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Role of Protein and Energy in the Regulation of Energy Metabolism and Reproductive Performance of Large White Turkey Hens1
Role of Protein and Energy in the Regulation of Energy Metabolism and Reproductive Performance of Large White Turkey Hens1 R. W. ROSEBROUGH, 2 N. C. STEELE, J. P. McMURTRY, M. P. RICHARDS, and C. C. CALVERT US Department of Agriculture, Beltsvitte, Maryland 20705 (Received for publication January 24, 1983)
1983 Poultry Science 62:2452-2459 INTRODUCTION A l t h o u g h t h e National Research Council ( 1 9 7 7 ) r e c o m m e n d s a 14% protein diet containing 2 9 0 0 kcal of ME/kg for t h e t u r k e y breeder h e n , m a n y w o r k e r s reported t h a t this dietary formulation does n o t satisfy t h e daily protein r e q u i r e m e n t . Cherms et al. ( 1 9 7 6 ) reported a decrease in daily feed intake b y t u r k e y breeder hens over t h e course of a 28-week trial. T h u s , a 12% protein diet resulted in a protein intake of 25 g/day during t h e first week of egg p r o d u c t i o n , whereas a 15% p r o t e i n diet gave this p r o t e i n intake during t h e 2 8 t h week of egg p r o d u c t i o n . F u t h e r m o r e , Cherms et al. ( 1 9 7 8 ) r e p o r t e d t h a t a protein intake of less t h a n 25 g/day b y t h e b r e e d e r hen did n o t s u p p o r t reproductive function. Carter et al. ( 1 9 5 7 ) reported t h a t 18% p r o t e i n diets were superior t o 16% protein diets regardless of dietary energy levels. In contrast, Menge et al. ( 1 9 7 9 ) reported t h a t diets containing 18% protein were equal t o t h o s e containing 14% protein. Robblee
1 Mention of a trade name does not constitute a guarantee or warranty by the US Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable. J USDA, Agricultural Research Service, Beltsville Agricultural Research Center, Animal Science Institute, Nonruminant Animal Nutrition Laboratory, Beltsville, Maryland 20705.
and Clandinin ( 1 9 5 9 ) reported t h a t 17% p r o t e i n diets, regardless of energy level, were equal t o 15% protein diets. Subsequent w o r k b y Jensen and McGinnis ( 1 9 6 1 ) d e m o n s t r a t e d t h a t 10% protein diets were a d e q u a t e for t h e breeder hen. More recent w o r k b y Krueger et al. ( 1 9 7 8 ) also d e m o n s t r a t e d t h a t 18 and 14% p r o t e i n diets were equal w h e n fed t o t u r k e y breeder hens. Overall, a low protein diet coupled t o high feed intake gave an a d e q u a t e daily p r o t e i n intake during t h e early phase of egg p r o d u c t i o n . In contrast, as daily feed cons u m p t i o n decreased, either t h e p r o t e i n o r energy level was changed t o m e e t daily req u i r e m e n t s for p r o t e i n . T h e e x p e r i m e n t in this r e p o r t was designed t o measure responses of t h e t u r k e y breeder hen t o t w o levels of protein (12 and 17%) and t w o levels of energy ( 2 4 0 0 and 2 8 5 0 kcal ME/kg). Diets were formulated so as t o avoid complications of either high fat or inert filler materials. Measures of in vitro metabolism were also d e t e r m i n e d to compare reproductive param e t e r s t o changes in intermediary m e t a b o l i s m . MATERIALS AND METHODS Animals. Nicholas Large White t u r k e y females from a F e b r u a r y hatch were reared t o 8 weeks of age on a 30% protein starter diet. F r o m 8 t o 12 weeks of age, t h e females were fed 26% protein diets and from 12 t o 2 6 weeks
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ABSTRACT An experiment was conducted with Nicholas Large White turkey hens to determine the roles of energy and protein in energy efficiency, reproductive performance, and in vitro metabolism. Hens were fed diets containing 2400 or 2850 kcal ME/kg and either 12 or 17% protein. The experiment was conducted in environmentally controlled rooms maintained at 21 C and 50% dew point. The efficiency of energy utilization for both egg and poult production was greater in the 2400 kcal diets, and the efficiency of protein utilization was greater in the 12% protein diets. In vitro lipogenesis was less and plasma uric acid was greater (P<.05) in hens fed diets containing 17% protein. In contrast, plasma phosphorous and calcium were greater (P<.05) in hens fed diets containing 2400 kcal ME/kg. Plasma zinc and copper were unaffected by either dietary energy or protein levels. Because the 2400 kcal diets contained 30% wheat middlings, the higher metal levels observed in hens may be related to some component in middlings. Broodiness and mortality were also lower (P<.05) in the groups fed diets containing 2400 kcal ME/kg. (Key words: turkey hen, lipogenesis, energy, protein)
PROTEIN AND ENERGY IN REPRODUCTION
were assigned to each of four dietary treatments (Table 1). A 2 X 2 factorial arrangement of treatment consisted of two levels of crude protein (12 and 17%) and two levels of metabolizable energy (2400 and 2850 kcal/kg). Feed consumption for each hen was determined on a weekly basis. In Vitro Metabolic Studies. At the end of the 16-week experiment, 14 hens from each dietary treatment group were selected from hens producing eggs. A blood sample was obtained from the brachial vein and the hen was killed by cervical dislocation. The liver was rapidly excised and divided in half. One-half was rapidly frozen in liquid nitrogen and the other half placed in a warm solution of 50 mM phosphate-buffered saline (pH 7.4). The blood sample was centrifuged at 600 X g and the plasma was drawn off and stored at —80 C until analyzed. The frozen liver sample was also stored at —80 C until analyzed for lipid according to Folch et al. (1957). A portion of the fresh liver was sliced (50 to 75 mg), and duplicate slices were incubated at 37 C in 25 ml Erlenmeyer flasks containing a balance salt solution (Hanks and Wallace, 1949) supplemented with 10 mM HEPES [N-2hydroxyethylpiperazine-N'-ethane sulfonic acid
TABLE 1. Composition of the experimental diets Ingredient
2400-12
2400-17
2850-12
2850-17
\y *&/ Soybean meal, 49% protein Corn meal Distillers dried grains with solubles (corn) Wheat middlings Glucose monohydrate Cellulose Dicalcium phosphate Limestone Vitamin mix a Mineral mix" Selenium mix c Magnesium oxide Iodized salt Calculated analyses ME, kcal/kg Protein, g/kg Lysine, g/kg Methionine, g/kg Cystine, g/kg
450 130 300
180 415
115 735
229 658
300
20 33.3 54.7 5 1 1 1 4
5 33.3 54.7 5 1 1 1 4
14 37 33.3 54.7 5 1 1 1 4
10 3 33.3 54.7 5 1 1 1 4
2405 123 4.1 2.3 2.2
2402 173 8.8 2.8 2.9
2850 121 5.4 2.3 1.9
2850 170 8.8 2.9 2.7
a,b,c For composition of mixes see Rosebrough et al. (1982).
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of age 19% protein diets. The prebreeding diet contained 15% protein. From 19 to 30 weeks, the females were restricted to 6 hr of light. At 30 weeks of age, the hens were transferred to individual cages in two environmentally controlled rooms (21 C and 50% dew point). At this time, the hens were assigned to one of four dietary treatments (Table 1) and were allowed to consume feed and water ad libitum. At 32 weeks of age, the hens were exposed to a 14 hr light 10 hr dark (14L: 10D) cycle. All hens were initially inseminated with .05 ml of diluted (1:2) semen at the time of 50% egg production (approximately 21 days following initial lighting). The lighting schedule was modified to a 16 L:8D cycle after 8 weeks of egg production. Individual body weights of the hens were recorded at the time of lighting and after 16 weeks of egg production. Egg collection began at the onset of 50% egg production. Eggs were collected twice daily and weighed. Eggs were stored at 13 C and 50% dew point until set on a weekly basis. After 7 days of incubation, the eggs were candled and fertility noted. Following incubation, the number of poults were recorded and expressed on the basis of fertile eggs produced. Diets. Thirty-nine individually caged hens
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RESULTS AND DISCUSSION
Feed Intake and Reproductive Performance. Neither protein nor energy levels of the diets
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(pH 7.4)] and 10 mM (1- 14 C) sodium acetate (specific activity of 74 DPM/nmoe). The reaction was terminated after 2 hr and the slices were extracted for 24 hr with 2:1 chloroform methanol. The extract was then fractionated with .2 vol of .88% KC1 and the bottom phase (chloroform) was washed twice with the theoretical upper phase (5:3 methanolic (KCl) and then evaporated to dryness (Folch et al., (1957). The extract was dispersed in Scintillene and radioactivity determine by liquid scintillation spectroscopy. A slice of liver for each sample was also placed in a flask that contained .5 ml of 4N H 2 S 0 4 to stop metabolic activity and the nonmetabolic addition of acetate to lipid was determined. In practice, little radioactivity was noted in the lipid fraction of previously acid-treated slices. The extracted slices were weighed and rates of lipogenesis were expressed on the basis of wet tissue and dry, fat-free weights. The portion of the liver remaining after slicing was homogenized in 100 raM Tris (pH 7.4) buffer containing 150 m/W MgCl2, and .5% Triton X-100 and centrifuged at 50,000 X g for 60 min. The supernatants were kept at 0 C until analyzed for malic enzyme, nicotinamide adenine dinucleotide phosphate NADP linked isocitrate dehydrogenase, and glutamic-aspartic amino transferase activities (Rosebrough et al., 1982). Liver lipid content was determined gravimetrically (Folch et al., 1957). Plasma zinc, copper, iron, calcium, and magnesium were determined by atomic absorption spectroscopy. Uric acid (Schmidt, 1957) and phosphorus (Fiske and SubbaRow, 1925) were determined on deproteinated (10% trichloroactic acid) plasma samples. Plasma immunoreactive insulin (IRI) was determined by a modification of the method of Morgan and Lazarow (1963). Highly purified chicken insulin was used as both the 12S iodinated hormone and as the standard. The primary antisera, anti-chicken insulin, was obtained from immunized guinea pigs. Data were analyzed as a 2 X 2 factorial arrangement of treatments with protein and energy levels as the main treatment effects. Differences for significance are presented for these two effects and were derived from the residual standard deviation resulting from the analysis of variance (Kirk, 1968).
PROTEIN AND ENERGY IN REPRODUCTION
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prevented the usual weight loss of hens during the reproductive cycle (Table 2). In contrast, Meyer et al. (1980) reported weight gains in hens during egg production, although hens used in their experiment were generally lighter in body weight at the initiation of egg production than were hens in the present study. Daily feed intakes were converted to appropriate energy intakes because of the difference in caloric density between energy levels. Although differences were not statistically significant, hens receiving the 2850 kcal diets tended to consume more energy. The failure to reject the overall null hypothesis indicates that the turkey hens can regulate feed intake to compensate for differences in dietary energy density. The significant differences in protein intake were due to the differences in energy level of the diets and the parallel change in the calorie: protein ratios (2400 to 12,21:1; 2400 to 17,14:1; 2850 to 12,24:1; 2850 to 17,17:1). Hens fed 2400 kcal diets produced more eggs than hens fed 2850 kcal diets (P<.05; Table 2). In calculating results, no attempt was made to correct for pauses in production or for hens in production at the end of 16 week (Table 3). As expected, the rate of egg production decreased in all treatment groups over the course of the experiment (Fig. 1). The rate of decrease across the four periods of production was similar for all dietary treatments. Thus, dietary treatments affected early egg production and persisted throughout the cycle. Dietary treatments did not affect the time to first egg following the onset of lighting. The overall average was 17.8 days from lighting to first egg and 21.5 days until 50% production. Likewise, diets did not affect egg weights, although eggs at the end of the 16 week were heavier (P<.05) than those at the beginning of egg production. Voitle et al. (1973) reported similar findings concerning egg weight during the production cycle. Dietary treatments did not affect any of the usual indices of reproductive performance (eggs set, fertility, or hatchability; Table 3). During 16 weeks of egg production, two hens died in each of the groups receiving the 2400 kcal diets. Mortality was equally low in the 2850 kcal diets; however, 15 hens from diet 2850 to 12 and 10 hens from diet 2850 to 17 were showing signs of broodiness or of molting. Thus, it is possible that the low energy dietary treatments exerted their influence by maintaining reproductive health of hens. The data
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from this experiment also demonstrated that because hens receiving 2400 kcal diets laid more eggs without a decrease in fertility or hatchability, they were more efficient in using dietary energy for poult production. In Vitro Metabolism. Dietary energy did not effect either liver lipid or rate of in vitro lipogenesis (Table 4). In contrast, there was a significant (P<.05) effect of protein on both of these variables. Expressing in vitro lipogenesis on the basis of fat-free, dry liver basis rather St. than on a wet tissue basis indicated that liver lipid levels did not mask the significant effect of protein. Yeh and Leveille (1969) reported w that higher dietary protein decreased lipo- < genesis in the chick, and Rosebrough et al. (1982) reported a similar finding in turkey poults. Regretfully, measurements of lipid metabolism in the turkey breeder hen did not explain differences in egg production. Maurice and Jensen (1979) studied lipid metabolism in the mature laying hen and Japanese quail and also reported that differences in egg pro-
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duction were not related to lipid metabolism. The differences in the rate of lipogenesis between the two levels of protein may also be related to the quality of the energy sources. The 12% protein diets contained more corn (as meal or distiller grains) than the 17% protein diets, and Bolton (1955) reported that corn polysaccharides were more available than were polysaccharides in other feeds. Maurice and Jensen (1978) discussed the feasibility of adding myoinositol to improve the availability of polysaccharides in other feed grains. Dietary treatments' did not affect the activities of either glutamic-aspartic aminotransferase or isocitrate dehydrogenase. This observation suggested that the turkey breeder hens in this study had the ability to adapt to differences in energy or protein without the need for new enzyme protein. Both protein and energy affected (P<.05, Table 5) plasma uric acid levels. Uric acid was the final point in the disposal route of protein and isocitrate dehydrogenase and glutamic-aspartic aminotransferase catalyzed a coupled beginning point in protein degradation (transamination and alpha-ketoglutarate utilization). Early work with rats illustrated that the ability to degrade protein accompanied the synthetic ability; therefore protein utilization was a delicate balance between synthesis and degradation (Maramatsu and Asheda, 1961; Waldorf et al., 1963). Protein, but not energy, significantly affected (P<.05) malic enzyme activity. As with in vitro lipogenesis the effect persisted even in defatted tissue. Therefore, endogenous liver lipid did not regulate reducing equivalent production or in vitro lipogenesis. Ballard and Hanson (1967) proposed the existence of a reducing equivalent shuttle that comprised mitochondrial NAD-malic dehydrogenase and pyruvate carboxylase and transferred malate to the cytoplasm for lipogenesis. Yeh and Leveille (1969) reported that dietary protein enhanced glucose production by the liver, used available reducing equivalents, and reduced shuttle activity. Further work by Leveille and Yeh (1972) and Rosebrough et al. (1982) indicated that malic enzyme activity paralleled lipogenic activity and did not regulate lipogenesis. Lipogenesis always changed before enzyme activity, although these groups did not measure substrate (cytoplasmic malate) turnover during periods of lipogenic change. The plasma uric acid levels in this study
Insulin
PROTEIN AND ENERGY IN REPRODUCTION
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ROSEBROUGH ET AL.
In contrast to its effect on plasma uric acid and insulin, dietary protein had no effect on plasma metal levels. Compared to 2400 kcal diets, 2850 kcal diets decreased (P<.05) plasma phosphorous and calcium. Several explanations were possible. Because hens ate more of the 2400 kcal diets, they consumed more metals, and this consumption pattern influenced circulatory values. Cereal grains contain phytates, which alter metal metabolism by chelation. Small quantities of phytates enchanced metal absorption and large quantities inhibited absorption (Kratzer et al, 1959). Suso and Edwards (1968) reported that the chelator EDTA (ethylenediaminetetraacetate) improved metal absorption; however, they also reported a range of chelator metal ratios unfavorable to absorption. Lastly, the egg production status of each hen may have regulated metal levels regardless of dietary treatments. Hens consuming the 2400 kcal diets produced more eggs and diets may have affected mineral metabolism as an indirect result of egg production. Thomason et al. (1976) reported on the confounding of egg production per se and egg production as a dietary function. Data in this study indicated that at an environmental temperature of 21 C, the turkey breeder hen required a 2400 kcal diet con-
taining only 12% protein to maintain reproductive performance. At this temperature, all hens consumed virtually the same amount of energy; therefore, the 2850 kcal diet containing 12% protein resulted in an inadequate daily protein intake. Previous research advocating higher levels of protein probably contained a safety margin for variations in feed intake due to reproductive age or changes in temperatures. The present report does not support a relationship between egg production and lipogenic capacity in the turkey breeder hen.
REFERENCES Ballard, F. J. and R. W. Hanson, 1967. The citrate cleavage pathway and Iipogenesis in rat adipose tissue: replenishment of oxaloacetate. J. Lipid Res 8 : 7 3 - 8 1 . Bolton, W, 1955. The digestibility of the carbohydrate complex of barley, wheat and maize by adult fowls. J. Agric. Sci. 46:119-122. Carter, R. D., J. W. Wyne, V. D. Chamberlin, and M. G. McCartney, 1957. The influence of dietary energy and protein on reproductive performance of turkey breeders. Poultry Sci. 36:1108-1109. Cherms, F. L., M. G. Stoller, and J. J. Macllraith, 1978. A comparison of 22 and 35 grams of daily protein intake and the effects on reproduction of turkey hens. Poultry Sci. 57:1126. (Abstr.) Cherms, F. L., M. G. Stoller, J. J. Macllraith, and H. R. Halloran, 1976. Reproduction in turkey hens as influenced by prebreeder and breeder protein intake and the environment. Poultry Sci. 55: 1678-1690. Fiske, C. H., and Y. SubbaRow, 1925. The colorimetric determination of phosphorus. J. Biol. Chem. 66:375. Folch, J., M. Lees, and G. M. Sloane-Stanley, 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497-509. Hanks, J. H., and R. E. Wallace, 1949. Relation of oxygen and temperature in the preservation of tissues by refrigeration. Proc. Soc. Exp. Biol. Med. 171:196-200. Hevia, P., and A. J. Clifford, 1977. Protein intake, uric acid metabolism and protein efficiency ratio in growing chicks. J. Nutr. 107:959-964. Jensen, L. S., and J. McGinnis, 1961. Nutritional investigations with turkey hens. 1. Quantitative requirements for protein. Poultry Sci. 40:288— 290. Kirk, J., 1968. Experimental Design Procedures for the Behavoral Sciences. Wadsworth Publ. Co., Belmont, CA. Kratzer, F. H., J. B. Allred, P. N. Davis, B. J. Marshall, and P. Vohra, 1959. The effect of autoclaving soybean protein and the addition of ethylenediamine tetraacetic acid on the biological availability of dietary zinc for turkey poults. J. Nutr. 68:313-322. Krueger, K. K., J. A. Owen, C. E. Krueger, and T. M.
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established a positive (P<.05) relationship between uric acid and the dietary protein status of the turkey breeder hen. That is, the higher values for uric acid were from hens consuming diets containing 17% protein. Miles and Featherston (1976) reported that uric acid excretion estimated the nutritional adequacy of dietary protein for chicks. Previous work with adult hens established a positive relationship between uric acid excretion and dietary protein quality (Teekell et al, 1969). Likewise, Okumura and Tasaki (1968) reported a positive relationship between plasma uric acid levels and dietary protein quality. Hevia and Clifford (1977) reported that an increase in dietary protein of 5 g/day increased plasma uric acid 1 mg/100 ml. The present study suggested a relationship (P<.05) between dietary protein levels and plasma insulin and possibly between plasma insulin and uric acid (R = .89; P<.05). Touchburn et al. (1981) reported no relationship between dietary protein and plasma insulin although these authors used chickens selected for abdominal fat to draw this conclusion. These birds were refractory to control of insulin because of excess fat.
PROTEIN AND ENERGY IN REPRODUCTION
relationship of energy and protein to reproductive performance in turkey breeders. Poultry Sci. 38:141-145. Rosebrough, R. W., N. C. Steele, and L. T. Frobish, 1982. Effect of protein and amino acid status on lipogenesis by turkey poults. Poultry Sci. 61: 731-738. Schmidt, G., 1957. Colorimetric and enzymatic methods for the determination of some purines and pyrimidines. Vol. III. Page 7 7 5 - 7 8 1 ' in Methods in Enzymology. Academic Press, New York, NY. Suso, F. A., and H. M. Edwards, 1968. Influence of various chelating agents on absorption of 6 0 Co, 59 Fe, 54Mn and 6S Zn by chickens. Poultry Sci. 47:1417-1422. Teekell, R. A., C. E. Richardson, and A. B. Watts, 1969. Dietary protein effects on urinary nitrogen components of the hen. Poultry Sci. 47:1260— 1266. Thomason, D. M., A. T. Leighton, Jr., and J. P. Mason, 1976. A study of certain environmental factors and mineral chelation on the reproductive performance of young and yearling turkey hens. Poultry Sci. 55:1343-1355. Touchburn, S., J. Simon, and B. Lechberg, 1981. Evidence of a glucose-insulin imbalance and effect of dietary protein and energy level in chickens selected for high abdominal fat content. J. Nutr. 111:325-335. Voitle, R. A., J. H. Walter, H. R. Wilson, and R. H. Harms, 1973. The effect of low protein and skip-a-day grower diets on the subsequent performance of turkey breeder hens. Poultry Sci. 52:543-548. Waldorf, M. S., M. C. Kirk, H. Linksweiller, and A. E. Harper, 1963. Metabolic adaptations in higher animals. VII. Responses of glutamic-oxalacetate and glutamate-pryuvate transaminases to diet. Proc. Soc. Exp. Biol. Med. 112:764- 768. Yeh, Y.-Y., and G. A. Leveille, 1969. Effect of dietary protein on hepatic lipogenesis in the growing chick. J. Nutr. 98: 356-366.
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Ferguson, 1978. Effect of feed and light regimens during the growing period on subsequent reproductive performance of broad breasted white turkeys fed two protein levels. Poultry Sci. 57:27-37. Leveille, G. A., and Y. Y. Yeh, 1972. Influence of intermittent fasting or protein-free feeding on lipid metabolism in young cockerals. J. Nutr. 102:733-740. Maramatsu. K., and N. D. Asheda, 1961. Effect of dietary protein level on growth and liver enzyme activity in rats. J. Nutr. 76:143—150. Maurice, D. V., and L. S. Jensen, 1978. Effect of dietary cereal on liver and plasma lipids in laying Japanese quail. Br. Poult. Sci. 19:199-205. Maurice, D. V., and L. S. Jensen, 1979. Hepatic lipid metabolism in domestic fowl as influenced by dietary cereal. J. Nutr. 109:872-882. Menge, H., L. T. Frobish, B. T. Weinland, and E. G. Geis, 1979. Effect of dietary protein and energy on reproductive performance of turkey hens. Poultry Sci. 58:419-426. Meyer, G. B., C. F. Props, A. T. Leighton, Jr., H. P. Van Krey, and L. M. Potter, 1980. Influence of dietary protein during the pre-breeder period on subsequent reproductive performance of Large White turkeys. 1. Growth, feed consumption, and female sex-linked reproductive traits. Poultry Sci. 59:353-357. Miles, R. D., and W. R. Featherston, 1976. Uric acid excretion by the chick as an indicator of dietary protein quality. Poultry Sci. 55:98-102. Morgan, C. R., and A. Lazarow, 1963. Immunoassay of insulin: two antibody systems. Diabetes 12:115-126. National Research Council, 1977. Nutrient Requirements of Poultry. Natl. Acad. Sci. Washington, DC. Okumura, J. and I. Tasaki, 1968. Effect of fasting, refeeding and dietary protein level on uric acid and ammonia content of blood, liver and kidney in chickens. J. Nutr. 97:316-320. Robblee, A. R., and D. R. Clandinin, 1959. The
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1983 Poultry Science 62:2452-2459 INTRODUCTION A l t h o u g h t h e National Research Council ( 1 9 7 7 ) r e c o m m e n d s a 14% protein diet containing 2 9 0 0 kcal of ME/kg for t h e t u r k e y breeder h e n , m a n y w o r k e r s reported t h a t this dietary formulation does n o t satisfy t h e daily protein r e q u i r e m e n t . Cherms et al. ( 1 9 7 6 ) reported a decrease in daily feed intake b y t u r k e y breeder hens over t h e course of a 28-week trial. T h u s , a 12% protein diet resulted in a protein intake of 25 g/day during t h e first week of egg p r o d u c t i o n , whereas a 15% p r o t e i n diet gave this p r o t e i n intake during t h e 2 8 t h week of egg p r o d u c t i o n . F u t h e r m o r e , Cherms et al. ( 1 9 7 8 ) r e p o r t e d t h a t a protein intake of less t h a n 25 g/day b y t h e b r e e d e r hen did n o t s u p p o r t reproductive function. Carter et al. ( 1 9 5 7 ) reported t h a t 18% p r o t e i n diets were superior t o 16% protein diets regardless of dietary energy levels. In contrast, Menge et al. ( 1 9 7 9 ) reported t h a t diets containing 18% protein were equal t o t h o s e containing 14% protein. Robblee
1 Mention of a trade name does not constitute a guarantee or warranty by the US Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable. J USDA, Agricultural Research Service, Beltsville Agricultural Research Center, Animal Science Institute, Nonruminant Animal Nutrition Laboratory, Beltsville, Maryland 20705.
and Clandinin ( 1 9 5 9 ) reported t h a t 17% p r o t e i n diets, regardless of energy level, were equal t o 15% protein diets. Subsequent w o r k b y Jensen and McGinnis ( 1 9 6 1 ) d e m o n s t r a t e d t h a t 10% protein diets were a d e q u a t e for t h e breeder hen. More recent w o r k b y Krueger et al. ( 1 9 7 8 ) also d e m o n s t r a t e d t h a t 18 and 14% p r o t e i n diets were equal w h e n fed t o t u r k e y breeder hens. Overall, a low protein diet coupled t o high feed intake gave an a d e q u a t e daily p r o t e i n intake during t h e early phase of egg p r o d u c t i o n . In contrast, as daily feed cons u m p t i o n decreased, either t h e p r o t e i n o r energy level was changed t o m e e t daily req u i r e m e n t s for p r o t e i n . T h e e x p e r i m e n t in this r e p o r t was designed t o measure responses of t h e t u r k e y breeder hen t o t w o levels of protein (12 and 17%) and t w o levels of energy ( 2 4 0 0 and 2 8 5 0 kcal ME/kg). Diets were formulated so as t o avoid complications of either high fat or inert filler materials. Measures of in vitro metabolism were also d e t e r m i n e d to compare reproductive param e t e r s t o changes in intermediary m e t a b o l i s m . MATERIALS AND METHODS Animals. Nicholas Large White t u r k e y females from a F e b r u a r y hatch were reared t o 8 weeks of age on a 30% protein starter diet. F r o m 8 t o 12 weeks of age, t h e females were fed 26% protein diets and from 12 t o 2 6 weeks
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ABSTRACT An experiment was conducted with Nicholas Large White turkey hens to determine the roles of energy and protein in energy efficiency, reproductive performance, and in vitro metabolism. Hens were fed diets containing 2400 or 2850 kcal ME/kg and either 12 or 17% protein. The experiment was conducted in environmentally controlled rooms maintained at 21 C and 50% dew point. The efficiency of energy utilization for both egg and poult production was greater in the 2400 kcal diets, and the efficiency of protein utilization was greater in the 12% protein diets. In vitro lipogenesis was less and plasma uric acid was greater (P<.05) in hens fed diets containing 17% protein. In contrast, plasma phosphorous and calcium were greater (P<.05) in hens fed diets containing 2400 kcal ME/kg. Plasma zinc and copper were unaffected by either dietary energy or protein levels. Because the 2400 kcal diets contained 30% wheat middlings, the higher metal levels observed in hens may be related to some component in middlings. Broodiness and mortality were also lower (P<.05) in the groups fed diets containing 2400 kcal ME/kg. (Key words: turkey hen, lipogenesis, energy, protein)
PROTEIN AND ENERGY IN REPRODUCTION
were assigned to each of four dietary treatments (Table 1). A 2 X 2 factorial arrangement of treatment consisted of two levels of crude protein (12 and 17%) and two levels of metabolizable energy (2400 and 2850 kcal/kg). Feed consumption for each hen was determined on a weekly basis. In Vitro Metabolic Studies. At the end of the 16-week experiment, 14 hens from each dietary treatment group were selected from hens producing eggs. A blood sample was obtained from the brachial vein and the hen was killed by cervical dislocation. The liver was rapidly excised and divided in half. One-half was rapidly frozen in liquid nitrogen and the other half placed in a warm solution of 50 mM phosphate-buffered saline (pH 7.4). The blood sample was centrifuged at 600 X g and the plasma was drawn off and stored at —80 C until analyzed. The frozen liver sample was also stored at —80 C until analyzed for lipid according to Folch et al. (1957). A portion of the fresh liver was sliced (50 to 75 mg), and duplicate slices were incubated at 37 C in 25 ml Erlenmeyer flasks containing a balance salt solution (Hanks and Wallace, 1949) supplemented with 10 mM HEPES [N-2hydroxyethylpiperazine-N'-ethane sulfonic acid
TABLE 1. Composition of the experimental diets Ingredient
2400-12
2400-17
2850-12
2850-17
\y *&/ Soybean meal, 49% protein Corn meal Distillers dried grains with solubles (corn) Wheat middlings Glucose monohydrate Cellulose Dicalcium phosphate Limestone Vitamin mix a Mineral mix" Selenium mix c Magnesium oxide Iodized salt Calculated analyses ME, kcal/kg Protein, g/kg Lysine, g/kg Methionine, g/kg Cystine, g/kg
450 130 300
180 415
115 735
229 658
300
20 33.3 54.7 5 1 1 1 4
5 33.3 54.7 5 1 1 1 4
14 37 33.3 54.7 5 1 1 1 4
10 3 33.3 54.7 5 1 1 1 4
2405 123 4.1 2.3 2.2
2402 173 8.8 2.8 2.9
2850 121 5.4 2.3 1.9
2850 170 8.8 2.9 2.7
a,b,c For composition of mixes see Rosebrough et al. (1982).
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of age 19% protein diets. The prebreeding diet contained 15% protein. From 19 to 30 weeks, the females were restricted to 6 hr of light. At 30 weeks of age, the hens were transferred to individual cages in two environmentally controlled rooms (21 C and 50% dew point). At this time, the hens were assigned to one of four dietary treatments (Table 1) and were allowed to consume feed and water ad libitum. At 32 weeks of age, the hens were exposed to a 14 hr light 10 hr dark (14L: 10D) cycle. All hens were initially inseminated with .05 ml of diluted (1:2) semen at the time of 50% egg production (approximately 21 days following initial lighting). The lighting schedule was modified to a 16 L:8D cycle after 8 weeks of egg production. Individual body weights of the hens were recorded at the time of lighting and after 16 weeks of egg production. Egg collection began at the onset of 50% egg production. Eggs were collected twice daily and weighed. Eggs were stored at 13 C and 50% dew point until set on a weekly basis. After 7 days of incubation, the eggs were candled and fertility noted. Following incubation, the number of poults were recorded and expressed on the basis of fertile eggs produced. Diets. Thirty-nine individually caged hens
2453
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RESULTS AND DISCUSSION
Feed Intake and Reproductive Performance. Neither protein nor energy levels of the diets
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(pH 7.4)] and 10 mM (1- 14 C) sodium acetate (specific activity of 74 DPM/nmoe). The reaction was terminated after 2 hr and the slices were extracted for 24 hr with 2:1 chloroform methanol. The extract was then fractionated with .2 vol of .88% KC1 and the bottom phase (chloroform) was washed twice with the theoretical upper phase (5:3 methanolic (KCl) and then evaporated to dryness (Folch et al., (1957). The extract was dispersed in Scintillene and radioactivity determine by liquid scintillation spectroscopy. A slice of liver for each sample was also placed in a flask that contained .5 ml of 4N H 2 S 0 4 to stop metabolic activity and the nonmetabolic addition of acetate to lipid was determined. In practice, little radioactivity was noted in the lipid fraction of previously acid-treated slices. The extracted slices were weighed and rates of lipogenesis were expressed on the basis of wet tissue and dry, fat-free weights. The portion of the liver remaining after slicing was homogenized in 100 raM Tris (pH 7.4) buffer containing 150 m/W MgCl2, and .5% Triton X-100 and centrifuged at 50,000 X g for 60 min. The supernatants were kept at 0 C until analyzed for malic enzyme, nicotinamide adenine dinucleotide phosphate NADP linked isocitrate dehydrogenase, and glutamic-aspartic amino transferase activities (Rosebrough et al., 1982). Liver lipid content was determined gravimetrically (Folch et al., 1957). Plasma zinc, copper, iron, calcium, and magnesium were determined by atomic absorption spectroscopy. Uric acid (Schmidt, 1957) and phosphorus (Fiske and SubbaRow, 1925) were determined on deproteinated (10% trichloroactic acid) plasma samples. Plasma immunoreactive insulin (IRI) was determined by a modification of the method of Morgan and Lazarow (1963). Highly purified chicken insulin was used as both the 12S iodinated hormone and as the standard. The primary antisera, anti-chicken insulin, was obtained from immunized guinea pigs. Data were analyzed as a 2 X 2 factorial arrangement of treatments with protein and energy levels as the main treatment effects. Differences for significance are presented for these two effects and were derived from the residual standard deviation resulting from the analysis of variance (Kirk, 1968).
PROTEIN AND ENERGY IN REPRODUCTION
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prevented the usual weight loss of hens during the reproductive cycle (Table 2). In contrast, Meyer et al. (1980) reported weight gains in hens during egg production, although hens used in their experiment were generally lighter in body weight at the initiation of egg production than were hens in the present study. Daily feed intakes were converted to appropriate energy intakes because of the difference in caloric density between energy levels. Although differences were not statistically significant, hens receiving the 2850 kcal diets tended to consume more energy. The failure to reject the overall null hypothesis indicates that the turkey hens can regulate feed intake to compensate for differences in dietary energy density. The significant differences in protein intake were due to the differences in energy level of the diets and the parallel change in the calorie: protein ratios (2400 to 12,21:1; 2400 to 17,14:1; 2850 to 12,24:1; 2850 to 17,17:1). Hens fed 2400 kcal diets produced more eggs than hens fed 2850 kcal diets (P<.05; Table 2). In calculating results, no attempt was made to correct for pauses in production or for hens in production at the end of 16 week (Table 3). As expected, the rate of egg production decreased in all treatment groups over the course of the experiment (Fig. 1). The rate of decrease across the four periods of production was similar for all dietary treatments. Thus, dietary treatments affected early egg production and persisted throughout the cycle. Dietary treatments did not affect the time to first egg following the onset of lighting. The overall average was 17.8 days from lighting to first egg and 21.5 days until 50% production. Likewise, diets did not affect egg weights, although eggs at the end of the 16 week were heavier (P<.05) than those at the beginning of egg production. Voitle et al. (1973) reported similar findings concerning egg weight during the production cycle. Dietary treatments did not affect any of the usual indices of reproductive performance (eggs set, fertility, or hatchability; Table 3). During 16 weeks of egg production, two hens died in each of the groups receiving the 2400 kcal diets. Mortality was equally low in the 2850 kcal diets; however, 15 hens from diet 2850 to 12 and 10 hens from diet 2850 to 17 were showing signs of broodiness or of molting. Thus, it is possible that the low energy dietary treatments exerted their influence by maintaining reproductive health of hens. The data
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from this experiment also demonstrated that because hens receiving 2400 kcal diets laid more eggs without a decrease in fertility or hatchability, they were more efficient in using dietary energy for poult production. In Vitro Metabolism. Dietary energy did not effect either liver lipid or rate of in vitro lipogenesis (Table 4). In contrast, there was a significant (P<.05) effect of protein on both of these variables. Expressing in vitro lipogenesis on the basis of fat-free, dry liver basis rather St. than on a wet tissue basis indicated that liver lipid levels did not mask the significant effect of protein. Yeh and Leveille (1969) reported w that higher dietary protein decreased lipo- < genesis in the chick, and Rosebrough et al. (1982) reported a similar finding in turkey poults. Regretfully, measurements of lipid metabolism in the turkey breeder hen did not explain differences in egg production. Maurice and Jensen (1979) studied lipid metabolism in the mature laying hen and Japanese quail and also reported that differences in egg pro-
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duction were not related to lipid metabolism. The differences in the rate of lipogenesis between the two levels of protein may also be related to the quality of the energy sources. The 12% protein diets contained more corn (as meal or distiller grains) than the 17% protein diets, and Bolton (1955) reported that corn polysaccharides were more available than were polysaccharides in other feeds. Maurice and Jensen (1978) discussed the feasibility of adding myoinositol to improve the availability of polysaccharides in other feed grains. Dietary treatments' did not affect the activities of either glutamic-aspartic aminotransferase or isocitrate dehydrogenase. This observation suggested that the turkey breeder hens in this study had the ability to adapt to differences in energy or protein without the need for new enzyme protein. Both protein and energy affected (P<.05, Table 5) plasma uric acid levels. Uric acid was the final point in the disposal route of protein and isocitrate dehydrogenase and glutamic-aspartic aminotransferase catalyzed a coupled beginning point in protein degradation (transamination and alpha-ketoglutarate utilization). Early work with rats illustrated that the ability to degrade protein accompanied the synthetic ability; therefore protein utilization was a delicate balance between synthesis and degradation (Maramatsu and Asheda, 1961; Waldorf et al., 1963). Protein, but not energy, significantly affected (P<.05) malic enzyme activity. As with in vitro lipogenesis the effect persisted even in defatted tissue. Therefore, endogenous liver lipid did not regulate reducing equivalent production or in vitro lipogenesis. Ballard and Hanson (1967) proposed the existence of a reducing equivalent shuttle that comprised mitochondrial NAD-malic dehydrogenase and pyruvate carboxylase and transferred malate to the cytoplasm for lipogenesis. Yeh and Leveille (1969) reported that dietary protein enhanced glucose production by the liver, used available reducing equivalents, and reduced shuttle activity. Further work by Leveille and Yeh (1972) and Rosebrough et al. (1982) indicated that malic enzyme activity paralleled lipogenic activity and did not regulate lipogenesis. Lipogenesis always changed before enzyme activity, although these groups did not measure substrate (cytoplasmic malate) turnover during periods of lipogenic change. The plasma uric acid levels in this study
Insulin
PROTEIN AND ENERGY IN REPRODUCTION
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ROSEBROUGH ET AL.
In contrast to its effect on plasma uric acid and insulin, dietary protein had no effect on plasma metal levels. Compared to 2400 kcal diets, 2850 kcal diets decreased (P<.05) plasma phosphorous and calcium. Several explanations were possible. Because hens ate more of the 2400 kcal diets, they consumed more metals, and this consumption pattern influenced circulatory values. Cereal grains contain phytates, which alter metal metabolism by chelation. Small quantities of phytates enchanced metal absorption and large quantities inhibited absorption (Kratzer et al, 1959). Suso and Edwards (1968) reported that the chelator EDTA (ethylenediaminetetraacetate) improved metal absorption; however, they also reported a range of chelator metal ratios unfavorable to absorption. Lastly, the egg production status of each hen may have regulated metal levels regardless of dietary treatments. Hens consuming the 2400 kcal diets produced more eggs and diets may have affected mineral metabolism as an indirect result of egg production. Thomason et al. (1976) reported on the confounding of egg production per se and egg production as a dietary function. Data in this study indicated that at an environmental temperature of 21 C, the turkey breeder hen required a 2400 kcal diet con-
taining only 12% protein to maintain reproductive performance. At this temperature, all hens consumed virtually the same amount of energy; therefore, the 2850 kcal diet containing 12% protein resulted in an inadequate daily protein intake. Previous research advocating higher levels of protein probably contained a safety margin for variations in feed intake due to reproductive age or changes in temperatures. The present report does not support a relationship between egg production and lipogenic capacity in the turkey breeder hen.
REFERENCES Ballard, F. J. and R. W. Hanson, 1967. The citrate cleavage pathway and Iipogenesis in rat adipose tissue: replenishment of oxaloacetate. J. Lipid Res 8 : 7 3 - 8 1 . Bolton, W, 1955. The digestibility of the carbohydrate complex of barley, wheat and maize by adult fowls. J. Agric. Sci. 46:119-122. Carter, R. D., J. W. Wyne, V. D. Chamberlin, and M. G. McCartney, 1957. The influence of dietary energy and protein on reproductive performance of turkey breeders. Poultry Sci. 36:1108-1109. Cherms, F. L., M. G. Stoller, and J. J. Macllraith, 1978. A comparison of 22 and 35 grams of daily protein intake and the effects on reproduction of turkey hens. Poultry Sci. 57:1126. (Abstr.) Cherms, F. L., M. G. Stoller, J. J. Macllraith, and H. R. Halloran, 1976. Reproduction in turkey hens as influenced by prebreeder and breeder protein intake and the environment. Poultry Sci. 55: 1678-1690. Fiske, C. H., and Y. SubbaRow, 1925. The colorimetric determination of phosphorus. J. Biol. Chem. 66:375. Folch, J., M. Lees, and G. M. Sloane-Stanley, 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497-509. Hanks, J. H., and R. E. Wallace, 1949. Relation of oxygen and temperature in the preservation of tissues by refrigeration. Proc. Soc. Exp. Biol. Med. 171:196-200. Hevia, P., and A. J. Clifford, 1977. Protein intake, uric acid metabolism and protein efficiency ratio in growing chicks. J. Nutr. 107:959-964. Jensen, L. S., and J. McGinnis, 1961. Nutritional investigations with turkey hens. 1. Quantitative requirements for protein. Poultry Sci. 40:288— 290. Kirk, J., 1968. Experimental Design Procedures for the Behavoral Sciences. Wadsworth Publ. Co., Belmont, CA. Kratzer, F. H., J. B. Allred, P. N. Davis, B. J. Marshall, and P. Vohra, 1959. The effect of autoclaving soybean protein and the addition of ethylenediamine tetraacetic acid on the biological availability of dietary zinc for turkey poults. J. Nutr. 68:313-322. Krueger, K. K., J. A. Owen, C. E. Krueger, and T. M.
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established a positive (P<.05) relationship between uric acid and the dietary protein status of the turkey breeder hen. That is, the higher values for uric acid were from hens consuming diets containing 17% protein. Miles and Featherston (1976) reported that uric acid excretion estimated the nutritional adequacy of dietary protein for chicks. Previous work with adult hens established a positive relationship between uric acid excretion and dietary protein quality (Teekell et al, 1969). Likewise, Okumura and Tasaki (1968) reported a positive relationship between plasma uric acid levels and dietary protein quality. Hevia and Clifford (1977) reported that an increase in dietary protein of 5 g/day increased plasma uric acid 1 mg/100 ml. The present study suggested a relationship (P<.05) between dietary protein levels and plasma insulin and possibly between plasma insulin and uric acid (R = .89; P<.05). Touchburn et al. (1981) reported no relationship between dietary protein and plasma insulin although these authors used chickens selected for abdominal fat to draw this conclusion. These birds were refractory to control of insulin because of excess fat.
PROTEIN AND ENERGY IN REPRODUCTION
relationship of energy and protein to reproductive performance in turkey breeders. Poultry Sci. 38:141-145. Rosebrough, R. W., N. C. Steele, and L. T. Frobish, 1982. Effect of protein and amino acid status on lipogenesis by turkey poults. Poultry Sci. 61: 731-738. Schmidt, G., 1957. Colorimetric and enzymatic methods for the determination of some purines and pyrimidines. Vol. III. Page 7 7 5 - 7 8 1 ' in Methods in Enzymology. Academic Press, New York, NY. Suso, F. A., and H. M. Edwards, 1968. Influence of various chelating agents on absorption of 6 0 Co, 59 Fe, 54Mn and 6S Zn by chickens. Poultry Sci. 47:1417-1422. Teekell, R. A., C. E. Richardson, and A. B. Watts, 1969. Dietary protein effects on urinary nitrogen components of the hen. Poultry Sci. 47:1260— 1266. Thomason, D. M., A. T. Leighton, Jr., and J. P. Mason, 1976. A study of certain environmental factors and mineral chelation on the reproductive performance of young and yearling turkey hens. Poultry Sci. 55:1343-1355. Touchburn, S., J. Simon, and B. Lechberg, 1981. Evidence of a glucose-insulin imbalance and effect of dietary protein and energy level in chickens selected for high abdominal fat content. J. Nutr. 111:325-335. Voitle, R. A., J. H. Walter, H. R. Wilson, and R. H. Harms, 1973. The effect of low protein and skip-a-day grower diets on the subsequent performance of turkey breeder hens. Poultry Sci. 52:543-548. Waldorf, M. S., M. C. Kirk, H. Linksweiller, and A. E. Harper, 1963. Metabolic adaptations in higher animals. VII. Responses of glutamic-oxalacetate and glutamate-pryuvate transaminases to diet. Proc. Soc. Exp. Biol. Med. 112:764- 768. Yeh, Y.-Y., and G. A. Leveille, 1969. Effect of dietary protein on hepatic lipogenesis in the growing chick. J. Nutr. 98: 356-366.
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Ferguson, 1978. Effect of feed and light regimens during the growing period on subsequent reproductive performance of broad breasted white turkeys fed two protein levels. Poultry Sci. 57:27-37. Leveille, G. A., and Y. Y. Yeh, 1972. Influence of intermittent fasting or protein-free feeding on lipid metabolism in young cockerals. J. Nutr. 102:733-740. Maramatsu. K., and N. D. Asheda, 1961. Effect of dietary protein level on growth and liver enzyme activity in rats. J. Nutr. 76:143—150. Maurice, D. V., and L. S. Jensen, 1978. Effect of dietary cereal on liver and plasma lipids in laying Japanese quail. Br. Poult. Sci. 19:199-205. Maurice, D. V., and L. S. Jensen, 1979. Hepatic lipid metabolism in domestic fowl as influenced by dietary cereal. J. Nutr. 109:872-882. Menge, H., L. T. Frobish, B. T. Weinland, and E. G. Geis, 1979. Effect of dietary protein and energy on reproductive performance of turkey hens. Poultry Sci. 58:419-426. Meyer, G. B., C. F. Props, A. T. Leighton, Jr., H. P. Van Krey, and L. M. Potter, 1980. Influence of dietary protein during the pre-breeder period on subsequent reproductive performance of Large White turkeys. 1. Growth, feed consumption, and female sex-linked reproductive traits. Poultry Sci. 59:353-357. Miles, R. D., and W. R. Featherston, 1976. Uric acid excretion by the chick as an indicator of dietary protein quality. Poultry Sci. 55:98-102. Morgan, C. R., and A. Lazarow, 1963. Immunoassay of insulin: two antibody systems. Diabetes 12:115-126. National Research Council, 1977. Nutrient Requirements of Poultry. Natl. Acad. Sci. Washington, DC. Okumura, J. and I. Tasaki, 1968. Effect of fasting, refeeding and dietary protein level on uric acid and ammonia content of blood, liver and kidney in chickens. J. Nutr. 97:316-320. Robblee, A. R., and D. R. Clandinin, 1959. The
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