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Metabolic and endocrine effects of anabolic compounds in growing beef steers Gopinath, Ramachandran 1983

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METABOLIC AND ENDOCRINE EFFECTS OF ANABOLIC COMPOUNDS IN GROWING BEEF STEERS by RAMACHANDRAN GOPINATH B.Sc, Annamalai University, India, 1974 .Sc., Tamil Nadu A g r i c u l t u r a l University, India, 1976 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES Department of Animal Science We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1983 ® Ramachandran Gopinath, 1983 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 -6 (3/81) ABSTRACT The metabolic and endocrine effects of anabolic compounds, v i z . , Zeranol, d iethyls t i lbestrol (DES) and Synovex-S were investigated in growing steers in order to understand their biochemical mechanisms of action. The growth promoting properties of certain anabolic compounds were established. A marked reduction in the plasma concentration of urea nitrogen, alpha amino nitrogen and amino acids was found in steers implanted with anabolic compounds. The results suggested that anabolic compounds signif icantly alter the nitrogen metabolism of steers by increasing the efficiency of u t i l i za t ion of absorbed nitrogenous compounds. Implantations of DES and Synovex-S were more effective than Zeranol in enhancing the growth rate and altering the nitrogen metabolism of the animals. The usefulness of measuring N T-methylhistidine in urine as a non-destructive, in vivo index of myofibril lar protein degradation and the developmental aspects of muscle protein metabolism in cattle were demonstrated. The implantation of anabolic compounds, in general, increased the efficiency of muscle protein synthesized and protein deposited by the steers. Implantations of DES and Synovex-S were more effective than Zeranol due to a reduction in the muscle protein degradation per unit synthesized. Hydroxyproline excretion in the urine was used as an in vivo indicator of collagen turnover in steers. As the animals gained weight - i i i -and advanced in maturity, i t s excretion in the urine decreased indicating a reduction in collagen turnover. Implantation of DES increased the hydroxyproline excreted by steers, while, Zeranol and Synovex-S had very l i t t l e effect. These results indicate that DES implantation increases the amount of collagen and i t s turnover in steers. DES and Synovex-S exerted a significant influence on the activity of the thyroid gland and caused an elevation in the circulating concentrations of free and total thyroxine. On the other hand, the implantation of Zeranol resulted in plasma thyroxine concentrations similar to or lower than in the controls. Plasma triiodothyronine concentrations were not influenced by any of the compounds studied. Increased plasma thyroxine concentration observed in the DES or Synovex-S implanted steers was shown to be due to an increase in the secretion and a decrease in the metabolic clearance rates of thyroxine. The implantation of Zeranol appeared to increase the secretion rate of thyroxine, and resulted in slightly depressed plasma thyroxine concentration due to a higher metabolic clearance rate. Implantations of anabolic compounds resulted in an increase in the circulating concentration of growth hormone. Implantations of DES and Synovex-S were more effective than Zeranol in increasing the plasma growth hormone concentration. The kinetic parameters of growth hormone metabolism in growing steers were determined. The anabolic compounds increased the secretion rate of growth hormone from the pituitary gland suggesting that these - iv -compounds evoke growth promotion in steers through changes in the endogenous growth hormone status. These changes involved an increased secretion rate of growth hormone with very l i t t l e alteration in the metabolic clearance rate. The metabolism of insulin was influenced to a significant extent in steers implanted with anabolic compounds. Zeranol increased the insulin secretion rate to a greater extent than Synovex-S or DES. The data indicated that the implantation of anabolic compounds altered the metabolism of steers significantly and enhanced the secretion rate of thyroxine, growth hormone and insulin. They altered the metabolism of the steers in such a way that there was an efficient u t i l i z a t i o n of absorbed nutrients. The mechanisms of action of anabolic compounds have been discussed in detail and the directions for future research suggested. - v -TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS v LIST OF TABLES ix LIST OF FIGURES x i i LIST OF APPENDICES xiv ACKNOWLEDGEMENTS xv INTRODUCTION 1 LITERATURE REVIEW 3 General concepts of animal growth 3 Concepts and definitions of growth 3 Composition of growth and the concept of nutrient partitioning 5 Hormonal control of growth and metabolism i n animals 7 Endogenous hormones affecting animal growth and metabolism 7 Growth hormone 7 Somatomedins 9 Prolactin 11 Insulin 12 Thyroid hormones 14 Gonadal and adrenal steroids 15 Miscellaneous endogenous growth factors 17 Exogenous hormones and compounds affecting growth, body composition and metabolism of animals 19 Anabolic compounds: Concepts and definitions 19 Aspects of growth and protein metabolism 20 Endogenous hormonal changes as influenced by the application of anabolic compounds 25 Methodological aspects to study protein metabolism in animals treated with anabolic compounds 28 Measurement of protein synthesis 28 Continuous infusion method 28 Feeding method 29 - v i -Measurement of muscle p r o t e i n degradat ion 29 I s o t op i c method • 29 Non - i s o t op i c method u t i l i z i n g n o n - r e u t i l i z a b l e amino a c i d 32 Co l l agen turnover 35 Measurement of c o l l a gen s yn thes i s 36 Measurement of c o l l a gen degradat ion 36 S T U D Y I : Metabolic aspects of anabolic compound action in growing beef steers 38 KXPT. I A 39 E f f e c t s of anabo l i c compounds on growth and n i t r o g e n metabolism i n growing beef s teer s 39 I n t r o d u c t i o n 39 M a t e r i a l s and Methods 40 Re su l t s 46 D i s cu s s i on 52 Conc lus ion 61 EXPT. I B 63 Muscle p r o t e i n metabolism i n s tee r s as determined by the u r i n a r y N T - m e t h y l h i s t i d i n e e x c r e t i o n as an i n v i v o index of m y o f i b r i l l a r p r o t e i n breakdown: Developmental aspects of muscle p r o t e i n metabolism and the e f f e c t of anabo l i c compounds 63 I n t r o d u c t i o n 63 M a t e r i a l s and methods 65 Re su l t s 68 D i s cu s s i on 80 Conc lus ion 93 K X P T . I C 94 Co l lagen metabolism i n growing beef s tee r s as assessed by the u r i n a r y hydroxypro l i ne e x c r e t i o n and the e f f e c t s of e s t r ogen i c anabo l i c compounds 94 I n t r o d u c t i o n 94 M a t e r i a l s and methods 95 Re su l t s 97 D i s cu s s i on 100 Conc lus ion 109 - v i i -STUDY I I : E f f e c t o f a n a b o l i c compounds on t h e m e t a b o l i s m o f hormones i n growing b e e f s t e e r s I l l EXPT. I I A : l 112 E f f e c t of anabo l i c compounds on plasma l e v e l s of t h y r o i d hormones i n growing beef s tee r s 112 I n t r o d u c t i o n 112 M a t e r i a l s and methods 113 Re su l t s 117 D i s cu s s i o n 122 Conc lu s ion 129 EXPT. I I A:2 130 K i n e t i c parameters of thy rox ine metabolism i n s tee r s implanted w i th anabo l i c compounds 130 I n t r o d u c t i o n 130 M a t e r i a l s and methods 131 Re su l t s 137 D i s cu s s i o n 145 C o n c l u s i o n . . . . . . . 152 EXPT. I I B 154 E f f e c t of anabo l i c compounds on plasma growth hormone l e v e l and i t s k i n e t i c parameters of metabolism i n growing s t e e r s . . . . . 154 I n t r o d u c t i o n 154 M a t e r i a l s and Methods 156 Re su l t s 163 D i s cu s s i on 173 Conc lu s ion 181 EXPT. I I C 183 E f f e c t of anabo l i c compounds on the k i n e t i c parameters of i n s u l i n metabolism i n growing s tee r s 183 I n t r o d u c t i o n 183 M a t e r i a l s and methods 184 Re su l t s 187 D i s cu s s i o n 191 Conc lus ion 199 - v i i i -GENERAL DISCUSSION AND CONCLUSIONS 200 LITERATURE CITED 207 APPENDIX I 232 APPENDIX I I 234 APPENDIX I I I 237 - ix -LIST OF TABLES Page Table 1 Serum hormone-like growth factors 18 Table 2 Anabolic compounds available for use in farm animals 22 Table 3 Changes in the endogenous hormone concentration following the administration of anabolic compounds 26 Table A In vitro and in vivo methods for the quantitation of protein turnover in different species (summary) 30 Table 5 Ration composition (EXPT. I A) 43 Table 6 Plasma free amino acid levels in steers on 28 days following the implantation of various anabolic compounds 51 Table 7 Daily urinary nitrogen excretion in steers implanted with various anabolic compounds 54 Table 8 Effect of anabolic compounds on hepatic tyrosine aminotransferase levels 55 Table 9 Daily urinary excretion of creatinine and N T-methylhistidine: Creatinine ratio during different periods of the experiment 71 Table 10 Fractional rate of protein breakdown and the h a l f - l i f e values of myofibrillar proteins in growing steers on different periods of the experiment 75 Table 11 Amount of muscle protein gained in steers implanted with various anabolic compounds 81 Table 12 Effect of anabolic compounds on the fractional rate of protein breakdown (FBR) and the h a l f - l i f e values of myofibrillar proteins in growing beef steers 82 Table 13 The efficiency of muscle protein synthesis i n steers implanted with various anabolic compounds... 83 - x -Table 1 4 Urinary hydroxyproline excretion in steers on different days following the implantation of anabolic compounds • 1 0 1 Table 1 5 Plasma total thyroxine concentration in steers on different days following the implantation of anabolic compounds 1 1 8 Table 1 6 Plasma total triiodothyronine concentration in steers following the implantation of anabolic compounds 1 2 0 Table 1 7 Thyroxine binding globulin capacity ( T B G R D ^ ) in steers on different days following the implantation of anabolic compounds.. 1 2 1 Table 1 8 Mean free thyroxine assessment (FTA) in steers on different days following the implantation of anabolic compounds. 1 2 3 Table 1 9 Summary of changes in the plasma thyroid hormone concentration in steers implanted with anabolic compounds 1 2 4 Table 2 0 Ration composition (EXPT. II A : 2 ) 1 3 2 Table 2 1 Kinetic parameters of thyroxine metabolism in growing beef steers 1 4 0 Table 2 2 Kinetic parameters of thyroxine metabolism in steers on 2 0 days following the implantation of anabolic compounds 1 4 1 Table 2 3 Kinetic parameters of thyroxine metabolism in steers on 4 0 days following the implantation of anabolic compounds.. 1 4 3 Table 2 4 Kinetic parameters of thyroxine metabolism in steers on 6 0 days following the Implantation of anabolic compounds 1 4 4 Table 2 5 Effect of anabolic compounds on plasma thyroxine status 1 4 6 Table 2 6 Kinetic parameters of thyroxine metabolism i n cattle.. 1 4 9 Table 2 7 Plasma G H concentration in steers on different days following the implantation of anabolic compounds 1 6 5 - 3d -Table 28 Kinetic parameters of GH metabolism in growing steers.. 169 Table 29 First-order rate constants for distribution and elimination of plasma GH in steers .. 170 Table 30 H a l f - l i f e and fractional disappearance rate of injected GH in steers 171 Table 31 Metabolic clearance and secretion rates of GH in steers on 20 days following the implantation of anabolic compounds 172 Table 32 H a l f - l i f e , fractional disappearance rate and metabolic clearance rate of GH in steers on 40 days following the implantation of anabolic compounds 174 Table 33 Kinetic parameters of insulin metabolism in steers.. 189 Table 34 Kinetic parameters of insulin metabolism in steers implanted with anabolic compounds 190 - x i i -LIST OF FIGURES Page F i gu re 1 S t r u c t u r a l formulae of c e r t a i n important e s t r ogen i c and androgenic anabo l i c compounds 21 F i gu re 2 Schematic summary of N T - m e t h y l h i s t i d i n e metabol ism 34 F i gu re 3 E f f e c t of anabo l i c compounds on growth i n s t ee r s 47 F igure 4 Plasma urea n i t r ogen (PUN) and alpha amino n i t r o g e n (otAN) concent ra t ions i n s tee r s implanted w i t h va r ious anabo l i c compounds 49 F i gu re 5 Plasma urea-N and a lpha amino-N concent ra t ions i n s t ee r s implanted w i t h anabo l i c compounds 50 F i gu re 6 Plasma t o t a l e s s e n t i a l amino a c i d ( To t a l EAA) concent ra t i ons i n growing s tee r s on 28 days f o l l o w i n g the imp l an t a t i o n of anabo l i c compounds... 53 F i gu re 7 Body weight and average d a i l y ga in of s teer s 69 F i gu re 8 U r i na r y N T - m e t h y l h i s t i d i n e e x c r e t i o n i n s teer s on d i f f e r e n t per iods of the experiment 70 F i gu re 9 Muscle p r o t e i n degraded and synthes i zed i n s tee r s on d i f f e r e n t per iods of the experiment 73 F i gu re 10 R e l a t i o n s h i p between s k e l e t a l muscle p r o t e i n s yn thes i s and degradat ion i n s teer s 76 F i gu re 11 U r i na r y e x c r e t i o n of N T - m e t h y l h i s t i d i n e i n c a t t l e 77 F i gu re 12 U r i na r y N/t-methylh i s t id ine e x c r e t i o n i n s teer s implanted w i t h anabo l i c compounds 78 F i gu re 13 Rates of muscle p r o t e i n degradat ion and s yn thes i s i n s teer s implanted w i t h anabo l i c compounds 79 F i gu re 14 U r i na r y t o t a l hydroxypro l i ne (HYPRO) excreted by s tee r s on d i f f e r e n t per iods of the experiment 98 - x i i i -Figure 15 Urinary hydroxyproline-N [HYPRO-N expressed as a per cent of urinary total nitrogen] excretion by steers on different periods of the experiment 99 Figure 16 Urinary total hydroxyproline (HYPRO) excretion by steers on different days following the implantation of anabolic compounds 102 Figure 17 Effect of anabolic compounds on urinary total hydroxyproline excretion by growing beef steers.... 103 Figure 18 A schematic summary of the relationship between urinary hydroxyproline excretion and the metabolism of collagen 107 Figure 19 The disappearance of thyroxine from the plasma after a single intravenous injection of 12 to 16 mg of L-thyroxine in steers 138 Figure 20 Metabolic clearance and secretion rates of thyroxine on 20, 40 and 60 days following the implantation of anabolic compounds in steers....... 147 Figure 21 Effect of anabolic compounds on plasma GH concentration in growing steers 166 Figure 22 The disappearance of GH from the plasma following the intravenous injection of 6-8 mg of bovine GH (NIH-GH-B18) 168 Figure 23 Metabolic clearance and secretion rates of GH in steers implanted with anabolic compounds 175 Figure 24 The disappearance of insulin from the plasma of steers following a single intravenous injection of one mg bovine insulin 188 Figure 25 Metabolic clearance and secretion rates of insulin in steers implanted with anabolic compounds 192 Figure 26 Summary of the metabolic and endocrine effects of anabolic compounds in steers 201 - xiv -LIST OF APPENDICES Page Appendix I Sources of materials 232 Appendix II Radioiodination of growth hormone 234 Appendix III Plasma GH concentration in steers on different days following the implantation of anabolic compounds (EXPT. II B) 237 ACKNOWLEDGEMENTS I wish to express my gratitude to my research supervisor, Dr. W.D. Kitts, the Dean, Faculty of Agricultural Sciences, for his constant encouragement, valuable advice and guidance. Appreciation is extended to Dr. C.R. Krishnamurti (Professor, Department of Animal Science) for his interest, advice and criticisms during this research. I would also wish to thank Drs. D.B. Bragg (Professor and Head, Department of Poultry Science), H.C. Nordan (Professor, Department of Zoology) and W.D. Powrie (Professor and Head, Department of Food Science) for serving in my graduate committee. I wish to thank Dr. S. Raiti (National Pituitary Agency, Baltimore, Maryland) and the National Institute of Health (Bethesda, Maryland) for donating large quantities of bovine growth hormone (-300 mg) and ovine growth hormone radioimmunoassay materials. I thank Dr. D. Lyster (Professor, Pharmaceutical Sciences) for allowing me to use the gamma counter. Appreciation is also extended to Mr. P. Willing and Mr. E. Mirehouse for their help with the animal studies and to Mr. J. Ciok for constructing the metabolism crates. I thank my uncle and a l l the family members for their help and support during my stay in Vancouver. Finally, I thank my wife, Chitra and my family members in India for their patience, encouragement and understanding which enabled me to pursue my objectives. - 1 -INTRODUCTION With the dramatic increase in the world human population and with the increasing demand for food, techniques have been developed within the last 20 years to increase the productivity of domesticated animals for meat. Among the techniques are the parenteral administration of substances that are capable of increasing the growth rate and the efficiency of meat produced from farm animals. These substances include anabolic compounds and antibiotics to name a few. Anabolic compounds are those which when administered to animals are capable of increasing the growth rate, feed conversion efficiency and in general improving the carcass quality and quantity. A number of compounds have been identified to have such anabolic properties; they include the natural or synthetic compounds that belong to the sex-steroid group or compounds with a modified steroidal or non-steroidal structure. The choice of an appropriate anabolic compound is important which i s related to the sex-hormone status of the animal. In beef cattle, the estrogenic anabolic compounds are found effective i n increasing the growth rate of steers, while androgenic anabolic compounds are effective in heifers. The growth promoting properties of some of the anabolic compounds, namely diethylstilbestrol (DES) (Clegg and Cole, 1954), Zeranol (Sharp and Dyer, 1971), Synovex-S (Kahl et a l . , 1978) and trenbolone acetate (Heitzman et a l . , 1977) have been reported. The administration of anabolic compounds to ruminants in general results in improved live-weight gain. The carcass from treated animals contains a higher percentage of protein and a lower percentage - 2 -of f a t than the unt reated an imal s . In t h i s regard and anabo l i c compounds prov ide a p r a c t i c a l means of i n c r e a s i n g the e f f i c i e n c y of meat produced from ruminants. Even though the growth promoting p r o p e r t i e s of anabo l i c compounds i n ruminants have been repeated ly documented, the b i ochemica l mechanism by which they e l i c i t anabo l i c a c t i o n i s not c l e a r l y understood. Only r e c e n t l y s e ve r a l p o s s i b l e mechanisms of a c t i on s of anabo l i c compounds have been suggested (But te ry et a l . , 1978; P r e s t on , 1975). I t was proposed that anabo l i c compounds e l i c i t changes both on the endocr ine system and on the genera l metabol ism of the an ima l . Understanding the mechanism of a c t i o n of anabo l i c compounds i s important f o r the i d e n t i f i c a t i o n of growth processes that are i n f l uenced by these a c t i v e compounds. In t h i s study, the b iochemica l e f f e c t s of c e r t a i n e s t r ogen i c anabo l i c compounds, v i z . , DES, Zerano l and Synovex-S on the endocr ine system and on the metabolism of ruminants have been i n v e s t i g a t e d . A t t e n t i o n was focussed on the e f f e c t s of anabo l i c compounds on n i t r o g e n , s k e l e t a l muscle p r o t e i n and co l l a gen metabolism and on the metabolism of endogenous hormones such as the growth hormone, i n s u l i n and thy rox ine i n growing beef s t e e r s . - 3 -LITERATURE REVIEW Animal growth is a complex phenomenon involving the interaction of hormonal, nutritional and genetic factors. A precise knowledge of the animal growth process i s important in order to improve the efficiency of meat production. In this respect anabolic compounds have been found effective in increasing the growth rate of farm animals. In this review, a brief account of the concepts of growth along with various endogenous and exogenous hormonal factors that influence growth and metabolism of meat producing animals w i l l be presented. Anabolic compounds have been included under the broad heading, 'exogenous hormones and compounds'. In addition a review on the recent methods for studying protein turnover in the whole body, muscle and collagen is also presented. These methodological advancements have fac i l i t a t e d a better understanding of the growth process in animals and in time w i l l widen the knowledge on the mechanism of action of anabolic compounds. I. GENERAL CONCEPTS OF ANIMAL GROWTH a. Concepts and Definitions of Growth Animal growth is characterized by an orderly increase In mass of tissue and organs as well as in form and body composition. It is a complex phenomenon involving nutritional, hormonal and genetic interrelationships and can be described according to allometric relationships (Brody, 1945; Huxley, 1932). Young (1950) defined growth as the addition of materials to that which has already organized into a living pattern. Seebeck (1968) in - 4 -his review defined growth as the increase in weight of an animal or any part of i t , as i t approaches mature size. Normal growth, however, is d i f f i c u l t to define due to the complexity of both the animal and i t s environment. Fowler (1968) indicated two aspects of animal growth, viz: (a) the increase in mass (weight) per unit time, and (b) the aspect involving changes in form and composition resulting from differential growth of the component parts of the body. In physiological terms most tissues appear to have three stages of growth: (a) increase in c e l l number with l i t t l e increase in c e l l size (hyperplasia), (b) increase i n both c e l l number and c e l l size (hyperplasia and hypertrophy), and (c) increase in c e l l size or hypertrophy (Enesco and Leblond, 1962; Winick and Noble, 1965; Winick and Noble, 1966; Winick et a l . , 1972). The early definitions of growth and development in farm animals have been reviewed by Brody (1945), Richards and Kavanagh (1945), Pomeroy (1955), Palsson (1955), Needham (1964) and Seebeck (1968). A significant contribution to the knowledge on the growth patterns in farm animals has been made by the members of the Hammond School (Palsson, 1955). Their work has revealed that during foetal l i f e , the head and lower limbs of the animal grow relatively faster than the rest of the body, so that, at birth, the growing animal has a relatively large head and long legs with the trunk being short and shallow. After birth the growth rate of the upper limbs and trunk overtake that of the head and lower limbs and the body develops, so that the head and lower limb become a progressively smaller proportion of the - 5 -whole. These differential growth pattern or the waves of growth in farm animals was suggested by Palsson (1955). Palsson (1955) also suggested the phenomenon of changing priorities for nutrient use by different tissues as the animals develop. These patterns of tissue growth determine eventually the composition of growth. b. Composition of Growth and the Concept of  Nutrient partitioning Muscle, fat, bone and the connective tissues are the major components of the dressed carcass. In meat producing animals the pattern of growth of these major tissues (muscle, bone, fat) is important. The various factors that influence relative growth of muscle, fat and bone have been discussed in detail by Berg and Butterfield (1976) and include: (a) genetics (Lohman, 1971), (b) sex of the animal (Galbraith and Topps, 1981), and (c) the plane of nutrition (Callow, 1961). The skeletal muscle constitutes a major proportion of the carcass of the meat animals. The diversion of nutrients towards increased muscle growth w i l l be beneficial to increase the amount of meat produced. Bauman and Currie (1980) drew attention to the partitioning of nutrients in animals as they advance through different physiological stages. The concept of homeorhetic regulation or the regulation of nutrient flow between body pool and tissues has been recently presented by Bauman et^ a l . (1982). Byers (1982b) indicated that the actual partitioning of absorbed nutrients between protein and fat deposition depends upon a number of factors such as sex, hormonal regulation, - 6 -stages of growth and the intake of required nutrients. Trenkle (1981) indicated that the endocrine system integrates the metabolism of different organs and establishes different priorities of available nutrients. Indeed, i t w i l l be desirable to regulate the nutrient flow towards more protein than fat synthesis. But achieving increased protein synthesis at the expense of fat could affect the overall efficiency of animal production as indicated by Webster (1977) that animals retaining higher proportion of energy as protein are energetically less efficient than those retaining more energy in the adipose tissue. The hormonal influence on nutrient partitioning for increased skeletal muscle growth can be further illustrated by the hormonal differences between different breeds of cattle. The larger and lean breeds of cattle tend to have more growth hormone and less insulin in the plasma than the smaller breeds and such a hormonal interrelationship was thought to favour more muscle growth than shifting the nutrients and energy to adipose tissue (Trenkle, 1981). Hart et^ a l . (1975) indicated further the differences in the circulating hormonal level between the lactating dairy and beef cattle. The higher concentration of growth hormone and the lower concentration of insulin in the plasma of the dairy than in beef cattle was thought to increase the flow of nutrients towards the mammary gland than the skeletal muscle or the adipose tissue. The role played by hormones such as growth hormone and prolactin (Bauman et a l . , 1982), insulin (Prior and Smith, 1982) and by other nutritional factors (Byers, 1982b) in regulating nutrient - 7 -ut i l i z a t i o n and body composition has been reviewed thoroughly. The endocrine system seems to play an importnt role in regulating growth in animals and i t s composition. The endocrine balance in turn could be favourably manipulated by the strategic use of nutrients or by the use of other external factors (such as anabolic compounds). The concept of 'growth engineering' was suggested by Elsley (1976). The concept of waves of growth in animals (Palsson, 1955) provides hope that 'growth engineering' could be achieved by inhibiting the growth of an undesirable component of the body at i t s most dynamic phase of growth. II HORMONAL CONTROL OF GROWTH AND METABOLISM IN ANIMALS a. Endogenous Hormones Affecting Animal Growth  and Metabolism The various aspects of endocrine control on growth in experimental animals and human subjects have been reviewed recently (Daughaday, 1981). Of the endogenous hormones, growth hormone, somatomedin, prolactin, insulin, thyroid hormones, adrenal and gonadal steroids and other hormone-like growth factors play a v i t a l role in regulating growth and metabolism. 1. Growth Hormone Growth hormone (GH), also called the somatotrophin, has a striking trophic effect on the soma resulting in growth and maturation of the organs of the body. Endogenous GH is a single-chain polypeptide with two interchain disulfide bridges and has a molecular weight of about 22000. Aspects of GH Chemistry ( L i , 1975), biological effects (Engel and Kostyo, 1964; Merimee and Rabin, 1973) and i t s effect on - 8 -amino a c i d t r a n s p o r t , tRNA metabolism and p r o t e i n s ynthes i s e s p e c i a l l y i n s k e l e t a l muscle (Young, 1970; Young and P l u s k a l , 1977) have been thoroughly rev iewed. The growth promoting a c t i on s of bovine GH i n r a t s was demonstrated as e a r l y as 1931 by Evans and Simpson (1931). Brumby (1959) repor ted the ga in i n l i v e - w e i g h t and height i n young c a t t l e r e c e i v i n g GH. B a i r d et a l . (1952) demonstrated the presence of g r ea te r amount of GH i n the p i t u i t a r y of r a p i d l y growing p igs than i n those w i t h a lower growth r a t e . Amstrong and Hansel (1956) repor ted a p o s i t i v e c o r r e l a t i o n between the growth ra te of F r i e s i a n h e i f e r s and the GH content of the a n t e r i o r p i t u i t a r y . F u r t he r , the a d m i n i s t r a t i o n of GH to farm animals has r e s u l t e d i n i nc reased growth ra te and feed e f f i c i e n c y i n p igs (Mach l i n , 1972) and wether lambs (Wagner and Veenhuizen, 1978), and inc reased n i t r o gen r e t e n t i o n i n sheep (Davis et a l . , 1970; Wheatley et a l . , 1966; Wallace and Basset t 1966; Struempler and Burroughs, 1959) and i n s tee r s (Moseley et a l . , 1982). These repo r t s demonstrate c o n c l u s i v e l y the growth promoting p r ope r t i e s of GH i n farm an imal s . The i n f l u e n c e of GH on p r o t e i n anabol ism i s important among i t s metabo l i c a c t i on s i n the animal body. The GH i nc rea se s the net p r o t e i n d e p o s i t i o n and decreases the weight of the adipose t i s s u e i n the body. Chron ic a d m i n i s t r a t i o n of GH has been shown to i nc rea se p r o t e i n a c c r e t i o n i n sheep (Wagner and Veenhuizen, 1978). A decrease i n the plasma concen t r a t i on of amino a c i d and n i t r o gen e x c r e t i o n i n u r i n e a f t e r exogenous GH a d m i n i s t r a t i o n (Davis et a l . , 1970) demonstrates the e f f e c t of GH on amino a c i d uptake by the body t i s s u e s . Moreover, an inc reased - 9 -retention of calcium in sheep injected with GH was also reported (Braithwaite, 1975). Cheek and H i l l (1970) showed that the postnatal DNA synthesis in the muscle of rats is dependent upon GH. Trenkle (1974) indicated that the DNA accumulation in the muscle is abolished by hypophysectomy and that GH is capable of partially restoring DNA to near normal level. Peel et a l . (1981) demonstrated that the administration of GH to high producing dairy cows resulted in a 10-15% increase in the milk yield and a 17-18% increase in milk energy secretion. The GH also provides a homeorhetic signal and seems to influence the co-ordination of the metabolism among body tissues in a manner to support the partitioning of the nutrients for muscle protein accretion (Bauman et a l . , 1982). These concepts were similar to those illustrated by Machlin (1972) who found that the growth rate of pigs fed a restricted ration and administered with exogenous GH was comparable to those fed ad libitum. GH thus plays a v i t a l role In determining the growth rate of the animal and co-ordination of metabolism of the body in favour of more protein production. Therefore, i t appears that a potential exists to improve growth rate and manipulate metabolism of the animal by the stimulation of endogenous GH secretion. i i . Somatomedins Somatomedins are a family of circulating peptides thought to contribute to growth by the stimulation of proliferating cartilage (Van Wyk et a l . , 1974). They are formed in the l i v e r under the influence of GH and mediate most of the actions of the latter (Chochinov and - 10 -Daughaday, 1976) i n the promotion of p o s t n a t a l s k e l e t a l growth ( P h i l l i p s and V a s s i l o p o u l o u - S e l l i n , 1980). Van Wyk and Underwood (1978) proposed s p e c i f i c c ond i t i o n s f o r substances to q u a l i f y as somatomedins; that the substance should be GH-dependent, posses i n s u l i n - l i k e a c t i on s on the e x t r a s k e l e t a l t i s s u e s , and promote the i n c o r p o r a t i o n of su lphate i n t o c h o n d r o i t i n of the c a r t i l a g e . App ly ing these c r i t e r i a , a number of pept ides have been i d e n t i f i e d and c l a s s i f i e d under the gene ra l term somatomedins: they i n c l ude somatomedin-A (SM-A: H a l l , 1972), somatomedin-B (SM-B: Uthene, 1973), somatomedin-C (SM-C: Van Wyk e t ^ a l . , 1974), i n s u l i n - l i k e growth f a c t o r - I ( IGF- I : R inderknecht and Humbel, 1978a), i n s u l i n - l i k e growth f a c t o r - I I ( IGF - I I : R inderknecht and Humbel, 1978b) and the m u l t i p l i c a t i o n s t i m u l a t i n g a c t i v i t y (MSA: P i e r s o n and Temin, 1972). The p roduc t i on of somatomedins i s thought to be under the i n f l u e n c e of n u t r i t i o n ( P h i l l i p s and Young, 1976) as w e l l as under the i n f l u e n c e of va r i ou s other hormones bes ides GH (Holder and P reece , 1981). A l a r ge body of evidence supports the r o l e of somatomedins i n r e g u l a t i n g the somatic growth of meat producing an ima l s . Olsen et^ a l . (1981) repor ted a p o s i t i v e r e l a t i o n s h i p between serum somatomedin- l ike a c t i v i t y and the r e l a t i v e weight ga in i n lambs. Wangsness et a l . (1981) found serum somatomedins to be h igher i n f a s t e r growing S u f f o l k - s i r e d lambs than i n the s lower growing F i n n - s i r e d lambs. Somatomedins were a l s o found to be r e l a t e d to the r a t e of ga in and l i n e a r growth of Red Danish b u l l s (Lund-Larsen e t a l . , 1977) and swine (Lund-Larsen and Bakke, 1975). The b i o l o g i c a l a c t i on s of somatomedins have been reviewed by Holder and Preece (1981). - 11 -i i i . Prolactin P r o l a c t i n i s a l a c t o g e n i c hormone and i t s r o l e i n rep roduc t i on has long been recognized i n exper imenta l an ima l s . I t i s u s u a l l y cons idered to have other ove r l app ing b i o l o g i c a l a c t i o n s w i t h GH (Turner and Bagnara, 1976). However, not much a t t e n t i o n has been focused on the growth promoting p r ope r t i e s of p r o l a c t i n i n mammals. Bovine p r o l a c t i n has been shown to i nc rease the growth ra te of S n e l l dwarf mice ( W a l l i s and Dew, 1973) and r a t s (Bates et^ a l . , 1964). The anabo l i c e f f e c t s of sex s t e r o i d s , te s to s te rone prop ionate and DES i n sheep were p a r t l y a t t r i b u t e d to the inc reased endogenous p r o l a c t i n sec re to ry pa t t e rn (Davis et a l . , 1978). I t was demonstrated that the p o s i t i v e growth response i n lambs and h e i f e r s t r e a t ed w i t h s y n t h e t i c estrogens or ch ron i c t h y r o t r o p i n r e l e a s i n g hormone (TRH) a d m i n i s t r a t i o n was accompanied by an e leva ted c i r c u l a t i n g concent ra t i on s of both GH and p r o l a c t i n (Davis et^ a l . , 1976; Davis e£ a l . , 1977b; Davis et a l . , 1978). These observat ions suggest the r o l e p layed by p r o l a c t i n i n r e g u l a t i n g the growth of mammals. R e c e n t l y , Bauman et a l . (1982) suggested that p r o l a c t i n might d i r e c t l y or i n d i r e c t l y a l t e r the capac i t y of muscle f o r net p r o t e i n a c c r e t i o n as w e l l as metabolism i n other t i s s u e s such as adipose t i s s u e s , i n a manner to a l l ow g rea te r p a r t i t i o n i n g of n u t r i e n t s towards i nc rea sed muscle growth. Fu r the r evidence f o r the involvement of p r o l a c t i n on growth processes of animals could be obta ined from the repo r t s on the pho tope r i od i c r e g u l a t i o n of growth i n animals (Forbes et a l . , 1975; Schanbacher and Crouse, 1981). Lambs have been shown to - 12 -gain rapidly, convert feed to live weight more eff i c i e n t l y and to produce acceptable carcasses when exposed to long photoperiods (Forbes et a l . , 1979; Schanbacher and Crouse, 1980) and these changes in growth were accompanied by an increased serum prolactin concentration (Forbes et a l . , 1975). However, Eisemann et a l . (1981) reported that wether lambs receiving daily injection of prolactin for 9 weeks had no increase in growth rate over the respective controls. It can be concluded that the growth rate in animals could be related to their prolactin status and manipulation of growth could be achieved by altering prolactin status. i v . Insulin Insulin plays a complex role in the growth process. Several similar actions between insulin and somatomedins suggest that these two hormones may be acting through a common mechanism. Insulin is required for the expression of f u l l anabolic effects of growth hormone. The growth stimulating effects of insulin were demonstrated by Gey and Thalhimer (1924). It is an important growth-regulating hormone during fetal development. At birth there is a positive correlation between plasma insulin and birth weight of human infants (Shima et a l . , 1966). The growth promoting effects of insulin are best demonstrated by the presence of detectable growth in subjects without measurable amount of immunoreactive growth hormone, such as that associated with removal of pituitaries or hypothalamic tumors. Martinovich and Margolin (1975) reported an improvement in the rate of gain and feed efficiency in cattle receiving insulin injections every ten days. - 13 -Even though direct findings, such as those reported for cattle, on the effect of insulin on the growth process are limited, a number of workers have indicated indirectly the growth promoting properties of insulin. The poor growth or growth retardation in situations characterized by low insulin production as in diabetes mellitus (Schiff et^al., 1972) has been reported. Phillips and Orawski (1977) demonstrated that diabetic rats grew normally when given an adequate dose of insulin. Insulin also stimulates growth and proliferation of many c e l l types (Straus, 1981) and i s required by a variety of cells for optimal growth in hormone-supplemented serum free medium (Barnes and Sato, 1980). Insulin is a powerful anabolic hormone increasing the synthesis of DNA, RNA, nucleic acids and protein in the target tissues (Manchester, 1972). The biochemical events regulated by insulin have been reviewed by Straus (1981). In meat producing animals insulin plays a v i t a l role i n regulating carbohydrate and protein metabolism. Trenkle (1970) reported that in cattle the plasma insulin concentration increased with the length of time they were fed grain. A positive correlation was observed between plasma insulin concentration and fatness of the carcass (Trenkle and Irvin, 1970). Jefferson et a l . (1974) reported that insulin increases the incorporation of labelled amino acid into muscle protein i n non-ruminants. Preliminary observations by Chrystie et a l . (1977) indicate that in fetal lambs insulin decreases protein breakdown without any change in protein synthesis. The role played by insulin on - 14 -partitioning of nutrients for tissue growth has recently been reviewed by Prior and Smith (1982). In his review, Trenkle (1981) indicated that larger and leaner breeds of cattle tend to have more growth hormone and less Insulin in the plasma and that the smaller breeds have more insulin. The concentration of insulin in plasma was positively related to the carcass adipose tissue and negatively related to the amount of carcass protein. v. Thyroid Hormones Thyroid hormones are involved i n the regulation of growth and act as an important regulator of metabolic rate (Tata, 1964). The participation of thyroid hormones in the growth process could be best illustrated by the presence of decreased growth during hypothyroidism (Falconer and Draper, 1968) and following thyroidectomy (Bray, 1964). Thyroxine (T4) participates in growth and differentiation of bones. It seems that T4 acts synergetically with- GH in inducing the growth of longitudinal bones (Thorngren and Hansson, 1973). Only a few attempts have been made so far to explore the growth promoting properties of thyroid hormones in farm animals (Brumby, 1959; Hart and Laffey, 1959). However, attempts to increase the endogenous thyroid hormones have been made to alter growth and body composition in animals (Wagner and Veenhuizen, 1978). Wagner and Veenhuizen (1978) fed thyroprotein to increase the thyroid hormone levels in wether lambs and found an increase in the amount of feed required per unit gain and a decrease in carcass fat. They have also reported increased growth rate and protein deposition when thyroprotein was fed to lambs receiving GH injections. - 15 -I t was specu lated that t h y r o i d hormones may have been l i m i t i n g when growth was i nc reased w i th exogenous growth hormone ( T r enk l e , 1981). I t i s ev ident that the ac t i on s of t h y r o i d hormone on the growth process are complex i n v o l v i n g the i n t e r a c t i o n between many of the endogenous anabo l i c hormones. The synergism between t h y r o i d hormones and somatomedins has rece i ved much a t t e n t i o n r e c e n t l y . Gaspard et^ a l . (1978) showed that the treatment of hypophysectomized r a t s w i t h thy rox ine r e s u l t e d i n r e s t o r i n g growth and somatomedin a c t i v i t y even i n the absence of GH. Bes ides , t h y r o i d hormones a l s o p a r t i c i p a t e i n the maintenance of p i t u i t a r y - a d r e n a l gland f u n c t i o n (Hellman et a l . , 1961), i n the normal p r o l a c t i n synthes i s (Augustine and Hymer, 1978; Chen and Me i te s , 1969), i n the maintenance of normal l e v e l s of p i t u i t a r y ACTH ( F o r t i e r et a l . , 1970) and i n the promotion of the synthes i s of GH i n the somatotrophic c e l l s through the s t i m u l a t i o n of c y t o d i f f e r e n t i a t i o n or maturat ion of GH c e l l s or both ( I sh ikawa ejt a l . , 1976). The presence of t e t r a i odo th y r on i ne (TA) and t r i i o d o t h y r o n i n e (T3) i n serum of embryonic ch i ck has been s t r ong l y i m p l i c a t e d i n the embryonic growth (K ing and K i n g , 1978). I t i s ev ident that t h y r o i d hormones are i n vo l ved i n the growth process of an ima l s , but t h e i r a c t i on s seem to be complex i n v o l v i n g the p a r t i c i p a t i o n of other endogenous anabo l i c hormones. v i . Gonadal and Adrenal Steroids Among the s t e r o i d hormones secreted by the adrena l co r tex and the gonads, the g l u c o c o r t i c o i d s , androgens and estrogens exer t a s u b s t a n t i a l i n f l u e n c e on the somatic growth and matu ra t i on . Whereas the i n f l u e n c e - 16 -of the l a t t e r two hormones are s t i m u l a t o r y , g l u c o c o r t i c o i d s exert an i n h i b i t o r y e f f e c t on animal growth. Androgens and Estrogens The i n f l u e n c e of gonadal s t e r o i d s on growth and metabolism i s w e l l i l l u s t r a t e d by the e f f e c t of c a s t r a t i o n on animal growth as w e l l as t h e i r i n f l u e n c e on growth promotion and maturat ion dur ing puberty and ado lescence. The i n f l u e n c e of both androgens and estrogens can be seen by the d i f f e r e n c e s i n the growth r a te and body compos i t ion between the ca s t r a te s and t h e i r e n t i r e c oun te rpa r t s . B u l l s ga in f a s t e r , convert food e f f i c i e n t l y i n t o l i v e weight and have h igher per cent of l ean and lower per cent of f a t i n the carcass than s t e e r s . A s i m i l a r d i f f e r e n c e e x i s t s between h e i f e r s and spayed h e i f e r s . The i n f l u e n c e of sex and c a s t r a t i o n on body growth and compos i t ion i n c a t t l e , sheep and the p i g ( G a l b r a i t h and Topps, 1981), the involvement of androgens and estrogens i n va r i ou s aspects of growth (Wiedemann, 1981) and t h e i r mo lecu la r mechanism of a c t i o n (Young and P l u s k a l , 1977) have been rev iewed. The advancements i n the knowledge of the e f f e c t of androgens and estrogens on growth processes of animals have lead to t h e i r a p p l i c a t i o n d i r e c t l y or through t h e i r s y n t h e t i c counterpar t s to manipulate growth, feed convers ion e f f i c i e n c y and body compos i t ion i n meat producing animals (Lu and Rendel , 1976). These compounds have gene r a l l y been des ignated as ' a n a b o l i c compounds'. A d e t a i l e d account of t h e i r a p p l i c a t i o n i n meat producing animals i s d e a l t s e p a r a t e l y . Glucocorticoids The involvement of g l u c o c o r t i c o i d s , e s p e c i a l l y , c o r t i c o s t e r o i d s - 17 -i n normal growth has been d i scussed by Loeb (1976). G l u c o c o r t i c o i d s exer t a c a t a b o l i c e f f e c t i n the animal body. As e a r l y as 1938, Ing le et a l . (1938) demonstrated that r a t s t r ea ted w i t h the adrena l co r tex e x t r a c t s f a i l e d to ga in weight no rma l l y . Wel l s and K e n d a l l (1940) observed a s i g n i f i c a n t r e t a r d a t i o n of growth and bone matura t ion i n weaning r a t s t r ea ted w i th 1 mg/d of e i t h e r C o r t i s o l or c o r t i c o s t e r o n e . The c a t a b o l i c e f f e c t s of g l u c o c o r t i c o i d s seem to be d i r e c t e d towards the growth and metabolism of s k e l e t a l muscle. The a d m i n i s t r a t i o n of c o r t i c o s t e r o i d s to animals has been shown to cause l o s s of s k e l e t a l muscle p r o t e i n (Munro, 1964) which i s a t t r i b u t e d to an i n h i b i t i o n of muscle p r o t e i n s ynthes i s and an a c c e l e r a t i o n of muscle p r o t e i n breakdown (Young, 1970). Bes ides , s e ve r a l workers have proposed impai red somatomedin generat ion or the d i r e c t i n h i b i t i o n of somatomedin a c t i v i t y i n the s k e l e t a l t i s s u e as p o s s i b l e mechanisms f o r the g rowth - re ta rd i ng e f f e c t of g l u c o c o r t i c o i d s ( E lde r s et a l . , 1975; Van Den Brande et a l . , 1975). v i i . Miscellaneous Endogenous Growth Factors Bes ides the major endogenous hormones that r egu l a te growth and metabolism d i scussed i n the prev ious s e c t i o n s , there are a l s o s e ve r a l o ther growth f a c t o r s i n the serum w i t h hormone- l ike a c t i v i t y that have been s t r ong l y i m p l i c a t e d i n the growth processes (Table 1 ) . Even though the i n f l u e n c e of growth f a c t o r s i n the serum on the growth s t i m u l a t i o n of c u l t u r e d c e l l s has been w ide l y r epo r ted , t h e i r s i g n i f i c a n c e i n c o n t r o l l i n g growth and metabolism i n farm animals has not been - 18 -Table 1. Serum Hormone-Like Growth Fac to r s Fac to r s Target C e l l 1. Nerve Growth Facto r (NGF) Sympathetic Nerves 2. Ep idermal Growth Facto r (EGF) Ep idermal C e l l s 3. F i b r o b l a s t Growth Fac to r (FGF) Mesodermal C e l l s A. Ovar ian Growth Fac to r Ovary 5. Myoblast Growth Fac to r Myoblasts 6. Thrombopoiet in Thrombocytes 7. Thymosin Thymocytes 8. Melanocyte S t i m u l a t i n g Fac to r Melanocytes XYoung (1980) - 19 -understood. The importance of growth factors, especially fibroblast proliferative activity (FPA) in lambs has been reported (Olsen et^ a l . , 1981; Wangsness et a l . , 1981). b. Exogenous Hormones and Compounds Affecting Growth,  Body Composition and Metabolism of Animals The term "exogenous hormones and compounds" has been used loosely. For the purposes of this study the various anabolic compounds that are commonly used in animal agriculture to alter growth and body composition are included under the broad term 'exogenous hormones and compounds'. As a vast body of information is available on this subject only the salient features of exogenous compounds on growth and body composition are presented. The historical and other background information on anabolic compounds such as, their usage, their effect on growth, body composition and various implications of their use can be obtained from reviews (Galbraith and Topps, 1981; Heitzman, 1980; Hoffman, 1980; Lu and Randel, 1976; Preston, 1975). 1. Anabolic Compounds: Concepts and Definitions When used in the context of animal production, the term anabolic compounds include the male and female sex hormones, the non-steroidal estrogens as well as GH and insulin or in general those compounds that promote growth and anabolism in animals. The term, however, when used by a cl i n i c i a n or endocrinologist denotes only those steroids whose chemistry is derived from testosterone and 19-nortestosterone. Estrogens, though possessing anabolic properties do not belong to this class according to the latter definition. In general, anabolic - 20 -compounds could be def ined as those which when admin i s tered to animals cause (1) i n c rea se i n growth r a t e , (2) i nc rea se i n muscle mass and p r o t e i n d e p o s i t i o n , (3) i nc rea se i n n i t r o gen r e t e n t i o n , (4) improve feed e f f i c i e n c y , (5) change i n f a t d e p o s i t i o n and d i s t r i b u t i o n and (6) improve a p p e t i t e . The expres s ion of these ba s i c c h a r a c t e r i s t i c s of anabo l i c compounds i s , of course dependent e n t i r e l y upon sex, environment i n c l u d i n g n u t r i t i o n , spec ies and age of the animals to which they are app l i ed t o . A number of exogenous compounds have been i d e n t i f i e d to e x h i b i t the above c r i t e r a and are b road ly c l a s s i f i e d i n t o the f o l l o w i n g groups: a. androgenic anabo l i c compounds b. e s t r ogen i c anabo l i c compounds A summary of e s t r ogen i c and androgenic compounds which are commonly i n use, t h e i r chemical s t r u c t u r e and the animal species or sex whereupon they favourab ly express t h e i r anabo l i c p r ope r t i e s i s presented i n F i gu re 1 and Table 2. i i . Aspects of Growth and Protein Metabolism The improved growth response, inc reased p r o t e i n depo s i t i o n e s p e c i a l l y of muscle p r o t e i n and decreased f a t a c c r e t i o n i n the carcasses of animals implanted w i t h anabo l i c compounds have played a s i g n i f i c a n t r o l e i n the wide s ca le a p p l i c a t i o n of anabo l i c compounds i n meat producing an imal s . Byers (1982 a) reported inc reased p r o t e i n growth i n y e a r l i n g s t ee r s from 90 and 106 g f o r non- implanted c a t t l e to 118 and 154 g f o r DES implanted c a t t l e . I t was concluded that DES increased the p r o t e i n - 21 -Androgenic Anabolic Compounds O H = acetate TESTOSTERONE TRENBOLONE ACETATE OH Estrogenic Anabolic Compounds OH HO Estradiol - j - c = 0 Progesterone ESTRADIOL- 170 SYNOVEX-S HO HEXOESTROL OH HO O C H 3 DIETHYLSTILBESTROL Z E R A N O L Figure 1. Structural formulae of certa in important estrogenic and androgenic anabolic compounds. - 22 -Table 2. Anabo l i c compounds a v a i l a b l e f o r use i n farm animals Anabo l i c Compound Method of A p p l i c a t i o n Species E f f e c t i v e i n 1. Androgenic: Testos terone Trenbolone aceta te Sc I, 0 Sc. I W, S, H C, B, Turkey E s t r o g e n i c : DES** DES-prop ionate* * E s t r a d i o l - 173 H e x o s t e r o l * * Z e r a n o l 2 * * * Phytoestrogens * Sc. I, F 0 Sc. I Sc. I Sc. I F S, B V S, H W, S S, H, V, L W Androgen + Estrogen Testosterone & e s t r a d i o l Testosterone & e s t r a d i o l benzoate 1* Trenbolone acetate & e s t r a d i o l Trenbolone acetate & h e x o e s t r o l Trenbolone acetate & ze rano l Sc. I Sc. I Sc. I Sc. I Sc. I H, V H S, B, V, W, Boars S S, V c o n t ' d . Table 2 c o n t ' d . . Method of Species E f f e c t i v e Anabo l i c Compound A p p l i c a t i o n i n 4. Progestogens Melongesterone acetate (MGA)** 0 H 5. Estrogens + Progestogens Progesterone & e s t r a d i o l benzoate 5 Sc. I S Progesterone & e s t r a d i o l Sc. I S t rade name = F i n a p l i x trade name = Ra l g ro trade name = Imp l i x Bf **Trade name = Synovex-H 5 t r a d e name = Synovex-S 6 t r a d e name = Imp l i x BM 7 though not s t r i c t l y used, i t s p o t e n t i a l , e s p e c i a l l y f o r coumestrol has been i n d i c a t e d ( O l d f i e l d et a l . , 1966) S = S tee r ; B = B u l l ; W = Wether Lambs C = Cu l l ed beef cow; V = Vea l c a l f ; H = H e i f e r * = n a t u r a l l y o c cu r r i n g i n p l an t s * * = s y n t h e t i c compounds * * * = mod i f i ed form of n a t u r a l l y o c cu r r i n g compounds Method of a d m i n i s t r a t i o n : 0, o r a l ; Sc. I, Subcutaneous imp lant ; F, i n feed - 24 -growth of c a t t l e and mimicked responses i n p r o t e i n growth of c a t t l e w i t h g rea te r genet i c p o t e n t i a l f o r p r o t e i n growth. Bes ides , Byers (1982 b) a l s o concluded that anabo l i c compounds d i v e r t e d energy to p r o t e i n storage which r e s u l t e d i n g rea te r p r o t e i n accumulat ion and l e s s f a t at any s e l e c ted weight. From a d e t a i l e d chemical a n a l y s i s of the carcass obta ined from DES implanted s t e e r s , Rumsey et a l . (1981) observed i nc reased p r o t e i n ga in and concluded that DES inc reased the r a te of ga in (17% h igher ) and p r o t e i n d e p o s i t i o n (28% h igher ) and reduced f a t d e p o s i t i o n . A s i m i l a r improvement i n the l i v e weight ga in and p r o t e i n d e p o s i t i o n due to Synovex-S i m p l a n t a t i o n i n s tee r s has a l s o been repor ted (Rumsey, 1982). Ze r ano l , though weaker i n i t s response, has a l s o been shown to improve l i v e weight ga in and p r o t e i n d e p o s i t i o n (Sharp and Dyer, 1971). Most of the research p e r t a i n i n g to the anabo l i c e f f e c t s of androgenic anabo l i c compounds, e s p e c i a l l y t renbolone aceta te e i t h e r alone or i n combinat ion w i t h other anabo l i c compounds has been c a r r i e d out i n the U.K. G r i f f i t h s (1982) concluded that a combination of t renbolone acetate and ze rano l i n s tee r s r e s u l t e d i n improved l i v e weight ga in and n i t r o gen r e t e n t i o n and the r e s u l t a n t carcass conta ined more lean meat (muscle mass) and p r o t e i n and l e s s f a t . Burch et_ a l . (1982) s tud ied f a t t y a c i d s ynthes i s i n wether lambs implanted w i t h anabo l i c compounds ( p repa ra t i on con ta i n i n g t renbolone acetate and e s t r a d i o l - 1 7 $ ) and found a r educ t i on i n the a c t i v i t i e s of enzymes a s soc i a ted w i th f a t t y a c i d s y n t he s i s , f a t t y a c i d synthetase and a c e t y l - c o A ca rboxy la se . The e f f e c t of va r i ou s anabo l i c compounds on - 25 -the body growth and p r o t e i n metabolism has been reviewed e x t e n s i v e l y ( G a l b r a i t h and Topps, 1981; P r e s t on , 1975). I t i s apparent that anabo l i c compounds a l t e r growth and body compos i t ion i n farm animals.. Most of the compounds seem to i n f l u e n c e muscle p r o t e i n d e p o s i t i o n and thus the muscle seems to be e i t h e r a d i r e c t or i n d i r e c t t a r g e t . The d i r e c t i n f l u e n c e of anabo l i c compound, t renbo lone aceta te on muscle p r o t e i n metabolism was s tud ied u s ing the n o n - r e u t i l i z a b l e amino a c i d , N T - M e t h y l h i s t i d i n e (Vernon and But te ry 1978a). S ince the major c o n t r o l over muscle p r o t e i n metabolism appears to be hormonal, the need f o r f u tu re research on the e f f e c t of a nabo l i c compounds on muscle p r o t e i n metabolism i s obv ious. i i i . Endogenous Hormonal Changes as Influenced by the  Application of anabolic compounds A number of hormones admin i s tered exogenously have been shown to a l t e r the c i r c u l a t i n g concen t ra t i on of endogenous hormones i n an ima l s . Thus the man ipu la t i on of growth and metabolism i n farm animals as i n f l u enced by the a d m i n i s t r a t i o n of exogenous hormones could p a r t l y be a t t r i b u t e d to the subsequent changes i n the endogenous hormones such as GH, i n s u l i n , t h y r o i d hormones, p r o l a c t i n , somatomedins and other serum growth f a c t o r s . The i n f l u e n c e of a l l the endogenous hormones on growth and body compos i t ion of animals was d i scussed e a r l i e r ( Sec t i on I I ) . A summary of the changes o c cu r r i n g i n the endogenous c i r c u l a t i n g hormone concent ra t i on s f o l l o w i n g the a d m i n i s t r a t i o n of c e r t a i n commonly used anabo l i c compounds i s presented i n Table 3. The changes i n the endogenous hormone concen t ra t i on f o l l o w i n g the a d m i n i s t r a t i o n of Table 3. Changes i n the endogenous hormone concent ra t i on f o l l o w i n g the a d m i n i s t r a t i o n of a nabo l i c compounds Endogenous Hormones Serum Anabo l i c Compound Growth Hormone I n s u l i n Thyroxine P r o l a c t i n b Somatomedins Growth-Factors Estrogenic Anabolic Compounds: a. Zerano l Increased ( 1 , 2, 3)* Increased ( 1 , 4, 5) Unchanged (2) Depressed (3) Unchanged or s l i g h t l y depressed (6) Depressed (3) Unchanged (4) Unchanged (4) i ON b. DES Increased (3 , 7, 8, 9, 10) Increased (7, 8, 9, 10) Increased Increased (11) N.A. d N.A. ' c . Synovex-S N.A. N.A. Increased (12) N.A. N.A. N.A. d . E s t r a d i o l - 1 7 0 Increased (13) Increased (13) Depressed (13) S l i g h t l y Increased (13) N.A. N.A. Androgenic Anabolic Compound: a. Trenbolone aceta te Unchanged (14) Unchanged (13 ,14) Depressed (13, 14) No Change (13) N.A. N.A. changes o c cu r r i n g i n farm animals are only cons idered i n the t a b l e c o n t 1 d . . . Table 3 cont'd. b somatomedin or somatomedin-like activity c numbers in the parenthesis indicate reference number d N.A. not available for farm species References: (Number corresponds to number in the parenthesis) 1. Olsen et a l . (1977) 2. Borger et a l . (1973) 3. Wiggins et a l . (1976) 4. Wangsness et a l . (1981) 5. Sharp and Dyer (1970) 6. Wiggins et a l . (1979) 7. Preston (1975) 8. Trenkle (1976b) 9. Trenkle (1970a) 10. Trenkle (1969) 11. Davis et a l . (1978) 12. Kahl et a l . (1978) 13. Donaldson et a l . (1981) 14. Heitzman et a l . (1977) - 28 -anabolic compounds have been reviewed recently by Buttery et a l . (1978) and Preston (1975). Whereas i t is evident that the application of exogenous hormones, such as anabolic compounds, alter endogenous anabolic hormone status, the mechanism by which they evoke such changes or the dynamics of endogenous hormone secretion is not clearly understood. I l l Methodological Aspects to Study Protein Metabolism i n Animals Treated with Anabolic Compounds a. Measurement of Protein Synthesis Protein synthesis in the body and tissues could be estimated by conventional nitrogen balance techniques or by the use of methods involving either the radiotracer or stable isotopes of amino acids. Whereas only the net balance between the dietary intake and nitrogen excretion could be obtained by the former method, insight into the dynamic aspects of protein turnover could be obtained by the latter. A variety of techniques are available to measure protein synthesis in the whole body or in selected tissues and have been exhaustively reviewed (Waterlow a l . , 1978). 1. Continuous Infusion Method The continuous infusion of a labelled amino acid intravenously into the metabolic amino acid pool of the body has thus far been the most commonly used method for the determination of protein synthesis in the whole body or in selected tissues of the body (Garlick et^ a l . , 1973; Garlick et a l . , 1976b). Briefly when a labelled amino acid ( 3H or ll*C) is introduced continuously into the blood, after some time the - 29 -s p e c i f i c a c t i v i t y of the i n t roduced amino a c i d i s r a i s e d to a p l a teau (steady s t a t e ) and remains e s s e n t i a l l y constant dur ing the re s t of the i n f u s i o n p e r i o d . From the s p e c i f i c r a d i o - a c t i v i t y of the f r ee and p r o t e i n bound amino a c i d at the end of the i n f u s i o n p e r i o d , the r a te of p r o t e i n s yn thes i s i n i n d i v i d u a l t i s s u e s can be est imated ( G a r l i c k et a l . , 1973)-. The whole-body r a te of p r o t e i n s ynthes i s can be c a l c u l a t e d from the amino a c i d f l u x (Waterlow and Stephen, 1968). The method has been d i scussed i n cons ide rab le d e t a i l by G a r l i c k et a l . (1973) and G a r l i c k et a l . , (1976b). The a p p l i c a t i o n of t h i s method to farm animals has been repor ted i n sheep by But te ry et a l . (1975), i n the p i g by G a r l i c k et a l . (1976a), and i n c a t t l e by Lobley et a l . (1980). i i . Feeding Method An a l t e r n a t i v e to the continuous i n f u s i o n method i s the method i n v o l v i n g the feed ing of the l a b e l l e d amino a c i d i nco rpo ra ted i n t o an agar g e l (Harney et a l . , 1976). Th is method avoids the cannu la t i on and r e s t r a i n i n g of the animal dur ing the long i n f u s i o n per iod and has been repor ted to y i e l d s l i g h t l y d i f f e r e n t est imates of s y n t h e t i c ra te s compared to the continuous i n f u s i o n method. A summary of methods a v a i l a b l e f o r s tudy ing p r o t e i n s yn thes i s i s presented i n Table 4. b. Measurement of Muscle Protein Degradation 1. Isotoplc Method I s o t o p i c methods have o f t en used the disappearance of l a b e l from the p r o t e i n ( s ) a f t e r l a b e l l i n g them w i t h r a d i o - l a b e l l e d t r a c e r s ( G a r l i c k - 30 -Table 4. In vitro and in vivo methods for the quantitation of protein turnover in different species (Summary) Protein Method Species Reference PROTEIN SYNTHESIS PULSE LABELLING Mixed Sarcoplasmic 1I+C - Na 2C0 3 Piglet Perry (1974) Mixed Myofibrillar ll+C - Na 2C0 3 Piglet Perry (1974) Mixed Muscle lkC - Lysine Rat Lajtha et a l . (1957) Intact Soleus U( 1 1 +C) - Tyrosine (in vitro) Rat L i and Goldberg (1976) CONTINUOUS INFUSION Mixed Muscle Lysine infusion Rat Waterlow & Stephen (1968) Mixed Muscle Glycine infusion Rat Garlick (1969) Mixed Muscle & Liver ll*C - Tyrosine infusion Pig Garlick et al.(1976a) Mixed Muscle & Whole 1 4C — Tyrosine infusion Rat Garlick et a l . (1973) Body Whole Body & Tissues 3H - Tyrosine & 3H -Leucine Infusion Cattle Lobley et a l . (1980) Tissues (Muscle & C - Leucine or 1 4C - Lysine Pig Simon et a l . (1978) Liver) - 31 -Table 4 c o n t ' d . . . . P r o t e i n Method Species Reference PROTEIN DEGRADATION Mixed Sarcoplasmic & M y o f i b r i l l a r lhC - Na 2 C0 3 Rat M i l l w a r d (1970) Mixed Muscle ***C - A s p a r t i c a c i d Rat Young et a l . (1971) A l d o l a s e , A c t i n & Myosin 1 4*C - Pheny la l an ine Rabb i t V e l i c k (1956) Mixed Sarcoplasmic lkC - Na 2 C0 3 P i g l e t Pe r ry (1974) Mixed Muscle Tyros ine r e l ea se ( i n v i t r o ) Rat L i & Goldberg (1976) M y o f i b r i l l a r P r o t e i n s N T - M e t h y l h i s t i d i n e T Rat , Rabb i t , Man Young & Munro (1978) M y o f i b r i l l a r P r o t e i n N - M e t h y l h i s t i d i n e C a t t l e H a r r i s & M i lne (1981a) M y o f i b r i l l a r P r o t e i n X N - M e t h y l h i s t i d i n e Sheep* H a r r i s & M i lne (1980) M y o f i b r i l l a r P r o t e i n T N - M e t h y l h i s t i d i n e P i g * H a r r i s & M i lne (1981b) * s i g n i n d i c a t e s i n v a l i d i t y of the method - 32 -e t a l . , 1973; Waterlow and Stephen, 1967). The r e s u l t s obta ined from such methods are o f ten compl icated by the i n t e r c e l l u l a r (between c e l l s ) and i n t r a c e l l u l a r ( w i t h i n c e l l s ) r e c y c l i n g of amino ac ids and a l s o due to the hete rogen ie ty of p r o t e i n degradat ion ra tes ( G a r l i c k et a l . , 1976 a ) . A comparison of degradat ion ra te s of m y o f i b r i l l a r p r o t e i n s us ing l a b e l l e d amino ac ids has shown great d i s c r epanc i e s i n h a l f - l i f e va lues which ranges from 20 to 145 days (Swick and Song, 1974). Fashak in and Hegsted (1970) found that r a t s g iven l l*C-amino a c i d l o s t the l a b e l from the plasma w i t h a h a l f - l i f e of about 18 days, wh i l e i n j e c t i n g the r a t s w i t h p r e l a b e l l e d plasma p r o t e i n y i e l d e d h a l f - l i f e va lues of 3 days or l e s s (Anker, 1960). S i m i l a r d i s c repanc i e s i n the degradat ion r a te s due to the k ind of t r a c e r used were repor ted by Swick and Ip (1974). The r a t e of decay of r a d i o a c t i v i t y from a albumin was about 30% slower when 1 4 C - g u a n i d i n o - a r g i n i n e was used than l l 4 C -b i c a r bona te (Swick and Ip, 1974). i i . Non-Isotopic Method U t i l i z i n g Non-Reutilizable  Amino Acid The t e c h n i c a l problems a s s oc i a t ed w i t h the r e u t i l i z a t i o n of amino ac id s have lead to the i d e n t i f i c a t i o n of an amino a c i d or a product of t i s s u e degradat ion which i s not r e u t i l i z e d f o l l o w i n g deg radat ion . To serve as an i n v i v o index of muscle p r o t e i n breakdown, the amino a c i d s e l e c t e d should have the f o l l o w i n g c h a r a c t e r i s t i c (Munro, 1970): 1. Should be chemica l l y mod i f ied a f t e r the pept ide bond s yn t he s i s . 2. Such chemica l l y mod i f ied groups should not undergo exchange once they appear i n the p r o t e i n bound amino a c i d . - 33 -3. The concentration of the amino acid should be known or should be constant in the muscle protein. 4. Should not be formed to an important extent in other proteins. 5. Should be released at the same time as other amino acids from the completed protein. 6. Should not undergo further metabolism in the body. 7. Should have a low renal threshold. 8. Should be quantitatively excreted in the urine. N TMethylhistidine (NTMH) or 3-Methylhistidine (3-MH) has been identified as one such amino acid satisfying a l l the above mentioned c r i t e r i a (Young and Munro, 1978). Briefly, NT-MH originates from the degradation of actin and some species of myosin heavy chains in which i t is present after the post-translational methylation of specific histidine residues (Munro and Young, 1978; Figure 2). Its synthesis, metabolism and validity to serve as an in_ vivo index of myofibrillar protein degradation in many species including man have been reviewed (Young and Munro, 1978; Ward and Buttery, 1978). This method has been shown to give reasonable estimation of myofibrillar protein degradation rates comparable to the ll*C-Na2C03 method of Millward (1970) and seems to be an attractive technique in large farm animals as i t does not require the use of isotopes and the destruction of the animal. Harris and his co-workers validated this technique for use in farm species and concluded that the urinary NT-MH excretion to be a - 34 -MUSCLE SAME t C H , -METHIONINE (RECYCLII PROTEIN BREAKDOWN NG) + N L M H Transamination'-'' & Oxidation ~\ (actin & . 1 myosin) AMINO ACIDS 1 SYNTHESIS 2. BREAKDOWN 3 RELEASE BLOOD N-METHYLHISTIDINE 5 EXCRETION KIDNEY N-AcetVI-NlMH LIVER 4 MODIFICATION (RAT) • URINE a N'-MH b N-Acetyl-N-MH Figure 2. Schematic summary of N T-methylhistidine metabolism. - 35 -valid index of myofibrillar protein degradation in cattle (Harris and Milne, 1981 a) but an invalid index in both sheep and the pig (Harris and Milne, 1980; Harris and Milne, 1981 b). In the latter species the large body pool of non-protein bound NT-MH has been attributed for i t s unsuitability to serve as an index of myofibrillar protein degradation (Harris and Milne, 1980, 1981b). It could be concluded that the technique of measuring NT-MH excretion in urine presents a great potential to study protein degradation in large farm animals and i t s regulation by endogenous and exogenous hormonal procedures. Since the major effect of anabolic compounds seem to be on the skeletal muscle protein, the technique of measuring NT-MH in urine presents potential for studying muscle protein metabolism in such conditions and to understand the mechanism of action of anabolic compounds on the skeletal muscle. The data obtained from this method, however, should be considered with caution. Recently Millward et a l . (1980b) raised serious doubts as to the validity of the method to serve as an index of myofibrillar protein degradation since N*-MH is also present in tissues other than the skeletal muscle. The validity of the method, however, was reappraised by Harris (1981). A summary of methods available for studying protein degradation is presented in Table 4. c. Collagen Turnover Collagen i s a hydroxyproline rich protein, representing about 30% of the total body protein. It is a predominant protein in the skin, - 36 -bone and tendon and performs mainly a s t r u c t u r a l r o l e . The chemist ry , b iochemi s t r y and the metabo l i c aspects of c o l l a gen have been reviewed by K i v i r i k k o (1970). i . Measurement of Collagen Synthesis During c o l l a gen b i o s y n t h e s i s , p r o l i n e i s i n co rpo ra ted i n t o the po l ypept ide cha in and i s then hydroxy la ted to hydroxypro l i ne by the p r o t o c o l l a g e n - p r o l i n e - h y d r o x y l a s e system (Peterko f sky and Uden f r i end , 1965). The i n c o r p o r a t i o n of r a d i o a c t i v e p r o l i n e i n t o c o l l a gen as l a b e l l e d hydroxypro l i ne has been w ide l y used to measure c o l l a gen s y n t h e t i c r a te s (Robins, 1981). The other methods f o r s tudy ing c o l l a gen s yn the s i s , i n c l ude (a) the measurement of s p e c i f i c r a d i o a c t i v i t y of the r e l e van t tRNA-bound amino a c i d (McKee et a l . , 1978), and (b) the measurement of s p e c i f i c r a d i o a c t i v i t y of the nascent po lypept ide removed from ribosomes ( I l a n and S inger , 1975). 11. Measurement of Collagen Degradation The enzymes capable of degrading co l l a gen have been found i n numerous t i s s u e s of l i v i n g animals (Horwitz e t a l . , 1977; L i o t t a et a l . , 1979). Dur ing the degradat ion of c o l l a g e n , hydroxypro l i ne i s re lea sed and excreted i n u r i ne as f r ee or bound to sma l l p r o te i n s ( K i v i r i k k o , 1970). Hence, the u r i n a r y e x c r e t i o n of hydroxypro l i ne has been wide ly used as an index of co l l a gen turnover In humans and exper imenta l animals ( K i v i r i k k o , 1970) and to a l i m i t e d extent i n farm animals (B lack and Capen, 1971; Gordon, 1982). - 37 -The complexity of growth processes in animals, the role played by various endogenous and exogenous hormonal factors on animal growth and the need for studies on the mechanism by which anabolic compounds influence growth and metabolism of meat producing animals are readily apparent. - 38 -STUDY I METABOLIC ASPECTS OF ANABOLIC COMPOUND ACTION IN GROWING BEEF STEERS Study I was conducted to explore the various metabolic effects of estrogenic anabolic compounds in growing beef steers and includes the following experiments: EXPERIMENT IA: Effect of anabolic compounds on growth and nitrogen metabolism in growing beef steers. EXPERIMENT IB: Muscle protein metabolism In steers as determined by the urinary N T-methylhistidine excretion as an in vivo index of myofibrillar protein breakdown: Developmental aspects of muscle protein metabolism and the effect of anabolic compounds. Collagen metabolism in growing beef steers as assessed by the urinary hydroxyproline excretion and the effect of estrogenic anabolic compounds. EXPERIMENT IC: - 39 -EXPERIMENT IA EFFECT OF ANABOLIC COMPOUNDS ON GROWTH AND NITROGEN METABOLISM IN GROWING BEEF STEERS INTRODUCTION Anabolic compounds are used extensively i n animal agriculture to increase the efficiency of meat production. The growth promoting effects of zeranol (Sharp and Dyer, 1971; Borger et a l . , 1973), diethylstilbestrol (Clegg and Cole, 1954; Oltjen et a l . , 1973), Synovex-S (Kahl et a l . , 1978; Rumsey, 1982) and trenbolone acetate (Heitzman et a l . , 1977) have been reported and the effect of hormones on the growth and body composition has been recently reviewed by Galbraith and Topps (1981). Even though a number of studies have established the growth promoting potentials of anabolic compounds, the mode of action has not been conclusively demonstrated. Recently, several possible mechanisms of action of anabolic compounds have been reported (Buttery et a l . , 1978). It was suggested through these studies that anabolic compounds e l i c i t changes both at the endocrine and at the metabolite level, the latter through changes In the intermediary metabolism. In ruminants, estrogenic anabolic compounds have been reported to increase the endogenous concentrations of growth hormone and insulin (Preston, 1975) and thyroid hormones (Kahl et a l . , 1978). The effects of anabolic compounds in altering nitrogen retention (Vanderwal, 1976), fractional rate of protein synthesis in different tissues (Vernon and Buttery, 1978b) and protein retention (Rumsey, 1982) have also been reported. - 40 -Whereas the e f f e c t s of DES on n i t r o g e n metabolism have been s tud ied e x t e n s i v e l y , on ly a few attempts have been made to e l u c i d a t e the metabo l i c e f f e c t s of more commonly used anabo l i c compounds, v i z . Zeranol and Synovex-S, e s p e c i a l l y on plasma f ree amino ac id s and hepa t i c enzymes of amino a c i d ca tabo l i sm. This study was t he re fo re conducted to i n v e s t i g a t e the e f f e c t of e s t r o gen i c anabo l i c compounds ( Ze rano l , DES, Synovex-S) on n i t r o gen metabol ism i n growing beef s t e e r s . A t t e n t i o n was focussed on the plasma l e v e l s of urea-N, a lpha amino-N, amino a c i d and hepa t i c l e v e l s of amino a c i d c a t a b o l i z i n g enzymes. MATERIALS AND METHODS Experimental Animals and their Management Herefo rd beef s t e e r s , weighing approx imately 380 kg, were used. Upon a r r i v a l at the U n i v e r s i t y of B r i t i s h Columbia Beef C a t t l e Teaching and Research U n i t , the animals were weighed and i n j e c t e d w i t h 1 2 Poten-ADE to c o r r e c t any v i t am in d e f i c i e n c y a long w i t h Penlong-S p lus to prevent i n f e c t i o n s due to p e n i c i l l i n or s t reptomyc in s e n s i t i v e 3 b a c t e r i a . At t h i s time Contravac was a l s o admin i s tered to prevent I n f e c t i o u s Bovine R h i n o t r a c h e i t i s and P a r a i n f l u e n z a ( P I - 3 ) . I d e n t i f i c a t i o n tags were attached to the ears of the s t e e r . The animals were he ld i n groups of four s teer s i n a 3.6 x 9.1 meter pen, a l l o w i n g 8.32 Sqm per s t e e r . The pens contained an ea r the rn f l o o r bedded w i t h wood shav ings. The wood shavings were p e r i o d i c a l l y 1 , 2 Rogar/STB, London, On ta r i o , Canada. 3Connaught L abo ra to r i e s L t d . , W i l l owda l e , O n t a r i o , Canada. - 41 -r e p l a c e d . Water was prov ided ad l i b i t u m through a water bowl p laced i n each pen. The animals were group fed by p l a c i n g the feed i n an open wooden t rough. I n i t i a l l y they were fed a r a t i o n composed of chopped hay and b a r l e y . The chopped hay content of the r a t i o n was reduced g r adua l l y and the animals were a l lowed to get used to a f u l l concentrate d i e t . When the s tee r s were f u l l y adapted to a h igh g r a i n r a t i o n as i n d i c a t e d by the l a ck of d i g e s t i v e d i s o rde r s and regu la r feed i n t a k e , the s tee r s were in t roduced to the exper imenta l d i e t . A l l feed troughs were prov ided w i t h f ree choice c o b a l t - i o d i z e d s a l t b l o c k s . Feeding was normal ly c a r r i e d out d a i l y at 0730 h and aga in at 1430 h. The body weight of the s tee r s was taken by p l a c i n g them i n d i v i d u a l l y on a weighing b r i d ge . Th i s procedure was done wi thout much excitement and the body weight was taken a f t e r an overn ight f a s t . Experimental Procedure S i x teen Hereford s tee r s were randomly d i v i d e d i n t o 4 groups and a l l o t t e d to one of the f o l l o w i n g four t reatments : 1. Unimplanted c o n t r o l 2. 36 mg implant of Zerano l 3. 36 mg implant of D i e t h y s t i l b e s t r o l (DES) 4. One implant of Synovex-S 1 The anabo l i c compounds were implanted i n the form of slow r e l e a s i n g p e l l e t s at the base of the ea r . Th i s procedure i nvo l ved the placement Synovex-S, one implant conta ins 200 mg progesterone and 20 mg e s t r a d i o l benzoate. - 42 -of implant-gun needle between the skin folds of the pinna and the slow deposition of the pellets. The needle, after the deposition of the pellets, was carefully removed and the presence of the pellets in the skinfold was assured. The control groups were also subjected to the same procedure but without the deposition of any hormone pellets. The steers were fed twice daily with a finishing ration to provide dry matter requirements according to NRC standards (NRC, 1976; Table 5). The body-weight after an overnight fast was taken at weekly intervals. Blood samples were collected on 14, 28, 42 and 56 days following the implantation of anabolic compounds by means of jugular vein puncture. The samples were collected in heparinized vacutainer tubes and were placed in an ice bath. Blood samples were collected from the steers after an overnight fast. The samples were centrifuged at 1500 x g (Sorvall Superspeed Centrifuge, Model RC 2B, Norwalk, Conn., U.S.A.) and the plasma was harvested and stored at -20°C. Urine samples were collected over a period of 24 h from two steers in each treatment group on day 28, 42 and 56 following the implantation of anabolic compounds (See Page 65 for details of urine collection). Urine samples were stored at -20°C until analyzed for metabolites. The experimental steers were sacrificed on the 105th day following the implantation of anabolic compounds. At the time of slaughter, hepatic tissues were removed from a l l the steers and frozen in liquid nitrogen within minutes after collection. The hepatic tissues were stored at -20°C until analyzed for enzymes of amino acid catabolism. - 43 -Table 5. Ration Composition (Expt. IA) Ingredient % of Ration (as Fed) Steam Rolled Barley 89.0 Chopped Grass Hay 4.0 Soybean Meal 4.0 Ground Limestone 2.0 Mineral - Vitamin Mixture 2 3 k 1.0 12.0% crude protein (calculated). Mineral-vitamin mixture was supplied as K-72 mineral and vitamin mixture: Buckerfields Ltd., Abbotsford, B.C., Canada Compostion of K-72 mixture (1000 Kg): Premixture (5 Kg), limestone (70 Kg), dicalcium phosphate (50 Kg), salt (80 Kg), barley (680 Kg), wheat shorts (100 Kg) and animal fat (15 Kg). Composition of premixture of K-72 mineral-vitamin mixture (5 Kg): Vitamin A (140 g, 324000 I.U./g), vitamin D2 (120 gm, 176000 I.U./g), CuSO^ (40 g), MnSOk (600 g), ZnS0L (1000 g), prodine 17% I (72 g, 170 mg I/g) and wheat shorts (to 5 Kg). - 44 -Analytical Procedures The source of chemicals used in this and subsequent studies is given in Appendix I. Metabolites The plasma samples collected from a l l the steers were thawed (at 4°C) and analyzed for alpha amino nitrogen (ctAN), plasma urea nitrogen (PUN) and plasma free amino acid concentrations (AA). Alpha Amino Nitrogen (aAN) Plasma aAN concentration was determined by the photometric measurement of the yellow colour produced by the reaction of amino acids with l-fluoro-2, 4-dinitrobenzene (FDNB) as described by (Goodwin, 1968). Briefly, the plasma was deproteinized using a hydrochloric acid-tungstate reagent. The protein free filtrate was incubated at 70°C for 15 min in the presence of l-fluoro-2, 4-dinitrobenzene in a water bath. The reaction mixture was cooled and allowed to react with acidified dioxane. The final colour produced was measured in a spectrophotometer at 420 nm and the ctAN concentration in the plasma was obtained from a standard curve (glutamic acid - glycine mixture) obtained under similar conditions. Plasma Urea Nitrogen (PUN) Plasma urea nitrogen concentration was determined by a colorimetric method described by Fawcett and Scott (1960) and Chaney and Marbach (1962) using the urea nitrogen reagent package obtained from Sigma Chemical Co., St. Louis, Mo. (Tech. Bull. 640, Sigma Chemical Co.). Briefly, urea was hydrolysed to ammonia using urease (from jack - 45 -bean) and the ammonia produced was a l lowed to reac t w i t h a l k a l i n e h y p o c h l o r i t e and phenol i n the presence of sodium n i t r o p r u s s i d e . The concen t r a t i on of the f i n a l r e a c t i o n product , indophenol (b lue chromogen), which i s d i r e c t l y p r o p o r t i o n a l to the ammonia c o n c e n t r a t i o n , was measured s pec t r opho tomet r i c a l l y at 670 nm. Plasma Free Amino Acids Blood samples c o l l e c t e d from two s tee r s i n each treatment group were used. The plasma concen t ra t i on of f r ee AA was determined by an ion-exchange chromatographic method us ing an amino a c i d ana l yze r [Dionex, Model D500], Amino a c i d a n a l y s i s was conducted at the AAA Laboratory (AAA Labora to ry , Mercer I s l a n d , Washington, U.S.A. ) . Urinary Total Nitrogen Ur ine samples were thawed at room temperature and the t o t a l n i t r o g e n content was determined by the K j e l d a h l method. Enzymes TYROSINE AMINOTRANSFERASE (TAT) The l i v e r samples were thawed at 4°C, homogenized i n 10 ml i c e co l d KC1 (0.14 M) and the t y r o s i n e aminotransferase [Ty ros ine : 2 -oxog lu ta ra te aminot rans ferase, E.C. 2.6.1.5] a c t i v i t y was determined accord ing to the method of Granner and Tomkins (1970). B r i e f l y , a 0.2 ml supernatant of the crude l i v e r homogenate at approp r i a te d i l u t i o n s was added to cuvettes con ta i n i ng the f o l l o w i n g reagent m ix tu re : t y r o s i n e (.007 M, 1.6 m l ) , a - k e t o g l u t a r a t e (.5M, pH 7.0, .04 m l ) , p y r i doxa l - 5 ' - pho spha te (.005M, pH6.5, .02 m l ) . The assay - 46 -mixture was incubated f o r 5 - 3 0 minutes at 37°C. The enzymatic r e a c t i o n was stopped by the a d d i t i o n of 10 N KOH (0.14 ml) w i t h immediate m i x i n g . The assay mixture was incubated f o r another 30 minutes at 37°C and the absorbance was read at 331 nm aga ins t a ' z e r o t i m e ' blank prepared by adding the KOH to the r e a c t i o n components before the a d d i t i o n of the enzyme. The p r o t e i n content i n the l i v e r homogenate was determined by the method of Lowry et a l . (1951). One u n i t of TAT i s de f ined as the quan t i t y which ca ta l y se s the format ion of one u mole of p-hydroxyphenyl pyruvate per minute at 37°C. S t a t i s t i c a l Analysis The data were subjected to the a n a l y s i s of va r i ance ( S t e e l and T o r r i e , 1960) and the means were separated by the Newman-Keul's m u l t i p l e range t e s t . The s lopes of the body weight of the i n d i v i d u a l treatment groups over time were subjected to a n a l y s i s of cova r i ance . The amino a c i d data were subjected to s i n g l e degree of freedom orthogonal con t ra s t t e s t ( S t e e l and T o r r i e , 1960) and the treatment d i f f e r e n c e s were compared f o r each amino a c i d . A l l s t a t i s t i c a l analyses were c a r r i e d out us ing UBC BMD:10V l i n e a r hypothes i s computer program. RESULTS The i n f l u e n c e of Ze rano l , DES, and Synovex-S on body weight of the s tee r s i s presented i n F i gu re 3. Steers t r ea ted w i t h anabo l i c compounds g ra ined more r a p i d l y (P < .01) than the c o n t r o l s . The s lopes of the body weight of the treatments were s i m i l a r (P = .15) . However, the body weight over time reg re s s i on equat ions obta ined f o r the treatments were found to be d i f f e r e n t (P < .01 ) . The growth due to DES O = CONTROL A r ZERRNOL + = OES • s STNOVEX 14 28 42 56 70 84 98 DAYS AFTER IMPLANTATION 112 E f f e c t of anabo l i c compounds on growth i n s t e e r s . Each po int represents mean of four ob se r va t i on s . Regress ion equat ions : C o n t r o l , Y = 370.9 + 1.27x; Ze rano l , Y = 379.3 + 1.38x; DES, Y = 377.1 + 1.56x; Synovex-S, Y - 373.1 + 1.54x. Test of hypothes i s f o r common s l ope , P = 0.15; Test of hypothes i s f o r common equat i on , P = 0.0006. - 48 -and Synovex-S was s l i g h t l y g reate r than that induced by Ze rano l . The PUN and aAN concent ra t ions as i n f l uenced by d i f f e r e n t a n a b o l i c treatments on d i f f e r e n t per iods f o l l o w i n g the i m p l a n t a t i o n are presented i n F i gu re 4. The concent ra t i on of both the metabo l i te s due to the anabo l i c treatments was lower than that i n the c o n t r o l group (P < .05 ) . The e f f e c t of anabo l i c compounds was very much pronounced on day 28 f o l l o w i n g t h e i r i m p l a n t a t i o n . A s i g n i f i c a n t (P < .01) treatment x pe r i od i n t e r a c t i o n was no t i ced f o r both PUN and aAN. When a l l the time per iods (14, 28, 42, & 56 days) were taken i n t o c o n s i d e r a t i o n (F igure 5 ) , there was approx imately 15-23% r educ t i on i n PUN and a 17-19% r e d u c t i o n i n aAN concent ra t ions due to the imp l an t a t i o n of Ze rano l , DES and Synovex-S. However, there was no d i f f e r e n c e (P > .05) among the d i f f e r e n t anabo l i c compounds ( Ze rano l , DES andSynovex-S) i n a l t e r i n g PUN and/or aAN. Plasma l e v e l s of f r ee AA as i n f l u e n c e d by the treatments are presented i n Table 6. Cons ider ing the marked r educ t i on i n the plasma l e v e l s of PUN and aAN on day 28 f o l l o w i n g the i m p l a n t a t i o n , i t appeared that a corresponding AA p r o f i l e on t h i s day w i l l r e v e a l f u r t h e r the e f f e c t of anabo l i c compounds on n i t r ogen metabolism i n growing s t e e r s . The concen t ra t i on of most of the AA, both e s s e n t i a l and n o n - e s s e n t i a l , was found to be s i g n i f i c a n t l y lower (P < .05) i n s teer s implanted w i t h Z e r a n o l , DES and Synovex-S than i n the c o n t r o l s t e e r s . In g e n e r a l , the concen t ra t i on of plasma AA was more reduced due to DES and Synovex-S than due to Zerano l Imp lan ta t i on . There was no d i f f e r e n c e (P > .05) between DES and Synovex-S i n terms of a l t e r i n g the plasma AA p a t t e r n . - 49 -]0 14 28 42 56 70 DAYS AFTER IMPLANTATION F i gu re 4. Plasma urea n i t r o gen (PUN) and a lpha amino n i t r o gen (aAN) concent ra t i on s i n s teer s implanted w i t h va r ious anabo l i c compounds. Each po in t represents mean va lues obta ined from four s t e e r s . 16r 14 Q12 10 O ) E d UREA-N (P<-os) • • • • • • • • • • • • •••• O/^  ••••oxd • • • • •••• A 4**** oxc • • • • L T O / - S i I t t t f f » M . 10r 8 8 6 E4 0 ALPHA AMINO-N (P<.01) 4& • < TT •••• +••• •••+ • • • • J ^ V . •••• j _ C • • • • ~ 0 ^ ••••} C •••• • 'n .•••• v ^ f ! • • • • » • « 6 e a • • • • • • • • • • • • CONTROL ZERANOL DES SYNOVEX-S o F i gu re 5. Plasma urea-N and a lpha amino-N concent rat ions i n s tee r s implanted w i t h anabo l i c compounds. Each bar represents the mean ± SE of va lues f o r four s t ee r s on days 14, 28, 42 and 56 f o l l o w i n g the imp l an ta t i on (n=16). a » b B a r s w i t h d i f f e r e n t alphabet d i f f e r s i g n i f i c a n t l y . Table 6. Plasma free amino add levels in steers on 28 days1 following the lmplantlon of various anabolic compounds ANABOLIC TREATMENTS AMINO ACID CONTROL (1) ZERANOL (2) DES (3) SYNOVEX-S (4) SIGNIFICANCE OF DIFFERENCE 1 Vs 2 1 Vs 3 1 Vs 2, Vs 4 2 Vs 3 2 Vs 4 3 Vs 4 3, 4 NS NS NS NS * ** NS NS NS ** NS NS NS NS NS * NS NS NS * * NS NS NS * ** NS NS NS ** * * * NS ** ** * NS NS ** * NS NS NS ** * NS NS NS * ** NS NS NS ** * NS NS NS ** NS NS NS NS NS ** NS NS NS ** ** * NS NS ** ** ** * NS ** ** NS NS NS ** NS NS NS NS NS ** NS NS NS ** Alanine (ALA) Arglnine (ARG) Aspartic Acid (ASP) Glutamic Acid (GUI) Glycine (GLY) Histldine (HIS) Isoleucine (ILE) Leucine (LEU) Lysine (LYS) Methionine (MET) {Phenylalanine (PHE) Proline (PRO) Serine (SER) Threonine (THR) [Tyrosine (TYR) Valine (VAL) Glutamine (GLN) Citrulline (CIT) Ornithine (ORN) jimole/100 ml NS 26.2 ± 6.7 15.0 ± 2.7 9.2 ± 3.8 10.5 ± 3.0 NS 14.0 ± 3.0 5.1 ± .5 3.08 ± 1.0 2.4 ± 1.3 * * 2.5 ± .9 1.8 ± .4 1.8 ± .7 1.8 ± .2 NS NS 14.9 ± 3.7 6.5 ± 1.0 5.6 ± 1.8 5.6 ± 1.6 NS * 24.8 ± .8 11.8 ± 2.1 11.2 ± 3.8 11.5 ± 2.9 * * 7.5 ± 1.3 2.8 ± .5 1.9 ± .7 1.5 ± .5 * ** 12.0 ± .1 7.2 ± .8 4.6 ± .9 4.5 ± .3 ** * 18.8 ± 1.4 10.2 ± 1.1 5.6 ± .9 6.5 ± .8 ** ** 15.9 ± 4.4 6.0 t 1.0 3.0 ± .8 2.5 ± .7 * * 2.8 ± .1 1.7 ± .1 & 1.3 1.3 * * 6.2 ± .4 3.5 ± .5 1.9 ± .5 2.4 ± 5 * ** 20.3 ± 4.2 10.8 ± .9 7.1 ± .7 6.4 t 1.7 * * 13.2 ± 2.0 6.3 ± .8 7.0 ± 3.9 4.8 t 1.4 NS NS 9.3 ± .3 4.6 ± .2 3.0 ± .7 3.0 ± .7 ** ** 5.6 ± .2 3.7 * .4 2.0 ± .1 2.7 t .6 * ** 32.2 t 1.5 19.6 ± 2.2 9.6 ± .5 11.7 ± .8 ** ** 25.7 ± 2.4 11.3 ± 1.9 5.7 ± .4 9.0 ± 2.7 ** ** 9.5 ± 2.7 6.4 ± .1 3.8 ± .8 4.4 ± .7 NS NS 10.4 ± 1.0 4.5 ± .8 2.8 ± .6 2.9 ± .5 ** ** 'Each value is the mean (± SE) of data obtained from two steers. 2Single degree of freedom orthogonal comparisons data from one animal *P < .05 **P < .01 N.S. " Non-significant - 52 -Plasma l e v e l s of e s s e n t i a l AA exc lud ing t ryptophan were t o t a l l e d f o r each treatment and are presented i n F i gu re 6. When expressed as a per cent of that found i n the c o n t r o l s t e e r s , the l e v e l s were 51, 29 and 30 i n Ze r ano l , DES and Synovex-S implanted s teer s r e s p e c t i v e l y . The amount of n i t r o g e n excreted i n the u r i ne over a pe r i od of 24 h by the s teer s i s presented i n Table 7. There was approx imately 8%, 11% and 12% r educ t i on i n the u r i n a r y n i t r o gen e x c r e t i o n due to Ze rano l , DES and Synovex-S i m p l a n t a t i o n , r e s p e c t i v e l y , when compared to the c o n t r o l s t e e r s . Hepat i c l e v e l s of t y r o s i n e aminotransferase as i n f l uenced by d i f f e r e n t anabo l i c compounds are presented i n Table 8. TAT l e v e l s were s i m i l a r (P > .05) In the c o n t r o l and i n the implanted s t e e r s . DISCUSSION The growth promoting p o t e n t i a l s of anabo l i c compounds have been very w e l l documented but the mechanism of a c t i o n has not been c o n c l u s i v e l y demonstrated. The r e s u l t s of t h i s study prov ide f u r t h e r i n s i g h t i n t o the e f f e c t s of e s t rogen i c anabo l i c compounds, v i z . , Z e r a n o l , DES and Synovex-S on d i f f e r e n t aspects of n i t r o gen metabol i sm. Plasma l e v e l s of u rea-N, aAN and f r ee AA were a l l found to be s i g n i f i c a n t l y reduced i n s teer s Implanted w i t h Ze rano l , DES and Synovex-S (F igure 4 and Table 6 ) . A s i m i l a r r educ t i on i n the plasma concent ra t i on s of urea-N i n c a t t l e implanted w i t h DES ( O l t j e n and Lehmann, 1968), lambs implanted w i t h Zeranol (Wiggins et a l . , 1976) and DES ( P re s t on , 1968) and i n h e i f e r s t r ea ted w i t h t renbolone acetate ( G a l b r a i t h , 1980) has been r epo r t ed . The r educ t i on i n PUN observed i n t h i s study may be due to a r educ t i on i n the hepa t i c ca tabo l i sm of - 53 -|120r °100| O E80 ai _,40| § 20I 0 • • * • • • • • • • • • • • • >o CONTROL ZERANOL DES SYNOVEX-S > • • • • • • • • • • F i gu re 6. Plasma t o t a l e s s e n t i a l amino a c i d ( T o t a l EAA) concent ra t i on s i n growing s tee r s on day 28 f o l l o w i n g the imp l an t a t i o n of anabo l i c compounds. Each bar represents the mean of va lues obta ined from two s t e e r s . T o t a l e s s e n t i a l amino ac ids does not i n c l ude t ryptophan. - 54 -Table 7. D a i l y u r i n a r y n i t r ogen e x c r e t i o n of s t ee r s implanted w i t h va r i ou s anabo l i c compounds 0.75 Treatment U r i na r y -N (mg/d/kg ) C o n t r o l 765.4 ± 7 5 . 3 b Zerano l 705.0 ± 41.4 DES 685.2 ± 64.1 Synovex-S 673.1 ± 55.4 Mean of va lues obta ined on 28, 42 and 56 days f o l l o w i n g i m p l a n t a t i o n . Same group of s teer s were used oh a l l the three u r i ne c o l l e c t i o n p e r i o d s . 'Mean ± SE of va lues obta ined from two s teer s on a l l the three u r i ne c o l l e c t i o n per iods (n = 6 ) . - 55 -Table 8. Effect of various anabolic compounds on hepatic tyrosine aminotransferase levels in steers Tyrosine aminotransferase (micromoles p-hydroxyphenylpyruvate Treatment formed/min./mg protein) Control 0.9725 ± 0.07* Zeranol 1.1730 ± 0.04 DES 0.9885 ± 0.19 Synovex-S 0.9047 ±0.03 *Mean ± SE of values obtained from four steers - 56 -n i t rogenous compounds. Sykes (1978) suggested that the endogenous p roduct ion of urea depends l a r g e l y on the deaminat ion of amino a c i d s . Reduct ions i n the plasma aAN concent ra t i ons may be a s c r i bed to the inc reased requirements of amino ac id s f o r p r o t e i n d e p o s i t i o n , or t o a decrease i n the amino ac ids produced dur ing p r o t e i n turnover (M i l lwa rd et a l . , 1976). These reduct ions i n the plasma aAN were f u r t h e r supported by the reduct ions i n both e s s e n t i a l and n o n - e s s e n t i a l AA c oncen t r a t i o n s . A r educ t i on of plasma e s s e n t i a l and n o n - e s s e n t i a l amino a c i d concent ra t i on s was a l so no t i c ed by O l t j e n and Lehmann (1968) i n s teer s implanted w i t h DES. McLaren et a l . (1960) reported a r e d u c t i o n i n both blood ammonia and urea and suggested that DES f a c i l i t a t e s a b e t t e r u t i l i z a t i o n of non -p ro te in n i t r o g e n . Plasma growth hormone (GH) concent ra t i on s were measured i n a l l the s tee r s used i n t h i s study and found to be 130%, 231% and 219% h igher i n Ze r ano l , DES and Synovex-S implanted s tee r s than i n the c o n t r o l s t e e r s , r e s p e c t i v e l y on 28th day f o l l o w i n g imp l an t a t i o n ( r e s u l t s of Expt . I I B ) . Furthermore e s t r ogen i c anabo l i c compounds have been shown to i nc rease plasma l e v e l s of i n s u l i n i n ruminants ( P re s ton , 1975). Both GH and i n s u l i n have been shown to i n c rea se the t i s s u e uptake of amino ac ids (R iggs, 1970; Manchester, 1972). Growth hormone a l s o s t imu l a te s N r e t e n t i o n and favours d e p o s i t i o n of carcass -N by s t i m u l a t i n g amino a c i d t r an spo r t i n t o the body t i s s u e s (Kostyo, 1973). Hence the r educ t i on i n PUN, aAN and plasma AA observed i n the implanted s tee r s used i n t h i s study may be a t t r i b u t e d to the i n d i r e c t e f f e c t s brought about by the changes i n endogenous growth hormone and i n s u l i n s t a t u s . I t may a l s o be hypothes ized that - 57 -a n a b o l i c compounds enhance the a b i l i t y of the animal to draw nitrogenous substances from the plasma f o r b u i l d i n g p r o t e i n s . The Increased amount of carcass -N depos i ted i n s teer s implanted w i t h Zerano l (Sharp and Dyer, 1971), DES (Rumsey et a l . , 1981) and Synovex-S (Rumsey, 1982) adds support to the above mentioned hypothes i s . The f r ee AA concent ra t i ons (Table 6) i n the plasma of the implanted s tee r s are much lower than those found i n the unimplanted s tee r s and are e s p e c i a l l y lower than those compiled by Bergen (1975) f o r ruminants. However, i t should be po inted out that the f r ee amino a c i d concen t r a t i on of the c o n t r o l s t ee r s were s i m i l a r to that reported by Bergen (1979). The reduced plasma f r e e AA p r o f i l e of the implanted s tee r s as compared to the values of Bergen (1979) could be due to i nc reased d i s t r i b u t i o n r a t i o (DR, concen t r a t i on i n t i s s u e to concen t r a t i on i n plasma) as a r e s u l t of i nc reased AA uptake by the t i s s u e s (due to hormonal changes as d i scus sed above). Bergen (1979) suggested that low plasma AA, e s p e c i a l l y e s s e n t i a l AA may be due to a r e s u l t of e i t h e r a d i e t a r y d e f i c i t or an inc reased uptake of e s s e n t i a l AA i n t o p r o t e i n and that h igh plasma l e v e l s of e s s e n t i a l AA may be due to a r e s u l t of d i e t a r y excess or ex tens i ve net ca tabo l i sm of body p r o t e i n s . A l l the s teer s used i n t h i s study were fed accord ing to the s p e c i f i c a t i o n s set by NRC f o r growing beef s t e e r s . Cons ide r ing the inc reased growth ra te of the implanted s tee r s i t i s suggested that the r e d u c t i o n i n plasma f r ee AA p r o f i l e of the implanted s tee r s compared to the va lues compiled by Bergen (1979) i s a r e f l e c t i o n of i nc reased t i s s u e uptake and not due to a d i e t a r y d e f i c i t . - 58 -Cons ide r i ng the inc reased amount of p r o t e i n depos i ted i n the implanted s teer s as reported i n s e v e r a l s t u d i e s , the f o l l o w i n g genera l d i s c u s s i o n on p r o t e i n metabolism would be app rop r i a te to understand the a l t e r a t i o n s i n N metabol i sm. The s imple p r o t e i n - N metabol ism model desc r ibed by B ruckenta l et a l . (1980) was used to f u r t h e r de sc r i be the a l t e r a t i o n s i n n i t r ogen metabolism e x h i b i t e d by the implanted s t e e r s . In t h i s model the absorbed amino ac i d -N enters the f ree amino ac id -N poo l i n the body. Due to the g reate r demand of absorbed amino ac id s f o r t i s s u e p r o t e i n s y n t he s i s , on ly a l i m i t e d amount of amino ac i d -N w i l l be c a t a b o l i z e d and excreted as urea (see B ruckenta l et a l . , 1980 f o r the model ) . The r educ t i on i n plasma urea-N observed i n t h i s study Is a f u r t h e r i l l u s t r a t i o n . However, not a l l u rea i s formed as the r e s u l t of ca tabo l i sm of amino ac id -N p o o l . A s u b s t a n t i a l quan t i t y of urea i s de r i ved from ammonia absorbed from the gut (Nolan, 1975) and sma l l e r q u a n t i t y from the catabo l i sm of metabo l i t e s such as py r im id ine s (Mahler and Cordes, 1971). I t i s qu i t e probable that these a l t e r n a t e pathways of urea p roduct ion are a l s o under r egu l a to r y c o n t r o l i n the implanted s t e e r s . Decreased PUN observed i n the implanted s tee r s of t h i s study cou ld a l s o be due to the metabo l i c adapta t i on to decreased p roduc t i on of urea i n v o l v i n g s p e c i f i c a l l y the r a t e - l i m i t i n g steps i n the urea c y c l e . The r a t e - l i m i t i n g r e a c t i o n i n the urea c y c l e i n the ra t l i v e r i s the condensing enzyme of the a r g i n i ne - s yn the ta se system, a r g i no succ i na te synthetase (Brown and Cohen, 1960). I t has been e s t a b l i s h e d that i n r a t l i v e r there i s a s u b s t a n t i a l r educ t i on i n the a c t i v i t y of urea c y c l e enzymes due to growth hormone a d m i n i s t r a t i o n (McLean and Gurney, 1963). - 59 -I t i s l i k e l y that the a c t i v i t y of most of the enzymes a s soc i a ted w i t h the urea c y c l e could have been lowered i n the implanted s t e e r s . As most of the metabo l i c a c t i on s of a nabo l i c compounds are mediated through endogenous hormones, such as growth hormone, makes the above mentioned p o s s i b i l i t y very a t t r a c t i v e . However, the l i v e r b iopsy samples removed a f t e r the 28th day of DES treatment i n growing lambs revea led no d i f f e r e n c e i n a rg inase a c t i v i t y , an another enzyme i n vo l ved i n the urea c y c l e ( P re s ton , 1968). I t should be noted that on ly the a r g i n i ne - s yn the ta se system of the urea c y c l e was s i g n i f i c a n t l y reduced due to GH treatment (McLean and Gurney, 1963). Fu r the r work on the e f f e c t of anabo l i c compounds on the enzymes of urea c y c l e i s warranted to understand the mechanism behind the reduced urea p roduc t i on . The e f f e c t of anabo l i c compounds i n s t i m u l a t i n g p r o t e i n s yn the s i s and i n c r e a s i n g n i t r ogen r e t e n t i o n as repor ted by e a r l i e r workers was f u r t h e r conf irmed by the s i g n i f i c a n t reduct ions i n methionine (P < .05 ) , th reon ine (P < .01) and l y s i n e (P < .01) i n the implanted s t e e r s . Meth ion ine , threon ine and l y s i n e are thought to be l i m i t i n g i n ruminants (Bergen, 1979). In t h i s study a N balance experiment was not conducted to conf i rm N r e t e n t i o n i n the implanted s tee r s but the reduced amount of u r i n a r y n i t r o gen e x c r e t i o n (Table 7) i n d i c a t e d inc reased N r e t e n t i o n . S i g n i f i c a n t r educ t i on (P < .01) i n the plasma l e v e l s of the n o n - e s s e n t i a l AA, o r n i t h i n e , i n the implanted s t ee r s i s of importance w h i l e con s i de r i n g the growth promoting p o t e n t i a l of these compounds. O r n i t h i n e Is decarboxy lated by o r n i t h i n e decarboxylase to form pu t re s c i ne ( R u s s e l l and Snyder, 1968) and the l a t t e r forms the - 60 -diamlnobutane back bone of the polyamines. The polyamines have been strongly implicated in the growth process (Russell, 1973). The reduction in plasma ornithine levels indicates an enhanced u t i l i z a t i o n of ornithine for putrescine biosynthesis. Hence the results obtained i n this study tend to demonstrate Indirectly the effect of anabolic compounds on polyamine biosynthesis. Ornithine decarboxylase plays an important role in the estrogen induced changes in the growth of the target tissues such as chicken oviduct (Levy et a l . , 1981) and rat uterus (Kaye et al.,1971). Correlation between tissue growth and ornithine decarboxylase activity has been shown in many cases (Tabor and Tabor, 1976). Taken collectively the growth promoting potentials and the increased polyamine biosynthesis (assuming a relationship between plasma ornithine and putrescine biosynthesis) i t is suggested that anabolic compounds mediate their growth promoting properties through polyamine metabolism also. Further work is warranted to conclusively demonstrate this aspect of anabolic compound action. With regard to the level of hepatic amino acid catabolising enzyme, TAT, only a non-significant difference was obtained due to the implantation of Zeranol, DES and Synovex-S. A significant reduction in the hepatic TAT activity was reported by Rodway and Galbraith (1979) in female rats treated with the androgenic anabolic compound, trenbolone acetate and i t was concluded that such a reduction in the hepatic TAT activity would increase the availability of tyrosine for the synthesis of new proteins. It is not clear at present whether the lack of difference in TAT activities among the steers is due to an absolute - 61 -d i f f e r e n c e i n the mode of a c t i o n of e s t r ogen i c and androgenic anabo l i c compounds or due to the inc reased length of time which elapsed between the i m p l a n t a t i o n of these compounds and the sampling of hepa t i c t i s s ue s f o r TAT a c t i v i t y . A r educ t i on i n the concen t r a t i on of t y r o s i ne (P < .01) a long w i t h the reduced PUN and AN on day 28 f o l l o w i n g the i m p l a n t a t i o n i n d i r e c t l y suggests that there would have been a marked d i f f e r e n c e i n TAT a c t i v i t y at t h i s time per iod between the implanted and unimplanted s t e e r s . I t i s a l so po s s i b l e that other enzymes of amino a c i d ca tabo l i sm could p lay a very important r o l e i n r e g u l a t i n g the a v a i l a b i l i t y of amino ac ids ( e s p e c i a l l y e s s e n t i a l ) f o r p r o t e i n s y n t h e s i s . CONCLUSION The r e s u l t s of t h i s i n v e s t i g a t i o n have confirmed the growth promoting p r ope r t i e s of anabo l i c compounds and i n d i c a t e d that the imp l an t a t i on s of DES and Synovex-S are more e f f e c t i v e i n i n c r e a s i n g the growth of s tee r s than the imp l an t a t i o n of Ze rano l . A con s ide rab le r e d u c t i o n i n the plasma concent ra t i ons of n i t rogenous compounds was observed due to the imp l an t a t i on of anabo l i c compounds which i nc luded a 15 - 23% r educ t i on i n PUN, 17-19% reduc t i on i n AN and about 29-51% r educ t i on i n the e s s e n t i a l AA c o n c e n t r a t i o n . The r e s u l t s revea led a l a c k of d i f f e r e n c e i n the l e v e l of hepa t i c TAT between the implanted and unimplanted s t e e r s . I t i s concluded that anabo l i c compounds s i g n i f i c a n t l y a l t e r n i t r o gen metabolism i n s teer s by i n c r e a s i n g the e f f i c i e n c y of u t i l i z a t i o n of n i t rogenous compounds. The i r i m p l a n t a t i o n seems to - 62 -reduce the hepa t i c p roduct ion of u rea. The data on plasma o r n i t h i n e concen t r a t i on i n s tee r s f o l l o w i n g the i m p l a n t a t i o n of anabo l i c compounds suggested that these compounds may mediate t h e i r growth promoting p r o p e r t i e s through the metabolism of polyamines as w e l l . Implantat ions of DES and Synovex-S were concluded to be more e f f e c t i v e i n a l t e r i n g n i t r ogen metabolism of s teer s than the imp l an t a t i o n of Ze r ano l . - 63 -EXPERIMENT IB MUSCLE PROTEIN METABOLISM IN STEERS AS DETERMINED BY THE URINARY NT-METHYLHISTIDINE EXCRETION AS AN IN VIVO INDEX OF MYOFIBRILLAR PROTEIN BREAKDOWN: Developmental aspects of muscle protein metabolism and the Influence of estrogenic anabolic compounds INTRODUCTION A major factor of growth in farm animals is the increase i n muscle mass which contributes about 45% of the body weight in mature mammals, regardless of their size (Munro, 1969) and serves as an important tissue for protein synthesis, reserve and turnover (Cheek, 1971). The net deposition of protein in mammals is determined by the balance between the rates of synthesis and degradation. Millward et a l . (1975) reported that there is a direct correlation between growth rate and muscle protein breakdown rate and that rapid growth is always accompanied by high rates of protein breakdown. Since muscle protein is a major product harvested from farm animals and muscle mass is determined by the balance between muscle protein synthesis and degradation, i t becomes necessary to develop quantitative procedures to study muscle protein metabolism under various physiological conditions. Anabolic compounds, both androgenic and estrogenic have been shown to increase the amount of protein deposited in farm animals (Byers, 1982b; Rumsey et a l . , 1981; Rumsey, 1982; G r i f f i t h s , 1982; Sharp and Dyer, 1971). The mechanism of action of these compounds on muscle protein metabolism has not been clearly demonstrated. Attempts have - 64 -been made at least with the androgenic compound, trenbolone acetate, to elucidate their mechanism of action on the muscle protein metabolism (Vernon and Buttery 1978a,b). N T-Methylhistidine (NT-MH) or 3-Methylhistidine (3-MH) excretion in urine has been widely used as a useful non-destructive technique for measuring in vivo muscle protein degradation (Young and Munro, 1978). NT-MH originates from the degradation of actin and certain species of myosin heavy chain in which i t i s present after the post-translational methylation of specific histidine residues (Young and Munro, 1978). During the catabolism of myofibrillar proteins, i t i s released and excreted in urine and i s not reutilized for protein synthesis (Young et a l . , 1972) or metabolized oxidatively (Long et a l . , 1975). Hence urinary excretion of NT-MH was suggested as a valid in  vivo index of muscle protein degradation (Ward and Buttery, 1978). The validity of this method has been questioned recently by Millward et_ al_., (1980b). The determination of 24 h urinary excretion of NT-MH to measure muscle protein degradation has been used in man (Tomas et a l . , 1979), rats (Santidrian et^ a l . , 1981), rabbits (Harris et a l . , 1977) and recently validated for cattle (Harris and Milne, 1981a). This method, however, has been reported to be an invalid index of muscle protein degradation in sheep (Harris and Milne, 1980) and in the pig (Harris and Milne, 1981b). This experiment was conducted in growing steers with the following objectives: - 65 -(a) to study the developmental changes a s soc i a ted w i t h muscle p r o t e i n metabolism (b) to study the e f f e c t of c e r t a i n e s t r ogen i c anabo l i c compounds on va r ious aspects of muscle p r o t e i n metabol i sm, and (c) to demonstrate, i n g ene r a l , the u se fu lnes s of NT-MH e x c r e t i o n i n u r i n e as a means of q u a n t i t a t i n g muscle p r o t e i n degradat ion MATERIALS AND METHODS Animals and their Management Growing Hereford beef s teer s were used i n t h i s s tudy. The animals and t h e i r management are the same as desc r ibed i n EXPT. IA. Experimental Procedure Two s tee r s from each of the four anabo l i c treatments [ c o n t r o l , Z e r ano l , d i e t h y l s t i l b e s t r o l (DES), Synovex-S) desc r ibed i n Expt . IA were used. The anabo l i c compounds and t h e i r i m p l a n t a t i o n procedures have been desc r ibed i n EXPT. IA. The animals were fed twice d a i l y w i t h a f i n i s h i n g r a t i o n as desc r ibed i n ' T a b l e 5. The body weight was measured on every seventh day of the exper iment. Urine Collection Procedure Ur ine samples were c o l l e c t e d i n s p e c i a l l y designed metabolism c ra te s over a per iod of 24 h on days 24, 48, 56 and 63 f o l l o w i n g the Imp lan ta t i on of anabo l i c compounds. The s tee r s were a l lowed to adapt to the metabolism c ra tes f o r a per iod of 24 h before the a c t u a l u r i n e c o l l e c t i o n was begun. On the day of the experiment, the s tee r s were - 66 -weighed, fed t h e i r morning p o r t i o n of the d i e t and p laced i n the metabol ism c ra te s f o r t o t a l u r i ne c o l l e c t i o n . The s teer s r ece i ved feed i n the metabolism c r a te dur ing the c o l l e c t i o n pe r iod and water was prov ided ad l i b i t u m . Ur ine was c o l l e c t e d i n p l a s t i c ve s se l s con ta i n i n g 6M HCl or to luene as the p r e s e r v a t i v e . Dur ing the c o l l e c t i o n process , the u r i ne was passed through two l a ye r s of cheese c l o t h to avo id f e c a l con taminat ion . The c l e a r u r i ne samples were s to red at -20°C. Analytical Procedures NT-MH : U r i na r y NT-MH concent ra t i ons were determined by an ion-exchange chromatographic method us ing an automatic amino a c i d ana ly se r (Beckman 120 C, Beckman Instruments, Pa lo A l t o , CA. ) . U r i na r y c r e a t i n i n e concent ra t i ons were determined by a c o l o r i m e t r i c procedure us ing the c r e a t i n i n e determinat ion reagent package obta ined from Sigma Chemical Co. , S t . L o u i s , MO. (Tech. B u l l e t i n No.555, Sigma Chemical Co . ) . Calculation of Parameters of Muscle Protein Metabolism The d e s c r i p t i o n s and c a l c u l a t i o n s of the parameters of muscle p r o t e i n metabolism were accord ing to B a l l a r d et a l . (1979) and H a r r i s and M i lne (1981a). The r a te of muscle p r o t e i n degradat ion (MPD, g . d . - 1 ) was c a l c u l a t e d by the equat ion : = ymol NT-MH excreted per day ^ N T -MH content of s k e l e t a l muscle p r o t e i n - 67 -In the above equat ion the denominator was taken to be 3.5 ymol of NT-MH/g muscle p r o t e i n as repor ted by Nish izawa et a l . (1979) f o r growing s t e e r s . The f r a c t i o n a l r a te of muscle p r o t e i n breakdown (FBR, % . d - 1 ) was c a l c u l a t e d by the equat ion : F B R = NT-MH excreted per day (ymol) x 1 0 Q ( 2 ) t o t a l N T -MH i n s k e l e t a l muscle (ymol) The denominator i n equat ion 2 was c a l c u l a t e d as the product of s k e l e t a l muscle mass (33% of the body weight; A l l e n et a l . , 1968; Brannang, 1971), the p r o t e i n content of s k e l e t a l muscle (157 mg/g f r e s h weight ) and the l^-MH content of s k e l e t a l muscle p r o t e i n (Nish izawa et a l . , 1979). The h a l f - l i f e va lue of the m y o f i b r i l l a r p r o te i n s ( a c t i n and myosin) was c a l c u l a t e d from the breakdown ra te by assuming f i r s t - o r d e r k i n e t i c s (Ward and Bu t t e r y , 1978). The ra te of muscle p r o t e i n s ynthes i s (MPS) i n the s tee r s was c a l c u l a t e d as the d i f f e r e n c e between the r a te of muscle p r o t e i n ga in (MPG) and the ra te of MPD. THe MPG was c a l c u l a t e d as the product of the amount of muscle mass gained per d (33% of average d a i l y ga in) and the p r o t e i n content of the s k e l e t a l musc le. The c a l c u l a t i o n s f o r MPS were s i m i l a r to those used by M i l l w a r d et^ a l . (1975) who est imated muscle p r o t e i n breakdown i n r a t s as the d i f f e r e n c e between s y n t h e t i c ra te and growth r a t e . The e f f i c i e n c y of muscle p r o t e i n s yn thes i s i n s teer s implanted w i t h anabo l i c compounds was c a l c u l a t e d accord ing to the method desc r ibed by Bates and M i l lwa rd (1981). - 68 -E f f i c i e n c y of Muscle P r o t e i n Synthes i s = Q v e r - a l ^ S y n t h e s i s X Where: Net Synthes i s or p r o t e i n depos i ted = Muscle p r o t e i n g a i n , (MPG g/d) O v e r - a l l Synthes i s = Muscle p r o t e i n synthes ized/day (MPS, g/d) The parameters of s k e l e t a l muscle p r o t e i n metabolism i n a l l the s tee r s at d i f f e r e n t stages of growth were averaged and subjected to the a n a l y s i s of v a r i ance u s ing UBC BMD: 10V l i n e a r hypothes i s computer program. Simple c o r r e l a t i o n s among metabo l i c parameters were a l s o computed. RESULTS (1) Developmental Aspects of Muscle Protein Metabolism The body weight changes and average d a i l y ga in of s tee r s up to day 98 of the experiment are presented i n F igure 7. The animals gained r a p i d l y dur ing the i n i t i a l phase of the study. Up to day 56, the growth r a t e of s tee r s was h igher (P < .01) than up to day 42. By day 63, however, there was a decrease i n growth r a t e compared to day 56 (P < .01 ) . Subsequently, the s tee r s gained weight at a ra te of 1.4 Kg per day dur ing the r e s t of the exper imenta l p e r i o d . The mean d a i l y amount of NT-MH and c r e a t i n i n e excreted at d i f f e r e n t stages of growth i s presented i n F i gu re 8 and Table 9 r e s p e c t i v e l y . There was a g radua l i n c rea se i n the amount of NT-MH excreted up to day 56 of the study (P < .001 compared to day 42) f o l l owed by a - 69 550,-500 X 4501 0 LU ^ 400U >-Q O CQ 3501 1.5 oe ,0 g .5 0*- 14 28 42 oo — PERIOD O F E X P E R I M E N T (d) U J O < F i gu re 7. Body weight and average d a i l y ga in of s t e e r s . Each po int represents the mean of data obta ined from e igh t s t e e r s . The arrows i n d i c a t e the u r i ne c o l l e c t i o n p e r i o d . + P<.05 compared w i t h the preceding p e r i o d . * * P<.05 compared w i th day 28. 1 4 L 1 2 _ 1 . 0 -4 i i i i 0 2 8 4 2 5 6 7 0 PERIOD OF EXPERIMENT^) Figure 8. Urinary N T-methylhistidine excretion in steers on different periods of the experiment. Values are mean ± SE for eight steers. > I UJ + P<.05 compared with preceding period. * P<.05 compared with day 28. *** P<.001 compared with day 28. - 71 -Table 9. D a i l y u r i n a r y e x c r e t i o n of c r e a t i n i n e and N T - M e t h y l h i s t i d i n e : c r e a t i n i n e r a t i o du r i ng d i f f e r e n t per iod of the expe r iment 3 x Pe r i od of N - M e t h y l h i s t i d i n e U r i n a r y experiment e x c r e t i o n C r e a t i n i n e (d) (pmol/mg c r e a t i n i n e ) (g/d) 28 0.14 ± 0 . 0 1 b 11.46 ± 0.88 42 0.17 ± 0 . 0 1 b ° 11.39 ± 1.00 56 0.23 ± 0 . 0 3 C 12.74 ± 0.63 63 0.16 ± 0 . 0 2 b C 12.93 ± 0.42 Values are mean ± standard e r r o r s obta ined from e ight s tee r s i n each pe r i od of the experiment b c ' Means i n the same column w i t h d i f f e r e n t s u p e r s c r i p t s d i f f e r (P < .05) - 72 -decrease on day 63. These changes i n NT-MH e x c r e t i o n r e f l e c t the NT-MH a r i s i n g from the degradat ion of the s k e l e t a l muscle p r o t e i n and seem to resemble the growth curve of the exper imenta l an imal s . U n f o r t u n a t e l y , pa t te rns of NT-MH e x c r e t i o n were s tud ied on ly up to day 63 of the study. On an average, the d a i l y u r i n a r y NT-MH e x c r e t i o n by the s tee r s was 1957 ± 88 umol/d. The d a i l y amount of c r e a t i n i n e excreted at d i f f e r e n t per iods of the study was not s t a t i s t i c a l l y d i f f e r e n t (P > .05) but tended to i n c r ea se as the s tee r s i nc reased i n bodyweight. The r a t i o of NT-MH to u n i t c r e a t i n i n e was found to be h igher on day 56 (P < .05) than the other per iods (Table 9 ) . The mean ra te of MPD dur ing va r i ou s stages of growth was c a l c u l a t e d from the d a i l y amount of NT-MH excreted and the NT-MH content of s k e l e t a l muscle p r o t e i n (F igure 9 ) . The MPD was h igher (P < .05) on day 56 than on day 42 of the study. There was a decrease i n the ra te of p r o t e i n degraded from 676 ± 57 g/d on day 56 to 586 ± 25 g/d on day 63 (P < .05) . The mean r a te of MPD of the s tee r s was c a l c u l a t e d to be 557 ± 25 g/d dur ing the e n t i r e exper imenta l p e r i o d . The mean ra te of MPS (F igure 9) was c a l c u l a t e d as the d i f f e r e n c e between the ra te of muscle p r o t e i n gained per day and MPD. The MPS at d i f f e r e n t growth stages fo l l owed a s i m i l a r p a t t e r n to that of MPD, but was always h igher i n magnitude than MPD. The mean r a t e of MPS dur ing the e n t i r e exper imenta l per iod was c a l c u l a t e d to be 622 ± 27 g/d. The mean FBR (%/d) of the s tee r s was c a l c u l a t e d to be 2.44 ± .09 dur ing the e n t i r e exper imenta l p e r i o d . The FBR was h igher on day 56 - 73 -PERIOD OF EXPERIMENT (d) F i gu re 9. Muscle p r o t e i n degraded and synthes i zed i n s tee r s on d i f f e r e n t per iods of the exper iment. Va lues are mean ± SE f o r e i gh t s t e e r s . + P<.05 compared w i t h preced ing p e r i o d . * P<.05 compared w i th day 28. * * P<.01 compared w i t h day 28. - 74 -(P < .05) than on day 28 (Table 10). The h a l f - l i f e of the myofibrillar proteins i s presented in Table 10. It is to be noted that the shorter h a l f - l i f e of myofibrillar proteins was accompanied by higher MPD values, and that the h a l f - l i f e value of myofibrillar proteins on day 56 of the study (corresponds to 456 kg liveweight) was calculated to be 24 d. The relationship between MPD and MPS is shown in Figure 10. Similar correlation studies revealed a significant relationship between urinary NT-MH (umol/ml urine) and creatinine (mg/ml urine) (r = .71; n = 31; P < .01) and between muscle mass (kg) and NT-MH excretion (umol/d) (r = .68; n = 32; P < .01). The correlation between average daily gain of steers (kg/d) and MPD, (g/d) was low (r = .43; n = 32; P < .05). A comparison of the daily amount of NT-MH excreted in urine by cattle as reported by different workers is presented in Figure 11. (2) Effect of Anabolic Compounds on Muscle Protein Metabolism The daily amount of NT-MH excreted in urine was not different (P > .05) among the different treatment groups (Figure 12). The steers implanted with anabolic compounded exhibited a slight, but a non-significant increase in the amount of NT-MH excreted in urine. Urinary NT-MH excretion was slightly higher (P > .05) in Zeranol than in DES or Synovex-S implanted steers. The mean (±SE) daily rates of MPD (g/d) and MPS (g/d) as influenced by different treatments are presented in Figure 13. The treatment means for the two variables were not different (P > .05), however, the results indicate an increased rate of both degradation and - 75 -Table 10. F r a c t i o n a l r a te of p r o t e i n breakdown and the h a l f - l i f e Values of m y o f i b r i l l a r p r o te i n s i n growing s tee r s dur ing a d i f f e r e n t per iods of the experiment Pe r i od of Muscle p r o t e i n F r a c t i o n a l r a t e of H a l f - l i f e of experiment g a i n e d b p r o t e i n breakdown M y o f i b r i l l a r (d) (g/d) (%/d) p r o t e i n s 28 58.8 11.1 2.07 0.2 33.5 42 65.0 7.6 2.33 0.2 29.7 56 71.4 7 . 5 d 2.84 0 . 26 24.4 63 61.9 f 6.5 2.53 0.1 27.8 a V a l u e s are mean standard e r r o r s obta ined from e igh t s teer s on each pe r i od of the experiment ^ C a l c u l a t e d as the product of the amount of weight gained as muscle and the p r o t e i n content of s k e l e t a l muscle Assuming f i r s t - o r d e r k i n e t i c s d i f f e r e n t (P < .01) compared to va lues on day 28 e D i f f e r e n t (P < .05) compared to va lues on day 28 ^ D i f f e r e n t (P < .05) compared to va lues on the preceding day - 76 -F i gu re 1 0 . R e l a t i o n s h i p between s k e l e t a l muscle p r o t e i n synthes i s and degradat ion i n s t e e r s . - 77 -~ 2 5 _ _L _L o Nishizawa et §1.(1979) • Harris & Milne (1981a) • Present study 645 0.5l_fl_ 245 345 445 BODY-WEIGHT (kg) 545 Figure 11. Urinary excretion of N T-methylhistidine i n c a t t l e . . . . .1 • • • • • • • • CONTROL ZERANOL DES SYNOVEX-S I 1 • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 1 ••+•+ +++++ •++++ ••+++ «•++++ *•++++ +++++ •++•+ +++++ •••++ •+••• •+•++ +++++ +++++ •+++• +•++• •+++• ••••+ ++•++ ++••• ++••+ +++++ ++••• +••++ +++++ ••••• to *• y u • • }***** • ******* I • • «!••••• |» • • ++••• si I' •o° *0 0 >o° Ur i na r y N T - m e t h y l h i s t i d i n e e x c r e t i o n i n s tee r s implanted w i t h anabo l i c compounds. Each bar represents mean ± SE of values obta ined from two s teer s on days 28, 42, 56 and 63 f o l l o w i n g the imp l an t a t i o n (n=8). - 79 -650 r _ 600 Q Q. 550 500 0 1 -'"•X • • • • • o • • • • • • • • • o • • • • • • • • • • • • • • o • • • • • • • • • • • a • • • • • • • • » • • * • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • T e • • T t t * * * 1 • • i. • • • • CONTROL ZERANOL DES SYNOVEX-S CI >°o( ••••• v.* ••••• ••••• v.* ••'•I ••••• ••••• • V . •+••• ••••• ••••• ••••• ••••• ••••• ••••• ••••• ••••• ••••+ ••••• ***** ***** ***** ***** ***** ***** ***** ***** ••••• ••••• L » • L***W* I a »w»\***** >o° >°o' >%% ° 750 700 650 ^ bl) CO 600 g= 550 io F i gu re 13. Rates of muscle p r o t e i n degradat ion (MPD) and synthes i s (MPS) i n s teer s implanted w i t h anabo l i c compounds. Each bar represent s mean ± SE of va lues obta ined from two s tee r s on days 28, 42, 56 and 63 f o l l o w i n g the imp l an t a t i o n (n=8). - 80 -s yn the s i s of muscle p r o t e i n i n s teer s implanted w i t h anabo l i c compounds. Zeranol imp l an t a t i o n tended to r e s u l t i nc reased ra tes of both MPS and MPD than DES and Synovex-S i m p l a n t a t i o n s . The amount of muscle p r o t e i n gained was found to be s i g n i f i c a n t l y h igher (P < .01) i n DES and Synovex-S than i n Zerano l implanted or the unimplanted c o n t r o l s teer s (Table 11). Though s l i g h t l y lower than DES and Synovex-S imp l an t a t i o n s , the s tee r s implanted w i t h Zeranol e x h i b i t e d s i g n i f i c a n t l y h igher (P < .01) amount of MPG than the unimplanted s t e e r s . The FBR and the h a l f - l i f e va lues of m y o f i b r i l l a r p r o te i n s as i n f l u e n c e d by d i f f e r e n t treatment groups are presented i n Table 12. Zerano l imp l an t a t i o n r e s u l t e d i n s l i g h t l y h igher (P > .05) FBR and lower (P > .05) h a l f - l i f e va lues f o r the m y o f i b r i l l a r p ro te i n s than the other treatment groups. From the va lues of MPS, MPD and MPG an e s t i m a t i o n of the e f f i c i e n c y of muscle p r o t e i n s ynthes i s was c a l c u l a t e d and i s presented i n Table 13. The e f f i c i e n c y was s l i g h t l y h igher (12%) i n s teer s implanted w i t h DES and Synovex-S than i n Zeranol (9%) implanted or the unimplanted c o n t r o l s t ee r s (8%). DISCUSSION Validity of the method N T - M e t h y l h i s t i d i n e e x c r e t i o n i n u r i ne has been used to determine the r a te of MPD and to est imate parameters of muscle p r o t e i n metabol ism i n growing beef s t e e r s . Most of the assumptions f o r u s ing NT-MH as an index of muscle p r o t e i n breakdown have been expe r imenta l l y - 8 1 -Table 11. Amount of muscle p r o t e i n gained (MPG) i n s tee r s implanted w i t h va r i ous anabo l i c compounds 1 Treatment MPG (g/d ) 2 C o n t r o l 45.2 + 4 . 0 a Zerano l 60.1 + 4 . 5 b DES 73.8 + 2 . 9 C Synovex-S 77.9 + 1 2 . 3 C Mean of va lues obta ined on 28, 42, 56 and 63 days f o l l o w i n g i m p l a n t a t i o n Weight ga in as muscle x p r o t e i n content of s k e l e t a l muscle i b c ' ' Means w i t h d i f f e r e n t s upe r s c r i p t s d i f f e r (P < .01) - 82 -Table 12. Effect of anabolic compounds on the fractional rate of protein breakdown (FBR) and the h a l f - l i f e values of myofibrillar proteins in growing beef steers 1 FBR2 Half-Life 3 Treatment (%/d) (d) Control 2.4 ± 0.2 29.5 Zeranol 2.6 ± 0.2 26.3 DES 2.3 ± 0.2 29.6 Synovex-S 2.4 ± 0.7 28.6 *Mean ± SE obtained on 28, 42, 56 and 63 days following the implantation ^-MH x 100/Total NT—MH content of skeletal muscle Assuming first-order kinetics - 83 -Table 13. The e f f i c i e n c y of muscle p r o t e i n s yn thes i s i n s tee r s implanted w i t h va r i ous anabo l i c compounds P r o t e i n P r o t e i n Net E f f i c i e n c y Synthes i s Degradat ion Synthes i s (Ne t/Ove ra l l ) Treatments (g/d) (g/d) (g/d) xlOO C o n t r o l 565 + 50 519 + 47 46 8.14 (' " 8%) Ze rano l 663 + 45 603 + 43 60 9.05 (' " 9%) DES 617 + 51 543 + 49 74 11.99 (' "12%) Synovex-S 641 + 74 563 + 67 78 12.17 (' "12%) Mean ± SE - 84 -v e r i f i e d f o r t h e i r v a l i d i t y i n c a t t l e by H a r r i s and M i l ne (1981a). The bas i s of the v a l i d a t i o n procedure i s that i n c a t t l e there i s a r a p i d recovery of i n j e c t e d r a d i o - l a b e l e d NT-MH w i t h i n 5 to 7 days i n u r i n e . Even though NT-MH e x c r e t i o n i n u r i ne has been accepted as a v a l i d index of MPD i n many spec ies i n c l u d i n g c a t t l e , se r i ous doubts have been r a i s e d by M i l l w a r d et a l . (1980b) as to the v a l i d i t y of t h i s method i n r a t s . I t was suggested that t i s s ue s other than s k e l e t a l muscle, v i z . , s k i n , i n t e s t i n e , e t c . might c on t r i bu t e to a cons ide rab le p o r t i o n of the u r i n a r y N T-MH. More r e c e n t l y , however, S a n t r i d r i a n et a l . (1981) d i s cu s sed that the u r i n a r y NT-MH o r i g i n a t e s l a r g e l y from s k e l e t a l musc le. N i sh izawa et a l . (1979) repor ted that more than 93% of the t o t a l NT-MH i n the analyzed c a t t l e t i s s u e occurred i n s k e l e t a l muscle p r o t e i n . I t i s f o r these reasons that i n t h i s study NT-MH e x c r e t i o n i n u r i ne was used as an i n v i vo index of MPD. One po in t which must be s t re s sed i s that the c a l c u l a t e d va lues of MPD, MPS and MPG were a l l based on c e r t a i n assumptions (See M a t e r i a l s and Methods) and may not n e c e s s a r i l y represent abso lute e s t ima te s . Because t h i s study was designed p r i m a r i l y to monitor the changes a s soc i a ted w i t h muscle p r o t e i n metabolism and develop a r a t i o n a l e f o r the mechanism of a c t i o n of anabo l i c compounds on muscle p r o t e i n metabol i sm, the c a l c u l a t e d va lues were taken as such f o r comparison among t reatments . The ease w i t h which the parameters of muscle p r o t e i n metabol ism cou ld be c a l c u l a t e d from the u r i n a r y NT-MH va lues alone makes t h i s technique very a t t r a c t i v e i n s tud ie s of t h i s na tu re . For the abso lu te est imates of muscle p r o t e i n s ynthes i s and muscle p r o t e i n g a i n , - 85 -one has to r e s o r t to l abo r i ou s and ted ious methodologies as desc r ibed i n the l i t e r a t u r e . Developmental Aspects of Muscle Protein Metabolism Urinary Nr-MH excretion The va lues of NT-MH e x c r e t i o n repor ted f o r s teer s i n t h i s study are s l i g h t l y h igher than those repor ted by Nish izawa et_ a l . (1979) and H a r r i s and Mi lne (1981a) (F i gu re 11). At a growth stage of 411, 432, 456 and 455 kg , the r a t e of NT-MH excreted i n u r i ne was 3.64, 4.27, 5.21 and 4.52 umol/d/kg r e s p e c t i v e l y , as compared to the va lues of 3.68 to 2.75 umol/d/kg at growth stages of 217 to 312 kg f o r H o l s t e i n s t ee r s (N i sh izawa et a l . , 1979) and 3.74 umol/d/kg c a l c u l a t e d f o r 250 kg body weight f o r purebred F r i e s i a n male c a t t l e ( H a r r i s and M i l n e , 1981a). The l a r g e r mean d a i l y e x c r e t i o n of NT-MH by the s tee r s no t i ced i n t h i s study suggests that i n these animals a l a r ge p r o p o r t i o n of the body weight may be con t r i bu ted by muscle t i s s u e and thus r e f l e c t changes i n the m y o f i b r i l l a r p r o t e i n breakdown. The u r i n a r y e x c r e t i o n of a compound could be of e i t h e r endogenous or exogenous i n o r i g i n . I t has been unequ i voca l l y demonstrated that NT-MH o r i g i n a t e s endogenously from the ca tabo l i sm of m y o f i b r i l l a r p r o t e i n s (Young and Munro, 1978). I t i s p o s s i b l e that d i e t a r y sources of NT-MH may a l s o c o n t r i b u t e to a c e r t a i n p r opo r t i on of the u r i n a r y N T-MH. The feed used was ce rea l - ba sed and devoid of any animal product and was s i m i l a r to that used by H a r r i s and M i lne (1981a). No attempt was made to analyze the feed f o r i t s NT-MH c o n c e n t r a t i o n . Recen t l y , however, Nish izawa et  a l . (1979) repor ted the NT-MH content of hay and concent rate to be - 86 -2.4 and 7.7 mg/kg, r e s p e c t i v e l y , and suggested that about 30% of the u r i n a r y NT-MH output could be con t r i bu ted by f eed . I t i s , thus, p o s s i b l e that d i e t a r y NT-MH could have c on t r i bu ted to c e r t a i n po r t i on s of the u r i n a r y NT-MH repor ted here. I t i s t he re fo re suggested that d i e t a r y sources of NT-MH should be taken i n t o c o n s i d e r a t i o n wh i l e r e p o r t i n g NT-MH values i n u r i n e . The e f f e c t of d i e t upon u r i n a r y NT-MH e x c r e t i o n has a l s o been repor ted by Ward and Bu t te ry (1980). The u r i n a r y c r e a t i n i n e concen t r a t i on as w e l l as N T-MH: c r e a t i n i n e r a t i o (Table 9) tended to i nc rea se as the animal gained we ight . The r a t i o of NT-MH to that of c r e a t i n i n e i n u r i ne i n d i c a t e s the l e v e l o f MPD r e l a t i v e t o that of muscle mass (Ward and B u t t e r y , 1980). Growth and Muscle Protein Degradation As shown i n F i gu res 7 and 9, there appears to be a r e l a t i o n s h i p between growth of the s teer s and the r a te of MPD. These developmental changes i n MPD are i n c l o se agreement w i t h those repor ted by M i l l w a r d et a l . (1975). As the s tee r s gained weight there was an i nc rea se i n MPD. The inc rea sed growth r a te and u r i n a r y NT-MH e x c r e t i o n are s i m i l a r to the a n a b o l i c i n c rea se i n MPD dur ing growth (M i l lwa rd et a l . , 1980a). They have suggested that the r a te of MPD may be e leva ted dur ing growth ( anabo l i c i n c rea se ) or dur ing muscle w a s t i n g ( c a t a b o l i c i n c r e a s e ) . Exces s i ve MPD does not always r e f l e c t c a t a b o l i c s t a te as i n the case of muscle wast ing d i sease or a f t e r c o r t i c o s t e r o i d a d m i n i s t r a t i o n . Contrary to the data reported he re , Vernon and Bu t te r y (1978a) found a decrease - 87 -i n NT-MH e x c r e t i o n i n r a p i d l y growing r a t s t r e a t ed w i t h the anabo l i c compound, t renbolone a ce t a t e . These workers suggested that a decrease i n MPD was r e spon s i b l e f o r the inc reased amount of muscle p o r t e i n depo s i t ed . Th i s r e l a t i o n s h i p between growth and MPD obtained by Vernon and Bu t te r y (1978a) could be s i m i l a r to the anabo l i c decrease i n MPD du r ing growth shown by M i l l w a r d et a l . (1980a). The inc reased r a te of MPD observed dur ing r ap id growth (F igure 9) cou ld a l s o be due to m y o f i b r i l l a r s p l i t t i n g and remode l l i ng . Inc reased r a te s of m y o f i b r i l l a r p o r t e i n breakdown dur ing growth were i m p l i c a t e d i n m y o f i b r i l l a r p r o l i f e r a t i o n (M i l lwa rd et a l . , 1975). Dur ing r a p i d growth, i nc reased m y o f i b r i l l a r s p l i t t i n g r e s u l t e d i n inc reased p r o t e i n turnover (Go ldsp ink , 1970). The h igh c o r r e l a t i o n obta ined between MPD and MPS ( r = .9 , F i gu re 10) of the animals conf i rms r ap id muscle p r o t e i n tu rnove r . The mean FBR of m y o f i b r i l l a r p r o te i n s was c a l c u l a t e d to be 2.4%/d (Table 12) . The FBR va lues of 1.22%/d at a growth stage of 217 kg to 1.02%/d at 312 kg (Nish izawa et a l . , 1979) and 1.4%/d at a growing stage of 236 kg ( H a r r i s and M i l n e , 1981a) were reported f o r c a t t l e . The average FBR reported here corresponds to a h a l f - l i f e of approx imately 29 d f o r the m y o f i b r i l l a r p r o t e i n s , a c t i n and myosin. Us ing NT-MH e x c r e t i o n as an index of m y o f i b r i l l a r p r o t e i n breakdown, h a l f - l i f e va lues of 21 to 41 d f o r the m y o f i b r i l l a r p r o te i n s were obta ined by Ward and But te ry (1980) f o r r a t s . I t i s d i f f i c u l t to compare the FBR va lues obta ined i n t h i s study w i th those of the l i t e r a t u r e because the MPD i s under the i n f l u e n c e of a number of f a c t o r s such as hormonal, n u t r i t i o n a l - 88 -and developmental stage of the animal (M i l lwa rd elt a l . , 1980a). The r educ t i on i n the h a l f - l i f e of m y o f i b r i l l a r p ro te i n s I nd i ca te s that there i s a r ap i d turnover of these p r o te i n s i n the s teer s used i n t h i s s tudy. Muscle Protein Synthesis (MPS) The es t imates of MPS were obta ined as the d i f f e r e n c e between the r a te of muscle p r o t e i n gained and degraded. P r o t e i n s yn thes i s i n t i s s u e s of c a t t l e was est imated by Lob ley et a l . (1980) from the i r r e v e r s i b l e l o s s from the blood of r a d i o a c t i v e amino a c i d . P r o t e i n s ynthes i s i n s k e l e t a l muscle of c a t t l e at bodyweight 236, 263 and 628 kg was repor ted to be 316, 355 and 449 g/d, r e s p e c t i v e l y (Lobley et a l . , 1980) and was based on the assumption that the s p e c i f i c a c t i v i t y of f r ee amino a c i d ( l y s i n e ) i n blood de f i ne s c l o s e l y the s p e c i f i c a c t i v i t y of amino a c i d p recur sor f o r p r o t e i n s y n t h e s i s . As i t can be seen, the MPS va lue obta ined i n the present study (622 g/d) i s h igher than those repor ted by Lobley et a l . (1980). The reason f o r the inc reased est imates of MPS observed i n t h i s study may be due to the f a c t that growing s teer s were used wh i l e the above researcher s used h e i f e r s and a dry cow. Secondly, the s p e c i f i c r a d i o a c t i v i t y of p r o t e i n bound amino a c i d i n m^ long i s s imus d o r s i was taken as r e p r e s e n t a t i v e of the t o t a l s k e l e t a l muscle by Lobley et^ a l . (1980). NT-MH has been repor ted to r e f l e c t the average p r o t e i n degradat ion i n whole musculature ( S a n t i d r i a n et a l . , 1981). Hence, MPS va lues obta ined i n t h i s study may r e f l e c t p r o t e i n s yn thes i s i n the musculature of the s teer s as a whole. - 89 -Effect of Anabolic Compounds Though the mechanism of a c t i o n of anabo l i c compounds i s not w e l l understood i t has been e s t a b l i s h e d that they are e f f e c t i v e i n enhancing growth and p r o t e i n d e p o s i t i o n i n the s k e l e t a l muscle. The main i n f l u e n c e on the ra te of p r o t e i n synthes i s and degradat ion i n muscle seems to be hormonal (Young 1980) which has been e x t e n s i v e l y reviewed (Goldberg et a l . , 1980). Bes ides , p r o t e i n degradat ion i t s e l f has been repor ted to p lay a major r o l e i n the r e g u l a t i o n of p r o t e i n balance i n s k e l e t a l muscle (M i l lwa rd et_ a\_., 1980a). There fo re , i t becomes necessary to study the e f f e c t of anabo l i c compounds on muscle p r o t e i n degradat ion and other parameters of s k e l e t a l muscle p r o t e i n metabol i sm. Urinary NT-MH and Muscle Protein Degradation There was a tendency, though not s i g n i f i c a n t , towards inc reased m y o f i b r i l l a r p r o t e i n degradat ion i n s tee r s implanted w i t h e s t rogen i c anabo l i c compounds (F igure 12 and 13). As t h i s i s the f i r s t repor t on the e f f e c t of e s t r ogen i c anabo l i c compounds on MPD us ing NT-MH e x c r e t i o n as an Index, no d i r e c t comparison of the r e s u l t s cou ld be made w i t h va lues i n the l i t e r a t u r e . Recen t l y , G r i f f i t h s (1982) demonstrated a n o n - s i g n i f i c a n t decrease i n NT-MH e x c r e t i o n i n u r i ne from s tee r s r e c e i v i n g a combinat ion of androgenic (Trenbolone ace ta te ) and e s t r ogen i c (Ra lgro) anabo l i c compounds. In an e a r l i e r s tudy, Vernon and Bu t te r y (1978a), demonstrated a s i g n i f i c a n t r educ t i on i n m y o f i b r i l l a r p r o t e i n degradat ion as judged by the reduced u r i n a r y NT-MH e x c r e t i o n , i n female r a t s t r ea ted w i t h the androgenic anabo l i c compound, t renbolone a c e t a t e . The observat ions on MPD of the present study and those - 90 -repo r ted by Vernon and But te ry (1978a) r e v e a l c e r t a i n d i f f e r e n c e s i n the mechanism of a c t i o n of androgenic and e s t r ogen i c anabo l i c compounds on the s k e l e t a l muscle. Whereas e s t r ogen i c anabo l i c compounds seem to have a tendency to i nc rease m y o f i b r i l l a r p r o t e i n degradat ion , the androgenic anabo l i c compounds seem to reduce I t (Vernon and B u t t e r y , 1978a). I n t e r e s t i n g l y , the observat ions of G r i f f i t h s (1982) seem to demonstrate a balance between the a c t i o n s of androgenic and e s t r ogen i c compounds i n c a s t r a t e d males as evidenced by a n o n - s i g n i f i c a n t decrease i n NT-MH e x c r e t i o n i n u r i ne by the s tee r s implanted w i t h both t renbolone aceta te and Ra lgro (Con t r o l = 1.10 m mol N T-MH/d; implanted =0.89 mmol N T-MH/d). Among the anabo l i c compounds used i n t h i s experiment, Zerano l seems to exer t a n o n - s i g n i f i c a n t i nc rea se i n both the u r i n a r y NT-MH e x c r e t i o n and MPD per day than that e x h i b i t e d by s teer s implanted w i t h DES or Synovex-S. The molecular mechanism of a c t i o n ( s ) of p r o t e i n degradat ion as i n f l u e n c e d by the anabo l i c compounds used i n t h i s study i s not c l e a r l y understood. Receptors f o r s t e r o i d hormones i n s k e l e t a l muscle, e s p e c i a l l y androgens have been demonstrated (M iche l and B a u l i e u , 1976; Dahlberg et a l . , 1981) and that the mechanism of a c t i o n of androgenic anabo l i c compounds has been po s tu l a ted to be due to the b i nd i n g of te s to s te rone to g l u c o c o r t i c o i d receptor s and the i n h i b i t i o n of p r o t e i n ca tabo l i sm induced by g l u c o c o r t i c o i d s (Mayer and Rosen, 1975). However, the presence of estrogen receptor s i n the s k e l e t a l muscle or the a l t e r a t i o n of g l u c o c o r t i c o i d induced p r o t e i n degradat ion by e s t r o gen i c anabo l i c compounds remains to be s t u d i e d . The other p o s s i b l e mechanisms by which e s t r ogen i c anabo l i c compounds could - 91 -i n f l u e n c e MPD are by d i r e c t l y a f f e c t i n g muscle p ro te ina se a c t i v i t i e s ( e . g . lysosomal enzymes such as ca theps in B, ca theps in D, a c i d p ro teases , e t c . ) or i n d i r e c t l y by t h e i r e f f e c t on endogenous t h y r o i d hormone s t a t u s . The involvement of muscle p ro te inases on p r o t e i n degradat ion and the p a r t i c i p a t i o n of t h y r o i d hormones i n the r e g u l a t i o n of lysosomal contents have been e x t e n s i v e l y reviewed (M i l lwa rd et^ a l . , 1980a; Goldberg et a l . , 1980). Muscle Protein Synthesis and Gain Both MPS and MPD (F i gu re 13) seem to have been i nc reased i n s teer s implanted w i t h anabo l i c compounds when compared to the unimplanted c o n t r o l s t e e r s . Among the anabo l i c compounds . i n te re s t i ng l y , Zerano l i m p l a n t a t i o n causes a r ap i d turnover of muscle p r o t e i n s . Though muscle p r o t e i n turnover seems to be r ap i d i n a l l the implanted ( Ze rano l , DES & Synovex-S) when compared to the c o n t r o l s teer s i t seems to be s lower i n s tee r s implanted w i t h DES or Synovex-S than Ze rano l . The r a p i d turnover r a te of muscle p r o t e i n i n Zerano l implanted s tee r s cou ld be s ub s t an t i a t ed f u r t h e r by the h igh FBR va lues (2.6% vs 2.3 to 2.4%d) and the lower h a l f - l i f e va lues f o r the m y o f i b r i l l a r p ro te i n s (26d vs 28.6 to 29.6d) . The s i g n i f i c a n t l y h igher (P < .01) amount of MPG by the s tee r s implanted w i t h Ze rano l , DES and Synovex-S than the c o n t r o l s tee r s (Table 11) i s i n agreement w i th e a r l i e r repor t s on the e f f e c t of these compounds on r a te of p r o t e i n ga in (Sharp and Dyer, 1971; Byers , 1982b; Rumsey et a l . , 1981; Rumsey, 1982). The decreased amount of muscle - 92 -p r o t e i n gained by the Zerano l implanted s tee r s could be due to the r e l a t i v e i n c rea se i n MPD compared to DES and Synovex-S implanted s t e e r s . Contrary to the t rends of MPS and MPD observed (namely i nc rea se i n both MPS & MPD) i n t h i s exper iment, androgenic anabo l i c compounds have been shown to decrease both MPS and MPD compared to the unimplanted c o n t r o l an ima l s , w i t h the decrease i n MPS being l e s s than the decrease i n the r a t e of MPD (Vernon and B u t t e r y , 1978b). A l t e r n a t i v e l y , the i n f l u e n c e of anabo l i c compounds on the parameters of muscle p r o t e i n metabol i sm, namely MPS and MPD cou ld be b e t t e r exp la i ned by t h e i r e f f e c t on the e f f i c i e n c y of MPS (Table 13). The e f f i c i e n c y of MPS was found to be h igher i n s t ee r s implanted w i t h DES and Synovex-S (12%) than Zerano l (9%) or unimplanted c o n t r o l s t ee r s (8%) . The h i gher r a t e of muscle p r o t e i n gained by the s tee r s implanted w i t h DES and Synovex-S than that observed i n Zerano l implanted s tee r s cou ld be p a r t l y a t t r i b u t e d to the lower r a te s of MPD (as i n d i c a t e d by the h igher e f f i c i e n c y f i g u r e s ) . Bates and M i l l w a r d (1981) concluded that the i nc reased muscle growth of the r e l a t i v e l y f a s t growing r a t s t r a i n compared to that of a slow ga in i ng hooded s t r a i n (M i l lwa rd et a l . , 1975) cou ld be due to the involvement of l e s s MPD per u n i t s y n t h e s i s . I t i s suggested from the r e s u l t s of the present experiment that both the r a te s of MPS and MPD have to be s imu l taneous l y cons idered w h i l e s tudy ing the e f f e c t of anabo l i c compounds on muscle p r o t e i n metabol ism as both are i n vo l ved i n governing the amount of muscle p r o t e i n depo s i t ed . - 93 -CONCLUSION The r e s u l t s of t h i s study demonstrated the u se fu lnes s of measuring N T - M e t h y l h i s t i d i n e i n u r i n e as a r a p i d , non -de s t r u c t i v e method f o r s tudy ing muscle p r o t e i n metabol ism i n l a r ge animals such as beef c a t t l e . The r a p i d growth r a t e of s tee r s was accompanied by a r a p i d r a t e of muscle p r o t e i n tu rnover . The i m p l a n t a t i o n of anabo l i c compounds r e s u l t e d i n an i nc reased amount of muscle p r o t e i n gained by the s t e e r s . A gene r a l , n o n - s i g n i f i c a n t i n c rea se i n muscle p r o t e i n degradat ion was observed i n s t ee r s implanted w i t h anabo l i c compounds, but muscle p r o t e i n d e p o s i t i o n was f a c i l i t a t e d by a h igher r a te of muscle p r o t e i n s yn thes i s than deg radat i on . Anabo l i c compounds were found to be e f f e c t i v e i n i n c r e a s i n g the e f f i c i e n c y of muscle p r o t e i n s y n t h e s i s . The amount of muscle p r o t e i n gained was h igher i n s tee r s implanted w i t h DES and Synovex-S than Ze r ano l . The d i f f e r e n t i a l e f f e c t s of anabo l i c compounds were concluded to be due to the involvement of l e s s muscle p r o t e i n degradat ion per u n i t s y n t he s i s . - 94 -EXPT. IC COLLAGEN METABOLISM IN GROWING BEEF STEERS AS ASSESSED BY THE URINARY HYDROXYPROLINE EXCRETION AND THE EFFECT OF ESTROGENIC ANABOLIC COMPOUNDS INTRODUCTION A l t e r a t i o n s i n p r o t e i n metabol i sm seem to be one of the major metabo l i c consequences of anabo l i c compounds i n ruminants and non-ruminants. A number of s tud ie s have demonstrated the e f f e c t of anabo l i c compounds i n enhancing p r o t e i n a c c r e t i o n i n an imal s : DES (Byers , 1982b; Rumsey et a l . , 1981); Synovex-S (Rumsey, 1982); Zerano l (Sharp and Dyer, 1971). Even though the genera l p r o t e i n anabo l i c p r o p e r t i e s of anabo l i c compounds have been e s t a b l i s h e d , only a few experiments have been conducted to study t h e i r e f f e c t on s p e c i f i c and mixed p r o te i n s of the body: e . g . m y o f i b r i l l a r p r o t e i n metabolism (Vernon and B u t t e r y , 1978a) but no such attempts have been made to exp lo re t h e i r e f f e c t on the metabolism of c o l l a g e n , a major s t r u c t u r a l p r o t e i n of the mammalian body. Co l l agen i s a hyd roxypro l i ne (HYPRO) r i c h p r o t e i n , r ep re sen t i ng about 30 per cent of the t o t a l body p r o t e i n s . I t has been shown to i n f l u e n c e muscle development (De La Haba et a l . , 1975; Ke t l e y et a l . , 1976) and has been suggested to have d i r e c t f u n c t i o n i n d e f i n i n g growth (Robins, 1977). Dur ing the degradat ion of co l l a gen f i b r i l s , HYPRO i s re lea sed and the quan t i t y of HYPRO i n u r i ne has been cons idered as an index of co l l a gen metabolism ( Z i f f et a l . , 1956; K i v i r i k k o , 1970). The e x c r e t i o n of HYPRO i n u r i ne has been reported to be under the i n f l u e n c e - 95 -of different hormones (Jasin et a l . , 1962;) and several other physiological conditions (See Review by Kivirikko, 1970). This experiment was conducted to study the effect of estrogenic anabolic compounds on collagen metabolism in vivo in growing steers. Urinary total HYPRO (free and peptide bound) excretion has been uti l i z e d as an in_ vivo index of collagen metabolism. The main objectives of this investigation were: (a) to study collagen metabolism at different growth stages in rapidly growing steers (b) to study the effect of estrogenic anabolic compounds on urinary HYPRO excretion and thus on collagen metabolism, and (c) to establish urinary HYPRO excretion patterns and values for steers. MATERIALS AND METHODS Animals The animals, their management and the procedure for the implantation of anabolic compounds were the same as described in EXPT. IA. Two steers from each of the four treatment groups [control Zeranol, Diethylstilbestrol (DES) and Synovex-S ] described in EXPT.IA were used. The steers were fed a finishing ration (Table 5) and the body weight measurements were taken at weekly intervals. - 96 -Urine Collelction Procedure Ur i na r y HYPRO e x c r e t i o n was monitored from the 24 h u r i n e c o l l e c t e d from i n d i v i d u a l s t ee r s over a per iod of 24 h on days 28, 42, 56 and 63 f o l l o w i n g the i m p l a n t a t i o n of anabo l i c compounds. The method of u r i ne c o l l e c t i o n and storage were s i m i l a r to those desc r ibed i n Expt . IB. Analytical Procedure  Hydroxyproline (HYPRO) T o t a l HYPRO ( f r e e and peptide-bound) concen t r a t i on i n the u r i ne samples was determined accord ing to the method desc r ibed by F i r s c h e i n and S h i l l (1966). B r i e f l y , u r i n e samples were a c i d hydro lyzed w i t h equal volume of concent ra ted h y d r o c h l o r i c a c i d (12N) a t 110°C f o r 24 h. The r e s u l t a n t hyd ro l y za te was f i l t e r e d through g l a s s wool and n e u t r a l i z e d w i t h 5% Na 2 C03. The pH of the hyd ro l y za te was then adjus ted to 6.0 w i t h 0.1 M c i t r a t e - b u f f e r . The HYPRO i n the a c i d hyd ro l y za te was then o x i d i z e d to p y r r o l e i n the presence of i s o p r o p y l a l c o h o l and ch loramine-T. The reagent mixture was a l lowed to stand at room temperature f o r 4 minutes and then reacted w i t h p-dimethylamlno-benzaldehyde ( E h r l i c h ' s Reagent) i n a b o i l i n g water bath f o r 2 minutes. The reagent mixture was then coo led immediately i n an i c e bath and kept at room temperature f o r 90 minutes . The f i n a l c o l o r was read at 575 nm aga in s t a reagent b l ank . Total Nitrogen T o t a l n i t r o g e n concen t r a t i on i n the u r i ne samples was determined by the K j e l d h a l procedure. - 97 -Creatinine C r e a t i n i n e concen t r a t i on i n the samples was determined by a c o l o r i m e t r i c procedure as desc r ibed e a r l i e r (Expt . I B ) . Calculations D a i l y u r i n a r y HYPRO e x c r e t i o n was c a l c u l a t e d from the concen t ra t i on of HYPRO and the 24 h u r i ne volume. HYPRO e x c r e t i o n per day = HYPRO (mg/ml) x 24 h u r i ne volume (ml) (mg/d) D a i l y HYPRO e x c r e t i o n data was expressed as mg/d, mg/d/kg^'^^, and HYPRO-N (as % of t o t a l N i n u r i n e ) . For the purposes of the l a t t e r e xp re s s i on , n i t r ogen content of HYPRO was taken as 10.68 per c e n t . The va lues of d a i l y HYPRO e x c r e t i o n by a l l the s tee r s on any one of the c o l l e c t i o n dates were averaged and subjected to l e a s t squares a n a l y s i s of va r i ance us ing UBC BMD:10V l i n e a r hypothes i s computer program. RESULTS Urinary HYPRO Excretion at Different Stages of Development Ur i na r y HYPRO e x c r e t i o n at d i f f e r e n t growth stages i n s tee r s i s presented i n F i gu res 14 and 15. The HYPRO e x c r e t i o n (mg/d; mg/d/ k g 0 * 7 5 ) i n u r i n e decreased (P < .01) g r adua l l y as the animals advanced i n matu r i t y and gained weight . U r i na r y HYPRO e x c r e t i o n decreased u n t i l day 42 (P < .01) and remained at s i m i l a r l e v e l s on day 56 and 63. Though a s i m i l a r p a t t e r n was observed when HPYRO was expressed as HYPRO-N (as a % of t o t a l n i t r o g e n i n u r i ne ) there was a r educ t i on i n u r i n a r y HYPRO e x c r e t i o n even beyond - 98 -F i gu re 14. U r i na r y t o t a l hyd roxypro l i ne (HYPRO) excreted by s tee r s on d i f f e r e n t per iods of the exper iment. Each po in t represents mean ± SE of va lues obta ined from eight s t e e r s . * * P < .01 compared w i t h day 28. - 99 -Figure 15. Urinary hydroxyproline-N (HYPRO-N expressed as a per cent of urinary total nitrogen) excretion by steers on different periods of the experiment. Each point represents mean ± SE of values obtained from eight steers. +P < .05 compared with day 42. *P < .05 compared with day 28. ***P < .001 compared with day 28. - 100 -day 42 w i t h no change beyond day 56 (F igure 15) . The r a t i o of HYPRO:creat in ine i n u r i ne on d i f f e r e n t stages of development i s presented i n Table 14. The r a t i o of HYPRO: C r e a t i n i n e decreased (P < .05) u n t i l day 42 and remained constant dur ing the r e s t of the exper imenta l p e r i o d . Effect of Anabolic Compounds on Urinary HYPRO Excretion T o t a l HYPRO e x c r e t i o n i n u r i ne by the s tee r s on d i f f e r e n t times f o l l o w i n g the imp l an t a t i o n of anabo l i c compound i s presented i n F i gu re 16. The treatment x pe r i od i n t e r a c t i o n was not s t a t i s t i c a l l y s i g n i f i c a n t (P > .05) but a g radua l r educ t i on i n the amount of HYPRO excreted beyond day 28 was observed i n a l l the treatment groups. In a l l the per iods s tud ied HYPRO e x c r e t i o n was found to be s l i g h t l y e l eva ted i n s t ee r s implanted w i t h DES when compared to other compounds. When a l l the per iods (28, 42, 56 & 63 d) were taken i n t o c o n s i d e r a t i o n , u r i n a r y HYPRO e x c r e t i o n was h igher (P < .05) i n s tee r s implanted w i t h DES than Zeranol or Synovex-S or the unimplanted c o n t r o l s t ee r s (F i gure 17) . There was no d i f f e r e n c e (P > .05) i n the u r i n a r y HYPRO e x c r e t i o n among Ze rano l , Synovex-S or unimplanted c o n t r o l s t e e r s . The u r i n a r y HYPRO:creat in ine r a t i o was found to be s i m i l a r among a l l the treatment groups (P > .05, Table 14). DISCUSSION Hydroxypro l ine (HYPRO) e x c r e t i o n i n u r i ne has been used i n t h i s experiment to monitor co l l a gen turnover i n s teer s implanted w i t h v a r i ou s anabo l i c compounds. - 101 -Table 14. U r i na r y hydroxypro l i ne e x c r e t i o n (mg/g c r e a t i n i n e ) i n s teer s on va r i ou s times f o l l o w i n g the i m p l a n t a t i o n of anabo l i c compounds U r i na r y HYPRO: c r e a t i n i n e days a f t e r i m p l a n t a t i o n Mean Treatment 28 42 56 63 (± SE) C o n t r o l 67.66 42.99 39.56 53.02 50 . 81 a (5.24) Zerano l 63.22 49.93 58.35 29.29 50 .20 3 (6.76) DES 85.41 72.48 38.96 57.86 67 . 21 3 (7.84) Synovex-S 70.14 53.11 49.3 39.14 52 .92 3 (5.83) Mean (± SE) 7 1 . 61 a 54 .63 b 47 .63 b 44 .83 b (7.04) (4.89) (5.22) (5.19) i ,b Columns w i t h d i f f e r e n t s u p e r s c r i p t s d i f f e r (P < .05) Rows w i t h d i f f e r e n t s u p e r s c r i p t s d i f f e r (P < .05) - 102 -JZ o ? 101-O cr CL > i >-cc < 8 6-4 2 0 JJ X %% 44 • 4 44 ^ 4 4 4 4 44 44 44 44 4 4 4 4 4 4 •\** 4 4 • 4 4 ' 4 4 • 4 4 4 4 4 4 4 * , ' «ltt rl+t< O X o o o X 28 4 4 4 4 4 4 4 4 4 4 4-4 4 4 4 4 4 4 4 4 4 4 a 42 X?1 • • • • • • • • __ m _QJ_\ CONTROL ZERANOL DES SYNOVEX-S X o I o 0 4 4 4 4 4 4 4 4 44 4 4 4 4 44 44 0 4 4 • 4 4 44 44 < 0 • 44 • • 44 • • 44 44 0 • 44 < o • • 44 44 • 44 56 63 DAYS AFTER IMPLANTATION Figure 16. Urinary total.hydroxyproline (HYPRO) excretion by steers on different days following the implantation of anabolic compounds. Each bar represents mean ± SE of values obtained from two steers. - 103 -10 r— O _ $2 8 6 > ^ 4 DC CvJ 0 X b X I > • • « » c • . • i •++ •++ •++ •+• •++ •+• •++ ••+ •++ +++ •++ ••+ +++ •++ +•+ • • • *++ >o oc o oc o oc o DC o i V . • • • • CONTROL ZERANOL fp&SYNOVEX-S F i gu re 17. E f f e c t of anabo l i c compounds on u r i n a r y t o t a l hyd roxypro l i ne e x c r e t i o n by growing beef s t e e r s . Each bar represents mean ± SE of va lues obta ined from two s tee r s on days 28, 42, 56 and 63 f o l l o w i n g the i m p l a n t a t i o n (n=8). a » t B a r s w i t h d i f f e r e n t alphabet d i f f e r s i g n i f i c a n t l y (P < .05) . - 104 -Validity of the Method In collagen biosynthesis, proline is incorporated into the peptide chain and is then hydroxylated to HYPRO (Peterkofsky and Udenfriend, 1965) by the protocollalgen-proline-hydroxylase system. During the degradation of collagen, HYPRO is released and it s excretion reflects changes in the turnover of collagen in the body (Kivirikko, 1970; Robins, 1977). Besides, free HYPRO present in the biological f l u i d has been demonstrated to originate from the degradation of collagen and not from the direct hydroxylation of proline or prolyl-t RNA (Review by Kivirikko, 1970) and has been utilized to study collagen metabolism to a large extent in man (Jasin et a l . , 1962; Kivirikko, 1970). Though HYPRO excretion was used as an index, i t s presence in the N-terminal extension peptides Of protocollagen (Horlein et a l . , 1978) and the contribution to the urinary HYPRO by Clq plasma protein constituent (Kohler and Muller-Eberhard, 1972) pose a problem to the application of this method. While the N-terminal extension peptides are released and presumably degraded during normal fibrillogenesis the Clq plasma protein constituent exhibits a rapid turnover rate. An alternative pathway unrelated to collagen metabolism, but contributing small amounts of HYPRO from glyoxylate and pyruvate in liver was also reported (Goldstone and Adam, 1965; Efron et a l . , 1968). Developmental Aspects of Collagen Metabolism Data in Figure 14 demonstrate the developmental aspects associated with collagen turnover. It tended to decrease as the steers - 105 -gained weight as evidenced by the decrease in urinary HYPRO. The developmental changes in collagen content In the bovine carcass from birth to maturity were presented i n detail by Boccard (1978). It was reported that the synthesis of collagen decreased, as the activity of prolyloxylase responsible for the hydroxylation f e l l during the ageing process (Boccard, 1978). The reduction in the urinary HYPRO excretion with advancing maturity observed in the present study could be interpreted as due to a reduction in the synthesis of new collagen or due to increased resistance of collagen f i b r i l s to breakdown by collagenolytic enzymes. Kivirikko (1970) reported that during the maturation process more and more intermolecular cross-links were formed and the collagen became increasingly resistant to solubilization and collagenase attack. Goll et_ a l . (1964) reported the presence of more frequent or strong cross-linkages in the collagen of mature than young bovine and a decreased collagen solubility with advancing maturity. The developmental changes in collagen turnover observed in this study are similar to those reported by Wu et a l . (1981) in steers. However, in the latter study plasma non-protein HYPRO (NPH) was used as an index of collagen turnover. When the steers were fed a high concentrate diet, NPH in plasma increased upto 6 weeks (or t i l l 410 kg -indicating rapid collagen turnover) and decreased thereafter (Wu et a l . , 1981). It is possible that animals used in the present study could have exhibited rapid collagen turnover had they been studied at an earlier stage of growth. Besides, the steers used in this experiment could have begun to accumulate more fat with continual reduction in muscle - 106 -deposition thus exhibiting reductions in urinary HYPRO excretion. Kivirikko (1970) suggested that both the rate of collagen synthesis (ki and k 2; Figure 18) and degradation (k 5, k 6 and k 7) are considerably slower in older animals in spite of their greater collagen content of the body. In conclusion, the developmental reduction in the urinary HYPRO excretion and hence reduction in collagen turnover could be attributed to a reduction in the total amount of soluble and metabolically active collagen and the increased resistance of the collagen f i b r i l s to collagenase attack as the steers advance in maturity. Anabolic Compounds and Collagen Turnover The effect of anabolic compound on collagen metabolism in growing steers is shown in Figure 16 and 17. Among the compounds, DES seem to increase collagen turnover as indicated by increased (Figure 17) HYPRO excretion in urine compared to other treatments. In fact, urinary HYPRO excretion observed in control and steers implanted with Zeranol and Synovex-S was similar (P > .05). These differential actions of anabolic compounds on collagen metabolism are noteworthy, and since this is the f i r s t paper on the effect of anabolic on urinary HYPRO excretion, no direct comparison with the values in the literature could be made. Increased HYPRO in urine by the steers Implanted with DES indicates an increased amount of collagen present and i t s rapid turnover. These observations are comparable to the early reports of Mcintosh et a l . (1961) on the effect of orally administered stilbestrol in increasing connective tissues of lamb skeletal muscle. Based on - 107 -PROLINE K1 Protocollagen •14 M NaCl k3 Less kn Insoluble Soluble Soluble Collagen k4 Collagen Collagen Peptide Hypro in tissues Pept ide Hypro in Plasma V14 Peptide Hy.proM . in Urine Free Hypro] in Tissues v11 K 1 0 Free Hypro in Plasma Free Hypro in Urine co 2 Urea Figure 18. A schematic summary of the relationship between urinary hydroxyproline excretion and the metabolism of collagen, k l to k!5 refers to conversion rate constants. - 108 -unpubl i shed da ta , they have a l s o i n d i c a t e d inc reased co l l a gen content of s k e l e t a l muscle a long w i t h mucoprotein and e l a s t i n from s t i l b e s t r o l t r e a t e d lambs. S i m i l a r i nc rea se i n s k i n c o l l a gen content was a l s o reported by Ana s t a s s i ad i s et a l . (1955) from estrogen t r ea ted immature p u l l e t s . As most of the anabo l i c a c t i on s of DES i n ruminants are mediated through changes i n endogenous p r o t e i n hormones, e s p e c i a l l y GH, one could hypothes ize that the inc reased HYPRO e x c r e t i o n i n DES implanted s t ee r s may be p a r t l y due to the i n d i r e c t e f f e c t s of GH, which has been repor ted to promote co l l a gen synthes i s in_ v i t r o (Daughaday and M a r i z , 1962) and p o s s i b l y i n f l u e n c e HYPRO e x c r e t i o n by i n c r e a s i n g o v e r a l l c o l l a gen s y n t h e s i s . But the l a c k of response on u r i n a r y HYPRO e x c r e t i o n due to Zerano l and Synovex-S imp lan ta t i on s makes the above theory i n v a l i d , as the l a t t e r anabo l i c compounds have a l s o been shown to a l t e r endogenous GH s ta tu s (Olsen et a l . , 1977; r e s u l t s of Expt . I I B ) . S i m i l a r (P > .05) HYPRO:creat in ine r a t i o (Table 14) among a l l the treatment groups suggests f u r t h e r that growth hormone may not be i n vo l v ed i n the d i f f e r e n t i a l responses of anabo l i c compounds on co l l a gen tu rnove r . Though a r a t i o n a l exp l ana t i on f o r the d i f f e r e n t i a l a c t i o n of DES as compared to Zeranol and Synovex-S on co l l a gen metabolism could not be g i ven , the data presented e s p e c i a l l y f o r DES, support the genera l hypothes i s on the e f f e c t of s t i l b e s t r o l i n a l t e r i n g connect ive t i s s u e metabol i sm. I t i s hypothes ized that the inc reased HYPRO e x c r e t i o n i n DES - 109 -implanted s tee r s may be a t t r i b u t e d to : a) i nc reased r a te of c o l l a gen synthes i s ( k i and k 2 ; F i gu re 18); b) r educ t i on i n the convers ion of s o l ub l e c o l l a g e n to l e s s s o l ub l e c o l l a gen (k3 and k n ) ; c) i nc reased r a t e of c o l l a g e n degradat ion ( k n , k^, k 5 , kg and k j ) , and d) the involvement of a l l the above f a c t o r s . Data i n d i c a t e a need f o r f u r t h e r research i n t h i s a rea . From a p r a c t i c a l view p o i n t , i f h igher HYPRO e x c r e t i o n ra te i s t \ as soc i a ted w i t h h igher c o l l a g e n content i t would he lp to e x p l a i n the l ower i ng of carcass q u a l i t y f o l l o w i n g s t i l b e s t r o l f eed i ng . Th is i n l i g h t of the l a ck of s i g n i f i c a n t e f f e c t on c o l l a gen metabolism due to Zerano l and Synovex-S imp lan ta t i on s suggests the l a t t e r compounds be b e t t e r replacements f o r DES. CONCLUSION The r e s u l t s of t h i s study i n d i c a t e d c e r t a i n developmental changes a s soc i a ted w i t h c o l l a g e n metabolism i n s t e e r s . Co l l agen turnover decreased as the s tee r s advanced i n ma tu r i t y and gained weight as i n d i c a t e d by the r educ t i on i n the u r i n a r y hyd roxyp ro l i ne e x c r e t i o n . I t i s concluded that the r educ t i on i n the u r i n a r y hydroxypro l i ne e x c r e t i o n w i t h advancing matu r i t y of s tee r s i s due to a r educ t i on i n the synthes i s of new co l l a gen f i b r i l s and due to the inc reased r e s i s t a n c e of the c o l l a g e n f i b r i l s to the breakdown by the c o l l a g e n o l y t i c enzymes. The d i f f e r e n t i a l e f f e c t of anabo l i c compounds on co l l a gen metabol ism was a l so e s t a b l i s h e d i n t h i s study. Imp lan ta t i on of DES had a profound e f f e c t on the i n v i v o c o l l a gen turnover as i n d i c a t e d by the inc reased u r i n a r y hydroxypro l i ne e x c r e t i o n wh i l e the imp l an t a t i o n w i t h - 110 -Zerano l and Synovex-S had no e f f e c t on co l l a gen metabol i sm. I t i s concluded that DES i nc reases the amount of c o l l a gen and i t s turnover In s t e e r s . These a c t i on s of DES on connect ive t i s s u e metabolism cou ld be r e spon s i b l e f o r the genera l l ower ing of the carcass q u a l i t y observed by other r e sea rche r s . - I l l -STUDY II EFFECT OF ANABOLIC COMPOUNDS ON THE METABOLISM OF HORMONES IN GROWING BEEF STEERS Studies were conducted to explore certain aspects of the effects of anabolic compounds on the endocrine system in growing beef steers. Attention was focussed on the metabolism of thyroxine, growth hormone and insulin. EXPERIMENT IIA: Effect of anabolic compounds on thyroid hormone status in growing beef steers. (1) plasma thyroid hormone levels (2) kinetic parameters of thyroxine metabolism. EXPERIMENT IIB: Effect of anabolic compounds on plasma growth hormone level and i t s kinetic parameters of metabolism in growing steers. EXPERIMENT IIC: Effect of anabolic compounds on the kinetic parameters of insulin metabolism in growing steers. - 112 -EXPERIMENT I I A:l EFFECT OF ANABOLIC COMPOUNDS ON PLASMA LEVELS OF THYROID HORMONES IN GROWING BEEF STEERS INTRODUCTION Increased growth rate of animals implanted with anabolic compounds has been established (Preston, 1975; Galbraith and Topps, 1981). Since thyroid hormones are involved in the regulation of growth and metabolism (Thorngren and Hansson, 1973) i t appears that they might also mediate, at least in part, the growth promoting properties of anabolic compounds. The increased weight of the thyroid gland (Clegg and Cole, 1954) and the height of the thyroid f o l l i c u l a r epithelial cells (Davey et a l . , 1959) of steers treated with DES add further support to this hypothesis. However, the effect of anabolic compounds on the thyroid gland seems to be inconsistent as indicated by the lack of increase in the thyroid gland weight and the concentration of plasma protein-bound iodine in DES treated steers (Trenkle, 1969; Clegg and Carroll, 1956; Trenkle, 1970a). The effect of DES in increasing the fasting metabolic rate of cattle (Rumsey et a l . , 1973; Tyrrell et a l . , 1975) and sheep (Louw et^ a l . , 1964) strongly suggests the involvement of thyroid hormones in the estrogen induced growth processess in farm animals. This study was conducted to investigate the effect of certain estrogenic anabolic compounds (Zeranol, DES and Synovex-S) on the activity of the thyroid gland. Attention was focussed on the plasma levels of thyroxine (T4) and triiodothyronine (T3), on the binding - 113 -c a p a c i t y of thy rox ine b ind ing g l o b u l i n (as TBG RDA.) a n < * o n t n e s t a t u s o f f r e e and m e t a b o l i c a l l y a c t i v e f r a c t i o n of thy rox ine (as Free Thyrox ine Assessment, FTA) i n the plasma. The major o b j e c t i v e was to study the involvement of t h y r o i d hormones i n the est rogen mediated growth i n s t e e r s . MATERIALS AND METHODS Animals and Experimental Procedure S i x t e e n Hereford s teer s which were used i n Expt . IA were used i n t h i s exper iment. The anabo l i c compounds, ( C o n t r o l , Ze rano l , DES, Synovex-S) t h e i r imp l an t a t i o n procedures and the exper imenta l r a t i o n used were the same as been desc r ibed i n Expt . IA. Blood samples were c o l l e c t e d from i n d i v i d u a l s t ee r s on days 14, 28, 42 and 56 f o l l o w i n g the imp l an t a t i o n of anabo l i c compounds by means of j u g u l a r v e i n puncture i n hepa r i n i z ed evacuated tubes ( vacuta ine r tubes, Beck ton -D ick in son , D i ck in son and Co., New J e r s e y ) . The tubes were kept i n i c e and were cen t r i f u ged immediately at 1000 x g; the plasma was c o l l e c t e d and s to red at -20°C. Blood samples were c o l l e c t e d a f t e r an overn ight f a s t . Analytical Procedure The plasma samples were thawed at 4°C and the concen t r a t i on of t h y r o i d hormones was then determined. (1) T o t a l Thyroxine (T4) The plasma concen t ra t i on of t o t a l T4 was determined by a s p e c i f i c radioimmunoassay procedure (Chopra, 1972) us ing a s o l i d -pha se - 114 -radioimmunoassay system (Immuchemical Co . ) ; 8 - a n i l i n o , 1 -naphtha lene - su l fon i c a c i d (ANS) was used to d i s r up t the b i nd ing of T4 to the t h y r o i d b ind ing g l o b u l i n (TBG). The t e s t sample or T4 standard (0 to 20 ug/100 ml plasma) or the q u a l i t y c o n t r o l sample was added to ant ibody coated tubes fo l l owed by 125J_-J>4 i n 0.01 M phosphosal ine b u f f e r con ta i n i n g ANS. The tubes were v o r t e x mixed and incubated f o r 1 h at 37°C. A f t e r the i n cuba t i on s tep , the s o l u t i o n con ta i n i n g the f r e e 125I-T4 was decanted and the bound r a d i o a c t i v i t y was counted i n an automatic gamma-counter (Model 1185, Nuc lear Ch icago) . The concen t r a t i on of T4 i n the unknown samples was c a l c u l a t e d from the standard curve us ing the r ap i d procedure desc r ibed by Rodbard et a l . (1969). The i n t e r - and i n t r aa s s ay c o e f f i c i e n t s of v a r i a t i o n (Rodbard, 1974) were 8.1% and 12.1%, r e s p e c t i v e l y , f o r a sample w i t h a mean concen t ra t i on of 6.7 ug/100 m l . A l l the samples c o l l e c t e d at any one time per iod were analysed i n the same assay, to minimize assay v a r i a t i o n s . (2) T o t a l T r i i o d o t h y r o n i n e (T3) The plasma t o t a l T3 concent ra t i ons were determined by a s p e c i f i c radioimmunoassay procedure descr ibed by Chopra et a l . (1972) us ing a s o l i d phase radioimmunoassay package. ANS was used to d i s r u p t the b ind ing of T3 to t h y r o i d b ind ing g l o b u l i n (TBG). The t e s t sample or standard (0-80 ng/100 ml) or the q u a l i t y c o n t r o l sample was added to T3 - ant ibody coated tubes. A s o l u t i o n of 125j _ T 3 £ n o.Ol M phosphosal ine bu f f e r con ta i n i ng ANS was added and - 115 -the tubes were incubated f o r 2 h at 37°C. At the end of the i n cuba t i on p e r i o d , the s o l u t i o n con ta i n i n g f r e e 125 T _ T 3 was decanted and the bound r a d i o a c t i v i t y was counted i n an automatic gamma counter (Model 1185, Nuc lea r -Ch i cago ) . The c a l c u l a t i o n of T3 concen t r a t i on i n the unknown samples and the i n t e r - and i n t r aa s s ay c o e f f i c i e n t s of v a r i a t i o n s were s i m i l a r to those de sc r i bed f o r T4 - radioimmunoassay. A l l the samples c o l l e c t e d at any one time per iod were analysed i n the same assay to minimize assay v a r i a t i o n s . (3) T o t a l Thyroxine B ind ing G l o b u l i n (TBG) B ind ing Capac i ty The t o t a l TBG b ind ing capac i t y was determined by a r ad i o displacement assay (NMS, 1980a). The technique measured the t o t a l t hy rox ine b i nd ing g l o b u l i n c apac i t y of the s p e c i f i c thy rox ine b ind ing i n t e r a l p h a g l o b u l i n . A l a r ge excess of l a b e l l e d ( ^ 5 i _ x4) and u n l a b e l l e d (T4) thy rox ine was added to tubes c on ta i n i n g the t e s t sample or re fe rence standards (h igh standard = 20.8 ug/100 ml ; low standard = 10.1 ug/100 ml) or b u f f e r . The tubes were incubated at room temperature f o r 30 min and the f r e e * 2 5 i _ x4 was separated from the bound f r a c t i o n by a c ha r coa l - dex t r an s l u r r y . The bound f r a c t i o n was counted i n an automatic gamma counter (Model 1185, Nuc lea r -Ch i cago ) . Calculation The b i nd i n g c apac i t y of the thy rox ine b i nd i ng g l o b u l i n i n the unknown samples was c a l c u l a t e d accord ing to the fo rmu la : - 116 -C ? M U N K - B (RDA) A Where: TBG(RDA)= Thyroxine binding globulin capacity by radio-displacement assay C J ? % N K • Bound radioactivity in the unknown tube C P M ( H i g h standard) C P M(Low standard) Assayed V a l u e ( H i g h s t d > ) - Assayed V a l u e ( L o w ^ C P M / T - » - A (Assayed value, , . (Low std.) 3 Low std.) Thyroxine binding globulin opacity was expressed as ug T4 bound per 100 ml plasma. (4) Free Thyroxine Assessment (FTA) The FTA, which is an index of the free, unbound thyroxine in the circulation was calculated from the total T4 concentration in the circulation and the total T4 binding capacity, according to the formula: T4 FTA T B G(RDA) where FTA = Free thyroxine assessment T4 = Total T4 concentration (ug/100 ml) TIH*(RDA) = Total thyroxine binding globulin capacity determined by the radio-displacement assay (ug/100 ml) - 117 -S t a t i s t i c a l A n a l y s i s The plasma concent ra t i ons of t h y r o i d hormone were averaged f o r each treatment group on a l l the sampling per iods and subjected to l e a s t squares a n a l y s i s of va r i ance ( S t e e l and T o r r i e , 1960) us ing the UBC BMD:10V l i n e a r hypothes i s computer program. The s i n g l e degree of freedom or thogona l con t ra s t t e s t ( S t e e l and T o r r i e , 1960) was used to compare t reatments . RESULTS Plasma Total Thyroxine (T4) The plasma concen t r a t i on of t o t a l T4 was determined from samples c o l l e c t e d on days 14, 28, 42 and 56 f o l l o w i n g the i m p l a n t a t i o n of a nabo l i c compounds (Table 15). T o t a l T4 concen t r a t i on i n the plasma was about 23 (P < .05) and 18 per cent (P = .08) h igher i n s t ee r s implanted w i t h Synovex-S and DES, r e s p e c t i v e l y , than i n the unimplanted c o n t r o l s t e e r s . On the c o n t r a r y , on ly a s l i g h t i n c rea se i n the plasma T4 concen t r a t i on (2.2%, n o n - s i g n i f i c a n t ) was observed i n s tee r s implanted w i t h Zerano l compared to the c o n t r o l s t e e r s . There was no d i f f e r e n c e (P > .05) i n the plasma T4 concen t r a t i on i n s tee r s implanted w i t h Synovex-S and DES. However, compared to Zerano l i m p l a n t a t i o n , s i g n i f i c a n t l y h igher (P < .05) c oncen t r a t i on of plasma T4 was observed i n s tee r s Implanted w i t h Synovex-S. Though the treatment X pe r i od i n t e r a c t i o n was not s i g n i f i c a n t , plasma T4 concen t r a t i on on a l l the time pe r i od s tud ied was h i gher i n Synovex-S and DES implanted than i n the c o n t r o l s t e e r s . The - 118 -Table 15. Plasma t o t a l thy rox ine (T4) c oncen t r a t i on i n s tee r s on d i f f e r e n t days f o l l o w i n g the Imp lanta t ion of anabo l i c compounds Days A f t e r Imp lan ta t i on Treatment 14 28 42 56 O v e r a l l Treatment Mean SE J C o n t r o l Ze rano l DES Synovex-S 4.50 5.33 5.51 5.53 3.65 4.26 5.01 4.30 ug/100 ml 4.68 4.78 3.89 4.5 5.11 5.14 4.95 6.84 4.40 4.50 5.19 5.40 0.30 0.30 0.27 0.44 Orthogonal Cont ras t s f o r O v e r a l l Treatment Means Cont ra s t s P C o n t r o l vs . Zerano l N.S C o n t r o l v s . DES 0.08 C o n t r o l v s . Synovex-S 0.03 Zerano l v s . DES N.S Ze rano l v s . Synovex-S 0.04 DES v s . Synovex-S N.S C o n t r o l vs. Zerano l + DES + Synovex-S N.S Pooled S.E. of the mean. S i ng l e degree of freedom orthogona l con t ra s t were c a l c u l a t e d u s i ng UBC BMD:10V l i n e a r hypothes i s computer program. - 119 -e f f e c t of Zerano l on the t h y r o i d gland was only ma rg i na l , w i t h s l i g h t l y i nc reased response no t i ced on day 14 f o l l o w i n g i t s i m p l a n t a t i o n . The o v e r - a l l mean plasma T4 concen t r a t i on i n the s tee r s used i n t h i s experiment was found to be 4.87 ± 0.17 ug/100 m l . Plasma Concentration of Total Triiodothyronine The plasma concen t ra t i on of t o t a l T3 was determined i n samples c o l l e c t e d on days 14, 42 and 56 f o l l o w i n g the i m p l a n t a t i o n of a nabo l i c compounds (Table 16). Plasma T3 concen t r a t i on was found to be s i m i l a r (P > .05) i n implanted and unimplanted c o n t r o l s tee r s on a l l the time pe r i od s t u d i e d . O v e r - a l l mean plasma concen t ra t i on of T3 i n the s tee r s used i n t h i s study was found to be 133.3 ± 6.0 ng/100 m l . Thyroxine Binding Globulin Capacity (as T B G R D A ) The b i nd i ng c apac i t y of thy rox ine b i nd ing g l o b u l i n was determined on days 42 and 56 f o l l o w i n g the i m p l a n t a t i o n of anabo l i c compounds by a r a d i o displacement assay ( T B G R D A , Table 17) . O v e r - a l l mean T B G R D A va lues of the s tee r s used i n t h i s experiment was found to be 15.22 ± .8 ug/100 ml plasma and was i n agreement w i t h the normal va lues reported u s i ng the rad io -d i sp l acement assay (NMS, 1980a). When both the time per iods (days 42 and 56) were taken i n t o account, TBGjyj^ was found to be s i g n i f i c a n t l y reduced (P < .001, or thogona l comparison f o r c o n t r o l vs a l l the anabo l i c treatments) i n s teer s implanted w i t h anabo l i c compounds. Free Thyroxine Assessment (FTA) An assessment of the FTA i n the plasma was c a r r i e d out as the - 120 -Table 16 - Plasma t o t a l t r i i o d o t h y r o n i n e (T3) c oncen t r a t i on i n s teer s on d i f f e r e n t days f o l l o w i n g the i m p l a n t a t i o n of anabo l i c compounds Days A f t e r Imp lan ta t i on O v e r a l l Treatment 14" 42 56 Treatment SE Mean ng/100 ml C o n t r o l 132.7 128.4 150.5 137.1 15.4 Zerano l 144.8 90.7 124.6 120.7 13.7 DES 138.0 137.5 129.4 135.0 7.7 Synovex-S 149.2 109.0 165.7 139.2 11.5 Orthogonal Cont ras t s f o r O v e r a l l Treatment Means Cont ra s t s P C o n t r o l vs . Zerano l N.S C o n t r o l vs . DES N.S C o n t r o l v s . Synovex-S N.S Zerano l v s . DES N.S Zerano l v s . Synovex-S N.S DES v s . Synovex-S N.S Pooled standard e r r o r of the mean. S i n g l e degree of freedom orthogonal con t r a s t were c a l c u l a t e d u s i ng BMD:10V l i n e a r hypothes i s computer program. - 121 -Table 17. Thyroxine binding globulin capacity (TBG R D A) in steers on different days following the implantation of anabolic compounds Treatment Days After Implantation 42 56 Overall Treatment Mean Control Zeranol DES Synovex-S 19.41 13.08 17.21. 16.86 yg/100 ml . 18.59 12.85 13.36 10.39 19.00 12.97 15.28 13.63 1.36 0.84 1.40 1.89 Orthogonal Comparisons for Overall Treatment Means Contrasts P Control vs. Zeranol 0.001 Control vs. DES 0.02 Control vs. Synovex-S 0.002 Zeranol vs. DES N.S Zeranol vs. Synovex-S N.S DES vs. Synovex-S N.S Control vs. Zeranol + DES + 0.001 Synovex-S ug T4 bound per 100 ml plasma determined by radio displacement assay. Pooled standard error of the mean. Orthogonal comparisons were calculated using BMD:10V computer program. - 1 2 2 -r a t i o of plasma T4 and T B G R D A (Table 18) . O v e r - a l l mean FTA i n the s t ee r s used i n t h i s experiment was found to be 0.3281 ± .021 and was i n agreement w i t h the normal va lues of FTA i n the plasma (NMS, 1980a). When both the time per iods (days 42 and 56) were taken i n t o account, the mean FTA value was found to be s i g n i f i c a n t l y h igher (P < .05, o r thogona l comparison f o r c o n t r o l vs a l l the anabo l i c compounds) i n s tee r s implanted w i t h anabo l i c compounds than the c o n t r o l s . The FTA was found to be 53 (P < .02 ) , 37 (N.S.) and 29 (N.S.) per cent h igher i n s tee r s implanted w i t h Synovex-S, DES and Z e r a n o l , r e s p e c t i v e l y , than the unimplanted c o n t r o l s t e e r s . Increased FTA va lues i n the implanted s tee r s i n d i c a t e an e l e v a t i o n i n the plasma concen t ra t i on of f r e e , d i a l y z a b l e and m e t a b o l i c a l l y a c t i v e form of c i r c u l a t i n g T4. A summary of changes i n plasma t h y r o i d hormone concen t ra t i on due to anabo l i c compound imp lan ta t i on s i s presented i n Table 19. DISCUSSION In t h i s exper iment, the e f f e c t of e s t r ogen i c anabo l i c compounds on the plasma t h y r o i d hormone l e v e l s was i n v e s t i g a t e d i n order to understand the mechanism of growth induced by anabo l i c compounds and eva luate the involvement of t h y r o i d hormones i n such growth p roces ses . Re su l t s of t h i s experiment i n d i c a t e an Increase i n the plasma t o t a l T4 and f r ee T4 (as FTA) l e v e l s i n Synovex-S (P < .05) and DES (N.S.) implanted s t e e r s . These e l e v a t i o n s i n the plasma T4 concen t r a t i on are i n l i n e w i t h the e a r l i e r f i n d i n g s on the changes i n the plasma T4 s ta tu s f o l l o w i n g estrogen a d m i n i s t r a t i o n i n man - 123 -Table 18. Mean f r e e thy rox ine assessment (FTA) i n s tee r s on d i f f e r e n t days f o l l o w i n g the i m p l a n t a t i o n of anabo l i c compounds Treatment Days A f t e r Imp lan ta t i on 42 56 O v e r a l l Treatment Mean SE' C o n t r o l Ze rano l DES Synovex-S 0.2498 0.2996 0.3066 0.2951 0.2578 0.3592 0.3908 0.5119 0.2538 0.3294 0.3487 0.3880 0.0241 0.0281 0.0320 0.0623 Orthogonal Comparisons f o r O v e r a l l Treatment Means Cont ra s t s P C o n t r o l v s . Zerano l N.S C o n t r o l v s . DES N.S C o n t r o l v s . Synovex-S 0.02 Zerano l v s . DES N.S Zerano l v s . Synovex-S N.S DES v s . Synovex-S N.S C o n t r o l v s . Zerano l + DES + 0.03 Synovex-S 1 m T o t a l T4 yg/100 ml T B G ^ ug/100 ml o Pooled standard e r r o r of the mean. - 124 -Table 19. Summary of changes l n the plasma t h y r o i d hormone concen t ra t i on i n s tee r s implanted w i t h anabo l i c compounds Treatment T4J T3 i TBG- FTA C o n t r o l Zerano l DES Synovex-S 4.40 4.50 5.19 5.40 137.1 120.7 135.0 139.2 19.00 0.2538 12.97 0.3294 15.28 0.3487 13.63 0.3880 Orthogonal Comparisons C o n t r o l v s . Zerano l N.S N.S 0.001 N.S C o n t r o l v s . DES 0.08 N.S 0.02 N.S C o n t r o l v s . Synovex-S 0.03 N.S 0.002 0.02 Zerano l v s . DES N.S N.S N.S N.S Zerano l v s . Synovex-S 0.04 N.S N.S N.S DES v s . Synovex-S N.S N.S N.S N.S C o n t r o l v s . Zerano l + DES + Synovex-S N.S. N.S. 0.001 0.03 mean of va lues (ug/100 ml) obta ined on 14, 28, 42 & 56 days mean of va lues (ng/100 ml) obta ined on 14, 42 and 56 days mean of va lues (ug/100 ml) obta ined on 42 and 56 days mean of va lues obta ined on 42 and 56 days - 125 -( Zan i nov i ch , 1973) and i n Synovex-S implanted s tee r s (Kahl et a l . t 1978). Increased plasma T4 concen t r a t i on was proposed to be r e spon s i b l e f o r the i nc rea sed growth r a t e f o l l o w i n g DES i m p l a n t a t i o n i n s t ee r s (Burgess and Lamming, 1960) and a t rend towards i nc reased T4 s e c r e t i o n r a t e was repor ted i n DES-fed lambs (T renk l e , 1969). Bes ides , an i n c rea se i n the heart r a te and ba sa l metabo l i c r a t e of s teer s implanted w i t h DES (Rumsey et a l . , 1973; T y r r e l l et a l . , 1975) suggests the involvement of T4. I t would appear that the inc reased T4 l e v e l s i n the Synovex-S implanted s tee r s used i n t h i s experiment cou ld have r e s u l t e d i n s i m i l a r changes i n energy metabol i sm. E leva ted T4 l e v e l observed i n the s tee r s cou ld a l s o be due to a r educ t i on i n T4 degradat ion r a te and an i n c rea se i n TBG b i nd i ng c a p a c i t y ( Zan inov i ch , 1973). Thyroxine b i nd ing g l o b u l i n c a p a c i t y which exer t s a r a te l i m i t i n g e f f e c t upon the p e r i p h e r a l metabol ism of t h y rox i ne (Dowling et a l . , 1960) was reduced i n both Synovex-S (P < .05) and DES (N.S.) implanted s t e e r s . Estrogen a d m i n i s t r a t i o n was a s s oc i a t ed w i t h i nc rea sed b i nd i ng c apac i t y of TBG (Sawhney et a l . , 1978; Zan i nov i ch , 1973) an obse rva t i on which was not supported i n t h i s exper iment. The TBG b i nd i ng c apac i t y i n t h i s s tudy, however, was measured by a rad io -d i sp lacement assay and i t s r educ t i on i n the implanted s t ee r s cou ld not be e x p l a i n e d . Based on T 3 - r e s i n uptake r a t i o , Kah l et a l . (1978) have repor ted enhanced b ind ing capac i t y of the plasma t h y r o i d hormone b ind ing p ro te i n s i n Synovex-S implanted s t e e r s . I t i s not c l e a r whether the inc reased plasma T4 observed i n the implanted s t ee r s i s a f u n c t i o n of i nc reased t h y r o i d a l T4 s e c r e t i o n r a te - 126 -or decreased metabolic clearance from the circulation or due to relative changes in both the parameters. Increased plasma T4 concentration observed in the implanted steers, especially with Synovex-S and DES was similar to the increased growth response (Figure 3, Expt. IA). The changes in plasma T4 and body weight gain of steers suggest that T4 could be involved In the growth process induced by estrogenic anabolic compounds. A relationship between T4 secretion rate and growth has been reported in pigs(Marple et a l . , 1981). The thyroid stimulating effects of estrogenic anabolic compounds, especially Synovex-S and DES, established in this study are in contradiction to that reported for androgenic anabolic compounds (Donaldson et a l . , 1981; Heitzman et a l . , 1977). Trenbolone acetate, either alone or in combination with estradiol has been shown to cause marked reduction (up to 45%) in the plasma T4 concentration in wether lambs (Donaldson et a l . , 1981). The thyroid stimulating effects of estrogenic and the depressing influence of androgenic anabolic compounds indicate the differential mechanisms by which they mediate growth in farm animals. Diethylstilbestrol has been shown to stimulate thyrotropin (TSH) secretion from the pituitary gland both in in vivo (Davis et a l . , 1978) and in vitro (Miller et a l . , 1977). The T4 concentration in the plasma was not altered by Zeranol implantation, however, a decreasing trend in i t s concentration was observed beyond day 28 following the implantation. A similar reduction in plasma T4 level (Wiggins et^ a l . , 1979) and a decrease in the thyroid - 127 -g land a c t i v i t y (Rothenbacher j i t a l_ . , 1975) were reported i n Zerano l implanted lambs. However, t h y r o i d gland weight of Zerano l implanted lambs was found to be inc reased (Wiggins ^ a l . , 1976) or decreased (Wiggins e t ^ a l . , 1979). I t was po s tu l a ted by Wiggins et a l . (1979) that the decreased T4 l e v e l s observed i n Zerano l implanted lambs as compared to c o n t r o l s could lower the basa l metabo l i c r a te and thus could i n c rea se the p r opo r t i on of absorbed n u t r i e n t s a v a i l a b l e f o r growth. A t rend towards decreased plasma T4 concen t r a t i on i n Zeranol implanted s t ee r s used i n t h i s experiment suggests that p o s s i b i l i t y . The i m p l a n t a t i o n of DES on the other hand seems to i nc rease the ba sa l metabo l i c r a te i n s t ee r s (Rumsey et a l . , 1973; T y r r e l l et a l . , 1975). The r e s u l t s of t h i s exper iment, i n con junc t i on w i th the observat ions of e a r l i e r workers (Kah l et a l . , 1978; Rumsey et a l . , 1973; T y r r e l l et a l . , 1975; Wiggins et a l . , 1979) suggest that the mechanism of a c t i o n of Ze r ano l , DES, and Synovex-S as f a r as t h y r o i d gland a c t i v i t y i s concerned may be d i f f e r e n t . Plasma T3 l e v e l s i n the implanted and c o n t r o l s tee r s were s i m i l a r (P > .05) on a l l the per iods s tud ied and the r e s u l t s demonstrate the l a c k of i n f l u e n c e on T3 s ta tus i n s tee r s due to anabo l i c compound i m p l a n t a t i o n s . I t i s not c l e a r whether T3 i s i n vo l ved i n the mechanism of growth promotion mediated by the anabo l i c compounds. S i m i l a r n o n - s i g n i f i c a n t d i f f e r e n c e i n the plasma T3 l e v e l s between the c o n t r o l and Synovex-s implanted s teer s was reported by Kah l et a l . (1978) who have concluded that i n the bov ine, T4 r a the r than T3 might r e f l e c t p h y s i o l o g i c a l and metabo l i c a l t e r a t i o n s . - 128 -About one t h i r d or l e s s of the c i r c u l a t i n g T3, i s of t h y r o i d a l o r i g i n and the r e s t i s de r i ved from the e x t r a t h y r o i d a l monodeiodinat ion of T4 to T3 ( S t e r l i n g and Lazarus , 1977). The presence of the l a t t e r mechanism was reported i n man by Braverman et a l . (1970), i n the ra t by Schwartz et _ a l . (1971), and i n sheep by F i s he r et a l . (1972). The concen t r a t i on of T3 i n the plasma i s t he r e f o r e a f u n c t i o n of T4 to T3 conver s ion r a t e , t h y r o i d a l s e c r e t i o n r a t e , d i s p o s a l r a t e , and the concen t r a t i on of serum b ind ing p r o t e i n s . The f o l l o w i n g a l t e r n a t i v e s are suggested f o r the l a c k of response i n plasma T3 f o l l o w i n g the i m p l a n t a t i o n of anabo l i c compounds: (a) there cou ld be a r educ t i on i n the p e r i p h e r a l convers ion of T4 to T3 i n the implanted s t ee r s ; enhanced b ind ing c apac i t y of the thy rox ine b i nd i n g g l o b u l i n and reduced f r a c t i o n a l turnover ra te of T4 (Dowling £ t a l . , 1960; Engbr ing and Engstrom, 1959) f o l l o w i n g DES and n a t u r a l es t rogen a d m i n i s t r a t i o n and the i n h i b i t i o n of d e i o d i n a t i o n of T4 to T3 i n the i n t a c t c e l l u l a r p repara t ions by estrogens (Yamazaki and S l i n g e r l a n d , 1959) add support to t h i s p o s s i b i l i t y ; (b) In the implanted s tee r s the metabol ism of T3 i t s e l f could be r a p i d , namely i t s convers ion to t r i i o d o t h y r o a c e t i c a c i d ; the t i s s u e s that are s u s c e p t i b l e to the s t i m u l a t i o n of t h y r o i d hormones ( hea r t , k idney and l i v e r ) have been shown to con ta i n enzymes f o r the conver s ion of T3 to t r i i o d o t h y r o a c e t i c a c i d i n t h e i r mi tochondr ia ( A l b r i g h t et_ a l . , 1959) and that the l a t t e r me tabo l i t e was more a c t i v e than T3. The r e s u l t s of t h i s i n v e s t i g a t i o n , suggest the need f o r f u r t h e r r e sea rch on the p e r i p h e r a l convers ion of T4 to T3 i n s t e e r s , e s p e c i a l l y f o l l o w i n g anabo l i c compound i m p l a n t a t i o n . - 129 -CONCLUSION The r e s u l t s of t h i s study i n d i c a t e d that one of the mechanisms by which e s t r ogen i c anabo l i c compounds evoke growth promotion i n ruminants i s by the a l t e r a t i o n of the endogenous c i r c u l a t i n g t o t a l and f r ee T4. Implantat ions of Synovex-S and DES r e s u l t e d i n i nc reased growth ra te and plasma T4 c oncen t r a t i o n . Only a s l i g h t e l e v a t i o n In the plasma T4 concen t r a t i on was observed due to Zerano l i m p l a n t a t i o n . The r e s u l t s of t h i s study a l s o suggested that T3 may not be i n vo l ved i n the growth processes mediated by the anabo l i c compounds as no change i n i t s concen t ra t i on was observed. Fu r the r s tud ie s are warranted to i n v e s t i g a t e the e f f e c t of anabo l i c compounds on T 3 metabol ism i n s t e e r s . Cons ider ing the p a r t i c i p a t i o n of T4 i n the r e g u l a t i o n of growth and metabolism of an imal s , i t i s concluded that T4 i s i nvo l ved i n the mechanism of a c t i o n of anabo l i c compounds. - 130 -EXPERIMENT II A:2 KINETIC PARAMETERS OF THYROXINE METABOLISM IN STEERS IMPLANTED WITH ANABOLIC COMPOUNDS INTRODUCTION Resu l t s of Expt . I IA: 1 (see page 117) i n d i c a t e d a h igher plasma T4 concen t r a t i on i n s tee r s f o l l o w i n g Synovex-S and DES i m p l a n t a t i o n than i n the c o n t r o l s and suggested the involvement of T4 i n the growth processes mediated by anabo l i c compounds. On the other hand, plasma T4 concen t r a t i on was not i n f l uenced by Zerano l i m p l a n t a t i o n s . Though i t was concluded that the changes i n the concen t r a t i on of plasma T4 i n s tee r s f o l l o w i n g Synovex-S, DES and Zerano l Implantat ions could be due to t h e i r d i f f e r e n t i a l mechanism of a c t i o n , the exact mechanism f o r such a l t e r a t i o n s could not be e xp l a i ned . A t rend towards i nc reased thy rox ine s e c r e t i o n r a te f o l l o w i n g DES i m p l a n t a t i o n had been reported e a r l i e r ( T renk le , 1969). The concen t r a t i on of T4 i n plasma at a g iven time i s a f u n c t i o n of s e c r e t i o n r a te and c learance r a t e . I t i s thus p o s s i b l e that a l t e r a t i o n s i n plasma T4 concent ra t i on s f o l l o w i n g the i m p l a n t a t i o n of anabo l i c compounds could be due to a f u n c t i o n of e i t h e r i t s s e c r e t i o n r a t e , c lea rance r a t e or both. Estrogen a d m i n i s t r a t i o n has been shown to decrease d a i l y thy rox ine degradat ion r a te ( Zan inov i ch , 1973) and the p a r t i c i p a t i o n of t h y r o i d hormone b ind ing g l o b u l i n s In such processes has been d i scussed (Dowling et a l . , 1960). Thus an experiment on the k i n e t i c parameters of T4 metabolism i n s teer s e s p e c i a l l y f o l l o w i n g the i m p l a n t a t i o n of anabo l i c compounds assumes great importance. Th i s study was t he re f o re conducted to i n v e s t i g a t e the e f f e c t of e s t r ogen i c - 131 -a n a b o l i c compounds on the k i n e t i c parmeters of T4 metabolism i n growing beef s t e e r s . MATERIALS AND METHODS Animals: Twenty four Hereford s t e e r s , weighing approx imately 270 kg, were used i n t h i s s tudy. The animal management and the feed ing procedure were s i m i l a r to those desc r ibed i n Expt. IA. Anabolic Treatments and Feeding Proceudre: The s tee r s were ass igned to one of the f o l l o w i n g treatment groups (n = 6 per t rea tment ) : 1. C o n t r o l 2. 36 mg implant of DES 3. 36 mg implant of Zerano l 4. One implant of Synovex-S The method f o r the i m p l a n t a t i o n of the anabo l i c compound has been desc r ibed e a r l i e r (Expt . I A ) . The s tee r s were weighed at weekly i n t e r v a l s and fed twice d a i l y w i t h a f i n i s h i n g r a t i o n (Table 20 ) . Water and s a l t b locks were prov ided f r ee cho i ce . Animal preparation for the Kinetic Experiments: The s tee r s were f i t t e d w i t h an i n d w e l l i n g po l ye thy lene j u g u l a r ca the te r (P .E . 90, I.D = 0.86 mm; O.D = 1 . 2 mm) a day before the k i n e t i c experiments began. The f r e e end of the ca the te r was taped i n p o s i t i o n and the ca the te r was f l u shed w i t h a 4% sodium c i t r a t e s o l u t i o n . - 132 -Table 20. Ra t i on Composit ion (Expt. I IA:2) % of Ra t i on I ng red ien t s (as fed) Steam r o l l e d ba r l e y 79.0 Chopped grass hay 15.0 Soybean meal 3.5 Ground L imestone 1.5 M i n e r a l - V i t a m i n M i x t u r e 1 , 2 1.0 Supp l ied by B u c k e r f i e l d s L i m i t e d , Abbo t s fo rd , B.C., Canada ^Minera l - v i t am in mixture compos i t ion: s a l t (40.0%), phosphorus (10.0%), f l u o r i n e (0.2%), i r o n (0.42%), z i n c (0.8%), manganese (0.2%) , coba l t (0.016%), copper (0.15%), v i t am in A (700,000 I .U./kg), v i t a m i n D (70,000 I.U./kg). - 133 -Kinetic Experiments: The k i n e t i c parameters of T4 metabolism were est imated on days 20, 40 and 60 f o l l o w i n g the i m p l a n t a t i o n . Two s tee r s from each treatment group were used at any one exper imenta l p e r i o d . The s tee r s used i n one exper imenta l pe r i od were never used i n the subsequent pe r i od s t u d i e d . K i n e t i c parameters were obta ined from e i gh t s t ee r s on each of the three per iods mentioned above. Experimental Procedure: A s i n g l e bolus i n j e c t i o n , n o n - r a d i o a c t i v e T4 k i n e t i c s method was used (Fox et a l . , 1974; Post and Mixner , 1961). On the day of the experiment, the s tee r s were weighed, fed the morning p o r t i o n of the r a t i o n and were p laced i n metabolism c ra te s which a l lowed some freedom of movement but prevented them from t u r n i n g around. Fo l l ow ing a pe r iod of adjustment, two blood samples were c o l l e c t e d v i a the c a the te r to o b t a i n the b a s a l T4 c o n c e n t r a t i o n . The animals were then r a p i d l y i n j e c t e d w i t h a c a l c u l a t e d dose of L-T4 (4.5 mg f o r the f i r s t 45 kg body weight and 1.5 mg f o r each a d d i t i o n a l 45 kg) through the c a t h e t e r . The ca the te r was immediately f l u shed w i t h 0.9% s a l i n e to make sure that a l l the i n j e c t e d dose of T4 enter the b lood stream f o r immediate mix ing and to avo id the contaminat ion of I n j e c t ed T4 w i t h the subsequent b lood samples to be c o l l e c t e d through the same c a t h e t e r . S e r i a l blood samples were then c o l l e c t e d at 6, 12, 24, 30, 48, 54, 72, 78 and 96 h f o l l o w i n g T4 i n j e c t i o n to monitor the d isappearance of the i n j e c t e d T4 from the c i r c u l a t i o n . Each time a f t e r wi thdrawing blood samples, the ca the te r was f l u shed w i t h s a l i n e (0.9% - 134 -w/v) or sodium c i t r a t e (4% w/v). The b lood samples were kept i n tubes c on t a i n i n g hepar in and were cen t r i f u ged to ob t a i n the plasma. The plasma samples were s to red at -20°C. Analytical Procedure T4 radioimmunoassay: The concen t r a t i on of T4 i n the samples c o l l e c t e d on d i f f e r e n t pe r i ods f o l l o w i n g T4 i n j e c t i o n was determined by a s p e c i f i c radioimmunoassay procedure. The assay d e s c r i p t i o n , c a l c u l a t i o n of T4 c oncen t r a t i on i n the t e s t samples and the de te rm ina t i on of i n t e r - and i n t r a a s s a y v a r i a t i o n s were s i m i l a r to that desc r ibed e a r l i e r (T4-Radioimmunoassay, Expt . I IA: 1) w i t h the except i on that a double ant ibody radioimmunoassay procedure was used (NMS, 1980b) and the ® r a d i o a c t i v i t y was counted i n an automatic gamma counter (Auto-Gamma 500 C count ing system, Packard Instruments, I l l i n o i s , U.S.A). The sheep a n t i - r a b b i t gamma g l o b u l i n was used to p r e c i p i t a t e the f i r s t ant ibody . The i n t e r - and i n t r aa s s a y c o e f f i c i e n t of v a r i a t i o n s were 3.8% and 5.0%, r e s p e c t i v e l y , f o r a sample w i t h a mean concen t r a t i on of 8.0 ug/100 m l . Analysis of Kinetic Data The plasma T4 d i l u t i o n curve was analysed u s ing a d e c i s i o n making F o r t r a n computer program, AUTOAN (Sedman and Wagner, 1976). From the r e s u l t of the l e a s t square e s t imate s , the observed data was found to be best desc r ibed by a s i n g l e exponen t i a l equa t i on . Subsequently, the k i n e t i c data from a l l the exper imenta l s t ee r s were analysed by an one-compartment open-model (Wagner, 1975). - 135 -Model Description The one compartmental model d e s c r i b i n g T4 disappearance from the blood plasma i s dep ic ted below. One Compartment Model d e s c r i b i n g T4 K i n e t i c s i n Steers Where: D = dose i n j e c t e d (mg) CQ = T4 concen t r a t i on at time t = 0 (yg/100 ml) C = T4 concen t ra t i on at time t = t (yg/100 ml) k = f i r s t - o r d e r e l i m i n a t i o n ra te constant ( h - 1 ) Calculations 1. Thyroxine Fractional Turnover Rate (TFTR or k, h " 1 ) : de f i ned as the f r a c t i o n of T4 degraded and rep laced per u n i t time - 136 -- c a l c u l a t e d from the exponent i a l equat ion : C = C Q e - k t where: k = f r a c t i o n a l turnover ra te ( h - 1 ) . . . Z i l v e r s m i t (1960) 2. Biological H a l f - l i f e (Tl/2, h): T 1 / 2 . iB_(21 Where: Tl/2 - b i o l o g i c a l h a l f - l i f e in 2 = n a t u r a l l o g . of 2 = 0.693 k = f r a c t i o n a l turnover r a te ( h - 1 ) 3. Thyroxine Volume of Distribution (TVD, t ) : JD Post and Mixner (1961) C n and F r e i n k e l and Lewis (1957) TVD = where: TVD = volume of d i s t r i b u t i o n (A) D = dose of T4 i n j e c t e d (ug) CQ = concen t r a t i on of T4 at time zero (ug/100 ml) 4. Metabolic Clearance Rate (MCR, l/d): - de f i ned as the volume of blood from which thy rox ine was complete ly and i r r e v e r s i b l y removed i n u n i t t ime . MCR {llA) = TVD x TFTR . . . T a i t and B u r s t e i n (1964) where: TVD = thy rox ine volume of d i s t r i b u t i o n (i) - 137 -TFTR = f r a c t i o n a l turnover r a te (d ) 5. Thyroxine Secretion Rate (TSR, yg/d) TSR (ug/d) TVD x TFTR X Basa l T4 . . . Post and Mixner (1961) and F r e i n k e l and Lewis (1957) where: TVD volume of d i s t r i b u t i o n (1) TFTR f r a c t i o n a l turnover r a te (d ) Ba sa l T4 = ba s a l T4 concen t r a t i on (ug/100 ml) The k i n e t i c parameters obta ined from two s tee r s from each treatment on each of the per iods s tud ied were averaged and expressed as mean ± S.E. The treatment d i f f e r e n c e s were te s ted by S tudent ' s t - t e s t ( S t e e l and T o r r i e , 1960). Plasma Thyroxine Disappearance Plasma T4 concen t ra t i on i n s tee r s immediately f o l l o w i n g the bolus i n j e c t i o n of L - thy rox ine i s presented i n F i gu re 19. The T4 concen t r a t i on inc reased to about 40-50 ug/100 ml f o l l o w i n g the i n j e c t i o n and g r adua l l y decreased. The plasma T4 disappearance curve was best de sc r i bed by an one compartment open model and the data obta ined from a l l the s tee r s were f i t t e d to t h i s model and the k i n e t i c parameters were c a l c u l a t e d from the s l ope , and the ze ro - t ime i n t e r c e p t of the RESULTS - 138 -100c steer: 3 E no] o o < 100E c/) < 10 steeMO • observed • predicted 0 15 30 45 60 /b 90 I05 ^ TIME POST INJECTION!h) F i gu re 19. The disappearance of thy rox ine from the plasma a f t e r a s i n g l e int ravenous i n j e c t i o n of 12 to 16 mg of L - t h y r ox i ne i n s t e e r s . Arrow i n d i c a t e s the time of T4 i n j e c t i o n . Basa l T4 concen t r a t i on i s i n d i c a t e d by the dotted l i n e s . - 139 -concen t r a t i on vs time curve (C = C° The k i n e t i c parameters of T4 metabol ism i n s tee r s obta ined by the method used i n t h i s experiment are presented i n Table 21. T4 Kinetics: Effect of Anabolic Compounds S ince T4 k i n e t i c parameters on d i f f e r e n t dates f o l l o w i n g the i m p l a n t a t i o n were obta ined from separate group of s teer s (n = 8 per p e r i o d ) , the data obta ined on each per iod were analysed sepa ra te l y (Table 22, 23 and 24 ) . On day 20 f o l l o w i n g the i m p l a n t a t i o n (Table 22) , plasma T4 b i o l o g i c a l h a l f - l i f e was s i g n i f i c a n t l y i nc reased (P < .05) i n s t ee r s implanted w i t h Synovex-S compared to other treatment groups. A s l i g h t , but n o n - s i g n i f i c a n t i nc rease i n T4 b i o l o g i c a l h a l f - l i f e was a l s o no t i c ed i n DES implanted s teer s compared to the unimplanted c o n t r o l s . On the c on t r a r y , b i o l o g i c a l h a l f - l i f e of T4 i n s tee r s implanted w i t h Zerano l was s i m i l a r (P > .05) to that of the unimplanted c o n t r o l s . The T4 f r a c t i o n a l turnover ra te was about 22 per cent lower (P < .05) i n Synovex-S implanted than the c o n t r o l s t e e r s . T4 volume of d i s t r i b u t i o n was s i m i l a r (P > .05) i n both the implanted and unimplanted c o n t r o l group; however, i t was s l i g h t l y l a r g e r i n s tee r s implanted w i t h Zerano l than w i t h other treatment groups. The MCR of T4 was 20 and 37 per cent lower (P < .05) i n DES and Synovex-S implanted s t e e r s , r e s p e c t i v e l y , than the unimplanted c o n t r o l s t e e r s . On the cont ra ry MCR was s l i g h t l y e l eva ted (N.S.) due to Zerano l i m p l a n t a t i o n . - 140 -Table 21. K i n e t i c parameters of thy rox ine metabolism i n growing beef s t e e r s 1 K i n e t i c Parameters Mean + SE 2 B i o l o g i c a l H a l f - L i f e (h) 41.13 + 1.07 F r a c t i o n a l Turnover Rate ( d - 1 ) 0.41 + 0.01 Volume of D i s t r i b u t i o n (£) 24.20 + 0.68 Me tabo l i c C learance Rate lid 9.97 + 0.40 Jt/d/kg body weight 0.03 + 0.01 J l /d/kg^ " 7 ^ body weight 0.13 + 0.01 Thyroxine S e c r e t i on Rate ug/day 835.10 + 42.90 ug/d/kg body weight 0.75 ug/d/kg body weight 2.43 + 0.12 10.87 + 0.52 Mean of va lues obta ined on 20, 40, 60 days f o l l o w i n g the i m p l a n t a t i o n . Mean ± °SE of va lues obta ined from 24 s t e e r s . Thyroxine Fractional Thyroxine Turnover Volume of Metabolic Clearance Rate ( M C R ) T h y r o x i n e Secretion Rate T4SR 0.75 Treatment1 Biological Half-Life Control DES Zeranol Synovex-S 36.94 ± 1.181 39.78 ±0.51 35.98 ±3.40 0.42 ±0.01 0.47 ±0.05 47.35*» t +' + 0.35*'+t ±0.24 ±0.002 21.74 ±1.00 26.95 ±4.30 21.66 ±0.68 9.09 ±0.28 12.77 ±3.20 0.0331 ±0.002 0.0472 ±0.013 0.14 ±0.01 0.19 ±0.05 *- t **. t * 7.81 , f 0.0257 , T ±.08 ±-001 .11 ±0.01 * * , t " 837.40 ±33.20 807.21 ±21.60 875.31 ±123.50 3.07 ±0.42 2.97 ±0.13 3.20 ±0.68 m^ean ± S.E. *>**P < .05, P < .01 respectively, compared to control. t»ttp < .05, P < .01, respectively compared to DES. +P < .05 compared to Zeranol. "Sign Indicates statistical significance when the calculated t value was compared with tabular t value (sign considered) (Table A.3, Steel.and Torrie, I960). 12.46 ±1.41 12.06 ±0.47 12.24 ±1.79 - 142 -The TSR was inc reased i n a l l the implanted s t e e r s . But the treatment d i f f e r e n c e s were not s i g n i f i c a n t (P > .05) due to a l a r ge an imal v a r i a t i o n observed i n the c o n t r o l group. Among the anabo l i c compounds, TSR was s l i g h t l y h igher due to DES and Synovex-S than Zerano l i m p l a n t a t i o n s . On day 40 f o l l o w i n g the Imp lanta t i on (Table 23) , a s i m i l a r t rend towards i nc reased TSR was observed i n Synovex-S implanted s t e e r s . A l l the k i n e t i c parameters, v i z . , T 1/2, TFTR, TDS, MCR, TSR, however, were s i m i l a r (P > .05) i n both the implanted and unimplanted s t e e r s . The MCR, on t h i s p e r i o d , was a l s o s l i g h t l y e levated i n a l l the implanted s t ee r s w i t h the except ion o f that implanted w i t h DES. On day 60 f o l l o w i n g the Imp lanta t i on (Table 24) , volume of T4 d i s t r i b u t i o n was s i g n i f i c a n t l y h igher (P < .05) i n a l l the implanted s tee r s than the c o n t r o l group of s t e e r s . There was no change i n T 1/2 and TFTR due to the imp l an t a t i o n of anabo l i c compounds. Although the mean MCR of T4 f o r a l l the implanted s tee r s were h igher than f o r c o n t r o l s t ee r s these d i f f e r e n c e s were not s t a t i s t i c a l l y s i g n i f i c a n t (P > 0 .05 ) . The TSR was 43 (P < .05), 51 (P < .05) and 68 (P < .001) per cent h i gher i n DES, Ze rano l , and Synovex-S implanted s t e e r s , r e s p e c t i v e l y , than the c o n t r o l s t e e r s . Among the anabo l i c compounds, TSR was s l i g h t l y h igher i n s t ee r s implanted w i t h Synovex-S than DES or Ze rano l . Though separate groups of s tee r s were u t i l i z e d on the d i f f e r e n t per iods s tud ied (20, 40 and 60 days a f t e r i m p l a n t a t i o n ) , i n order to f a c i l i t a t e a b e t t e r a p p r e c i a t i o n of the f l u c t u a t i o n s i n MCR and TSR (and Control DES Table 23. Kinetic parameters of thyroxine (T4) metabolism in steers on 40 days following the implantation anabolic compounds 45.06 ±3.30 0.373 ±0.027 21.16 ±0.79 7.86 ±0.53 0.024 ±.001 0.102 ±0.005 733.83 ±49.07 2.22 ±0.10 9.49 ±0.48 O J Zeranol 42.09 ±3.33 0.397 ±0.022 24.42 ±2.38 9.63T ±0.18 0.02841 ±.0002 0.121' ±0.0001 843.30 ±19.22 2.48 ±0.12 10.65 ±0.46 'mean ± SE tp < .05 compared to DES +P < .05 compared to Zeranol "Sign indicates statistical significance when the calculated t value was compared with tabular t value (sign considered) (Table A.3, Steel and Torrie, 1960) Table 24. Kinetic parameters of thyroxine (T4) metabolism ln steers on 60 days following the Implantation of anabolic compounds Biological Thyroxine Fractional Thyroxine Volume of Metabolic Clearance Rate (MCR) Thyroxine Secretion Rate T4SR Treatment Half-Life Tl/2 (h) Turnover Rate TFTR, per d Distribu-tion <*> i/d (MCR) t/d/kg (MCRK) „..„ 0.75 t/d/kg (MCRM) Mg/d ug/d/kg ug/d/kg Control 39.57 ± 4.571 0.426 ±0.05 20.64 ± 0.29 8.81 ± 1.14 0.0243 ±0.002 0.106 ±0.010 633.46 ± 0.28 1.76 ±0.08 7.67 ± 0.25 DES 38.64 ±4.67 0.437 ±0.07 24.40* ±0.48 10.63 ±1.08 0.0284 ±0.002 0.124 ±0.011 ** 904.49 ±9.67 * 2.42 ±0.02 ** 10.63 ±0.03 Zeranol 37.2 ±5.84 0.458 ±0.07 **+ 26.04 T ±0.29 11.92 ±1.74 0.0314 ±0.005 0.138 ±0.023 953.78* ±91.9 2.51 ±0.30 11.08 ±1.27 Synovex-S 37.97 ±0.31 0.438 ±.004 ** 24.65 ±0.55 10.80 ±0.33 0.0276 ±0.0001 0.122 ±0.001 ***++ 1063.95 T T ±8.35 * 2.72 ±0.10 12.08* ±0.36 'mean ± SE 't**»***p ^ ^ p ^ ^ p ^ .001, respectively, compared to control. »tt,p < .05, P < .01, respectively compared to DES. 'Sign indicates statistical significance when the calculated t value was compared with tabular t value (sign considered) (Table A.3, Steel and Torrle, 1960). - 145 -hence the changes i n plasma T4 l e v e l s ) , the va lues obta ined on d i f f e r e n t per iods f o l l o w i n g the imp l an t a t i o n were pooled and are presented i n Table 25. The TSR (per kg metabo l i c body s i z e ) was found to be e leva ted (32 - 59% h igher than the c o n t r o l group) i n a l l the implanted s t e e r s . I n s p i t e of the inc reased TSR, plasma T4 concen t ra t i on was on ly s l i g h t l y e l e va ted (4%) i n Zeranol implanted s t e e r s . The MCR on the other hand, was decreased due to DES and Synovex-S i m p l a n t a t i o n and inc reased due t o the i m p l a n t a t i o n of Ze rano l . These f l u c t u a t i o n s i n TSR and MCR were r e f l e c t e d i n 20, 41, and 4 per cent inc reased plasma T4 concen t r a t i on i n DES, Synovex-S and Ze rano l , r e s p e c t i v e l y , when compared to the unimplanted c o n t r o l s t e e r s . The e f f e c t of anabo l i c compounds on MCRM and TSRM on a l l the per iods s tud ied i s summarized i n F i gu re 20 f o r b e t t e r c l a r i t y . DISCUSSION Thyroxine K i n e t i c s : V a l i d i t y of the Method The concen t ra t i on of T4 i n the blood i s due to the r e s u l t of I t s s e c r e t i o n from the t h y r o i d g land, i t s c lea rance from the c i r c u l a t i o n and i s i n genera l governed by i t s k i n e t i c parameters. In t h i s experiment T4 k i n e t i c parameters were determined by the n o n - r a d i o a c t i v e , bolus i n j e c t i o n chemical turnover method (Post and Mixner, 1961). Apart from the method used i n t h i s exper iment, other approaches f o r the de te rm ina t i on of T4 k i n e t i c s have a l s o been repor ted i n the l i t e r a t u r e : s u b s t i t u t i o n method (P ipes et a l . , 1957); poo l turnover method ( Ingbar - 146 -Table 25. E f f e c t of anabo l i c compounds on plasma thy rox ine s t a t u s 3 Treatment TSRM MCRM Plasma T4 L e v e l (% i n c rea se over c o n t r o l ) C o n t r o l 8.22 0.13 — DES 10.86 0.12 20.0 (32%) (-7.67%) Zerano l 11.27 0.15 3.8 (37%) (15.35%) Synovex-S 13.12 0.12 41.0 (59%) (-5.8%) aMean of va lues obta ined on 20, 40 and 60 days f o l l o w i n g the i m p l a n t a t i o n . ^Thyroxine s e c r e t i o n r a t e per kg metabo l i c body s i z e (ug/d/kg^*^^) va lue s i n the parenthes i s i n d i c a t e percent i nc rea se over c o n t r o l . c M e t a b o l i c c learance r a t e per kg metabo l i c body s i z e (1/d/kg^*^ ) va lues i n the paranthes i s i n d i c a t e percent i nc rea se (+) or decrease (-) over the c o n t r o l . - 147 -\ 250 200 150 100 ++ •• •+ +• •• •+ +• METABOLIC CLEARANCE RATE Sit >C O )C o •+ ****¥.' o li 1 T T«r-M ° o 20 15 2 10 \ XJ \ O) 5 SECRETION RATE t « o • K • t « O • • < )C • • t O • )C • • 1 O • )C • O • • < X • • i O • • < X • V O •« > c - -T T •+ •+ ++ •• •• •+ ++ ++ •• •+ 1 o _6_ CONTROL ZERANOL DES SYNOVEX-S JLI -TT + T • + • + • + *• o 20 40 60 DAYS AFTER IMPLANTATION Figure 20. Metabolic clearance and secretion rates of thyroxine on days 20, 40 and 60 following the implantation of anabolic compounds in steers. Each bar represents mean ± SE of values obtained from two steers. - 148 -and F r e i n k e l , 1955). In farm an imals , e s p e c i a l l y i n c a t t l e and sheep, both the r ad i o t h y r ox i ne (*^I or 1311 l a b e l l e d ) or the chemical ( u n l a b e l l e d L-Thyroxine) thy rox ine turnover methods have been u t i l i z e d to study T4 k i n e t i c s (Wi lson et_al., 1977; Yousef and Johnson, 1967; Anderson, 1971; Post and Mixner, 1961; Fox et a l . , 1974). The chemical r a the r than r ad i o t h y r ox i ne disappearance method was adopted i n t h i s study due to i t s s i m p l i c i t y and the non -de s t r uc t i ve nature (as the animals are not contaminated w i t h r a d i o a c t i v e t h y r o x i n e ) . Bes ides the k i n e t i c parameters obta ined us ing t h i s method were shown to be s i m i l a r to those us ing the r ad i o thy rox i ne disappearance method (Post and M ixner , 1961). The v a l i d a t i o n of the chemical thy rox ine turnover method has been d i scus sed i n d e t a i l by Post and Mixner (1961). However, Swanson and M i l l e r (1973) have shown that aberrant r e s u l t s could be obta ined u s ing the chemica l turnover method. The plasma T4 d i l u t i o n curve was analysed mathemat i ca l l y , t r e a t i n g the system s tud ied as a s i n g l e homogeneous r a p i d l y e q u i l i b r i a t i n g compartment i n t o which T4 i s d i s t r i b u t e d and removed at a constant r a t e . S i m i l a r mathematical treatment of the plasma T4 d i l u t i o n curve f o l l o w i n g T4 i n j e c t i o n was reported by others i n farm animals (Post and Mixner, 1961; Fox et^ a l . , 1974; Marple eit a l . , 1981; Ingram and Evans, 1980). In a d d i t i o n , the a p p l i c a t i o n of two (Sawhney et a l . , 1978) and mult icompartmental (Wi l son et_ a l . , 1977; DI Stefano et a l . , 1982) procedures has a l so been reported r e c e n t l y . The va lues of T 4 - k i n e t i c parameters obta ined i n t h i s experiment were comparable to those repor ted f o r c a t t l e (Table 26 ) . The s l i g h t Table 26. K i n e t i c parameters of T4 metabolism i n c a t t l e TFTR 2 ( d - 1 ) Thyroxine Sec re t i on Rate (TSR) Sex Method 1 yg/d yg/d/kg y g / d / k g 0 , 7 5 Reference Calves C.T. 0.40 344 6.83 N.A. Post and M ixner , 1961 Cows C.T. 0.40 1590 2.89 N.A. Post and Mixner , 1961 Cow R.T. 0.30 1580 3.16 14.9 Yousef and Johnson, 1967 B u l l s R.T. 0.23 to 0.32 N.A. N.A. N.A. T v e i t and A l m i d , 1980 Steers C.T. N.A. 810 to 1210 3.24 to 10 to 15.2 3.70 Fox et a l . , 1974 Steers C.T. 0.41 835 2.43 10.8 Expt . I IA:2 method used fo r the thyrox ine k i n e t i c s C.T. = Chemical thy rox ine ( L - thy rox ine ) turnover R.T. = Rad iothyrox ine ( 1 2 5 I or 1 3 l I - T4) turnover 2 Thy rox i ne f r a c t i o n a l turnover ra te N.A = not a v a i l a b l e or reported by the workers - 150 -v a r i a t i o n i n the est imates could have been due to the k i n e t i c method used and the p h y s i o l o g i c a l s t a te of the animals used. From a d e t a i l e d spec ies comparison, Marple et a l . (1981) have concluded t h a t , TSR expressed per kg metabo l i c body weight ( k g 0 , 7 5 ) was w i t h i n a narrow range (3.53 p g / d / k g 0 , 7 5 i n man to 20.55 u g / d / k g 0 * 7 5 i n ch icken) among d i f f e r e n t spec i e s . The va lues of TSR (10.87 u g / d / k g 0 * 7 5 ) obta ined i n t h i s experiment are s i m i l a r to those repor ted f o r beef s tee r s (Fox et^ a l . , 1974) and are w i t h i n the i n t e r s p e c i e s range. Thyroxine Kinetics: Effect of Estrogenic Anabolic Compounds Re su l t s of t h i s experiment show c e r t a i n important f i n d i n g s on the e f f e c t of anabo l i c compounds on T4 k i n e t i c s i n s t e e r s . There appears to be an o v e r - a l l i n c rea se i n TSR and a decrease i n MCR i n s teer s implanted w i t h DES or Synovex-S and an i n c rea se i n both the parameters i n Zerano l implanted s t e e r s . The r e s u l t s a l s o i n d i c a t e that d i f f e r e n t i a l pa t te rn s of T4 k i n e t i c s f o l l o w i n g Synovex-S and DES as aga ins t Zerano l i m p l a n t a t i o n and i n genera l r e v e a l the e f f e c t of estrogens on t h y r o i d f u n c t i o n . Burgess and Lamming (1960) have proposed that Increased TSR cou ld be r e spon s i b l e f o r the inc reased growth r a te f o l l o w i n g estrogen a d m i n i s t r a t i o n i n farm an imal s . D i e t h y l s t i l b e s t r o l fed lambs have been shown to e x h i b i t a t rend towards i nc reased TSR (T renk l e , 1969). The r e s u l t s obta ined i n t h i s experiment are thus i n l i n e w i t h the f i n d i n g s of the e a r l i e r workers and i n d i c a t e that the inc reased plasma T4 concent ra t i on s f o l l o w i n g Synovex-S or DES imp lan ta t i on s ( r e s u l t s of Expt . I IA: 1) could be due to the r e l a t i v e changes i n T4 s e c r e t i o n and - 151 -c lea rance r a t e s . S ince the c learance of t h y r o i d hormone i s r e l a t e d to the s t r eng th of the plasma t h y r o i d hormone b i nd ing p r o t e i n s , o v e r a l l r educ t i on i n MCR f o l l o w i n g Synovex-S and DES imp l an ta t i on s cou ld be a t t r i b u t e d to an i nc rea se i n TBG b ind ing c a p a c i t y . A s i g n i f i c a n t i n c r ea se i n TBG b ind ing c apac i t y f o l l o w i n g est rogen therapy was repor ted i n rhesus monkeys (Sawhney et a l . , 1978). F u r t he r , a r educ t i on i n T4 degradat ion r a t e was observed i n p a t i e n t s t r ea ted w i t h estrogens ( Zan i nov i ch , 1973). Serum b ind ing of T4 was a t t r i b u t e d i n par t to the reduced T4 degradat ion f o l l o w i n g est rogen a d m i n i s t r a t i o n (Robbins and Ne l son, 1958; Engbr ing and Engstrom, 1959). The extent to which TBG has p a r t i c i p a t e d i n reduc ing the MCR i n the Synovex-S and DES implanted s tee r s used i n t h i s experiment i s not c l e a r l y understood as s imultaneous measurements of TBG were not c a r r i e d out . E a r l i e r f i n d i n g s (Expt I IA: 1 ) , however, do not support the above theory as TBG(RDA) (Table 17) was s i g n i f i c a n t l y reduced i n a l l the implanted s t e e r s . But , u s ing T3 r e s i n uptake as the c r i t e r i o n , Kah l et a l . (1978) have repor ted enhanced b i nd i n g c apac i t y of the plasma T4 b i nd ing p r o t e i n i n Synovex-S implanted s t e e r s . Re su l t s of t h i s experiment r e v e a l c e r t a i n aspects on the mechanism of a c t i o n f o r Zerano l on the t h y r o i d gland and on the p e r i p h e r a l metabol ism of T4. The TSR, i n genera l was s l i g h t l y h igher i n Zerano l implanted than the c o n t r o l s t ee r s w i t h s i g n i f i c a n t l y h igher va lues (51 percent i nc rea se over c o n t r o l ) obta ined on day 60 f o l l o w i n g i t s i m p l a n t a t i o n . Thus i n t h i s respect the mechanism of a c t i o n of Ze rano l seems to be s i m i l a r to that of Synovex-S and DES, however - 152 -Zerano l e x h i b i t s a weaker i n f l u e n c e on the t h y r o i d gland compared to the l a t t e r two compounds. The r e s u l t s , t h e r e f o r e , do not support the t h y r o i d depress i ve a c t i on s of Zerano l proposed by Rothanbacher et a l . (1975). But the mechanism of a c t i o n of Zerano l d i f f e r s from that of DES and Synovex-S i n that i t s i m p l a n t a t i o n i nc reases MCR as w e l l . Increased MCR of T4 could thus be a t t r i b u t e d to the s i m i l a r T4 concen t ra t i on i n both the c o n t r o l and Zeranol implanted s teer s (Expt. I IA: 1) i n s p i t e of the i nc reased TSR due to Zerano l i m p l a n t a t i o n . Plasma T4 concen t r a t i on i n Zerano l implanted animals was found to be unchanged or s l i g h t l y decreased compared to the c o n t r o l animals (Wiggins et a l . , 1979) but the e f f e c t of Zerano l on the t h y r o i d gland seems to be i n c o n s i s t e n t as both an i nc rea se (Wiggins et a l . 1976) and a decrease (Wiggins et a l . , 1979) i n i t s weight have been r epo r ted . In t h i s study a bas i s f o r the l a ck of d i f f e r e n c e i n T4 l e v e l between Zerano l implanted and the unimplanted c o n t r o l s teer s was f u r t h e r e s t a b l i s h e d . CONCLUSION The r e s u l t s of t h i s study revea led that the inc reased plasma T4 of the s teer s implanted w i t h anabo l i c compounds i s due to an i nc reased s e c r e t i o n r a t e of T4. Up to 31-59% i nc rea se i n the T4 s e c r e t i o n r a te was observed due to the i m p l a n t a t i o n of anabo l i c compounds. Both DES and Synovex-S were found to be more e f f e c t i v e i n i n c r e a s i n g T4 concen t r a t i on i n the plasma and i t s s e c r e t i o n ra te than Z e r a n o l . Imp lantat ions w i t h Zerano l a l s o r e s u l t e d i n an inc reased T4 s e c r e t i o n r a te but the p a r t i c i p a t i o n of inc reased metabo l i c c learance r a te - 153 -r e s u l t e d i n e i t h e r a s l i g h t e l e v a t i o n or no change i n the plasma T4 c o n c e n t r a t i o n . I t i s concluded that the inc reased T4 s e c r e t i o n r a t e cou ld be one of the mechanisms by which anabo l i c compounds evoke growth response i n s t e e r s . - 154 -EXPERIMENT I I B EFFECT OF ANABOLIC COMPOUNDS ON PLASMA GROWTH HORMONE LEVEL AND ITS KINETIC PARAMETERS OF METABOLISM IN GROWING STEERS INTRODUCTION Changes i n the endogenous c i r c u l a t i n g growth hormone (GH) concen t r a t i on have been proposed as the major mechanism by which a n a b o l i c compounds mediate t h e i r metabo l i c and growth promoting p r o p e r t i e s i n ruminants (Bu t te ry et a l . , 1978; P re s t on , 1975). These hypotheses are very a t t r a c t i v e con s i de r i ng the inc reased somatic growth, p r o t e i n a c c r e t i o n and reduced f a t synthes i s i n ruminants f o l l o w i n g the i m p l a n t a t i o n of anabo l i c compounds and the e f f e c t of GH on a l l the above processes . However, the l a ck of c o r r e l a t i o n between plasma GH and growth r a te i n c a t t l e (Joakimsen and Blom, 1976; K e l l e r et a l . , 1979; Ohlson et^ a l . , 1981; Purchas et^ a l . , 1970; T r e n k l e , 1977; Trenk le and I r v i n , 1970; Trenk le and Tope l , 1978) r a i s e s quest ions as to the r e a l i n t e r r e l a t i o n s h i p between blood GH concen t r a t i on and growth r a te and suggests perhaps other growth f a c t o r s mediated by GH, such as somatomedins, p lay an important r o l e . Many of the anabo l i c a c t i on s of GH are now be l i e ved to be mediated by somatomedins (Chochinov and Daughaday, 1976). A r e l a t i o n s h i p between serum somatomedins or somatomedin- l ike a c t i v i t y and growth of b u l l s (Lund-Larsen _t_ a l . , 1977), Swine (Lund-Larsen and Bakke, 1975) and lambs (Olsen et a l . , 1981; Wangsness et a l . , 1981) has been r epo r t ed . - 155 -A number of workers have repor ted i nc reased weight of the p i t u i t a r y gland (Burgess and Lamming, 1960; Clegg and Co l e , 1954; Wiggins et a l . , 1976; Wiggins et a l . , 1979), number of a c i d o p h i l s i n the p i t u i t a r y g land (Clegg and Co l e , 1954) and plasma GH concen t r a t i on (Borger et_ a l . , 1973; Donaldson et a l . , 1981; Olsen et^ a l . , 1977; Wiggins e£ a l . , 1976; T renk l e , 1970a; T renk le 1976b) i n ruminants t r e a t e d w i t h e s t r o gen i c anabo l i c compounds (DES, Ze rano l , e s t r a d i o l ) . No such i n f o r m a t i o n , however, i s a v a i l a b l e f o r Synovex-S which has been shown e f f e c t i v e i n i n c r e a s i n g the growth r a t e (Kahl et a l . , 1978) and thy rox ine s e c r e t i o n r a te from the t h y r o i d gland ( r e s u l t s of Expt . I IA: 2 ) . Fu r the r the mechanism by which anabo l i c compounds i n f l u e n c e plasma GH concen t r a t i on has not been c l e a r l y understood. Though the inc reased p i t u i t a r y g land weight and number of a c i d o p h i l s i n the est rogen t r e a t ed animals i n d i r e c t l y i n d i c a t e enhanced s e c re to r y a c t i v i t y of the p i t u i t a r y , abso lu te va lues of GH s e c r e t i o n r a t e or GH k i n e t i c parameters are not known. S ince the plasma GH s ta tu s i s a r e s u l t of i t s s e c r e t i o n r a t e , metabo l i c c learance r a t e and Is i n genera l determined by i t s k i n e t i c parameters, s tud ie s on GH k i n e t i c s i n estrogen t r e a t ed animals w i l l be advantageous i n understanding the mechanism of a c t i o n of anabo l i c compounds. Th i s experiment was t he re fo re conducted to study the k i n e t i c parameters of GH metabolism i n s tee r s implanted w i t h the e s t r o gen i c a n a b o l i c compounds i n order to understand the mechanism by which they i n f l u e n c e plasma GH s t a t u s . The major o b j e c t i v e s of t h i s experiment i n v e s t i g a t i o n were t o : - 156 -a . E s t a b l i s h plasma GH l e v e l s i n s tee r s implanted w i t h DES, Zerano l and Synovex-S, and b. Determine k i n e t i c parameters of GH metabol ism i n s tee r s implanted w i t h anabo l i c compounds. Ob jec t i v e s (a) and (b) were s tud ied i n separate group of s t e e r s . MATERIALS AND METHODS 1. Effect of Anabolic Compounds on Plasma Growth Hormone Level  l n Steers Animals and Experimental Procedure S i x t een Hereford s t ee r s which were used i n Expt. IA were u t i l i z e d i n t h i s experiment to study the e f f e c t of anabo l i c compounds on plasma GH l e v e l s . The anabo l i c compounds used ( Ze rano l , DES, Synovex-S), t h e i r i m p l a n t a t i o n procedure and the exper imenta l d i e t used were the same as de sc r i bed e a r l i e r (Expt. I A ) . Blood samples were c o l l e c t e d from i n d i v i d u a l s t ee r s on 14, 28, 42 and 56 days f o l l o w i n g the i m p l a n t a t i o n by means of j u g u l a r v e i n puncture i n p r e c h i l l e d hepa r i n i zed evacuated tubes (Vacuta iner tubes , Beck ton -D i ck in son , D i ck in son and Co., New J e r s e y ) . B lood samples were c o l l e c t e d before feed ing the animal at 0730 h and at 1400 h. Care was taken not to e x c i t e the animals dur ing the sampling p e r i o d . The samples were kept i n an i c e bath and were cen t r i f u ged at 1000 x g ( S o r v a l l superspeed C e n t r i f u g e , Model RC 2B, Norwalk, Conn., U.S.A.) to ob ta i n the plasma which was then s to red at -20°C. - 157 -2. Effect of Anabolic Compounds on the Kinetic Parameters of GH Metabolism Animals S i x teen Hereford s tee r s were used i n t h i s experiment to study the e f f e c t of anabo l i c compounds on GH k i n e t i c s . The animlas were s e l e c t e d from a group of 24 s teer s that were used i n Expt. I IA: 2. The s tee r s used i n t h i s study represented a l l the four treatment groups ( c o n t r o l , DES, Ze rano l , Synovex-S) desc r ibed i n Expt. I IA: 2. The method of anabo l i c compound i m p l a n t a t i o n , animal feed ing procedure and the p r epa r a t i on of s teer s f o r GH k i n e t i c s tud ie s were desc r ibed i n Expt. I IA: 2. K i n e t i c parameters were est imated on days 20 and 40 f o l l o w i n g the i m p l a n t a t i o n (n = 2 s teer s per treatment group f o r each pe r i od s t u d i e d ) . Experimental Procedure Preparation of GH Solution for Kinetic Studies NIH-GH-B 18 (Na t i ona l P i t u i t a r y Agency, Ba l t imo re , Maryland) was used i n a l l the GH k i n e t i c exper iments . The hormone was c a r e f u l l y weighed, d i s s o l v e d i n 0.9% Nac l and the pH was adjusted between 9 and 10 w i t h IN NaOH. An a l i q u o t of 3 to 4 ml of the GH s o l u t i o n , con ta i n i n g 6-8 mg NIH-GH-B18 was s to red f rozen i n 5 ml sy r inges at -20°C. One sy r inge con ta i n i n g the GH p repa ra t i on was thawed on the day of the experiment and used as such. GH Kinetics A s i n g l e bolus i n j e c t i o n , n on - r ad i o a c t i v e GH k i n e t i c procedure was adopted (T renk l e , 1976a; T r enk l e , 1977). - 158 -On the day of the experiment the s tee r s were weighed, fed the morning p o r t i o n of the r a t i o n and were p laced i n metabolism c ra tes t ha t a l lowed some freedom of movement but prevented the animal from t u r n i n g around. The animals were a l lowed to recover i n the c r a t e f o r an hour and b lood samples were c o l l e c t e d through the ca the te r every 30 min f o r 3.5 h f o r a t o t a l of e i gh t blood samples from each animal to a s c e r t a i n b a s a l GH sec re to r y p a t t e r n . The s tee r s were then r a p i d l y i n j e c t e d w i t h 6-8 mg of bovine GH (NIH-GH-B18; one sy r inge as desc r ibed above) and the c a t he te r was immediately f l u shed w i t h 0.9% s a l i n e . To measure the disappearance of the i n j e c t e d GH, s e r i a l blood samples were taken at 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 90 and 120 minutes f o l l o w i n g the GH i n j e c t i o n . The blood samples were kept i n i c e i n hepa r i n i zed tubes and were c e n t r i f u g e d to o b t a i n the plasma. The plasma samples were s to red at -70°C t i l l they were ana l yzed . Two s tee r s r ep re sen t i ng a treatment group were s tud ied s imu l taneous l y . 3. Analytical Procedure Plasma GH concen t ra t i on i n a l l the samples c o l l e c t e d on days 14, 28, 42 and 56 f o l l o w i n g the i m p l a n t a t i o n (analyzed on both the 0730h and 1400 h sample) and i n a l l the samples c o l l e c t e d f o l l o w i n g GH i n j e c t i o n (GH K i n e t i c s ) was analysed by a h i g h l y s p e c i f i c double ant ibody radioimmunoassay procedure. GH Radioimmunoassay GH concen t r a t i on i n the bovine plasma was determined by the radioimmunoassay method desc r ibed by Niswender et a l . (1969) and i t s m o d i f i c a t i o n o u t l i n e d by Davis (1972) u s i ng the ov ine GH radioimmuno-- 159 -assay m a t e r i a l s obta ined from NIAMDD, Bethesda, Mary land. Ovine GH (NIAMDD-oGH-I-1) was i od i na ted by the chloramine-T method (Greenwood ejt a l . , 1963) and was p u r i f i e d as desc r ibed by Purchas et a l . (1970). A d e t a i l e d account of the GH i o d i n a t i o n procedure, and the p u r i f i c a t i o n of 125J_ 0Q H i s presented i n Appendix I I . B r i e f l y , the reagents were added to d i sposab le po l y s t y rene tubes (12 x 75 mm, Amersham Co., Canada) i n the f o l l o w i n g sequence: 200 y l of PBS - 1% BSA (0.01 M phosphate bu f fe red s a l i n e , pH 7.0 c on ta i n i n g 1% bovine serum albumin, f r a c t i o n V) or 200 y l t e s t sample or app rop r i a te amount of oGH standard (0 to 20 ng NIAMDD-oGH-I-1/tube) and 200 y l of r a b b i t ant i serum to ovine GH ( F i n a l tube d i l u t i o n 1:40000). The tubes were then incubated at 4°C. Twenty four hours a f t e r the a d d i t i o n of r a b b i t ant i serum to oGH, 100 y l of 1 2 5 I - o G H ( p u r i f i e d m a t e r i a l from the Sephadex G-100 Column, 35000 cpm per ml of PBS-1% BSA) was dispensed (us ing Eppendorf repeater p i p e t t e , Model 4780, Brlnkmann Instruments, I n c . , New York) to each tube and the i n cuba t i on was cont inued at 4°C. Twenty four hours a f t e r the a d d i t i o n of 125T- 0GH, 100 y l of goat ant i serum to r a b b i t gamma g l o b u l i n was added to each tube and the i n cuba t i on was cont inued at 4°C. A f t e r 72 h of f u r t h e r i n c u b a t i o n , 2.5 ml of PBS (pH 7.0) was added to each tube and the tubes were cen t r i f u ged at 1000 x g i n a r e f r i g e r a t e d c e n t r i f u g e (Beckman, Model TJ6) f o r about 30 minutes. The supernatant con ta i n i n g the f r ee 125J._0GH was decanted and the r a d i o a c t i v i t y a s soc i a ted w i t h the p r e c i p i t a t e was counted i n an ® automat ic gamma counter (Auto-Gamma 500 C Countimg System, Packard - 160 -Inst ruments, I l l i n o i s , U.S.A.) . A l l assays were performed i n d u p l i c a t e . Blood samples c o l l e c t e d on any one sampling per iod or from any one GH k i n e t i c experiment were analysed i n the same assay. The procedure f o r the c a l c u l a t i o n of GH concen t ra t i on i n the plasma samples and f o r the de te rm ina t i on of i n t e r - and i n t r aa s s a y v a r i a t i o n was s i m i l a r to that desc r ibed f o r the T4-radioimmunoassay (Expt . I IA: 2 ) . Under the cond i t i on s desc r ibed above, the i n t e r and i n t r a a s s a y c o e f f i c i e n t s of v a r i a t i o n were 3.2% and 9.6%, r e s p e c t i v e l y , f o r a sample w i t h a mean concen t ra t i on of 16.5 ng/ml. Analysis of Kinetic Data The plasma growth hormone disappearance curve (0 to 120 min post i n j e c t i o n ) was analysed u s ing a d e c i s i o n making F o r t r a n Computer Program, AUTOAN (Sedman and Wagner, 1976). From the r e s u l t s of the l e a s t square est imates the observed data was found to be best desc r ibed by the b i e x p o n e n t i a l equat ion : C = A e " 0 * * B e " 3 1 Where: C = concen t r a t i on of GH at time = t A = Y i n t e r c e p t of the f i r s t component of the disappearance curve (Fast Phase) (ng/ml) a = d i s p o s i t i o n constant i n the r ap i d phase ( m i n - 1 ) B = Y i n t e r c e p t of the second component of the disappearance curve (Slow Phase) (ng/ml) 3 = d i s p o s i t i o n constant i n the slow phase ( m i n - 1 ) - 161 -Model Description The two compartment model describing GH disappearance from the blood plasma is depicted below: where: k 1 2 k21 Model used to describe GH Metabolism in Steers Cj ™ Central compartment (represents the plasma) C^ • Peripheral compartment (sites of GH u t i l i z a t i o n and degradation) K._ « the first-order rate constant for the elimination of GH from the central compartment (min - 1) K,. - the first-order rate constant for the distribution 12 of GH from the central to the peripheral compartment (min - 1) • the first-order rate constant for the distribution of *21 GH from the peripheral to central compartment (min - 1). - 162 -Calculations: 1. GH Volume of Distribution (£) GH Volume of d i s t r i b u t i o n was c a l c u l a t e d from the equat ion : Volume of d i s t r i b u t i o n = D A/ot2 + B/32 (A/a + B/P) 2 Where: D = dose of GH i n j e c t e d A, B, a , 6 = see above model . . . R i z k a l l a h et a l . (1969) 2. Metabolic Clearance rate of GH (MCR, ml. min - 1): MCR of GH from the c i r c u l a t i o n was c a l c u l a t e d from the equat ion : MCR (ml. m i n - 1 ) = A Q D ° g p . . . T a i t and B u r s t e i n (1964) A p T Bot 3. GH secretion rate or production rate (GH - SR, ng. min-*) S e c r e t i o n r a te of GH was c a l c u l a t e d from the metabo l i c c learance r a t e and ba sa l GH concen t ra t i on as desc r ibed i n the equat ion : GH - SR (ng. m i n - 1 ) = MCR x basa l GH concen t ra t i on . . . Gurpide and Mann (1970) 4. Half-Life of GH T 1/2 (a) = 0.693/a T 1/2 (3) = 0.693/3 . . . Sh ip ley and C l a r k (1972) - 163 -where: a, 3 are the r a te constants of the r a p i d and slow components of the disappearance curve , r e s p e c t i v e l y . S t a t i s t i c a l Analysis Plasma GH concent ra t i ons on days 14, 28, 42 and 56 f o l l o w i n g the i m p l a n t a t i o n were averaged (GH l e v e l i n the 0730 h and 1400 h was averaged f o r each s t ee r on each sampling pe r i od ) and were subjected to a n a l y s i s of va r i ance ( S t e e l and T o r r i e , 1960). The means were separated by the Newman-Keuls m u l t i p l e range t e s t . The hormone concent ra t i on s were transformed to n a t u r a l logar i thms p r i o r to s t a t i s t i c a l a n a l y s i s . Bas se t t and Thorburn (1971) repor ted that l o g a r i t h m i c t r an s fo rmat i on of p r o t e i n hormones i n the plasma conformed more c l o s e l y to a normal s t a t i s t i c a l d i s t r i b u t i o n than the abso lute c oncen t r a t i o n . A s i m i l a r treatment of data was repor ted by Donaldson et a l . (1981). The a n a l y s i s of the data us ing the abso lute GH concen t r a t i on was a l s o c a r r i e d out and the r e s u l t s obta ined from such a n a l y s i s are presented i n Appendix I I I . The k i n e t i c parameters were averaged and are presented as mean ± SE. The treatment d i f f e r e n c e s were te s ted by S tudent ' s t - t e s t ( S t e e l and T o r r i e , 1960). RESULTS 1. Effect of Anabolic Compounda on Plasma GH Levels; GH concen t ra t i on i n the plasma was expressed as ng NIAMDD-oGH-I-l/ml. Mean plasma GH concen t r a t i on i n the s tee r s used i n t h i s experiment was 33 ± 7.5 ng/ml (n = 64) . Us ing the radioimmunoassay - 164 -procedure de sc r i bed i n t h i s exper iment, a GH concen t r a t i on of about 13.9-18.6 ng/ml was obta ined f o r a bovine plasma poo l (from 10 a s say s ) . The e f f e c t of anabo l i c compounds on plasma GH concent ra t i on s on d i f f e r e n t days f o l l o w i n g the i m p l a n t a t i o n i s presented i n Table 27 ( a l s o see Appendix I I I ) and the o v e r - a l l e f f e c t of t reatments on plasma GH i s presented i n F i gu re 21. There was a s i g n i f i c a n t i nc rea se i n the plasma GH concen t r a t i on i n s t ee r s due to the i m p l a n t a t i o n of anabo l i c compounds (P < .001, C o n t r o l vs a l l the anabo l i c compounds). When a l l the sampling per iods (14, 28, 42 and 56 days) were taken i n t o c o n s i d e r a t i o n , plasma GH concen t r a t i o n was about 76, 153 and 135 per cent h i gher i n Ze rano l , DES and Synovex-S implanted s t e e r s , r e s p e c t i v e l y , than i n the c o n t r o l s t e e r s . S teers implanted w i t h DES and Synovex-S had s l i g h t l y h igher plasma GH concen t r a t i on than those implanted w i t h Ze r ano l . The treatment x pe r i od i n t e r a c t i o n was not s i g n i f i c a n t (P > 0 .05 ) . Plasma GH was mainta ined at a h igher concen t r a t i on on a l l the per iods s t ud i ed i n s tee r s implanted w i t h DES and Synovex-S than the unimplanted c o n t r o l s t e e r s . On the con t r a r y , e l eva ted plasma GH i n Zerano l implanted s tee r s was observed on day 14 a f t e r the i m p l a n t a t i o n and the concen t r a t i on decreased beyond day 14 to about 20 ng/ml on day 56. 2. Effect of Anabolic Compounds on the Kinetic Parameters  of Growth Hormone Metabolism The disappearance of GH i n the plasma was s tud ied a f t e r i n j e c t i n g the s tee r s r a p i d l y w i t h 6 mg of N1H-GH-B18. A f t e r the i n j e c t i o n , GH disappeared from the c i r c u l a t i o n i n a m u l t i e x p o n e n t i a l f a sh i on over the - 165 -Table 27. Plasma GH concent ra t ions i n s tee r s on d i f f e r e n t days f o l l o w i n g the imp l an t a t i o n of e s t r o gen i c anabo l i c compounds 1 Treatment Days A f t e r Imp lan ta t i on 14 28 42 56 Mean S E ' ng NIAMDD-oGH-I-l/ml C o n t r o l 14.04 J ±0.18 12.70 ± 0.22 22.98 ± 0.37 23.61 ± 0.24 17.98 0.14 Zerano l 40.28 ±0.45 29.31 ±0.24 28.77 ±0.2 20.51 ±0.26 29.72 0.14 DES 37.59 ±0.39 42.10 ±0.15 49.65 ±0.36 44.65 ±0.14 43.49 0.13 Synovex-S 39.45 ±0.26 40.52 ±0.29 45.7 ±0.50 34.92 ±0.44 40.15 ' 0.17 S t a t i s t i c a l a n a l y s i s based on n a t u r a l l o g . transformed va l ue , a b ' means i n the same column w i t h d i f f e r e n t s u p e r s c r i p t s d i f f e r (P < .05) 4 s tee r s per t reatment. 2 p o o l e d n a t u r a l l o g standard e r r o r . 3 The mean of GH concen t ra t i on i n 0730h and 1400 h sample from 4 s t e e r s . - 166 -50 30 20 =; 10 40 • • • • • • • • ±±± >od CONTROL ZERANOL DES SYNOVEX-S 0 I • • • • • • • • • • • • • • • • • • • • • • • • • • • • . V • • • ••••• • • • • • • • • • • • • +• 4+ 4 4 4 4 4 4 4 4 4 4 ++••4 • • • • • +++•• +•••• 4 4 4 4 4 ***** ***** ***** ***** ***** 4 4 4 4 4 4 4 4 4 4 4+++4 • 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 444+4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 yzol Figure 21. Effect of anabolic compounds on plasma GH concentration in growing steers. Each bar represents mean ± SE of values obtained from four steers on days 14, 28, 42 and 56 following the implantation (n=16). a> DBars with different alphabet differ significantly (P < .05). - 167 -next 120 min (F igure 22 ) . The plasma GH disappearance curve d i s p l a yed the presence of two components: an i n i t i a l h i g h l y r a p i d phase l a s t i n g 5.0 ± 0.8 min, and a slow disappearance phase l a s t i n g 42 ± 3 min. The va lues of MCR and s e c r e t i o n r a t e s , c a l c u l a t e d from the parameters obta ined by f i t t i n g the disappearance curve to a two-compartment model, are summarized i n Table 28. F r a c t i o n a l r a t e s of GH disappearance from the r a p i d l y t u r n i n g over and s l ow l y t u r n i n g over compartment, r e s p e c t i v e l y , were 0.167 and 0.017 min - 1 . The average volume of d i s t r i b u t i o n of GH i n the s tee r s used i n t h i s experiment was found to be 6.1 ± 0.6 per cent of the body we ight . Mean MCR and s e c r e t i o n ra te s of GH were 21 1/h and 252 pg/h or 74.5 ml/h/kg and 0.91 ug/h/kg. The e f f e c t of anabo l i c compounds on the k i n e t i c parameters of GH metabol ism on 20 days f o l l o w i n g the i m p l a n t a t i o n i s presented i n Table 29 to 31. The h a l f - l i f e of GH i n the r a p i d l y t u r n i n g over compartment ranged from 3.64 to 8.02 min i n s t e e r s . A s i g n i f i c a n t i nc rea se i n the h a l f - l i f e of GH i n the r a p i d phase was no t i c ed i n Synovex-S implanted s tee r s when compared to the c o n t r o l s t e e r s . Steers from d i f f e r e n t treatment groups had s i m i l a r h a l f - l i f e of d isappearance of GH i n the s l ow l y t u r n i n g over phase. H a l f - l i f e of GH i n the the s l ow ly t u r n i n g over phase ranged from 34 - 49 min. The MCR of GH i n s tee r s ranged from 18.3 - 23.2 1/h (Table 31) . There was no s i g n i f i c a n t (P > .05) d i f f e r e n c e i n the MCR of GH i n s tee r s from d i f f e r e n t treatment groups. However, MCR was n o n - s i g n i f i c a n t l y l e s s i n DES (64 vs 73 ml/h/kg) and h igher i n Zerano l (83 vs 73 ml/h/kg) - 168 -F igure 22. The disappearance of GH from the plasma f o l l o w i n g the intravenous i n j e c t i o n of 6 to 8 mg of bovine GH (NIH-GH-B18). Arrow i n d i c a t e s the time of GH i n j e c t i o n . Basa l GH concen t ra t i on i s i n d i c a t e d by the dotted l i n e . - 169 -Table 28. Kinetic parameters of GH metabolism in growing steers Kinetic Parameters Mean ± S. .E.1 H a l f - l i f e (min) Fast component (T 1/2 a) 5.02 + 0.78 Slow component (T 1/2 g) 41.81 + 3.06 Fractional Turnover Rate (min - 1) Fast component (a) 0.1674 + 0.0297 Slow component (3) 0.0172 + 0.0013 Volume of distribution (% of body weight) 6.11 + 0.59 Metabolic Clearance Rate li t e r s / h 20.68 + 2.09 ml/h/kg body weight ml/h/kg * body weight 74.47 + 6.36 303.85 + 27.03 Secretion Rate Pg/h 252.14 + 27.72 ug/h/kgQ 7 5 ug/h/kg * body weight 0.91 + 0.09 3.70 + 0.37 m^ean ± S.E. of values obtained from 8 steers - 170 -Table 29. F i r s t - o r d e r r a t e constants f o r d i s t r i b u t i o n and e l i m i n a t i o n of plasma GH i n s t e e r s 1 Treatment F i r s t - O r d e r Rate Constants 1 k l 2 k 2 l kio min _1 C o n t r o l 0.06800 ± 0.00215 2 0.05400 ± 0.00896 0.04189 ± 0.00610 DES 0.08762 ± 0.04375 0.05161 ± 0.01463 0.10130 ± 0.07246 Zerano l 0.06800 ± 0.00215 0.12891 ± 0.06721 0.03617 ± 0.00453 Synovex-S 0.02132 ± 0.00468 0.02465 ± 0.00454 0.02899 ± 0.00427 1 C a l c u l a t e d from the two compartment model. k^2 " F i r s t - o r d e r r a te constant f o r the d i s t r i b u t i o n of GH from the c e n t r a l to the p e r i p h e r a l compartment. k 2 1 - F i r s t - o r d e r ra te constant f o r the d i s t r i b u t i o n of GH from the p e r i p h e r a l to the c e n t r a l compartment. kio - F i r s t - o r d e r r a t e constant f o r the e l i m i n a t i o n of GH from the c e n t r a l compartment. 2Mean ± standard e r r o r . *p < .05 compared to c o n t r o l . - 171 -Table 30. H a l f - l i f e and f r a c t i o n a l d isappearance r a t e of i n j e c t e d GH i n s t ee r s Fast Component Slow Component Treatment H a l f - L i f e (min) F r a c t i o n a l Turnover Rate ( m i n - 1 ) («> H a l f - L i f e (min) F r a c t i o n a l Turnover Rate ( m i n - 1 ) (3) C o n t r o l 4.78 0.1460 49.00 0.0143 ± 0.002 ±0 .37 1 ±0.011 ±5.18 DES 3.64 0.2219 41.50 0.0167 ± 0.001 ±1.37 ±0.084 ±2.35 Zerano l 3.65 0.2145 34.65 0.0204 ± 0.003 ±1.24 ±0.073 ±4.80 Synovex-S 8 .02* " 0 .0873* " 42.07 0.0175 ± 0.004 ±0.80 ±0.009 ±10.19 ^mean ± standard e r r o r *P < .05 compared to c o n t r o l - 172 -Table 31. Me tabo l i c c learance and s e c r e t i o n r a te s of GH i n s teer s on 20 days f o l l o w i n g the i m p l a n t a t i o n of anabo l i c compounds Metabo l i c C learance Rate S e c r e t i on Rate Treatment 1/h ml/h/kg m l / h / k g 0 ' 7 5 ug/h ug/h/kg /wi. ° '7 5 Ug/h/kg C o n t r o l 18.51 72.64 290.16 135.01 0.53 2.12 ± 2 .07 1 ± 4.30 ± 20.90 ± 11.05 ±0.02 ±0.09 DES 18.33 63.95 262.90 290.24*" 1.04* 4.25* ±8.22 ±23.40 ±101.80 ±50.88 ±0.08 ±0.44 Zerano l 22.74 83.30 338.60 299.60* 1.10* 4.48* ±4.90 ±16.90 ±69.90 ±14.30 ±0.07 ±0.27 Synovex-S 23.15 77.98 323.70 283.72* 0.955** 3.96* ±2.50 ±8.90 ±36.20 ±13.60 ±0.04 ±0.17 Mean ± s tandard e r r o r . P < .05 compared to c o n t r o l . * * P < .01 compared to c o n t r o l . S ign i n d i c a t e s s t a t i s t i c a l s i g n i f i c a n c e when the c a l c u l a t e d t va lue was compared w i t h t abu l a r t va lue ( s i g n cons idered) (Table A . 3 , S t e e l and T o r r i e , 1960). - 173 -and Synovex-S (78 vs 73 ml/h/kg) than i n the c o n t r o l s t e e r s . A s i m i l a r change i n MCR of GH was observed i n s tee r s on day 40 f o l l o w i n g the i m p l a n t a t i o n (Table 32) . The s e c r e t i o n ra te of GH was c a l c u l a t e d as the product of MCR and ba s a l GH concen t r a t i on s . The s e c r e t i o n r a te of GH was h igher i n s t ee r s implanted w i t h anabo l i c compounds than i n the c o n t r o l s . (P < .05, C o n t r o l , 135 ug/h vs a l l the anabo l i c compounds combined, 291 ug/h). The GH s e c r e t i o n r a t e (ug/h) was about 115, 122 and 101 per cent h i ghe r i n s t ee r s implanted w i t h DES, Zerano l and Synovex-S r e s p e c t i v e l y , than the c o n t r o l s and the d i f f e r e n c e s among anabo l i c compounds were not s i g n i f i c a n t (P > .05) . The r e l a t i v e changes i n GH s e c r e t i o n r a te and MCR, both expressed per kg metabo l i c body s i z e , are presented i n F i gu re 23. DISCUSSION The plasma GH concen t r a t i on and the k i n e t i c parameters of GH metabol ism i n s teer s implanted w i t h anabo l i c compounds were s tud ied i n t h i s exper iment. Plasma GH Concentration l n Steers and the Effect of  Anabolic Compounds Plasma GH was measured from the samples c o l l e c t e d at 0730 h and 1400 h and not from hour l y samples. The e p i s o d i c nature of GH s e c r e t i o n i n ruminants (Anf inson et a l . , 1975; Davis et a l . , 1977a) makes i t ve ry d i f f i c u l t to ob t a i n r e p r e s e n t a t i v e samples and the de te rminat i on of GH from s i n g l e samples could thus be c r i t i c i z e d . Due to the d i f f i c u l t y i n ana l y s i ng the l a r ge number of samples generated i n s t ud i e s as repor ted Table 32. H a l f - l i f e , f r a c t i o n a l disappearance r a t e and metabo l i c c learance ra te of GH i n s teers on AO days f o l l o w i n g the imp l an ta t i on of anabo l i c compounds Fast Component Slow Component Me tabo l i c C learance Rat (MCR) Treatment H a l f - l i f e ( t 1/2 a , min) F r a c t i o n a l Turnover Rate ( a , per min) F r a c t i o n a l H a l f - l i f e Turnover ( t 1/2 0, Rate (6, per 1/h min) min) (MCR) ml/h/kg m l / h / k g 0 , 7 5 Body-weight Body-weight (MCRK) (MCRM) C o n t r o l DES 4.55 ± 0.22 1 3.05 ±0.10 0.1527 ± 0.007 0.2276" ±0.007 34.44 ±1.30 37.9 ±0.93 0.0201 ±0.001 0.0182 ±0.0004 22.51 ±0.39 16.96 ±3.16 68.93 ±0.20 51.39 ±8.50 292.79 ±1.96 219.00 ±37.52 Zerano l Synovex-S 8.02 ±0.13 4.44'' ±0.36 **,tt 0.0864 ±0.001 0.1572 ±0.013 *,tt t,+ 43.10 ±2.32 40.77 ±3.30 0.0161 ±0.001 0.0171 ±0.001 24.30 ±4.00 23.83 ±3.80 71.18 ±9.95 74.08 ±7.14 305.92 ±44.76 313.60 ±35.17 mean ± standard e r r o r . *>**P < .05, P < .01 r e s p e c t i v e l y , compared to c o n t r o l . " r»'l" rP < .05, P < .01, r e s p e c t i v e l y compared to DES. "hp < .05 compared to Ze rano l . - 1 7 5 -400 300 METABOLIC CLEARANCE RATE 200 \ \ E 100 • • • • • • • • • • • • DO » OC x* • • • • l& • • • • • • • • • • V . • • • • V . • • • •Pt Jr\y. • • • • CONTROL ZERANOL DES SYNOVEX-S 5 r SECRETION RATE Figure 23. Metabolic clearance and secretion rates of GH in steers implanted with anabolic compounds. Each bar represents mean ± SE of values obtained from two steers. - 176 -he re , I t becomes d i f f i c u l t to f o l l o w the s e r i a l b lood sampling methodology (Davis et a l . , 1977a; Ohlson et a l . , 1981) to o b t a i n s e c re to r y pat terns of GH. I n s tead , blood samples were c o l l e c t e d from s tee r s at 0730 h and 1400 h on each sampling pe r i od and the average concen t r a t i on of GH from these two samples was taken to represent ba sa l GH c o n c e n t r a t i o n . Recen t l y , Donaldson et a l . (1981) measured GH i n s i n g l e samples c o l l e c t e d around 0900 - 1000 h and argued that a l o g - t r a n s f o r m a t i o n of the r e s u l t s would suppress o u t l y i n g va lues and the va lues thus obta ined were r e p r e s e n t a t i v e of ba sa l GH l e v e l s . S ince a s i m i l a r procedure w i t h sampling both at morning (0730 h) and a f te rnoon (1400 h) f o l l owed by the l o g - t r an s f o rma t i on of the mean concent ra t i ons was performed, the va lues repor ted i n Table 27 are cons idered to be r e p r e s e n t a t i v e of ba sa l GH concen t ra t i on of the s t e e r s . Mean plasma GH concen t ra t i on f o r c a t t l e repor ted i n t h i s experiment was g rea te r than that repor ted by Davis and h i s co-workers (Davis et a l . , 1977a; Ohlson et a l . , 1981). Whereas the method of sample c o l l e c t i o n as d i scussed above could have played a r o l e , i t should be mentioned that the plasma GH concen t ra t i on was obta ined from the radioimmunoassay us ing NIAMDD-oGH-I-1 as the s tandard . A l a r ge v a r i a t i o n i n the plasma GH of c a t t l e and sheep has been repor ted i n the l i t e r a t u r e . Mean GH va lues obta ined i n t h i s study have to be compared w i t h the va lues of 3.5 - 5.2 ng/ml i n c a t t l e (Ohlson elt_ a l . , 1981), 3.6 - 5.7 ng/ml i n wether lambs (Olsen et a l . , 1977), 5 - 2 5 ng/ml i n wether lambs (Donaldson et a l . , 1981), l e s s than 3.07 ng/ml i n lambs (Wiggins et a l . , 1976) up to 25 ng/ml i n s teer s (Sharp and Dyer, 1971), more than - 177 -40 ng/ml i n c a t t l e (Dean et a l . , 1982) and 19 - 37 ng/ml i n c a t t l e ( T renk le , 1970a). These d i s c repanc i e s i n the l i t e r a t u r e on plasma GH l e v e l s i n ruminants suggest that the r e l a t i v e changes i n the concen t ra t i on r a the r than the abso lute va lues should be the c r i t e r i o n f o r comparison between the t reatment s . Effect of Anabolic Compounds on Plasma GH Concentration Plasma l e v e l s of GH were s i g n i f i c a n t l y e leva ted i n s tee r s implanted w i t h anabo l i c compounds and the r e s u l t s e s t a b l i s h e d i n t h i s study support the theory of GH p a r t i c i p a t i o n i n t h e i r mechanism of growth promotion (Bu t te r y et a l . , 1978; P res ton 1975). The plasma GH concen t r a t i on was s i m i l a r to the growth response (F igure 3, Expt. IA) obta ined due to the imp l an t a t i o n of anabo l i c compounds used i n t h i s s tudy. These observat ions suggest that GH along w i t h other hormonal f a c t o r s i n the plasma (somatomedins, p r o l a c t i n , i n s u l i n ) could have played a s i g n i f i c a n t r o l e i n enhancing the growth ra te of s t e e r s . Increased plasma GH concen t r a t i on i n s tee r s implanted w i t h DES ( T r enk l e , 1976b), Zeranol (Borger et a l . , 1973; Sharp and Dyer, 1971) and i n lambs implanted w i t h DES (Wiggins et a l . 1976) and Zeranol (Olsen et a l . , 1977) has been r epo r t ed . Olsen et a l . (1977) concluded that a ch ron i c exposure to Zeranol was necessary to i nc rease the serum concen t ra t i on of GH and i n s u l i n . The e f f e c t of Synovex-S on plasma GH concen t ra t i on i s a s i g n i f i c a n t f i n d i n g i n t h i s exper iment. Whereas no i n f o rmat i on i s a v a i l a b l e on i t s e f f e c t on the p i t u i t a r y g l and , inc reased growth r a t e (Kahl et a l . , 1978) and p r o t e i n r e t e n t i o n (Rumsey, 1982) f o l l o w i n g i t s - 178 -i m p l a n t a t i o n i n s t ee r s would i n d i r e c t l y suggest the p a r t i c i p a t i o n of endogenous anabo l i c hormones, such as GH and i n s u l i n , i n i t s mechanism of a c t i o n . Thus the inc reased plasma GH and growth r a t e (Expt . IA) of s tee r s implanted w i t h Synovex-S demonstrated In t h i s experiment p rov ide a b i ochemica l mechanism of a c t i o n of Synovex-S i n ruminants. Be s ides , i n ruminants, Synovex-S seems to exe r t a s i g n i f i c a n t i n f l u e n c e on the t h y r o i d g land (Kahl et a l . , 1978; Expt . I IA: 1 and 2 ) . The i n t e r a c t i o n of GH and t h y r o i d hormones i n r e g u l a t i n g growth r a t e and p r o t e i n d e p o s i t i o n i n sheep has been emphasized by Wagner and Veeinhuzen (1978) and Trenk le (1981). Increased GH s e c r e t i o n observed i n s t ee r s f o l l o w i n g the i m p l a n t a t i o n of DES, Synovex and Zerano l i s i n genera l agreement w i t h the e f f e c t of e s t r ogen i c anabo l i c compounds on the p i t u i t a r y g land weight (Burgess and Lamming, 1960; Clegg and Co l e , 1954; Wiggins et a l . , 1978; Wiggins et a l . , 1979). In male r a t s a s i n g l e dose of s t i l b e s t r o l p rop ionate has been shown to i nc rea se p i t u i t a r y m i t o t i c a c t i v i t y , p i t u i t a r y weight and serum GH (L loyd et a l . , 1971). Thus the i nc reased plasma GH f o l l o w i n g the i m p l a n t a t i o n of Ze r ano l , DES and Synovex-S l n s t ee r s could be due to the r e s u l t of i t s i nc reased s e c r e t i o n from the p i t u i t a r y . The extent to which anabo l i c compounds i n f l u e n c e MCR of GH i n s t ee r s i s not c l e a r l y known. GH Kinetics The k i n e t i c parameters of GH metabol ism i n s t ee r s were determined by a s i n g l e , bolus i n j e c t i o n k i n e t i c procedure (us ing 6 mg of N1H-GH-B 18). Though a l a r ge i n j e c t i o n dose was employed i n t h i s - 179 -experiment s t ud i e s i n r a t s (Frohman and Be rna rd i s , 1970) and humans (Tay lo r et a l . , 1969) have shown that the metabo l i c c learance of GH i s independent of the amount of GH i n j e c t e d over a wide range of dose. A s i m i l a r l a c k of d i f f e r e n c e i n the c learance of GH between d i f f e r e n t doses of GH i n j e c t e d was repor ted f o r mice (S inha et a l . , 1979) and sheep ( T renk l e , 1976a). The dose of GH used i n t h i s experiment was s i m i l a r to that used by Trenk le (1977) i n s t e e r s . The mathematical treatment of the plasma GH disappearance curve, however, was d i f f e r e n t from the procedure used by T renk l e , (1976a). In both sheep (T renk le , 1976a) and c a t t l e ( T renk l e , 1977), the turnover r a te of the GH poo l and o ther k i n e t i c parameters was c a l c u l a t e d from the data obta ined up to 40 min post GH i n j e c t i o n . However, i n t h i s s tudy, va lues obta ined up to 120 min post GH i n j e c t i o n were used to c a l c u l a t e GH k i n e t i c parameters by f i t t i n g the disappearance curve to a two-compartment model. The i n j e c t e d GH has been shown to e q u i l i b r i a t e i n two compartments i n s tee r s ( T renk l e , 1977) and i n multicompartments i n mice (S inha et a l . , 1979) man (Bauman, 1979), r a t s (Frohman and B e r n a r d i s , 1970) and i n sheep (Wal lace et a l . , 1972). I n s tead , u s i ng 1 2 5 I - G H , Yousef et a l . (1969) have repor ted a constant disappearance of r a d i o a c t i v i t y from the c i r c u l a t i o n i n cows. In s p i t e of the d i f f e r e n c e s i n the approaches used, the ca l cua ted va lues GH s e c r e t i o n and c learance ra te s obta ined i n t h i s experiment were s i m i l a r to those repor ted by Trenk le (1977) f o r s t e e r s . GH Kinetics: Effect of Anabolic Compounds The r e s u l t s of t h i s study demonstrate the i nc reased s e c re to r y - 180 -a c t i v i t y of the p i t u i t a r y g land of s tee r s i n response to anabo l i c compound imp lan ta t i on s and suggest that the i nc reased plasma GH concen t r a t i on observed i n the e a r l i e r experiment (F igure 21) and that repor ted by others ( T renk l e , 1976b; Borger et a l . , 1973; Olsen et a l . , 1977) i s indeed due to an i nc reased GH s e c r e t i o n from the p i t u i t a r y g l and . The MCR of GH i n the implanted and c o n t r o l s t ee r s was found to be s i m i l a r (P > .05) . Thus i t appears that the i nc rea sed plasma GH observed i n s t ee r s implanted w i t h anabo l i c compounds i s more due to a f u n c t i o n of i t s s e c r e t i o n r a the r than c learance from the c i r c u l a t i o n . Bes ides a l a c k of d i f f e r e n c e i n the MCR of GH between the implanted and c o n t r o l s t ee r s on day 40 f o l l o w i n g the i m p l a n t a t i o n (Table 32) tends to con f i rm the above hypo thes i s . T renk le (1981) r e c e n t l y concluded that the changes i n the concen t r a t i on of hormone i n the plasma were i n f l u enced more by s e c r e t i o n than by c l e a r ance . F u r t h e r , from two independent s t ud i e s Sinha and h i s co-workers have demonstrated that the i nc rea sed plasma GH concen t ra t i on i n the pregnant mice (S inha et a l . , 1974) i s more a f u n c t i o n of s e c r e t i o n r a te wi thout much change i n MCR (S inha et a l . , 1979). In the r a t s , S t ro s se r and M ia lhe (1975) repor ted tha t the MCR i s independent of serum GH concen t r a t i on and that the change i n serum GH depends p r i m a r i l y on i t s s e c r e t i o n r a t e from the p i t u i t a r y g l and . The r e s u l t s of the present experiment are i n l i n e w i t h the f i n d i n g s of the above works and suggest, that s e c r e t i o n r a te of GH i s the pr imary k i n e t i c parameter i n f l u e n c e d by anabo l i c compounds i n ruminants . Growth hormone s e c r e t i o n r a te was s i m i l a r (P > .05) among the - 181 -a n a b o l i c compounds used i n t h i s exper iment, however, marked d i f f e r e n c e s i n t h e i r growth promoting p r o p e r t i e s were repor ted e a r l i e r ( F i gu re 3, Expt . I A ) . The k i n e t i c parameters of GH metabol ism was obta ined on day 20 f o l l o w i n g the i m p l a n t a t i o n of a nabo l i c compounds. I t i s l i k e l y that i n the e a r l y phases of hormone i m p l a n t a t i o n , the anabo l i c compounds are e q u a l l y e f f e c t i v e i n s t i m u l a t i n g GH s e c r e t i o n from the p i t u i t a r y g l and . The r e s u l t s a l s o suggest that complex processes such as animal growth, might I nvo lve the i n t e r a c t i o n of a number of hormonal f a c t o r s r a t h e r than the e f f e c t of e i t h e r one a lone. In sheep, Wagner and Veeinhuzen (1978) repor ted that t h y r o i d hormones may have been l i m i t i n g when growth r a t e was i nc reased w i t h exogenous growth hormone. Thus the decreased t h y r o i d hormone s t a t u s i n the Ze rano l implanted s tee r s as observed i n the e a r l i e r experiments (Expt. I IA, page 117) tends to e x p l a i n the decreased growth response obta ined due to i t s i m p l a n t a t i o n compared to Synovex-S and DES i n s p i t e of i t s comparable GH s e c r e t i o n r a t e i n s tee r s implanted w i t h the l a t t e r compounds. A low c o r r e l a t i o n between T4 and GH s e c r e t i o n r a te on day 20 f o l l o w i n g the i m p l a n t a t i o n a l so tends to i n d i c a t e the i n t e r a c t i o n between the two hormones (GH s e c r e t i o n r a t e , ug/h/kg0.75 V s t hy rox i ne s e c r e t i o n r a t e , ug/d/kg0.75 ) r = 0 # 7 1 > P = o.05, n - 8 ) . CONCLUSION The i m p l a n t a t i o n of e s t r o gen i c anabo l i c compounds r e s u l t e d i n an i n c rea se i n the GH concen t ra t i on i n s t e e r s . The growth promoting p r o p e r t i e s of Synovex-S was a s soc i a ted w i t h an inc reased plasma GH c o n c e n t r a t i o n . Thus the r e s u l t s of t h i s study demonstrate f o r the f i r s t - 182 -time the e f f e c t of Synovex-S on the p i t u i t a r y g l and . Implantat ions of DES and Synovex-S r e s u l t e d i n h igher plasma GH concen t r a t i on i n s t ee r s than Ze r ano l . In the second p a r t , a ba s i s f o r the changes i n the plasma GH s t a t u s i n the implanted s tee r s was e s t a b l i s h e d . In s t e e r s , the i n j e c t e d GH was e l im i na t ed i n a mult icompartmental f a s h i o n , e x h i b i t i n g a h a l f - l i f e of 5 min i n the f a s t and 42 min i n the slow phases of the disappearance curve. The r e s u l t s I nd i ca ted inc reased GH s e c r e t i o n r a t e i n s t ee r s implanted w i t h anabo l i c compounds. The MCR of GH was not i n f l u e n c e d by anabo l i c compound i m p l a n t a t i o n s . In the e a r l y per iods a f t e r the i m p l a n t a t i o n (20 days) a l l the anabo l i c compounds were found to be e q u a l l y e f f e c t i v e i n s t i m u l a t i n g p i t u i t a r y GH s e c r e t i o n . I t i s concluded that the e s t r o gen i c anabo l i c compounds mediate t h e i r growth promoting p r o p e r t i e s through the changes i n the endogenous GH s t a t u s . Such changes i n tu rn are a s soc i a ted w i t h i nc reased GH s e c r e t i o n r a t e w i t h l i t t l e changes i n the MCR. - 183 -EXPERIMENT I I C EFFECT OF ANABOLIC COMPOUNDS ON THE KINETIC PARAMETERS OF INSULIN METABOLISM IN GROWING STEERS INTRODUCTION I n s u l i n p lay s an important r o l e i n an imal growth and metabol i sm. I t s e f f e c t on growth r a t e and feed convers ion e f f i c i e n c y i n c a t t l e (Ma r t i n o v i c h and Ma rgo l i n , 1975), growth and p r o l i f e r a t i o n of many c e l l types i n v i t r o ( S t r au s , 1981), p a r t i t i o n i n g of n u t r i e n t s f o r t i s s u e growth ( P r i o r and Smith, 1982) and p r o t e i n s ynthes i s (Manchester, 1972) have been w e l l documented. The growth promoting p r o p e r t i e s of e s t r o gen i c anabo l i c compounds are proposed to be mediated through changes i n the endogenous GH and i n s u l i n concent ra t i on s (Bu t te ry et^ a l _ . , 1978; P r e s t on , 1975; T renk l e , 1976b). In accordance w i t h the proposed hypo the s i s , i nc reased plasma i n s u l i n concent ra t i on s i n animals f o l l o w i n g the i m p l a n t a t i o n of DES (Davis et a l . , 1970; T renk l e , 1970a), Zerano l (Olsen et_ a l . , 1977; Sharp and Dyer, 1970; Wangsness et a l . , 1981) and e s t r a d i o l (Donaldson et a l . , 1981) have been r epo r ted ; but e i t h e r the mechanism f o r such changes i n i n s u l i n s ta tus or the a l t e r a t i o n i n the k i n e t i c parameters of i n s u l i n metabolism i n animals was not c l e a r l y demonstrated. Th i s experiment was, t h e r e f o r e , conducted to study the k i n e t i c parameters of I n s u l i n metabol ism i n s tee r s implanted w i t h anabo l i c compounds i n order to f u r t h e r understand the mechanism(s) by which they a l t e r plasma i n s u l i n s t a tu s i n s t e e r s . - 184 -MATERIALS AND METHODS Animals E i gh t Hereford s teer s were used i n t h i s experiment to study the e f f e c t of anabo l i c compounds on the k i n e t i c parameters of i n s u l i n metabol i sm. The animals were s e l e c t ed from a group o f 24 s t ee r s that were used i n Expt . I I A :2 . The s tee r s used i n t h i s study represented a l l the four treatment groups ( C o n t r o l , Ze rano l , DES and Synovex-S) desc r ibed i n Expt I IA: 2. The method of anabo l i c compound i m p l a n t a t i o n , f eed ing procedure and the p repa ra t i on of animals f o r i n s u l i n k i n e t i c s tud ie s were desc r ibed i n Expt . I IA: 2. K i n e t i c parameters were est imated i n s tee r on day 20 f o l l o w i n g the i m p l a n t a t i o n of anabo l i c compounds. Experimental Procedure Preparation of Insulin for Kinetic Studies: Bovine i n s u l i n (Sigma, Lot # 49 C - 0197; 23.6 I.U./mg) was c a r e f u l l y weighed and d i s s o l v e d i n 0.9% NaCl at a concen t r a t i on of 1 mg/ml. One ml a l i q u o t s of t h i s s tock s o l u t i o n were s to red i n 1 cc sy r inges at -20°C and were used i n the k i n e t i c exper iments . Kinetic Procedure A s i n g l e bolus i n j e c t i o n of non - r ad i oac t i v e i n s u l i n k i n e t i c procedure was used (Madigan and Evans, 1973). On the day of the exper iment, the s tee r s were weighed, fed the morning p o r t i o n of the r a t i o n and were p laced i n the metabolism c ra te s which a l lowed some freedom of movement but prevented the s teer s from tu rn i n g around. The - 185 -animals were a l lowed to remain i n the c r a t e f o r an hour before two blood samples at 10-15 min i n t e r v a l were c o l l e c t e d to o b t a i n ba s a l i n s u l i n l e v e l . The s tee r s were then i n j e c t e d w i t h 1 mg of bovine i n s u l i n (one sy r inge as desc r ibed above) r a p i d l y through the j u g u l a r v e i n c a t h e t e r . The c a the te r was f l u shed immediately w i t h 0.9% s a l i n e . In order to measure the disappearance of i n j e c t e d i n s u l i n s e r i a l blood samples were c o l l e c t e d at 5, 10, 15, 20, 25, 30, 35, 40, 50 and 60 min a f t e r i n j e c t i n g the hormone. The blood samples were kept i n i c e i n h e p a r i n i z e d tubes and were c e n t r i f u g e d immediately to o b t a i n the plasma. The plasma was s to red at -70°C. Two s t e e r s , r ep re sen t i ng one treatment group were s tud ied s imu l taneous l y . Analytical Procedure Insulin Plasma i n s u l i n concent ra t i ons were determined by a h i g h l y s p e c i f i c double ant ibody radioimmunoassay procedure desc r ibed by Hales and Randle (1963) w i t h s l i g h t m o d i f i c a t i o n s u s ing an i n s u l i n radioimmunoassay package (Amersham Co., O a k v i l l e , O n t a r i o , Canada; Code # IM 78) . The reagents were added to d i spo sab le po l y s t y rene tubes (12 x 75 mm) i n the f o l l o w i n g sequence: 100 y l phosphate b u f f e r or bovine plasma or app rop r i a te amount of i n s u l i n standard (0 to 80 yU of bovine i n s u l i n / m l ) and 100 y l a l i q u o t of i n s u l i n b i nd i ng reagent ( gu inea -p i g ant i serum to I n s u l i n p r e c i p i t a t e d w i t h r a b b i t a n t i - g u i n e a p i g serum). The tubes were incubated at 2 - 4°C and 45 min a f t e r the a d d i t i o n of i n s u l i n b i nd i ng reagent, 100 y l of ^ ^ I - I n s u l i n was dispensed to each - 186 -tube. The tubes were vo r t ex mixed and incubated at 2 - 4°C. A f t e r 2 h of f u r t h e r i n c u b a t i o n , 700 y l of phosphate b u f f e r was added; the tubes were vo r t ex mixed and cen t r i f u ged at 1500 x g f o r 25 min i n a r e f r i g e r a t e d c e n t r i f u g e (Beckman Model T J6 ) . The s o l u t i o n c on t a i n i n g the f r e e 1 J I - i n s u l i n was decanted and the r a d i o a c t i v i t y a s soc i a ted w i t h the p r e c i p i t a t e was counted i n an automat ic gamma counter (Model 1185, Nuc l ea r -Ch i cago ) . I n s u l i n concen t r a t i on i n the plasma and the assay c o e f f i c i e n t of v a r i a t i o n s were c a l c u l a t e d as desc r ibed i n the T4 radioimmunoassay (Expt . I IA: 2 ) . The i n t e r - and i n t r a a s s a y c o e f f i c i e n t s of v a r i a t i o n were 5% and 10%, r e s p e c t i v e l y , f o r a sample w i t h a mean concent ra t i ono f 15.2 yU/ml. Analysis of Kinetic Data The plasma i n s u l i n disappearance curve (0 to 60 min) was f i t t e d to an one compartment model. The model d e s c r i p t i o n has been presented e a r l i e r (See T4 K i n e t i c s , Expt . I IA: 2 ) . Calculations The c a l c u l a t i o n s of i n s u l i n turnover r a t e , b i o l o g i c a l h a l f - l i f e , volume of d i s t r i b u t i o n , metabo l i c c learance r a t e and s e c r e t i o n r a te were c a r r i e d - o u t u s i ng the equat ions desc r ibed e a r l i e r (See T4 K i n e t i c s Expt . I IA: 2 ) . The k i n e t i c parameters were averaged and expressed as mean ± SE. The treatment d i f f e r e n c e s were te s ted by S tudent ' s t - t e s t ( S t e e l and T o r r i e , 1960). - 187 -R E S U L T S The disappearance of i n s u l i n from the plasma f o l l o w i n g the int ravenous i n j e c t i o n i s presented i n F i gu re 24. Immediately f o l l o w i n g the i n j e c t i o n , i n s u l i n concen t ra t i on i n the plasma inc reased to about 300 - 400 uU/ml and g r adua l l y decreased. The k i n e t i c parameters of i n s u l i n metabolism are summarized i n Table 33. Mean ba sa l concen t ra t i on of i n s u l i n of the s teer s used i n t h i s study was 16.2 uU/ml. I n s u l i n was r a p i d l y c l ea red i n s teer s w i t h a h a l f - l i f e of 13.7 min and a f r a c t i o n a l turnover r a t e of 0.0505 m i n - 1 . The metabo l i c c learance and s e c r e t i o n r a t e s , of i n s u l i n expressed per kg body we ight , were 13 ml per min and 208 uU per min, r e s p e c t i v e l y . The e f f e c t of anabo l i c compounds on the k i n e t i c parameters of i n s u l i n metabolism i n s teer s i s presented i n Table 34. The h a l f - l i f e of i n s u l i n i n the plasma was not a f f e c t e d e i t h e r by Zerano l or DES i m p l a n t a t i o n s . However, there was a s i g n i f i c a n t (P < .05) r educ t i on i n the h a l f - l i f e of i n s u l i n In s tee r s due to Synovex-S i m p l a n t a t i o n when compared to the c o n t r o l s t e e r s . There was a corresponding i nc rea se i n the f r a c t i o n a l turnover ra te (11 per cent h igher than c o n t r o l , P < .05) of i n s u l i n i n s teer s due to the i m p l a n t a t i o n of Synovex-S. Though not s i g n i f i c a n t l y d i f f e r e n t (P > .05) from the c o n t r o l s t e e r s , f r a c t i o n a l turnover ra te of i n s u l i n was s l i g h t l y e levated i n s tee r s implanted w i t h Zerano l or DES. The volume of d i s t r i b u t i o n of i n s u l i n i n s teer s implanted w i t h Zerano l was about 20 per cent h igher (P < .05) than the unimplanted c o n t r o l s tee r s and about 25 per cent h igher than the s teer s implanted - 188 -F i gu re 24. The disappearance of i n s u l i n from the plasma of s teer s f o l l o w i n g a s i n g l e I.V. i n j e c t i o n of one mg of bovine i n s u l i n . Arrow i n d i c a t e s the time of hormone i n j e c t i o n . Basa l i n s u l i n concen t ra t i on i s i n d i c a t e d by the dotted l i n e . - 189 -Table 33. K i n e t i c parameters of i n s u l i n metabolism i n s tee r s K i n e t i c Parameters Mean + SE 1 H a l f - l i f e (min) 13.72 + 0.34 F r a c t i o n a l Turnover Rate (min - 1 ) 0.0505 + 0.001 Volume of D i s t r i b u t i o n (ml/kg) 249.77 + 9.16 Me tabo l i c Clearance Rate (MCR) ml/min/kg body weight 12.69 + 0.55 ml/min/kg 0 , 7 5 body weight 51.62 + 2.40 S e c r e t i on Rate (SR) u U/min/kg body weight 207.73 + 16.87 u U/min/kg 0 , 7 5 body weight 3.08 + 0.25 mean ± SE of va lues obta ined from 8 s t e e r s . Table 34. Kinetic parametes of Insulin metabolism in steers Implanted with anabolic compounds Fractional Biological Turnover Half-Life Rate Treatment Control (min) Metabolic Clearance Volume of Rate Dlstribu- _ — tlon Secretion Rate Basal Insulin min" (ml/kg) ml/min/kg ml/min/kg0'75 uU/min/kg uU/min/kg0,73 u H/ml 14.83 0.0467 234.99 10.98 43.84 ± 0.001 ±.000004 ±10.9 ± 0.51 ±2.60 151.83 ± 31.27 2.37 ±0.40 13.73 ± 2.21 DES 13.32 ±1.07 0.0524 ±0.004 224.11 ±10.78 11.69 ±0.38 47.61 ±0.36 215.07 ± 7.60 3.20 ±0.35 17.72 ±0.73 Zeranol 13.60 ±0.46 0.0510 ±0.007 280.07 ±10.01 *,t 14.27 ±0.02 57.96 ±0.15 '.tt 263.57 ± 4.18 *".t 3.94 ±0.01 18.47 ±0.19 Synovex-S 13.14 ±0.18 0.0519 ±0.001 259.89 ±10.74 13.77 ±0.7 57.06 ±2.69 *,t 200.44 ± 23.96 2.80 ±0.35 14.8 ±0.70 mean ± SE from 2 steers. 20 days following Implantation. *P < .05 compared to control. t.ttP < .05 compared to DES. *P < .05 compared to Zeranol "Sign indicates statistical significance when the calculated t value was compared with tabular t-value (sign considered) (Table A.3, Steel and Torrie, 1960). - 191 -w i t h DES. Volume of d i s t r i b u t i o n of i n s u l i n was not a f f e c t e d by e i t h e r DES or Synovex-S imp lan ta t i on s when compared to the c o n t r o l s t e e r s . The MCR of i n s u l i n from the plasma was s i g n i f i c a n t l y i nc reased (P < .05) i n s tee r s implanted w i t h Zerano l and Synovex-S when compared to the c o n t r o l s t e e r s . On the con t r a r y , when compared to the c o n t r o l s t e e r s , MCR was not i n f l uenced by DES i m p l a n t a t i o n . Among the anabo l i c compounds, however, MCR ( m l / m i n / k g 0 , 7 5 ) was s i g n i f i c a n t l y reduced (P < 0.05) i n s t ee r s implanted w i t h DES. S ec re t i on ra te of i n s u l i n i n s t ee r s was c a l c u l a t e d as the product of MCR and the p r e - i n j e c t i o n ba sa l i n s u l i n c o n c e n t r a t i o n . Steers implanted w i t h anabo l i c compounds had h igher i n s u l i n s e c r e t i o n r a te than the unimplanted c o n t r o l s t e e r s . The s e c r e t i o n r a t e (uU/ml/kg) when expressed as a percent i nc rease over the c o n t r o l va lue was about 74 (P < 0 .05) , 42 (N.S . ) , and 32 (N.S.) per cent h igher i n s t ee r s implanted w i t h Z e r a n o l , DES, and Synovex-S, r e s p e c t i v e l y . The ba sa l i n s u l i n c oncen t r a t i on was s l i g h t l y h igher i n s tee r s implanted w i t h Zeranol and DES than the unimplanted c o n t r o l s t e e r s . The r e l a t i v e changes i n MCR ( m l / m i n / k g 0 , 7 5 ) and s e c r e t i o n r a te ( u U / m i n / k g 0 , 7 5 ) of i n s u l i n i n s tee r s implanted w i t h va r i ou s anabo l i c compounds are presented i n F i gu re 25. DISCUSSION The k i n e t i c parameters of i n s u l i n metabolism i n s teer s implanted w i t h anabo l i c compounds were determined us ing a n o n - r a d i o a c t i v e , bolus i n j e c t i o n k i n e t i c procedure. - 192 -IC \ C 60 r-50 E 40 E 0^  METABOLIC CLEARANCE RATE 1 +••• ••++ ++•• ++++ ++++ ++•• ••+• ••++ •••+ •••• +••• ••++ ****** *+*+ • : 5°; 3 ° • • * •••tl •••• m >od CONTROL ZERANOL DES SYNOVEX-S SECRETION RATE ID \ C E D 4 4 r 3U I I TTT+ •+•• ••++ •+•• +++• •••+ •+++ ••++ •+++ +++• ••++ ••+• •++• •••• ••+• •+•• ••+• ++++ •+++ «•••• •••• •••• t*** •••• 1 O O O O O o o F i gu re 25. Metabo l i c c learance and s e c r e t i o n r a te s of i n s u l i n i n s teer s implanted w i th anabo l i c compounds. Each bar represents mean ± SE of values obta ined from two s t e e r s . - 193 -Insulin Kinetics The use of u n l a b e l l e d or l a b e l l e d or 125j) i n s u l i n to determine k i n e t i c parameters of i n s u l i n metabolism i n man (Genuth, 1972; Orskov and Ch r i s t en sen , 1969; Sherwin et a l . , 1974; S r i v a s t a va et a l . , 1975; Stern et a l . , 1968), dog (Nava le s i et a l . 1976), horse (Madigan and Evans, 1973) r a t (Wi r th et^ a l . , 1978) and i n mice (Cresto et a l . , 1977) has been r epo r t ed . The plasma i n s u l i n disappearance curve up to 60 min p o s t - i n j e c t i o n was analyzed by an one compartment model. Both i n man and i n exper imenta l an ima l s , i n s u l i n disappearance i n the plasma has been desc r ibed by one (Cresto et a l . , 1977), two ( P i e r l u i s s i et a l . , 1978) and m u l t i e x p o n e n t i a l (Sherwin et a l . , 1974; S i l v e r s e t a l . , 1970) equat i on s . S ince the o b j e c t i v e of t h i s experiment was to study changes i n i n s u l i n k i n e t i c s f o l l o w i n g the i m p l a n t a t i o n of anabo l i c compounds, no attempt was made to develop i n s u l i n k i n e t i c models i n s t e e r s . However, when the plasma i n s u l i n disappearance up to 120 min p o s t - i n j e c t i o n was analysed mathemat ica l l y (as attempted i n few animals — data not presented) the presence of more than one exponent i a l i n the disappearance equat ion was observed and these observat ions suggest that i n f u tu re a mul t i -compartmenta l approach should be undertaken to de sc r i be i n s u l i n k i n e t i c s i n s t e e r s . The shape of the plasma i n s u l i n d isappearance up to 60 min p l o t t e d on a semi - log paper (F igure 24) c l o s e l y resembles that reported by (Madigan and Evans, 1973) i n horses up to 40 min f o l l o w i n g the i n j e c t i o n of equine i n s u l i n . H a l f - l i f e of i n s u l i n (13.7 min) f o r s teer s obta ined i n t h i s experiment i s comparable to that reported by Madigan and Evans (1973) i n - 194 -horses us ing a s i n g l e i n j e c t i o n of equine or po rc ine i n s u l i n ; but was con s i de rab l y lower than that obta ined u s i ng a cont inuous i n f u s i o n of l ^ l l - p o r c i n e i n s u l i n (Madigan and Evans, 1973). A con s ide rab le spec ie s v a r i a t i o n i n the h a l f - l i f e of i n s u l i n i n the plasma has been r epo r t ed : 4.5 min f o r ch icken (Langslow, 1976), 10 min f o r r a t s (Cres to et a l . , 1977) and between 10 to 13 min f o r man (Orskov and Ch r i s t en sen , 1969). The f r a c t i o n a l turnover r a te and the MCR of i n s u l i n c a l c u l a t e d f o r s t ee r s (Table 33) are comparable to 0.053 to 0.054 m i n " 1 and 10-13 ml/min/kg, r e s p e c t i v e l y , reported f o r dogs ( P i e r l u i s s i et a l . , 1978). S i m i l a r va lues of MCR have been reported i n dogs (15 ml/min/kg, Sherwin et^ a l . , 1974; 10.5 ml/min/kg, Nava l e s i e£ a l . , 1975). These s i m i l a r i t i e s i n i n s u l i n k i n e t i c parameters between c a t t l e and the above spec ies repor ted are of i n t e r e s t con s i de r i n g the l a r ge d i f f e r e n c e s i n the carbohydrate metabolism between the ruminants and the non-ruminants. The s e c r e t i o n r a t e of i n s u l i n , 207 uU/min/kg, c a l c u l a t e d f o r s t ee r s i n t h i s experiment i s comparable to the turnover r a te or r a te of disappearance of 290 uU/min/kg (Nava le s i et a l . , 1976) and 267 uU/min/kg ( P i e r l u i s s i et a l . , 1978) repor ted f o r normal dogs. The turnover r a t e represents the amount of i n s u l i n sec reted by the pancreas per u n i t t ime (Nava l e s i et a l . , 1976). I n s u l i n s e c r e t i o n r a t e , however, was c a l c u l a t e d from the plasma disappearance of p e r i p h e r a l l y i n t roduced i n s u l i n and the va lues presented i n Table 33 may not prov ide i n f o rma t i on about the a c t u a l p anc r ea t i c s e c r e t i o n r a t e as the p e r i p h e r a l l y i n t roduced i n s u l i n does not f o l l o w the endogenous i n s u l i n s ec re to r y - 195 -pathway. I n s u l i n sec re ted by the 3 c e l l s of the pancreas enter s the p o r t a l c i r c u l a t i o n p r i o r to mix ing i n the genera l c i r c u l a t i o n and the l i v e r forms an important s i t e f o r i t s degradat ion (Kaplan and Madison, 1959); f o r example i n dogs, about 48 per cent of the secreted i n s u l i n has been shown to be degraded by the l i v e r (Nava le s i et a l . , 1976). The measurement of i n s u l i n k i n e t i c s f o l l o w i n g the i n j e c t i o n of i n s u l i n i n t o the p e r i p h e r a l and p o r t a l v e i n has been suggested as the other a l t e r n a t i v e (Nava le s i et a l . , 1976). Effect of Anabolic Compounds The r e s u l t s of t h i s experiment demonstrate, f o r the f i r s t t ime, c e r t a i n aspects of i n s u l i n k i n e t i c s i n s tee r s f o l l o w i n g the i m p l a n t a t i o n of a nabo l i c compounds. Increased I n s u l i n s e c r e t i o n r a te s i n s t ee r s implanted w i t h Ze r ano l , DES and Synovex-S demonstrate the e f f e c t of anabo l i c compounds on the pancreas. The r e s u l t s support the genera l mechanism of a c t i o n proposed f o r e s t r ogen i c anabo l i c compounds i n ruminants (Bu t te ry et_ a l . , 1978; P r e s t on , 1975), namely the involvement of GH and i n s u l i n i n t h e i r growth promoting p r o p e r t i e s . Increased plasma i n s u l i n concen t r a t i on has been repor ted i n lambs f o l l o w i n g DES (Wiggins et a l . , 1976), Zerano l (Olsen et a l . , 1977; Wangsness et a l . , 1981), e s t r a d i o l (Donaldson et a l . , 1981) and i n s t ee r s f o l l o w i n g Zerano l (Sharp and Dyer, 1970) i m p l a n t a t i o n s . On the con t ra r y decreased plasma i n s u l i n concen t r a t i on was a l s o repor ted i n lambs implanted w i t h Zerano l (Wiggins et a l . , 1976). These e f f e c t s of e s t r ogen i c anabo l i c compounds on i n s u l i n s e c r e t i o n are s i m i l a r to the i nc reased i n s u l i n concen t r a t i on normal ly - 196 -n o t i c e d i n pregnant women ( Spe l l a cy and Goetz, 1963) and the r e s u l t s of t h i s experiment e s t a b l i s h the e f f e c t of e s t r o gen i c compounds on the pancreas. However, i t i s not apparent immediate ly , whether such an i n c rea se i n i n s u l i n s e c r e t i o n observed i n s t ee r s f o l l o w i n g the i m p l a n t a t i o n of e s t r ogen i c anabo l i c compounds i s due to a d i r e c t e f f e c t exer ted on the pancreas or due to an i n d i r e c t e f f e c t brought about by the changes i n the endogenous hormones and m e t a b o l i t e s . Both i n c a t t l e and i n sheep, plasma GH was inc reased f o l l o w i n g DES ( T r enk l e , 1976b) and Zerano l imp lan ta t i on s (Borger et a l . , 1973; Olsen et a l . , 1977; Wiggins et a l . , 1976). When the e f f e c t of estrogens on GH s e c r e t i o n (F rantz and Rabk in , 1965; P r e s t on , 1975) and the In f luence of GH on i n s u l i n s e c r e t i o n (Rab inow i t z , 1972) were taken i n t o account, i t appears that the inc reased plasma i n s u l i n s e c r e t i o n f o l l o w i n g the i m p l a n t a t i o n of e s t r o g e n i c anabo l i c compounds i s a r e s u l t of e l eva ted GH i n the c i r c u l a t i o n . A p o s i t i v e r e l a t i o n s h i p between GH and i n s u l i n s e c r e t i o n r a te s observed i n the s tee r s used i n t h i s experiment f u r t h e r supports the above hypothes i s (GH s e c r e t i o n r a t e , ug/hr/kg vs i n s u l i n s e c r e t i o n r a t e uU/min/kg; r = 0.77, P < .05, n = 8 ) . F u r t he r , among the m e t a b o l i t e s , g lucose and prop ionate have been shown to be potent s t i m u l a t o r s of p anc rea t i c i n s u l i n s e c r e t i o n i n c a t t l e and sheep (Manns et a l . , 1967; T r enk l e , 1970b). A r e l a t i o n s h i p between plasma g lucose and i n s u l i n s e c r e t i o n thus seems to e x i s t i n animals t r ea ted w i t h anabo l i c compounds. Increased plasma g lucose concen t ra t i on i n s teer s implanted w i t h DES and Zerano l (Davis et a l . , 1970; Sharp and Dyer, - 197 -1970) has a l s o been r epo r t ed . Increased s e c r e t i o n r a te of i n s u l i n i n the implanted s teer s could a l s o be due to the d i r e c t e f f e c t of anabo l i c compounds on panc rea t i c i n s u l i n s e c r e t i o n . I n t e r e s t i n g l y , the presence of est rogen receptor s i n the mammalian pancreas was demonstrated by Sandberg et a l . (1973). The e s t r o gen i c a c t i v i t y of DES and Zerano l and t h e i r a b i l i t y to i n t e r a c t w i t h the est rogen recepto r s (Dodds et a l . , 1938; H o s p i t a l et a l . , 1972; Katzenel lenbogen et a l . , 1979) i n d i c a t e p o s s i b i l i t i e s f o r the d i r e c t i n t e r a c t i o n of a nabo l i c compounds w i t h the panc r ea t i c est rogen r e c e p t o r s . In a d d i t i o n s t e r o i d s such as progesterone have been shown to i n c r ea se the a b i l i t y of p anc r ea t i c (5-cel ls to sec re te i n s u l i n (Ashby a l . , 1978). Synovex-S seems to exe r t s i m i l a r e f f e c t s on i n s u l i n metabol ism when compared to the other anabo l i c compounds s t u d i e d . Though not s i g n i f i c a n t , i t s i m p l a n t a t i o n i n s tee r s r e s u l t e d i n i nc reased i n s u l i n s e c r e t i o n r a t e when compared to the c o n t r o l s t ee r s but the r a te s were s l i g h t l y lower than i n those Implanted w i t h Zerano l or DES. Whereas the e f f e c t of Synovex-S on plasma GH and i n s u l i n has not been documented, i nc rea sed GH observed i n Synovex-S implanted s tee r s ( r e s u l t s of Expt . I IB) would i n d i c a t e a s i m i l a r mechanism (based on GH) f o r the i nc rea sed i n s u l i n s e c r e t i o n r a t e as suggested f o r DES and Ze rano l . A s i g n i f i c a n t r educ t i on i n the i n s u l i n T l / 2 and an i n c rea se i n the f r a c t i o n a l tu rnover r a t e were a l s o observed due to Synovex-S i m p l a n t a t i o n . O r a l c on t r a cep t i v e s which are s i m i l a r i n t h e i r chemical compos i t ion to Synovex-S have been shown to exer t marked a l t e r a t i o n s i n i n s u l i n - 198 -metabol ism i n p a t i e n t s ( S r i v a s t a va et a l . , 1975). But , the h a l f - l i f e of i n s u l i n was found to be i nc reased (5.6 vs 4.4 min) i n p a t i e n t s t r e a t ed w i t h o r a l c on t r acep t i ve s compared to normal c o n t r o l s . Bes ides the e f f e c t on i n s u l i n s e c r e t i o n r a t e , anabo l i c compounds a l s o seem to i n f l u e n c e the MCR of i n s u l i n . When compared to the c o n t r o l s t e e r s , MCR was e leva ted (P < .05) i n Zerano l and Synovex-S and was s i m i l a r i n DES implanted s t e e r s . The mechanism behind the inc reased i n s u l i n c learance i n Zeranol and Synovex-S implanted s tee r s i s not c l e a r l y understood. But the r e s u l t s i n d i c a t e that the plasma concen t r a t i on of i n s u l i n i s the r e s u l t of r e l a t i v e changes In both the s e c r e t i o n and c learance r a t e (F i gu re 25 f o r d e t a i l s ) . The l i v e r i s the major s i t e of i n s u l i n degradat ion (Kaplan and Madison, 1959). Thus the i n c r ea sed MCR observed i n Ze rano l and Synovex-S implanted s t ee r s cou ld be a f u n c t i o n of inc reased c a t a b o l i c a c t i v i t y of the l i v e r . I t i s p o s s i b l e that the p e r i p h e r a l s i t e s of i n s u l i n degradat ion are more a c t i v e i n s t ee r s implanted w i t h Zerano l and Synovex-S than the c o n t r o l s t e e r s . Such mechanisms, however, have been proposed f o r the inc reased MCR of i n s u l i n observed i n p a t i e n t s r e c e i v i n g o r a l c on t r acep t i ve s c o n t a i n i n g est rogen and progesterone ( S r i v a s t a va et a l . , 1975). The pa t te rn s of i n s u l i n s e c r e t i o n observed i n t h i s experiment are not c on s i s t en t w i t h the e f f e c t of the compounds on the growth r a t e of s tee r s ( F i gu re 3, Expt . I A ) . Among the anabo l i c compounds used i n t h i s exper iment, Zerano l was found to be l e s s e f f e c t i v e i n s t i m u l a t i n g the body growth of s teer s than DES or Synovex-S (F i gu re 3 ) . Yet i t s i m p l a n t a t i o n r e s u l t s i n h igher i n s u l i n s e c r e t i o n r a t e than the - 199 -i m p l a n t a t i o n of the l a t t e r compounds. These observat ions suggest that animal growth i s complex, i n v o l v i n g the i n t e r a c t i o n of a number of hormonal f a c t o r s r a t he r than the e f f e c t of a s i n g l e hormone a l one . CONCLUSION The i m p l a n t a t i o n of a nabo l i c compounds r e s u l t e d i n an e l e v a t i o n i n the r a t e of i n s u l i n sec reted from the pancreas. The a l t e r e d i n s u l i n s e c r e t i o n r a t e was suggested as a r e s u l t of changes i n the endogenous GH s t a t u s . A p o s i t i v e c o r r e l a t i o n between GH s e c r e t i o n r a te and i n s u l i n s e c r e t i o n r a t e was e s t a b l i s h e d . The p o s s i b i l i t y of a d i r e c t i n t e r a c t i o n of anabo l i c compounds w i t h the panc rea t i c estrogen recepto r s was a l s o proposed. Among the anabo l i c compounds, imp l an ta t i on s w i t h Zerano l i n f l u enced the panc rea t i c i n s u l i n s e c r e t i o n r a te to a g rea te r ex tent than w i t h DES or Synovex-S. Anabo l i c compounds have a l s o been found to i n f l u e n c e the c learance ra te of i n s u l i n from the c i r c u l a t i o n . The enhanced a c t i v i t i e s of the l i v e r and other p e r i p h e r a l s i t e s of i n s u l i n degradat ion have been proposed as p o s s i b l e mechanisms f o r the i nc rea sed metabo l i c c lea rance r a t e of i n s u l i n observed i n s t ee r s implanted w i t h Zerano l and Synovex-S. Imp lanta t i on of DES, seemed to have l i t t l e i n f l u e n c e on the metabo l i c c learance r a t e of i n s u l i n . I t i s concluded that i n c r e a s i n g the i n s u l i n s e c r e t i o n r a t e Is one of the mechanisms by which anabo l i c compounds e l i c i t growth promotion i n s t e e r s . However, changes i n the metabolism of other hormones, such as GH and t h r y r o x i n e might govern the degree of growth promotion ach ieved . - 200 -GENERAL DISCUSSION AND CONCLUSIONS Imp lan ta t i on of anabo l i c compounds r e s u l t s i n inc reased growth r a te of meat producing ruminants. The carcass from such t r e a t ed animals conta ins a h igher percentage of p r o t e i n and a lower percentage of f a t than the unt reated an ima l s . The b iochemica l mechanism by which these compounds evoke growth promotion i n domestic animals i s not c l e a r l y understood. Th i s study was conducted to i n v e s t i g a t e the metabo l i c and endocr ine e f f e c t s of anabo l i c compounds i n ruminants. From the r e s u l t of t h i s i n v e s t i g a t i o n the f o l l o w i n g conc lus ions were drawn. In Study I, the metabo l i c e f f e c t s of anabo l i c compounds were i n v e s t i g a t e d . A t t e n t i o n was focussed on the metabolism of n i t r o g e n , muscle p r o t e i n and c o l l a g e n In growing beef s t e e r s . The metabo l i c e f f e c t s are summarized i n F i gu re 26. The r e s u l t s of EXPT. IA revea led the growth promoting p r o p e r t i e s of these compounds. Implantat ions w i t h DES and Synovex-S were found to be more e f f e c t i v e i n promoting the growth of s teer s than those w i t h Ze r ano l . Implantat ions of these anabo l i c compounds r e s u l t e d i n a s i g n i f i c a n t r educ t i on i n the plasma concent ra t ions of n i t rogenous compounds, e s p e c i a l l y PUN, aAN, e s s e n t i a l and n o n - e s s e n t i a l amino a c i d s . Anabo l i c compounds appear to a l t e r s i g n i f i c a n t l y the n i t r ogen metabol ism of s tee r s by i n c r e a s i n g the e f f i c i e n c y of u t i l i z a t i o n of absorbed n i t rogenous compounds. The data suggest that these compounds reduce the hepa t i c p roduct ion of urea and Increase the a v a i l a b i l i t y of amino ac id s f o r t i s s u e uptake. Implantat ions w i t h DES and Synovex-S were more e f f e c t i v e i n a l t e r i n g n i t r ogen metabolism of s teer s than w i t h z e r a n o l . S tud ies on muscle p r o t e i n metabolism (EXPT. IB) demonstrated the NITROGEN _ METABOLISM (EXPT.IA) 1.INCREASED UTILIZATION OF ABSORBED NITROGENOUS COMPOUNDS 2.OECREASED HEPATIC DEAMI NATION 3.INCREASED AVAILABILITY OF AA FOR PROTEIN SYNTHESIS MUSCLE PROTEIN _ METABOLISM (EXPT.IB) 1.INCREASED MUSCLE PROTEIN DEPOSITION 2.INCREASED AMOUNT ANU EFFICIENCY OF MUSCLE PROTEIN SYNTHESIS COLLAGEN _ METABOLISM (EXPT.IC) 1.INCREASED AMOUNT OF CONNECTIVE T1SSUES(DES ONLY) F i gu re 26. Summary of the metabol ic O _ l o CQ < EFFECTS OF ANABOLIC COMPOUNDS IN S T E E R S UJ z o o o z Ui . PITUITARY (EXPTIIB) 1. INCREASED PLASMA 6H 2.INCREASE0 CH SECRETION RATE 3.NO CHANCE IM SH METABOLIC CLEARANCE RATE THYROID (EXPT.UA) 1. INCREASED PLASM TOTAL I FREE T* 2.INCREASED T« SECRETION RATE 3.NO CHANGE IN PLASMA T3 _ PANCREAS (EXPT.IIC) 1.INCREASED PLASMA INSULIN 2.INCREASED INSULIN SECRETION RATE endocrine e f f e c t s of anabo l i c compounds i n growing beef s t e e r s . - 202 -use fu lne s s of measuring N T - m e t h y l h i s t i d i n e i n u r i n e as a n o n - d e s t r u c t i v e , i n v i v o index of m y o f i b r i l l a r p r o t e i n degradat ion i n l a r ge animals such as c a t t l e . The r ap id growth r a te of s tee r s was accompanied by a r a p i d r a t e of muscle p r o t e i n tu rnove r . Implantat ions of anabo l i c compounds r e s u l t e d i n an Increased amount of muscle p r o t e i n depos i ted and among the anabo l i c compounds used, DES and Synovex-S were more e f f e c t i v e than Ze r ano l . A gene r a l , n o n - s i g n i f i c a n t i nc rease i n muscle p r o t e i n degradat ion occurred i n s teer s implanted w i t h these compounds. Muscle p r o t e i n d e p o s i t i o n i n the implanted s t e e r s , however, was f a c i l i t a t e d by a h igher ra te of muscle p r o t e i n s ynthes i s than deg rada t i on . The anabo l i c compounds were e f f e c t i v e i n i n c r e a s i n g the e f f i c i e n c y of p r o t e i n synthes i zed i n the s k e l e t a l muscle. Imp lan ta t i on of DES and Synovex-S were more e f f e c t i v e i n i n c r e a s i n g muscle p r o t e i n d e p o s i t i o n and the e f f i c i e n c y of s yn thes i s than that of Ze rano l . The d i f f e r e n t i a l e f f e c t s of anabo l i c compounds were suggested due to the involvement of l e s s muscle p r o t e i n degradat ion per u n i t s yn the s i s . Hydroxypro l ine excreted i n the u r i n e , decreased i n d i c a t i n g a r e d u c t i o n i n c o l l a gen turnover as the s tee r s gained weight and advanced i n matu r i t y (EXPT. I C ) . The r educ t i on i n the u r i n a r y hyd roxyp ro l i ne e x c r e t i o n w i t h advancing matu r i t y may be due to a r educ t i on i n the s yn thes i s of new co l l a gen f i b r i l s and the inc reased r e s i s t a n c e of the c o l l a g e n f i b r i l s to the breakdown by the c o l l a g e n o l y t i c enzymes. Imp lanta t i on w i t h DES caused i nc reased u r i n a r y hyd roxyp ro l i ne exc reted by the s t e e r s , wh i l e Zeranol and Synovex-S had l i t t l e e f f e c t . These d i f f e r e n t i a l e f f e c t s of anabo l i c compounds on c o l l a gen metabolism are not c l e a r l y understood at p resent , but the r e s u l t s suggest that DES - 203 -may i nc rea se the amount of co l l a gen and i t s turnover i n s t e e r s . These e f f e c t s of DES could be re spon s i b l e f o r the genera l l ower ing of carcass q u a l i t y observed by va r i ou s r e sea r che r s . In Study I I , the e f f e c t of anabo l i c compounds on the metabol ism of t h y r o x i n e , growth hormone, i n s u l i n was i n v e s t i g a t e d . The endocr ine e f f e c t s are summarized i n F i gu re 26. E s t r ogen i c anabo l i c compounds, e s p e c i a l l y DES and Synovex-S exer ted a s i g n i f i c a n t e f f e c t on the a c t i v i t y of the t h y r o i d gland (EXPT. I I A : 1 ) . An i nc rea se i n the plasma t o t a l and f r ee thy rox ine concen t ra t i on s was observed i n s tee r s implanted w i t h DES and Synovex-S. Ze r ano l , on the other hand, exer ted a d i f f e r e n t mechanism of a c t i o n as f a r as the a c t i v i t y of the t h y r o i d gland i s concerned. Plasma concen t r a t i on of thy rox ine was e i t h e r s i m i l a r or s l i g h t l y lowered i n s tee r s implanted w i t h Zerano l when compared to the c o n t r o l s . T r i i o d o t h y r o n i n e concen t ra t i on i n the plasma was not a f f e c t e d by any of the anabo l i c compounds; i t i s specu lated that i t may not be i nvo l ved i n the growth process mediated by these compounds. Th i s p o s s i b i l i t y warrants f u r t h e r i n v e s t i g a t i o n . S tud ies on the e f f e c t of anabo l i c compounds (EXPT. I IA:2) on the k i n e t i c parameters of thy rox ine metabolism revea led an inc rease and a decrease i n thy rox ine s e c r e t i o n and metabo l i c c learance r a t e s , r e s p e c t i v e l y , i n s teer s implanted w i t h DES and Synovex-S. Ze r ano l , on the con t r a r y , caused e l e v a t i o n of both the s e c r e t i o n and c learance ra tes of t h y r o x i n e . The inc reased s e c r e t i o n ra te of thy rox ine may be i n vo l ved i n the mechanisms of growth mediated by these compounds. I t i s suggested that Zerano l exe r t s a weak i n f l u e n c e on the t h y r o i d gland and - 204 -may not n e c e s s a r i l y be t h y r o i d depress ive as suggested by Rothenbacher et a l . (1975). In f a c t , i m p l a n t a t i o n of Zerano l appeared to i n c rea se the thy rox ine s e c r e t i o n r a t e and r e s u l t e d In e i t h e r a s i m i l a r or s l i g h t l y depressed plasma thy rox ine concen t ra t i on due to the p a r t i c i p a t i o n of e l eva ted metabo l i c c learance r a t e . Measurements of k i n e t i c parameters may be b e t t e r I nd i c a t o r s of plasma hormone s ta tu s than the abso lu te concen t r a t i on i n the plasma. E s t r o gen i c anabo l i c compounds are e f f e c t i v e i n i n c r e a s i n g the GH concen t r a t i on i n the plasma (EXPT. I I B ) . A s i g n i f i c a n t f i n d i n g of t h i s study i s the demonstrat ion of the e f f e c t of Synovex-S on the p i t u i t a r y g l and . Imp lan ta t i on w i t h DES and Synovex-S r e s u l t e d i n h igher plasma GH concen t r a t i on than w i t h Z e r a n o l . The k i n e t i c parameters of GH metabol ism were determined by a n on - r ad i o a c t i v e k i n e t i c procedure. The r e s u l t s i n d i c a t e d inc reased GH s e c r e t i o n r a te i n s tee r s implanted w i t h a n a b o l i c compounds. A l l the compounds were equa l l y e f f e c t i v e i n i n c r e a s i n g the GH s e c r e t i o n r a t e , e s p e c i a l l y at the e a r l y per iods (day 20) f o l l o w i n g t h e i r i m p l a n t a t i o n . The metabo l i c c learance r a te was not i n f l u e n c e d to a s i g n i f i c a n t extent by the anabo l i c compounds. I t i s concluded that e s t r ogen i c anabo l i c compounds mediate growth processes i n ruminants through changes In the endogenous GH s t a t u s ; such changes i n t u rn are a s soc i a ted w i t h an inc reased GH s e c r e t i o n r a te w i t h l i t t l e changes i n the metabo l i c c learance r a t e . The metabolism of i n s u l i n was i n f l uenced to a s i g n i f i c a n t extent i n s teer s implanted w i t h anabo l i c compounds (EXPT. I I C ) . The i r i m p l a n t a t i o n r e s u l t e d i n an Increase i n the panc r ea t i c i n s u l i n s e c r e t i o n . The a l t e r e d plasma i n s u l i n s ta tus was suggested due to a - 205 -r e s u l t of changes i n the concen t ra t i on of endogenous GH and metabo l i te s such as g l uco se . The chances of a d i r e c t i n t e r a c t i o n of the anabo l i c compounds w i t h the panc rea t i c estrogen receptor s were proposed. Th is I n v e s t i g a t i o n a l so demonstrated a r e l a t i o n s h i p between the s e c r e t i o n r a te s of GH and i n s u l i n i n c a t t l e . Among the a n a b o l i c compounds, Zerano l i n f l u enced the panc rea t i c i n s u l i n s e c r e t i o n to a g rea te r ex tent than Synovex-S or DES, wh i l e the metabo l i c c learance r a te of i n s u l i n was n o n - s i g n i f i c a n t l y reduced i n s tee r s implanted w i t h DES. From these data i t i s concluded that I nc reas ing the i n s u l i n s e c r e t i o n r a te i s one of the mechanism by which anabo l i c compounds e l i c i t growth promotion i n ruminants. However, changes i n the metabolism of other hormones, such as GH and thy rox ine (and po s s i b l y somatomedins and p r o l a c t i n ) might govern the degree of growth promotion ach ieved. Taken c o l l e c t i v e l y , the f i nd i n g s of t h i s i n v e s t i g a t i o n make s i g n i f i c a n t c o n t r i b u t i o n s to the sub jec t of the mechanism of a c t i o n of anabo l i c compounds i n ruminants. The f i nd i n g s of t h i s i n v e s t i g a t i o n i n d i c a t e that anabo l i c compounds i n f l u e n c e the metabolism of the animals f a vo r ab l y f o r an e f f i c i e n t u t i l i z a t i o n of absorbed n u t r i e n t s . The metabol ism i n tu rn i s i n f l uenced by the endogenous changes i n the s t a tu s of GH, i n s u l i n and t h y r o x i n e . Thus, the implanted animal i s i n a p h y s i o l o g i c a l s t a t e that i s conducive f o r b e t t e r p a r t i t i o n i n g of n u t r i e n t s towards inc reased growth ra te and p r o t e i n p r oduc t i on . In t h i s regard anabo l i c compounds seem to exer t a homeorhetic c o n t r o l on the metabol ism i n the ruminants. I t i s suggested that f u tu re s tud ie s are needed to i n v e s t i g a t e the e f f e c t of anabo l i c compounds on a) urea c y c l e enzymes and other enzymes - 206 -of amino a c i d ca tabo l i sm, b) polyamine metabol ism, c) somatomedin phys io logy , d) p r o l a c t i n metabol i sm, e) p r o t e i n s yn thes i s i n the whole-body and i n s e l e c t ed t i s s u e s , and f ) i d e n t i f i c a t i o n of other serum growth f a c t o r s that may be i n f l u e n c e d by them. I t i s hoped that such s t u d i e s , i n the l i g h t of knowledge gained from the present i n v e s t i g a t i o n w i l l widen the understanding of the mechanism of a c t i o n of a n a b o l i c compounds. 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The des ign and a n a l y s i s of i s o tope exper iments . Amer. J . Med. 29 : 832-848. - 232 -APPENDIX I SOURCES OF MATERIALS CHEMICAL ( i ) Radioactive Chemicals Sodium - 1 2 5 I - I o d i d e 1 2 5 1 - Thyroxine 1 2 5 1 - T r i i o d o t h y r o n i n e 1 2 5 1 - I n s u l i n SOURCE Amersham Corp. , O a k v i l l e , Ontar io Canada 1. Nuc lear Med i ca l Systems Inc. New Por t Beach, CA 2. Immuchem Corp. , Carson, CA Immuchem Corp. , Carson, CA Amersham Corp. , O a k v i l l e , Ontar io Canada 1 2 5 1 - Ovine Growth Hormone Prepared i n authors l a bo r a t o r y (11) Hormones and Antibody Preparations Bovine Growth Hormone (NIH - GH - B 18) Ovine Growth Hormone (NIH - GH - S l l ) Ovine Growth Hormone NIAMDD-oGH-I-1 ( f o r i o d i n a t i o n ) I n s u l i n , Bovine L-Thyrox ine Rabbi t Ant iserum to oGH Rabb i t Ant i serum to thy rox ine (T4) N a t i o n a l P i t u i t a r y Agency, B a l t imo re , MD (Same as above) NIAMDD, Bethesda, MD 1. Sigma Chemical Co., S t . L o u i s , MO 2. Dr. M.A. Root, E l i L i l l y & Co., G r e e n f i e l d , IN Sigma Chemical Co., S t . L o u i s , MO NIAMDD, Bethesda, MD 1. Nuc lear Med i ca l Systems, Inc. CA 2. Immuchemical Corp. , CA - 233 -Rabbi t Ant iserum to t r i i o d o t h y r o n i n e (T3) Gu inea-p ig Ant i serum to I n s u l i n Goat A n t i - R a b b i t Immunoglobulin (second an i tbody) Immuchemlcal Corp. , CA Amersham Corp . , O a k v i l l e , Ontar io Canada M i l e s Labora to ry , E l k h a r t , IN ( i i i ) Other Chemicals l - f l u o r o - 2 , 4 -d i n i t r obenzene P y r i d oxa l - 5 ' - p ho spha te Urease (from Jack Bean) Hydroxypro l i ne p-diamino benzaldehyde C r e a t i n i n e Reagent K i t Urea-N Reagent K i t Bovine serum albumin, F r a c t i o n V Sigma Chemical Co, S t . L o u i s , MO Chloramine-T Sodium m e t a b i s u l f i t e B i o Ge l P-60 Sephadex G 100 MCB Manufactur ing Chemists, I n c . , NJ F i s h e r S c i e n t i f i c Co., NJ Bio-Rad L a b o r a t o r i e s , Richmond, CA Pharmacia F ine Chemicals , Uppsa la , Sweden l v . Catheter Materials Po l ye thy lene Catheter (P .E . 90) C lay Adams D i v i s i o n of Beckton, D i ck in son Co., Par s ippany, NJ - 234 -APPENDIX I I RADIOIODINATION OF GROWTH HORMONE Materials: 125 - I, carrier-free - Phosphate buffer, 0.5M, pH 7.5. - Phosphate buffer, 0.05M, pH 7.5 - Phosphate buffered saline, pH 7.0 (PBS) - Phosphate buffered saline containing 1% bovine serum albumin (PBS-1% BSA). - Chloramine-T - 2.0 yg/ul/ 0.05M Phosphate buffer, pH 7.5. - prepared fresh immediately prior to iodination - Sodium metabisulfite - 2.5 ug/yl/ 0.05M phosphate buffer, pH 7.5. - prepared fresh immediately prior to iodination - Transfer solution - 10 mg/ml KI in 16% sucrose - Rinse solution 10 mg/ml KI in 8% sucrose - GH for iodination - aliquots of 2.5 ug NIAMDD-OGH-I-1 per 25 y l 0.01M NaHC03 were dispensed in iodination tubes (12 x 25 mm) and stored frozen at -70°C until the day of iodination Procedure: 1. Add 24 ul of 0.5M phosphate buffer, pH 7.5 to the iodination v i a l and mix. 2. Add 0.5 mCi of carrier-free Iodine-125. Add 15 y l choloramine-T solution. Mix v i a l contents for exactly 15 seconds. 3. Add 50 ul sodium metabisulfite. Mix. 4. Add 100 u l transfer solution, mix, and transfer contents of the v i a l to a column (0.9 cm x 12.0 cm) of Biogel P-60. 5. Add 100 u l of serum to the iodination v i a l , mix, and transfer v i a l contents to the column. 6. Add 700 u l rinse solution to the v i a l , mix and transfer the contents to the column. The column was eluted with 0.05M Phosphate buffer, pH 7.5 and 0.5 ml fractions were collected into tubes containing 0.1 ml PBS-1% - 235 -• «F IOC BSA. Two peaks r ep re sen t i ng I-oGH ( f r a c t i o n s 4-8) and f r ee I ( f r a c t i o n s 10-14) were ob ta i ned . The l abe l ed GH from the f i r s t column was p u r i f i e d f u r t h e r on a l a r ge column (1.5 x 44 cm) of Sephadex G-100 at 4°C. The column was e l u t ed w i t h PBS (pH 7.0) and 1.0 ml f r a c t i o n s were c o l l e c t e d i n tubes con ta i n i n g 0.5 ml PBS-1% BSA. Three peaks r ep re sen t i n g a po s s i b l e aggregat ion product (Peak I ) , immunoreactive GH (Peak I I ) and f r ee 1 2 5 I (Peak I I I ) were obta ined (Appendix F igure 1 ) . The f r a c t i o n s from the second peak (from the descending l imb) were poo led , s to red at 4°C and were used i n the radioimmunoassay on the same week of p u r i f i c a t i o n . - 236 -APPENDIX PEAK I 30 40 SO E L U T I O N V O L U M E ( m l ) 90 100 I O C F i gu re 1. E l u t i o n of I-oGH dur ing r e p u r i f i c a t i o n on 1.5 x 44 cm column of Sephadex G-100 at 4°C. The three peaks represent a p o s s i b l e aggregat ion product (Peak I ) , immunoreactive I-oGH (Peak I I ) , and f r ee I (Peak I I I ) . - 237 -APPENDIX I I I Plasma GH concent ra t i ons i n s tee r s on d i f f e r e n t days f o l l o w i n g the i m p l a n t a t i o n of e s t r ogen i c anabo l i c compounds (Expt . I IB) Days A f t e r Imp lanta t i on (d) Treatment 14 28 42 56 Mean SE 2 ng NIAMDD-oGH-I-l/ml C o n t r o l 14.04 3 12.70 22.98 23.61 17.98 a 2.9 ± 2.5 ± 2.5 ±8.9 ±7.2 2 9 . 7 2 a b Zerano l 40.28 29.31 28±77 20.51 4.5 ±14.9 ± 7.2 ±6.3 ±5.3 DES 37.59 42.1 49.65 44.65 43 .49 b 4.7 ±12.5 ± 7.0 ±13.9 ±5.8 Synovex-S 39.45 42.52 45.7 34.92 40 .15 b 6.8 ±8.2 ±9.1 ±25.9 ±9.8 S t a t i s t i c a l a n a l y s i s based on abso lute hormone concen t r a t i on a b ' means w i t h d i f f e r e n t s u p e r s c r i p t s d i f f e r (P < .05) Pooled standard e r r o r mean of GH concen t r a t i on i n 0730 h & 1400 h sample from 4 s t ee r s PUBLICATIONS Gopinath, R., F. E. Newsome and W.D. K i t t s . 1978. Phytoestrogen format ion i n c e l l suspension c u l t u r e s of a l f a l f a . Can. J . Anim. S c i . 58 : 16. Newsome, F. E., R. Gopinath and W.D. K i t t s . 1980. P r a c t i c a l aspects of forage phyto-est rogen p roduc t i on . Can. J . Anim. S c i . 60: 558. Gop inath , R. and W.D. K i t t s . 1981. E f f e c t of anabo l i c compounds on plasma l e v e l s of n i t rogenous compounds and hepa t i c l e v e l s of t y r o s i ne aminotransferase i n growing beef s t e e r s . J . Anim. S c i . 53 ( s upp l . 1 ) : 494. (P roc . West. Sec. Am. Soc. Animal S c i . 32 : 225 - 228). Gop inath , R. and W.D. K i t t s . 1981. E f f e c t of anabo l i c compounds on plasma l e v e l s of t h y r o i d hormones i n growing beef s t e e r s . J . Anim. S c i . 53 ( s upp l . 1) : 321. Gop inath , R. and W.D. K i t t s . 1981. N T - M e t h y l h i s t i d i n e e x c r e t i o n and muscle p r o t e i n turnover i n beef c a t t l e s tee r s _in v i v o . The P h y s i o l o g i s t 24 : 44. Gop inath , R and W.D. K i t t s . 1982. N T - M e t h y l h i s t i d i n e e x c r e t i o n and muscle p r o t e i n turnover i n beef c a t t l e s teer s ia v i v o : E f f e c t of anabo l i c compounds. J . Anim. S c i . 55 ( s upp l . 1) :219. Gop inath, R. and W.D. K i t t s . 1982. K i n e t i c parameters of t hy rox i ne metabol ism i n growing beef s teer s implanted w i t h anabo l i c compounds. J . Anim. S c i . 55 ( s upp l . 1):218. Gop inath , R. and W.D. K i t t s . 1982. Tyros ine f l u x and body p r o t e i n s yn thes i s i n growing lambs. J . Anim. S c i . 55 ( s upp l . 1):427. Gop inath , R. and W.D. K i t t s . 1982. E f f e c t of anabo l i c compounds on plasma l e v e l s of f r ee amino ac ids i n growing beef s t e e r s . Can. J . Anim. S c i . 62 : 1261. Gop inath, R. and W.D. K i t t s . 1982. U r i na r y hyd roxyp ro l i ne e x c r e t i o n and c o l l a gen metabolism i n growing beef s teer s and the i n f l u e n c e of anabo l i c compounds. Can. J . Anim. S c i . 62 : 1261. Gop inath, R. and W.D. K i t t s . 1982. Growth, N T - M e t h y l h i s t i d i n e e x c r e t i o n and muscle p r o t e i n turnover i n growing beef s t e e r s . J . Anim. S c i . ( submitted f o r p u b l i c a t i o n ) . Gop inath , R. and W.D. K i t t s . 1983. E f f e c t of anabo l i c compounds on parameters of n i t r o g e n metabolism i n growing beef s t e e r s . J . Nut r . ( submitted f o r p u b l i c a t i o n ) . 

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