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L 9... it . ‘ « .L.I\£LL£LLELE: .. . .X 2“! ~IL§§€K .1, tl‘ig Eli: :05 ~ ill grist-x54 ‘IL . ALEIIthlxsftuvft. . ‘ m5?- ’ LIBRARIES MICHIGAN STATE UNIVERSITY, EAST LANSING, MICH. 48824 This is to certify that the thesis entitled EFFECT OF TRENBOLONE ACETATE ON PERFORMANCE, CARCASS CHARACTERISTICS, BODY COMPOSITION AND MUSCLE PROTEIN DEGRADATION IN FINISHING HEIFERS presented by Donald R . Benner has been accepted towards fulfillment of the requirements for Master's degree in Animal Science %%g MA, Major professor Date May 25, 1983 0-7639 MSU is an Affirmative Action/Equal Opportunity Institution / MSU PIace in book drop to t ' eckout from . LIBRARIES . ’15-’— your record. FINES will be charged if book is returned after the date stamped below. /~- DO NOT (IRC LATE R OM USE O Y D NOT (IR IIIA'IE EFFECT OF TRENBOLONE ACETATE ON PERFORMANCE, CARCASS CHARACTERISTICS, BODY COMPOSITION AND MUSCLE PROTEIN DEGRADATION IN FINISHING HEIFERS BY Donald Ray Benner A THESIS Submitted to Michigan State University in partial fullment of the requirements for the degree of MASTER OF SCIENCE Department of Animal Science 1983 ABSTRACT EFFECT OF TRENBOLONE ACETATE ON PERFORMANCE, CARCASS CHARACTERISTICS, BODY COMPOSITION AND MUSCLE PROTEIN DEGRADATION IN FINISHING HEIFERS BY Donald R. Benner The effect of trenbolone of acetate (TBA) on feedlot performance was investigated at 0, 140, 200, or 300 mg per head during a 63 day period with 40 heifers per treatment. TBA at 200 and 300 mg resulted in small increases in average daily gain, dry matter intake and feed efficiency. Differen— ces in carcass characteristics were small and not signi- ficantly different (P>.05). Four successive measures of body composition of 24 subjects were determined in vivo by deuterium oxide tracer methodology. All three doses of TBA tended to increase empty body weight and empty body protein, but empty body fat was similiar to control. Fractional breakdown rates (FBR) of skeletal muscle protein were cal- culated from urinary 3—methylhistidine excretion. FBR was similiar among all treatments. These results fail to demon— strate TBA increases skeletal muscle accretion by a reduction in FBR. ACKNOWLEDGEMENTS I am indebted to many persons for their assistance, counsel and encouragement necessary to complete this thesis. To those I have forgotten, my sincere apologies for failing to offer a more personal thanks. Nevertheless, your help was always appreciated. My sincere appreciation is extended to Dr. John Waller, my advisor, for his guidance during my graduate study, the opportunities he provided to participate in extension programs and for conveying so many interesting experiences. I am also grateful for the unending contributions Dr. Werner Bergen made in the planning, analysis, and inter— pretation of this research. His unselfish assistance was invaluable. To Dr. Dave Hawkins I extend all my thanks for his aid during this experiment and for allowing me to instruct some of his undergraduate classes. Dr. Hawkins provided encour— agement of humor the moment it was needed most and often turned a dreary day into a brighter one. I express sincere gratitude to Dr. John Gunther for his endless help with this research, hiscareful review of this manuscript, and most of all for his frienship. I also thank Bill Rumpler, Kris Johnson, Donna Cox, Dennis Banks, Chuck Reid, Pete Sweeney, Betty Talcott, Dr. Harlan Ritchie and Dr. Farabee McCarthy for their friend— ship and contributions to my graduate study. A special thanks is extended to Gary Weber and Scott Barao for their comradeship and tireless aid in collecting samples. Gratitude is extended to Dr. Ron Nelson for financial retribution and use of facilities and experimental animals. His leadership in the Department of Animal Science has pro- vided the "environment" in which I have enjoyed participa— ting. Most of all I am grateful to Juliana King and my par- ents. Without their continued encouragement and understand- ing, I would have never completed my graduate program. ii TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . . . LIST OF FIGURES . . . . . . . . . . . INTRODUCTION . . . . . . . . . . . . LITERATURE REVIEW . . . . . . . . . . The Effect of Anabolic Agents on Growth. . Effect of Trenbolone Acetate on Growth . . Effect of Trenbolone Acetate on Blood Metabolites. . . . . . Tissue Residue Levels of Trenbolone . . . Methodology to Assess Body Composition . . Serial Slaughter Techniquies . . . . Whole Animal Chemical Analysis . . . 9-10-11 Rib Section Analysis . . . . Specific Gravity . . . . Use of Carcass Cuts to Predict Body Composition. . . . . . In Vivo Determination of Body Composition . Dilution Techniques . . . . . . . Isotope Dilution . . . . . . . . Urea Dilution . . . . . . . . Potassium 40 Counting . . . . . . Adipose- -Cell Size. . . . . Use of 3-Methylhistidine as an Index of In Vivo Muscle Protein Degradation Rates . Protein Turnover in Animals. . . . . . MATERIALS AND METHODS Feedlot Performance and Carcass Characteristics . . . . . . . . Experimental Animals. . . . . . . Experimental Design . . . . . Collection of Experimental Data . . . Statistical Analysis. . . . . . . iii PAGE ll l6 l7 19 2O 21 23 25 25 3O 32 33 35 39 Trial 2. Studies on Composition of Gain and Skeletal Muscle Degradation . . Experimental Animals. . . . . . Experimental Design . . . . . . Management Procedures . . . . . Body Composition Determination . . Urine Collection . . . . . 3-Methylhistidine Analysis. . . . Creatinine Analysis . . . . . . Statistical Analysis. . . . . . RESULTS AND DISCUSSION Trial 1.Feedlot Performance and Carcass Characteristics . . . . Trial 2. Effect of Trenbolone Acetate on Body Composition and Muscle Protein Degradation. . Effect of Trenbolone Acetate on 3— —Methyl— Histidine Excretion . . . . . . SUMMARY I O O O I Q 0 O I O 0 0 APPENDIX . . . . . . . . . . . . LITERATURE CITED . . . . . . . . . iv 119 121 l27 10. ll. 12. 13. 14. LIST OF TABLES Ingredients and Composition of Experimental Diet . . . . . . . . . . . . . . Effect of Trenbolone Acetate on Feedlot Performance of Finishing Heifers . . . . . Effect of Trenbolone Acetate on Carcass Characteristics of Finishing Heifers . . . Number of Animals by Treatment for Analysis of Body Composition . . . . . . . . . Number of Animals by Treatment for Analysis of Urine Metabolites . . . . . . . . . Effect of and Empty Effect of Water and Effect of Trenbolone Acetate on Live Weights Body Weights . . . . . . . . Trenbolone Acetate on Empty Body Protein 0 I O 0 0 O O O O C Trenbolone Acetate on Empty Body Fat Effect of Trenbolone Acetate on Body Composition Determined by Single Pool Deuterium Oxide Tracer Kinetics. . . . . . Effect of Trenbolone Acetate on Daily Empty Body Weight and Empty Body Water Gain . . . . . Effect of Trenbolone Acetate on Daily Accretion of Empty Body Protein and Fat . . . . . . Least Square Means for Daily Urine Excretion by Treatment and Period . . . . . . . . . Effect of Trenbolone Acetate on Excretion of 3-Methylhistidine . . . . . . . . . . Effect of Trenbolone Acetate on Excretion of Creatinine . . . . . . . . . . . . Page A8 64 66 7O 71 75 79 84 91 93 97 99 103 15. l6. l7. l8. Effect of Trenbolone Acetate on Urinary 3-Methylhistidine to Creatinine Ratio Effect of Trenbolone Acetate on Skeletal Muscle and Total Skeletal Muscle 3—Methylhistidine Pool Effect of Trenbolone Acetate on Fractional Excretion of 3— —Methylhistidine and Half Life of Skeletal Muscle Skeletal Muscle Protein Synthesis, Degra— dation, and Accretion o Page 105 110 112 116 FIGURE Model for Two Pool Open System Kinetic Equations for Two~Pool Methodology Effect of Trenbolone Acetate on Live Body Weight . Effect of Trenbolone Acetate on Empty Body Protein . Effect of Trenbolone Acetate on Empty Body Fat . . Effect of Samples Prior to Empty Body Equillibration on Dilution Kinetics Effect of Trenbolone Acetate on Urinary Excretion of 3—Methylhistidine Per Unit of Body Weight LIST OF FIGURES 0 vii Page 59 73 77 81 88 101 INTRODUCTION Comprehension of animal growth processes and subsequent manipulation of these phenomenon offer tremendous oppor- tunities to enhance animal production efficiency. Research nutrition, genetics, reproduction and disease has led to improved production and large gains in meat animal produc- tion efficiency. Further improvements in these areas are inevitable as more knowledge is attained. Manipulation of animal metabolic processes may be possible to enhance pro— tein production per unit of energy input, while minimizing lipid deposition. These processes are under complex meta- bolic control and in most cases only partially understood. An animal's ability to synthesize large amounts of protein is documented (Mulvaney, 1981; McCarthy, 1981). Net protein accretion is small, however, due to high rates of protein degradation relative to protein synthesis. Precise regula— tion of protein turnover without detriment to the animal would reduce energy requirements. Research in this area is hampered by inaccuracy or expense of measuring changes in rates of protein synthesis, degradation and accretion in the live animal. Manipulation of metabolic functions necessi- tates comprehension of their regulation. In this regard, our knowledge is inadequate. LITERATURE REVIEW The Effect of Anabolic Agents on Growth Exogenous administration of anabolic agents, primarily hormones, is practiced worldwide to enhance the efficiency of meat production. In general, anabolic agents stimulate live weight gain and protein deposition, primarily at the ex— pense of fat, and reduce feed required per unit of live weight gain. There are many exceptions to these broad generalizations depending on the type of anabolic agent, sex and age of the animal, the environmental conditions, and per— haps a host of other unknown factors. Hormones are known to regulate animal growth and parti- tioning of nutrients to various tissues. These processes are complex and undoubtably regulated by the balance and interaction of many hormones rather than the concentration of any one hormone in particular. A number of investi- gations have probed the changes in hormonal concentrations following treatment with anabolic agents. Interpretation of this of literature is complicated by failure of many investi— gators to control environmental conditions known to alter hormonal profiles. Trenkle (1978) has suggested a number of A considerations for this type of investigation, including proper animal adaptation, constancy in temperature and photo- period, scheduled feeding, and multiple sampling. Several authors have reviewed the effect of anabolic agents on growth and their possible mode of action (Heitzman, 1976; Trenkle, 1976; Michel and Balieu, 1976; Trenkle, 1978; Buttery et al., 1978; Broome, 1980; Heitzman, 1980; Galbraith and Topps, 1981). Anabolic agents are broadly classified as estrogens or androgens. Some commonly used estrogenic agents include diethylstilbestrol, hexestrol, zeranol and estradiol. Testosterone and tren— bolone acetate are commonly used androgens. The mechanism by which these compounds exert their effects is incompletely understood. It is clear, however, they impinge upon a variety of metabolic processes. In steers, improvement in growth rate and feed effi- ciency has been observed following treatment with diethyl— stilbestrol (Fowler et al., 1970), hexestrol (Perry et al., 1955), and estradiol (Hale and Ray, 1973). Fowler et a1. (1970), as well as others, demonstrated increases in carcass gain, protein deposition and nitrogen retention while car- cass fat and energy retention were reduced. similiar but less pronounced affects have been observed in heifers (Beeson, 1969). Diethylstibestrol (DES) has inconsistently improved growth or feed efficiency in bulls (Baker and Arthaud, 1972). Diethylstilbestrol treated bulls have often shown increases in carcass fat depostion. similiar results have been reported for zeranol (resorcylic acid lactone) treated intact males (Brethour, 1982). Although the mode of action of estrogenic agents is not fully elucidated, it is thought estrogenic compounds stimu— late growth and improve feed efficiency by altering the endo- genous hormonal status. Preston (1975) discusses several hypotheses postulated by various authors. He concludes the most plausible hypothesis is estrogens act upon the hypo- thalamus to stimulate secretion of growth hormone releasing factors, which cause a concomitant increase in plasma growth hormone concentrations. This hypothesis is strengthened by the findings of Davis (1969), who reported an intact pituitary is obligatory for estrogens to stimulate growth. Trenkle (1970) reported higher plasma insulin levels in DES treated animals. Since injections of growth hormone in sheep were shown to increase plasma insulin levels (Wallace and Bassett, 1966), elevated insulin levels may be a secondary effect stimulated by the estrogenic in— duced increase in growth hormone. Preston (1975) reviewed the metabolic changes following estrogen treatment, which included higher nitrogen retention, lower plasma urea and amino acid concentrations, and changes in weight of the liver, kidney and anterior pituitary. Apparently, growth hormone plays an important role in regulation of animal growth. However, Trenkle and Topel (1978) reported they were unable to observe any consistent relationship between growth hormone levels and rates of gain. Eversole et a1. (1981) reported serum growth hormone levels were not affected by cattle type or diet energy density, but that serum insulin levels were positively related to average daily live weight gain, protein gain, and fat gain. Broome (1980) suggested the effects of growth hormone may be to partition more metabolizable energy into protein synthesis rather than fat. Rogerson and Ledger (1970) reported higher heat production in hexestrol implanted steers. Oltjen et a1. (1973) reported DES treated subjects had faster weight gains, but had greater weight losses during restricted feeding than untreated subjects. It is not clear how these different observations are integrated to form a plausible mode of action for estrogenic anabolic agents. However, present knowledge indicates estrogen treated animals tend to partition greater energy toward protein and less energy to fat than untreated animals. This effect is likely mediated by increased growth hormone concentration and (to a smaller extent) increased insulin concentration. Androgens Far less research has been conducted on the anabolic effect of exogenously administered androgens. The majority of work has been reported with combinations of androgens and estrogens, particularly trenbolone acetate and estrogens, which will be reviewed in detail later. Burriss et a1. (1953) demonstrated weekly injections of testosterone in heifers and steers reduced carcass fat. Burgess and Lamming (1960) reported testosterone treated steers grew more rapidly and deposited more muscle and less fat in the twelfth rib than untreated steers. Combinations of estrogens and androgens have been shown to stimulate gain and protein depostion in steers (Beeson et al., 1956). A combination of 20 mg estradiol benzoate and 200 mg testosterone propionate has been shown to increase growth in heifers (Utley et al., 1976; Wilson and Burdette, 1973). This combination is available commercially. Androgens are thought to directly affect regulation of protein synthesis after binding to intracellular receptors (King and Mainwaring, 1974). Michel and Baulieu (1976) cite evidence of skeletal muscle androgen receptors for males and females. However, it is undetermined if the anabolic action of androgens is solely dependent on formation of the androgen—receptor complex (Young, 1980). Young (1980) discussed evidence androgens displace or inhibit gluco— corticoid binding to cellular receptors. In view of this finding, androgens may enhance protein metabolism by inhibiting glucocorticoid-dependent muscle catabolism. Heitzman (1976) has suggested the maximum stimulation of growth occurs when estrogens and androgens are present simultaneously. Thus, the sex of the animal determines which anabolic agents are appropriate for maximum response. In general, most of the experimental results reported in the literature support this claim. Heifers generally respond greatest response to trenbolone acetate or testosterone propionate with or without additional estrogen (Heitzman, 1976). Estrogen and androgen combinations were consistently superior to estrogen treatment alone in steers (Roche and Davis, 1978; Heitzman et al., 1981). In bulls, estrogens generally stimulated growth, but little or no response was observed following treatment with androgens (Heitzman, 1976). Effect of Trenbolone Acetate on Growth Trenbolone acetate (3-oxo-178-hydroxy 4,9,11 estra trien acetate), an anabolic steriod, was first synthesized by Velluz et a1. (1967). Trenbolone acetate (TBA) was shown to increase livewight gain in heifers (Best, 1972; Heitzman and Chan, 1974; Galbraith, 1980) and female rats (Vernon and Buttery, 1976, l978a,b). similiar results were obtained in steers (Heitzman et al., 1977; Galbraith and Watson, 1978) and bulls (Galbraith, 1979) when TBA was combined with an estrogen. Increased live weight gain following implantation with TBA and estradiol was reported in veal calves, feedlot bulls, wethers and barrows (Grandadam, et al., 1975). Response to TBA alone in steers or bulls was negligible compared to control subjects (VanderWal et al., 1975; Roche et al., 1978). Stollard and Jones (1979) measured a 10% increase in live weight gain involving 610 heifers in commercial prac- tice treated with 300 mg TBA. In two trials with fewer sub— jects, daily gain of TBA treated heifers was improved 23% (Galbraith, 1980) and 71% (Heitzman and Chan, 1974). Gal— braith (1980) also reported a 23% improvement in feed per unit of gain. Stollard and Jones (1979) reported 300 mg TBA and 60 mg hexestrol implanted simultaneously improved gain 10 an average of 30% in 2500 steers in commercial production. Experimental subjects were housed on 29 different farms either fed forage or concentrate diets. Chan et al., (1975) demonstrated TBA treated heifers retained significantly more nitrogen than controls without changes in dietary nitrogen digestibility. Later work by Galbraith (1980) also demonstrated increased nitrogen reten— tion by TBA treated heifers. Griffiths (1982) reported in— creased nitrogen retention in steers implanted with 300 mg TBA and 36 mg zeranol. This effect was unaltered by either low of high dietary protein. Griffiths (1982) also reported treated steers deposited significantly more protein, more water and less fat. Implanted steers deposited a higher pro— portion of meat in the forequarter. Increased nitrogen re- tention is consistent with greater protein accretion, how— ever studies regarding composition of gain in TBA treated heifers have not been published. Coelho et a1. (1978) and Yasin and Galbraith (1981) reported an increase in carcass protein and a reduction in carcass fat in TBA and estradiol treated wether lambs. 11 Effect of Trenbolone Acetate on Blood Metabolites Hormonal changes often accompany treatment of experi- mental subjects with anabolic agents. The disparity in res- ponse to TBA treatment in male and female subjects suggests the anabolic effect of TBA is dependent on the host endo— crine enviroment. Several investigators have measured changes in blood metabolites in TBA treated heifers. Heitzman and Chan (1974) reported reduced plasma urea and albumin concentrations and unchanged plasma concentrations of glucose, free fatty acids, insulin, magnesium, calcium and total protein. Weekly samples were drawn for analysis. Galbraith (1980) also reported lower plasma urea and albumin levels in TBA treated heifers while growth hormone, insulin and prolactin concentrations were unchanged. similiar results were reported with steers. Heitzman et al. (1977) demonstrated lower total plasma thyroxine, urea and albumin levels in TBA and estradiol treated steers. Plasma growth hormone, prolactin and insulin were unchanged. Galbraith and Watson (1978) reported increased growth hormone concen- trations in hexestrol and TBA—hexestrol treated steers, but serum growth hormone was unchanged with TBA treatment alone. Serum insulin, total protein, glucose and free fatty acids 12 were indifferent from controls. Galbraith (1982) measured blood metabolite changes in bulls treated with TBA and hex— estrol. Bi—hourly samples assayed for growth hormone, insulin, cortisol and prolactin were not different among control and implanted bulls. Galbraith and Geraghty (1982) reported higher blood glucose in TBA and hexestrol implanted steers. No changes in growth hormone, insulin, or prolactin were observed. Galbraith (1979) observed decreased plasma testosterone and dihydrotestosterone in TBA and hexestrol treated bulls when compared to controls or pretreatment values. These changes in hormone profiles are charac- teristic of exogenously administered androgens (Mainwaring, 1977) but are in contrast to those observed with estrogen administration (Preston, 1975). The mechanism of action of TBA has not been fully elucidated, but a number of observations have been made. Neumann (1976) concluded TBA has a three times stronger androgenic effect in rats than testosterone propionate. He also reported TBA has three times the antigaonadotropic activity of testosterone propionate measured in terms of inhibition of ovulation and testicular growth in rats. Vernon and Buttery (1976) demonstrated TBA reduced heart and muscle fractional synthesis rate as well as reducing the fractional degradation rate of hind limb muscle. In an sub— sequent experiment, Vernon and Buttery (1978a) reported a 13 reduced rate of myofibrillar protein degradation in TBA treated female rats. Pottier et a1. (1975) reported TBA is deacetylated in blood to trenbolone (TBOH), the metabo— lically active component. Since TBOH is structurally similiar to testosterone, Vernon and Buttery compared their effects on growth rate and uterine regression. They found testosterone stimulated uterine regression and growth rate. TBA also stimulated growth rate but did not alter uterine development. Thus, they concluded the metabolic action of these steriods differs. In a third study, Vernon and Buttery (l978b) reported decreased RNA activity and frac— tional synthesis rate of skeletal muscle in TBA treated female rats. These findings seem inconsistent with increased growth and protein deposition often observed in TBA treated subjects. However, simultaneous depression of fractional synthetic and degradative rates are indicative of reduced protein turnover, which could account partially or wholly for improved nitrogen retention and feed conversion. Griffiths (1982) failed to demonstrate significant changes in myofibrillar protein degradation rates as measured by 3-methylhistidine excretion in TBA and zeranol treated steers. In sheep, Sinnett—Smith et a1. (1983) found TBA reduced muscle protein synthesis rate and free cathepsin D activity in growing ewes. Nearly identical results were found for zeranol treated ewes. In a similiar study with 14 TBA treated steers, Lobley et a1. (1981) measured protein synthesis by infusion of labelled leucine and reported no detectable effect of the steriods. In contrast to their earlier experiments, Vernon and Buttery (1981) failed to detect differences in protein turnover in TBA treated female rats, as judged by 3—methylhistidine to creatinine ratios. Several other metabolic changes were reported in TBA treated subjects. Thomas and Rodway (1982) noted a sup— pression of adrenal activity in TBA treated female rats and ewe lambs. This observation lends support to the hypothesis TBA inhibits glucocorticoid-dependent muscle catabolism. In sheep, Scaife et al. (1982) has suggested TBA and estradiol reduce plasma triglyceride levels. TBA treatment also de- pressed, while estradiol elevated, plasma cholesterol concen- trations. Richardson et a1. (1982) studied the effect of TBA and estradiol on hepatic metabolism in perfused livers of suckling and ruminating lambs. He concluded TBA and estradiol maintain hepatic gluconeogensis from alanine at rates similiar to those found in suckling lambs. TBA in- duced increases in hepatic gluconeogensis may partially ex— plain the suppression of adrenal function observed by Thomas and Roadway (1982), since glucocorticoids tend to increase with low blood glucose concentrations. Donaldson and Heitzman (1983) reported reduced urea synthesis in TBA treated heifers. 15 The necessity of combining an estrogen with TBA to illicit a response in male cattle is not suprising in view of Heitzman's hypothesis that both androgens and estrogens must by present to obtain maximum growth. Riis and Suresh (1976) studied the effect of TBA on the rate of release and excretion of subcutaneously administered estradiol. They reported 95% of the estradiol was recovered within 20 days after estradiol implantation alone. However, 95% of the estradiol was recovered in 107 days when implanted simul— taneously with TBA. Since the relation between circulating activity and the rate of fecal and urinary activity of estradiol was similiar for both groups, Riis and Suresh (1976) concluded TBA remarkably retards estradiol release from subcutaneous implants with no apparent effect on estra— diol excretion. They speculate administration of subcu— taneous implants with estradiol and TBA results in rela- tively steady plasma estradiol concentrations near the physiological range necessary to illicit anabolic action. These findings imply one anabolic action, or perhaps only anabolic action, of TBA is to affect estradiol metabolism. Hendricks et al. (1982) reported TBA implantation in heifers increased plasma estradiol concentration and that trenbolone and estradiol varied directly. The stimulation of growth in heifers administered exogenous estrogens may imply estrogen is limiting maximum growth potential even in females. One 16 could postulate TBA stimulates estradiol synthesis or secretion in females to a more prolonged mean plasma concen- tration necessary to achieve maximum growth. The significance of many of these research findings regarding TBA is unclear. Obviously TBA stimulates growth in females or males if combined with an estrogen. Many of the observed metabolic changes in response to TBA are incon- sistent and may be secondary rather than primary effects. Further investigation of TBA will allow a broader compre- hension of its mechanism of action and hopefully lead to a better understanding of other anabolic agents. Tissue Residue Levels of Trenbolone Potential public health hazards from use of anabolic steriods in beef production necessitates an evaluation of trenbolone (TBOH) tissue residue levels. Hendricks et a1. (1982) reported substantial TBOH accumulation in adipose and liver tissue during implantation, but TBOH cleared rapidly during a 15 day withdrawl. Heitzman and Harwood (1977) found concentrations in liver, muscle, kidney and fat were less than 0.5 parts per billion at 63 days after implantation. l7 METHODOLOGY TO ASSESS BODY COMPOSITION Maynard and Loosli (1969) defined true growth as an increase in muscles, bones and organs which should be dis— tinguished from any increase resulting from fat deposition in adipose tissue. Live weight gain over time is an easily measured and widely used tool to assess animal growth. Selection criteria of animal breeding rely heavily on live weight gain to compare growth among animals. However, accretion rates of protein and fat can vary dramatically among animals with a similiar rate of live weight gain. Enhanced beef production efficiency is possible by maxi— mizing energy retention in protein while minimizing fat depostion to a level that will ensure quality. In order to manipulate or select for energetic efficiency and partioning among protein and lipid depots, measurement of body comp- osition is essential. Several methods are available (Haecker, 1920; Hankins and Howe, 1946; Garrett et al., 1959; Byers, 1979a; Meissner, l980a,b,c; Johnson and Charles, 1981). However, each method has shortcomings in regard to expense, labor, precision or applicability to commercial use. Furthermore, many of these methods require slaughter of the animal, thus preventing further measures of growth. A rapid, inexpensive, accurate, and in vivo l8 technique to measure body composition would greatly enhance research and animal breeding objectives. Serial Slaughter Techniques Terminal body compostion measured by chemical analysis, or partial chemical analysis, of carcass components elimi— nates further measures of growth. Thus, many researchers have employed serial slaughter techniques to evaluate changes in body composition over time or in response to a treatment regime. Successive body composition measures of representative animals at selected time intervals are assumed to represent changes in body composition for all experimental subjects. Large numbers of genotypically and phenotypically similiar subjects randomly allocated to each treatment should improve validity of composition estimates. However, Byers (1979a) questioned the underlying assumptions of serial slaughter techniques. He points out the absolute accuracy of the methodology may less important than varia- tions among animals in body composition during growth. 19 Whole Animal Chemical Analysis Gross chemical analysis of the whole body provides an accurate and precise measure of body composition. Present knowledge of water, protein, fat and mineral relationships as an animal grows is largely derived from experiments that measured whole animal chemical analysis. This procedure is often the method of choice in smaller animals. Whole chemi— cal analysis is limited with large animals because of car- cass economic value. Composition of specific tissues or parts have been determined by several workers by physical separation and chemical analysis (Haecker, 1920; Moulton et al., 1922; Jesse et al., 1976). This procedure provides sufficient accuracy in measuring body composition, but is limited due to labor and expense. Nevertheless, chemical analysis pro— vides a measure of reference to derive and evaluate more rapid and inexpensive methods to ascertain body composition in large animals. 2O 9-10—11 Rib Section Separation Lush (1926) credited Trowbridge and Moulton for first suggesting the wholesale rib out rather adequately repre- sented the carcass. Hopper (1944) presented numerous rela- tionships and estimating equations between whole rib and ninth—tenth—eleventh rib chemical composition and empty body, carcass, and edible portion of the carcass. From the 92 cattle sampled, Hopper found the rib cut highly cor- related with several empty body and carcass paramaters. Hankins and Howe (1946), in a cooperative study with many agricultural experiment stations, investigated the use of the whole rib and the ninth-tenth-eleventh rib cut to reliably predict body composition. They found separable fat and lean of the ninth—tenth—eleventh rib highly correlated with carcass fat and lean. They provided detailed rib separation guidelines to improve accuracy of carcass comp- osition. Rib section analysis has provided reasonable results of composition in scores of investigations and is often the method of choice in serial slaughter experiments when the expense of chemical analysis is prohibitive. 21 Specific Gravity Since there is ample evidence the fat—free body is fairly constant in composition in mature animals (Murray, 1922; Messinger and Steele, 1949; Huff and Feller, 1956), determination of fat content allows calculation of water, protein and ash. Fatness or leaness can be determined from density measurement by conceptually separating the carcass or subject into lean tissue with a density of 1.10 and fat tissue with a density of 0.90. Thus, the proportion of lean and fat can be estimated from density of the whole body. Use of density measurement to predict composition was first reported in humans (Boyd, 1933; Behnke et al., 1942). A number of investigators have reported relationships between carcass density and carcass fatness in bovines (Kraybill et al., 1952; Garrett and Hinman, 1969; Preston et al., 1974; Ferrell et al., 1976). However, Jones et a1. (1978) pointed out procedures for estimating actual carcass fatness by these investigators have varied widely, and in some cases are unclear. Theoretical considerations and usefullness of body density to predict composition was reviewed by Pearson et a1. (1968) and more recently by Jones et a1. (1978). In practice, bovine carcass specific gravity has been deter- mined by hydrostatic weighing. Thus, specific gravity is 22 calculated as weight in air divided by the difference be- tween weight in air and weight in water. Adjustments to calculations are necessary if carcass or water temperature differ or water density changes upon successive carcass measures. Garrett (1968) offered several guidelines for bovine carcass density determination. Pearson et a1. (1968) reviewed evidence of differences in density of fat and lean among and within species. He suggests it may by difficult to use equations derived from one population to estimate body fatness of another popu- lation. However, single equation values may be meaningful for comparative purposes. The reduction in water content of the fat free mass in young animals (Moulton et al., 1922; Gnaeding et al., 1963) may limit the validity of carcass density predictions of fatness in very young growing animals. Garrett (1968) has suggested individual estimates of fatness from density have unacceptable standard errors; thus, density measures are more appropiate in large rep— licated experiments. Despite obvious shortcomings, measures of composition from carcass density can provide treatment comparisons in ap- propiately designed experiments, particularly when obtained in light of theoretical considerations. 23 Use of Carcass Cuts to Predict Body Composition Several investigators have studied the use of retail cuts to assess beef carcass composition (Allen, et al., 1969; Johnson and Charles, 1981). Prediction of composition from retail cuts, if sufficiently precise, is easily adapted to widespread commercial use and would facilitate carcass trade, experimental research and animal breeding. Allen et a1. (1969) investigated chemical composition of one side of the carcass to retail cuts in the other side in eighty steers. They reported retail cuts and fat trim accounted for less than 71% of muscle and fat variation. They con- cluded some selected parts of separable components predict composition more precisely than retail cuts. Johnson and Charles (1981) investigated prediction of carcass compo- sition by determining the percentage of muscle and fat of specific cuts. They offer an array of regression equations with standard errors to predict carcass composition from specific primal cuts. Johnson and Charles provide a table of man-hours required for each partial dissection to allow researchers to choose among predictive accuracy, labor requirements, and carcass loss when conducting experiments. More recently, Marchello et a1. (1983) evaluated the use of the chuck, rib, plate, shank and brisket, loin, and flank 2n chemical composition to predict whole carcass composition. Composition of wholesale cuts accurately predicted carcass moisture, fat and protein with four exceptions when compared 00 actual values. Marchello et al. (1983) suggests easy accessibility and low economic value of the flank and plate may enhance their use in estimating carcass composition. IN VIVO DETERMINATION OF BODY COMPOSITION Compared to serial slaughter techniques, in vivo measures of body composition allow repeated measurements over time on the same subject, composition estimates of indispensable animals, and fewer subjects per treatment in research investigations. However, in vivo techniques can be generally characterized as either expensive or imprecise. A number of methods have been developed including potassium 40 counting (Zobrisky et al., 1959; Lohman et al., 1966), ultra- sonic probes (Kempster and Arnall- 1982), isotopic dilution (Hevesy and Hofer, 1934; Searle, 1970a,b; Crabtree et al., 1974; Byers, 1979a), antipyrine dilution (Soberman, 1949) and adipose—cell size (Robelin, 1982a). No single method has overcome all the problems of expense, labor, precision and analytical difficulty. Although some procedures have potential for practical application (Meissner, 1980a,b,c; Robelin, 1982a), further documentation is necessary. Dilution Techniques Estimation of body composition from dilution techniques is based on the premise the fat free body is constant in regard to water, protein and ash. Moulton (1923) defined 25 26 the point at which water, protein and ash comprised a con— stant proportion of the fat free mass as chemical maturity. He estimated most mammals reached chemical maturity after approximately four percent of their lifespan had elapsed. Thus, in vivo measures of water or fat allow determination of composition. In short, dilution techniques involve addition of a known quantity (or count) of a tracer into the test subject and sampling after tracer equillibration with metabolic pools. The volume is calculated as the amount of tracer injected divided by tracer concentration in equillibrated samples. The tracer should be rapidly and uniformly dis— tributed with body pools, be non—toxic and slowly excreted. Isotope Dilution Deuterium oxide and tritiated water are commonly used to study body water volume and turnover as well as body composition. Deuterium oxide is a naturally occuring stable isotype of hydrogen with an atomic mass of three. Thus, deuterium oxide weighs approximately 11% more than water. Tritium is a radioactive isotope of hydrogen with atomic mass of three. Both tritium and deuterium oxide are assumed to equillibrate uniformly with body water. One might 27 suspect deuterium oxide will overestimate body water volume due to partial exchange of deuterium with hydrogen of water. Conversely, tritium may underestimate body water volume due to tritium incorporation into molecules other than water. Indeed, Trigg et a1. (1978) found deuterium oxide over— estimated total body water by 2.4% and tritium underesti- mated total body water by 4.6% in an experiment with 13 preg— nant ewes. Each of the isotopes has a number of advantages and disadvantages. Tritiated water is relatively inex- pensive and is accurately measured at low concentrations. Animals treated with radioisotopes cannot be sold and present a large disposal expense. Deuterium oxide avoids these difficulties, but is expensive and less accurately measured at low concentrations. A number of prediction equations have been developed to assess body compostion in farm animals from tritiated water space. In ruminants, the majority of studies were confined to sheep (Panaretto, 1968; Reardon, 1969; Searle, 1970a,b; Smith and Sykes, 1974). Meissner et a1. (1980a,b,c), Chigaru and Topps (1981) Carnegie and Tulloh (1968) inves— tigated tritiated water space in cattle. However, only Meissner et a1. (l980a,b,c) physically separated and chemi— cally analyzed empty body components to compare body comp- osition to tritiated water space. They found water, pro— tein, and inorganic matter can be predicted from tritiated 28 water and urea dilution with a mean absolute error of 4—10% at 400 kg mass. The mean absolute error for fat estimation at 400 kg live weight was 25 to 30%. Meissner and col— leagues concluded more than one measurement of water space may be required to quantify body composition differences among animals. Furthermore, they suggested prediction of fat, muscle, and bone in vivo by water space is less accurate than prediction of these tissues by prime rib cut in the slaughtered animal. Searle (1970b) used 33 wethers to evaluate tritiated water space as a predictor of body composition in regression equations previously published (Searle, 1970a). He found the variance for each body component was less than the residual variance of the original regression analysis from which the prediction equations were obtained. Since pre- dicted values closely agreed with actual values, Searle (1970b) concluded tritium dilution may be a useful tool in estimating body composition. Trigg et a1. (1978) reported tritiated water dilution provided more precise estimates of body composition in sheep than either potassium 42 or deuterium oxide dilution techniques. Researchers have developed prediction equations rela— ting deuteruium oxide dilution to body compostion in sheep (Trigg et al., 1978) and cattle (Crabtree et al., 1974; Byers, 1979a; Robelin, 1982a,b,c). Crabtree et al. (1974) 29 reported fat-free mass and empty body water highly corre- lated with deuterium oxide space (DOS) in an experiment with six Fresian steers and six Fresian x Hereford steers. Other empty body components were less correlated to DOS. Robelin (1982b) discussed his experiences using deuterium oxide to estimate body composition. He proposes heavy water dilu- tuion gives a good estimate of composition and discusses means to control sources of error, particularly variations in gastrointestinal fill. Byers (1979a) has considered empty body water and alimentary water as two separate pools. Thus, he developed predictive equations to assess body composition from deuterium oxide dilution based on two pool tracer kinetic techniques (Shipley and Clark, 1972). In theory, elimi- nating gastrointestinal water from total body water should improve estimates of composition from dilution techniques. Smith and Sykes (1974) reported 99 percent of the variation in fat content was accounted for by the variation in empty body weight and water content. Only 88% of the variation in fat content was attributable to body weight and water con- tent when gastrointestinal water was included. Robelin (1982b) reported total body water can vary by 3 to 5 percent daily. Much of this variation is likely due to fluctuations in gastrointestinal water, which may affect measures of live weight and empty body water. Byers (1979a) reported empty 30 body components measured by two pool deuterium oxide methodology highly correlated to empty body components determined by chemical analysis. However, high correlations may be inevitable over the range in weight and age of experi— mental subjects used by Byers (l979a). Although derivation of predictive equations from diverse populations allows broader application, it does so with less precision. Potassium has been suggested as a measure of body composition due to its association with the fat free body mass. Trigg et a1. (1978) compared potassium 42, deuterium oxide and tritiated water dilution in sheep. They found 89.4% of body potassium was exchangeable and that a positive relationship existed between exchangeable potassium and total body potassium. Inclusion of potassium 42 space with live weight in multiple regressions improved composition estimates, but were less accurate than tritiated water alone. Remenchik et a1. (1968) reviewed the use of potas- sium to assess body composition. He presents evidence po— tassium tissue content among tissues differs as much as 30%. Urea Dilution Preston and Koch (1973) and Koch and Preston (1979) found a single measure of urea space highly correlated with carcass specific gravity. Based on these results, several 31 authors suggested urea dilution as a rapid and economical tool to estimate body composition. Jones et a1. (1982) compared urea dilution and ultrasonic backfat thickness to chemical analysis of physically separated carcasses with 38 lambs, 25 Holstein cows and 30 steers. They found urea space and fat thickness less than 30% correlated with car- cass lean mass in lambs. Urea space was 55% and 14% cor— related with lean mass in cows and steers, respectively. Jones and colleagues propose urea space and ultrasonic fat thickness measurements appear limited for precise evaluation of body composition. Meissner et a1. (l980a,b,c) reported urea space alone poorly predicted body components as comp— ared to chemical analysis of half carcasses. Bennett et al. (1982) found urea space was no better estimating body compo- sition than ultrasonic measures of backfat thickness over an wide range of breeds. These studies indicate urea dilution has limited if any usefullness in estimating body composition in ruminants; however, precision of body composition estimates may be improved by measuring urea space in conjunction with other methodologies. Potassium 40 Counting Potassium 40 is a naturally occurring radioactive isotope in relatively constant proportion to potassium. Potassium 40 has a long half life of 3 billion years and is measured in live animals by whole body counters. Since body potassium is primarily associated with the skeleton, muscle and gastrointestinal tract, several investigators have suggested body potassium, as measured by potassium 40 counting in live animals, may predict body composition (Zobrisky et al., 1959; Clark et al., 1976; Belyea et al., 1978; Rider et al., 1981). Technical and biological errors associated with counting potassium 40 have been described (Bennink et al., 1968; Lohman et al., 1970). The fallacies associating body potassium to lean body mass have been previously discussed. Furthermore, potassium counting is hampered by equipment calibration, background interference, inaccuracy and poor repeatability. These problems have been discussed in detail by several authors (Lohman et al., 1968; Martin et al.,l968). Despite many of these shortcomings, potassium 40 counting has shown reasonable accuracy estima— ting lean body mass in some experiments (Clark et al., 1976; Belyea et al., 1978; Rider et al., 1981). The necessity of 32 33 a whole body counter restricts use of potassium 40 counting methodology to assess body composition. Estimation of Body Composition by Adipose-Cell Size Robelin (1982a,b) reported the weight of body lipids is fairly well correlated with mean adipose size, since 70% of the increase in body fat following birth is due to hyper- tropyhy of fat cells. Robelin (1982a) evaluated the use of mean adipose—cell size to estimate body fat and found the residual standard deviations of the technique similiar to those for deuterium oxide methodology. Further work is needed to validate this procedure with different breeds of cattle, with variations in fat content, and under different enviromental conditions. These results are promising since adipose—cell size is easily and economically determined in vivo and is independent of gastrointestinal fill, a large source of error in tracer dilution methodology. Combining Methodology to Estimate Body Composition Simultaneous use of two or more in vivo techniques to estimate body composition has been shown to increase 34 accuracy of composition estimates. Trigg et a1. (1978) compared deuterium oxide, tritiated water and potassium 42 dilution alone and in combination to estimate body components. Meisssner et a1. (l980a,b,c) estimated composition from urea and tritiated water space. Several authors have reported simultaneous use of ultrasonic fat measures and urea dilution (Jones et al., 1982; Bennett et al., 1982). In general, these investigators found including two estimates of composition in predictive equations improved accuracy; however, research in combining techniques is rather limited. Coupling of two methods that are complimentary should increase accuracy of composition estimates. For example, deuterium oxide predicts body water, ash, and protein more accurately than fat, since fat is determined by difference. Conversely, adipose—cell size may predict fat more accurately than other body components. Further work is warranted in combining several methodologies to more accurately measure in vivo body composition. USE OF 3-METHYLHISTIDINE AS AN INDEX OF IN VIVO MUSCLE PROTEIN DEGRADATION RATES Extensive recycling of amino acids creates difficulties in measuring rates of protein synthesis and degradation in live animals. Asatoor and Armstrong (1967) and Young et a1. (1973) suggested use of 3-methylhistidine (3—MeHis) as an index of muscle protein degradation. Young and Munro (1978) suggested urinary excretion of 3—MeHis appears to be a reliable measure of myofibrillar protein turnover in rats and humans. Harris and Milne (1980, 1981a,b) reported 3eMeHis as a reliable index for protein degradation in cattle, but not in swine or sheep. The quantitative con- tribution of non—skeletal muscle tissue to urinary 3-MeHis has been questioned (Millward et al., 1980). Wohlt et a1. (1982) recently reviewed the use of 3—MeHis as an index of myofibrillar protein turnover in ruminants. Young et al. (1972b) reported t-RNA was not charged with 3-MEHIS when administered in rats. He concluded 3-MeHis is not reutilized for protein synthesis. Cowgill and Freeberg (1957) demonstrated the methyl group of 3—MeHis is not oxidized or transferred to adjacent nitrogens or methyl group receptors. From these investigations, it appears 3-MeHis is stable once synthesized and subsequently 35 36 not reutilized for protein synthesis once degraded from protein. Quantitative excretion of labelled 3-MeHis in urine has been reported in rats (Young et al., 1972), man (Long et al., 1975) and cattle (Harris and Milne, 1981b; McCarthy, 1981). Harris and Milne (1981b) reported essentially quan- titative recovery of 3-MeHis in five to seven days. McCarthy (1981) recovered 89.7% of an orginal dose of 3-MeHis in 120 hours. These investigations indicate 3-MeHis is apparently excreted rapidly after degradation from myo— fibrillar protein. The major portion of 3-MeHis must be confined to skeletal muscle if 3-MeHis is a valid index of myofibrillar protein degradation. In skeletal muscle, 3—MeHis is a normal constituent of actin and myosin (Johnson et al., 1967). Haverberg (1975) reported skeletal muscle contained 97.8% of whole body 3—MeHis in rats with exception of skin and intestine. Nishazawa et al. (1977b) reported the skin and intestines comprise 10% of the total body 3-MeHis pool in rats. In cattle, Nishizawa et a1. (1979) found greater than 93.4% of total 3-MeHis in skeletal muscle protein. The contribution of non—skeletal muscle tissues to urinary excretion of 3-MeHis has been questioned. In theory, skin and.intestinal tissue would comprise a larger portion of urinary 3—MeHis concentration compared to their 36 not reutilized for protein synthesis once degraded from protein. Quantitative excretion of labelled 3—MeHis in urine has been reported in rats (Young et al., 1972), man (Long et al., 1975) and cattle (Harris and Milne, 1981b; McCarthy, 1981). Harris and Milne (1981b) reported essentially quan- titative recovery of 3-MeHis in five to seven days. McCarthy (1981) recovered 89.7% of an orginal dose of 3—MeHis in 120 hours. These investigations indicate 3-MeHis is apparently excreted rapidly after degradation from myo- fibrillar protein. The major portion of 3-MeHis must be confined to skeletal muscle if 3—MeHis is a valid index of myofibrillar protein degradation. In skeletal muscle, 3-MeHiS is a normal constituent of actin and myosin (Johnson et al., 1967). Haverberg (1975) reported skeletal muscle contained 97.8% of whole body 3-MeHis in rats with exception of skin and intestine. Nishazawa et al. (1977b) reported the skin and intestines comprise 10% of the total body 3-MeHis pool in rats. In cattle, Nishizawa et al. (1979) found greater than 93.4% of total 3-MeHis in skeletal muscle protein. The contribution of non-skeletal muscle tissues to urinary excretion of 3-MeHis has been questioned. In theory, skin and.intestinal tissue would comprise a larger portion of urinary 3-MeHis concentration compared to their 37 percent contribution to whole body 3-MeHis content due to a higher turnover rate than skeletal muscle. Millward et a1. (1980) reported skeletal muscle in rats may contribute only 25% of urinary 3—MeHis excretion. However, Harris (1981) points out technical and conceptual errors made by Millward and colleagues. In further experiments, Bates and Millward (1981) reported skeletal muscle accounted for less than 41% of 3-MeHis excretion in rats. Nishizawa et al. (1977a) concluded intestine and skin account for 10% of total body 3-MeHis and approximately 17% of 3-MeHis urinary excretion in rats. The most interesting investigation regarding 3—MeHis was recently conducted in a single paralyzed patient with neither macroscopic nor microscopic detectable skeletal muscle (Afting et al., 1981). Since 3—MeHis excretion by this patient was 28% of normal control subjects, the inves— tigators concluded skeletal muscle appears to contribute approximately 75% of urinary 3—MeHis excretion. Young and Munro (1978) suggested the contribution of skin and intes— tine to 3—MeHis excretion will decline with increasing size of the animal due to the surface area law. This principle may have less importance in ruminants, which have larger gastrointestinal tracts in proportion to body size than non—ruminants. The validity of 3-MeHis as an index of muscle degra- dation in rats, humans, and cattle has not been fully 38 resolved. Despite remaining questions, Young and Munro (1978) and McCarthy (1981) have found muscle degradation rates calculated from 3—MeHis excretion and concentration in muscle to agree well with values determined by other techniques. PROTEIN TURNOVER IN ANIMALS Protein turnover is the continual synthesis and degra— dation of body proteins. Although few measures of protein turnover are published in large animals, this process appears quantitatively large in relation to the total pro— tein pool (Millward et al., 1975; Lobely et al., 1980; McCarthy, 1981; Mulvaney, 1981; Lanka and Broderick, 1981). Protein turnover may exceed the daily requirement for pro— tein by five fold (Millward et al., 1975), thus 80% of amino acids are reutilized (Bergen, 1979). Van Es (1980) reviewed the energy costs of protein deposition and concluded the efficiency of metabolizable enery use for synthesis of tissue proteins to range from 40 to 60 percent. Webster (1981) has suggested, on a between species basis, protein synthesis may account for 20—25% of the total heat produc— tion in an animal at rest. Clearly, protein turnover is an energetically expensive process. In a recent review, Swick (1982) suggests protein turn— over insures redistribution of amino acids into proteins essential to life at any particular moment. In undomesti- cated non—ruminants, protein turnover provides a ready pool of amino acids which buffers the variability in dietary protein quantity and quality. Undoubtably, this mechanism 39 40 assumes reduced importance in ruminants due to rumen fermen— tation and the semi-continuous supply of high quality bac- terial protein. Swick (1982) discussed the importance of protein turnover in repair of damaged proteins, and the restructuring and remodeling of tissues and organs as an animal grows. When synthesis exceeds degradation, growth occurs. In mature animals, synthesis and degradation are equal, thus protein turnover continues but there is no net gain or loss in total body protein. In growing animals, accretion of body protein is commonly less than 30% of the protein syn— thesis rate (Mulvaney, 1981; McCarthy, 1981). The frac— tional rate of protein synthesis and degradation declines as the animal matures (Waterlow et al., 1978). Thus, pro— tein synthesis and degradation tend to move in synchrony. A notable exception is during prolonged fasting (Millward et a1. 1976). The low rate of protein accretion relative to protein synthesis is unfortunate in regard to the efficiency of meat production. Reducing protein turnover without detri— ment to the animal would spare energy and enhance meat prod— uction efficiency. Protein breakdown and synthesis in skeletal muscle are likely subject to precise regulation since muscle serves as the primary store of amino acids. A number of factors are known to affect protein synthesis and degradation including A1 hormones, nutrition ,age, sex, species, trauma, excercise and perhaps numerous unknown factors. Millward et al. (1976) suggested regulation of muscle mass is primarily controlled by changes in protein synthesis. Changes in catabolism appear to occur only in response to exceptional situations. He based this conclusion on the suppression of protein synthesis in rats after receiving a range of inadequate diets and in diabetic, hypophysectomized, and glucocorticoid—treated rats. Protein degradation was largely unchanged except in fasted, diabetic, and protein deficient treatments. Other authors have shown in vitro intracellular protein degradation is under physiological regulation and varies with physiological demand (Bradley,l977). Several investigators have questioned whether protein degradation is a selective or random process. Dreyfuss et a1. (1960) suggested muscle proteins have a finite lifespan. More recently, Schreurs et a1. (1981) demonstrated mutual differences in turnover rates of actin and myosin. Several investigators have suggested protein degradation is a random process (Goldberg, 1969; Millward et al., 1976). Several authors have reviewed in detail the role of hor— mones in regulation of protein synthesis and degradation, particularly in reference to skeletal muscle (Goldberg et al., 1980; Young, 1980). The importance and complexity of the endocrine system in regulation of protein metabolism 42 needs little emphasis. Unfortunately, these processes are incompletely understood. Insulin, growth hormone, thyroid hormones, adrenal steriods, and sex steriods are known to have important roles in muscle turnover. Insulin is characterized as anabolic due to its stimlation of protein synthesis and inhibition of protein degradation. Uptake and release of amino acids from muscle parallel the rise and fall of insulin concen- trations (Felig, 1975). However, Goldstein and Reddy (1967) were unable to demonstrate insulin stimulated protein syn— thesis after insulin—induced amino acid uptake. Several in— vestigators have reported reduced rates of protein syn— thesis in diabetic subjects (Hay and Waterlow, 1967; Sender and Garlick, 1973). Jefferson et a1. (1974) and Rothig et a1. (1978) provided evidence insulin may inhibit protein deg— radation by stabilizing lysosomal membranes. Young (1980), concluded growth hormone affects amino acid transport, RNA metabolism and ribosomal aspects of pro— tein synthesis. No evidence exists implementing growth hor- mone in regulation of protein catabolism. Bergen (1975) has suggested growth hormone may stimulate protein synthesis. Glucocorticoid effects on muscle are generally chara- cterized as catabolic. Glucocorticoids apparently have important action in facilitating mobilization of amino acids from muscle. A number of investigators have observed 143 reduced muscle protein synthesis after glucocorticoid treat- ment (Kostyo and Redmond, 1966; Goldberg, 1969; Tomas et al., 1979). In addition, Tomas et al. (1979) observed in- creased protein degradation as measured by 3-methylhis- tidine excretion. The role of thyroid hormones on protein metabolism is attracting increased interest. Thyroid hormones have been implemented in stimulating protein degradation by activating intracellular proteases. DeMartino and Goldberg (1978) reported treatment of hypothyroid subjects with thyroxine caused a two to three fold increase in activity of cathepsin B and D. They also found levels of these enzymes were approximately 50% in thyroidectomized subjects compared to controls. Apparently these effects are restricted to skeletal muscle and liver. More recently, Simon et a1. (1981) reported thyroid hormone therapy increased protein turnover in pigs. Millward and Waterlow (1978) recently reviewed the effect of nutrition on protein turnover in skeletal muscle. Rates of protein synthesis and degradation appear sensitive to frequency of feeding and quality of the diet. Garlick et a1. (1973) reported muscle protein synthesis was increased after feeding and fell to approximately half as the fasting period lengthened. The degradation rate was lowest follow- ing a meal and rose to its highest levels 12 to 16 hours H4 after feed removal. Millward et a1. (1976) found the frac— tional degradation rate did not increase appreciably in rats until the fourth day of fasting. Millward and Waterlow (1978) have proposed fractional synthesis is related to the ratio of protein:DNA, which describes the size of the DNA unit. As the size of the DNA unit increases, a fall in the fractional synthesis rate is expected. Likewise, RNA acti- vity is defined as the grams of protein synthesized per gram of RNA. These relationships have been compared during nu- tritional insult by a number of investigators. Millward et a1. (1975) found proteianNA three times higher in rats fed protein free diets compared to controls. Millward and Waterlow (1978) concluded RNA is rapidly lost during fasting and that muscle RNA concentrations respond to acute dietary changes. Apparently, protein synthesis is limited by ribo- somal activity when nutrition is unlimited. Since protein synthesis and degradation rates are large in proportion to protein accretion, protein turnover is rapid and energetically expensive. The goal of reducing muscle degradation, rather than increasing muscle synthesis, appears to have more promise to enhance meat production efficiency. Although some protein turnover is undoubtably essential for life, small reductions in the rate of protein turnover will result in large increases in energetic efficiency. MATER IALS AND METHODS Feedlot Performance and Carcass Characteristics Experimental Animals A feedlot trial was initiated on October 20, 1981 and terminated February 10, 1982 to investigate the effect of three doses of trenbolone acetate (TBA) on feedlot perform— ance and carcass characteristics of finshing heifers during a sixty-three day treatment period. One hundred and sixty predominately large—framed crossbred yearling heifers obtained from Kentucky and Tennessee were used in both trials. Experimental animals were largely crosses of Simmental, Angus or Charolais. Experimental Design One hundred and sixty heifers were blocked by initial weight into one of five blocks. From each block of 32, a group of 8 heifers was randomly allotted to each of four treatments: (1) control; (2) 140mg TBA; (3) 200mg TBA; (4) 300mg TBA. Experimental animals were fed in pens of 8 head on the north side of a covered, open sided, slotted-floor 46 barn. Treatment-block pens were randomized within the feed— ing facility to eliminate experimental bias with regard to pen location. The heifers were not implanted during the initial 35 days of the trial to insure blocking was successful in re~ ducing experimental error and that no inherent differences in performance existed between designated treatment groups. The experimental animals were subcutaneously implanted in the middle third of the ear on Day 35. The implants were surgically removed on Day 98. Following a 2 week withdrawl period, heifers were slaughtered and carcass data obtained. Management Procedures All subjects were vaccinated within 24 hours for Infectious Bovine Rhinotracheitis, Parainfluenza-Type 3, Bovine Viral Diarrhea and Pasteurella. All cattle were eartagged, tattooed, and injected with 2 million inter— national units of Vitamin A. Two weeks following arrival, all heifers were wormed, treated for grubs and examined for pregnancy by rectal palpation. Pregnant heifers were aborted by an intramuscular injection of 35 mg prostaglandin F (PGF) if estimated as less than 100 day pregnant or 40 mg PGF and 24 mg of dexamethasone if estimated as greater than 100 days pregnant. Subjects were examined again in two 47 weeks. All heifers remaining pregnant after re-examination were deleted from the investigation. Experimental Diet Incoming heifers were started on a corn silage diet supplemented with soybean meal for the initial 10 days. The level of high moisture corn was slowly increased during the next four weeks to allow animals to adjust to the experi- mental diet. All heifers were fed a diet formulated to contain 20% corn silage, 69% high moisture corn and 11% protein-mineral supplement to provide on a dry basis, 13% crude protein, 2.07 Mcal/kg net energy for maintenance and 1.34 Mcal/kg net energy for gain. Composition of the diet is listed in Table 1. High moisture corn was stored in an oxygen limiting silo and fed whole. Corn silage was stored in a concrete stave silo. The experimental diet was mixed fresh daily in a horizontal mixer and delivered by belt feeder to indi- vidual pens. Experimental cattle were fed once daily ad libitum. Collection of Experimental Data Individual weights were obtained after cattle were without 48 Table 1. Ingredients and Composition of Experimental Dieta Item IFN % of dry matter Corn, aerial pt., w-ears, w—husks, ensiled mx.50% mn. 30% dry matter 3-08—154 20.00 Corn, dent, yellow grain, gr ZUS (68-75% dry matter) 4-02-931 68.80 Supplement Soybean seeds, meal 5-04-604 9.60 solv—extd Limestone, grnd 6—02—632 1.30 Dicalcium phosphate 6-01—080 0.05 Trace mineral saltb 0.25 Composition Crude protein 12.90 Acid detergent fiber 7.20 aExperimental diet contained 275 IU vitamin D per kg. 0 bTrace mineral salt contained a minimum of .356 Zn, .20% Fe, .03% Cu, .005% Co, .007% I and 96% salt. 49 feed and water overnight. One unshrunk weight was obtained prior to slaughter following the two week withdrawl period. Dry matter intake was determined for each pen of eight heifers. Stale or weather damaged feed was removed and weighed weekly or more frequently as conditions dictated. Feed intake was adjusted accordingly. Feed refusals were not noticeably different in composition and dry matter from the fresh ration. Feed samples were obtained twice weekly from a number of pens, mixed thoroughly and represen— tative subsamples obtained. Samples from each two week period were composited and frozen until analyzed. Equal numbers of cattle from each treatment were slaughtered on three consecutive days at Dinner Bell Meats in Archibold, Ohio. Experimental animal carcasses were individually identified and carcass data obtained following a 24 hour chill. Hot carcass weight, ribeye area, adjusted backfat thickness, kidney, pelvic and heart fat, maturity and degree of marbling were determined by MSU personnel. Quality grade and yield grade as determined by a USDA grader were also obtained. Statistical Analysis The results were analyzed as a completely randomized block design with heifer within dose x block as the error 50 term to test individual gains and carcass data. Dry matter intake and feed/gain were analyzed with dose x block as the appropriate error term since only pen intakes were avail- able. Dunnett's test was used to make treatment comparisons with control. Statistical analysis was obtained by Genstat statistical program on a Control Data Cyber 170 Model 750 computer. 51 TRIAL 2. STUDIES ON COMPOSITION OF GAIN AND SKELETAL MUSCLE DEGRADATION Experimental Animals Twenty-four large frame crossbred yearling heifers were selected for similiarity in frame, type, and condition to study the effect of three doses of trenbolone acetate (TBA) on body composition and skeletal muscle degradation. These heifers were contemporaries of experimental subjects used in Trial 1. All 24 heifers were halter—broken during a four week adjustment period to the experimental diet. The diet was previously described (Table 1). Experimental Design All experimental subjects were randomly allocated to one of four treatments: (1) control; (2) 140mg TBA; (3) 200mg TBA; (4) 300mg TBA. Heifers were not implanted during the initial 34 days of the trial. Experimental subjects were implanted subcutaneously at the middle third of the ear on Day 34. Implants were surgically removed on Day 99. Following a three-week withdrawl period, experimental sub— jects were slaughtered and carcass data obtained. The trial 52 was divided into three periods consisting of 34, 31 and 32 days. The second and third periods were treatment periods. Body composition was determined at the beginning of period 1 and the end of periods 1, 2 and 3. Urine was collected on 16 heifers at the end of each period to determine 3-methyl- histidine and creatinine excretion. Management Procedures All subjects were subjected to the incoming processing regime as previously described in Trial 1. Due to limita- tions in facilities, heifers were not individually fed; thus each treatment group was allotted equally among three adjacent pens. Animals were fed in concrete floored pens approximately 40% sheltered. Body Composition Determination Body composition was determined on 24 heifers by deu— terium oxide dilution using the methodology of Byers (1979a). Experimental animals were infused with deuterium oxide four times approximately 32 days apart to ascertain body composition during three experimental periods. Each subject was restrained and the jugular vein catheterized. A 50 cm piece of polyethlene tubing (PE200) was passed through 53 a 12 gauge needle into the jugular vein. The 12 gauge needle was removed and a 17 gauge tubing adaptor was attached to the polyethylene tubing and plugged. Deuterium oxide (99.8%) was used as a tracer of body water. Deuterium oxide was brought to physiological osmolarity by adding 9 grams of sodium chloride/1000 m1 of deuterium oxide. Pre- weighed syringes (60ml) of deuterium oxide were prepared to infuse each animal at the rate of 9-1lg of deuterium oxide per 45 kg of body weight. Ten heparinized bloodtubes (12ml) were prepared for each animal. After collection of an ini— tial blood sample to ascertain background levels, deuterium oxide was infused quickly and time recorded. The catheter was flushed with 40 ml of saline solution to insure total infusion of the tracer and then was flushed with 10-15 ml of 4.0% sodium citrate solution to prevent catheter blood clots. Prior to each blood sample, 15—20 ml of blood was drawn and discarded. A total of nine blood samples post- infusion were taken at approximately 20, 30, 40, 50, 80, and 240-360 minutes. Remaining samples were obtained once daily over the next three days. Actual bleeding times were re— corded. All animals were weighed daily as blood samples were collected and averaged for the experimental period. Heifers were allowed free access to feed and water prior to infusion. Deuterium oxide samples were analyzed with methods described by Byers (1979b). Deuterium oxide and water were isolated from heparinized blood samples by vacuum sublima— tion. Methanol was supercooled (-80 degrees C) by a liquid refrigeration unit equiped with immersion probe. In order to shell samples, supercooled methanol was circulated through aluminum tubing to an adjacent container. Blood samples were placed in volumetric flasks and connected to cold finger condensers partially submerged in the super— cooled methanol. Five mm Hg vacuum was applied by a vacuum pump through a 60mm x 1m manifold with 20 control valves. Condensers were connected to a vacuum manifold by poly- ethylene tubing (Tygon). Deuterium oxide and water were recovered in approximately 3 hours. Samples were immedi— ately thawed and collected from condensers and frozen in serum bottles until analyzed. Deuterium oxide concentration was determined by a Wilks Scientific Miran I Fixed Filter Infrared Analyzer with a Gilford Model 410 digital display unit. A 4.0 micron filter and .l879mm spacer calcium fluoride cell provided the appro- priate wavelength to measure deuterium oxide infrared absorb- ance with minimun interference by water. Since infrared ab— sorbance by deuterium oxide and water exhibits strong temp- erature dependence, a water bath circulated fluid at con- stant temperature (25 degrees C) through a jacketed cell 55 holder. Samples were analyzed with an absorbance range of 0 to 0.1, high gain and 10 second response integration. The cell was filled and flushed three times with aliquots of deuterium oxide—water samples and absorbance recorded after sample-cell temperature equillibration. Equillibration time was rapid (15-30 seconds) if sample and cell temperature were nearly identical. Large disparities in sample (room) temperature and cell temperature lengthened equillibration time considerably and were avoided. Analysis of samples with large differences in deuterium oxide concentration necessitated five or six cell flushes to avoid preceeding sample carryover. Deuterium oxide was measured to the nearest 2 ppm. Estimates of body composition were obtained by analysis of deuterium oxide dilution according to the methods out- lined by Byers (l979a) utilizing two—pool open kinetics described by Shipley and Clark (1972). Figure 1 illustrates the model for a two—pool open system. The tracer dose is placed in Pool A, which is closely related to empty body water. Similarly, addition of water into the system is assumed through Pool B, which is closely related to gastro- intestinal fill. Loss of deuterium oxide and water is assumed only through Pool A. The model is mathematically described on the following pages. Figure 1. Model for two pool open system. 57 k Overall equation for the system; SAt=SAO(l)e_ 1t + SA0(2)e Normalized equation as a fraction of a dose: qa/an=Hle-g1t + H e-th Where: = quantity of tracer in pool A at any one time qa qa0 = dose placed in pool A at time zero a Q = Pool A Qb = Pool B H1 = (intercept l) (Qa) H2 = (interecpt 2) (Oh) Rate Constants Kaa + Kbb = gl + g2 KaaKbb " Kabea = glgz K = k ab bb since pool A is the only outlet for pool B ao Kaa-Kba Kaa’ Kbb = overall turnover for pools A and B,respectively Flow Rates: Fab = Kabe Fba = KbaQa Fab = Faa = KaaQa Foa = Kab Qb Pool Sizes Qa = l/SAao Qb = Fab/Kab = Pool B Body composition estimates from pool sizes: Empty body water, kg = 1.038(Pool A)-17.918 Gastrointestinal fill(GIT), kg = 0.832(Pool B)-3.10 Empty body weight, kg = live weight-GIT weight Empty body minerals, kg = empty body water(.00689) Empty body protein, kg = empty body water(.3017) Empty body fat, kg = live wt.-(GIT wt.+ empty body water/.7296) The slopes and intercepts of the overall equation are derived by plotting the log of deuterium oxide concentration versus time. As described by Shipley and Clark (1972), "curve peeling" allows separation of an early and late equil— librating pool and mathematical describtion of the dilution curve by summation of two exponential functions. Late time samples, which correspond to deuterium oxide equillibration with total body water, are extrapolated back to time zero to derive intercept (2) and slope (2). Intercept (l) and slope (1) are derived by subtracting values along the extrapolated line from the log of deuterium oxide concentrations of early samples. These equations are demonstrated in Figure 2. From the overall equation of the dilution curve, the norma- lized equation as a fraction of the dose is determined by dividing each intercept by the tracer dose. The intercepts from the normalized equation are summed and inversed to LOG 020 Figure 2. 59 _ —kt -kt SAt - SAO(1)e 1 + SAO(2)e 2 s.zxc)(2)e'k2t TIME Kinetic Equations for Two—Pool Methodology 6O determine Pool A. Pool B is determined by solving for the given rate constants and flow rates. Example calculations have been given by Byers (1979a) and McCarthy (1981). Urine Collection Three urine collections were conducted at the end of each period on four heifers from each treatment. Heifers were placed in individual 92 x 244 cm stalls for a 2 day adaptation and 3 day urine collection. An indwelling Foley catheter (24 French with 75cc balloon) was placed through the urethra and into the bladder. Urine was collected in 10 liter containers by connecting polyethylene tubing (Tygon) to the Foley catheter. Approximately 300ml of 50% sulfuric acid was added daily to collection vessels to acidify urine samples to a pH of 2-3. Urine volumes were recorded and 10% aliquots were obtained twice daily and stored at 5 degrees centigrade. Aliquots were composited in proportion to daily excretion after each 3 day collection and frozen until analyzed. 61 3-Methylhistidine Analysis Eight m1 of urine was deproteinized with 0.8 ml 50% sulfosalicylic acid in ice for 20 minutes and then centri- fuged at 27,000 x g for 15 minutes. Urine samples were filtered through Metricel 0.2 micron membrane filter with a milli—pore filtering system. One ml of 1M pyridine was added to a 4 m1 urine sample and applied to a 1.5 x 7.5 cm column which contained Dower 50W—X8 200—400 mesh cation exchange resin. The column was desalinized with 30 ml of water and equillibrated with 40 ml of 0.2 M pyridine. The 3-MeHis fraction was eluted with 125 ml of 1M pyridine. Remaining amino acids were eluted with an additional 150 m1 1M pyridine. The 3—MeHis fraction was evaporated to dryness, dis— solved in 5 ml of 0.01 N HCl, and filtered through a Metricel 0.2 micron membrane filter. Samples were analyzed by ion exchange chromotography (Dionex) with lithium citrate buffers. L—a-amino-B—guanidinopropionic acid (Pierce Chemical) was used as the internal standard. 62 Creatinine Analysis Urinary creatinine was determined using Sigma Kit No. 555-A. Statistical Analysis Body composition estimates and urine metabolites were analyzed as a split—plot design by least square regression. The model used for analysis of all traits included fixed effects of treatment, time, animal within treatment, and the interaction of time and treatment. Animal within treatment mean square was used to test the effect of treatment and the residual mean square was used to test the effect of time and the time and treatment interaction. Dunnett's test was em— ployed to compare each treatment to control (Gill, 1978). Scheffe's procedure was used to test differences over time. Genstat (1980) statistical program was used to analyze experimental data at the MSU Computer Center. RESULTS AND DISCUSSION Trial 1. Feedlot Performance and Carcass Characteristics Although all heifers were examined for pregnancy at the beginning of the trial, three heifers were detected pregnant at slaughter and stage of gestation estimated at 150 days. Results are presented intact due to lack of appropriate ad— justment factors, the fact pregnant heifers gained simi- liarly to the average of all heifers, and that fetal growth is small during the first 150 days. One subject was removed from the trial due to illness. The effect of three doses of trenbolone acetate (TBA) on performance and carcass characteristics is presented in Table 2 and Table 3. Dry matter intake (DMI), average daily gain (ADG) and feed/gain (F/G) were nearly identical among all designated treatment groups dring the untreated initial 35 days. During the 63 day implant period, comparisons of each dose versus control for all performance and carcass data yielded no significant differences (P>.05); however, certain trends are evident. Average daily gain was Table 2. Effect of Trenbolone Acetate on Feedlot Performance of Finishing Heifersa Item Day 0—35 73-98 36-98 Day 0-35 36—72 73-98 36—98 I—IOI—‘I—J Day 0-35 36—72 73—98 36-98 \IGDOU'I 3 Days 7.83 36-72 8. 7 7 34 .08 .89 .44 .24 .86 .09 .43 .83 .42 .27 O to 35 were a non—treatment period. 7.82 8.39 7.12 7.87 Average daily gain, .41 .26 .72 .04 I—IOI-‘H Dry matter/gain .56 .67 .32 .58 \IOCNU'I 7.68 8.46 7.51 8.07 1.41 1.33 0.89 1.14 5.46 6.38 8.65 7.03 Trenbolone acetate implant, mg 0 I40 200 300 Dry matter intake, kg/d 7.85 8.48 7.08 8.03 kg/d .40 .34 .84 .15 I—JOI—‘b—J .60 .32 .57 .97 O‘CXDOU'I 0000 0000 0000 SE .37 .37 .62 .42 .07 .03 .07 .03 .16 .15 .69 .16 65 increased approximately 5% by 200 mg and 300 mg of TBA. This is less than the 10% increase reported by Stollard and Jones (1979) in commercial practice with 300 mg TBA on medium to low energy diets. The 140 mg TBA treatment group gained similiar to controls during the initial treatment period, but gained less weight during the entire implant period. Substantial reduction in gains during the final 25 days for all treatments may be partially a consequence of fattening, but is more likely due to subzero temperatures experienced during January. These results were pooled with two identical trials and reported by Brown (1982). ADG was improved an average of 6.9% for combined data of all three doses of TBA. In this experiment, DMI was slightly higher for 200 mg TBA and 300 mg TBA treatments compared to controls. This contrasts the depression of DMI by TBA reported in other trials (Brown, 1982). Feed efficiency was improved approximately 4.0% by 200 mg and 300 mg TBA. However, 140 mg TBA treated heifers required slightly more feed per unit of gain than controls. Brown (1982) reported TBA treatment improved feed efficiency an average of 7.0%. Carcass data is presented in Table 3. No significant differences (P>.05) were found comparing each dose to con- trol. However, TBA treated heifers tended to have larger ribeyes and less fat, thus lower yield grades. Carcass 66 .ANEO .moea ozonne x cm.o0 - flux .20: x swoo.0 + mazae x ON.O0 + man .mmocxonaa use .fie< x wa.o0 + m.N n ovate SASS» a .mms-ooe .nmoeoe msome? .Hsasm MamN-ooN .namAHm mass-oos .maomeu mam-o .eno>oe sHHaOnpumna ”canmflem> msoscflpcoo m we ponxflmcm was wopoom mmz wcfifiasme wo ooewom w mN.0 mN.N HH.N 0H.N H0.N nowmgm vfiofl> em.sfi Nom emm efim 0am moeoom mcflflnaaz ea.0 m.N N.N m.N o.N a .Omw OA>Hoa .nsao: .socenx 0m.H m.Nw N.mw m.m0 m.0n NEO .mogm oxonflm 00.0 00.0 00.0 s0.0 H0.H Eo .mmocxoficu paw .h0< No.0 N.m0 H.mo m.mo H.m0 pcooeom wcflmmogo e.m Nwm mwm Nwm owm we .namfloz mmmopmo so: mm 00m 00m 00H 0 EouH E .ucm EH ouwpoom ocofio cogs H . n whomeo: wcflsmflcflm wo moflumfieouomemgu mmmoewu co ounpoo< ocoHoQCOSH mo poowwm .m ombmb 67 weight, dressing percent and marbling score were similiar for all groups. The 10% increase in ADG reported by Stollard and Jones (1979) in commercial practice is greater than reported here or by Brown (1982). However, these results are consistent with increases in performance and feed efficiency in heifers treated with anabolic agents (Preston, 1975; Heitzman, 1976; Galbraith and Topps, 1981). In this experiment, improve- ments in ADG and F/G were nearly identical for 200 mg and 300 mg TBA treatments, suggesting 200 mg TBA may be the optimum dose. 68 Trial 2. Effect of Trenbolone Acetate on Body Composition and Muscle Protein Degradation Number of subjects by treatment and period for analysis of body composition is listed in Table 4. One heifer died from unknown causes during the initial 35 days. Six heifers were deleted from the periods shown in Table 4 either due to contraction of anaplasmosis or with urinary infections. Table 5 lists the number of animals by cell for urine collec— tions and subsequent data analysis of 3-methylhistidine, creatinine and muscle protein degradation. The effect of TBA on live weight and empty body weight is shown in Table 6. Figure 3 graphically depicts changes in live weight. Live body weight was similiar for all groups on Day 0 and Day 35 prior to TBA implantation. After the 63 day treatment period, heavier weights were evident for all 3 doses of TBA. However, treatment comparisons to control were not significantly different (P>.05). All live weights over time were significantly different (P<.05). Results for empty body weight were similiar to those for live weight. Empty body weight was determined as pro— posed by Byers (1979a) and described previously. Empty body Table 4. Number of Animals by Treatment for Analysis of Body Composition Trenbolone acetate, m Time 0 I40 200 300 hmOON bwrQI—l J>O’\O\O\ #mmu; 01000 70 Table 5. Number of Animals by Treatment for Analysis of Urine Metabolites _ Trenbolone acetate, mg Time 0 14 200 300 (AND—1 DIAL): tuba Lid-(>5 (Nb-b 71 Table 6. Effect of Trenbolone Acetate on Live Weights and Empty Body Weightsa Trenbolone acetate im lant, mg Item 0 140 200 300 Liveweight, kgbd Day 0 317 .317 325 320 35 365 360 371 364 67 405 403 426 411 99 429 444 462 452 Empty body weight, kng Day 0 280 266 284 268 35 340 327 340 329 7 376 369 401 378 99 400 411 432 421 a Least square means b Standard error ranged from 4.6 to 5.6 kg. C Standard error ranged from 6.2 to 7.5 kg. 9 All means within columns differ (P<.05). 72 Figure 3. Effect of Trenbolone Acetate on Live Body Weight. 73 .m unease DOA om ponmz w>H4 20 amp do pomumm [0M1 leJIBM A008 3AI'I weight is live weight minus gastrointestinal weight deter- mined by two-pool deuterium oxide methodology. TBA treated subjects tended to have greater empty body weights than con— trols at the end of the trial. The 200 mg and 300 mg TBA groups were an average of 32 kg and 21 kg heavier than con— trols. These differences were not significantly different (P>.05). It is interesting to note empty body weight became a larger percent of live weight as animals approached ma- turity. Table 7 shows measures of empty body water and empty body protein by treatment over time. Protein is assumed constant in proportion to body water, thus comparisons of means are identical for empty body water and empty body protein. Treatment differences were not significant (P>.05), however certain trends were evident. All treatmets gained significant amounts of protein and water from Day 0 to Day 35. Empty body water and empty body protein measures of control subjects were not significantly different during the last three determinations (P>.05). As illustrated in Figure 4, measures of empty body protein and water were virtually identical on Day 67 and Day 99 for control and 200 mg TBA treatments. All TBA treatments contained more empty body water and protein on Day 99 than controls. Further, all treatment groups contained significantly more empty body water and protein on Day 99 compared to pre-treatment values 75 Table 7. Effect of Trenbolone Acetate on Empty Body Water and Protein3 Trenbolone acetate implant, mg Item 0 140 200 300 Empty body water, k0 Day 0 155.1d 144.4d 156.8d 137.1d 35 179.76 175.58f 177.2d 167.4e_ 67 190.36 191.0: 203.96 188.6:f 99 190.16 200.3 203.96 203.1 Empty body protein, kgC Day d e d 0 46.8 43.6: 47.3 41.4 35 54.2e 52.7f 53.5: 50.53f 67 57.46 57.6 g 61.6f 57.0? 99 57.4e 60.4g 61.6 61.31 a Least square means. b Standard error of the mean ranged from 5.3 to 6.5 kg C Standard error of the means ranged from 1.6 to 2.0 kg d,e,f Means in the same column without a common super— script differ (P<.05). Figure 4. Effect of Trenbolone Acetate on Empty Body Protein 77 Ov-‘Nm .4 meanna w»¢0 so“ on o 999*1 szeomm >oom >pmzu 20 amp mo howmmm (0M) NIBlOMd A008 AldHB 78 (Day 35). On a percent basis, TBA subjects contained 6—7% more empty body protein at the end of the trial. Each dose of TBA seemed to have similiar effects. Rates of empty body protein accretion slow over time for all treatments, as pre- viously reported in steers by other investigators (Byers and Rompala, 1979; McCarthy, 1981). A note on the precision of these measures of body comp— osition is in order. Byers (1979a) indicated two-pool deu- terium oxide methodology values of empty body water to have a standard deviation of 10.89 kg and an average coefficient of variation of 5.1%. In this experiment, the standard error of empty body water was similiar. If we assume TBA actually increases protein accretion 6-7%, a large number of subjects would be necessary to detect this difference with good confidence. Estimates of empty body fat (EBF) are presented by treatment and time in Table 8 and Figure 5. No significant differences among treatments in EBF were evident (P>.05), although 200 mg TBA and 300mg TBA treatments tended to con— tain slightly more fat on Day 99. Changes in fat deposition were similiar over time for all treatments. It is inter- esting to note total EBF comprised approximately 25% of empty body weight on Day 0 and exceeded empty body protein. similiar results were found by Haecker et al. (1920) and McCarthy (1981) in steers of approximately 300 kg. These 79 Table 8. Effect of Trenbolone Acetate on Empty Body Fat8 Trenbolone acetate implant, mg Item 0 14 00 300 ________________ kg-—----------—-—— Day 0 7.6: 68.2: 69.6: 79.8: 35 93.6 87.8 97.3 99.7 67 115.19. 106.9d 121.56 118.96 99 139.1I 136.9e 152.5f 143.0f a Least square means. b Standard error of the mean ranged from 4.6 to 5.6 kg. c,d,e,r Means in the same column without a common superscript differ (P<.05). 80 Figure 5. Effect of Trenbolone Acetate on Empty Body Fat 81 OfiNm w»¢o cog am e EMHH .m 6»5Mae Ham >oom rpmzm 20 amp mo Hommmm (SM) 185 A008 AldNE 82 findings support other observations that fat constitutes a larger percent of empty body weight than protein at a relatively early age (Haecker, 1920; Byers and Rompala, 1979; McCarthy, 1981). Differences among treatments were small on Day 99 for percentage of fat and protein in the empty body, which ranged from 33-35% and l4.2-l4.5%, re- spectively. Body composition was also estimated by equations pub- lished by Crabtree et al. (1974), who considered body water a single pool. Deuterium oxide space is determined by plot— ting the log of deuterium oxide blood concentration as a fraction of dose versus time after tracer equillibration with total body water. Deuterium oxide normally equilli- brates with total body water in 4 to 6 hours (Crabtree et al., 1974; Byers, 1979a; Robelin, 1982b). The extrapolated intercept at time zero is considered the deuterium oxide space (DOS). Theoretically, the intercept is an estimate of total body water. However, since deuterium oxide dilu— tion commonly overestimates total body water, it is more appropriately named DOS. Equations presented by Crabtree et al. (1974) relating DOS to empty body components determined by chemical analysis are as follows: Total Body Water 87.14 + 0.59(DOS) Empty Body Water = 66.64 + 0.53(DOS) Total Nitrogen = 3.09 + O.75(DOS) Empty Body Fat -99.69 + (0.86LW-0.68DOS) where DOS is deuterium oxide space and LW is live weight. Equations were derived from six Friesian and six Friesian x Hereford steers at 340 and 420 kg live weight. Estimates of empty body protein and empty body fat based on single pool tracer kinetics are presented in Table 9. Treatments comparisons with control were not significant (P>.05) and were similiar to results obtained with two-pool methodology. However, absolute amounts of protein and fat were different as well as differences over time. No signi- ficant changes in empty body protein were evident in any treatment on Day 35 compared to Day 0 or Day 99 compared to Day 67. Empty body protein measures of control were simi— liar on Day 99 and Day 67, while TBA treatments contained more empty body protein with each estimate over time. Within treatments, empty body fat (EBF) was signifi- cantly different over time (P<.05). Few differences among treatments were evident. Empty body protein expressed as a percent of empty body weight is initially higher and changes less during the trial with single—pool methodology equations of Crabtree et al. Table 9. Item Day 35 .7 l 99 Day 35 67 99 84 Effect of Trenbolone Acetate on Body Composition Determined by Single Pool Deuterium Oxide Tracer Kineticsa Trenbolone acetate implant, ma 0 ________________ ko----—---------- 59. 60. 64. 63. 27. 62. 86. 107. I‘QOO\1'~O 140 Empty body protein 60. 61. 65. 66. Empty body fatC 7 a Least square means. r3. 55. 80. 110. 2 3 2 8 200 O 60. 62. 65. 66. 30. 64. 99. 126. b Standard error of the mean ranged 0.86 kg. C Standard error of the mean ranged 4.07 kg. 300 b 73 59. 3 61. 5e 64. 8e 66. f 4 29. 7 61. 6 89. 1 117. from 0.71 from 3.32 O‘\J>~LOJ> t0 t0 d,e Means within columns without a common superscript differ (P<.05). f All means within columns differ (P<.05). (1974) compared to two—pool methodology predictive equations of Byers (1979a). Conversely, EBF on Day 0 was measured as less than half, but comparisons over time were similiar for both methods. Fat and protein ranged from 25.8 to 29.0% and 15.8 to 16.2% of empty body weight, respectively, on Day 99. These results indicate experimental subjects contained an average of approximately 2.0% more protein and 5—6% less fat than estimates from two—pool methodology. A number of reasons for this disparity may exist. First, the population upon which the predictive equations were derived may have a large influence on composition estimates. Crabtree et al. (1974) compared DOS to chemical analysis in 12 Friesian and Friesian x Hereford steers in a weight range of 340-420 kg. Byers (1979a) utilized 23 cattle including calves, yearlings, slaughter steers and cows. Second, pre-infusion handling of animal may be im- portant in the single pool model to reduce variability of gastrointestinal fill. Crabtree et al. (1974) did not indicate if feed and water were restricted prior to in- fusion. It seems logical to assume the predictive equations of Crabtree et al. (1974) adjust DOS related to empty body water to account for an "average" gastointestinal fill weight. Errors may arise from large variations among animals in gastrointestinal fill or if the average gut fill of experimental animals in the study largely differed from 86 those used by Crabtree et al. (1974). This may have occur— red if Crabtree and workers restricted feed prior to in— fusion, since subjects in this investigation were allowed free access to feed and water prior to tracer infusion. Third, errors may arise quantitating pool sizes by methods of Byers. Although separating early and late equilli- brating pools and relating them to empty body and gastro- intestinal water seems conceptually sound, interpretation of dilution curves creates technical problems. Figure 6 illustrates graphically the log of deuterium oxide concen- tration versus time. Line B describes the least squares regression of all samples obtained five hours after tracer infusion. The intercept of line B represents the log of deuterium oxide space. Mathematical description of the data is completed by subtracting points along regression line B from actual early samples to obtain line C. All sample points used to determine line B should ensue whole body tracer equillibration. Likewise, samples used to determine line C should only include samples after empty body equillibration. Assuming the sample marked A preceeds empty body equillibration and should be excluded from early samples, the predictive dilution curve becomes the summation of line Figure 6. Effect of Samples Prior to Empty Body Equillibration on Dilution Kinetics 88 NSZP de 020 901 B with D rather than C. To illustrate the differences this situation may create, body composition is listed below with and without the 20 minute sample for an actual dilution curve. Time, min. Deuterium oxide, ppm 20.1 413.0 30.3 360.0 40.3 353.0 50.3 347.0 80.0 339.0 346.0 316.0 1568.0 268.0 3041.0 246.0 4406.0 218.0 Item, kg All samples Without 20 min. sample Pool A 165.4 182.7 Pool B 51.6 34.9 EB weight 319.8 333.8 EB water 153.8 171.7 EB protein 46.4 51.8 EB fat 109.0 98.5 90 As illustrated, body composition differs if samples prior to empty body equillibration are included. In this experiment equillibration had occurred by 25 minutes, but frequently after 20 minutes as judged by graphical inspec— tion of dilution curves. Thus, all 20 minute samples were excluded for determination of pool sizes. The question remains whether composition estimates by single-pool methodology or double—pool methodology are more correct. Although the standard error of the mean is less for the single-pool model, comparative accuracy of these methods necessitates whole body chemical analysis. A larger number of samples than acquired in this investigation should more precisely define deuterium oxide dilution with time and facillitate a more accurate measure of pool sizes and sub— sequent estimates of body composition. Average daily empty body weight (EBWT) gains are shown in Table 10. EBWT gains exceeded live weight gains during the entire trial for each treatment. Although this seems unusual, trends in pool partitioning with increasing weight were evident and explain observed differences in live weight gain and EBWT gain. There was a tendency for the weight attributed to the gastrointestinal tract to decrease with each measure of body composition. Weight attributed to gut 91 Table 10. Effect of Trenbolone Acetate on Daily Empty Body Weight and Empty Body Water Gaina Trenbolone acetate im lant, m Item 0 140 200 300 Empty body weight gain, kg/db Day 0-34 1.75d 1.79 1.64df 1.81 35-67 1.16de 1.74 1.96df 1.57 67-99 0.608 1.57 0.8468 1.19 Empty body water gain, g/dC Day 0-34 725 888 602 892 35-67 341 508 864 684 67-99 —43 305 -78 442 a Least square means. b Standard error of the mean ranged from .22 to .30 kg/d. C Standard error of the mean ranged from 253 to 341 g/d. d,e Means within columns without a common superscript differ (P<.05). f’g Means within columns without a common superscript differ (P<.10). 92 fill differed greatest between the first and second tracer infusions with fewer differences among the second, third and fourth infusions. It is not clear if this disparity rep— resents a bias in this investigation or in two-pool tracer methodology, or actually reflects variations within in animals in gastrointestinal weight over time. Experimental subjects were handled similiarly during each tracer in— fusion. Since all animals were fed and allowed water on days of tracer infusion according to the normal feeding regime, gastrointestinal weight differences were possible and are assumed correct. Treatment comparisons to control were not significantly different (P>.05). EBWT gain significantly declined over time in control and 200 mg TBA treated subjects (P<.05). It is interesting to note this large decline in EBWT corre- sponds to the cessation of empty body protein accretion in these two groups. EBWT gain was increased in excess of 35% by all three doses of TBA in both treatment periods compared to control. Average daily gains in empty body water (EBWAT) and empty body protein (EBP) are shown in Table 10 and Table 11, respectively. These values were derived by two—pool metho— dology. Daily gain in EBWAT and EB? were not significantly different over time or among treatment comparisons (P>.05). These measures are characterized by large standard errors 93 Table 11. Effect of Trenbolone Acetate on Daily Accretion of Empty Body Protein and Eata Trenbolone acetate implant, mg Item 0 140 200 300 -------------- g/d-----------’---- Empty body protein gainb Day 0-34 219 268 185 270 35-66 103 257 264 209 67-99 -11 172 -59 103 Empty body fat gainC Day 0-34 766 576 817 588 35-66 700 567 780 619 67-99 694 794 1118 725 3 Least square means. b Standard error of the mean ranged from 66.6 to 89.4 g/d. C Standard error of the mean ranged from 182 to 245 g/d. due to substantial animal variation in rates of growth and due to coupling of two variable measures of EBWAT over short time intervals. Rates of protein accretion were less dis— similiar during the untreated initial 34 days than following TBA implantation. During the second period, rates of pro- tein accretion for all 3 doses of TBA were 2 fold higher than control. The 140 mg TBA and 300 mg TBA treatments con- tinued to gain protein during the final 32 days. Negative values for control and 200 mg TBA treatments likely arise from indifference of measures of empty body protein on Day 67 and Day 99, inequality of cell sizes over time, and least squares regression analysis of the data. Rates of EBP accretion during the first two periods for all treatments were higher than reported by Byers and Rompala (1979). These findings support the idea anabolic agents can stimu— late protein accretion beyond the normal biological limit of an animal (Byers, 1982). As an animal matures protein accretion should slow and eventually stop, reaching an equillibrium when protein synthesis and degradation are equal (Millward et al., 1975). This general pattern is evident when examining within treatments over time. Cessation of empty body protein gain in 2 treatments during the final 32 days suggests protein accretion constitutes a small portion of live weight gain as animals approach 32 to 35% body fat. 95 Empty body fat (EBF) gains are reported in Table 11. Treatment and time comparisons were not significantly dif- ferent (P>.05). With the exception of controls, fat gains generally increased as the feeding period lengthened and protein accretion declined. Fat deposition exceeded protein deposition by greater than two fold during all periods of the investigation. Thus, periods of rapid animal growth and protein accretion were accompanied by even larger rates of fat deposition. Suprisingly, TBA did not reduce fat accre- tion as commonly reported with anabolic agents (Preston, 1975: Galbraith and Topps, 1981: Byers, 1982). In fact, EBF gains tended to be slightly larger in TBA treatments. These results indicate TBA increased EBWT gain and EBP gain, but with little effect on fat gain. Although individual feed con— sumption was unavailable, increases in ADG, DMI, and feed efficiency reported in Trial I are consistent with changes in body composition in this trial. Effect of Trenbolone Acetate on 3—Methylhistidine Excretion Anabolic agents commonly increase lean body mass at the expense of fat accretion. The mode of action of anabolic agents may be direct, or indirect through changes in hor— monal profiles as previously addressed. Few attempts have been made to ascertain rates of protein synthesis and degra— dation in vivo in large animals, particularly cattle. Vernon and Buttery (1976) reported TBA reduced protein synthesis and degradation in female rats. Since protein degradation was depressed more than protein synthesis, protein accretion was increased. In a similiar study with ewes, Sinnett-Smith et a1. (1983) demonstrated reduced muscle fractional synthesis rates following TBA or zeranol treatment. These findings suggest anabolic agents may increase lean body mass, performance and feed efficiency by reducing protein degradation, and perhaps to a smaller extent, protein synthesis. In light of these findings, an experiment was designed to measure protein accretion and in vivo rates of myofibrillar protein degradation as judged by 3—methylhistidine excretion in TBA treated heifers. Average daily urine excretion from three day collection periods is shown in Table 12. Urine excretion was highly variable among heifers and was not different among treat— ments or time (P>.05). However, urine excretion tends to decline over time, which may reflect problems encountered during the third urine collection period. Several subjects were uncomfortable with urinary catheters during this period. These subjects continually strained against Foley catheters, which in some cases resulted in loss of the 97 Table 12. Least Squares Means for Daily Urine Excretion by Treatment and Perioda b Trenbolone acetate implant/mg 0__TT—___140_—_‘—__200_'____—300 ______________ l/d?-—-—--—---—---—- Period 1 12.4 7 0 9.5 7.3 2 10.9 6 0 7.2 9.4 3 8.8 5 3 7.5 8.3 a Standard error of the mean ranged from 1.6 to 2.1 1/d. b Least squares means. catheter. These problems were negligible during the first two periods. Table 13 summarizes the effect of TBA on 3—methyl- histidine (3—MeHis) excretion and 3-MeHis output per unit of body weight. During the first urine collection, 3—MeHis output was similiar among control, 140 mg TBA and 300 mg TBA treatments and slightly elevated for the 200 mg TBA group. After implantation, 3-MeHis excretion increased for all doses of TBA as well as control. Treatment comparisons to control were not different (P>.05). However, all three doses of TBA corresponded with higher 3-MeHis output than untreated subjects during the second period. Mean values of 3-MeHis excretion during the third period do not fit into the general pattern of 3-MeHis output among treatments and time, particulary in control and 300 mg TBA groups, which may reflect the smaller number of subjects (due to missing data) and the apparent stress on collected subjects during this period. Total protein degradation increases over time as evidenced by increased 3-MeHis and is consistent with a larger protein pool as an animal grows. However, these differences are less pronounced over time when expressed per unit of body wight as illustrated in Figure 7. The trend among treatments is similiar as for total 3-MeHis excretion since differences in body weight were small. These values agree with results of Nishizawa et a1. (1979), who reported 99 Table 13. Effect of Trenbolone Acetate on Excretion of 3-Methylhistidinea Trenbolone acetate implant, mo 0 Item 0 140 200 300 - . . b 3—MeH15 excretion,mm01€5/d Period 1 1.08 2.17 1.38 1.02d 2 1.27 1.40 1.49 1.56de 3 1.79 1 35 1.50 1.97e 3 MeHis excretion/kg bodyweight, umoles/dC Period 1 2.98 3.11 3.76 3 2.83 2 3.04 3.48 3.54 3.80 3 4.09 2.99 3.24 4.46 a Least squares means b Standard error of the mean ranged from 0.22 to 0.30 mmoles/d. C Standard error of the mean ranged from 0-55 to 0.78 nmoles/d. d,e Means in the same column without a common superscript differ (P<.05). 100 Figure 7. Effect of Trenbolone Acetate on Urinary Excretion of 3—Methylistidine Per Unit of Body Weight 101 Dawn w>¢o no“ om o 19: .ow.N .wa. IE: 1? ozuSMJ .om.m .a mesm2a .om.¢ HIonz >Dom mum onHmmoxw wHIMZIm N011380X3 3N10I181H1AH13N-8 102 3-MeHis output of 2.75 micromoles/d/kg body weight in 300 kg steers. Harris and Milne (1981b) reported 3.62 micromoles/d/kg body weight in 600 kg subjects. However, these values are lower than those reported by McCarthy (1981). Nishizawa et al. (1979) reported a decline in 3—MeHis excretion per unit of body weight as animals became larger. Harris and Milne (1981b) suggested little decline is evident after 400 kg. In this experiment, 3-MeHis/body weight is nearly the same or higher during the first two periods. A decline over time in each period is evident only for the 200 mg TBA treatment. Table 14 summarizes the effect of TBA on urinary creati- nine output. Creatinine excretion was largely indifferent among treatments and increased over time. Creatinine output per unit of body weight was similiar across all treatments and time. These results are consistent with evidence creati- nine is excreted in constant proportion to muscle mass. Total creatinine excretion or creatinine excretion per unit of body weight was slightly higher for TBA treatments during the second period. It is interesting to note creatinine and 3—MeHis output were quite high relative to other means for control and 300 mg TBA treatments during the third period. Although these values may be correct, errors may have arisen obtaining a urine sample from "normal" subjects. Changes in normal animal metabolism in response to individual 103 Table 14. Effect of Trenbolone Acetate on Excretion of Creatininea Trenbolone Acetate implant, mg Item 0 140 200 300 Creatinine excretion, g/d Period 1 8.36 8.66 8.85 7.29d 2 9.70 10.80 10.37 10.50de 3 13.34 11.84 11.24 13.16e Creatinine excretion/kg body weight, mg/dC Period 1 24.7 23.1 23.9 20.4 2 23.8 26.7 24.7 25.7 3 30.7 26.1 24 2 29.7 a Least squares means. b Standard error of the mean ranged from 1.47 to 2.10 g/d. C Standard error of the mean ranged from 3.75 to 5.30 mg/d. d,e Means in the same column without a common superscript differ (P<.10). 104 housing and urinary catherization are of primary concern in this investigation. These changes are difficult or impos— sible to quantify and are assumed negligible. However, rates of protein degradation are known to change in response to frequency and level of intake as well as in response to stress. If animals are stressed, catabolic processes are presumably accelerated and biased estimates of muscle protein degradation will result. In this experiment, effort was made to assure as near normal animal state as possible. This attempt seemed successful during the first two urine collections as judged by level of intake and visual ap— pearance. Less success was considered during the third period. If we assume urinary 3—MeHis excretion is pro- portional to muscle degradation and urinary creatinine excretion is proportional to muscle mass, the ratio of 3-MeHis to creatinine should approximate skeletal muscle protein turnover. Table 15 and Figure 8 summarize the effect of time and TBA on 3MeHis/creatinine. Treatment comparisons to control and comparisons over time were not significantly different (P>.05). There was a tendency for the ratio to decline over time in the two low doses of TBA and slightly increase over time in the control and 300 mg TBA treatments. Protein degradation per unit of muscle mass was higher for all doses of TBA compared to control during 105 Table 15. Effect of Trenbolone Acetate on Urinary 3-Methylhistidine to Creatinine Ratioa Trenbolone acetate implant, mg Item 0 140 200 300 Period 1 .125 .138 .169 .141 2 .130 .131 .141 .148 3 .135 .114 .132 .154 a Least square means. b Standard error of the mean ranged from .012 to .017. 106 Figure 8. Effect of Trenbolone Acetate on the Ratio of Urinary Excretion of 3-Methylhistidine to Creatinine 107 wxmo cod cm a .oowd. ozuoum .w manage .oo¢«. .ooma. .oom—. oHpcm mszHpcmmo op mzHoHHwHIJ>Ipmzum .ooou. 01108 3NIN110383 01 3N101181H1AH13N-8 108 the first two periods. Thus, TBA seemed to have no consis— tent effect on protein turnover measured in this fashion. Skeletal muscle protein degradation can be estimated from urinary excretion of 3—MeHis. Haecker (1920) physi— cally separated and chemically analyzed carcasses of 63 steers at 45 kg increments into several body components, including skeleton, blood, offal and flesh. Flesh was total body muscle and fat combined. Haecker found the protein content of flesh (soft tissue) approximately 54% of total body protein in cattle weighing 300 to 500 kg. Kertz et a1. (1982) reported the protein content of adipose tissue ranged from 2-6%. Thus, skeletal muscle protein has been estimated to comprise approximately 51% of total body protein. Obviously, this is a best guess approach and assumes skele- tal muscle is constant in proportion to total body protein over the weight range in this investigation. Haecker's results generally support this assumption for 350-550 kg weight cattle. Further differences among treatments in total body protein are assumed proportional among skeletal muscle protein and non—skeletal muscle protein. This assumption may not be valid, since the effect of TBA is not limited to skeletal muscle protein in rats (Vernon and Buttery, 1976, l978a,b). However, these affects are assumed to be negligible. It is conceivable a particular treatment therapy may affect only skeletal muscle protein or 109 non-skeletal muscle protein at the exclusion of the other. Biased estimates of muscle protein turnover would result. Table 16 illustrates total skeletal muscle by treatment and time for subjects used in urine collections. Skeletal muscle protein was assumed 51% of total empty body protein. Total skeletal muscle was similiar but slightly higher for TBA treated subjects compared to controls during period 3 and tended to increase over time for all treatments. The skeletal muscle 3—MeHis pool was calculated assuming 3.5 mmoles 3—MeHis/kg muscle protein (Nishizawa et a1. 1979) and is shown in Table 16. Fractional excretion of 3-MeHis should equal the frac— tional breakdown rate (FBR) of myofibrillar protein if skeletal muscle turnover accounts for most of the excreted 3-MeHis. As previously discussed, total body 3—MeHis is predominately confined to skeletal muscle as a component of actin and myosin, which are assumed to turnover at similiar rates. The contribution of organs other than skeletal muscle to urinary 3-MeHis, if larger than their percent of total body 3-MeHis pool, may lead to an overestimation of FBR. Skeletal muscle is thought to contribute 60—83% of urinary 3-MeHis in rats (Nishizawa et al. 1977a; Wassner and Li, 1982) and approximately 75% in man (Afting et al., 1981). In this investigation, skeletal muscle was assumed to contribute 80%. This value has not been determined in 110 Table 16. Effect of Trenbolone Acetate on Skeletal Muscle and Total Skeletal Muscle 3—Methy1histidine Poola Trenbolone acetate implant, mg Item 0 1 0 200 300 Skeletal muscle, kgb Period - 1 27.3 28.5 26.8d 25.1d 2 30.2 29.0 30.9e 29.0e 3 29.9 30.7 30.96 30.0e Skeletal muscle 3 MeHis pool, mm01€SC Period d d 1 95.2 100.0 93.6 87.53 2 105.5 101.8 107.86 101.2 3 104.3 108.0 108.0e 104.88 a Least square means. b Standard error ranged from 1.04 to 1.46 kg. C Standard error ranged from 3.6 to 5.1 mmoles. d,e Means within columns without a common superscript differ (P<.05). 111 cattle, but evidence is growing in other species that skeletal muscle contributes substantially less than 100%. Even less is known regarding changes in the relative con- tribution of skeletal muscle and non-skeletal muscle tissues to urinary 3-MeHis excretion in response to changes in physiological state. These changes may be important when investigating 3—MeHis excretion during feeding and fasting, but are assumed negligible in this experiment. Mulvaney (1981) suggested turnover rates may vary among muscles depending on maturation rate. Thus, degradation rates calculated from 3-MeHis excretion may represent only an average FBR for skeletal muscle. Fractional excretion of 3-MeHis is shown in Table 17 and is assumed an appropriate measure of FBR. FBR was similiar among all treatments during the untreated first period. Following implantation, TBA treated subjects had a slightly higher FBR than controls in Period 2. FBR of 140 mg and 200 mg TBA groups were similiar during both implant periods and to pre-treatment values. Control and the high dose of TBA tended to have a higher rate of protein degra— dation over time. Nishizawa et al. (1979) reported an FBR of 1.02% in 312 kg cattle, assuming 94% of urinary 3-MeHis originated from skeletal muscle. Harris and Milne (1981b) reported FBR of 1.37% and 1.23% in cattle weighing 263 kg and 376 kg, respectively. 112 Table 17. Effect of Trenbolone Acetate on Fractional Excretion of 3-Methy1histidine and Half Life of Skeletal Musclea Trenbolone acetate implant, mg Item 0 140 200 300 Fractional excretion of 3MeHis, %/db Period 1 0.92 0.93 1.18 0.93 2 0.96 1.13 1.11 1.23 3 1.37 1.03 1.13 1.51 Half life of skeletal muscle, dC Period 1 75 75 59 75 2 72 61 62 56 3 51 67 61 46 a Least squares means. b Standard error of the mean ranged from 0.19 to 0.26%/d. C Standard error of the mean ranged from 18 to 27 d. 113 The fact FBR does not appreciably decline in all treat— ments over time was suprising, since measures of FBR are commonly lower in mature animals compared to growing animals (Garlick, 1980). This may be a consequence of the small weight range investigated in this trial and the summation of errors determining 3—MeHis and total empy body protein. Skeletal muscle half—life values were calculated from fractional excretion rates of 3-MeHis and are presented in Table 17. Naturally, the relationship between half-life means among treatments is identical to that for fractional excretion of 3-MeHis by virtue of the calculation. However, the data illustrates the rapid turnover of skeletal muscle protein. These rapid turnover rates confirm the large energetic expense of maintaining skeletal muscle (Webster, 1980). These results contradict the findings by Sinnett—Smith et al. (1983) with female sheep and Vernon and Buttery (1976, l978a,b) with female rats, who reported TBA treatment reduces skeletal muscle protein turnover. Small increases in muscle accretion with TBA implantation in this investi- gation, particularly in Period 2, were not due to a reduc- tion in muscle protein degradation. This finding appears contradictory to the known stimulatory effects of TBA on animal performance and improvement in feed conversion efficiency. However, an increase in protein turnover 114 concomitant with a reduction in fat accretion may account for observed improvements in performance and feed efficiency. For example, assume the fractional synthesis rate (FSR) and FBR are increased, but FSR by a larger magnitude. Thus, muscle protein accretion and turnover are increased. Further assume fat accretion is decreased and that a conservative estimate of the efficiency of fat and protein synthesis is 70% and 35% , respectively (Webster, 1980). Since the energy density of fat is approximately eight fold higher than protein on a wet basis, each 1 kg reduction in fat deposition spares energy potentially available for over 4 kg of protein (lean) deposition, when differences in the efficiency of fat and protein synthesis and energy density are considered. Higher rates of skeletal muscle protein turnover would require more energy to support a larger FBR and FSR for a given skeletal muscle mass. However, if energy is shunted to protein deposition at the expense of fat accretion, improved weight gain and feed conversions will result even with substantial increases in skeletal muscle protein turnover. Few experiments with cattle have been conducted to investigate changes in skeletal muscle turnover in response to anabolic agents. Gopinath and Kitts (1982) reported non—significant increases in 3-MeHis excretion and FBR in zeranol and Synovex-S treated steers. Griffiths (1982) failed to detect 115 significant differences in 3-MeHis output in TBA and zeranol (Ralgro) implanted steers. These preliminary investigations have failed to clearly demonstrate an affect of anabolic agents on protein turnover in cattle. However, any effects may differ dramatically between sex, age, and particular anabolic agent. Rates of skeletal muscle protein synthesis, degradation and accretion are shown in Table 18. Muscle protein accre- tion was calculated by difference of least square means for skeletal muscle protein on Day 0, 35, 67, and 99. Muscle protein accretion was considered constant within each period. Degradation rates were calculated from FBR (measured during the final 8 days of each period) and the average quantity of skeletal muscle during each period. Muscle degradation rates were assumed constant within each period. Synthesis rates were estimated by summing rates of degradation and accretion. Since all calculations are from mean values, statistical analyses are not provided. Muscle protein degradation is variable among treatments during the untreated first period. During the second period, degradation rates are higher in all TBA treatments compared to controls. This pattern is reversed for the 140 mg TBA and 200 mg TBA treatments during the final period. Skeletal muscle protein accretion is initially similiar among control, 200 mg TBA and 300 mg TBA treatments. The 116 Table 18. Skeletal Muscle Protein Synthesis, Degradation and Accretion Trenbolone acetate implant, mg Item 0 140 200 300 ---------------- g/d—--—-----——---—-- Muscle protein synthesis Days 0-34 330 436 374 338 35-66 370 341 452 458 67-99 403 360 349 476 Muscle protein degradation Days 0-34 236 232 303 214 35-66 276 325 320 333 67-99 412 307 349 445 Muscle protein accretion Days 0-34 94 204 71 124 35-66 94 16 132 125 67-99 —9 53 0 31 117 two high doses of TBA tended to increase rates of muscle protein deposition after implantation. For the 140 mg TBA treatment, accretion rates were higher than all treatments during the first and third periods, but lower during the second period. Rates of protein synthesis are similiar to those re— ported by Lobley et al. (1980) in two heifers and one dry cow by constant infusion of labelled leucine and tyrosine. They reported total muscle protein synthesis for the two heifers weighing 236 kg and 263 kg to range from 284.5 to 354.9 g/d, depending on whether rates were calculated from the specific radioactivity of the tracers in the blood or the tissue homogenate. In the 628 kg dry cow, muscle pro- tein synthesis was 448.8 g/d and 400.7 g/d when calculated from tissue homogenate specific radioactivity and blood specific radioactivity, repectively. Thus, quite similiar results are obtained in this investigation and that reported by Lobley et al., (1980) utilizing different methodologies, but variation was large in both experiments. The variation over time and among treatments in degra— dation and accretion is compounded when calculating skeletal muscle protein synthesis. Thus, any differences or trends are likely obscured. These values illustrate the problems encountered measuring muscle protein accretion in vivo over short periods of time. Also, standard errors for 3—MeHis [uni-khak— 118 excretion in this experiment as well as others (Griffiths, 1982; Gopinath and Kitts, 1982) often exceeded 10% of the mean value. Serious consideration is warranted with regard to use of 3-MeHis excretion to measure FBR and tracer dilu— tion to measure fat and protein accretion. These methods appear to be appropriate in vivo, non-invasive techniques, but necessitate careful consideration of frequency of measures over time and number of animals required to detect small differences. 1. SUMMARY Trenbolone acetate at 200 mg and 300 mg per head resulted in small increases in average daily gain, dry matter intake and feed efficiency. Trenbolone acetate did not affect carcass characteristics. All three doses of trenbolone acetate tended to increase empty body weight and empty body protein gains, but empty body fat gains were similiar to control. Empty body protein gains tended to decline over time while empty body fat gains increased. Empty body fat gains exceeded empty body protein gains during all periods. Urinary excretion of 3-methylhistidine was not significantly different over time or among treatments. 119 10. 11. 120 Creatinine excretion increased with body weight, but differences over time were small when expressed per unit of live weight. The ratio of 3-methylhistidine to creatinine was similiar among treatments. Fractional breakdown rate of skeletal muscle protein calculated from urinary 3—methylhistidine excretion was similiar among treatments and ranged from 0.92% to 1.51% per day. Skeletal muscle half—life values were estimated as 59 to 75 days. Methodologies employed in this investigation to measure in vivo rates of protein accretion and degradation seem appropriate, but warrant serious consideration of sample size to detect small differences. APPENDIX 121 .mmo.vmv Hosucoo Eosw msowwflm p .ANEU moan osonua x omo.e - flux .20: x swoo.s + hwzae x ON.OC + nap .mmoaxuaau use Bap gums x wa.oC + m.N n weeps 6362» u .006 .aam-ocN .aemflam lama-ooa .moumpp ”ma-o .eao>6e kHHmOHuumsm ”oflnwfisw> msozcfluCOO m mm poNxfimcm paw wosoom mcflfibsme wo ooswoo b .mcmoE osmscm ummoq w N.N o.N o.N 4.N posses 6260s fiom .zo:pflx N.Nw mm.flm o.Nm H.4s ~50 .8608 oxobum HH.H cw.o 05.0 No.0 Eu .umw nap :pNH owm Hem BAN mON ms .uEMsz mmmupmo no: m p q v m mfimsflcm mo sobssz EmpH meHumflsouomsmnu mmmusmu co oumuoo< oonOQCosb mo uoowwm H< oabme 122 A.2 Abbreviations and Units for Individual Data Abbreviation Anim. TBA Dose Urine output 3-MeHis Excre. Creat. Excre. LBW EBWT EBWAT EBP EBAI EBF DOS Name Individual identification Trenbolone acetate implant bLNNl—l Ave. daily urine excretion Ave. daily 3—methy1histidine excretion Ave. daily creatinine excretion Live body weight Empty body weight Empty body water Empty body protein Empty body minerals Empty body fat Deuterium oxide space 0 mg 140 mg 200 mg 300 mg liters/d mmoles/d g/d 123 INDIVIDUAL ESTIMATES OF BODY COMPOSITION A.3 467 31858325144301986006794.687424691076936475214621 S ................................................ 086010919431788616863985382276279570068845367351373 D214590214710112454011244693333913344.0/1240031331233 2222122222222222?—222222222222212222212222222222222 754256686037 672271975048262906927996AU61420283247440 F oooooooooooo o u o o ooooooooooo n o c ........... 398426677493179986481570314754282170530679906865583 E492448989178037903603470414893569255689024885825827 001.1000001_0011001~|_01¢l1001100110011.0000110000010011 88126585020336596877784667178070764788873342969709 Mo... oooooo coo-coo oooooooooo coco. ..... 0.0.0.. ..... 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W 53548625302610857814817416988120387974093478471869 B83825139705059059885807605648183921190495947275180 E23342337.34.233333332334234.4233423342323332232332334 4.0379035325166ng430891708630718073126323333155986 W O O O I O O Q 0 O O O O I I 0 O O C C O D I C I I O O O O I O O O O O I O I O O O O I O I D O I O O B10616020776038594030654290920657413494778723496168 L261».57464940370482317024930382305150514728271609514. 334.42333343334334433443345334433443333342333342344. E M1234.1231231234123412341234123412341212341231231234 I T E Sll111111111111111111112222222222222222222223333333 O D 00000001118888666699998888444455553377772221117777 D77772229991111444.4.11.1l4.44.4666633338888882228882222 I66667776666666666677776666666677776666666666667777 124 CONTINUED A.3 38383160476459257984242975590760752 S oooooooooooooooooooooooooooooooooo 094017520995101945570761741216364120 D13551256325712252568004434678012133 22222222222222222222222222221222222 00324948522872760964919655340096805 F o ooooooooooooooo o c o oooooooooo B1648139033931359451428576287577814s E91368904600368016835588392348911235 011100110l110011001100010111001111l 20946508297565191789961861067217180 M OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 803440254214502321224904393459125913 EllllllllllllllllllllOlllOlllOllOOll 79313760334867655425579492225213880 P ............................. B4.65364.55324764768565361017182935917 E4566456655664555455644664.5664452355 Tl7635333844763848841172386999115980 A oooooooooooooooooooooooo o n o u n o a o - W88693175633441070367445089250363119 84810581177l258986881450038024678378 E11221].22112211111112.112211221110111 06218339896856244810412174410520755 T ...... u... u scone-co oooooo no W44151275501131764772007262678093294 B97359403040473678385506188157253061 E23442344334.423332334233423442332334. 02288781448332239809643714823249461 w OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO B122027613666184816644.017145563315688 L405836264727058228499384.51580483503 34A...43344334.43393433442334.34443333344 E . M1234.123412341.2341234123412341231234. I T E 533333333333344444444444444444444444. O D 66660000777766668888555555553331111 D3333ll11777755556r066666611113330000 166667777666666666666666677776667777 125 , 3-METHYLHISTIDINE, AND CREATININE INDIVIDUAL URINE EXCRETION A.4 URINE OUTPUT TBA TIME DOSE ANIM ID 7101809708609942356155245034.73091.8584966428 343542Q.402124074439234.6135921242lelnw6243666 1111110111111111110111154111111110.1121111111 5886020266721655795810414631640467197789504 7nnwl554.842964.34556520107116109163_D7210429995 recto-no.0... ooooooo 0000 000.000.0000.. 8187CBC/029433386121024244109275079283803481 0110100110111100101110100110111100101011001 087 4.617 32701979375824878656211946511500114.4. 2631509522769449630265085791511341471159849 ooooooo OooooooOInooooooooo on... once... 112111011llll1.10101110200111122101112111011 80007533—30755705807072073002003337557600700 5900691493150455495095004411430379432036993 to... oooooooooooooooooooooooooooooooo Cot-o 8049004p3685986574555678770265567523329720618 0002100000000000000000011100000000010011010 1232312312312312312121212312312312312123123 1111111111122222222223333333333344444444444 000l7.8886668884A.455331177700077766688555111 7770991.1.1.444I».4466633888822211177755566666000 6.066666666666666677666677777766666666666777 1.. 126 Table A. 5 Least Squares Mean Square Errors for Animal Within Dose and Residual Error Item Live body weight, kg Two-pool model Empty body weight, kg Empty body water, kg Empty body protein, kg Empty body minerals, kg Empty body fat, kg Deuterium oxide space, kg Single pool model Empty body water, kg Empty body protein, kg Empty body fat, kg Body Composition Changes By Periods Live body weight, kg Empty body weight, kg Empty body water, kg Empty body protein, kg Empty body fat, kg Urine Metabolites Urine excretion, l/d Creatinine excretion, g/d 3—MeHis excretion, mmoles/d 3—MeHis excretion/LBW, p/d Creatinine/LBW, mg/kg/d 3-MeHis/creatinine, mmoles/g Protein Turnover Skeletal muscle, kg 3—MeHis fractional excretion Muscle half life, days Anim/Dose 3108 2800 734 66. 3. 968 985 276 34. 1008 158. 339. 436. 20. 217. 45. 18. 1.72 94. .0014 12. 2109' moo O‘LN\!OJ> 32 7 61 67 125. 228. 169. 15. 125. 85. 23. 3. 66. 159 347. 447. 30. 230. 1418 0000-500 0000 Residual Nob NNOO .19 .22 .0006 .25 .57 LITERATURE C ITED LITERATURE CITED Afting, Ernest-Gunter, Wolfgang Bernhardt, Rudolf W. 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