Energy and Protein Relationships in the Broiler

Energy and Protein Relationships in the Broiler. 1. Effect of Protein Levels and Feeding Regimens on Growth, Body Composition, and In Vitro Lipogenesis of Broiler Chicks R. W. ROSEBROUGH1 and N. C. STEELE US Department of Agriculture,2 Beltsville, Maryland 20705 (Received for publication March 1, 1984) ABSTRACT Male broiler chicks were fed ad libitum diets containing 18, 23, or 30% protein for 3 weeks. In addition, chicks were fed diets containing 12, 23, or 30% protein on a 3-day rotation. Finally, chicks were fed either the 23 or 30% protein on a schedule of 2 days on feed and 1 day off feed. These feeding schemes allowed chicks to consume low to excess protein coupled with either adequate or inadequate energy intake. Feed intakes were equal for the 18, 23, and rotational percent protein dietary groups and lower (P<.05) for the 30% ad libitum group. Chicks fed the 18% protein diet ad libitum were the least efficient (P<.05) at utilizing feed and the most efficient (P<.05) at utilizing protein for weight gain. Compared to the ad libitum situation, restricted feeding improved feed and protein efficiency of chicks fed 30% protein diets but not of chicks fed 23% protein diets. Body composition data on a dry matter basis supported a positive relationship between dietary protein and percent lean tissue. Conversely, expressing data on a whole bird basis indicated that lean body mass was favored by feeding the diet containing 23% protein. In vitro lipogenesis was greatest (P<.05) in chicks fed a 18% protein diet ad libitum and least (P<.05) in chicks fed a 30% protein diets ad libitum. The overall rate of in vitro lipogenesis for the rotational group (12 plus 23 plus 30/3) was less than that for the 18% ad libitum group and more than that for 23% ad libitum group. Compared with ad libitum fed controls, feed withdrawal decreased (P<.05) and refeeding increased (P<.05) in vitro lipogenesis in chicks fed diets containing either 23 or 30% protein. The overall rate of lipogenesis (1 off plus 1 on plus 2 on/3) for each of these two levels of protein was greater than for the respective ad libitum feeding. Malic enzyme activity paralleled changes in the rates of in vitro lipogenesis while glutamic oxaloacetic transaminase and isocitrate dehydrogenase activities were more indicative of the protein status of the chick. (Key words: energy:protein, body composition, growth lipogenesis, broilers) 1985 Poultry Science 64:119-126 INTRODUCTION Obesity occurs in birds when the caloric intake exceeds that required for lean tissue accretion and for maintenance of life. Forced feeding and hypothalamic lesions override natural controls on energy intake and can cause obesity. In contrast, genetic selection for obesity results in excess body fat unrelated to dietary energy. In fact, obese lines of chickens were similar in size to their lean counterparts

1 USDA, Agricultural Research Service, Beltsville Agricultural Research Center, Animal Science Institute, Nonruminant Animal Nutrition Laboratory, Beltsville, MD 20705. 2 Mention of trade name, propriety product, or vendor does not constitute a guarantee or warranty of the product by the USDA and does not imply its approval to the exclusion of other products or vendors that may also be suitable.

early in life (Lilburn et al., 1982). These lines of chickens did not differ in energetic efficiencies but did seem to differ in lean body mass potential. However, Griffiths et al. (1977) questioned the role of genetic involvement in fat pad development in commercial broiler chickens and suggested that diet regulated lipid deposition. These authors suggested that diets containing a wide calorie: protein ratio forced an overconsumption of energy as broilers ate to meet a protein requirement. Bartov and Bornstein (1976) reported similar findings. Likewise, diets containing a narrow calorie:protein ratio decreased carcass fat because broilers consumed less energy in meeting a protein requirement (Donaldson et al., 1956; Summers et al., 1965; Deatonet al, 1974). The level of dietary nitrogen and of limiting amino acids for lean tissue accretion can also regulate lipid metabolism in broiler chicks. For example, Khalil et al. (1968) produced obese

119

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ROSEBROUGH AND STEELE

chicks by feeding low protein diets; however, they reversed the obesity by refeeding a balanced diet to these same chicks. The purpose of the present research was to determine the effect of different protein intakes coupled to different energy intakes on both the in vitro lipogenesis and body composition of male broiler chicks. MATERIALS AND METHODS Animals and Diets. Groups of eight 1day-old male broiler chicks were allocated to pens in heated battery brooders and fed a diet containing 23% protein for 1 week. At this time, eight pens were allocated to diets containing 18, 23, or 30% protein and fed on an ad libitum basis for 21 days. Groups of eight pens were also fed either the 23 or 30% protein diets on a schedule of 2 days on feed 1 day off feed. The group consuming the 23% protein diet ad libitum was considered the control group for both protein and energy intake. Preliminary work demonstrated that when pens of eight chicks were fed on a cycle of 2 days with and 1 day without feed, they consumed approximately 75% of the feed of ad libitum controls. The 75% figure could be applied to protein levels to adjust intake. In comparison with controls, the 23% restricted group was restricted in both protein and energy, the 30%

restricted group was restricted in energy, and the 18% ad libitum group was restricted in protein. The 30% ad libitum group received an excess in protein but an adequate energy intake. Finally, a schedule was devised to feed diets containing 12, 23, and 30% protein diets on a 3-day protein rotation. Thus, the chicks were fed an adequate level of energy in the process of receiving either low and high protein diets on 2 days of a 3-day cycle. These 3-day cycles were continued for 21 days. Four diets were formulated to contain approximately the same energy levels (Table 1). Additional amino acids other than methionine were not added to the diets, because we have found that crystalline amino acids affect lipid metabolism differently than "natural" amino acids in the form of feedstuffs (Rosebrough et al, 1982). In Vitro Metabolic Studies. Two chicks from each pen were selected during each day of the 8th cycle to determine characteristics of in vitro metabolism. Chicks were killed by cervical dislocation and the livers were rapidly excised and weighed. Portions of each liver were sliced with a Stadie-Riggs hand microtome. The slices were incubated for 2 hr at 39 C in 25-ml Erlenmeyer flasks that contained a balanced salt solution (Hanks and Wallace, 1949) supplemented with 29 mAf HEPES (N-2-hydrox-

TABLE 1. Composition of the experimental diets Protein Ingredient

12%

18%

23%

30%

tg/Kg; Soybean meal, 49% protein Alfalfa meal, 17% protein Corn meal L-Methionine Soybean oil Cellulose Dicalcium phosphate Limestone Selenium mix 1 Mineral mix 1 Vitamin mix 1 Iodized salt Calculated analysis Metabolizable energy kcal/kg Protein, g/kg Lysine, g/kg Methionine, g/kg Cystine, g/kg 1

92 50 797

236 33 656 2 13

40 10 1 1 5 3 2995 121 5.2 2.4 2.0

For composition of mixes see Rosebrough et al. (1982).

375

550 350 1 40

40 10 1 1 5 3

525 3 20 17 40 10 1 1 5 3

2954 179 9.3 5.1 2.8

3005 231 13.2 6.8 3.6

2913 300 18.3 6.6 4.5

40 10 1 1 5 3

PROTEIN AND LIPOGENESIS IN BROILERS

yethylpiperazine-N-2-ethane sulfonic acid; pH 7.4). In addition, the flasks also contained 20 mM (1—14C) sodium acetate (specific activity = 37 DPM/nmole). After the 2-hr incubation, the slices were extracted for 24 hr in 15 ml of 2:1 chloroform:methanol. The extract was fractionated with .3 volume of .88% KC1, and the bottom phase (chloroform) was washed once with 8 ml of 5:3 methanol: .88% KC1 according to Folch et al. (1957). The bottom phase was evaporated to dryness, dispersed in Scintiverse,2 and counted by liquid scintillation spectroscopy. In vitro lipogenesis was calculated as micromoles of sodium acetate incorporated into lipids during the 2-hr incubation. The value was corrected for changes in liver size by expressing it on the basis of activity per 100 g of body weight. Enzyme Assays. Livers were homogenized in 10 volumes of 100 mM Tris (pH 7.5) that contained 150 mM KC1, 10 mM MgCl 2 , 10 mM mercaptoethanol, and .3% Triton X 100 and centrifuged at 50,000 X g for 45 min. The supernatant fraction was used to determine the activities of malic enzyme (ME; E.C. 1.1.1.40) nicotinamide-adenine dinucleotide phosphatelinked isocitrate dehydrogenase (NADP-ICD; E.C. 1.1.1.42) and glutamic-oxaloacetic transaminase (GOT; E.C. 2.6.1.1). Malic enzyme was determined by a modification of the method of Hsu and Lardy (1969). The reaction medium contained 50 mM glycylglycine (pH 7.5), 1 mM NADP, 7 mM MnCl 2 , and 1 mM malate. Isocitrate dehydrogenase activity was determined by a modification of the method of Cleland et al. (1969). The reaction medium contained 100 mM glycylglycine (pH 7.5), 1 mM MgCl 2 , 5 mM mercaptoethanol, 1 mM NADP, and 2.2 mM DL-isocitrate. Glutamic oxaloacetic transaminase was determined by a modification of the method of Martin and Herbein (1976). The reaction medium contained 50 mM potassium phosphate 9pH 7.6), 50 mM asparate, 20 mM alpha-ketoglutarate, 1 mM NADH, 5000 U/liter malate dehydrogenase, and 5000 U/liter lactate dehydrogenase. Enzyme activities were expressed as micromoles of nucleotide utilized per minute under the assay conditions. Final activities were expressed on the basis of relative liver size (g of liver + body weight X 100). Carcasses were dried to constant weights by heating at 100 C for 2 days in a forced air oven or by lyophilization for 2 wk. Body composition was estimated by the method of Summers

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and Fisher (1961) as described by Yeh and Leville (1969). Statistical Analysis. For all variables measured, the experimental unit was the individual pen of chicks. Therefore, treatment means were means of pens treated alike (eight pens/dietary treatment). Data were analyzed according to Kirk (1968). RESULTS

This experiment demonstrated no advantage in chick growth by feeding diets containing more than 23% protein (Table 2). Growth data also indicated that chicks tolerated a 18% protein diet better than a 30% protein diet, although both diets resulted in lighter (P<.05) chicks than did 23% protein. Feed intakes were similar for chicks consuming the 18, 23, and rotational protein diets, although protein intakes were obviously different. In contrast, chicks consuming 30% protein diets on an ad libitum basis ate less (P<.05) feed than the other ad libitum groups, Feed intake data for the group fed the rotational series of protein indicated that protein level regulated feed intake. The average chick consumed 583 g of the 12% diet, 556 g of the 23% diet, and 519 g of the 30% diet. The chicks consuming the 18% protein diet ad libitum were the most efficient (P<.05) in utilizing protein for weight gain but the least efficient (P<.05) in utilizing feed. Compared to the ad libitum condition, restricted feeding improved feed efficiency of chicks fed diets containing 30% protein. Body composition data, expressed on a unit of dry matter, seemed to support a positive relationship between dietary protein and protein deposition in the bird. Likewise, there appeared to be an opposite relationship for body fat (Table 3). Total body protein and fat data supported a somewhat different conclusion. Total protein deposition was greater (P<.05) in birds fed the diet containing 23% protein than in birds fed the diet containing 30% protein. In contrast, total body fat was lowest (P<.05) in that group fed the diet containing 30% protein. The group fed protein on a rotational basis and the group fed ad libitum the diet containing 30% protein had similar quantities of total body protein; however, body fat was greater (P<.05) in the rotational group. Restricted feeding of either the 23 or 30% protein diet decreased total body protein compared to feeding these diets on an

ROSEBROUGH AND STEELE

122

TABLE 2. Effect of protein and energy restriction upon growth and feed consumption of broiler chicks1

Protein

Wt

Rotational 2 18% Ad libitum 23% Ad libitum 30% Ad libitum 23%, 2 days on, l o f f 30%, 2 days on, l o f f

1106 c 1095 c 1163 d 1025 b 927a 933 a

Pooled SE

19

(g)

Feed intake

Feed/gain

1658 c 1708 c 1642 c 1513 b 1262 a 1248 a

174bcd 1.80 d 1.63 a b 1.75 c d 1.63 a b 1.61 a

Gain/ protein intake

Protein intake (g)

24

.04

354 b 307 a 377 e 454 d 302 a 377 c

2.69 c 3.09 d 2.67 c 1.91 a 2.56 c 2.06 b

6

.05

' ' ' Means within a column followed by a common superscript are not different (P<.05). 1

Values presented are means of eight pens of eight male chicks each per treatment fed experimental diets from 1 to 4 weeks of age. 2 Fed diets containing 12, 23, and 30% protein on a 3-day rotation. Overall protein level calculated from intakes of the three diets was 20%.

ad libitum basis and also did away with the previously mentioned differences in body protein and fat due to dietary protein. Chicks fed the diet containing 23% protein had a smaller (P<.05) relative liver size than chicks fed either the 18 or 30% protein diet (Table 4). When chicks were fed on a rotational basis, the relative liver size was greatest following consumption of the 12% protein diet. When chicks were fed on an ad libitum basis, in vitro lipogenic capacity was greatest (P<.05) in chicks fed diets containing 18% protein and least (P<.05) in those fed diets containing 30%

protein. The overall average of the group fed diets containing 12, 23, and 30% protein on a rotational basis was less (P<.05) than that for the 18% group but greater (P<.05) than that for the 23% group. The liver sizes of chicks subjected to the two restriction regimens changed significantly (P<.05) over the course of the 3-day cycles; however, there was no effect of dietary protein on this pattern (Table 5). Both feeding regimens and protein levels significantly (P<.05) affected in vitro lipogenesis. The overall mean of the group fed 23% protein was greater than

TABLE 3. Effect of protein and energy restriction upon body consumption of broiler chicks1 Protein

Moisture

(% DM) 3

(%) Rotational 2 18% Ad libitum 23% Ad libitum 30% Ad libitum 23%, 2 days on, 1 off 30%, 2 days on, l o f f Pooled SE

b

62.4 60.7 a 62.3 b 66.8 d 64.1c 64.4 C .4

b

41.7 38.5 a 42.lb 51.ld 45.0C 45.lc .5

Fat

(g/bird) c

173 166 b 184 d 174 c 150 a 149 a 2

(% DM) C

49.3 53.9 d 49.5 C 39.4 a 46.4 b 48.0bc .7

(g/bird) 205 c 232 e 217 d 134 a 154 b 159 b 3

3 h p d p 1

' ' ' ' Means within a column followed by a common superscript are not different (P<.05). Values presented are means of eight pens per treatment.

2 Fed diets containing 12, 23, and 30% protein on a 3-day rotation. Overall protein level calculated from intakes of the three diets was 20%. 3

DM = Dry matter.

PROTEIN AND LIPOGENESIS IN BROILERS

123

TABLE 4. Effect of protein level on in vitro lipogenesis and liver enzyme activities of broiler chicks fed ad libitum' E n z y m e activities 2 ' 3 ( u n i t s / 1 0 0 g B W )

Liver weight

In vitro lipogenesis

(g/lOOg BW) 5

(Mmol/lOOg BW)

R o t a t i o n a l overall 4 Post 12% Post 2 3 % Post 30%

2.59cd 3.11e 2.43bc 2.25b

18% 23% 30%

2.73d 1.83a 2.55cd

Protein

Pooled SE

.08

GOT

NADP-ICD

ME

88c 151e 82c 32a

219cd 188b 234d 235d

65b 71b 44a 80c

12e gcd

106d 5lb 21a

101a 194b

37a 42a 46a

265e

5

8

3

9

d

7C 8cd b

4

2a .4

3h cdp

' ' ' ' Means within a column followed by a common superscript are not different (P<.05).

1

Values presented are means of eight pens per treatment.

2

One unit is equal to the oxidation or reduction of the appropriate nucleotide at 25 C.

3 GOT = Glutamic oxaloacetic transaminase; NADP-ICD = nicotinamide adenine dinucleotide phosphate isocitrate dehydrogenase: ME = malic enzyme.

"Fed diets containing 12, 23, and 30% protein on a 3-day rotation. Overall protein level calculated from intakes of the three diets was 20%. 5

BW = Body weight.

that for the group fed 30% protein. Within each group, refed values were greater than for the 1 day without feed. The overall mean for each

protein level was also greater than for the ad libitum feeding of that same level of protein (Table 4).

TABLE 5. Effect of protein level on in vitro lipogenesis and liver enzyme activities of broiler chicks fed on a 2 on, I off schedule1

Protein

Liver weight

In vitro lipogenesis

(g/100g BW)

(jumol/lOOg BW)

23%, overall loff 1 on 2 on

2.89 b 2.04 a 3.68 d 2.96 b

30%, overall loff 1 on 2 on

2.81b 2.20a 3.24 c 2 .99bc

Pooled SE

99c 5a 156e 136d 79b 3a 118d 117cd

Enzyme activities2'3 (units/100 g BW) GOT

NADP-ICD

ME

305bc 27 7 a 31lbc 327c

6lb 41a 69bc 75c 79cd 39 a 110 e 87d

10 c 7b 14 d 10 c

355d 302b 404e 358d

.09

6b 3a gbc 6b .7

*t h c d p

' ' ' ' Means within a column followed by a common superscript are not different (P<.05).

1

Values presented are means of eight pens per treatment.

2

One unit is equal to the oxidation or reduction of the appropriate nucleotide at 25 C.

3 GOT = Glutamic oxaloacetic transaminase; NADP-ICD = nicotinamide adenine dinucleotide phosphate isocitrate dehydrogenase; ME = malic enzyme.

ROSEBROUGH AND STEELE

124

Increasing protein increased (P<.05) GOT activity but decreased (P<.05) ME activity in chicks fed ad libitum (Table 4). Within the rotational series, GOT activities were similar following the feeding of the 23 or 30% protein diets and greater (P<.05) than the activity following the feeding of the 12% protein diet. Malic enzyme activity, following the feeding of the 12% protein diet was also higher (P<.05) than after the feeding of the two higher protein diets. The NADP-ICD activity was inconsistently affected by dietary protein. Likewise, chicks placed on the restriction regimens also exhibited the same effect of protein on overall enzyme activities (Table 5). Within regimens, the day off feed reduced (P<.05) the activities of all three enzymes while the first day on feed produced the greatest activities during the cycle. Overall activities for both protein groups were greater than for the respective ad libitum groups (Table 4). DISCUSSION

Although many groups have reported that diets with wide calorie:protein ratios enhanced lipid deposition and that diets with narrow calorie: protein ratios enhanced lean tissue accretion, these groups did not offer a biochemical relationship between dietary protein and in vitro lipogenesis and between in vitro lipogenesis and body composition of broilers. Yeh and Leveille (1969) found inverse relationships between dietary protein and in vitro lipogenesis and between dietary protein and body fat of older chicks. Furthermore, these authors found that improving the quality of low protein diets by adding lysine increased the growth rate of chicks but did not decrease in vitro lipogenesis. In the present study there was a slight, although significant, increase in growth and a twofold decrease in the rate of in vitro lipogenesis accompanying an increase in dietary protein (18 vs. 23% protein). Although feeding a diet containing 30% protein further decreased in vitro lipogenesis, growth did not improve. Combinations of feeding regimens and dietary protein levels gave different protein and energy intakes relative to those attained with the ad libitum feeding of the 23% protein diet. Adequate protein but low energy (30% protein; 2 on, 1 off) resulted in no more lean tissue in the chick than a low protein intake coupled to a low energy intake (23% protein; 2 on, 1 off). Both restriction regimens also resulted in

similar growth rates and feed efficiencies although overall in vitro lipogenesis was less in the 30% restricted group. In contrast, coupling a low protein intake to a slight excess in energy (18% fed ad libitum) resulted in only a 10% increase in total body protein but a 50% increase in total body fat compared to the two restriction regimens. Conversely, an excess in protein coupled with an adequate energy intake (30% ad libitum) decreased body fat 40% compared with an adequate protein and energy intake (23% ad libitum). Bartov (1979) hypothesized that excessive protein intake forced the bird to expend energy to excrete excess nitrogen as uric acid. Thus, less energy was available for fat synthesis (Buttery and Boorman, 1976). The overall variation in total protein per bird was less than that for total body fat (8.4 vs. 22%) and suggested that the genetic predisposition of the broiler chick was to first meet some body protein content. At this point, dietary energy shifted to either uric acid production (excess dietary protein) or to fat synthesis. The rotational feeding regimen (12, 23, and 30% protein) demonstrated that these adaptations to variations in protein intake were rapid yet resulted in carcass characteristics similar to those attained by feeding the 23% protein diets on an ad libitum basis. This finding was not surprising, because the overall dietary protein of the rotational dietary series was 20%. The diets used in the present study contained a low enough level of protein to test the hypothesis that wide calorie: protein ratios forced overconsumption of energy. In addition, one dietary treatment (30% protein, ad libitum fed) may have redirected energy from fat synthesis to excretion of excess nitrogen. Touchburn et al. (1981) also reported that within a narrow range of dietary protein levels, energy intakes for selected and lean lines of chickens were identical and that body composition reflected protein intake. Those authors concluded, however, that obesity was not related to growth or feed intake. Tepperman and Tepperman (1958) demonstrated controls on lipogenesis in starvedrefed rats, and more recently, Leveille et al. (1975) reported that lipogenic enzyme activity lagged behind the hyperlipogenesis following refeeding. Our data demonstrated that cycles of starvation and refeeding produced lipogenic rates that were higher than ad libitum values regardless of protein level. Likewise, the feeding

PROTEIN AND LIPOGENESIS IN BROILERS

of rotational dietary protein series produced an effect which was similar to starvation-refeeding. Thus, two general conditions for the regulation of lipogenesis were present in the current study. Protein levels regulated lipogenesis were present in the current study. Protein levels regulated lipogenesis in ad libitum-fed chicks and the availability of lipogenic substrates regulated the process during starvation-refeeding. The exact mechanism responsible for the decrease and the subsequent increase in lipogenesis following starvation-refeeding or protein sequencing remains unknown although the availability of reducing equivalents cannot be discounted. Increasing protein at the expense of carbohydrates restricted the already limited flow of substrates through the glycolytic pathway and enhanced glucose production (gluconeogenesis) from noncarbohydrate precursors such as dietary protein (Yeh and Leveille, 1969). We hypothesized that GOT activity in chicks would increase as these chicks were fed diets containing higher protein levels. The activity would then indicate amino acid turnover and a requirement for disposal of the carbon fragments from these amino acids. The ubiquitous nature of isocitrate dehydrogenase suggests a role in the provision of alpha-ketoglutorate to be used as a nitrogen acceptor during transamination and as a source of reducing equivalents to be used during gluconeogenesis. Thus, it is possible that citrate would be used as a source of alpha-ketoglutarate for transamination rather than as a reactant in the citrate cleavage reaction. Furthermore, the depletion of both citrate and isocitrate through the aconitase equilibria and subsequent transamination removes these two compounds as potential allosteric activators of acetyl coenzyme A carboxylase, a proposed rate limiting step in de novo lipogenesis. Finally, the inverse relationship between malic enzyme activity and dietary protein is important for two reasons. This enzyme functions as a major source of reducing potential due to a lack of pentose phosphate dehydrogenases in the avian liver and also as a disposal route for four carbon skeletal fragments produced via the citrate cleavage system. REFERENCES Bartov, I., 1979. Nutritional factors affecting quantity and quality of carcass fat in chickens. Fed. Proc. 38:2627-2639.

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Bartov, I., and S. Bornstein, 1976. Effect of degree of fatness in broilers and other carcass characteristics: relationship between fatness and the composition of carcass fat. Br. Poult. Sci. 17: 29-38. Buttery, P. J., and K. N. Boorman, 1976. The energetic efficiency of amino acid metabolism. Pages 197—204 in Protein metabolism and nutrition. Butterworths, London, England. Cleland, W. W., V. W. Thompson, and R. E. Barden, 1969. Isocitrate dehydrogenase (TPN-specific) from pig heart. Pages 3 0 - 3 3 in Methods in enzymology. Academic Press, New York, NY. Deaton, J. W., L. F. Kubena, T. C. Chen, and F. N. Reece, 1974. Factors influencing the quantity of abdominal fat in broilers. 2. Cage versus floor rearing. Poultry Sci. 35:1100-1105. Donaldson, W. E., G. F. Combs, and G. L. Romoser, 1956. Studies on energy levels in poultry rations. 1. The effect of calorie-protein ratio of the ration on growth, nutrient utilization and body composition of chicks. Poultry Sci. 35:1100-1105. Folch, J., M. Lees, and G. H. Sloane-Stanley, 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497-509. Griffiths, L., S. Leeson, and J. D. Summers, 1977. Fat deposition in the broiler: effect of dietary energy to protein balance, and early life caloric restriction on productive performance and abdominal fat pad size. Poultry Sci. 56:638—646. 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. 71:196-199. Hsu, R. Y., and H. A. Lardy, 1969. Malic enzyme. Pages 230—235 in Methods in Enzymology. Academic Press, New York, NY. Khalil, A. A., O. P. Thomas, and G. F. Combs, 1968. Influence of body composition, methionine deficiency on toxicity and ambient temperature on feed intake in the chick. J. Nutr. 96:337— 341. Kirk, J., 1968. Experimental Design Procedures for the Behavioral Sciences. Wadsworth Publ. Co., Belmont, CA. Leveille, G. A., D. R. Romsos, Y-Y Yeh, and E. K. O'Hea, 1975. Lipid biosynthesis in the chick. A consideration of site of synthesis influence of diet and possible regulatory mechanisms. Poultry Sci. 54:1075-1093. Lilburn, M. S., F. D. Morrow, R. M. Leach, E. G. Buss, and R. J. Martin, 1982. A comparison of the in vitro lipogenic rates and other physiologic parameters in two strains of lean and obese chickens. Growth 46:163-170. Martin, R. J., and J. H. Herbein, 1976. A comparison of the enzyme levels and in vitro utilization on various substrate for lipogenesis in pair-fed lean and obese pigs. Proc. Soc. Exp. Biol. Med. 151:231-235. Rosebrough, R. W., N. C. Steele, and L. T. Frobish, 1982. Effects of protein and amino acid status on lipogenesis by turkey poults. Poultry Sci. 6 1 : 731-738. Summers, J. D., and H. Fisher, 1961. Net protein value of the growing chicken as determined by

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carcass analysis:exploration of the method. J. Nutr. 75:435-446. Summers, J. D., S. J. Slinger, and G. C. Ashton, 1965. The effect of dietary energy and protein on carcass composition with a note on a method for estimating carcass composition. Poultry Sci. 44:501-509. Tepperman, J. M., and J. Tepperman, 1958. The hexosemonophosphate shunt and adaptive hy-

perlipogenesis. Diabetes 7:478—490. Touchburn, S., J. Simon, and B. Leclercq, 1981. Evidence of a glucose-insulin imbalance and effect of dietary protein and energy level in chickens selected for high abdominal fat. J. Nutr. 111:325-335. Yeh, Y-Y, and G. A. Leveiile, 1969. Effect of dietary protein on hepatic lipogenesis in die growing chick. J. Nutr. 356-366.