 0.05) when PBB was in the diet at levels greater than 30 ppm. Most interesting to note is that following withdrawal of PBB diets, egg production returned to normal within 2 weeks if the hens had been fed PBB at 120 ppm or less, within 3 to 4 weeks if formerly fed 125 ppm, and within 5 to 6 weeks if formerly fed 625 or 3125. The minimum level of PBB to cause a decline in egg production was determined to be between 30 and 45 ppm. Hatchability No effect on hatchability occurred in experiment I when PBB was in the diet at 25 ppm or less (Fig. 3). Eggs obtained during the first week of the experiment from hens fed 125 ppm PBB hatched normally; but in subsequent hatches the values for hatchability were progressively lower reaching zero at the 5th week. A few eggs were obtained during the first week from hens given PBB in their diets at 625 or 3125 ppm. Already, an adverse effect on hatchability was detected. Upon withdrawal of the PBB diets, hatchability of eggs from hens previously fed 125 ppm PBB recovered rapidly (Fig. 3). On the other hand, those eggs obtained from the hens previously fed 625 or 3125 ppm PBB exhibited a high embryo mortality, although egg production by this time was normal. The data procurred from experiment 2 revealed that a borderline effect on hatchability was detected at 30 ppm and a definite effect at 45 ppm PBB in the (Fig. 4). Eggs did not hatch when collected from the fourth week on PBB through the first week off PBB when their source was the hens that had been fed 120 ppm levels of PBB, hatchability returned to the range of control values. The minimum effective level of PBB for an effect on hatchability was established to be greater than 30 ppm but less than 45 ppm in the diet of laying chickens. Offspring Mortality Chicks that hatched from eggs of hens fed PBB were raised using a chick ration without PBB. Treatments applied to the hens in experiment 2 were PBB levels of 30 to 120 ppm for 5 weeks. Eggs require about 9 days for PBB to reach a plateau (based on the weekly analysis of the eggs from experiment 1) and require about 9 days following withdrawal of PBB for a change to occur in PBB concentrations. (Yolks are formed in about 9 days and PBB is lipotropic.) Thus, the offspring obtained from the hens treated with PBB during weeks 2 to 5, and the first week after withdrawal (-1) were evaluated for carryover through the egg. lists the percent mortality of chicks according to the treatment applied to the hens. Offspring mortality increased linearly with increasing levels of PBB in the hens' diets according to the equation Y = -54.42 + 50.OOX, where X = log of ppm PBB in diet, and Y = arcsin \/% of the mortality. Allowing for 2oas a difference between control and a treated group for a significant effect at p = 0.05, the estimate for a minimum detectable effect for PBB on offspring mortality was estimated at 42 ppm PBB in the hens' diets. An average (but nonsignificant) increase in chick mortality was detected with 30 ppm of PBB in the hens' diets, a significant (p -0.05) increase at 45 ppm. Generally there was a tendency for chick mortality to be higher when orginally from eggs collected during weeks 2 to 4, than 5, and -1. Tissue Residues Muscle, liver, whole egg, and adipose samples obtained during the time PBB was fed had PBB residues which were linearly related to dietary PBB levels in a log-log relationship (Figs. 5, 6, and 7). Generally, the ratios of tissue PBB:diet PBB averaged 3:1 for adipose tissue, 1.5:1 for whole egg, 0.8:1 for liver, and 0.008:1 for muscle. The trend at which PBB accumulated in these tissues and whole egg with increasing levels of PBB in the diet, as based on slope values, were highest for muscle and egg (b = 1.015), intermediate for adipose (b = 0.923), and lowest for liver (b = 0.765). Controls had detectable PBB of 0.04, 0.08, 0.07, and 0.21 for muscle, liver, whole egg, and adipose tissues, respectively. This indicated that PBB dust from the contaminated diets was settling on the feed given to the control hens housed in the same room. At a time subsequent to the experiment, three hens housed in a different area of the campus were sacrificed and adipose tissues included in the samples submitted for analysis. Their value of 0.07 ppm represented a clean adipose tissue sample for comparison to samples from hens on the experiment. The withdrawal curves for PBB from muscle, liver, whole egg, and adipose tissue are plotted in Figures 8, 9, 10, and l1, respectively. Those for muscle indicate a PBB half-life (tj) of approximately 17 days, based on the average slopes for the former treatment levels of 5 to 625 ppm PBB in the diet and a withdrawal time of 56 days. When treatments had been 0.2 to 125 ppm PBB in the hens' diets, a biological half-life of 17 days was calculated also for whole eggs analyzed during the 56 days of withdrawal. The variability of the data on liver PBB levels following withdrawal made an estimated ti rather tenuous. A value was calculated based on the average slope for the lines depicting 1 to 125 ppm, and the 625 ppm line for days 28 to 56. That value for liver ti was estimated at 31 days, as based on the 56 days of withdrawal. Analysis of the data on adipose tissue (Fig. l1) revealed that PBB levels remained unchanged over the 56 days of withdrawal. 0/'<,/ ,/' Data from the USDA laboratory (4) indicated that 64 ppm PBB had no effect on feed intake, substantiating our data from experiment 2, and that 640 , / t ppm caused a refusal of feed, substantiating our data from experiment 1. Babish et al. (7) noted that ,' :0.1762 ,,' 1.012100 ppm PBB in the diet to Japanese quail had no // / / :0.1763 + 1.012 Xl effect, whereas 500 or 1000 ppm produced inanition. The USDA laboratory in two studies (2,4) reported that 20 ppm PBB produced an adverse effect on production and hatchability of eggs from chickens. This is a level slightly below the minimum effective level reported by us and is also in contrast to control 0.065 ( 0101) the data reported by Babish et al. (7). The latter 2 reported that no such adverse effects were detected 0. (4) and Japanese quail (7). The carryover of PBB in the egg was at a level approximately 1.5 times the dietary level when both are considered on an "'as is" basis. As pointed out previously (5), Babish et al. (7) reported an egg:diet ratio of 1.3 when their data are converted from a dry weight to an "'as is" basis. Dose-response curves for diet level of PBB versus PBB in whole egg allowed an estimate of 63 ppm (range 25-150 ppm) in egg at the estimated minimum effective level of 40 ppm for an adverse effect on hatch and offspring from PBB in the diet. On the estimate that a 59 g egg contents yields a chick of 45 g and that all of the PBB resides in the chick at hatching, then the body burden of PBB at hatch would be estimated at 3.72 mg/45 g chick or 82.6 mg/kg body weight. This would change markedly as the chick grew and would be reduced nearly 5-fold by day 21. Yet one has to recognize that this amount is producing a borderline toxicity. Despite very high levels of PBB in the adipose tissue of hens during the entire withdrawal period, eggs laid by hens previously fed levels of PBB up to 125 ppm showed a recovery to normal hatches and egg production within 2 to 4 weeks. PBB in the egg declined during this time but were shown to be present at substantial levels averaging 10 and 30 ppm in eggs from hens previously fed 25 and 125 ppm PBB in the diet. Therefore, the presence per se of PBB in eggs was not indicative that an embryotoxicity would ensue. Fries (/) showed that hepta-BB underwent a faster withdrawal from eggs than hexa-BB upon withdrawal of PBB from the diets. Although additional data are required before definite conclusions can be stated about particular PBB isomers and toxicity, one must recognize from the data just discussed, that such relationships must be considered. Body burdens of PBB can exist without any evidence of toxicity. Thus, PBB is similar to other poisons in that it too has a toxicity which is titratable down to a minimum.

v3-fos

2018-05-30T22:47:25.417Z

1919-04-01T00:00:00.000Z

215465543

s2

Dermatology covered a staining reagent, which he calls "dopa," obtained from c ertain plants such as " vicia faba," or synthetically from vanillin and hippuric acid. More elaborately it is called 3*4 dioxyphenylalanine, and is a combination of orthodioxybenzene (pyrocatechin) with ?-amino-propionic acid. An oxidation of the dopa takes place by means of a ferment called dopa-oxidase. This ferment is not affected by prussic acid, chloroform, acetone, benzole, or alcohol, but is destroyed by reducing and oxidising agents?sulphuretted hydrogen, toluol, heat, drying, etc. The skin is therefore obtained fresh, embedded in agar, and cut by the freezing microtome. The sections are placed for twenty-four hours at 37? C. in a 1 per cent, watery solution of dopa, then washed well, and stained with Unna's Pappenheim stain. The result shows dark staining of the basal layers of the epidermis, and the cutis vera is little affected. In the stained cells the nucleus is unaltered and the protoplasm alone stained. In animals the ferment is not found in the white patches of the skin, but only in the pigmented areas. Destruction or damage to the suprarenals produces increase^ supply of the substance plasm alone stained. In animals the ferment is not found in the white patches of the skin, but only in the pigmented areas. Destruction or damage to the suprarenals produces increase^ supply of the substance from which the ferment is made, but the quartz lamp, X-rays, and thorium increase the action of the ferment. In the presence of leucoderma the dopa oxidase disappears, but in the hyperpigmented area around it is in excess. Psoriasis. Heidingsfeld {Urol, incl Cut. Rcvieio, May 1918) discusses this in a thoroughly practical manner, giving it as his experience that, while every new form of treatment brought an increase of clientele at first, as certainly the patients disappeared when the results of treatment became evident. The host of remedies proposed is a natural outcome of our ignorance of the etiology of the disease, and the writer's statement that " few of these are without virtue, but none are specific," is generally accepted. " The psoriatic is prone to be the most disappointed of all dermatological patients. Like all patients he desires results, and results not at the cost of too disagreeable personal experience." Balm of Duret, which is a swan-shot preparation containing coal tar, chrysarobin, pyrogallic and salicylic acids, sulphur, green soap, resorcin, acetone, camphor, and guaiacol cleared up old inveterate patches, but was too disagreeable. White precipitate ointment 10 per cent., with 1 to 3 per cent, of chrysarobin, is still, he considers, very valuable in generalised cases. In 1914 human serum injections were commenced, and are of undoubted value; 5 to 10 c.c. of heterogenous serum from a non-psoriatic patient are given semi-weekly. X-rays are useful when given in moderate and infrequent doses, more especially for chronic patches. Other forms of radiotherapy are well adapted for psoriasis. The fact that the eruption affects the face and hands less frequently, that it disappears with sea-bathing and outdoor sports, and that the worst attacks occur in winter, when the helio-activity is lowest, is probably related to this. The disease being classified by the writer as a localised acidosis, he employs the following lotion successfully :? is due to a combination of etiological factors, the author then takes up these points as affecting treatment:? 1. Infective Theory.?This being well known in psoriatic individuals, the use of chrysarobin and sulphur as parasiticides is sound. 2. Nervous Theory.?This he does not believe in, but admits that some of the well-defined attacks and relapses have been ushered in bynervous exhaustion and worry. 3. Diet is very important, but treatment based on it is empirical? the elimination of substances which have a deleterious effect on the skin in general, and more particularly sweets and acid substances. Since intestinal intoxication cannot be ignored, he prescribes 4 to 16 minims of a 2 per cent, solution of phenol well diluted with water. 4. Rheumatic Theory.?Both diseases may have the same intestinal or obscure local infection as a factor. 5. Lastly, the clinical appearance is of important prognostic value. " As a rule, the smaller the lesions, the more favourable; the larger the lesions, the less favourable is the therapeutic outlook. Of much greater prognostic import is the tendency, or lack of tendency, of the lesions to undergo spontaneous central involution. -Psoriasis annulata, or gyrata, even when abundantly present and covering a wide area, offer a favourable prognosis for prompt and early disappearance with treatment. On the other hand, lesions with diffused erythematous infiltration, scaly bases, and slowly spreading borders, which show no central retrogressive changes offer the least favourable prognosis from a therapeutic standpoint." Ringworm of the Groins. Saboraud (La Presse Med., 20th May 1918) reiterates the importance of this condition at the present time in the Army. Very few realise that the disease may simultaneously affect the toes. As a result of this the eruption is half cured, and when marching is resumed there is a rapid spread, and the soldier has to be returned to hospital. All the interdigital spaces may be infected, and even the dorsum of the foot, but most commonly it is the fourth and fifth interspaces. The epidermophyton is easily killed, but it is hidden in masses of thickened epithelium. Thorough scraping with a sharp spoon to the extent even of producing oozing and bleeding is the most important item, and after this the parts are firmly rubbed with a 20 per cent, solution of iodine in alcohol. A zinc paste is now applied, and the whole process is repeated daily for eight days. This generally removes all the trouble, but, if not, then 10 per cent, of chrysarobin in lard is recommended. Dermatitis Venenata. Strickler (Amer. Journ. of Cut. Dis., June 1918) sounds a hopeful note when he discusses the question of the treatment of these by vegetable toxins. The active principle of poison ivy is of a glucosidal nature, yielding on analysis gallic acid, fixtin, and rhamnose, and is ?non-volatile. It is obtained from the leaves by extracting with alcohol, and subsequently filtering and precipitating. The precipitate is dried, then extracted with Soxhlet's extractor for ten hours. This extract is then dried at low temperature, weighed, and dissolved in absolute alcohol and water. Poison ivy, sumac, and nettle were all treated thus, and used in the experiments. When a case came under observation, c-c-ea?h ?f these was injected endermically, and the case examined at twenty-four and forty-eight hours' interval. A positive reaction was indicated by the formation of a papule, erythema, and tenderness, and a patient so differentiated was then used for treatment. Twelve patients suffering from dermatitis venenata, whose history indicated plant irritation, were given 0-3 to 0-7 c.c. of the toxin intramuscularly, and all were cured after one or two doses. Unfortunately, the immunity was found to be very fleeting. The possibilities of this method are manifold if subsequent experience gives as good results. Cases are always numerous, more so in war time, and many are very resistant to treatment. The use of tin salts, an old method revived, is often satisfactory. Burnier {La Presse Med., 2nd May 1918) finds that the root of bardane (lappa officinal.) is more useful for furunculosis in the cases under his care, although he still prefers the tin salts in folliculitis. The root must be collected in spring, dried at a low temperature, and 0-60 gr. of the soft extract is given in pills thrice daily. He states that in twenty-four to forty-eight hours the pain ceases, and that in three to four days the abscess evacuates spontaneously. M'Donagh {Med. Press and Circ., 5th December 1917) has been investigating the colloidal metals in this connection. Colloidal copper intravenously and intramuscularly did no good. Colloidal manganese given intramuscularly in 3 c.c. doses cleared up boils in three days. Smaller doses cause no inconvenience, and larger doses may cause a severe reaction, therefore he prefers to commence with 1*5 c.c. and then go to 3 c.c. in a few days if necessary. Out of 100 cases 50 had the usual treatment with vaccines, etc., and the rest were treated with manganese alone ; the first took fifty days on the average to be cured, and the latter only seven days. Auld (Brit. Med. Joum., 16th February 1918) is not so satisfied with the efficacy of the colloids. Manganese given intravenously was, in his opinion, more reliable in its action. Gold, silver, and copper in doses of 2 to 10 c.c. gave favourable results, especially if followed by a rise of temperature. In conclusion he states that the protective solution is an active ingredient in all the preparations. F. G.

v3-fos

2014-10-01T00:00:00.000Z

1978-04-15T00:00:00.000Z

2609280

s2

On the heredity of water intake and feed efficiency in the Fowl Several workers have, recently, studied the phenomenon of excessive water intake by hens housed in cages during the productive period (see review by LEESON et al., 197 6). SUNDE (cfBRETH, 19 64) showed the importance of water as alimiting factor of other physiological functions. Besides, the convenience of selecting poultry populations with a scarce water content in their excrements is considered necessary for good management. It would be interesting to demonstrate the Introduction Several workers have, recently, studied the phenomenon of excessive water intake by hens housed in cages during the productive period (see review by L EESON et al.,197 6). S UNDE (cfBRETH, 19 6 4 ) showed the importance of water as alimiting factor of other physiological functions. Besides, the convenience of selecting poultry populations with a scarce water content in their excrements is considered necessary for good management. It would be interesting to demonstrate the As in many studies of genetic factors, an adequate measurement of the trait requires considerable attention. Water intake is closely correlated with food intake and factors affecting food intake indirectly influence water consumption (ZW Gr,!R et al., 1971 ;BWR!R et al., zg66). Accordingly to assess any influence increased water intake may have on feed intake, water /feed ratio was considered as a determining criterion in two populations studied during the last five years. The objective of this work is to point out the existence in our populations of two genotypes namely (di) for excessive water intake and (Di) for normal water intake. This may be worthy for indirect selection, and to shorten the period required to perform the phenotypic classification of the flock. Material and methods 1. -Ex!erimental populations and traits measured Two populations have been studied: an experimental flock segregating for several marker genes (« Jouy » strain, years 1970(« Jouy » strain, years , 1971(« Jouy » strain, years , 1975 strain. In each year chicks were raised on floor till the age 1 6 weeks. Then a sample of females (5 9 to 94 according to year) obtained from several sire and dam families were set in individual cages. Several layingand egg traits were recorded on this sample. Moreover, from about 8 months age, feed intake of each bird (with a 1 6 p. 100 protein and 2 5 20 kcal /Kg ME ration (or 10 .55 MJ /kg), the formula being the same for all years) was controlled during 3 successive 2 8 day periods. During i 4 consecutive days in the last period, individual water consumption was also measured (including possible waste of water). For each individual, only its average value is considered for the traits of which the measurement is repeated, and only birds having all measurements recorded are considered. Table I summarizes the definition of the observed traits and abbreviated symbols designating them in the following tables. 2 . -Test of major gene hypothesis for water intake: definition of genotypes and tests In each examined year for the two populations, some individuals with abnormally high water consumption appeared. From preliminary analysis the water / feed ratio (on the basis of daily means for each bird) was considered the most discriminating between individuals and is used here. Each year the distribution of the sample for this ratio was obviously asymmetric. About 10 to 15 p. 100 of the individuals had values exceeding (quite appreciably for most of them) the mean of the whole sample augmented by twice its standard deviation. The average value of these &dquo; abnormal &dquo; birds was about double that of the other ( & d q u o ; normal & d q u o ; ) birds, of which the distribution seemed approximately normal. This suggested the presence of two distinct populations for the trait studied, possibly corresponding to different genotypes at a &dquo; major &dquo; locus. To check further this possibility, in each year from each strain the mean value x and standard deviation s of the whole sample for water /feed ratio were estimated. Individuals with a value exceeding z + 2S were considered &dquo; abnormals &dquo; and excluded from the distribution. After this exclusion, a new mean -i, and standard deviation Sl were computed. If in the corresponding sample there remained a few individuals with value appreciably superior to X3 -! 2S ,, these individuals were also considered &dquo; abnormals &dquo; and excluded from the distribution of &dquo; normal &dquo; birds of which the new mean x 2 and the new standard deviation S2 were calculated. It was never necessary to go further, as x 2 and s, were always close respectively to X l and s,. The value !e2 -E-2 s., (or x l -f-2SI ) finally separated two groups of individuals, those with a lower value ( & d q u o ; normals & d q u o ; ) and those with a higher value (&dquo; abnormals &dquo;). The normality of the distribution of the two groups separated in this way was tested. Then the genetic hypothesis of a major gene acting on the trait studied was checked by comparing, within full-sib families containing at least one &dquo; abnormal &dquo; hen, the observed proportion of &dquo; abnormals &dquo; to that expected, (chi-square test) taking account of the frequency of the postulated genotypes and of the limited family size. . -Statistical analysis on quantitative traits Pairs of full sisters were formed, one &dquo; normal & d q u o ; , the other &dquo; abnormal &dquo; (or &dquo; polydipsic & d q u o ; ) according to the criterion described above. From all pairs formed in each population, the mean value of &dquo; normals &dquo; and &dquo; polydipsics &dquo; is compared for each trait by a t-test (pair method). On the other hand, an analysis of variance is drawn from the same data with &dquo; genotype &dquo; (normal vs polydipsic) and year as sources of variation. The same data allow, finally, estimating the phenotypic correlation coefficients between all variables within each genotype after testing homogeneity of the correlations between populations. Anticipating on the following, as &dquo; normal &dquo; birds may be either homozygote for the supposed &dquo; major &dquo; gene (DiDi) or heterozygote (Didi) they will be designated in abbreviated way as (Di), and accordingly (di) for polydipsic. The term &dquo; genotype &dquo; will be used for each of these two groups. Results 1. -Presence and identification of a major gene for &dquo; !0!'!'Mt! &dquo; Table 2 gives the numbers and average values for the water /feed ratio of &dquo; normals &dquo; and &dquo; abnormals &dquo; (polydipsics) by year and strain. Figure I shows, on pooled years and strains, the distribution for the same trait expressed in deviations from means by strain and year, for &dquo; normals &dquo; and &dquo; abnormals & d q u o ; , considering only families containing both types of individuals (part overlapping of -the two distributions comes from the pooling of several years; according to the procedure used for definition of the two groups these is no overlapping within years). Within these families the total numbers of &dquo; nor-.mal &dquo; and &dquo; abnormal &dquo; birds are respectively 72 and 37 in the &dquo; Jouy &dquo; strain, 8 5 and 43 for the &dquo; M 99 &dquo; and 157 and 8 0 for both together. 2 . -Mean Pe y l py mance of genotypes and correlations within genotype Tab!es 3 , ¢ and 5 contain the mean values, t tests and analyses of variances for each population and &dquo; genotype &dquo; for water intake, feed intake, water /feed ratio, egg mass, gain in body weight, R and R'. Tables 6, 7 , 8 point out the mean values, t tests and analyses of variance by year and &dquo; genotype &dquo; for each population for sexual maturity, egg number, egg weight, Haugh units, shell thickness, wattle and shank length. Table 9 shows the pooled estimates, on a within-strain and year basis, of the correlations between the above mentioned characters within each genotype. The significances of the differences between the correlation values within genotypes are presented in table 10 , with the overall estimates of correlations for the two &dquo; genotypes &dquo;. Discussion i. -The &dquo; major gene &dquo; hypothesis for polydipsia a) Existence of two populations of individuals To summarize the above mentioned results, in a first step a discrimination between two types of birds is made thanks to the marked asymmetry of withinyear distributions. There-after it appears that the distribution of each type, years and strains being pooled, is visibly unimodal (Fig. i). According to the means and standard deviations of the distribution of &dquo; normals &dquo; and &dquo; polydipsics & d q u o ; , the probability for the distribution of one type containing individuals of the other type appears to be low enough so as not to bias appreciably the numbers of each of the two types. b) Test of the major gene hypothesis This likely existence of two distinct populations suggested in both populations the presence of several genotypes at a locus with &dquo; major &dquo; effect. The fact that families contained either only &dquo; normal &dquo; daughters, or a mixture of &dquo; normal &dquo; and &dquo; abnormal &dquo; ones with, on the whole, predominance of the former, suggests then the hypothesis of a single recessive being responsible for &dquo; polydipsia & d q u o ; , the ( * ) Which may come for a small part from year means being based mainly on &dquo; normal &dquo; birds with a somewhat variable contribution from &dquo; abnormal &dquo; ones. majority of parents being either &dquo; normal &dquo; homozygotes or heterozygotes. Of course this hypothesis was suggested on the other hand by analogy with the proven existence of such a recessive gene in other populations (Buss and MURPHY, 19 65). In full-sib families where at least one &dquo; polydipsic female was detected, the number of &dquo; normal &dquo; and &dquo; polydipsic &dquo; hens is respectively 72 vs 37 in the &dquo; Jouy &dquo; strain, 8 5 vs 43 in the &dquo; Mgg & d q u o ; , and 157 vs 8 0 for both pooled, as has been already mentioned. The expected numbers to which these figures have to be compared depend on the genotypes of the parents. Calling Di the dominant allele and di the postulated recessive allele causing high water intake (this being or not identical with that described by Buss and MURPHY), abnormal daughters can appear when the two parents are Didi or when one is Didi, the other didi (the case of two abnormal parents is expected to be rare and does not seem to have been realized). The frequency of &dquo; abnormal &dquo; females on the whole is . .174 in the &dquo; Jouy &dquo; population and . n5 in the &dquo; Mgg &dquo;. This corresponds to an estimated frequency for di q = . q. 2 in the &dquo; Jouy &dquo; strain and q = . .3 4 in the &dquo; Mgg &dquo;. The expected frequency of heterozygotes is then respectively . 4 8 and . 44 , which leads to estimate the proportion of the Didi and didi genotypes among parents having at least one di allele, respectively to . 7 .¢ and . 2 6 (for &dquo; Jouy & d q u o ; ) and to . 7 g and . 21 (for &dquo; Mgg &dquo;): from this the expected proportion of the matings susceptible of giving didi progeny (Didi X Didi or Didi x didi) may be estimated. Accordingly the expected ratio of progeny of (Di) and (di) phenotype on the whole would be 2 .58/ 1 for &dquo; Jouy &dquo; and 2.48 /1 for &dquo; Mgg & d q u o ; , corresponding respectively to the expected numbers 7 8.6 and 3 o.q in the first strain, 91 . 2 and 3 6.8 in the second. The difference with observed numbers is not significant, although some lack of (Di) appears (corrected x 2 = z . 7 o for &dquo; Jouy &dquo; and 1 .24 for M 99 &dquo;). It must be added that the limited size of dam families ( 3 . 95 progeny on average) is one more factor tending to underestimate the proportion of (Di) progeny, as families with no didi progeny but corresponding to mating types capable of giving it are discarded in our procedure. This may be estimated to represent around 79 additional (Di) birds ( * ), which would lead to the numbers 23 6 (Di) vs 8 0 (di) birds on total. Compared to the expected ratios estimated before ( * ) Based on 4 daughters, the probability of no didi progeny in Didi x Didi matings is 3/4 )'; in Didi x didi matings it is (r / 2 ) 4 . Each of these probabilities multiplied by expected frequency of mating type gives our gross estimation. (giving 2 .5 3/1 for both strains together) this gives a non-significant chi-square of 1 . 2 6. Finally the concordance between the hypothesis and available observations seems acceptable. The constitution of a line homozygote for the postulated di allele is being worked out so as to further confirm our hypothesis. . -Means o genotypes and analyses o variance The wide difference between the two &dquo; genotypes &dquo; (di) and (Di) for the mean values within years given in table 3 (a and b) for the two populations and the overall means for water intake proves to be highly significant, this being confirmed by t-test and analysis of variance in tables 4 and 5 . The same holds true for percentage of water intake related to body weight. The average values for water /feed ratio for (di), within populations and years, are about twice those of (Di). Meanwhile, the difference between &dquo; genotypes &dquo; for feed intake proved to be insignificant. That means that the variation between the two genotypes for water /feed ratio is essentially due to water intake. This finding is in agreement with the result pointed out by Buss and MURPHY ( 19 6 5 ) on polydipsic birds. The variation, within our two populations, for water intake proves to be of the same order, and the same for water /feed ratio. Both genotypes have no effect on body weight, gain in body weight and egg mass, their mean values being almost the same. On the other hand, the average difference between genotypes for the deviations of feed intake from regression on body weight, egg mass (R') or from regression on these same two traits and in addition body weight variation (R) prove also to be significant, with a lower mean value of (Di) birds as compared to (di), especially for R. It may be worthy to note, accordingly, a possibility of indirect selection on this last trait by discarding (di) birds, detected by their watery droppings. There are no significant differences between the two genotypes for egg number and egg weight overall the estimates on the two populations. The mean values for wattle length prove to be significantly different, for both populations, but in opposite directions. Another difference, for shank length and Haugh units, is found only in the M 99 population. Finally, it is interesting to mention that no significant interaction is observed for any of the traits studied. . -Correlation estimates As concerns the pooled estimates of correlations for the two populations, Table 9 shows that water /feed ratio is positively correlated, as expected, with both water intake and percentage of water to body weight for (Di) birds, and negatively correlated with feed intake, egg mass and egg number. The same can be observed for the estimates on the (di) birds; moreover, a negative correlation can be observed between water /feed ratio and body weight, variation in body weight, and also R and R', egg weight and wattle length. Buss and MURPHY ( 19 65) dis not find statistically significant correlations for water /feed ratio and egg number, egg weight, albumen quality, shell thickness or body weight. Water intake is positively and highly significantly correlated with both body weight and feed intake for (Di) birds. On the other hand, shank length is correlated positively, also, with water intake, egg weight and shell thickness. Meanwhile haugh units correlate negatively with both water intake and shell thickness. The gain in weight and body weight are highly positively correlated for the (Di) birds but not for (di) genotype. The correlations estimated on the (di) birds show that R and feed intake are highly positively correlated. R is positively correlated will wattle length while R' is negatively correlated with shell thickness. The same negative correlation is observed between water intake and wattle length. Haugh units for (di) genotype is positively correlated with sexual maturity. The significant differences between the two genotypes, with respect to correlation estimates within each of them (table io), are in some way genetic differences. This is the case for correlations between water intake and feed intake, between water /feed ratio and feed intake, R, R', egg weight, Haugh units and wattle length. These differences may be explained by the different variance in water intake between the two genotypes. Other correlations which appear significantly different between (Di) and (di) birds are those between body weight and variation in weight, between AW and R', F and R, EN and R, shell thickness and Haugh units, shell thickness and shank length, egg weight and shank length. It may be safe to confirm further these differences with more numerous data. Conclusion From the above mentioned results, it appears that the excessive water intake found for some birds in our two populations is achieved by genetic factors including probably a major gene, with apparently similar effect to that described previously by Buss and MURPHY ( 19 65). As mentioned above, the significant difference found in the present work between &dquo; normal &dquo; and &dquo; polydipsic &dquo; hens for R and R' (characterizing the part of feed efficiency independent from body weight and egg production) may suggest selecting against the di allele in such populations as ours, for improvement of the R trait, as our &dquo; polydipsic &dquo; birds are in general easily detected in cages according to their watery droppings. This effect associated to the Di locus may partly or totally account for the overall positive correlation (ignoring any variation at a particular locus), found previously between water intake and R, at fixed total feed intake, in the &dquo; Jouy &dquo; population (B ORDAS and ME RA T, 1974 ). On the other hand, the same remark as in this previous paper can be made as to a possible explanation of the effect of supplementary water intake on &dquo; residual &dquo; feed intake by the additional caloric requirement caused by the raise of temperature of ingested water to body temperature: estimating around 20 °C the mean temperature of ingested water, this explanation can give account of only a limited part of the observed effect. Re f u pour publication en juillet 197 8.

v3-fos

2017-07-01T23:30:56.317Z

1978-04-15T00:00:00.000Z

8560470

s2

Genetic and phenotypic parameters of the components parts of egg weight in Fayoumi and Rhode Island Reds The nineth and tenth eggs laid by each of 1593 Fayoumi and 465 R.I.R. pullets (i.e., a total of 3.x86 Fayoumi eggs and 93 o R.I.R. eggs) were used to study the weight of the egg and its three components. The Fayoumi pullets represent 80 sire families and 345 dam families, and the R.LR. ones represent 40 sire families and 141 dam families. Data were corrected for hatch effects before performing the variance and covariance analysis to estimate the heritability values beside the genetic and phenotypic correlations. The combined estimates of heritability were o.2 93+ 0.54 , 0. 22 6 :!: 0.0 49 , 0.310 :!: 0.054> and 0.251 --E 0.47 for Fayoumi egg weight, yolk weight, albumen weight and shell weight respectively. The corresponding figures for R.I.R. were 0.365 :!: 0.1I0 , o.3z6 ! 0.10 6, 0.5 36 0.115 and 0.578 ± o.1I6. It was shown that each gram of genetic gain in egg weight obtained through direct selection for this trait is composed of: Introduction The inheritance of the weights of the three components of the egg has not received the attention paid to egg weight. Studying the genetics of the three components of the egg may help the breeder in meeting the consumers demands and some requirements of good marketing. In this country (Egypt) the consumer pays much attention to yolk percentage, and it is of interest to know the consequences of breeding for higher egg weight on the relative weights of the egg components. The breaking strength of the egg is a function of shell percentage and shell thickness (MORG AN , I g 3 2; HALE, 1954;RICHARD and STALEY, 1967). Shell weight and percentage have their considerable significance from the marketing point of view, and genetic selection for egg weight may have indirect effect on these two characteristics of the shell. The present study was carried out to investigate the inheritance of the weight of the egg and its components. The work was done on Fayoumi, a native breed of chicken characterized by its high percentage of egg yolk and egg shell (MOSTAG!!R and K AMAR , ig6i), and the Rhode Island Red breed. Material and methods This work was carried out in the Poultry B y eeding Farm, Faculty of Ag y iculture, Cairo University. The nineth and tenth eggs laid by each of 1593 Fayoumi and 4 6 5 R.I.R. pullets (that is; a total of 3 1 86 Fayoumi eggs and 930 R.I.R. eggs) were used for this study. The Fayoumi pullets represent 8 0 sire families and 345 dam families and were obtained in z 5 different hatches in two different seasons. The R.I.R. birds were the daughters of 40 sires and 141 dams and were obtained in five hatches in one season. The Fayoumi and R.I.R. stocks used were not previously subjected to genetic selection and are considered non inbred. Eggs were weighed before breaking, and the yolk and shell (with its membranes) were freed and weighed as described by H AFEZ , BAnR!r,Drrr and K AMAR ( 1955 ). Albumen weight was obtained by difference. Data were corrected for hatch effects before performing the genetic analyses. Sire heritabilities (h 2 8 ), dam heritabilities (h 2 d ), combined heritabilities (h 2 ,) and combined genetic correlations (r 9 ) beside the phenotypic correlations were estimated by the variance and covariance component analysis. The standard errors of heritability values were estimated by the method suggested by Woo!F (ig6i), and those of the genetic correlations were calculated after RoB!xTSOrr ( 1959 ). Results and discussion a) The means and variances The means, genetic variances (taken as double the sum of the sire and the dam components of variance) and phenotypic variances of egg weight and its component parts in both Fayoumi and R.I.R. are given in table i. A large difference can be observed between the two breeds with respect to egg weight (more than 10 grams). Most of this difference (ca 8 grams) is attributed to the difference in albumen weight. The difference between the two breeds in shell weight is small ( 0 . 15 grams). Studying three different breeds, MAY and STan!r,-MAN ( 19 6 0 ) observed no significant differences between breeds with respect to shell weight, although differences did exist with respect to egg weight. Differences in egg weight, however, were not of the magnitude observed here between Fayoumi and R.LR. It may be of interest here to note that the percentages of yolk, white and shell in the Fayoumi egg are respectively: 31 . 11 , 57 .86 and 11 . 03 . The corresponding percentages in the R.I.R. egg are 29 . 09 , 6 1 .8 2 and 9 . 09 . The difference between the two breeds in the percentage of yolk as such is not much pronounced to be noticed and appraised by the consumer. Rather, the attention of the consumeris paid, it seems, to the percentage of yolk to albumen, a ratio which reaches the value of 5 3 .7 7 P . 100 in the Fayoumi egg and is only 47 . 0 6 p. 100 in the R.LR. egg. The estimates of the genetic variance of the weight of the egg and its three components were all higher in R.LR. than in Fayoumi, though the difference with respect to yolk weight was not very high. It can also be noticed that the R.LR. breed, compared to Fayoumi, showed significantly higher estimates of phenotypic variability in egg and albumen weights and lower estimates for yolk weight. The difference between the two breeds in the phenotypic variance of shell weight was not very high ( 0 . 347 in Fayoumi and 0 . 3 8 5 in R.I.R.). b) The heritability estimates The heritability estimates of egg weight and the weights of its three components in the two breeds under investigation are presented in table z. It can be observed in Fayoumi that the sire heritability estimates of egg, yolk and albumen weights are higher than their corresponding dam heritabilities, indicating thepossibility of sex linked genes involved in the inheritance of these characters. Such was not the case in R. L R., in which dam heritabilities were higher than the sire heritabilities in the four traits studied. It must be stated, however, that differences between sire and dam heritabilities in both breeds did not reach the level of significance for all the characters studied. The combined heritability estimates of egg weight and the weights of itsthree component parts are all higher in R.LR. than their corresponding estimates. in Fayoumi. In both breeds, yolk weight showed the lowest value of combined_ heritability compared to the weights of egg, white and shell. Estimates of heritability of the component parts of the egg were reported by SC H E INB E RG , WARD and N ORDSKO G ( 1953 ) and YAO and SKINNER (1959). The heritability estimates obtained by HILL, K RUEGER and Q UIS E NB E RRY ( I g66) for yolk, white and shell weights are closer to the estimates obtained in this study for R.I.R. c) Genetic and phenotypic correlations The genetic and phenotypic correlations between the four characters studied are given in table 3 . In Fayoumi, egg weight is genetically highly correlated with the weights of its three components; rg of egg weight reached the value of 0 . 9 6 with albumen weight and o.8 4 with yolk and shell weights. The three components of the Fayoumi egg are also significantly correlated with each other, the highest correlation ( 0 .8) was between albumen and shell weights. The R.I.R. showed a different picture. The yolk weight of the R.I.R. egg was not significantly correlated genetically with egg weight or with its other two components. Shell weight showed lower genetic correlations with egg weight and albumen weight ( 0 .5 1 and 0 . 35 resp.) compared with those observed in Fayoumi. The genetic correlations observed by Y AO and SKINNER ( 1959 ) between egg weight and each of yolk and albumen weight ( 0 . 71 and 0 . 9 8 resp.) are much higher than those obtained in R.I.R. in this study and closer to those of Fayoumi. The phenotypic correlations in Fayoumi are all a bit lower than their corresponding genetic correlations. In R.I.R., however, the reverse is true. Yolk weight in R.I.R. showed significant phenotypic correlations with the other three traits studied. Using the estimates shown in tables 1 , 2 and 3 , it is of interest to predict the consequences of genetic selection on the component parts of the egg. In Fayoumi, if we select for egg weight, it can be shown that each gram of genetic increase in egg weight will be distributed between the three components of egg as follows: 0 . 302 gm. yolk, 0 .6 02 gm. albumen and 0 . 09 6 gm. shell. Comparing these values with the percentages described above of the three components of the Fayoumi egg, it can be seen that direct selection for higher egg weight in this breed will result in increasing white percentage on the expense of yolk and shell percentages. With similar argument, it can be shown that the genetic increase of one gram in the R.I.R. egg obtained through direct selection for this trait will be distributed as follows: o. I I 3 gm. yolk, o.8 1 o gm. albumen and 0 . 077 gm. shell. Again the genetic increase of egg weight by direct selection in this breed will be mostly in albumen, but the increase in white percentage in the R. L R. egg will be much more pronounced than in the Fayoumi egg. The genetic increase in egg weight could, however, be obtained indirectly through selection for one of the three components of the egg. The relative efficiency of gain in egg weight arrived at through direct selection to that obtained through indirect selection is h l : rgh2, where h 2 I is the heritability of egg weight and h 2 2 is the heritability of the other trait. Thus, in Fayoumi, if selection is directed to the weight of the yolk rather than the weight of the egg, it can be shown that every gram of genetic gain in egg weight obtained through this indirect selection is distributed as follows: 0 . 425 gm. yolk, 0 . 4 8 9 gm. albumen and 0 . 0 86 gm. shell. Clearly, here we gain in yolk percentage on the expense of albumen and shell percentages, but the efficiency of genetic gain in egg weight will be only 74 p. 100 of the efficiency of direct selection. If yolk has its economic significance in Fayoumi, as the case seems to be in this country, a proper evaluation of this significance should be done. This will enable the breeder to evaluate the economic consequences of different selection indices and choose the most appropriate one. It can be also shown that the one gram indirect genetic increase in Fayoumi egg weight gained through selection on albumen weight is distributed as follows: 0 . 24 6 gm. yolk, 0 .658 gm. albumen and o.o 9 6 gm. shell, with a relative efficiency of 9 8 p. 100 , and the distribution when selecting on shell weight will be: 0.270 gm. yolk, 0.5 95 gm. albumen and 0 . 135 gm. shell, (with a relative efficiency of 7 8 p. 100 ). Selection on shell weight will increase the percentages of both shell and albumen on the expense of yolk percentage. Il est montré que chaque gramme de gain génétique sur le poids de l'oeuf obtenu par sélection directe pour ce caractère est composé de 0 , 302 g de jaune, o,6 0 2 g d'albumen et 0 , 09 6 g de coquille chez la Fayoumi, et de 0 , 113 g de jaune, o,8 1 o g d'albumen et 0 , 077 g de coquille chez la R.I.R.

v3-fos

2016-05-12T22:15:10.714Z

1978-01-15T00:00:00.000Z

11399631

s2

The effect of inflation and form of investment on the estimated value of genetic improvement in farm livestock High rates of interest (8 to 15%) commonly used in discounted cash flow analysis of genetic improvement schemes have tended to underestimate the value of the returns and to favour breeding programmes with short-term returns. It is shown that inflation should be removed from the interest rate, and an inflation-free rate used in discounting future returns to present-day value. Various methods for choosing discount rates are discussed. It is concluded that the appropriate rate is the real opportunity cost rate, that is the market cost of borrowing in real terms, in terms of goods rather than money. Taking account of inflation the average cost of borrowing in real terms in the United Kingdom over the past 32 years (1945 to 1976) is estimated as 2 to 3%. This also provides the best available estimate for discounting future returns, and it brings the opportunity cost rate into line with the social time preference rate often proposed for public investments. The contrast is made between the value of improvements made in the national interest and of those made by breeders or firms. The former benefit from returns on all national commercial production of improved stocks and the returns accumulate and are recouped over many years. On the other hand breeders and firms benefit only from the extra sales of breeding stock due to their temporary marginal superiority over competition and they are often at high risk of getting no returns. Implications to the form and amount of investment in animal improvement are discussed. 3 6, resp. However due to an indication of preferential treatment of daughters after the best cows and due to the relative few daughters yet available in the material, final conclusions concerning the efficiency of the two procedures have to wait for more extensive studies. II. - High rates of interest (8-15 p. ioo) commonly used in discounted cash flow analysis of genetic improvement schemes have tended to underestimate the value of the returns and to favour breeding programs with short-term returns. It is shown that inflation should be removed from the interest rate, and an inflation-free rate used in discounting future returns to present day values. Various methods for choosing discount rates are discussed. It is concluded that the appropriate rate is the veal opportunity cost rate, that is the market cost of borrowing in real terms, in terms of goods rather than in money terms. Taking account of inflation the average cost of borrowing in real terms in the U.K. over the past 32 years ( 1945 -197 6) is estimated as 2 -3 p. 100 . This also provides the best available estimate for discounting future returns, and it brings the opportunity cost rate into line with the social time preference rate often proposed for public investments. The contrast is made between the value of improvements made in the national interest and of those made by breeders or firms. The former benefit from returns on all national commercial production of improved stocks and the returns accumulate and are recouped over many years. On the other hand breeders and firms benefit only from the extra sales of breeding stock due to their temporary marginal superiority over competitors and they are often at high risk of getting no returns. Implications to the form and amount of investment in animal improvement are discussed. Using examples from sheep populations, the relative importance of male, female and slaughter traits is discussed in relation to different ways of obtaining replacement animals. Three types of population are distinguished, namely nucleus populations with self-supply of breeding animals of both sexes, subnucleus populations with self-supply of females only, and commercial populations buying all breeding animals. Nucleus populations which supply various other populations with breeding animals are also considered. SENSITIVITY OF OVERALL ECONOMIC The examples given indicate that the appropriate breeding objectives need not be identical for both sexes, even if the animals selected are to be used in the same population. The way in which replacement is recruited influences the relative importance of the various traits to be considered within the same sex as well as the differences in appropriate breeding objectives for the two sexes. Dpt of Animal Breeding, Agricultural College, S. 750 0 7 Uppsala, Sweden An investigation concerning the economic influence on the production cost of various traits in pig breeding has been carried out. The terms " economic influence " or " economic value " of a trait in this work have been used to indicate the effect on the production cost per kg lean

v3-fos