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Requirement of White Leghorn Laying and Breeding Hens for Methionine as Influenced by Stage of Production Cycle and Inorganic Sulfate1
Requirement of White Leghorn Laying and Breeding Hens for Methionine as Influenced by Stage of Production Cycle and Inorganic Sulfatel L . S. J E N S E N , 2 L . FALEN A N D G . W . SCHUMAIER
Department of Animal Sciences, Washington State University, Pullman 99163 (Received for publication June 2, 1973)
POULTRY SCIENCE 53: 535-544, 1974
INTRODUCTION
day. Harms and Damron (1969) concluded ETHIONINE is the first limiting amino that the methionine requirement was between acid in most practical rations fed to 250 and 280 mg. per day, provided that the laying hens and following the first study of diet contained a total of 530 mg. of sulfur Leong and McGinnis (1952) several labora- amino acids. Using a diet dilution technique, tories have investigated the quantitative re- Fisher and Morris (1970) estimated the requirement of hens for this amino acid as well quirement to be 275 mg. per bird per day as for total sulfur-containing amino acids. The for maximum egg yield of pullets during the National Research Council (1971) recom- early stages of lay. They also applied different mends a level of 0.28% methionine and 0.25% statistical procedures to the data of Bray cystine in a ration containing 2,850 kcal. (1965) and suggested that Bray has underestimetabolizable energy per kilogram. Data of mated the requirement. Several experiments Combs (1964) indicated a requirement of 295 conducted by Carlson and Guenthner (1969) mg. methionine per hen per day. Bray (1965) indicated that the requirement was in excess estimated the requirement to be 233 mg. per of 300 mg. per day during the first four months of production, but between 289 and 328 mg. per day during the later stages of the laying 1. Scientific Paper No. 4080. College of Agriculture cycle. Research Center, Washington State University. ProjLess attention has been paid to the requireect 1985. 2. Current Address: Dept. of Poultry Science, Uni- ment of breeding hens for methionine for obtaining maximum hatchability. Mehring et versity of Georgia, Athens.
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ABSTRACT Four experiments were conducted with Single Comb White Leghorn hens fed diets deficient in methionine to determine the effect of such a deficiency on hatchability and to estimate the requirement of methionine for egg production at different stages of the laying cycle and the influence of inorganic sulfate on the requirement. In two experiments hatchability of fertile eggs was not affected by a methionine deficiency, while in two others an indication of lower hatchability was observed at the lowest level of methionine, but the differences were not statistically significant. The results indicate that a level of methionine adequate for optimum egg production and egg weight is also adequate for maximum hatchability. Adding methionine to a corn-soybean meal ration containing 16% protein failed to improve any of the parameters measured. In one experiment a significant response in egg production was obtained by adding methionine to a corn-soybean ration containing 14% protein, but not in a second experiment. Egg production and egg weight was always improved by the addition of methionine to a corn-pea ration containing 16% protein. An evaluation of the four experiments indicated that a methionine requirement of between 290 and 300 mg./hen/day was adequate for optimum egg yield. The requirement was higher for obtaining maximum egg weight than for obtaining maximum egg production when the corn-pea ration was used. A significant response to methionine supplementation of the corn-pea ration was obtained in the presence or absence of 0.2% potassium sulfate, and there was no statistically significant difference in egg production and egg weight between the unsupplemented diets with or without sulfate. These results suggest that the methionine requirement estimated in these experiments was for methionine per se rather than for methionine plus inorganic sulfate derived from methionine.
536
L. S. JENSEN, L. FALEN AND G. W. SCHUMAIER
The inorganic sulfate content of the ration and the water supply is a factor which can influence the methionine requirement of laying hens. Machlin et al. (1953) showed that radioactive labeled inorganic sulfate fed to laying hens was transferred to the egg, part of it in the form of cystine. Recently, Harms (1973) has indicated that inorganic sulfate addition to laying diets can reduce the requirement for total sulfur containing amino acids. The present studies were conducted (1) to obtain further information on the methionine requirement of laying hens during different stages of the laying cycle; (2) to determine if a methionine deficiency will affect hatchability of eggs; and (3) to obtain information on the sparing effect of inorganic sulfate on the methionine requirement of hens. PROCEDURE Four experiments were conducted with Single Comb White Leghorn pullets of a commercial strain that were housed in group cages, 0.6 x 1.2 meter in size. The pullets
were reared in floor pens and fed a practical type chick starter ration -from 0-8 weeks of age containing corn, soybean meal, fish meal and meat meal and a developer ration from 8 weeks to time of housing in the laying cages composed of similar ingredients and containing approximately 13.5% protein. The pullets were housed in the group cages at 20 weeks of age, at which time they were either placed on the experimental rations or fed a practical layer ration containing corn, wheat, soybean meal, fish meal, meat meal and alfalfa meal and calculated to contain approximately 16% crude protein. All birds were given feed and water ad libitum. The birds were group-weighed at the beginning and end of each experiment to determine the effect of dietary treatments on body weight change. Egg production was recorded daily and calculated on a hen-day basis. Feed consumption was determined bi-weekly. All eggs laid every other week were weighed by pens to determine average egg weight. In order to study the effect of dietary treatments on fertility and hatchability, one Single Comb White Leghorn male was placed in each group cage and the males were rotated from pen to pen weekly. It was assumed that the male consumed feed about equivalent to one hen and when determining feed consumption, the total feed consumed by the pen was divided by the number of hens plus the male in the pen. Eggs that were clear by candling at 10 days of age of incubation were broken to distinguish early embryo deaths from infertile eggs. In Experiment 3, one week's collection of eggs toward the end of the experiment was used to determine the incidence of blood spots in the eggs and to determine albumen quality as expressed in Haugh units. Composition of three basal diets used in these experiments is presented in Table 1. The ration containing dry peas (Pisum sativum) was formulated to be deficient in methionine, but still to contain a level of protein
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al. (1954) observed that supplementing a corn-soybean meal ration for New Hampshire pullets with methionine significantly increased the hatchability of eggs. Daghir et al. (1964) also reported that hatchability of fertile eggs was significantly improved by supplementing a corn-soybean meal ration with 0.1 or 0.15% methionine hydroxy analog. Barred Plymouth Rock pullets were used in their study. Neither Mehring et al. (1954) nor Daghir et al. (1964) observed any effect of methionine of MHA supplementation on egg production, suggesting that there was a higher requirement for hatchability than for egg production. On the other hand, Leong and McGinnis (1952) failed to demonstrate any effect on hatchability of eggs of Single Comb White Leghorn pullets, even though a marked response in egg production was obtained by supplementing a pea-based diet with methionine.
537
METHIONINE REQUIREMENT TABLE 1.-—Composition
Ingredient Corn Soybean meal (48.5% protein) Peas (23.4% protein) Dehydrated alfalfa Limestone Dicalcium phosphate Iodized salt Vitamin premix' Trace mineral premix2
of basal rations Corn-soy-14 (%) 74.4 14.0
2.5 6.7 1.6 0.5 0.25 0.05
Diet Corn-soy-16 (%) 69.4 19.1 2.5 6.7 1.5 0.5 0.25 0.05
Corn-pea-16 (%) 35.9 52.8 2.5 6.7 1.3 0.5 0.25 0.05
commonly used under commercial conditions. Rations containing soybean meal were formulated to contain approximately 16 or 14% crude protein. Both the pea and the lower protein soybean ration contained calculated values of methionine and methionine plus cystine below that recommended by the National Research Council. The values were calculated on the basis of composition data presented by Scott et al. (1969). The basal diets were analyzed by use of a Technicon TSM Amino Acid Analyzer by a method similar to Spackman et al. (1958). Methionine was determined by a method developed by Hedwig et al. (1972), using thin-layer chromatography. The analyzed values for methionine were similar to the calculated values, but those for cystine were considerably higher than the calculated values. In Experiment 1, 8 pullets were placed in each group cage and three groups were fed
each of the experimental diets. Graded levels of DL-methionine from 0 to 0.12% were added to the soybean meal ration containing 16% protein and levels of 0 and 0.20% to the pea ration. The pullets were placed on the experimental diets after reaching peak production and were fed the diets from 32 to 45 weeks of age. In Experiment 2, 8 hens were placed in each group and three groups were fed each of the diets. All three basal rations were used in this study with 0 and 0.04% DL-methionine added to the two soybean rations and 0, 0.02, 0.04 and 0.08% DL-methionine to the pea ration. The hens were placed on the experiment during the latter part of the laying cycle (53 to 65 weeks of age). In Experiment 3, 8 pullets were placed in each group and four groups were fed each of the experimental diets. The pullets were placed on experimental diets at time of housing and were fed the diets from 20 to 36
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Calculated analysis: 16.0 13.9 16.0 Protein, % 2.99 3.01 2.99 Calcium, % .60 .60 .61 Phosphorus, % 2882 2922 2640 M.E., kcal./kg. .267 .240 .235 Methionine, % .280 .251 .163 Cystine, % .547 .491 .398 Methionine + cystine, % Determined analysis, % .248 .280 .249 Methionine, % .254 .330 .278 Cystine, % 'Vitamin premix supplied the following per kg.: vitamin A, 4,000 I.U.; vitamin D3, 500 I.C.U. riboflavin, 4 mg.; calcium pantothenate, 10 mg.; niacin, 10 mg.; folic acid, 0.3 mg.; vitamin B ] 2 5 meg.; choline chloride, 250 mg.; and ethoxyquin, 125 mg. 2 Trace mineral mix supplied the following mg./kg.: manganese, 50; iron, 50; zinc, 50; copper, 5 iodine, 1.5; and cobalt, 0.5.
Added meth. Egg prod.
Egg wt.
Change in body wt. 95ab 94ab 96a 99a 89b 96a 92ab 95ab 91ab 94ab
%
Fertility
Egg prod.
%
Added meth.
%
Egg wt.
Change in body wt.
gg61.9a 0 46.9c Corn-soy-14 -104 Corn-soy-14 .04 64.7ab 61.4abc -236 Corn-soy-16 0 72.7a 60.6abc -28 Corn-soy-16 .04 67.5a 61.7ab -132 Corn-pea-16 0 50.6bc 58.0c -343 .02 58.4bc 58.7abc Corn-pea-16 -113 .04 62.1bc 58.4bc Corn-pea-16 -95 Corn-pea-16 .08 65.6ab 61.1abc -254 'Values not having a common letter are statistically different (P < 0.05).
Diet
68a 78a 89a 76a 83a 72a 78a 56a
%
Fertility
TABLE 3.—Response of hens from 53 to 65 weeks of age to methionine supplementation of thre
0 80.3a 56.5a + 205 .04 74.4ab 57.4a + 115 ,08 82.7a 57.3a + 136 .12 79.0a 57.3a + 85 0 66.3b 51.4b -30 ,04 79.0a 56.8a +79 ,08 81.1a 55.9a + 57 .12 75.5a 56.4a + 116 ,16 77.9a 57.4a +94 .20 81.7a 57.0a + 103 'Values not having a common letter are statistically different (P < 0.05).
Corn- soy-16 Corn- soy-16 Corn- soy-16 Corn- soy-16 Corn- pea-16 Corn- pea-16 Corn- pea-16 Corn- pea-16 Corn- pea-16 Corn- pea-16
Diet
TABLE 2.—Response of pullets from 32 to 45 weeks of age to methionine supplementation of tw
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METHIONINE REQUIREMENT
RESULTS In Experiment 1, adding methionine to the soybean ration containing 16% protein failed to significantly affect any of the parameters measured in the experiment (Table 2). On the other hand, adding 0.04% or higher levels of DL-methionine to the pea ration significantly improved egg production and egg weight. Furthermore, the hens fed the pea diet with no methionine were the only ones to lose weight during the experiment. Fertility of the eggs was not greatly affected by methionine supplementation. Although hatchability of fertile eggs was slightly lower for the pea ration containing the two lowest levels of methionine supplementation, the differences were not statistically significant from the diets containing higher levels. Hatchability of fertile eggs from hens fed the pea diet tended to be slightly lower than that of eggs from hens fed the soybean diet. In Experiment 2, again no statistically significant improvement in any of the parameters measured was obtained by adding methi-
onine to the soy ration containing 16% protein (Table 3). On the other hand, a significant improvement in egg production was observed by adding methionine to the soy ration containing 14% protein. Although the differences were not statistically significant, adding methionine to the pea ration appeared to improve egg production and egg weight. Fertility varied considerably and did not appear to be improved by adding methionine to any of the diets. Hatchability of fertile eggs again tended to be lower for diets low in methionine, but the differences were not significant. In contrast to the previous experiment, results obtained in Experiment 3 showed no improvement in egg production by adding methionine to the soy ration containing 14% protein (Table 4). Egg weight also was not significantly affected, and adding methionine to the soybean ration containing 16% protein again failed to significantly improve the parameters measured. Adding 0.02% DL-methionine to the pea ration significantly improved egg production and higher levels did not increase egg production further. On the other hand, a level of 0.08% added methionine appeared to be necessary to obtain maximum egg weight during this period of the laying cycle when the birds were reaching peak egg production. Fertility was not affected by the dietary treatments and no evidence was obtained in this experiment that diets low in methionine resulted in depression in hatchability of fertile eggs. Albumen quality, as measured by Haugh units and incidence of blood spots were not significantly affected by the dietary treatments. In Experiment 4, adding potassium sulfate to the ration failed to modify the response of hens to methionine supplementation of the pea ration (Table 5). Adding 0.04% or higher levels of methionine significantly improved egg production in both series. A level of 0.08% added methionine was necessary to obtain maximum egg weight. Fertility and hatchability of eggs were not significantly affected
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weeks of age. All three basal rations were again used with varying levels of added methionine. In Experiment 4, 10 pullets were placed in each group and 4 groups were fed each of the experimental diets. Only the pea basal ration was used in this experiment and levels of 0, 0.04, 0.08 and 0.12% added DL-methionine were compared with and without 0.2% added potassium sulfate. Data were calculated for two phases of the experiment (21-45 weeks and 45-67 weeks) in order to see if major differences in requirement for methionine were evident during the two phases of the laying cycle. The sulfate content of the water supply for the hens in this experiment was determined by barium precipitation method and was found to be approximately 42 p.p.m.
539
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L. S. JENSEN, L. FALEN AND G. W. SCHUMAIER
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L . S. JENSEN, L. FALEN AND G. W.
TABLE 6.-
SCHUMAIER
-Summary of the range of daily methionine intakes that were adequate or inadequate for optimum performance
Basal diet Corn-soy-16 Corn-pea-16 Corn-soy-14 Corn-soy-16 Corn-pea-16 Corn-pea-16 Corn-soy-14 Corn-soy-16 Corn-pea-16 Corn-pea-16 Corn-pea-16 Corn-pea-16
Weeks of age 32-45 32-45 53-65 53-65 53-65 20-36 20-36 20-36 21-45 21-45 45-67 45-67
Daily methionine intake (mg.) Adequate' Inadequate' 333 — 349 244 308 247 280 — 293 265 312 268 250 — 264 — 302 267 299 261 343 289 334 294
Inadequate or adequate to support maximum daily egg yield.
by the dietary treatments. During the second half of the treatment, there were no statistically significant differences in egg production and egg weight between the groups fed no added methionine, with and without potassium sulfate, although egg size appeared to be slightly higher for those with sulfate. Again 0.04% added methionine appeared to be adequate for maximum egg production but a higher level was necessary for maximum egg weight. Fertility of eggs appeared to be lower for the low methionine diets without added sulfate. Hatchability of fertile eggs varied some but the variation did not appear to be related to methionine supplementation. DISCUSSION Hatchability of fertile eggs did not appear to be markedly affected by methionine level of the diets in any of the experiments. In Experiments 3 and 4, no statistically significant improvement in hatchability of fertile eggs was observed by adding methionine. In Experiments 1 and 2 the differences were also not statistically significant, but there was some indication that the hatchability was slightly lower with low methionine diets. Even if the differences observed in Tables 1 and 2 were of biological significance, the level of methionine adequate for maximum
egg production and egg weight would supply enough methionine to meet the requirements for maximum hatchability. These results are in general agreement with Leong and McGinnis (1952) where no effect on hatchability was observed by adding methionine to the diet, but are in contrast to results obtained by Mehring et al. (1954) and Daghir et al. (1964) who obtained significant responses in hatchability by adding methionine even though egg production was not improved. These investigators used New Hampshire or Barred Plymouth Rock chickens in contrast to the White Leghorn chickens used in our study and in that of Leong and McGinnis (1952). Perhaps there are breed differences in response to methionine for obtaining maximum hatchability. The results of these experiments again demonstrate that a corn-soybean meal ration formulated to contain 16% crude protein is adequate in methionine and cystine for obtaining optimum egg yield in laying hens. Others who have used corn-soybean meal rations to study the methionine requirements of laying hens have had to go to lower protein levels in order to obtain a response. In Experiment 2, a response in egg production was obtained by adding methionine to a corn-soy ration containing 14% protein, but no such response was observed in Experiment 3.
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Exp. No. 1 1 2 2 2 3 3 3 4 4 4 4
543
METHIONINE REQUIREMENT
The results obtained in Experiment 4 shows that a statistically significant response in egg production and egg weight was obtained to methionine supplementation even though the diet was supplemented with 0.2% potassium sulfate. These results suggest that the chickens were obtaining enough inorganic sulfate from their diet plus the water supply and that the requirement determined under the conditions of our laboratory was for methionine to serve as an amino acid in egg protein
synthesis rather than methionine to serve as both an amino acid and as a source of sulfate. Harms (1973) indicated that sulfate can spare part of the methionine requirement of laying hens. In his studies the sulfate forms of mineral elements were not included in the diet and the water supply contained 28 p.p.m. of sulfate. A higher level of total sulfate in the diet and water supply under our conditions may explain the lack of response to sulfate supplementation. REFERENCES Bray, D. J., 1965. The methionine requirement of young laying pullets. Poultry Sci. 44: 1173-1180. Carlson, C. W., and E. Guenthner, 1969. Response of laying hens fed typical corn-soy diets to supplements of methionine and lysine. Poultry Sci. 48: 137-143. Combs, C. F., 1964. Further studies of protein and amino acid needs of broilers and laying hens. Proc. Maryland Nutr. Conf. : 45-71. Daghir, N. J., S. S. Akrabawi and K. Rottensten, 1964. Methionine supplementation of breeder diets. Poultry Sci. 43: 1106-1109. Fisher, C , and T. R. Morris, 1970. The determination of the methionine requirement of laying pullets by a diet dilution technique. Brit. Poultry Sci. 11: 67-82. Harms, R. H., 1973. Sparing effect of sulfate and other factors on total sulfur amino acid requirements. Proc. Georgia Nutr. Conf. : 1-10. Harms, R. H., and B. L. Damron, 1969. Protein and sulfur amino acid requirement of the laying hen as influenced by dietary formulation. Poultry Sci. 48: 144-149. Hedwig, H. E., J. M. Lawrence and D. R. Cochran, 1972. Simple and rapid methods for determination of methionine and cystine in legume seeds. Anal. Biochem. 48: 353-364. Leong, K. C , and J. McGinnis, 1952. An estimate of the methionine requirement for egg production. Poultry Sci. 31: 692-695. Machlin, L. J., P. B. Pearson, C. A. Denton and H. R. Bird, 1953. The utilization of sulfate sulfur by the laying hen and its incorporation into cystine. J. Biol. Chem. 205: 213-219. Mehring, A. L., Jr., H. W. Titus and J. Waddell, 1954. The effect of adding methionine and vitamin B l2, singly and together, to a corn-soy diet for laying chickens. Poultry Sci. 33: 1191-1197. National Research Council, 1971. Nutrient requirements of domestic animals. No. 1. Nutrient require-
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Perhaps this reflects a slight difference in quantitative requirement between birds at two different stages of the laying cycle. It could also be due to variation in the ingredients used in the two experiments. It was interesting that even though methionine supplementation to the 14% protein corn-soy diet used in Experiment 2 significantly improved egg production, egg size was not affected. In all experiments involving the pea ration, the requirement for methionine for obtaining maximum egg size was higher than for maximum egg production. To aid in estimating the methionine requirement of laying hens from these studies, a summary of the range of daily methionine intakes that were adequate or inadequate for optimum performance is presented in Table 6. Some of these ranges would be lower if one only considered the rate of egg production. The summary indicates that a range between 290 and 300 mg. of methionine per day should be adequate to meet the requirements of laying hens at different stages of the laying cycle. This requirement is in fairly good agreement with that reported by Combs (1964), Harms and Damron (1969), Fisher and Morris (1970), and Carlson and Guenthner (1969). Although it is difficult to determine if there was any significant difference in requirement for daily methionine intake between stages of the laying cycle, the summary in Table 6 suggests that if there is a difference, it would be higher for the later stages of production than for the early stages.
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L. S. JENSEN, L. FALEN AND G. W. SCHUMAIER
ments of poultry. Washington, D.C. Scott, M. L., M. C. Nesheim and R. J. Young, 1969. Nutrition of the Chicken. Scott and Associates, Ithaca, N.Y.
Spackman, D. H., W. H. Stein and S. Moore, 1958. Automatic recording apparatus for use in the chromatography of amino acids. Anal. Chem. 30: 1190— 1206.
Maintenance of a Specific Pathogen Free Turkey Flock Y. MOHAMED SAIF
(Received for publication June 2, 1973)
ABSTRACT The procedures used to maintain a specific pathogen free turkey flock are dscribed. Seven generations of SPF turkeys were raised over the last six years. All birds were free of Mycoplasma, Salmonella, and Arizona infections. No evidence of airsacculitis or other infectious diseases was detected. POULTRY SCIENCE 53: 544-546,
report (Mohamed and Bohl, IN1969)a previous the procedures used for establishing; a specific pathogen free (SPF) turkey flock: were described. The main objective was to> develop a flock free of detectable Mycoplasma, Salmonella, and Arizona infections. This> report presents information on seven generations of SPF turkeys reared over the lastt six years at Ohio Agricultural Research andI Development Center. The incidence of diseases in conventional flocks derived from the: same parent flock and maintained under conventional conditions over the last six yearsi are discussed. MATERIALS AND METHODS The parent SPF flock was derived fromi a flock of heavy White turkeys naturally' infected with Mycoplasma meleagridis. The : flock was maintained at Ohio AgriculturalI Research and Development Center. A series> of serologic and cultural examinations was> conducted to establish the status of the flocki relative to infection with certain pathogens. Testing Procedure for Mycoplasma and Salmonella. The different generations were:
1974
tested according to a standard procedure. All birds were tested serologically at five weeks of age for M. meleagridis, Mycoplasma gallisepticum, and Mycoplasma synoviae. Tube agglutination tests were conducted using antigens prepared in this laboratory according to Mohamed and Bohl 1968, and Olson et al. (1963). At this time the rectum of each bird was swabbed and bedding samples were collected and inoculated into culture media for the isolation of Salmonella and Arizona organisms. The swabs and bedding material were incubated at 37° C. in selenite broth and transfers were made to brilliant green agar plates at 6 and 24 hours. The agar plates were incubated for 3 days and examined daily for Salmonella or Arizona-like colonies. Colonies suspected to be of Salmonella or Arizona were transferred into different media for identification. The following media were used, lactose broth, dulcital broth, malonate broth, urea broth, triple sugar iron agar, citrate agar, dextrose agar, lysine decarboxylase, ornithine decarboxylase, and phenyl deaminase. At 30 weeks of age the same procedure used at five weeks of age was followed. In addition, serum samples were
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Department of Veterinary Science and Poultry Science, Ohio Agricultural Research and Development Center, Wooster, Ohio 44691
Department of Animal Sciences, Washington State University, Pullman 99163 (Received for publication June 2, 1973)
POULTRY SCIENCE 53: 535-544, 1974
INTRODUCTION
day. Harms and Damron (1969) concluded ETHIONINE is the first limiting amino that the methionine requirement was between acid in most practical rations fed to 250 and 280 mg. per day, provided that the laying hens and following the first study of diet contained a total of 530 mg. of sulfur Leong and McGinnis (1952) several labora- amino acids. Using a diet dilution technique, tories have investigated the quantitative re- Fisher and Morris (1970) estimated the requirement of hens for this amino acid as well quirement to be 275 mg. per bird per day as for total sulfur-containing amino acids. The for maximum egg yield of pullets during the National Research Council (1971) recom- early stages of lay. They also applied different mends a level of 0.28% methionine and 0.25% statistical procedures to the data of Bray cystine in a ration containing 2,850 kcal. (1965) and suggested that Bray has underestimetabolizable energy per kilogram. Data of mated the requirement. Several experiments Combs (1964) indicated a requirement of 295 conducted by Carlson and Guenthner (1969) mg. methionine per hen per day. Bray (1965) indicated that the requirement was in excess estimated the requirement to be 233 mg. per of 300 mg. per day during the first four months of production, but between 289 and 328 mg. per day during the later stages of the laying 1. Scientific Paper No. 4080. College of Agriculture cycle. Research Center, Washington State University. ProjLess attention has been paid to the requireect 1985. 2. Current Address: Dept. of Poultry Science, Uni- ment of breeding hens for methionine for obtaining maximum hatchability. Mehring et versity of Georgia, Athens.
M
535
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ABSTRACT Four experiments were conducted with Single Comb White Leghorn hens fed diets deficient in methionine to determine the effect of such a deficiency on hatchability and to estimate the requirement of methionine for egg production at different stages of the laying cycle and the influence of inorganic sulfate on the requirement. In two experiments hatchability of fertile eggs was not affected by a methionine deficiency, while in two others an indication of lower hatchability was observed at the lowest level of methionine, but the differences were not statistically significant. The results indicate that a level of methionine adequate for optimum egg production and egg weight is also adequate for maximum hatchability. Adding methionine to a corn-soybean meal ration containing 16% protein failed to improve any of the parameters measured. In one experiment a significant response in egg production was obtained by adding methionine to a corn-soybean ration containing 14% protein, but not in a second experiment. Egg production and egg weight was always improved by the addition of methionine to a corn-pea ration containing 16% protein. An evaluation of the four experiments indicated that a methionine requirement of between 290 and 300 mg./hen/day was adequate for optimum egg yield. The requirement was higher for obtaining maximum egg weight than for obtaining maximum egg production when the corn-pea ration was used. A significant response to methionine supplementation of the corn-pea ration was obtained in the presence or absence of 0.2% potassium sulfate, and there was no statistically significant difference in egg production and egg weight between the unsupplemented diets with or without sulfate. These results suggest that the methionine requirement estimated in these experiments was for methionine per se rather than for methionine plus inorganic sulfate derived from methionine.
536
L. S. JENSEN, L. FALEN AND G. W. SCHUMAIER
The inorganic sulfate content of the ration and the water supply is a factor which can influence the methionine requirement of laying hens. Machlin et al. (1953) showed that radioactive labeled inorganic sulfate fed to laying hens was transferred to the egg, part of it in the form of cystine. Recently, Harms (1973) has indicated that inorganic sulfate addition to laying diets can reduce the requirement for total sulfur containing amino acids. The present studies were conducted (1) to obtain further information on the methionine requirement of laying hens during different stages of the laying cycle; (2) to determine if a methionine deficiency will affect hatchability of eggs; and (3) to obtain information on the sparing effect of inorganic sulfate on the methionine requirement of hens. PROCEDURE Four experiments were conducted with Single Comb White Leghorn pullets of a commercial strain that were housed in group cages, 0.6 x 1.2 meter in size. The pullets
were reared in floor pens and fed a practical type chick starter ration -from 0-8 weeks of age containing corn, soybean meal, fish meal and meat meal and a developer ration from 8 weeks to time of housing in the laying cages composed of similar ingredients and containing approximately 13.5% protein. The pullets were housed in the group cages at 20 weeks of age, at which time they were either placed on the experimental rations or fed a practical layer ration containing corn, wheat, soybean meal, fish meal, meat meal and alfalfa meal and calculated to contain approximately 16% crude protein. All birds were given feed and water ad libitum. The birds were group-weighed at the beginning and end of each experiment to determine the effect of dietary treatments on body weight change. Egg production was recorded daily and calculated on a hen-day basis. Feed consumption was determined bi-weekly. All eggs laid every other week were weighed by pens to determine average egg weight. In order to study the effect of dietary treatments on fertility and hatchability, one Single Comb White Leghorn male was placed in each group cage and the males were rotated from pen to pen weekly. It was assumed that the male consumed feed about equivalent to one hen and when determining feed consumption, the total feed consumed by the pen was divided by the number of hens plus the male in the pen. Eggs that were clear by candling at 10 days of age of incubation were broken to distinguish early embryo deaths from infertile eggs. In Experiment 3, one week's collection of eggs toward the end of the experiment was used to determine the incidence of blood spots in the eggs and to determine albumen quality as expressed in Haugh units. Composition of three basal diets used in these experiments is presented in Table 1. The ration containing dry peas (Pisum sativum) was formulated to be deficient in methionine, but still to contain a level of protein
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al. (1954) observed that supplementing a corn-soybean meal ration for New Hampshire pullets with methionine significantly increased the hatchability of eggs. Daghir et al. (1964) also reported that hatchability of fertile eggs was significantly improved by supplementing a corn-soybean meal ration with 0.1 or 0.15% methionine hydroxy analog. Barred Plymouth Rock pullets were used in their study. Neither Mehring et al. (1954) nor Daghir et al. (1964) observed any effect of methionine of MHA supplementation on egg production, suggesting that there was a higher requirement for hatchability than for egg production. On the other hand, Leong and McGinnis (1952) failed to demonstrate any effect on hatchability of eggs of Single Comb White Leghorn pullets, even though a marked response in egg production was obtained by supplementing a pea-based diet with methionine.
537
METHIONINE REQUIREMENT TABLE 1.-—Composition
Ingredient Corn Soybean meal (48.5% protein) Peas (23.4% protein) Dehydrated alfalfa Limestone Dicalcium phosphate Iodized salt Vitamin premix' Trace mineral premix2
of basal rations Corn-soy-14 (%) 74.4 14.0
2.5 6.7 1.6 0.5 0.25 0.05
Diet Corn-soy-16 (%) 69.4 19.1 2.5 6.7 1.5 0.5 0.25 0.05
Corn-pea-16 (%) 35.9 52.8 2.5 6.7 1.3 0.5 0.25 0.05
commonly used under commercial conditions. Rations containing soybean meal were formulated to contain approximately 16 or 14% crude protein. Both the pea and the lower protein soybean ration contained calculated values of methionine and methionine plus cystine below that recommended by the National Research Council. The values were calculated on the basis of composition data presented by Scott et al. (1969). The basal diets were analyzed by use of a Technicon TSM Amino Acid Analyzer by a method similar to Spackman et al. (1958). Methionine was determined by a method developed by Hedwig et al. (1972), using thin-layer chromatography. The analyzed values for methionine were similar to the calculated values, but those for cystine were considerably higher than the calculated values. In Experiment 1, 8 pullets were placed in each group cage and three groups were fed
each of the experimental diets. Graded levels of DL-methionine from 0 to 0.12% were added to the soybean meal ration containing 16% protein and levels of 0 and 0.20% to the pea ration. The pullets were placed on the experimental diets after reaching peak production and were fed the diets from 32 to 45 weeks of age. In Experiment 2, 8 hens were placed in each group and three groups were fed each of the diets. All three basal rations were used in this study with 0 and 0.04% DL-methionine added to the two soybean rations and 0, 0.02, 0.04 and 0.08% DL-methionine to the pea ration. The hens were placed on the experiment during the latter part of the laying cycle (53 to 65 weeks of age). In Experiment 3, 8 pullets were placed in each group and four groups were fed each of the experimental diets. The pullets were placed on experimental diets at time of housing and were fed the diets from 20 to 36
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Calculated analysis: 16.0 13.9 16.0 Protein, % 2.99 3.01 2.99 Calcium, % .60 .60 .61 Phosphorus, % 2882 2922 2640 M.E., kcal./kg. .267 .240 .235 Methionine, % .280 .251 .163 Cystine, % .547 .491 .398 Methionine + cystine, % Determined analysis, % .248 .280 .249 Methionine, % .254 .330 .278 Cystine, % 'Vitamin premix supplied the following per kg.: vitamin A, 4,000 I.U.; vitamin D3, 500 I.C.U. riboflavin, 4 mg.; calcium pantothenate, 10 mg.; niacin, 10 mg.; folic acid, 0.3 mg.; vitamin B ] 2 5 meg.; choline chloride, 250 mg.; and ethoxyquin, 125 mg. 2 Trace mineral mix supplied the following mg./kg.: manganese, 50; iron, 50; zinc, 50; copper, 5 iodine, 1.5; and cobalt, 0.5.
Added meth. Egg prod.
Egg wt.
Change in body wt. 95ab 94ab 96a 99a 89b 96a 92ab 95ab 91ab 94ab
%
Fertility
Egg prod.
%
Added meth.
%
Egg wt.
Change in body wt.
gg61.9a 0 46.9c Corn-soy-14 -104 Corn-soy-14 .04 64.7ab 61.4abc -236 Corn-soy-16 0 72.7a 60.6abc -28 Corn-soy-16 .04 67.5a 61.7ab -132 Corn-pea-16 0 50.6bc 58.0c -343 .02 58.4bc 58.7abc Corn-pea-16 -113 .04 62.1bc 58.4bc Corn-pea-16 -95 Corn-pea-16 .08 65.6ab 61.1abc -254 'Values not having a common letter are statistically different (P < 0.05).
Diet
68a 78a 89a 76a 83a 72a 78a 56a
%
Fertility
TABLE 3.—Response of hens from 53 to 65 weeks of age to methionine supplementation of thre
0 80.3a 56.5a + 205 .04 74.4ab 57.4a + 115 ,08 82.7a 57.3a + 136 .12 79.0a 57.3a + 85 0 66.3b 51.4b -30 ,04 79.0a 56.8a +79 ,08 81.1a 55.9a + 57 .12 75.5a 56.4a + 116 ,16 77.9a 57.4a +94 .20 81.7a 57.0a + 103 'Values not having a common letter are statistically different (P < 0.05).
Corn- soy-16 Corn- soy-16 Corn- soy-16 Corn- soy-16 Corn- pea-16 Corn- pea-16 Corn- pea-16 Corn- pea-16 Corn- pea-16 Corn- pea-16
Diet
TABLE 2.—Response of pullets from 32 to 45 weeks of age to methionine supplementation of tw
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METHIONINE REQUIREMENT
RESULTS In Experiment 1, adding methionine to the soybean ration containing 16% protein failed to significantly affect any of the parameters measured in the experiment (Table 2). On the other hand, adding 0.04% or higher levels of DL-methionine to the pea ration significantly improved egg production and egg weight. Furthermore, the hens fed the pea diet with no methionine were the only ones to lose weight during the experiment. Fertility of the eggs was not greatly affected by methionine supplementation. Although hatchability of fertile eggs was slightly lower for the pea ration containing the two lowest levels of methionine supplementation, the differences were not statistically significant from the diets containing higher levels. Hatchability of fertile eggs from hens fed the pea diet tended to be slightly lower than that of eggs from hens fed the soybean diet. In Experiment 2, again no statistically significant improvement in any of the parameters measured was obtained by adding methi-
onine to the soy ration containing 16% protein (Table 3). On the other hand, a significant improvement in egg production was observed by adding methionine to the soy ration containing 14% protein. Although the differences were not statistically significant, adding methionine to the pea ration appeared to improve egg production and egg weight. Fertility varied considerably and did not appear to be improved by adding methionine to any of the diets. Hatchability of fertile eggs again tended to be lower for diets low in methionine, but the differences were not significant. In contrast to the previous experiment, results obtained in Experiment 3 showed no improvement in egg production by adding methionine to the soy ration containing 14% protein (Table 4). Egg weight also was not significantly affected, and adding methionine to the soybean ration containing 16% protein again failed to significantly improve the parameters measured. Adding 0.02% DL-methionine to the pea ration significantly improved egg production and higher levels did not increase egg production further. On the other hand, a level of 0.08% added methionine appeared to be necessary to obtain maximum egg weight during this period of the laying cycle when the birds were reaching peak egg production. Fertility was not affected by the dietary treatments and no evidence was obtained in this experiment that diets low in methionine resulted in depression in hatchability of fertile eggs. Albumen quality, as measured by Haugh units and incidence of blood spots were not significantly affected by the dietary treatments. In Experiment 4, adding potassium sulfate to the ration failed to modify the response of hens to methionine supplementation of the pea ration (Table 5). Adding 0.04% or higher levels of methionine significantly improved egg production in both series. A level of 0.08% added methionine was necessary to obtain maximum egg weight. Fertility and hatchability of eggs were not significantly affected
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weeks of age. All three basal rations were again used with varying levels of added methionine. In Experiment 4, 10 pullets were placed in each group and 4 groups were fed each of the experimental diets. Only the pea basal ration was used in this experiment and levels of 0, 0.04, 0.08 and 0.12% added DL-methionine were compared with and without 0.2% added potassium sulfate. Data were calculated for two phases of the experiment (21-45 weeks and 45-67 weeks) in order to see if major differences in requirement for methionine were evident during the two phases of the laying cycle. The sulfate content of the water supply for the hens in this experiment was determined by barium precipitation method and was found to be approximately 42 p.p.m.
539
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L. S. JENSEN, L. FALEN AND G. W. SCHUMAIER
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L . S. JENSEN, L. FALEN AND G. W.
TABLE 6.-
SCHUMAIER
-Summary of the range of daily methionine intakes that were adequate or inadequate for optimum performance
Basal diet Corn-soy-16 Corn-pea-16 Corn-soy-14 Corn-soy-16 Corn-pea-16 Corn-pea-16 Corn-soy-14 Corn-soy-16 Corn-pea-16 Corn-pea-16 Corn-pea-16 Corn-pea-16
Weeks of age 32-45 32-45 53-65 53-65 53-65 20-36 20-36 20-36 21-45 21-45 45-67 45-67
Daily methionine intake (mg.) Adequate' Inadequate' 333 — 349 244 308 247 280 — 293 265 312 268 250 — 264 — 302 267 299 261 343 289 334 294
Inadequate or adequate to support maximum daily egg yield.
by the dietary treatments. During the second half of the treatment, there were no statistically significant differences in egg production and egg weight between the groups fed no added methionine, with and without potassium sulfate, although egg size appeared to be slightly higher for those with sulfate. Again 0.04% added methionine appeared to be adequate for maximum egg production but a higher level was necessary for maximum egg weight. Fertility of eggs appeared to be lower for the low methionine diets without added sulfate. Hatchability of fertile eggs varied some but the variation did not appear to be related to methionine supplementation. DISCUSSION Hatchability of fertile eggs did not appear to be markedly affected by methionine level of the diets in any of the experiments. In Experiments 3 and 4, no statistically significant improvement in hatchability of fertile eggs was observed by adding methionine. In Experiments 1 and 2 the differences were also not statistically significant, but there was some indication that the hatchability was slightly lower with low methionine diets. Even if the differences observed in Tables 1 and 2 were of biological significance, the level of methionine adequate for maximum
egg production and egg weight would supply enough methionine to meet the requirements for maximum hatchability. These results are in general agreement with Leong and McGinnis (1952) where no effect on hatchability was observed by adding methionine to the diet, but are in contrast to results obtained by Mehring et al. (1954) and Daghir et al. (1964) who obtained significant responses in hatchability by adding methionine even though egg production was not improved. These investigators used New Hampshire or Barred Plymouth Rock chickens in contrast to the White Leghorn chickens used in our study and in that of Leong and McGinnis (1952). Perhaps there are breed differences in response to methionine for obtaining maximum hatchability. The results of these experiments again demonstrate that a corn-soybean meal ration formulated to contain 16% crude protein is adequate in methionine and cystine for obtaining optimum egg yield in laying hens. Others who have used corn-soybean meal rations to study the methionine requirements of laying hens have had to go to lower protein levels in order to obtain a response. In Experiment 2, a response in egg production was obtained by adding methionine to a corn-soy ration containing 14% protein, but no such response was observed in Experiment 3.
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Exp. No. 1 1 2 2 2 3 3 3 4 4 4 4
543
METHIONINE REQUIREMENT
The results obtained in Experiment 4 shows that a statistically significant response in egg production and egg weight was obtained to methionine supplementation even though the diet was supplemented with 0.2% potassium sulfate. These results suggest that the chickens were obtaining enough inorganic sulfate from their diet plus the water supply and that the requirement determined under the conditions of our laboratory was for methionine to serve as an amino acid in egg protein
synthesis rather than methionine to serve as both an amino acid and as a source of sulfate. Harms (1973) indicated that sulfate can spare part of the methionine requirement of laying hens. In his studies the sulfate forms of mineral elements were not included in the diet and the water supply contained 28 p.p.m. of sulfate. A higher level of total sulfate in the diet and water supply under our conditions may explain the lack of response to sulfate supplementation. REFERENCES Bray, D. J., 1965. The methionine requirement of young laying pullets. Poultry Sci. 44: 1173-1180. Carlson, C. W., and E. Guenthner, 1969. Response of laying hens fed typical corn-soy diets to supplements of methionine and lysine. Poultry Sci. 48: 137-143. Combs, C. F., 1964. Further studies of protein and amino acid needs of broilers and laying hens. Proc. Maryland Nutr. Conf. : 45-71. Daghir, N. J., S. S. Akrabawi and K. Rottensten, 1964. Methionine supplementation of breeder diets. Poultry Sci. 43: 1106-1109. Fisher, C , and T. R. Morris, 1970. The determination of the methionine requirement of laying pullets by a diet dilution technique. Brit. Poultry Sci. 11: 67-82. Harms, R. H., 1973. Sparing effect of sulfate and other factors on total sulfur amino acid requirements. Proc. Georgia Nutr. Conf. : 1-10. Harms, R. H., and B. L. Damron, 1969. Protein and sulfur amino acid requirement of the laying hen as influenced by dietary formulation. Poultry Sci. 48: 144-149. Hedwig, H. E., J. M. Lawrence and D. R. Cochran, 1972. Simple and rapid methods for determination of methionine and cystine in legume seeds. Anal. Biochem. 48: 353-364. Leong, K. C , and J. McGinnis, 1952. An estimate of the methionine requirement for egg production. Poultry Sci. 31: 692-695. Machlin, L. J., P. B. Pearson, C. A. Denton and H. R. Bird, 1953. The utilization of sulfate sulfur by the laying hen and its incorporation into cystine. J. Biol. Chem. 205: 213-219. Mehring, A. L., Jr., H. W. Titus and J. Waddell, 1954. The effect of adding methionine and vitamin B l2, singly and together, to a corn-soy diet for laying chickens. Poultry Sci. 33: 1191-1197. National Research Council, 1971. Nutrient requirements of domestic animals. No. 1. Nutrient require-
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Perhaps this reflects a slight difference in quantitative requirement between birds at two different stages of the laying cycle. It could also be due to variation in the ingredients used in the two experiments. It was interesting that even though methionine supplementation to the 14% protein corn-soy diet used in Experiment 2 significantly improved egg production, egg size was not affected. In all experiments involving the pea ration, the requirement for methionine for obtaining maximum egg size was higher than for maximum egg production. To aid in estimating the methionine requirement of laying hens from these studies, a summary of the range of daily methionine intakes that were adequate or inadequate for optimum performance is presented in Table 6. Some of these ranges would be lower if one only considered the rate of egg production. The summary indicates that a range between 290 and 300 mg. of methionine per day should be adequate to meet the requirements of laying hens at different stages of the laying cycle. This requirement is in fairly good agreement with that reported by Combs (1964), Harms and Damron (1969), Fisher and Morris (1970), and Carlson and Guenthner (1969). Although it is difficult to determine if there was any significant difference in requirement for daily methionine intake between stages of the laying cycle, the summary in Table 6 suggests that if there is a difference, it would be higher for the later stages of production than for the early stages.
544
L. S. JENSEN, L. FALEN AND G. W. SCHUMAIER
ments of poultry. Washington, D.C. Scott, M. L., M. C. Nesheim and R. J. Young, 1969. Nutrition of the Chicken. Scott and Associates, Ithaca, N.Y.
Spackman, D. H., W. H. Stein and S. Moore, 1958. Automatic recording apparatus for use in the chromatography of amino acids. Anal. Chem. 30: 1190— 1206.
Maintenance of a Specific Pathogen Free Turkey Flock Y. MOHAMED SAIF
(Received for publication June 2, 1973)
ABSTRACT The procedures used to maintain a specific pathogen free turkey flock are dscribed. Seven generations of SPF turkeys were raised over the last six years. All birds were free of Mycoplasma, Salmonella, and Arizona infections. No evidence of airsacculitis or other infectious diseases was detected. POULTRY SCIENCE 53: 544-546,
report (Mohamed and Bohl, IN1969)a previous the procedures used for establishing; a specific pathogen free (SPF) turkey flock: were described. The main objective was to> develop a flock free of detectable Mycoplasma, Salmonella, and Arizona infections. This> report presents information on seven generations of SPF turkeys reared over the lastt six years at Ohio Agricultural Research andI Development Center. The incidence of diseases in conventional flocks derived from the: same parent flock and maintained under conventional conditions over the last six yearsi are discussed. MATERIALS AND METHODS The parent SPF flock was derived fromi a flock of heavy White turkeys naturally' infected with Mycoplasma meleagridis. The : flock was maintained at Ohio AgriculturalI Research and Development Center. A series> of serologic and cultural examinations was> conducted to establish the status of the flocki relative to infection with certain pathogens. Testing Procedure for Mycoplasma and Salmonella. The different generations were:
1974
tested according to a standard procedure. All birds were tested serologically at five weeks of age for M. meleagridis, Mycoplasma gallisepticum, and Mycoplasma synoviae. Tube agglutination tests were conducted using antigens prepared in this laboratory according to Mohamed and Bohl 1968, and Olson et al. (1963). At this time the rectum of each bird was swabbed and bedding samples were collected and inoculated into culture media for the isolation of Salmonella and Arizona organisms. The swabs and bedding material were incubated at 37° C. in selenite broth and transfers were made to brilliant green agar plates at 6 and 24 hours. The agar plates were incubated for 3 days and examined daily for Salmonella or Arizona-like colonies. Colonies suspected to be of Salmonella or Arizona were transferred into different media for identification. The following media were used, lactose broth, dulcital broth, malonate broth, urea broth, triple sugar iron agar, citrate agar, dextrose agar, lysine decarboxylase, ornithine decarboxylase, and phenyl deaminase. At 30 weeks of age the same procedure used at five weeks of age was followed. In addition, serum samples were
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Department of Veterinary Science and Poultry Science, Ohio Agricultural Research and Development Center, Wooster, Ohio 44691