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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 I N GROWING BEEF  STEERS  by  RAMACHANDRAN GOPINATH B.Sc,  Annamalai  University,  India,  .Sc., T a m i l Nadu A g r i c u l t u r a l U n i v e r s i t y ,  A THESIS SUBMITTED  1974 India,  IN PARTIAL FULFILLMENT OF  THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Department o f Animal S c i e n c e  We accept t h i s t h e s i s as conforming to the r e q u i r e d  standard  THE UNIVERSITY OF BRITISH COLUMBIA April,  1983  ® Ramachandran Gopinath, 1983  1976  In p r e s e n t i n g requirements  this thesis f o r an  of  British  it  freely available  agree that for  I agree that for reference  permission  understood  that  h i s or  be  her  copying or  f i n a n c i a l gain  shall  (3/81)  shall  and  study.  I  the  not  be  of  further this  thesis  this  my  It is thesis  a l l o w e d w i t h o u t my  Columbia  make  head o f  representatives.  of  The U n i v e r s i t y o f B r i t i s h 1956 Main Mall V a n c o u v e r , Canada V6T 1Y3  -6  Library  publication  the  University  the  g r a n t e d by  permission.  Department  the  f o r extensive copying of  s c h o l a r l y p u r p o s e s may by  f u l f i l m e n t of  advanced degree a t  Columbia,  department or for  in partial  written  ABSTRACT The metabolic and endocrine effects of anabolic compounds, v i z . , Zeranol, d i e t h y l s t i l b e s t r o l  (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 i n the plasma concentration of  urea nitrogen, alpha amino nitrogen and amino acids was found i n steers implanted with anabolic compounds.  The results suggested that anabolic  compounds s i g n i f i c a n t l y a l t e r the nitrogen metabolism of steers by increasing the efficiency compounds.  of u t i l i z a t i o n of absorbed nitrogenous  Implantations of DES and Synovex-S were more effective than  Zeranol i n enhancing the growth rate and a l t e r i n g the nitrogen metabolism of the animals. The usefulness of measuring N -methylhistidine i n urine as a T  non-destructive,  i n vivo index of m y o f i b r i l l a r protein degradation and  the developmental aspects of muscle protein metabolism i n c a t t l e were demonstrated.  The implantation of anabolic compounds, i n general,  increased the efficiency deposited by the steers.  of muscle protein synthesized and protein Implantations of DES and Synovex-S were more  effective than Zeranol due to a reduction i n the muscle protein degradation per unit  synthesized.  Hydroxyproline excretion i n the urine was used as an i n vivo indicator of collagen turnover i n steers.  As the animals gained weight  - iii and advanced i n maturity, i t s excretion i n the urine i n d i c a t i n g a reduction i n collagen turnover. increased the hydroxyproline  decreased  Implantation of DES  excreted by steers, while, Zeranol and  Synovex-S had very l i t t l e e f f e c t .  These results indicate that DES  implantation increases the amount of collagen and i t s turnover i n steers. DES and Synovex-S exerted a s i g n i f i c a n t influence on the a c t i v i t y of the thyroid gland and caused an elevation i n the c i r c u l a t i n g concentrations of free and t o t a l thyroxine.  On the other hand, the  implantation of Zeranol resulted i n plasma thyroxine similar to or lower than i n the controls.  concentrations  Plasma triiodothyronine  concentrations were not influenced by any of the compounds studied. Increased plasma thyroxine concentration observed i n the DES or Synovex-S implanted  steers was shown to be due to an increase i n the  secretion and a decrease i n the metabolic clearance rates of thyroxine. The implantation of Zeranol appeared to increase the secretion rate of thyroxine, and resulted i n s l i g h t l y depressed plasma thyroxine concentration due to a higher metabolic clearance rate. Implantations  of anabolic compounds resulted i n an increase i n  the c i r c u l a t i n g concentration of growth hormone.  Implantations  of DES  and Synovex-S were more e f f e c t i v e than Zeranol i n increasing the plasma growth hormone concentration. The k i n e t i c parameters of growth hormone metabolism i n growing steers were determined.  The anabolic compounds increased the secretion  rate of growth hormone from the p i t u i t a r y gland suggesting that these  - iv compounds evoke growth promotion i n steers through changes i n the endogenous growth hormone status.  These changes involved an increased  secretion rate of growth hormone with very l i t t l e a l t e r a t i o n i n the metabolic clearance rate. The metabolism of i n s u l i n was influenced to a s i g n i f i c a n t i n steers implanted with anabolic compounds. i n s u l i n secretion rate to a greater extent  extent  Zeranol increased the  than Synovex-S or DES.  The data indicated that the implantation of anabolic compounds altered the metabolism of steers s i g n i f i c a n t l y and enhanced the secretion rate of thyroxine, growth hormone and i n s u l i n .  They altered  the metabolism of the steers i n such a way that there was an e f f i c i e n t u t i l i z a t i o n of absorbed nutrients. The mechanisms of action of anabolic compounds have been discussed i n d e t a i l and the directions f o r future research suggested.  - v TABLE OF CONTENTS Page ABSTRACT  i i  TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES LIST OF APPENDICES ACKNOWLEDGEMENTS  v ix xii xiv xv  INTRODUCTION  1  LITERATURE REVIEW  3  General concepts of animal growth  3  Concepts and d e f i n i t i o n s of growth Composition of growth and the concept of  3  nutrient p a r t i t i o n i n g  5  Hormonal control of growth and metabolism i n animals  7  Endogenous hormones affecting animal growth and metabolism Growth hormone Somatomedins Prolactin Insulin Thyroid hormones Gonadal and adrenal steroids Miscellaneous endogenous growth factors  7 7 9 11 12 14 15 17  Exogenous hormones and compounds affecting growth, body composition and metabolism of animals  19  Anabolic compounds: Concepts and d e f i n i t i o n s Aspects of growth and protein metabolism Endogenous hormonal changes as influenced by the application of anabolic compounds  19 20 25  Methodological aspects to study protein metabolism i n animals treated with anabolic compounds  28  Measurement of protein synthesis  28  Continuous infusion method Feeding method  28 29  - vi Measurement  -  of muscle p r o t e i n d e g r a d a t i o n  I s o t o p i c method N o n - i s o t o p 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  29 •  Collagen turnover Measurement of c o l l a g e n s y n t h e s i s Measurement of c o l l a g e n d e g r a d a t i o n STUDY  Metabolic aspects of anabolic compound action i n growing beef steers  I :  KXPT. I A  29 32 35 36 36  38 39  E f f e c t s of a n a b o l i c compounds on growth and n i t r o g e n metabolism i n growing beef s t e e r s Introduction M a t e r i a l s and Methods Results Discussion Conclusion  39 39 40 46 52 61 63  EXPT. I B Muscle p r o t e i n m e t a b o l i s m i n s t e e r s as determined by the u r i n a r y N - 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 i n d e x of m y o f i b r i l l a r p r o t e i n breakdown: Developmental a s p e c t s of muscle p r o t e i n metabolism and the e f f e c t of a n a b o l i c compounds T  Introduction M a t e r i a l s and methods Results Discussion Conclusion K X P T .  I  63 63 65 68 80 93 94  C  C o l l a g e n metabolism i n growing beef s t e e r s as a s s e s s e d by the u r i n a r y h y d r o x y p r o l i n e e x c r e t i o n and the e f f e c t s of e s t r o g e n i c a n a b o l i c compounds Introduction M a t e r i a l s and methods Results Discussion Conclusion  94 94 95 97 100 109  - vii -  STUDY I I :  Effect  of anabolic  compounds o n 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 EXPT. I I A : l  E f f e c t of a n a b o 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 Introduction M a t e r i a l s and methods Results Discussion Conclusion E X P T . I I A:2  K i n e t i c parameters of t h y r o x i n e m e t a b o l i s m i n s t e e r s i m p l a n t e d w i t h a n a b o l i c compounds Introduction M a t e r i a l s and methods Results Discussion Conclusion....... EXPT. I I B  E f f e c t of a n a b o 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 m e t a b o l i s m i n growing s t e e r s . . . . . Introduction M a t e r i a l s and Methods Results Discussion Conclusion EXPT. I I C  E f f e c t of a n a b o l i c compounds on the k i n e t i c parameters of i n s u l i n m e t a b o l i s m i n growing s t e e r s Introduction M a t e r i a l s and methods Results Discussion Conclusion  I l l 112  112 112 113 117 122 129 130  130 130 131 137 145 152 154  154 154 156 163 173 181 183  183 183 184 187 191 199  - viii -  GENERAL  D I S C U S S I O N AND  LITERATURE  CITED  CONCLUSIONS  200 207  APPENDIX  I  232  APPENDIX  II  234  APPENDIX  III  237  - ix LIST OF TABLES Page Table 1  Serum hormone-like growth factors  18  Table 2  Anabolic compounds available for use i n farm animals  22  Changes i n the endogenous hormone concentration following the administration of anabolic compounds  26  In v i t r o and i n vivo methods for the quantitation of protein turnover i n d i f f e r e n t species (summary)  30  Table 5  Ration composition (EXPT. I A)  43  Table 6  Plasma free amino acid levels i n steers on 28 days following the implantation of various anabolic compounds  51  Daily urinary nitrogen excretion i n steers implanted with various anabolic compounds  54  E f f e c t of anabolic compounds on hepatic tyrosine aminotransferase l e v e l s  55  Daily urinary excretion of creatinine and N -methylhistidine: Creatinine r a t i o during d i f f e r e n t periods of the experiment  71  F r a c t i o n a l rate of protein 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 proteins i n growing steers on d i f f e r e n t periods of the experiment  75  Amount of muscle protein gained i n steers implanted with various anabolic compounds  81  E f f e c t of anabolic compounds on the f r a c t i o n a l rate of protein breakdown (FBR) and the h a l f - l i f e values of m y o f i b r i l l a r proteins i n growing beef steers  82  The e f f i c i e n c y of muscle protein synthesis i n steers implanted with various anabolic compounds...  83  Table 3  Table A  Table 7 Table 8 Table 9  T  Table 10  Table 11 Table 12  Table 13  - x Table 1 4  Table 1 5  Urinary hydroxyproline excretion i n steers on d i f f e r e n t days following the implantation of anabolic compounds  •  101  Plasma t o t a l thyroxine concentration i n steers on d i f f e r e n t days following the implantation of anabolic compounds  118  Plasma t o t a l triiodothyronine concentration i n steers following the implantation of anabolic compounds  120  Thyroxine binding globulin capacity ( T B G R D ^ ) i n steers on d i f f e r e n t days following the implantation of anabolic compounds..  121  Mean free thyroxine assessment (FTA) i n steers on d i f f e r e n t days following the implantation of anabolic compounds.  123  Summary of changes i n the plasma thyroid hormone concentration i n steers implanted with anabolic compounds  124  Table 2 0  Ration composition (EXPT. II A : 2 )  132  Table 2 1  K i n e t i c parameters of thyroxine metabolism i n growing beef steers  140  Table 2 2  K i n e t i c parameters of thyroxine metabolism i n steers on 2 0 days following the implantation of anabolic compounds  141  K i n e t i c parameters of thyroxine metabolism i n steers on 4 0 days following the implantation of anabolic compounds..  143  K i n e t i c parameters of thyroxine metabolism i n steers on 6 0 days following the Implantation of anabolic compounds  144  Table 2 5  E f f e c t of anabolic compounds on plasma thyroxine status  146  Table 2 6  K i n e t i c parameters of thyroxine metabolism i n cattle..  149  Table 2 7  Plasma G H concentration i n steers on d i f f e r e n t days following the implantation of anabolic compounds  165  Table 1 6  Table  17  Table 1 8  Table 1 9  Table 2 3  Table 2 4  -  Table 28 Table 29 Table 30 Table 31  Table 32  Table 33 Table 34  3d  -  K i n e t i c parameters of GH metabolism i n growing steers.. First-order rate constants for d i s t r i b u t i o n and elimination of plasma GH i n steers  169 ..  170  H a l f - l i f e and f r a c t i o n a l disappearance rate of injected GH i n steers  171  Metabolic clearance and secretion rates of GH i n steers on 20 days following the implantation of anabolic compounds  172  H a l f - l i f e , f r a c t i o n a l disappearance rate and metabolic clearance rate of GH i n steers on 40 days following the implantation of anabolic compounds  174  K i n e t i c parameters of i n s u l i n metabolism i n steers..  189  K i n e t i c parameters of i n s u l i n metabolism i n steers implanted with anabolic compounds  190  - xii  -  LIST OF FIGURES Page Figure 1  S t r u c t u r a l formulae of c e r t a i n i m p o r t a n t e s t r o g e n i c and a n d r o g e n i c a n a b o l i c compounds  21  Schematic summary of N - m e t h y l h i s t i d i n e metabolism  34  E f f e c t of a n a b o l i c compounds on growth i n steers  47  Plasma urea n i t r o g e n (PUN) and a l p h a amino n i t r o g e n (otAN) c o n c e n t r a t i o n s i n s t e e r s i m p l a n t e d w i t h v a r i o u s a n a b o l i c compounds  49  Figure 5  Plasma urea-N and a l p h a amino-N c o n c e n t r a t i o n s s t e e r s i m p l a n t e d w i t h a n a b o l i c compounds  50  Figure 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) c o n c e n t r a t i o n s i n growing s t e e r s on 28 days f o l l o w i n g the i m p l a n t a t i o n of a n a b o l i c compounds...  53  Figure 7  Body weight and average d a i l y g a i n of s t e e r s  69  Figure 8  Urinary N -methylhistidine excretion in on d i f f e r e n t p e r i o d s of the experiment  70  Figure 9  Muscle p r o t e i n degraded and s y n t h e s i z e d i n s t e e r s on d i f f e r e n t p e r i o d s of the experiment  73  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 y n t h e s i s and d e g r a d a t i o n i n s t e e r s  76  U r i n a r y e x c r e t i o n of N - m e t h y l h i s t i d i n e cattle  in 77  Urinary N/t-methylhistidine e x c r e t i o n i n i m p l a n t e d w i t h a n a b o l i c compounds  steers  Figure 2  Figure 3 Figure 4  F i g u r e 10 F i g u r e 11 F i g u r e 12 F i g u r e 13 F i g u r e 14  T  T  T  in  steers  78  Rates of muscle p r o t e i n d e g r a d a t i o n and s y n t h e s i s i n s t e e r s i m p l a n t e d w i t h a n a b o l i c compounds  79  U r i n a r y t o t a l h y d r o x y p r o l i n e (HYPRO) e x c r e t e d by s t e e r s on d i f f e r e n t p e r i o d s of the experiment  98  - xiii -  Figure 15  Figure 16  Figure 17  Figure 18  Figure 19  Figure 20  Figure 21 Figure 22  Figure 23 Figure 24  Figure 25 Figure 26  Urinary hydroxyproline-N [HYPRO-N expressed as a per cent of urinary t o t a l nitrogen] excretion by steers on d i f f e r e n t periods of the experiment  99  Urinary t o t a l hydroxyproline (HYPRO) excretion by steers on d i f f e r e n t days following the implantation of anabolic compounds  102  E f f e c t of anabolic compounds on urinary t o t a l hydroxyproline excretion by growing beef steers....  103  A schematic summary of the relationship between urinary hydroxyproline excretion and the metabolism of collagen  107  The disappearance of thyroxine from the plasma a f t e r a single intravenous i n j e c t i o n of 12 to 16 mg of L-thyroxine i n steers  138  Metabolic clearance and secretion rates of thyroxine on 20, 40 and 60 days following the implantation of anabolic compounds i n steers.......  147  Effect of anabolic compounds on plasma GH concentration i n growing steers  166  The disappearance of GH from the plasma following the intravenous i n j e c t i o n of 6-8 mg of bovine GH (NIH-GH-B18)  168  Metabolic clearance and secretion rates of GH i n steers implanted with anabolic compounds  175  The disappearance of i n s u l i n from the plasma of steers following a single intravenous i n j e c t i o n of one mg bovine i n s u l i n  188  Metabolic clearance and secretion rates of i n s u l i n i n steers implanted with anabolic compounds  192  Summary of the metabolic and endocrine effects of anabolic compounds i n steers  201  - xiv LIST OF APPENDICES Page Appendix Appendix  I  Sources of materials  I I Radioiodination of growth hormone  Appendix I I I Plasma GH concentration i n steers on d i f f e r e n t days following the implantation of anabolic compounds (EXPT. II B)  232 234  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. extended to Dr. C.R. Krishnamurti  Appreciation i s  (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 i s 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 i n the world human population and with the increasing demand for food, techniques  have been developed within  the l a s t 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 e f f i c i e n c y of meat produced from farm animals.  These substances include  anabolic compounds and a n t i b i o t i c s to name a few. Anabolic compounds are those which when administered are capable of increasing the growth rate, feed conversion and i n general improving the carcass quality and quantity.  to animals efficiency A number of  compounds have been i d e n t i f i e d to have such anabolic properties; they include the natural or synthetic compounds that belong to the sex-steroid group or compounds with a modified s t e r o i d a l or non-steroidal structure.  The choice of an appropriate anabolic compound  i s important which i s related to the sex-hormone status of the animal. In beef c a t t l e , the estrogenic anabolic compounds are found e f f e c t i v e i n increasing the growth rate of steers, while androgenic anabolic compounds are e f f e c t i v e i n h e i f e r s .  The growth promoting properties of  some of the anabolic compounds, namely d i e t h y l s t i l b e s t r o l (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 i n general r e s u l t s i n improved live-weight gain.  The carcass from treated animals  contains a higher percentage of protein and a lower percentage  - 2 -  o f f a t than the u n t r e a t e d a n i m a l s .  I n t h i s r e g a r d and a n a b o l i c  compounds p r o v i d e 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 r u m i n a n t s . a n a b o l i c compounds  Even though the growth promoting p r o p e r t i e s of  i n ruminants have been r e p e a t e d l y documented, the  b i o c h e m i c a l mechanism by which they e l i c i t a n a b o l i c a c t i o n i s c l e a r l y understood.  not  Only r e c e n t l y s e v e r a l p o s s i b l e mechanisms  of  a c t i o n s of a n a b o l i c compounds have been suggested ( B u t t e r y e t a l . , 1978; P r e s t o n , 1975).  I t was proposed t h a t a n a b o l i c compounds e l i c i t  changes  b o t h on the e n d o c r i n e system and on the g e n e r a l m e t a b o l i s m of the animal.  U n d e r s t a n d i n g the mechanism o f a c t i o n o f a n a b o l i c compounds  i m p o r t a n t f o r the i d e n t i f i c a t i o n of growth p r o c e s s e s by these a c t i v e  is  t h a t are i n f l u e n c e d  compounds.  I n t h i s s t u d y , the b i o c h e m i c a l e f f e c t s of c e r t a i n e s t r o g e n i c a n a b o l i c compounds, v i z . , DES,  Z e r a n o l and Synovex-S on the e n d o c r i n e  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 f o c u s s e d on the e f f e c t s of a n a b o 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 c o l l a g e n metabolism and on the metabolism of endogenous hormones growing beef  steers.  such as the growth hormone, i n s u l i n and t h y r o x i n e  in  - 3 -  LITERATURE REVIEW Animal growth i s a complex phenomenon involving the i n t e r a c t i o n of hormonal, n u t r i t i o n a l and genetic factors. the  A precise knowledge of  animal growth process i s important i n order to improve the  e f f i c i e n c y of meat production.  In t h i s respect anabolic compounds have  been found e f f e c t i v e i n increasing the growth rate of farm animals. In this review, a b r i e f 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 f o r studying protein turnover i n the whole body, muscle and collagen i s also presented.  These methodological advancements have  f a c i l i t a t e d a better understanding of the growth process i n animals and i n 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 D e f i n i t i o n s of Growth  Animal growth i s characterized by an orderly increase In mass of tissue and organs as well as i n form and body composition.  It i s a  complex phenomenon involving n u t r i t i o n a l , hormonal and genetic i n t e r r e l a t i o n s h i p s 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 l i v i n g pattern.  Seebeck (1968) i n  - 4 -  his  review defined growth as the increase i n weight of an animal or any  part of i t , as i t approaches mature s i z e .  Normal growth, however, i s  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, v i z : (a) the increase i n mass (weight) per unit time, and (b) the aspect involving changes i n form and composition r e s u l t i n g from d i f f e r e n t i a l growth of the component parts of the body.  In physiological terms most  tissues appear to have three stages of growth:  (a) increase i n c e l l  number with l i t t l e increase i n 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 i n c e l l size or hypertrophy (Enesco and Leblond, 1962; Winick and Noble, 1965; Winick and Noble, 1966; Winick et a l . , 1972). early d e f i n i t i o n s of growth and development  The  i n farm animals have been  reviewed by Brody (1945), Richards and Kavanagh (1945), Pomeroy (1955), Palsson (1955), Needham (1964) and Seebeck (1968). A s i g n i f i c a n t contribution to the knowledge on the growth patterns i n farm animals has been made by the members of the Hammond School (Palsson, 1955).  Their work has revealed that during f o e t a l  l i f e , the head and lower limbs of the animal grow r e l a t i v e l y faster than the  rest of the body, so that, at b i r t h , the growing animal has a  r e l a t i v e l y large head and long legs with the trunk being short and shallow.  After b i r t h 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 d i f f e r e n t i a l growth pattern or the waves of growth i n farm  animals was  suggested by Palsson (1955).  Palsson (1955) also suggested  the phenomenon of changing p r i o r i t i e s for nutrient use by d i f f e r e n t tissues as the animals develop.  These patterns of tissue growth  determine eventually the composition of growth. b.  Composition o f Growth and the Concept of Nutrient p a r t i t i o n i n g  Muscle, f a t , 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) i s important.  The various factors that influence r e l a t i v e growth of  muscle, fat and bone have been discussed B u t t e r f i e l d (1976) and include:  i n d e t a i l by Berg and  (a) genetics  the animal (Galbraith and Topps, 1981), and  (Lohman, 1971), (b) sex of  (c) the plane of n u t r i t i o n  (Callow, 1961). The s k e l e t a l muscle constitutes a major proportion of the of the meat animals.  carcass  The diversion of nutrients towards increased  muscle growth w i l l be b e n e f i c i a l to increase the amount of meat produced.  Bauman and Currie (1980) drew attention to the p a r t i t i o n i n g  of nutrients i n animals as they advance through d i f f e r e n t 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  p a r t i t i o n i n g of absorbed nutrients between protein and f a t 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 d i f f e r e n t organs and establishes d i f f e r e n t p r i o r i t i e s of available nutrients.  Indeed, i t w i l l be desirable to regulate the nutrient flow  towards more protein than f a t synthesis.  But achieving  increased  protein synthesis at the expense of fat could a f f e c t the o v e r a l l e f f i c i e n c y of animal production  as indicated by Webster (1977) that  animals retaining higher proportion of energy as protein are energetically less e f f i c i e n t than those retaining more energy i n the adipose t i s s u e . The hormonal influence on nutrient p a r t i t i o n i n g f o r increased s k e l e t a l muscle growth can be further i l l u s t r a t e d by the hormonal differences between d i f f e r e n t breeds of c a t t l e .  The larger and lean  breeds of c a t t l e tend to have more growth hormone and less i n s u l i n i n the plasma than the smaller breeds and such a hormonal i n t e r r e l a t i o n s h i p was thought to favour more muscle growth than s h i f t i n g the nutrients and energy to adipose tissue (Trenkle, 1981).  Hart et^ a l . (1975) indicated  further the differences i n the c i r c u l a t i n g hormonal l e v e l between the l a c t a t i n g dairy and beef c a t t l e .  The higher concentration  hormone and the lower concentration  of growth  of i n s u l i n i n the plasma of the  dairy than i n beef c a t t l e was thought to increase the flow of nutrients towards the mammary gland than the s k e l e t a l muscle or the adipose tissue.  The role played by hormones such as growth hormone and  p r o l a c t i n (Bauman et a l . , 1982), i n s u l i n (Prior and Smith, 1982) and by other n u t r i t i o n a l factors (Byers, 1982b) i n regulating nutrient  - 7-  u t 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 i n regulating growth i n animals and i t s composition.  The endocrine balance i n 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 E l s l e y (1976). The concept of waves of growth i n animals (Palsson, 1955) provides hope that 'growth engineering' could be achieved by i n h i b i t i n g 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 A f f e c t i n g Animal Growth and Metabolism  The various aspects of endocrine control on growth i n experimental animals and human subjects have been reviewed recently (Daughaday, 1981).  Of the endogenous hormones, growth hormone,  somatomedin, p r o l a c t i n , i n s u l i n , thyroid hormones, adrenal and gonadal steroids and other hormone-like growth factors play a v i t a l role i n regulating growth and metabolism. 1.  Growth Hormone  Growth hormone (GH), also called the somatotrophin, has a s t r i k i n g trophic effect on the soma resulting i n growth and maturation of the organs of the body.  Endogenous GH i s a single-chain polypeptide  with two interchain d i s u l f i d e bridges and has a molecular weight of about 22000.  Aspects of GH Chemistry ( L i , 1975), b i o l o g i c a l 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 y n t h e s i s  i n s k e l e t a l muscle (Young, thoroughly  1970; Young and P l u s k a l , 1977)  especially  have been  reviewed.  The growth promoting a c t i o n s of bovine GH i n r a t s  was  demonstrated as e a r l y as 1931 by Evans and Simpson ( 1 9 3 1 ) .  Brumby  (1959) r e p o r t e d the g a i n i n l i v e - w e i g h t and h e i g h t i n young c a t t l e r e c e i v i n g GH.  B a i r d e t a l . (1952) demonstrated the presence o f g r e a t e 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 i g s than i n those w i t h a lower growth r a t e .  Amstrong and Hansel  (1956) r e p o r t e d a p o s i t i v e  c o r r e l a t i o n between the growth r a t e of F r i e s i a n h e i f e r s and the GH c o n t e n t of the a n t e r i o r p i t u i t a r y .  F u r t h e r , the a d m i n i s t r a t i o n of GH t o  farm a n i m a l s has r e s u l t e d i n i n c r e a s e d growth r a t e and feed e f f i c i e n c y i n p i g s ( M a c h l i n , 1972)  and wether lambs (Wagner and V e e n h u i z e n ,  and i n c r e a s e d n i t r o g e n r e t e n t i o n i n sheep ( D a v i s e t a l . , 1970; e t a l . , 1966; W a l l a c e and B a s s e t t and i n s t e e r s  1978),  Wheatley  1966; S t r u e m p l e r and B u r r o u g h s ,  (Moseley e t a l . , 1982).  These r e p o r t s  1959)  demonstrate  c o n c l u s i v e l y the growth promoting p r o p e r t i e s o f GH i n farm a n i m a l s . The i n f l u e n c e of GH on p r o t e i n a n a b o l i s m i s i m p o r t a n t among m e t a b o l i c a c t i o n s i n the a n i m a l body. d e p o s i t i o n and decreases Chronic  The GH i n c r e a s e s  the net p r o t e i n  the weight of the a d i p o s e t i s s u e i n the  a d m i n i s t r a t i o n of GH has been shown to i n c r e a s e  a c c r e t i o n i n sheep (Wagner and V e e n h u i z e n , 1978).  its  body.  protein  A decrease i n the  plasma c o n c e n t r a t i o n of amino a c i d and n i t r o g e n e x c r e t i o n i n u r i n e exogenous GH a d m i n i s t r a t i o n ( D a v i s e t a l . , 1970)  demonstrates  of GH on amino a c i d uptake by the body t i s s u e s .  Moreover,  after  the e f f e c t  an i n c r e a s e d  - 9 -  retention of calcium i n sheep injected with GH was (Braithwaite, 1975). DNA  also reported  Cheek and H i l l (1970) showed that the postnatal  synthesis i n the muscle of rats i s dependent upon GH.  (1974) indicated that the DNA  Trenkle  accumulation i n the muscle i s abolished  hypophysectomy and that GH i s capable of p a r t i a l l y restoring DNA  by  to near  normal l e v e l . Peel et a l . (1981) demonstrated that the administration of GH to high producing dairy cows resulted i n a 10-15% increase i n the milk y i e l d and a 17-18% increase i n milk energy secretion.  The GH also  provides a homeorhetic signal and seems to influence the  co-ordination  of the metabolism among body tissues i n a manner to support the p a r t i t i o n i n g of the nutrients for muscle protein accretion (Bauman et a l . , 1982).  These concepts were s i m i l a r to those i l l u s t r a t e d by  Machlin (1972) who  found that the growth rate of pigs fed a r e s t r i c t e d  r a t i o n and administered libitum.  with exogenous GH was  comparable to those fed ad  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 i n 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.  ii.  Somatomedins  Somatomedins are a family of c i r c u l a t i n g peptides  thought to  contribute to growth by the stimulation of p r o l i f e r a t i n g c a r t i l a g e (Van Wyk  et a l . , 1974).  They are formed i n the l i v e r under the influence of  GH and mediate most of the actions of the l a t t e r (Chochinov and  - 10 -  Daughaday, 1976) i n the p r o m o t i o n o f 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 o n d i t i o n s f o r s u b s t a n c e s t o q u a l i f y as somatomedins; t h a t the s u b s t a n c e s h o u l d be GH-dependent, posses i n s u l i n - l i k e a c t i o n 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 s u l p h a t e 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 .  A p p l y i n g these c r i t e r i a , a number of  p e p t i d e s 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 g e n e r a l term somatomedins:  they i n c l u d e somatomedin-A (SM-A:  somatomedin-B (SM-B: et^al.,  Hall,  1972),  Uthene, 1973), somatomedin-C (SM-C:  1974), i n s u l i n - l i k e growth f a c t o r - I  Humbel, 1978a), i n s u l i n - l i k e growth f a c t o r - I I  (IGF-I:  R i n d e r k n e c h t and  (IGF-II:  R i n d e r k n e c h t 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 P i e r s o n and Temin, 1972).  Van Wyk  (MSA:  The p r o d u c t i o n of somatomedins i s thought t o  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 v a r i o u s o t h e r hormones b e s i d e s GH ( H o l d e r and Preece,  1981). A l a r g e body of e v i d e n c e s u p p o r t s the r o l e of somatomedins  r e g u l a t i n g the s o m a t i c growth of meat p r o d u c i n g a n i m a l s .  in  O l s e n et^ a l .  (1981) r e p o r t e d a p o s i t i v e r e l a t i o n s h i p between serum s o m a t o m e d i n - l i k e a c t i v i t y and the r e l a t i v e weight g a i n i n lambs.  Wangsness e t a l . (1981)  found serum somatomedins t o be h i g h e r 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 l o w e r growing F i n n - s i r e d lambs.  Somatomedins were  a l s o found t o be r e l a t e d t o the r a t e of g a i n and l i n e a r growth of Red D a n i s h b u l l s ( L u n d - L a r s e n e t a l . , 1977) and swine ( L u n d - L a r s e n and Bakke, 1975).  The b i o l o g i c a l a c t i o n s of somatomedins have been r e v i e w e d  by H o l d e r and Preece ( 1 9 8 1 ) .  -  iii.  11  -  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 r e p r o d u c t i o n has l o n g been r e c o g n i z e d i n e x p e r i m e n t a l a n i m a l s .  It  is  usually  c o n s i d e r e d t o have o t h e r o v e r l a p p i n g b i o l o g i c a l a c t i o n s w i t h GH ( T u r n e r and Bagnara,  1976).  However, not much a t t e n t i o n has been focused on the  growth promoting p r o p e r t i e s of p r o l a c t i n i n mammals.  Bovine  prolactin  has been shown to i n c r e a s e the growth r a t e of S n e l l dwarf mice and Dew, 1973) sex s t e r o i d s ,  and r a t s (Bates et^ a l . , 1964).  (Wallis  The a n a b o l i c e f f e c t s o f  t e s t o s t e r o n e p r o p i o n a t e and DES i n sheep were p a r t l y  a t t r i b u t e d to the i n c r e a s e d endogenous p r o l a c t i n s e c r e t o r y p a t t e r n ( D a v i s et a l . , 1978). response  I t was demonstrated t h a t the p o s i t i v e  growth  i n lambs and h e i f e r s t r e a t e d w i t h s y n t h e t i c e s t r o g e n s  or  c h r o n 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 l e v a t e d c i r c u l a t i n g c o n c e n t r a t i o n s of both GH and p r o l a c t i n ( D a v i s et^ a l . , 1976; D a v i s e £ a l . , 1977b; D a v i s et a l . , 1978).  These o b s e r v a t i o n s  suggest the r o l e p l a y e d 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 e t a l . (1982) suggested t h a t 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 c a p a c 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 o t h e r t i s s u e s tissues,  such as  adipose  i n a manner to a l l o w g r e a 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  i n c r e a s e d muscle g r o w t h .  F u r t h e r e v i d e n c e f o r the i n v o l v e m e n t  p r o l a c t i n on growth processes  of  of animals c o u l d be o b t a i n e d from t h e  r e p o r t s on the p h o t o p e r i o d i c r e g u l a t i o n of growth i n a n i m a l s et a l . , 1975; Schanbacher  towards  and Crouse,  1981).  (Forbes  Lambs have been shown t o  - 12 -  gain r a p i d l y , convert feed to l i v e weight more e f f i c i e n t l y and to produce acceptable carcasses when exposed to long photoperiods et a l . , 1979;  Schanbacher and Crouse, 1980)  and these changes i n growth  were accompanied by an increased serum prolactin concentration et a l . , 1975).  (Forbes  (Forbes  However, Eisemann et a l . (1981) reported that wether  lambs receiving daily i n j e c t i o n of p r o l a c t i n for 9 weeks had no increase i n growth rate over the respective controls. It can be concluded  that the growth rate i n animals could be  related to their p r o l a c t i n status and manipulation  of growth could be  achieved by a l t e r i n g p r o l a c t i n status.  iv.  Insulin  I n s u l i n plays a complex role i n the growth process.  Several  similar actions between i n s u l i n and somatomedins suggest that these hormones may  be acting through a common mechanism.  I n s u l i n i s required  for the expression of f u l l anabolic effects of growth hormone. growth stimulating effects of i n s u l i n were demonstrated by Gey Thalhimer (1924). f e t a l development.  It i s an important  two  growth-regulating  The and  hormone during  At b i r t h there i s a positive correlation between  plasma i n s u l i n and b i r t h weight of human infants (Shima et a l . , 1966). The growth promoting effects of i n s u l i n are best demonstrated by the presence of detectable growth i n subjects without measurable amount of immunoreactive growth hormone, such as that associated with removal of p i t u i t a r i e s or hypothalamic tumors.  Martinovich and Margolin (1975)  reported an improvement i n the rate of gain and feed e f f i c i e n c y i n c a t t l e receiving i n s u l i n i n j e c t i o n s every ten days.  -  13  -  Even though direct findings, such as those reported for c a t t l e , on the effect of i n s u l i n on the growth process are l i m i t e d , a number of workers have indicated i n d i r e c t l y the growth promoting properties of insulin.  The poor growth or growth retardation i n situations  characterized by low i n s u l i n production as i n diabetes mellitus (Schiff e t ^ a l . , 1972)  has been reported.  P h i l l i p s and Orawski (1977)  demonstrated that diabetic rats grew normally when given an adequate dose of i n s u l i n .  I n s u l i n also stimulates growth and p r o l i f e r a t i o n of  many c e l l types (Straus, 1981)  and i s required by a variety of c e l l s for  optimal growth i n hormone-supplemented serum free medium (Barnes and Sato, 1980). Insulin i s a powerful anabolic hormone increasing the synthesis of DNA,  RNA,  nucleic acids and protein i n the target tissues  (Manchester, 1972).  The biochemical events regulated by i n s u l i n have  been reviewed by Straus (1981). In meat producing  animals i n s u l i n plays a v i t a l role i n  regulating carbohydrate and protein metabolism.  Trenkle (1970) reported  that i n c a t t l e the plasma i n s u l i n concentration increased with the length of time they were fed grain.  A p o s i t i v e c o r r e l a t i o n was  observed  between plasma i n s u l i n concentration and fatness of the carcass  (Trenkle  and I r v i n , 1970).  Jefferson et a l . (1974) reported that i n s u l i n  increases the incorporation of labelled amino acid into muscle protein i n non-ruminants.  Preliminary observations by Chrystie et a l . (1977)  indicate that i n f e t a l lambs i n s u l i n decreases protein breakdown without any change i n protein synthesis.  The role played by i n s u l i n on  - 14 -  p a r t i t i o n i n g of nutrients for tissue growth has recently been reviewed by P r i o r and Smith (1982).  In his review, Trenkle  (1981) indicated that  larger and leaner breeds of c a t t l e tend to have more growth hormone and less Insulin i n the plasma and that the smaller breeds have more insulin.  The concentration of i n s u l i n i n plasma was  p o s i t i v e l y related  to the carcass adipose tissue and negatively related to the amount of carcass protein.  v.  T h y r o i d Hormones  Thyroid hormones are involved i n the regulation of growth and act as an important regulator of metabolic rate (Tata, 1964).  The  p a r t i c i p a t i o n of thyroid hormones i n the growth process could be best i l l u s t r a t e d by the presence of decreased growth during hypothyroidism (Falconer and Draper, 1968)  and following thyroidectomy (Bray, 1964).  Thyroxine (T4) participates i n growth and d i f f e r e n t i a t i o n of bones.  It  seems that T4 acts synergetically with- GH i n 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 i n farm animals (Brumby, 1959;  Hart and Laffey, 1959).  However, attempts to increase the endogenous thyroid hormones have been made to a l t e r growth and body composition i n animals (Wagner and Veenhuizen, 1978).  Wagner and Veenhuizen (1978) fed thyroprotein to  increase the thyroid hormone l e v e l s i n wether lambs and found an increase i n the amount of feed required per unit gain and a decrease i n carcass f a t .  They have also reported increased growth rate and  deposition when thyroprotein was  protein  fed to lambs receiving GH i n j e c t i o n s .  - 15  -  I t was s p e c u l a t e d t h a t t h y r o i d hormones may have been l i m i t i n g when growth was i n c r e a s e d w i t h exogenous growth hormone ( T r e n k l e , It process  1981).  i s e v i d e n t t h a t the a c t i o n s of t h y r o i d hormone on the growth  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 a n a b o l i c hormones. and somatomedins  The s y n e r g i s m between t h y r o i d  has r e c e i v e d much a t t e n t i o n r e c e n t l y .  (1978) showed t h a t the t r e a t m e n t of hypophysectomized  hormones  Gaspard et^ a l . rats with  t h y r o x i n e 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.  Besides,  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 g l a n d f u n c t i o n (Hellman e t a l . , 1961), i n the normal p r o l a c t i n s y n t h e s i s Meites,  (Augustine  and Hymer, 1978; Chen and  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 s y n t h e s i s  of GH i n  the s o m a t o t r o p h i c 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 m a t u r a t i o n of GH c e l l s or b o t h ( I s h i k a w a ejt a l . , 1976).  The  presence  of t e t r a i o d o t h y r o n i n e (TA) and t r i i o d o t h y r o n i n e (T3) i n serum of embryonic c h i c k has been s t r o n g l y ( K i n g and K i n g , It process  i m p l i c a t e d i n the embryonic  growth  1978).  i s e v i d e n t t h a t t h y r o i d hormones  are i n v o l v e d i n the growth  of a n i m a l s , but t h e i r a c t i o n s seem to be complex i n v o l v i n g  p a r t i c i p a t i o n of o t h e r endogenous a n a b o l i c  vi.  the  hormones.  Gonadal and Adrenal Steroids  Among the s t e r o i d hormones  s e c r e t e d by the a d r e n a l c o r t e x and the  gonads, the g l u c o c o r t i c o i d s , androgens and e s t r o g e n s i n f l u e n c e on the s o m a t i c growth and m a t u r a t i o n .  exert a s u b s t a n t i a l  Whereas  the i n f l u e n c e  -  16  -  o f 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 e x e r t an i n h i b i t o r y e f f e c t on a n i m a l g r o w t h .  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 a n i m a l 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 m a t u r a t i o n d u r i n g p u b e r t y and adolescence.  The i n f l u e n c e o f both androgens and e s t r o g e n s  can be seen  by the d i f f e r e n c e s i n the growth r a t e and body c o m p o s i t i o n between the c a s t r a t e s and t h e i r e n t i r e c o u n t e r p a r t s .  B u l l s gain 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 i g h e r per cent of l e a n and lower per cent o f f a t i n the c a r c a s s  than s t e e r s .  e x i s t s between h e i f e r s and spayed h e i f e r s .  A similar difference  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 c o m p o s i t i o n 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 i n v o l v e m e n t of androgens and  estrogens  i n v a r i o u s a s p e c t s o f growth (Wiedemann, 1981) and t h e i r m o l e c u l a r mechanism of a c t i o n (Young and P l u s k a l , 1977) have been r e v i e w e d . The advancements i n the knowledge of the e f f e c t of androgens and estrogens  on growth p r o c e s s e s  of animals have l e a d 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 o r through t h e i r s y n t h e t i c c o u n t e r p a r t s t o m a n i p u l a t e g r o w t h , f e e d c o n v e r s i o n e f f i c i e n c y and body c o m p o s i t i o n i n meat p r o d u c i n g animals  (Lu and  d e s i g n a t e d as  R e n d e l , 1976).  These compounds have g e n e r a l l y been  ' 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 p r o d u c i n g a n i m a l s i s d e a l t s e p a r a t e l y .  Glucocorticoids The i n v o l v e m e n t 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  -  i n normal growth has been d i s c u s s e d  17  -  by Loeb ( 1 9 7 6 ) .  e x e r t a c a t a b o l i c e f f e c t i n the a n i m a l body.  Glucocorticoids  As e a r l y as 1938,  Ingle  e t a l . (1938) demonstrated t h a t r a t s t r e a t e d w i t h the a d r e n a l c o r t e x e x t r a c t s f a i l e d t o g a i n weight n o r m a l l y .  W e l 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 m a t u r a t i o n weaning r a t s t r e a t e d w i t h 1 mg/d of e i t h e r 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 growth and m e t a b o l i s m of s k e l e t a l m u s c l e .  Cortisol  in  or c o r t i c o s t e r o n e .  seem t o be d i r e c t e d towards  the  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 t o animals has been shown t o 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 t o an i n h i b i t i o n of muscle p r o t e i n s y n t h e s 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,  s e v e r a l workers have proposed  1970).  Besides,  impaired  somatomedin g e n e r a t i o n 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 e f f e c t of g l u c o c o r t i c o i d s  (Elders  f o r the  growth-retarding  et a l . , 1975; Van Den Brande et a l . ,  1975).  vii.  Miscellaneous Endogenous Growth Factors  Besides  the major endogenous hormones  metabolism discussed  i n the p r e v i o u s  t h a t r e g u l a t e growth and  sections,  t h e r e are a l s o  several  o t h e r growth f a c t o r s i n the serum w i t h h o r m o n e - l i k e a c t i v i t y t h a t have been s t r o n g l y  i m p l i c a t e d i n the growth p r o c e s s e s  (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 o f c u l t u r e d c e l l s has been w i d e l y r e p o r t e d , t h e i r s i g n i f i c a n c e c o n t r o l l i n g growth and metabolism i n farm a n i m a l s has not been  in  -  T a b l e 1.  18  -  Serum Hormone-Like Growth F a c t o r s  Target  Factors  Cell  Sympathetic Nerves  1.  Nerve Growth F a c t o r  2.  E p i d e r m a l Growth F a c t o r  3.  F i b r o b l a s t Growth F a c t o r  A.  O v a r i a n Growth F a c t o r  Ovary  5.  M y o b l a s t Growth F a c t o r  Myoblasts  6.  Thrombopoietin  Thrombocytes  7.  Thymosin  Thymocytes  8.  Melanocyte S t i m u l a t i n g F a c t o r  Melanocytes  X  Young (1980)  (NGF) (EGF) (FGF)  Epidermal C e l l s Mesodermal  Cells  - 19 -  understood.  The importance of growth factors, especially f i b r o b l a s t  p r o l i f e r a t i v e a c t i v i t y (FPA) i n lambs has been reported (Olsen et^ a l . , 1981; b.  Wangsness et a l . , 1981).  Exogenous Hormones and Compounds A f f e c t i n g 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 i n animal agriculture to a l t e r growth and body composition compounds'.  are included under the broad term 'exogenous hormones and As a vast body of information i s available on this subject  only the salient features of exogenous compounds on growth and body composition  are presented.  The h i s t o r i c a l 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; Hoffman, 1980; 1.  Lu and Randel, 1976;  Anabolic Compounds:  Heitzman,  1980;  Preston, 1975).  Concepts and D e f i n i t i o n s  When used i n 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 i n s u l i n or i n general those compounds that promote growth and anabolism i n animals.  The term, however, when used  by a c l i n i c i a n or endocrinologist denotes only those steroids whose chemistry i s derived from testosterone and  19-nortestosterone.  Estrogens, though possessing anabolic properties do not belong to this class according to the l a t t e r d e f i n i t i o n .  In general, anabolic  - 20  compounds  -  c o u l d be d e f i n e d as those which when a d m i n i s t e r e d t o animals  cause (1) i n c r e a s e i n growth r a t e , (2) i n c r e a s e i n muscle mass and p r o t e i n d e p o s i t i o n , (3) i n c r e a s e i n n i t r o g e n 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  improve a p p e t i t e .  (6)  The e x p r e s s i o n of these b a s i c c h a r a c t e r i s t i c s of  a n a b o 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 , s p e c i e s and age o f t h e a n i m a l s to which they are a p p l i e d t o .  A number of exogenous compounds have been  i d e n t i f i e d t o e x h i b i t the above c r i t e r a and are  broadly c l a s s i f i e d  i n t o the f o l l o w i n g g r o u p s : a.  androgenic a n a b o l i c  compounds  b.  estrogenic anabolic  compounds  A summary of e s t r o g e n i c and a n d r o g e n i c compounds which are commonly i n u s e , t h e i r c h e m i c a l s t r u c t u r e and the a n i m a l s p e c i e s o r sex whereupon they f a v o u r a b l y express  their anabolic properties i s  presented  i n F i g u r e 1 and T a b l e 2. ii.  Aspects of Growth and Protein Metabolism  The improved growth r e s p o n s e ,  increased protein deposition  e s p e c i a l l y o f muscle p r o t e i n and d e c r e a s e d f a t a c c r e t i o n i n the carcasses  of animals i m p l a n t e d w i t h a n a b o l i c compounds have p l a y e d a  s i g n i f i c a n t r o l e i n the wide s c a l e a p p l i c a t i o n of a n a b o l i c compounds meat p r o d u c i n g Byers  in  animals.  (1982 a) r e p o r t e d i n c r e a s e d p r o t e i n growth i n y e a r l i n g  s t e e r s from 90 and 106 g f o r n o n - i m p l a n t e d c a t t l e t o 118 and 154 g f o r DES i m p l a n t e d c a t t l e .  I t was c o n c l u d e d t h a t DES i n c r e a s e d the p r o t e i n  - 21 -  Androgenic  Anabolic  Compounds  OH  = acetate  TESTOSTERONE Estrogenic  TRENBOLONE  Anabolic  ACETATE OH  Compounds  OH HO Estradiol  -j-  c  =0  Progesterone  E S T R A D I O L - 170  SYNOVEX-S  HO  HO DIETHYLSTILBESTROL  HEXOESTROL OH  O  CH  3  ZERANOL  Figure 1. S t r u c t u r a l formulae o f c e r t a i n important estrogenic and androgenic anabolic compounds.  - 22  T a b l e 2.  A n a b o l i c compounds a v a i l a b l e f o r use i n f a r m a n i m a l s  Anabolic 1.  -  Method o f Application  Compound  Species E f f e c t i v e in  Androgenic: Testosterone Trenbolone a c e t a t e  Sc I, 0 Sc. I  W, S, H C, B, Turkey  Estrogenic: DES** DES-propionate** E s t r a d i o l - 173 Hexosterol** Zeranol *** Phytoestrogens *  Sc. I, 0 Sc. I Sc. I Sc. I F  2  Androgen +  F  S, V S, W, S, W  B H S H, V, L  Estrogen  Testosterone & e s t r a d i o l T e s t o s t e r o n e & e s t r a d i o l benzoate * Trenbolone acetate & e s t r a d i o l Trenbolone a c e t a t e & h e x o e s t r o l Trenbolone a c e t a t e & z e r a n o l 1  Sc. Sc. Sc. Sc. Sc.  I I I I I  H, V H S, B, V, W, Boars S S, V  cont'd.  Table 2 c o n t ' d . .  Method of Application  A n a b o l i c Compound 4.  Progestogens Melongesterone  5.  a c e t a t e (MGA)**  0  H  Sc. I Sc. I  S S  E s t r o g e n s + Progestogens Progesterone Progesterone  & e s t r a d i o l benzoate & estradiol  5  t r a d e name  =  Finaplix  t r a d e name  =  Ralgro  t r a d e name  =  Implix  **Trade name  =  Synovex-H  5  t r a d e name  =  Synovex-S  6  t r a d e name  =  Implix  7  t h o u g h 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 c o u m e s t r o l has been i n d i c a t e d ( O l d f i e l d et a l . ,  S = Steer;  B  =  Bf  BM  Bull; W =  C = C u l l e d beef cow; V  =  * = naturally occurring i n ** = synthetic ***  Species E f f e c t i v e in  1966)  Wether Lambs Veal c a l f ; H  =  Heifer  plants  compounds  = m o d i f i e d form of n a t u r a l l y o c c u 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 i m p l a n t ; 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 greater genetic p o t e n t i a l f o r p r o t e i n growth. a l s o c o n c l u d e d t h a t a n a b o l i c compounds  Besides,  Byers  (1982  b)  d i v e r t e d energy t o p r o t e i n  s t o r a g e which r e s u l t e d i n g r e a t e r p r o t e i n a c c u m u l a t i o n and l e s s  f a t at  any s e l e c t e d w e i g h t .  carcass  From a d e t a i l e d c h e m i c a l a n a l y s i s  of the  o b t a i n e d from DES i m p l a n t e d s t e e r s , Rumsey e t a l . (1981)  observed  i n c r e a s e d p r o t e i n g a i n and c o n c l u d e d t h a t DES i n c r e a s e d the r a t e of  gain  (17% h i g h e r )  and p r o t e i n d e p o s i t i o n (28% h i g h e r )  and reduced f a t  deposition.  A s i m i l a r improvement i n the l i v e weight g a i n and p r o t e i n  d e p o s i t i o n due t o Synovex-S i m p l a n t a t i o n i n s t e e r s has a l s o r e p o r t e d (Rumsey, 1982).  been  Z e r a n o l , though weaker i n i t s r e s p o n s e ,  has  a l s o been shown t o improve l i v e weight g a i n 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 r e s e a r c h p e r t a i n i n g t o the a n a b o l i c e f f e c t s of a n d r o g e n i c a n a b o l i c compounds,  e s p e c i a l l y trenbolone acetate e i t h e r  a l o n e or i n c o m b i n a t i o n w i t h o t h e r a n a b o 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 t h a t a c o m b i n a t i o n of  t r e n b o l o n e a c e t a t e and z e r a n o l i n s t e e r s r e s u l t e d i n improved l i v e weight g a i n and n i t r o g e n r e t e n t i o n and the r e s u l t a n t c a r c a s s more l e a n meat (muscle mass) and p r o t e i n and l e s s (1982) s t u d i e d f a t t y a c i d s y n t h e s i s a n a b o l i c compounds estradiol-17$)  fat.  contained  Burch et_ a l .  i n wether lambs i m p l a n t e d w i t h  ( p r e p a r a t i o n c o n t a i n i n g t r e n b o l o n e a c e t a t e and  and found a r e d u c t i o n i n the a c t i v i t i e s o f  associated with f a t t y acid synthesis, acetyl-co A carboxylase.  f a t t y a c i d synthetase  The e f f e c t of v a r i o u s  enzymes and  a n a b o l i c compounds  on  - 25  -  t h e body growth and p r o t e i n m e t a b o l i s m 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, It  1981; P r e s t o n ,  1975).  i s apparent t h a t a n a b o l i c compounds  c o m p o s i t i o n i n farm animals..  a l t e r growth and body  Most of the compounds  seem t o 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 t o 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 a n a b o l i c  compound,  t r e n b o l o n e a c e t a t e on muscle p r o t e i n m e t a b o l i s m was s t u d i e d u s i n g n o n - r e u t i l i z a b l e amino a c i d , N - M e t h y l h i s t i d i n e T  1978a).  (Vernon and B u t t e r y  S i n c e the major c o n t r o l over muscle p r o t e i n metabolism  to be hormonal, the need f o r f u t u r e r e s e a r c h on the e f f e c t of compounds  on muscle p r o t e i n m e t a b o l i s m i s  iii.  the  appears  anabolic  obvious.  Endogenous Hormonal Changes as Influenced by the Application of anabolic compounds  A number of hormones  a d m i n i s t e r e d exogenously  have been shown t o  a l t e r the c i r c u l a t i n g c o n c e n t r a t i o n of endogenous hormones  in  animals.  Thus the m a n i p u l a t i o n of growth and metabolism i n farm animals i n f l u e n c e d by the a d m i n i s t r a t i o n of exogenous hormones a t t r i b u t e d t o the subsequent  c o u l d p a r t l y be  changes i n the endogenous hormones  GH, i n s u l i n , t h y r o i d hormones, growth f a c t o r s .  as  p r o l a c t i n , somatomedins  and o t h e r  such as serum  The i n f l u e n c e o f a l l the endogenous hormones on growth  and body c o m p o s i t i o n of a n i m a l s was d i s c u s s e d  e a r l i e r (Section II).  A  summary of the changes o c c u r r i n g i n the endogenous c i r c u l a t i n g hormone c o n c e n t r a t i o n 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 a n a b o l i c compounds  i s p r e s e n t e d i n T a b l e 3.  The changes i n the  endogenous hormone c o n c e n t r a t i o n f o l l o w i n g the a d m i n i s t r a t i o n o f  T a b l e 3.  Changes i n the endogenous hormone c o n c e n t r a t i o n 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 n a b o l i c compounds  Endogenous Hormones Serum A n a b o l i c Compound  Growth Hormone  Insulin  Thyroxine  Prolactin  Somatomedins  b  Growth-Factors  Estrogenic Anabolic Compounds: a.  Zeranol  Increased ( 1 , 2, 3)*  Increased ( 1 , 4, 5)  Unchanged or slightly depressed (6)  Depressed (3)  Unchanged (4)  Unchanged (4)  Unchanged (2)  i  Depressed (3) b.  DES  c.  Synovex-S  d.  Estradiol-170  Increased ( 3 , 7, 8, 9 , 10) N.A. Increased (13)  Increased ( 7 , 8, 9, 10) N.A. Increased (13)  ON  Increased  Increased (11)  N.A.  d  N.A.  N.A.  N.A.  N.A.  N.A.  (13)  Slightly Increased (13)  Depressed ( 1 3 , 14)  No Change (13)  N.A.  N.A.  N.A.  Increased (12) Depressed  '  Androgenic Anabolic Compound: a.  Trenbolone acetate  Unchanged (14)  Unchanged (13 ,14)  changes o c c u r r i n g i n farm animals are o n l y c o n s i d e r e d i n the t a b l e cont d... 1  Table 3 cont'd. b somatomedin or somatomedin-like a c t i v i t y c numbers i n the parenthesis indicate reference number d N.A. not available for farm species References: (Number corresponds to number i n 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 i s evident that the a p p l i c a t i o n of exogenous hormones, such as anabolic compounds, a l t e r endogenous anabolic hormone status, the mechanism by which they evoke such changes or the dynamics of endogenous hormone secretion i s not c l e a r l y understood.  Ill  a.  Methodological Aspects to Study Protein Metabolism i n Animals Treated with Anabolic Compounds Measurement of Protein Synthesis Protein synthesis i n 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 l a t t e r . A v a r i e t y of techniques  are available to measure protein synthesis i n  the whole body or i n selected tissues and have been exhaustively reviewed (Waterlow  1.  a l . , 1978).  Continuous Infusion Method  The continuous  infusion of a labelled amino acid intravenously  into the metabolic amino acid pool of the body has thus f a r been the most commonly used method for the determination of protein synthesis in the whole body or i n selected tissues of the body (Garlick et^ a l . , 1973; Garlick et a l . , 1976b).  B r i e f l y when a labelled amino acid ( H or  *C) i s introduced continuously into the blood, after some time the  ll  3  -  29  -  s p e c i f i c a c t i v i t y of the i n t r o d u c e d amino a c i d i s r a i s e d t o a p l a t e a u (steady  s t a t e ) and remains e s s e n t i a l l y c o n s t a n t d u r i n g the r e s t of the  infusion period.  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 e e 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 t e of protein synthesis al.,  1973)-.  i n individual tissues  can be e s t i m a t e d ( G a r l i c k e t  The whole-body r a t e of p r o t e i n s y n t h e s i s  from the amino a c i d f l u x (Waterlow and Stephen, been d i s c u s s e d  can be c a l c u l a t e d  1968).  The method has  i n c o n s i d e r a b l e d e t a i l by G a r l i c k e t a l . (1973) and  G a r l i c k et a l . , ( 1 9 7 6 b ) .  The a p p l i c a t i o n of t h i s method t o farm a n i m a l s  has been r e p o r t e d i n sheep by B u t t e r y et a l . ( 1 9 7 5 ) , i n the p i g by G a r l i c k e t a l . ( 1 9 7 6 a ) , and i n c a t t l e by L o b l e y e t a l . ( 1 9 8 0 ) .  ii.  Feeding Method  An a l t e r n a t i v e to the c o n t i n u o u s  i n f u s i o n method i s the method  i n v o l v i n g the f e e d i n g of the l a b e l l e d amino a c i d i n c o r p o r a t e d i n t o an agar g e l (Harney et a l . , 1976).  T h i s method a v o i d s  the c a n n u l a t i o n and  r e s t r a i n i n g of the a n i m a l d u r i n g the l o n g i n f u s i o n p e r i o d and has r e p o r t e d t o y i e l d s l i g h t l y d i f f e r e n t e s t i m a t e s of s y n t h e t i c compared t o the c o n t i n u o u s  been  rates  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 t u d y i n g p r o t e i n s y n t h e s i s  is  p r e s e n t e d i n T a b l e 4. b.  Measurement of Muscle Protein Degradation 1.  Isotoplc Method  I s o t o p i c methods have o f t e n used the d i s a p p e a r a n c e 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  (Garlick  - 30 -  Table 4.  In v i t r o and i n vivo methods f o r the quantitation of protein turnover i n d i f f e r e n t species (Summary)  Species  Method  Protein  Reference  PROTEIN SYNTHESIS PULSE LABELLING Mixed  Sarcoplasmic  1I+  C - Na C0  3  Piglet  Perry (1974)  C - Na C0  3  Piglet  Perry (1974)  Rat  Lajtha et a l . (1957)  Rat  L i and Goldberg (1976)  2  Mixed M y o f i b r i l l a r  ll+  Mixed Muscle  lk  Intact  Soleus  2  C  - Lysine  U( C) - Tyrosine (in v i t r o ) 11+  CONTINUOUS INFUSION Mixed Muscle  Lysine infusion  Rat  Waterlow & Stephen (1968)  Mixed Muscle  Glycine infusion  Rat  Garlick (1969)  *C - Tyrosine infusion  Pig  Garlick et al.(1976a)  C — Tyrosine infusion  Rat  Garlick et a l . (1973)  Cattle  Lobley et a l . (1980)  Pig  Simon et a l . (1978)  Mixed Muscle & Liver  ll  Mixed Muscle & Whole Body  1 4  Whole Body & Tissues  3  Tissues (Muscle & Liver)  H - Tyrosine & H Leucine Infusion  1 4  3  C - Leucine or C - Lysine  -  31  -  Table 4 c o n t ' d . . . .  Protein  Reference  Method  Species  Na C0  Rat  M i l l w a r d (1970)  Rat  Young et a l . (1971)  PROTEIN DEGRADATION Mixed S a r c o p l a s m i c & Myofibrillar Mixed  lh  C  -  2  3  ***C - A s p a r t i c a c i d  Muscle  Aldolase, Actin & Myosin Mixed  Sarcoplasmic  Mixed  Muscle  *C -  Phenylalanine  Rabbit  Velick  C  Na C0  Piglet  Perry  Tyrosine release (in vitro)  Rat  L i & Goldberg  Methylhistidine  Rat, Rabbit, Man  Young & Munro (1978)  Methylhistidine  Cattle  Harris & Milne  (1981a)  Methylhistidine  Sheep*  Harris & Milne  (1980)  Methylhistidine  Pig*  Harris & Milne  (1981b)  14  lk  Myofibrillar  Proteins  N -  Myofibrillar  Protein  N -  Myofibrillar  Protein  N -  Myofibrillar  Protein  N -  T  -  2  3  T  X  T  * 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  (1956) (1974) (1976)  - 32  -  e t a l . , 1973; Waterlow and Stephen, 1967).  The r e s u l t s o b t a i n e d from  such methods are o f t e n c o m p l i c a t e d 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 a c i d s and a l s o due t o the h e t e r o g e n i e t y of p r o t e i n d e g r a d a t i o n r a t e s ( G a r l i c k e t a l . , 1976 a).  A comparison of d e g r a d a t i o n r a t e s of m y o f i b r i l l a r p r o t e i n s  l a b e l l e d amino a c i d s has shown g r e a t d i s c r e p a n c i e s i n h a l f - l i f e w h i c h ranges  from 20 t o 145 days (Swick and Song, 1974).  Hegsted (1970) found t h a t r a t s g i v e n  ll  using values  F a s h a k i n and  *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 d a y s , w h 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 v a l u e s o f 3 days o r less  (Anker, 1960).  S i m i l a r d i s c r e p a n c i e s i n the d e g r a d a t i o n r a t e s due  t o the k i n d of t r a c e r used were r e p o r t e d by Swick and Ip ( 1 9 7 4 ) .  The  r a t e of decay of r a d i o a c t i v i t y from a a l b u m i n was about 30% s l o w e r when 1 4  C-guanidino-arginine  was used than  l l 4  C - b i c a r b o n a t e (Swick and  Ip,  1974).  ii.  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 o c i a t e d w i t h the r e u t i l i z a t i o n of amino a c i d s have l e a d 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 d e g r a d a t i o n which i s not r e u t i l i z e d f o l l o w i n g d e g r a d a t i o n .  To  s e r v e as an i n v i v o i n d e x of muscle p r o t e i n breakdown, the amino a c i d s e l e c t e d s h o u l d 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, 1.  1970):  Should be c h e m i c a l l y m o d i f i e d a f t e r the p e p t i d e bond synthesis.  2.  Such c h e m i c a l l y m o d i f i e d groups s h o u l d not undergo once they appear i n the p r o t e i n bound amino a c i d .  exchange  - 33 -  3.  The concentration of the amino acid should be known or should be constant i n the muscle protein.  4.  Should not be formed to an important  extent i n other  proteins. 5.  Should be released at the same time as other amino acids from the completed protein.  6.  Should not undergo further metabolism i n the body.  7.  Should have a low renal threshold.  8.  Should be q u a n t i t a t i v e l y excreted i n the urine.  N Methylhistidine  (N MH) or 3-Methylhistidine (3-MH) has been  T  T  i d e n t i f i e d as one such amino acid s a t i s f y i n g a l l the above mentioned c r i t e r i a (Young and Munro, 1978).  B r i e f l y , N -MH originates from the T  degradation of a c t i n and some species of myosin heavy chains i n which i t i s present after the p o s t - t r a n s l a t i o n a l methylation of s p e c i f i c h i s t i d i n e residues (Munro and Young, 1978;  Figure 2).  I t s synthesis,  metabolism and v a l i d i t y to serve as an in_ vivo index of m y o f i b r i l l a r protein degradation  i n 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 m y o f i b r i l l a r protein degradation rates comparable to the *C-Na2C03 method of Millward (1970) and seems ll  to be an a t t r a c t i v e technique  i n large farm animals as i t does not  require the use of isotopes and the destruction of the  animal.  Harris and h i s co-workers validated this technique farm species and concluded  for use i n  that the urinary N -MH excretion to be a T  - 34 -  MUSCLE  SAME t  -METHIONINE 1  CH,  PROTEIN BREAKDOWN ~\ (actin & . 1 myosin) (RECYCLING) (RECYCLII Transamination'-'' & Oxidation  +  AMINO ACIDS  N  L  M  2. BREAKDOWN 3  H  SYNTHESIS  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 -methylhistidine metabolism. T  - 35 -  v a l i d index of m y o f i b r i l l a r protein degradation i n c a t t l e (Harris and Milne, 1981 a) but an i n v a l i d index i n both sheep and the pig (Harris and Milne, 1980; Harris and Milne, 1981 b).  In the l a t t e r species the  large body pool of non-protein bound N -MH has been attributed for i t s T  u n s u i t a b i l i t y to serve as an index of m y o f i b r i l l a r protein degradation (Harris and Milne, 1980,  1981b).  It could be concluded that the technique of measuring N -MH excretion i n urine presents a great potential to study protein T  degradation i n 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 s k e l e t a l muscle protein, the technique of measuring N -MH i n urine presents potential for studying muscle T  protein metabolism i n such conditions and to understand the mechanism of action of anabolic compounds on the s k e l e t a l 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 v a l i d i t y of the method to serve as an index of m y o f i b r i l l a r protein degradation since N*-MH i s also present i n tissues other than the s k e l e t a l muscle.  The v a l i d i t y of the method, however, was  reappraised by Harris (1981). A summary of methods available for studying protein degradation i s presented i n Table 4.  c.  Collagen Turnover Collagen i s a hydroxyproline r i c h protein, representing about 30%  of the t o t a l body protein.  It i s a predominant protein i n the skin,  - 36  -  bone and tendon and performs m a i n l y a s t r u c t u r a l r o l e .  The  chemistry,  b i o c h e m i s t r y and the m e t a b o l i c a s p e c t s of c o l l a g e n have been reviewed by Kivirikko  (1970).  i.  Measurement of Collagen Synthesis  During  collagen biosynthesis,  p o l y p e p t i d e c h a i n and i s  p r o l i n e i s i n c o r p o r a t e d i n t o the  then h y d r o x y l a t e d to h y d r o x y p r o l i n e  protocollagen-proline-hydroxylase  by the  system ( P e t e r k o f s k y and U d e n f r i e n d ,  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 g e n as l a b e l l e d h y d r o x y p r o l i n e has been w i d e l y used t o measure synthetic rates synthesis,  (Robins,  amino a c i d (McKee et a l . , 1 9 7 8 ) , and (b)  ( I l a n and S i n g e r ,  1979).  removed  Measurement of Collagen Degradation  tissues  c o l l a g e n have been found  in  of l i v i n g a n i m a l s ( H o r w i t z e t a l . , 1977; L i o t t a e t a l . ,  D u r i n g the d e g r a d a t i o n of c o l l a g e n , h y d r o x y p r o l i n e  and e x c r e t e d i n u r i n e as f r e e or bound to s m a l l p r o t e i n s 1970).  the  1975).  The enzymes capable of d e g r a d i n g numerous  collagen  of s p e c i f i c r a d i o a c t i v i t y of the  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 p o l y p e p t i d e  from ribosomes 11.  The o t h e r methods f o r s t u d y i n g  i n c l u d e (a) the measurement  r e l e v a n t tRNA-bound measurement  1981).  collagen  is  released  (Kivirikko,  Hence, the u r i n a r y e x c r e t i o n of h y d r o x y p r o l i n e has been w i d e l y  used as an i n d e x of c o l l a g e n t u r n o v e r In humans and e x p e r i m e n t a l a n i m a l s ( K i v i r i k k o , 1970)  and to a l i m i t e d e x t e n t i n farm animals  Capen, 1971; Gordon,  1982).  (Black  and  - 37 -  The complexity of growth processes i n animals, the role played by various endogenous and exogenous hormonal factors on animal growth and the need f o r studies on the mechanism by which anabolic compounds influence growth and metabolism of meat producing animals are readily apparent.  - 38 -  STUDY I  METABOLIC  A S P E C T S OF ANABOLIC COMPOUND I N GROWING B E E F STEERS  ACTION  Study I was conducted to explore the various metabolic  effects of  estrogenic anabolic compounds i n growing beef steers and includes the following experiments: EXPERIMENT IA:  Effect of anabolic compounds on growth and nitrogen metabolism i n growing beef steers.  EXPERIMENT IB:  Muscle protein metabolism In steers as determined by the urinary  N -methylhistidine T  excretion as an i n vivo index of m y o f i b r i l l a r protein breakdown: Developmental aspects of muscle protein metabolism and the effect of anabolic compounds. EXPERIMENT IC:  Collagen metabolism i n growing beef steers as assessed  by the urinary  hydroxyproline  excretion and the effect of estrogenic anabolic compounds.  - 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 e f f i c i e n c y of meat production.  The growth promoting  e f f e c t s of zeranol (Sharp and Dyer, 1971;  Borger et a l . , 1973),  d i e t h y l s t i l b e s t r o l (Clegg and Cole, 1954;  Oltjen et a l . , 1973),  Synovex-S (Kahl et a l . , 1978; (Heitzman et a l . , 1977)  Rumsey, 1982)  acetate  have been reported and the effect of hormones on  the growth and body composition and Topps (1981).  and trenbolone  has been recently reviewed by Galbraith  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 et a l . , 1978).  It was  (Buttery  suggested through these studies that anabolic  compounds e l i c i t changes both at the endocrine and at the  metabolite  l e v e l , the l a t t e r through changes In the intermediary metabolism. In ruminants, estrogenic anabolic compounds have been reported to increase the endogenous concentrations of growth hormone and (Preston, 1975)  and thyroid hormones (Kahl et a l . , 1978).  insulin  The  effects  of anabolic compounds i n a l t e r i n g nitrogen retention (Vanderwal, 1976), f r a c t i o n a l rate of protein synthesis i n d i f f e r e n t tissues (Vernon and Buttery, 1978b) and protein retention (Rumsey, 1982) reported.  have also been  - 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 t u d i e d e x t e n s i v e l y , o n l y a few a t t e m p t s have been made t o e l u c i d a t e t h e m e t a b o l i c e f f e c t s o f more commonly used a n a b o l i c compounds, v i z . Z e r a n o l and Synovex-S, e s p e c i a l l y on plasma f r e e amino a c i d s and h e p a t i c  enzymes  o f amino a c i d c a t a b o l i s m . This  study was t h e r e f o r e conducted t o i n v e s t i g a t e t h e e f f e c t o f  e s t r o g e n i c a n a b o l i c compounds  ( Z e r a n o l , DES, Synovex-S) on n i t r o g e n  m e t a b o l i s m i n growing beef s t e e r s .  A t t e n t i o n was f o c u s s e d on t h e plasma  l e v e l s o f u r e a - N , a l p h a amino-N, amino a c i d and h e p a t i c l e v e l s o f amino acid catabolizing  enzymes.  MATERIALS AND METHODS Experimental Animals and t h e i r Management H e r e f o r d beef s t e e r s , w e i g h i n g a p p r o x i m a t e l y 380 k g , were u s e d . Upon a r r i v a l at the U n i v e r s i t y o f B r i t i s h Columbia Beef C a t t l e T e a c h i n g and R e s e a r c h U n i t , t h e a n i m a l s were weighed and i n j e c t e d w i t h 1  Poten-ADE  t o c o r r e c t any v i t a m i n d e f i c i e n c y a l o n g w i t h P e n l o n g - S  t o prevent i n f e c t i o n s due t o p e n i c i l l i n o r s t r e p t o m y c i n  2  plus  sensitive  3  bacteria.  At t h i s time C o n t r a v a c  was a l s o a d m i n i s t e r e d t o p r e v e n t  Infectious  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  (PI-3).  I d e n t i f i c a t i o n tags were a t t a c h e d t o t h e ears o f the s t e e r . The a n i m a l s were h e l d i n groups o f f o u r s t e e r 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 . f l o o r bedded w i t h wood s h a v i n g s . 1  3  The wood s h a v i n g s were p e r i o d i c a l l y  , R o g a r / S T B , London, O n t a r i o , 2  Connaught  The pens c o n t a i n e d an e a r t h e r n  Canada.  Laboratories L t d . , Willowdale, Ontario,  Canada.  - 41  replaced.  -  Water was p r o v i d e d ad l i b i t u m through a water bowl p l a c e d i n  each pen. The a n i m a l s were group f e d by p l a c i n g the f e e d i n an open wooden trough.  I n i t i a l l y they were f e d a r a t i o n composed of chopped hay and  barley.  The chopped hay c o n t e n t of the r a t i o n was reduced  gradually  and the a n i m a l s were a l l o w e d to get used to a f u l l c o n c e n t r a t e  diet.  When the s t e e r s were f u l l y adapted to a h i g h 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 c k of d i g e s t i v e d i s o r d e r s  and r e g u l a r feed i n t a k e , the  were i n t r o d u c e d t o the e x p e r i m e n t a l d i e t .  A l l feed troughs  steers  were  provided with free 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  n o r m a l l y c a r r i e d out d a i l y at 0730 h and a g a i n at 1430  h.  The body weight of the s t e e 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 w e i g h i n g b r i d g e .  T h i s procedure was done w i t h o u t much  e x c i t e m e n t and the body weight was taken a f t e r an o v e r n i g h t  fast.  Experimental Procedure S i x t e e n H e r e f o r d s t e e 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 t o one of the f o l l o w i n g f o u r  treatments:  1.  Unimplanted c o n t r o l  2.  36 mg i m p l a n t of  3.  36 mg i m p l a n t of D i e t h y s t i l b e s t r o l  4.  One i m p l a n t of S y n o v e x - S  Zeranol (DES)  1  The a n a b o l i c compounds were i m p l a n t e d i n the form of slow p e l l e t s at the base of the e a r .  releasing  T h i s procedure i n v o l v e d the placement  Synovex-S, one i m p l a n t c o n t a i n s 200 mg p r o g e s t e r o n e e s t r a d i o l benzoate.  and 20 mg  - 42 -  of implant-gun needle between the skin folds of the pinna and the slow deposition of the p e l l e t s .  The needle, a f t e r the deposition of the  p e l l e t s , was c a r e f u l l y removed and the presence of the p e l l e t s i n the skinfold was assured.  The control groups were also subjected to the  same procedure but without the deposition of any hormone p e l l e t s .  The  steers were fed twice daily with a f i n i s h i n g 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 i n t e r v a l s .  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 i n heparinized vacutainer tubes  and were placed i n an i c e bath.  Blood samples were collected from the  steers a f t e r an overnight  The samples were centrifuged at  fast.  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 i n each treatment group on day 28, 42 and 56 following the implantation of anabolic compounds (See Page 65 for d e t a i l s of urine collection).  Urine samples were stored at -20°C u n t i l analyzed for  metabolites. The experimental  steers were s a c r i f i c e d 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 i n l i q u i d nitrogen within minutes after c o l l e c t i o n . were stored at -20°C u n t i l analyzed catabolism.  The hepatic tissues  for enzymes of amino acid  - 43 -  Table 5.  Ration Composition (Expt. IA)  % of Ration (as  Ingredient  Steam Rolled Barley  Fed)  89.0  Chopped Grass Hay  4.0  Soybean Meal  4.0  Ground Limestone  2.0  Mineral - Vitamin Mixture2 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 D (120 gm, 176000 I.U./g), CuSO^ (40 g), MnSO (600 g), ZnS0 (1000 g), prodine 17% I (72 g, 170 mg I/g) and wheat shorts (to 5 Kg). 2  k  L  - 44 -  A n a l y t i c a l 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 1968).  (FDNB) as described by (Goodwin,  Briefly, the plasma was deproteinized using a hydrochloric  acid-tungstate reagent.  The protein free f i l t r a t e was incubated at 70°C  for 15 min in the presence of l-fluoro-2, 4-dinitrobenzene bath.  in a water  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 t h e ammonia produced was a l l o w e d t o r e a c 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 t h e presence of sodium n i t r o p r u s s i d e . c o n c e n t r a t i o n of the f i n a l r e a c t i o n product, indophenol  The  (blue  chromogen), which i s d i r e c t l y p r o p o r t i o n a l t o t h e ammonia c o n c e n t r a t i o n , was measured s p e c t r o p h o t o m e t r i c a l l y a t 670 nm.  Plasma Free Amino Acids B l o o d samples c o l l e c t e d from two s t e e r s i n each t r e a t m e n t group were u s e d . ion-exchange  The plasma c o n c e n t r a t i o n o f f r e e AA was determined by an c h r o m a t o g r a p h i c method u s i n g an amino a c i d a n a l y z e r  [Dionex, Model D500],  Amino a c i d a n a l y s i s was conducted a t the AAA  L a b o r a t o r y (AAA L a b o r a t o r y , Mercer I s l a n d , Washington,  U.S.A.).  Urinary Total Nitrogen U r i n e samples were thawed a t room temperature and t h e t o t a l n i t r o g e n c o n t e n t 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 a t 4 ° C , homogenized i n 10 ml i c e c o l d KC1 (0.14 M) and the t y r o s i n e a m i n o t r a n s f e r a s e  [Tyrosine:  2 - o x o g l u t a r a t e a m i n o t r a n s f e r a s e , E.C. 2 . 6 . 1 . 5 ] a c t i v i t y was determined a c c o r d i n g t o the method of Granner and Tomkins ( 1 9 7 0 ) . B r i e f l y , a 0.2 ml s u p e r n a t a n t o f the crude l i v e r homogenate a t a p p r o p r i a t e d i l u t i o n s was added t o c u v e t t e s c o n t a i n i n g the f o l l o w i n g reagent mixture: 7.0,  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  .04 m l ) , p y r i d o x a l - 5 ' - p h o s p h a t e  (.005M, pH6.5,  .02 m l ) .  The a s s a y  - 46  m i x t u r e was i n c u b a t e d f o r 5 - 3 0  -  minutes a t 37°C.  The e n z y m a t i c  r e a c t i o n was stopped by the a d d i t i o n o f 10 N KOH (0.14 ml) w i t h immediate m i x i n g .  The assay m i x t u r e was i n c u b a t e d f o r a n o t h e r 30  minutes a t 37°C and the absorbance was read a t 331 nm a g a i n s t a  'zero  t i m e ' b l a n k prepared by adding the KOH t o the r e a c t i o n components b e f o r e the  a d d i t i o n of the enzyme.  The p r o t e i n c o n t e n t i n the l i v e r  was determined by the method of Lowry et a l . ( 1 9 5 1 ) .  homogenate  One u n i t of TAT i s  d e f i n e d as the q u a n t i t y which c a t a l y s e s the f o r m a t i o n of one u mole of p-hydroxyphenyl  p y r u v a t e per minute a t 37°C.  S t a t i s t i c a l Analysis The d a t a were s u b j e c t e d to the a n a l y s i s T o r r i e , 1960) range t e s t .  of v a r i a n c e ( S t e e l and  and the means were s e p a r a t e d by the Newman-Keul's m u l t i p l e The s l o p e s of the body weight of the i n d i v i d u a l t r e a t m e n t  groups over time were s u b j e c t e d t o a n a l y s i s  of c o v a r i a n c e .  The amino  a c i d d a t a were s u b j e c t e d t o s i n g l e degree of freedom o r t h o g o n a l t e s t ( S t e e l and T o r r i e , 1960) compared f o r each amino a c i d .  contrast  and the t r e a t m e n t d i f f e r e n c e s were A l l s t a t i s t i c a l a n a l y s e s were c a r r i e d out  u s i n g UBC BMD:10V l i n e a r h y p o t h e s i s  computer program.  RESULTS The i n f l u e n c e of Z e r a n o l , DES, the  s t e e r s i s p r e s e n t e d i n F i g u r e 3.  compounds  and Synovex-S on body weight of Steers treated w i t h anabolic  g r a i n e d more r a p i d l y (P < .01)  than the c o n t r o l s .  of the body weight of the t r e a t m e n t s were s i m i l a r (P = . 1 5 ) . the  The  slopes  However,  body weight over time r e g r e s s i o n e q u a t i o n s o b t a i n e d f o r the  t r e a t m e n t s were found t o be d i f f e r e n t (P < . 0 1 ) .  The growth due t o DES  O A + •  = CONTROL r ZERRNOL = OES s STNOVEX  14 28 42 56 70 84 98 DAYS AFTER IMPLANTATION  112  E f f e c t o f a n a b o l i c compounds on growth i n s t e e r s . Each p o i n t r e p r e s e n t s mean o f f o u r o b s e r v a t i o n s . Regression e q u a t i o n s : C o n t r o l , Y = 370.9 + 1.27x; Z e r a n o l , Y = 379.3 + 1.38x; DES, Y = 377.1 + 1.56x; Synovex-S, Y - 373.1 + 1.54x. T e s t o f h y p o t h e s i s f o r common s l o p e , P = 0 . 1 5 ; T e s t of h y p o t h e s i s f o r common e q u a t i o n , P = 0.0006.  - 48  -  and Synovex-S was s l i g h t l y g r e a t e r than t h a t induced by Z e r a n o l . The PUN and aAN c o n c e n t r a t i o n s as i n f l u e n c e d by d i f f e r e n t a n a b o l i c t r e a t m e n t s on d i f f e r e n t p e r i o d s presented i n Figure 4.  f o l l o w i n g the i m p l a n t a t i o n are  The c o n c e n t r a t i o n of both the m e t a b o l i t e s due t o  the a n a b o l i c t r e a t m e n t s was lower than t h a t i n the c o n t r o l group (P < .05).  The e f f e c t of a n a b o l i c compounds was v e r y 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 significant  (P < .01)  p e r i o d i n t e r a c t i o n was n o t i c e d f o r both PUN and aAN. periods  treatment x  When a l l the time  (14, 28, 42, & 56 days) were t a k e n i n t o c o n s i d e r a t i o n  (Figure  5 ) , t h e r e was a p p r o x i m a t e l y 15-23% r e d u c t i o n i n PUN and a 17-19% r e d u c t i o n i n aAN c o n c e n t r a t i o n s due t o the i m p l a n t a t i o n of Z e r a n o l , and Synovex-S.  However, t h e r e was no d i f f e r e n c e  (P > .05)  DES  among the  d i f f e r e n t a n a b o l i c compounds ( Z e r a n o 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 e e AA as i n f l u e n c e d by the t r e a t m e n t s are  p r e s e n t e d i n T a b l e 6.  Considering  the marked r e d u c t i o n i n t h e 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  t h a t a c o r r e s p o n d i n g 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 a n a b o l i c compounds on n i t r o g e n metabolism i n growing The c o n c e n t r a t i o n of most of the AA, both e s s e n t i a l and was found t o be s i g n i f i c a n t l y lower (P < .05)  steers.  non-essential,  i n steers 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  general,  the c o n c e n t r a t i o n of plasma AA was more reduced due t o DES and Synovex-S than due t o Z e r a n o l I m p l a n t a t i o n .  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 g u r e 4.  Plasma u r e a n i t r o g e n (PUN) and a l p h a amino n i t r o g e n (aAN) concentrations i n steers implanted w i t h various anabolic compounds. Each p o i n t r e p r e s e n t s mean v a l u e s o b t a i n e d from four steers.  16r  ALPHA AMINO-N  UREA-N  10r  (P<-os)  (P<.01) 6 e  10 d  6  E4  ••••oxd ••••  •••• 4****  F i g u r e 5.  8  •••• O/^  O)  E  •••• •••• ••••  a  •••• •••• ••••  8  14 Q12  CONTROL ZERANOL DES SYNOVEX-S  »• «  4& TT  •••• +••• •••+  • • • • J^V. • < •••• j _ C  ••••~0^ ••••} C •••• •'n .•••• v ^ f  !••••  A oxc  ••••LTO/ -Si Itttff » M .  0  Plasma urea-N and a l p h a amino-N c o n c e n t r a t i o n s i n s t e e r s i m p l a n t e d w i t h a n a b o l i c compounds. Each bar r e p r e s e n t s the mean ± SE o f v a l u e s f o r f o u r s t e e r s on days 14, 2 8 , 42 and 56 f o l l o w i n g t h e i m p l a n t a t i o n (n=16). » 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 . a  b  o  Table 6. Plasma free amino a d d levels i n steers on 28 days following the lmplantlon of various anabolic compounds 1  SIGNIFICANCE OF DIFFERENCE  ANABOLIC TREATMENTS AMINO ACID  CONTROL (1)  SYNOVEX-S (4)  DES (3)  ZERANOL (2)  Alanine (ALA) Arglnine (ARG) Aspartic Acid (ASP) Glutamic Acid (GUI) Glycine (GLY) Histldine (HIS) Isoleucine (ILE) Leucine (LEU) Lysine (LYS)  26.2 ± 14.0 ± 2.5 ± 14.9 ± 24.8 ± 7.5 ± 12.0 ± 18.8 ± 15.9 ±  6.7 3.0 .9 3.7 .8 1.3 .1 1.4 4.4  jimole/100 ml 15.0 ± 2.7 9.2 ± 5.1 ± .5 3.08 ± 1.8 ± .4 1.8 ± 6.5 ± 1.0 5.6 ± 11.8 ± 2.1 11.2 ± 2.8 ± .5 1.9 ± 7.2 ± .8 4.6 ± 10.2 ± 1.1 5.6 ± 6.0 t 1.0 3.0 ±  Methionine (MET) {Phenylalanine (PHE) Proline (PRO) Serine (SER) Threonine (THR) [Tyrosine (TYR) Valine (VAL) Glutamine (GLN) C i t r u l l i n e (CIT) Ornithine (ORN)  2.8 ± 6.2 ± 20.3 ± 13.2 ± 9.3 ± 5.6 ± 32.2 t 25.7 ± 9.5 ± 10.4 ±  .1 .4 4.2 2.0 .3 .2 1.5 2.4 2.7 1.0  1.7 ± .1 3.5 ± .5 10.8 ± .9 6.3 ± .8 4.6 ± .2 3.7 * .4 19.6 ± 2.2 11.3 ± 1.9 6.4 ± .1 4.5 ± .8  3.8 10.5 1.0 2.4 .7 1.8 1.8 5.6 3.8 11.5 .7 1.5 .9 4.5 .9 6.5 .8 2.5  &  1.3 1.9 7.1 7.0 3.0 2.0 9.6 5.7 3.8 2.8  ± .5 ± .7 ± 3.9 ± .7 ± .1 ± .5 ± .4 ± .8 ± .6  1.3 2.4 6.4 4.8 3.0 2.7 11.7 9.0 4.4 2.9  ± ± ± ± ± ± ± ±  3.0 1.3 .2 1.6 2.9 .5 .3 .8 ± .7 5  ±  t 1.7 t 1.4 ±  t ± ± ± ±  .7 .6 .8 2.7 .7 .5  'Each value i s the mean (± SE) of data obtained from two steers. 2  Single degree of freedom orthogonal comparisons data from one animal  *P < .05 **P < .01 N.S. " Non-significant  1 Vs 2  1 Vs 3  NS  NS  NS NS  NS  *  *  Vs 4 NS  **  NS  * * ** ** *  * * ** * ** *  * * ** * ** *  * * *  * ** *  * ** *  ** * ** **  ** ** ** **  ** ** ** **  **  **  NS  NS  **  NS  NS  NS  NS  1 Vs 2, 3, 4  2 Vs 3  2 Vs 4  3 Vs 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  **  - 52  -  Plasma l e v e l s of e s s e n t i a l AA e x c l u d i n g t r y p t o p h a n were t o t a l l e d f o r each t r e a t m e n t and are p r e s e n t e d i n F i g u r e 6.  When e x p r e s s e d as a per  cent o f t h a t 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 5 1 , 29 and 30 i n Z e r a n o l , DES and Synovex-S i m p l a n t e d s t e e r s  respectively.  The amount of n i t r o g e n e x c r e t e d i n the u r i n e over a p e r i o d of 24 h by the s t e e r s i s p r e s e n t e d i n T a b l e 7.  There was a p p r o x i m a t e l y  8%,  11% and 12% r e d u c t i o n i n the u r i n a r y n i t r o g e n e x c r e t i o n due t o Z e r a n o 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 t o the control  steers. H e p a t i c l e v e l s of t y r o s i n e a m i n o t r a n s f e r a s e  as i n f l u e n c e d by  d i f f e r e n t a n a b o l i c compounds are p r e s e n t e d i n Table 8. s i m i l a r (P > .05)  In the c o n t r o l and i n the i m p l a n t e d  TAT l e v e l s were steers.  DISCUSSION The growth promoting p o t e n t i a l s of a n a b o l i c compounds have been v e r y 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. insight  The r e s u l t s of t h i s study p r o v i d e  further  i n t o the e f f e c t s of e s t r o g e n i c a n a b o 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 a s p e c t s of n i t r o g e n m e t a b o l i s m . Plasma l e v e l s of u r e a - N , aAN and f r e e 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 t e e r s  Implanted w i t h Z e r a n o l , DES  Synovex-S ( F i g u r e 4 and T a b l e 6 ) .  and  A s i m i l a r r e d u c t i o n i n the plasma  c o n c e n t r a t i o n s of u r e a - N i n c a t t l e i m p l a n t e d w i t h DES ( O l t j e n and Lehmann, 1968), lambs i m p l a n t e d w i t h Z e r a n o l (Wiggins e t a l . , 1976) DES ( P r e s t o n , 1968)  and  and i n h e i f e r s t r e a t e d w i t h t r e n b o l o n e a c e t a t e  ( G a l b r a i t h , 1980) has been r e p o r t e d .  The r e d u c t i o n i n PUN observed  t h i s study may be due t o a r e d u c t i o n i n the h e p a t i c c a t a b o l i s m of  in  - 53 -  |120r °100|  •• •••• •••• ••••  O  >o  CONTROL *  ZERANOL DES SYNOVEX-S  E80  ai ••• •••  _,40| § F i g u r e 6.  0  •••  > •  20I  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) c o n c e n t r a t i o n s i n growing s t e e r s 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 o f a n a b o l i c compounds. Each b a r r e p r e s e n t s t h e mean o f v a l u e s o b t a i n e d from two s t e e r s . T o t a l e s s e n t i a l amino a c i d s does not i n c l u d e t r y p t o p h a n .  - 54  T a b l e 7.  -  D a i l y u r i n a r y n i t r o g e n e x c r e t i o n of implanted with various  anabolic  steers  compounds 0.75  Treatment  U r i n a r y - N (mg/d/kg  Control  765.4 ±  75.3  Zeranol  705.0 ±  41.4  DES  685.2 ±  64.1  Synovex-S  673.1 ±  55.4  b  Mean of v a l u e s o b t a i n e d 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 o f s t e e r s were used oh a l l the t h r e e u r i n e c o l l e c t i o n periods. 'Mean ± SE of v a l u e s o b t a i n e d from two s t e e r s on a l l the t h r e e u r i n e c o l l e c t i o n p e r i o d s (n = 6 ) .  )  - 55 -  Table 8.  Treatment  Effect of various anabolic compounds on hepatic tyrosine aminotransferase levels i n steers  Tyrosine aminotransferase (micromoles p-hydroxyphenylpyruvate formed/min./mg protein)  Control  0.9725 ± 0.07*  Zeranol  1.1730 ± 0.04  DES  0.9885 ± 0.19  Synovex-S  0.9047 ± 0 . 0 3  *Mean ± SE of values obtained from four steers  - 56  n i t r o g e n o u s compounds.  -  Sykes (1978) suggested  t h a t the  endogenous  p r o d u c t i o n of u r e a depends l a r g e l y on the d e a m i n a t i o n of amino a c i d s . R e d u c t i o n s i n the plasma aAN c o n c e n t r a t i o n s may be a s c r i b e d t o the i n c r e a s e d r e q u i r e m e n t s of amino a c i d 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 d e c r e a s e i n the amino a c i d s produced d u r i n g p r o t e i n t u r n o v e r ( M i l l w a r d e t a l . , 1976).  These r e d u c t i o n s i n the plasma aAN were f u r t h e r  s u p p o r t e d by the r e d u c t i o n s i n b o t h e s s e n t i a l and n o n - e s s e n t i a l AA concentrations.  A r e d u c t i o n 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 c o n c e n t r a t i o n s was a l s o n o t i c e d by O l t j e n and Lehmann (1968) s t e e r s i m p l a n t e d w i t h DES.  in  McLaren e t a l . (1960) r e p o r t e d a r e d u c t i o n  i n b o t h b l o o d ammonia and u r e a and suggested  t h a t DES f a c i l i t a t e s  b e t t e r u t i l i z a t i o n of n o n - p r o t e i n n i t r o g e n .  Plasma growth hormone  a (GH)  c o n c e n t r a t i o n s were measured i n a l l the s t e e r s used i n t h i s study and found to be 130%, 231% and 219% h i g h e r i n Z e r a n o l , DES and Synovex-S i m p l a n t e d s t e e 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 i m p l a n t a t i o n ( r e s u l t s of E x p t . I I B ) .  Furthermore e s t r o g e n i c  a n a b o l i c compounds have been shown t o i n c r e a s e plasma l e v e l s of i n s u l i n i n ruminants ( P r e s t o n , 1975).  Both GH and i n s u l i n have been shown to  i n c r e a s e the t i s s u e uptake of amino a c i d s ( R i g g s , 1970; 1972).  Manchester,  Growth hormone a l s o s t i m u l a t e s N r e t e n t i o n and f a v o u r s  d e p o s i t i o n of c a r c a s s - N by s t i m u l a t i n g amino a c i d t r a n s p o r t i n t o the body t i s s u e s  (Kostyo,  1973).  Hence the r e d u c t i o n i n PUN, aAN and plasma  AA observed i n the i m p l a n t e d s t e e r s used i n t h i s study may be a t t r i b u t e d t o the i n d i r e c t e f f e c t s brought about by the changes i n growth hormone and i n s u l i n s t a t u s .  endogenous  I t may a l s o be h y p o t h e s i z e d t h a t  - 57  a n a b o l i c compounds substances  -  enhance the a b i l i t y of the a n i m a l t o draw  from the plasma f o r b u i l d i n g p r o t e i n s .  nitrogenous  The I n c r e a s e d  amount  of c a r c a s s - N d e p o s i t e d i n s t e e r s i m p l a n t e d w i t h Z e r a n o l (Sharp and Dyer, 1971), DES support  (Rumsey e t a l . , 1981)  t o the above mentioned  and Synovex-S (Rumsey, 1982)  adds  hypothesis.  The f r e e AA c o n c e n t r a t i o n s  ( T a b l e 6) i n the plasma of the  i m p l a n t e d s t e e r s are much lower than those found i n the u n i m p l a n t e d s t e e 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 s h o u l d be p o i n t e d out t h a t the f r e e amino a c i d  c o n c e n t r a t i o n of the c o n t r o l s t e e r s were s i m i l a r to t h a t r e p o r t e d by Bergen  (1979).  The reduced plasma f r e e AA p r o f i l e of the i m p l a n t e d  s t e e r s as compared t o the v a l u e s of Bergen (1979) c o u l d be due to i n c r e a s e d d i s t r i b u t i o n r a t i o (DR, c o n c e n t r a t i o n i n plasma) tissues  concentration i n tissue  to  as a r e s u l t of i n c r e a s e d AA uptake by the  (due t o hormonal changes as d i s c u s s e d  above).  Bergen  (1979)  suggested t h a t 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 t o 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 i n c r e a s e d uptake of  essential  AA i n t o p r o t e i n and t h a t h i g h plasma l e v e l s of e s s e n t i a l AA may be due t o a r e s u l t of d i e t a r y excess or e x t e n s i v e net c a t a b o l i s m of proteins.  body  A l l the s t e e r s used i n t h i s study were f e d a c c o r d i n g t o the  s p e c i f i c a t i o n s s e t by NRC f o r growing beef s t e e r s .  Considering  the  i n c r e a s e d growth r a t e of the i m p l a n t e d s t e e r s i t i s  suggested t h a t the  r e d u c t i o n i n plasma f r e e AA p r o f i l e of the i m p l a n t e d s t e e r s compared to the v a l u e s  compiled by Bergen (1979) i s a r e f l e c t i o n of i n c r e a s e d  uptake and not due t o a d i e t a r y  deficit.  tissue  -  58  -  C o n s i d e r i n g the i n c r e a s e d amount of p r o t e i n d e p o s i t e d i n the i m p l a n t e d s t e e r s as r e p o r t e d i n s e v e r a l s t u d i e s , discussion  the f o l l o w i n g g e n e r a l  on p r o t e i n metabolism would be a p p r o p r i a t e t o understand  a l t e r a t i o n s i n N metabolism.  the  The s i m p l e p r o t e i n - N m e t a b o l i s m model  d e s c r i b e d by B r u c k e n t a l e t a l . (1980) was used t o f u r t h e r d e s c r i b e the a l t e r a t i o n s i n n i t r o g e n metabolism e x h i b i t e d by the i m p l a n t e d  steers.  In t h i s model the absorbed amino a c i d - N e n t e r s the f r e e amino  acid-N  p o o l i n the body.  Due to the g r e a t e r demand of absorbed amino a c i d s  tissue protein synthesis,  for  o n l y a l i m i t e d amount o f amino a c i d - N w i l l be  c a t a b o l i z e d and e x c r e t e d as u r e a (see B r u c k e n t a l e t a l . , 1980 f o r the model).  The r e d u c t i o n i n plasma u r e a - N observed i n t h i s study Is  further i l l u s t r a t i o n .  However, not a l l u r e a i s formed as the r e s u l t  c a t a b o l i s m of amino a c i d - N p o o l .  A s u b s t a n t i a l q u a n t i t y of u r e a  d e r i v e d from ammonia absorbed from the gut ( N o l a n , 1975)  1971).  It  of  is  and s m a l l e r  q u a n t i t y from the c a t a b o l i s m of m e t a b o l i t e s such as p y r i m i d i n e s and C o r d e s ,  a  i s q u i t e p r o b a b l e t h a t these a l t e r n a t e  (Mahler  pathways  o f u r e a p r o d u c t i o n are a l s o under r e g u l a t o r y c o n t r o l i n the i m p l a n t e d steers. Decreased PUN observed i n the i m p l a n t e d s t e e r s of t h i s  study  c o u l d a l s o be due t o the m e t a b o l i c a d a p t a t i o n t o decreased p r o d u c t i o n o f u r e a 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 u r e a 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 u r e a c y c l e i n the r a t l i v e r i s condensing  enzyme of the a r g i n i n e - s y n t h e t a s e  synthetase  (Brown and Cohen, 1960).  liver  system,  the  arginosuccinate  I t has been e s t a b l i s h e d t h a t i n r a t  t h e r e i s a s u b s t a n t i a l r e d u c t i o n i n the a c t i v i t y of u r e a 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).  -  It  59  -  i s l i k e l y t h a t the a c t i v i t y of most of the enzymes a s s o c i a t e d w i t h  the u r e a c y c l e c o u l d have been lowered i n the i m p l a n t e d s t e e r s . of the m e t a b o l i c a c t i o n s of a n a b o l i c compounds endogenous hormones,  are mediated  As most  through  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 i o p s y  samples  removed  a f t e r the 28th day of DES treatment i n growing lambs r e v e a l e d no d i f f e r e n c e i n arginase cycle (Preston,  1968).  arginine-synthetase  a c t i v i t y , an another enzyme i n v o l v e d i n the u r e a It  s h o u l d be noted t h a t o n l y the  system of the u r e a c y c l e was s i g n i f i c a n t l y  due t o GH t r e a t m e n t (McLean and Gurney, 1963). e f f e c t of a n a b o l i c compounds to understand  reduced  F u r t h e r work on the  on the enzymes of u r e a c y c l e i s  warranted  the mechanism behind the reduced urea p r o d u c t i o n .  The e f f e c t of a n a b o l i c compounds  in stimulating protein  synthesis  and i n c r e a s i n g n i t r o g e n r e t e n t i o n as r e p o r t e d by e a r l i e r workers  was  f u r t h e r c o n f i r m e d by the s i g n i f i c a n t r e d u c t i o n s i n m e t h i o n i n e (P < t h r e o n i n e (P < .01)  and l y s i n e (P < .01)  i n the i m p l a n t e d  M e t h i o n i n e , t h r e o n i n e and l y s i n e a r e thought (Bergen,  1979).  .05),  steers.  to be l i m i t i n g i n  In t h i s study a N b a l a n c e experiment was not  ruminants conducted  t o c o n f i r m N r e t e n t i o n i n the i m p l a n t e d s t e e r s but the reduced amount u r i n a r y n i t r o g e n e x c r e t i o n ( T a b l e 7) i n d i c a t e d i n c r e a s e d N r e t e n t i o n . Significant  r e d u c t i o n (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 i m p l a n t e d s t e e r s i s of w h i l e c o n s i d e r i n g the growth promoting p o t e n t i a l o f these O r n i t h i n e Is  d e c a r b o x y l a t e d by o r n i t h i n e d e c a r b o x y l a s e  p u t r e s c i n e ( R u s s e l l and Snyder,  1968)  importance compounds.  t o form  and the l a t t e r forms  the  of  - 60 -  diamlnobutane back bone of the polyamines. strongly implicated i n the growth process  The polyamines have been (Russell, 1973).  The  reduction i n 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  t h i s study tend to demonstrate I n d i r e c t l y the e f f e c t of anabolic compounds on polyamine biosynthesis. important  Ornithine decarboxylase  plays an  role i n the estrogen induced changes i n the growth of the  target tissues such as chicken oviduct (Levy et a l . , 1981) uterus (Kaye et al.,1971). ornithine decarboxylase Tabor, 1976).  and rat  Correlation between tissue growth and  a c t i v i t y has been shown i n many cases (Tabor and  Taken c o l l e c t i v e l y the growth promoting potentials and  the increased polyamine biosynthesis (assuming a r e l a t i o n s h i p between plasma ornithine and putrescine biosynthesis) i t i s suggested that anabolic compounds mediate their growth promoting properties through polyamine metabolism also.  Further work i s warranted to conclusively  demonstrate this aspect of anabolic compound action. With regard to the l e v e l of hepatic amino acid catabolising enzyme, TAT,  only a non-significant difference was  implantation of Zeranol, DES and Synovex-S. the hepatic TAT a c t i v i t y was  obtained due to the  A s i g n i f i c a n t reduction i n  reported by Rodway and Galbraith (1979) i n  female rats treated with the androgenic anabolic compound, trenbolone acetate and i t was  concluded  that such a reduction i n the hepatic  TAT  a c t i v i t y would increase the a v a i l a b i l i t y of tyrosine for the synthesis of new  proteins.  It i s not clear at present whether the lack of  difference i n TAT a c t i v i t i e s among the steers i s 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 o g e n i c and a n d r o g e n i c  anabolic  compounds or due t o the i n c r e a s e d l e n g t h of time which e l a p s e d between the i m p l a n t a t i o n of these compounds and the sampling o f h e p a t i c f o r TAT a c t i v i t y . .01)  tissues  A r e d u c t i o n i n the c o n c e n t r a t i o n of t y r o s i n e (P <  a l o n g 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 t h a t t h e r e 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 p e r i o d between the i m p l a n t e d and unimplanted s t e e r s .  It  is also possible  t h a t o t h e r enzymes of amino  a c i d catabolism could play a very important r o l e i n r e g u l a t i n g a v a i l a b i l i t y of amino a c i d s  (especially essential)  the  for protein  synthesis. 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 c o n f i r m e d the growth promoting p r o p e r t i e s of a n a b o l i c compounds and i n d i c a t e d t h a t the i m p l a n t a t i o n 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 growth of s t e e r s  than the i m p l a n t a t i o n of Z e r a n o l .  the  A considerable  r e d u c t i o n i n the plasma c o n c e n t r a t i o n s of n i t r o g e n o u s  compounds was  observed due t o the i m p l a n t a t i o n of a n a b o l i c compounds which i n c l u d e d a 15 - 23% r e d u c t i o n i n PUN, 17-19% r e d u c t i o n i n r e d u c t i o n 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 .  AN and about  29-51%  The r e s u l t s r e v e a l e d 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 h e p a t i c TAT between the i m p l a n t e d and unimplanted It  steers.  i s concluded t h a t a n a b o 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 g e n metabolism i n s t e e 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 n i t r o g e n o u s  compounds.  T h e i r i m p l a n t a t i o n seems t o  - 62 -  reduce the h e p a t i c p r o d u c t i o n of u r e a . concentration i n steers  The d a t a on plasma  f o l l o w i n g the i m p l a n t a t i o n of a n a b o l i c compounds  suggested t h a t these compounds may mediate t h e i r growth properties  ornithine  through the metabolism of polyamines  promoting  as w e l l .  Implantations  of DES and Synovex-S were concluded t o 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 o g e n metabolism of s t e e r s  than the i m p l a n t a t i o n of  Zeranol.  - 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 i n farm animals i s the increase i n muscle mass which contributes about 45% of the body weight i n mature mammals, regardless of t h e i r size (Munro, 1969)  and serves as an  important tissue for protein synthesis, reserve and turnover 1971).  (Cheek,  The net deposition of protein i n mammals i s determined by the  balance between the rates of synthesis and degradation.  Millward  et a l . (1975) reported that there i s a direct c o r r e l a t i o n between growth rate and muscle protein breakdown rate and that rapid growth i s always accompanied by high rates of protein breakdown. a major product harvested  Since muscle protein i s  from farm animals and muscle mass i s  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 i n farm animals (Byers, 1982b; Rumsey et a l . , 1981; and Dyer, 1971).  Rumsey, 1982;  G r i f f i t h s , 1982;  Sharp  The mechanism of action of these compounds on muscle  protein metabolism has not been c l e a r l y 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 -Methylhistidine (N -MH) or 3-Methylhistidine (3-MH) T  T  excretion i n urine has been widely used as a useful non-destructive technique for measuring i n vivo muscle protein degradation (Young and Munro, 1978).  N -MH originates from the degradation of a c t i n and T  c e r t a i n species of myosin heavy chain i n which i t i s present a f t e r the post-translational methylation of s p e c i f i c h i s t i d i n e residues (Young and Munro, 1978).  During the catabolism of m y o f i b r i l l a r proteins, i t i s  released and excreted i n urine and i s not r e u t i l i z e d f o r protein synthesis (Young et a l . , 1972) or metabolized oxidatively (Long et a l . , 1975).  Hence urinary excretion of N -MH was suggested as a v a l i d i n T  vivo index of muscle protein degradation (Ward and Buttery, 1978). The v a l i d i t y of this method has been questioned recently by Millward et_ al_., (1980b). The determination of 24 h urinary excretion of N -MH to measure T  muscle protein degradation has been used i n man (Tomas et a l . , 1979), rats (Santidrian et^ a l . , 1981), rabbits (Harris et a l . , 1977) and recently validated for c a t t l e (Harris and Milne, 1981a).  This method,  however, has been reported to be an i n v a l i d index of muscle protein degradation i n sheep (Harris and Milne, 1980) and i n the pig (Harris and Milne, 1981b). This experiment was conducted i n growing steers with the following objectives:  - 65  (a)  -  t o study the d e v e l o p m e n t a l changes a s s o c i a t e d w i t h muscle p r o t e i n metabolism  (b)  t o study the e f f e c t of c e r t a i n e s t r o g e n i c a n a b o l i c  compounds  on v a r i o u s a s p e c t s of muscle p r o t e i n m e t a b o l i s m , and (c)  t o d e m o n s t r a t e , i n g e n e r a l , the u s e f u l n e s s  of N -MH T  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 degradation  MATERIALS AND METHODS  Animals and t h e i r Management Growing H e r e f o r d beef s t e e r s were used i n t h i s s t u d y .  The  a n i m a l s and t h e i r management are the same as d e s c r i b e d i n EXPT.  IA.  Experimental Procedure Two s t e e r s from each of the f o u r a n a b o l i c t r e a t m e n t s [ c o n t r o l , Z e r a n o l , d i e t h y l s t i l b e s t r o l (DES), Synovex-S) d e s c r i b e d i n E x p t . IA were used.  The a n a b o l i c compounds and t h e i r i m p l a n t a t i o n procedures  been d e s c r i b e d i n EXPT. IA.  have  The a n i m a l s were fed t w i c e 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 d e s c r i b e d i n ' T a b l e 5.  The body weight was  measured on e v e r y seventh day of the e x p e r i m e n t .  Urine C o l l e c t i o n Procedure U r i n e samples were c o l l e c t e d i n s p e c i a l l y d e s i g n e d m e t a b o l i s m c r a t e s over a p e r i o d of 24 h on days 24, 48, 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 a n a b o l i c compounds. the  The s t e e r s were a l l o w e d to adapt to  m e t a b o l i s m c r a t e s f o r a p e r i o d of 24 h b e f o r e 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 e x p e r i m e n t , the s t e e r s were  - 66 -  weighed, f e d t h e i r morning p o r t i o n o f the d i e t and p l a c e d i n t h e metabolism crates f o r t o t a l u r i n e c o l l e c t i o n .  The s t e e r s r e c e i v e d f e e d  i n the m e t a b o l i s m c r a t e d u r i n g t h e c o l l e c t i o n p e r i o d and water was p r o v i d e d ad l i b i t u m .  U r i n e was c o l l e c t e d i n p l a s t i c v e s s e l s  6M H C l o r t o l u e n e as the p r e s e r v a t i v e .  containing  D u r i n g the c o l l e c t i o n p r o c e s s ,  the u r i n e was passed through two l a y e r s o f cheese c l o t h t o a v o i d contamination.  fecal  The c l e a r u r i n e samples were s t o r e d a t - 2 0 ° C .  A n a l y t i c a l Procedures N -MH : T  ion-exchange analyser  U r i n a r y N -MH c o n c e n t r a t i o n s were determined by an T  chromatographic method u s i n g an a u t o m a t i c amino a c i d  (Beckman 120 C, Beckman I n s t r u m e n t s , P a l o A l t o , C A . ) .  U r i n a r y c r e a t i n i n e c o n c e n t r a t i o n s were determined by a c o l o r i m e t r i c procedure u s i n g the c r e a t i n i n e d e t e r m i n a t i o n reagent package o b t a i n e d from Sigma Chemical C o . , S t . L o u i s , MO. (Tech. B u l l e t i n No.555, Sigma Chemical C o . ) .  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 m e t a b o l i s m were a c c o r d i n g t o B a l l a r d e t a l . (1979) and H a r r i s and M i l n e  (1981a).  The r a t e o f muscle p r o t e i n d e g r a d a t i o n (MPD, g . d . ) was - 1  c a l c u l a t e d by the e q u a t i o n :  ymol N -MH e x c r e t e d p e r day  ^  T  =  N  T  -MH content of s k e l e t a l muscle  protein  - 67  -  I n the above e q u a t i o n the denominator was t a k e n t o be 3.5 ymol of N -MH/g muscle p r o t e i n as r e p o r t e d by N i s h i z a w a e t a l . (1979) f o r T  growing  steers. The f r a c t i o n a l r a t e of muscle p r o t e i n breakdown (FBR, % . d )  was  - 1  c a l c u l a t e d by the e q u a t i o n :  N -MH e x c r e t e d per day (ymol) T  F  B  R  =  total N  T  x  -MH i n s k e l e t a l muscle  1 0 Q  (  2  )  (ymol)  The denominator i n e q u a t i o n 2 was c a l c u l a t e d as the p r o d u c t of s k e l e t a l muscle mass (33% of the body w e i g h t ; A l l e n e t a l . , 1968; Brannang,  1971), the p r o t e i n c o n t e n t of s k e l e t a l muscle (157 mg/g  w e i g h t ) and the l^-MH c o n t e n t of s k e l e t a l muscle p r o t e i n e t a l . , 1979). and myosin)  (Nishizawa  The h a l f - l i f e v a l u e of the m y o f i b r i l l a r p r o t e i n s  was c a l c u l a t e d from the breakdown r a t e by  f i r s t - o r d e r k i n e t i c s (Ward and B u t t e r y ,  fresh  (actin  assuming  1978).  The r a t e of muscle p r o t e i n s y n t h e s i s  (MPS)  i n the s t e e 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 t e of muscle p r o t e i n g a i n (MPG) and the r a t e of MPD.  THe MPG was c a l c u l a t e d as the product of t h e  amount of muscle mass gained per d (33% of average d a i l y g a i n ) and the p r o t e i n c o n t e n t of the s k e l e t a l m u s c l e . s i m i l a r t o those used by M i l l w a r d  The c a l c u l a t i o n s f o r MPS were  et^ a l . (1975) who e s t i m a t e d 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 r a t e 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 y n t h e s i s  i n steers  implanted  w i t h a n a b o l i c compounds was c a l c u l a t e d a c c o r d i n g to the method d e s c r i b e d by Bates and M i l l w a r d  (1981).  - 68 -  E f f i c i e n c y o f Muscle P r o t e i n S y n t h e s i s  = Qver-al^Synthesis  X  Where: Net S y n t h e s i s  o r p r o t e i n d e p o s i t e d = Muscle p r o t e i n g a i n , (MPG g/d)  O v e r - a l l Synthesis  = Muscle p r o t e i n s y n t h e s i z e d / d a y  (MPS, g/d)  The parameters o f s k e l e t a l muscle p r o t e i n metabolism i n a l l t h e s t e e r s a t d i f f e r e n t stages o f growth were averaged and s u b j e c t e d t o the a n a l y s i s program.  o f v a r i a n c e u s i n g UBC BMD: 10V l i n e a r h y p o t h e s i s  computer  Simple c o r r e l a t i o n s among m e t a b o 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 g a i n of s t e e r s up t o  day 98 o f t h e experiment a r e p r e s e n t e d i n F i g u r e 7. r a p i d l y during the i n i t i a l  phase o f t h e s t u d y .  The a n i m a l s  gained  Up t o day 56, t h e growth  r a t e of s t e e r s was h i g h e r (P < .01) than up t o day 4 2 .  By day 6 3 ,  however, t h e r e was a d e c r e a s e i n growth r a t e compared t o day 56 (P < .01).  Subsequently,  the s t e e r s gained weight a t a r a t e o f 1.4 Kg p e r  day d u r i n g t h e r e s t of t h e e x p e r i m e n t a l p e r i o d . The mean d a i l y amount of N -MH and c r e a t i n i n e e x c r e t e d a t T  d i f f e r e n t stages  of growth i s p r e s e n t e d i n F i g u r e 8 and T a b l e 9  respectively. There was a g r a d u a l i n c r e a s e i n t h e amount o f N -MH e x c r e t e d up T  t o day 56 o f t h e study (P < .001 compared t o day 42) f o l l o w e d by a  -  69  550,-  500  X  0  4501  ,0  LU  ^ >Q  oe  1.5  g  400U UJ  .5 O <  O CQ 3501 0*-  14  28 PERIOD  F i g u r e 7.  42 OF  oo  —  EXPERIMENT  (d)  Body weight and average d a i l y g a i n o f s t e e r s . Each p o i n t r e p r e s e n t s the mean of d a t a o b t a i n e d from e i g h t s t e e r s . The arrows i n d i c a t e t h e u r i n e c o l l e c t i o n p e r i o d . + **  P<.05 compared w i t h the p r e c e d i n g p e r i o d . P<.05 compared w i t h day 2 8 .  1  4 L  > I UJ  12  _  1.0-  4 0  i  i  i  i  28  42  56  70  PERIOD Figure 8.  OF EXPERIMENT^)  Urinary N -methylhistidine excretion i n steers on d i f f e r e n t periods of the experiment. Values are mean ± SE for eight steers. T  + * ***  P<.05 compared with preceding period. P<.05 compared with day 28. P<.001 compared with day 28.  - 71 -  T a b l e 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 -Methylhistidine:  creatinine ratio  T  d i f f e r e n t p e r i o d of the  P e r i o d of experiment (d)  experiment  x  N - Methylhistidine excretion (pmol/mg c r e a t i n i n e )  28  0.14  42  ± 0.01  during  3  Urinary Creatinine (g/d)  11.46 ±  0.88  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  b C  12.93  V a l u e s are mean ± s t a n d a r d e r r o r s o b t a i n e d from e i g h t s t e e r s p e r i o d 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 (P < .05)  differ  ± 0.42  i n each  - 72 -  d e c r e a s e on day 6 3 . These changes i n N -MH e x c r e t i o n r e f l e c t t h e T  N -MH a r i s i n g from the d e g r a d a t i o n o f t h e s k e l e t a l muscle p r o t e i n and T  seem t o resemble t h e growth curve o f t h e e x p e r i m e n t a l a n i m a l s . U n f o r t u n a t e l y , p a t t e r n s o f N -MH e x c r e t i o n were s t u d i e d o n l y up t o day T  63 o f t h e s t u d y .  On an average, t h e d a i l y u r i n a r y N -MH e x c r e t i o n by T  the s t e e r s was 1957 ± 88 umol/d. The d a i l y amount o f c r e a t i n i n e e x c r e t e d a t d i f f e r e n t p e r i o d s o f 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) b u t tended t o i n c r e a s e as t h e s t e e r s i n c r e a s e d i n bodyweight.  The r a t i o o f N -MH t o T  u n i t c r e a t i n i n e was found t o be h i g h e r on day 56 (P < .05) than t h e other periods (Table 9 ) . The mean r a t e o f MPD d u r i n g v a r i o u s  stages o f growth was  c a l c u l a t e d from t h e d a i l y amount o f N -MH e x c r e t e d and t h e N -MH T  T  c o n t e n t o f s k e l e t a l muscle p r o t e i n ( F i g u r e 9 ) . The MPD was (P < .05) on day 56 than on day 42 o f t h e s t u d y .  higher  There was a d e c r e a s e  i n t h e r a t e o f p r o t e i n degraded from 676 ± 57 g/d on day 56 t o 586 ± 25 g/d on day 63 (P < . 0 5 ) .  The mean r a t e o f MPD o f t h e s t e e r s was  c a l c u l a t e d t o be 557 ± 25 g/d d u r i n g t h e e n t i r e e x p e r i m e n t a l p e r i o d . The mean r a t e o f MPS ( F i g u r e 9) was c a l c u l a t e d as t h e d i f f e r e n c e between t h e r a t e o f muscle p r o t e i n g a i n e d p e r day and MPD.  The MPS a t  d i f f e r e n t growth s t a g e s f o l l o w e d a s i m i l a r p a t t e r n t o t h a t o f MPD, b u t was always h i g h e r i n magnitude t h a n MPD.  The mean r a t e o f MPS d u r i n g  the e n t i r e e x p e r i m e n t a l p e r i o d was c a l c u l a t e d t o be 622 ± 27 g / d . The mean FBR (%/d) o f t h e s t e e r s was c a l c u l a t e d t o be 2.44 ± .09 during the e n t i r e experimental p e r i o d .  The FBR was h i g h e r on day 56  -  PERIOD  F i g u r e 9.  OF  73 -  EXPERIMENT (d)  M u s c l e p r o t e i n degraded and s y n t h e s i z e d i n s t e e r s on d i f f e r e n t p e r i o d s of the e x p e r i m e n t . V a l u e s a r e mean ± SE for eight steers. + * **  P<.05 compared w i t h p r e c e d i n g p e r i o d . P<.05 compared w i t h day 28. P<.01 compared w i t h day 2 8 .  - 74 -  (P < .05) than on day 28 (Table 10). proteins i s presented  The h a l f - l i f e of the m y o f i b r i l l a r  i n Table 10. I t i s to be noted that the shorter  h a l f - l i f e of m y o f i b r i l l a r proteins was accompanied by higher MPD values, and that the h a l f - l i f e value of m y o f i b r i l l a r proteins on day 56 of the study (corresponds  to 456 kg liveweight) was calculated to be 24 d.  The r e l a t i o n s h i p between MPD and MPS i s shown i n Figure 10. Similar correlation studies revealed a s i g n i f i c a n t r e l a t i o n s h i p between urinary N -MH (umol/ml urine) and creatinine (mg/ml urine) (r = .71; T  n = 31; P < .01) and between muscle mass (kg) and NT-MH excretion (umol/d) (r = .68; n = 32; P < .01).  The c o r r e l a t i o n 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 N -MH excreted i n urine by T  c a t t l e as reported by d i f f e r e n t workers i s presented (2)  i n Figure 11.  E f f e c t o f Anabolic Compounds on Muscle Protein Metabolism The daily amount of N -MH excreted i n urine was not different T  (P > .05) among the d i f f e r e n t treatment groups (Figure 12). implanted  The steers  with anabolic compounded exhibited a s l i g h t , but a  non-significant increase i n the amount of N -MH excreted i n urine. T  Urinary NT-MH excretion was s l i g h t l y higher (P > .05) i n Zeranol than i n 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  i n Figure 13. The  treatment means for the two variables were not d i f f e r e n t (P > .05), however, the results indicate an increased rate of both degradation and  -  T a b l e 10.  P e r i o d of experiment  -  F r a c t i o n a l r a t e of p r o t e i n breakdown and the h a l f - l i f e V a l u e s of m y o f i b r i l l a r p r o t e i n s i n growing s t e e r s d u r i n g a d i f f e r e n t p e r i o d s of the experiment Muscle p r o t e i n gained  (d)  a  75  b  (g/d)  F r a c t i o n a l r a t e of  Myofibrillar  (%/d)  proteins  58.8  11.1  2.07  0.2  42  65.0  7.6  2.33  0.2  56  71.4  7.5  2.84  0.2  63  61.9  2.53  0.1  6.5  standard V a l u e s are mean p e r i o d of the experiment  f  of  p r o t e i n breakdown  28  d  Half-life  33.5 29.7 6  24.4 27.8  e r r o r s o b t a i n e d from e i g h t s t e e r s on each  ^ 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 c o n t e n t o f 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 different  (P < .01)  compared t o v a l u e s on day 28  Different  (P < .05)  compared t o v a l u e s on day 28  e  ^ D i f f e r e n t (P < .05)  compared t o v a l u e s on the p r e c e d i n g day  - 76 -  Figure 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 s y n t h e s i s and degradation i n s t e e r s .  - 77 -  ~  25_  0.5l_fl_  245  345  o  Nishizawa  •  Harris & Milne (1981a)  •  Present study  _L  _L  445  et §1.(1979)  545  BODY-WEIGHT (kg)  F i g u r e 11.  Urinary e x c r e t i o n of N - m e t h y l h i s t i d i n e T  i n cattle.  645  CONTROL . . . .1 ZERANOL •••• ••••  DES SYNOVEX-S  1 I  1 to  • • •••+•+ +++++ • • ••++++ ••+++ • • •«•++++ *•++++ • • •+++++ +++++ • ••++•+ •••++  • • ••+••• •+•++ +++++  • • •+++++ •+++• • • •+•++• •+++• • • •••••+ ++•++  si I'  • • •++••• ++••+ •o° +++++ • • •++•••  *•  y  • • •+••++ *0 • • •+++++ ••••• u • • }***** >o° 0  • ******* I|»•••• «!••••• ++•••  Urinary N - 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 steers implanted w i t h a n a b o l i c compounds. Each b a r r e p r e s e n t s mean ± SE o f v a l u e s o b t a i n e d from two s t e e r s on days 2 8 , 4 2 , 56 and 63 f o l l o w i n g t h e i m p l a n t a t i o n (n=8). T  -  79  -  750 CONTROL • • ZERANOL •• i.• • DES SYNOVEX-S  650 r _  600 550  v.* ••••• •••••  '"•X •  •  •  •  CI  o • • o• • • • • • o •  a •  ••»••*  500  • • •  • •  • • •  • •  • • •  •• • • • • Te • • 0 1 -  F i g u r e 13.  650  ••••• •  Q Q.  700  1  ••••• ••••• ••••• ••••• ••••• ••••• ••••• ••••• ••••• ••••• ••••• ••••• ••••• ••••• ••••• •••••  bl) •+••• •••••  ••••• >o° v.* ••••• ••••• ••••• • • ' • I ••••• ••••• ••••• ••••+ >°o' ••••• ••••• •••••  >°o  ••••• •••••  Ttt***  R a t e s of muscle (MPS) i n s t e e r s r e p r e s e n t s mean days 28, 42, 56  (  •V.  ***** ***** ***** ***** ***** *****>%% ***** ***** ••••• •••••  L  »  •  L***W*  I a » »\***** w  ^  °  CO  600 g= 550  io  p r o t e i n d e g r a d a t i o n (MPD) and s y n t h e s i s i m p l a n t e d w i t h a n a b o l i c compounds. Each b a r ± SE of v a l u e s o b t a i n e d from two s t e e r s on 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).  - 80  synthesis  of muscle p r o t e i n i n s t e e r s  compounds.  -  implanted w i t h anabolic  Z e r a n o l i m p l a n t a t i o n tended t o r e s u l t i n c r e a s e d r a t e s  of  b o t h 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 higher  (P < .01)  i n DES and Synovex-S than i n Z e r a n o l i m p l a n t e d or the  unimplanted c o n t r o l steers  (Table 11).  Though s l i g h t l y lower than DES  and Synovex-S i m p l a n t a t i o n s , the s t e e r s i m p l a n t e d w i t h Z e r a n o l e x h i b i t e d s i g n i f i c a n t l y higher  (P < .01) amount of MPG than the u n i m p l a n t e d  steers. The FBR 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 t e i n s  i n f l u e n c e d by d i f f e r e n t treatment groups are p r e s e n t e d i n T a b l e Zeranol implantation resulted i n s l i g h t l y higher (P > .05)  h a l f - l i f e values  as  12.  (P > .05) FBR and lower  f o r the m y o f i b r i l l a r p r o t e i n s than the o t h e r  treatment groups. From the v a l u e s 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 y n t h e s i s i n T a b l e 13.  was c a l c u l a t e d and i s  The e f f i c i e n c y was s l i g h t l y h i g h e r  (12%)  i m p l a n t e d w i t h DES and Synovex-S than i n Z e r a n o l (9%) unimplanted c o n t r o l steers  in  presented  steers  i m p l a n t e d or the  (8%).  DISCUSSION  V a l i d i t y of the method N -Methylhistidine T  e x c r e t i o n i n u r i n e has been used t o  d e t e r m i n e the r a t e of MPD and t o e s t i m a t e parameters of muscle m e t a b o l i s m i n growing beef s t e e r s .  Most of the assumptions  for  protein using  N -MH as an i n d e x of muscle p r o t e i n breakdown have been e x p e r i m e n t a l l y T  -  T a b l e 11.  81  -  Amount of muscle p r o t e i n gained implanted with various anabolic  (MPG) i n s t e e r s compounds 1  Treatment  MPG ( g / d )  Control  45.2 +  4.0  a  Zeranol  60.1 +  4.5  b  DES  73.8 +  2.9  C  Synovex-S  77.9 + 1 2 . 3  Mean of v a l u e s o b t a i n e d on 28, 42, 56 and 63 days implantation  following  Weight g a i n as muscle x p r o t e i n c o n t e n t of s k e l e t a l i b c ' ' 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  differ  (P <  muscle  .01)  2  C  - 82 -  Table 12. E f f e c t of anabolic compounds on the f r a c t i o n a l rate of protein breakdown (FBR) and the h a l f - l i f e values of m y o f i b r i l l a r proteins i n growing beef s t e e r s 1  FBR (%/d)  Half-Life (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  2  Treatment  *Mean ± SE obtained on 28, 42, 56 and 63 days following the implantation ^-MH  x 100/Total N —MH content of skeletal muscle T  Assuming f i r s t - o r d e r k i n e t i c s  3  - 83  T a b l e 13.  -  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 i n i m p l a n t e d w i t h v a r i o u s a n a b o l i c compounds  Protein Synthesis (g/d)  Protein Degradation (g/d)  Control  565 + 50  519 + 47  46  8.14  ('"  8%)  Zeranol  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%)  Treatments  Mean ± SE  Net Synthesis (g/d)  steers  Efficiency (Net/Overall) xlOO  - 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 n e ( 1 9 8 1 a ) .  The  b a s i s of the v a l i d a t i o n procedure i s t h a t i n c a t t l e t h e r e i s a r a p i d r e c o v e r y o f i n j e c t e d r a d i o - l a b e l e d N -MH w i t h i n 5 t o 7 days i n u r i n e . T  Even though N -MH e x c r e t i o n i n u r i n e has been a c c e p t e d as a v a l i d T  i n d e x of MPD i n many s p e c i e s i n c l u d i n g c a t t l e , s e r i o u s doubts have been r a i s e d by M i l l w a r d e t a l . (1980b) as t o the v a l i d i t y of t h i s method i n rats.  I t was suggested t h a t t i s s u e s  o t h e r than s k e l e t a l m u s c l e , v i z . ,  s k i n , i n t e s t i n e , e t c . might c o n t r i b u t e t o a c o n s i d e r a b l e p o r t i o n of the u r i n a r y N -MH. T  discussed muscle.  More r e c e n t l y , however, S a n t r i d r i a n e t a l . (1981)  t h a t t h e u r i n a r y N -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 T  N i s h i z a w a e t a l . (1979) r e p o r t e d t h a t more than 93% of t h e  t o t a l N -MH i n t h e a n a l y z e d c a t t l e t i s s u e o c c u r r e d i n s k e l e t a l muscle T  protein.  I t i s f o r these reasons t h a t i n t h i s study N -MH e x c r e t i o n T  i n u r i n e was used as an i n v i v o  i n d e x of MPD.  One p o i n t which must be s t r e s s e d i s t h a t the c a l c u l a t e d v a l u e s o f 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 r e p r e s e n t a b s o l u t e e s t i m a t e s . Because t h i s study was d e s i g n e d p r i m a r i l y t o m o n i t o r the changes a s s o c i a t e d 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 o f a c t i o n o f a n a b o l i c compounds on muscle p r o t e i n m e t a b o l i s m , t h e c a l c u l a t e d v a l u e s were t a k e n as such f o r comparison among t r e a t m e n t s .  The ease w i t h which the parameters o f muscle p r o t e i n  m e t a b o l i s m c o u l d be c a l c u l a t e d from t h e u r i n a r y N -MH v a l u e s T  alone  makes t h i s t e c h n i q u e v e r y a t t r a c t i v e i n s t u d i e s of t h i s n a t u r e . a b s o l u t e e s t i m a t e s o f muscle p r o t e i n s y n t h e s i s  For the  and muscle p r o t e i n g a i n ,  - 85 -  one has t o r e s o r t t o l a b o r i o u s and t e d i o u s m e t h o d o l o g i e s as d e s c r i b e d i n the l i t e r a t u r e . Developmental Aspects of Muscle Protein Metabolism Urinary N -MH excretion r  The v a l u e s of N -MH e x c r e t i o n r e p o r t e d f o r s t e e r s i n t h i s T  study  a r e s l i g h t l y h i g h e r than those r e p o r t e d by N i s h i z a w a et_ a l . (1979) and H a r r i s and M i l n e (1981a) ( F i g u r e 1 1 ) .  At a growth stage o f 4 1 1 , 432,  456 and 455 k g , t h e r a t e o f N -MH e x c r e t e d i n u r i n e was 3.64, 4 . 2 7 , T  5.21 and 4.52 umol/d/kg r e s p e c t i v e l y , as compared t o t h e v a l u e s of 3.68 t o 2.75 umol/d/kg at growth s t a g e s o f 217 t o 312 kg f o r H o l s t e i n  steers  ( N i s h i z a w a e t 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 N -MH by t h e s t e e r s n o t i c e d i n t h i s T  s t u d y s u g g e s t s t h a t i n these animals a l a r g e p r o p o r t i o n o f t h e body weight may be c o n t r i b u t e d by muscle t i s s u e and thus r e f l e c t i n t h e 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 o f a  compound c o u l d be of e i t h e r endogenous o r exogenous i n o r i g i n . been u n e q u i v o c a l l y demonstrated t h a t N -MH o r i g i n a t e s T  I t has  endogenously  from the c a t a b o l i s m o f m y o f i b r i l l a r p r o t e i n s (Young and Munro, It  changes  1978).  i s p o s s i b l e t h a t d i e t a r y sources of N -MH may a l s o c o n t r i b u t e t o a T  c e r t a i n p r o p o r t i o n of t h e u r i n a r y N -MH. T  The f e e d used was  c e r e a l - b a s e d and d e v o i d o f any a n i m a l p r o d u c t and was s i m i l a r t o t h a t used by H a r r i s and M i l n e ( 1 9 8 1 a ) .  No attempt was made t o a n a l y z e t h e  f e e d f o r i t s N -MH c o n c e n t r a t i o n .  R e c e n t l y , however, N i s h i z a w a  T  al.  (1979) r e p o r t e d t h e  et  N -MH content of hay and c o n c e n t r a t e t o be T  -  86  -  2.4 and 7.7 mg/kg, r e s p e c t i v e l y , and suggested t h a t about 30% of t h e u r i n a r y N -MH o u t p u t c o u l d be c o n t r i b u t e d by f e e d . T  possible  I t i s , thus,  t h a t d i e t a r y N -MH c o u l d have c o n t r i b u t e d t o c e r t a i n p o r t i o n s T  o f t h e u r i n a r y N -MH r e p o r t e d h e r e .  I t i s t h e r e f o r e suggested t h a t  T  d i e t a r y sources  of N -MH s h o u l d be t a k e n i n t o c o n s i d e r a t i o n w h i l e T  r e p o r t i n g N -MH v a l u e s T  i n urine.  The e f f e c t of d i e t upon  urinary  N -MH e x c r e t i o n has a l s o been r e p o r t e d by Ward and B u t t e r y T  (1980).  The u r i n a r y c r e a t i n i n e c o n c e n t r a t i o n as w e l l as N -MH: T  c r e a t i n i n e r a t i o ( T a b l e 9) tended t o i n c r e a s e as t h e a n i m a l weight.  The r a t i o o f N -MH t o t h a t of c r e a t i n i n e i n u r i n e T  gained indicates  t h e l e v e l o f MPD r e l a t i v e t o t h a t o f muscle mass (Ward and B u t t e r y , 1980).  Growth and Muscle Protein Degradation As shown i n F i g u r e s  7 and 9, t h e r e appears  between growth o f the s t e e r s and t h e r a t e o f MPD.  t o be a r e l a t i o n s h i p These  developmental  changes i n MPD a r e i n c l o s e agreement w i t h those r e p o r t e d by M i l l w a r d et a l . ( 1 9 7 5 ) . As the s t e e r s gained weight t h e r e was an i n c r e a s e i n MPD.  The  i n c r e a s e d growth r a t e and u r i n a r y N -MH e x c r e t i o n a r e s i m i l a r t o the T  a n a b o l i c i n c r e a s e i n MPD d u r i n g growth ( M i l l w a r d e t a l . , 1980a). have suggested t h a t the r a t e of MPD may be e l e v a t e d d u r i n g ( a n a b o l i c i n c r e a s e ) o r d u r i n g muscle w a s t i n g ( c a t a b o l i c Excessive  They  growth  increase).  MPD does not always r e f l e c t c a t a b o l i c s t a t e as i n the case o f  muscle w a s t i n g d i s e a s e o r 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 . t o the d a t a r e p o r t e d h e r e ,  Contrary  Vernon and B u t t e r y (1978a) found a d e c r e a s e  - 87  -  i n N -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 e d w i t h the a n a b o l i c T  compound, t r e n b o l o n e a c e t a t e . i n MPD was r e s p o n s i b l e deposited.  These workers  suggested t h a t a d e c r e a s e  f o r the i n c r e a s e d amount of muscle  portein  T h i s r e l a t i o n s h i p between growth and MPD o b t a i n e d by Vernon  and B u t t e r y (1978a) c o u l d be s i m i l a r t o the a n a b o l i c d e c r e a s e i n MPD d u r i n g growth shown by M i l l w a r d e t a l . ( 1 9 8 0 a ) . The i n c r e a s e d r a t e of MPD observed d u r i n g r a p i d growth ( F i g u r e c o u l d a l s o be due t o m y o f i b r i l l a r s p l i t t i n g and  9)  remodelling.Increased  r a t e s of m y o f i b r i l l a r p o r t e i n breakdown d u r i n g 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 l w a r d et a l . , 1975).  During  rapid  growth, increased 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 increased turnover (Goldspink,  1970).  protein  The h i g h c o r r e l a t i o n o b t a i n e d between MPD  and MPS ( r = . 9 , F i g u r e 10) of the a n i m a l s c o n f i r m s r a p i d muscle  protein  turnover. The mean FBR of m y o f i b r i l l a r p r o t e i n s was c a l c u l a t e d t o be 2.4%/d (Table 1 2 ) .  The FBR v a l u e s of 1.22%/d a t a growth stage of 217  kg to 1.02%/d at 312 kg ( N i s h i z a w a e t a l . , 1979) stage of 236 kg ( H a r r i s  and 1.4%/d at a  and M i l n e , 1981a) were r e p o r t e d f o r c a t t l e .  average FBR r e p o r t e d here corresponds  t o a h a l f - l i f e of  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.  The  approximately U s i n g N -MH  e x c r e t i o n as an i n d e x o f m y o f i b r i l l a r p r o t e i n breakdown, values  growing  T  half-life  of 21 t o 41 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 were o b t a i n e d by Ward  and B u t t e r y (1980) f o r r a t s .  It  is difficult  t o compare the FBR  values  o b t a i n e d i n t h i s study w i t h those of the l i t e r a t u r e because the MPD  is  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 d e v e l o p m e n t a l stage of the a n i m a l ( M i l l w a r d elt a l . , 1980a). r e d u c t i o n 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 r o t e i n s I n d i c a t e s  The  that there  i s a r a p i d t u r n o v e r of t h e s e p r o t e i n s i n the s t e e r s used i n t h i s  Muscle Protein Synthesis  study.  (MPS)  The e s t i m a t e s of MPS were o b t a i n e d as the d i f f e r e n c e between the r a t e of muscle p r o t e i n gained and degraded. Protein synthesis  in tissues  of c a t t l e was e s t i m a t e d by L o b l e y  e t a l . (1980) from the i r r e v e r s i b l e l o s s from the b l o o d of r a d i o a c t i v e amino a c i d .  Protein synthesis  i n s k e l e t a l muscle of c a t t l e a t  bodyweight 236, 263 and 628 kg was r e p o r t e d to be 316, 355 and 449  g/d,  r e s p e c t i v e l y ( L o b l e y 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 e e amino a c i d ( l y s i n e ) i n blood d e f i n e 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 r e c u r s o r f o r p r o t e i n synthesis.  As i t can be seen, the MPS v a l u e o b t a i n e d i n the p r e s e n t  s t u d y (622 g/d)  i s h i g h e r than those r e p o r t e d by L o b l e y et a l . ( 1 9 8 0 ) .  The reason f o r the i n c r e a s e d e s t i m a t e s of MPS observed i n t h i s study may be due t o the f a c t t h a t growing s t e e r s were used w h i l e the above r e s e a r c h e r 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^ l o n g i s s i m u s  d o r s i was  t a k e n 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 L o b l e y et^ a l . (1980).  N -MH has been r e p o r t e d t o r e f l e c t the average T  protein  d e g r a d a t i o n i n whole m u s c u l a t u r e ( S a n t i d r i a n e t a l . , 1981). v a l u e s o b t a i n e d i n t h i s s t u d y may r e f l e c t p r o t e i n s y n t h e s i s m u s c u l a t u r e of the s t e e r s as a w h o l e .  Hence, MPS i n the  - 89 -  E f f e c t of Anabolic Compounds Though the mechanism of a c t i o n o f a n a b o l i c compounds  i s not w e l l  u n d e r s t o o d i t has been e s t a b l i s h e d t h a t they a r e 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 t h e s k e l e t a l m u s c l e . i n f l u e n c e on the r a t e of p r o t e i n s y n t h e s i s  The main  and d e g r a d a t i o n i n muscle  seems t o be hormonal (Young 1980) w h i c h has been e x t e n s i v e l y reviewed ( G o l d b e r g e t a l . , 1980).  Besides,  p r o t e i n d e g r a d a t i o n i t s e l f has been  r e p o r t e d t o p l a y a major r o l e i n the r e g u l a t i o n o f p r o t e i n b a l a n c e i n s k e l e t a l muscle ( M i l l w a r d et_ a\_., 1980a). necessary  T h e r e f o r e , i t becomes  t o study the e f f e c t of a n a b o l i c compounds  on muscle p r o t e i n  d e g r a d a t i o n and o t h e r parameters of s k e l e t a l muscle p r o t e i n m e t a b o l i s m .  Urinary N -MH and Muscle Protein Degradation T  There was a tendency, though not s i g n i f i c a n t , towards  increased  m y o f i b r i l l a r p r o t e i n degradation i n steers implanted with estrogenic a n a b o l i c compounds  ( F i g u r e 12 and 1 3 ) .  As t h i s i s t h e f i r s t r e p o r t on  the e f f e c t of e s t r o g e n i c a n a b o l i c compounds on MPD u s i n g N -MH T  e x c r e t i o n as an I n d e x , no d i r e c t comparison o f t h e r e s u l t s c o u l d be made w i t h v a l u e s i n the l i t e r a t u r e .  R e c e n 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 N -MH e x c r e t i o n i n u r i n e from s t e e r s T  r e c e i v i n g a c o m b i n a t i o n of a n d r o g e n i c estrogenic (Ralgro)  (Trenbolone a c e t a t e ) and  a n a b o l i c compounds.  I n an e a r l i e r s t u d y , Vernon and  B u t t e r y ( 1 9 7 8 a ) , demonstrated a s i g n i f i c a n t r e d u c t i o n i n m y o f i b r i l l a r p r o t e i n d e g r a d a t i o n as judged by the reduced u r i n a r y N -MH e x c r e t i o n , T  i n female r a t s t r e a t e d w i t h t h e a n d r o g e n i c a n a b o l i c compound, t r e n b o l o n e acetate.  The o b s e r v a t i o n s  on MPD of t h e p r e s e n t study and those  - 90  -  r e p o r t e d by Vernon and B u t t e r y (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 a n d r o g e n i c and e s t r o g e n i c a n a b o l i c compounds the s k e l e t a l m u s c l e .  Whereas e s t r o g e n i c a n a b o l i c compounds  a tendency to i n c r e a s e m y o f i b r i l l a r p r o t e i n d e g r a d a t i o n , the a n a b o l i c compounds Interestingly,  seem t o reduce I t  on  seem t o have androgenic  (Vernon and B u t t e r y , 1978a).  the o b s e r v a t i o n s of G r i f f i t h s (1982) seem t o demonstrate  a b a l a n c e between the a c t i o n s of a n d r o g e n i c and e s t r o g e n i c compounds  in  c a s t r a t e d males as e v i d e n c e d by a n o n - s i g n i f i c a n t decrease i n N -MH T  e x c r e t i o n i n u r i n e by the s t e e r s i m p l a n t e d w i t h b o t h t r e n b o l o n e a c e t a t e and R a l g r o  ( C o n t r o l = 1.10 m mol N -MH/d; i m p l a n t e d = 0 . 8 9 mmol T  N -MH/d). T  Among the a n a b o l i c compounds used i n t h i s e x p e r i m e n t , Z e r a n o l seems t o e x e r t a n o n - s i g n i f i c a n t i n c r e a s e i n b o t h the u r i n a r y N -MH T  e x c r e t i o n and MPD per day than t h a t e x h i b i t e d by s t e e r s i m p l a n t e d w i t h DES o r Synovex-S.  The m o l e c u l a r mechanism of a c t i o n ( s )  of p r o t e i n  d e g r a d a t i o n as i n f l u e n c e d by the a n a b o l i c compounds used i n t h i s i s not c l e a r l y u n d e r s t o o d .  study  R e c e p t o r s f o r s t e r o i d hormones i n s k e l e t a l  m u s c l e , e s p e c i a l l y androgens have been demonstrated ( M i c h e l and B a u l i e u , 1976; D a h l b e r g e t a l . , 1981) and t h a t the mechanism of a c t i o n o f a n d r o g e n i c a n a b o l i c compounds has been p o s t u l a t e d t o be due t o the b i n d i n g of t e s t o s t e r o n e t o g l u c o c o r t i c o i d r e c e p t o r s and the i n h i b i t i o n of p r o t e i n c a t a b o l i s m i n d u c e d by g l u c o c o r t i c o i d s (Mayer and Rosen, 1975).  However, the presence of e s t r o g e n r e c e p t o r s i n the s k e l e t a l  muscle o r 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 i n d u c e d p r o t e i n d e g r a d a t i o n by e s t r o g e n i c a n a b o l i c compounds remains t o be s t u d i e d .  The o t h e r  p o s s i b l e mechanisms by which e s t r o g e n i c a n a b o 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 r o t e i n a s e a c t i v i t i e s (e.g.  l y s o s o m a l enzymes such as c a t h e p s i n B, c a t h e p s i n D,  proteases,  acid  e t c . ) o r 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 i n v o l v e m e n t of muscle p r o t e i n a s e s  d e g r a d a t i o n and the p a r t i c i p a t i o n of t h y r o i d hormones of l y s o s o m a l  on p r o t e i n i n the r e g u l a t i o n  c o n t e n t s have been e x t e n s i v e l y reviewed ( M i l l w a r d et^ a l . ,  1980a; Goldberg et a l . , 1980).  Muscle Protein Synthesis and Gain B o t h MPS and MPD ( F i g u r e 13) seem t o have been i n c r e a s e d  in  s t e e r s i m p l a n t e d w i t h a n a b o l i c compounds when compared to the unimplanted c o n t r o l s t e e r s .  Among the a n a b o l i c  compounds.interestingly,  Z e r a n o l i m p l a n t a t i o n causes a r a p i d t u r n o v e r of muscle p r o t e i n s . muscle p r o t e i n t u r n o v e r seems t o be r a p i d i n a l l the i m p l a n t e d DES & Synovex-S) when compared to the c o n t r o l s t e e r s  be s u b s t a n t i a t e d f u r t h e r by the h i g h FBR v a l u e s  The  implanted steers  could  (2.6% vs 2.3 to 2.4%d)  and the lower h a l f - l i f e v a l u e s f o r the m y o f i b r i l l a r p r o t e i n s 28.6 t o  (Zeranol,  i t seems t o be  s l o w e r i n s t e e r s i m p l a n t e d w i t h DES o r Synovex-S than Z e r a n o l . r a p i d t u r n o v e r r a t e of muscle p r o t e i n i n Z e r a n o l  Though  (26d  vs  29.6d). The s i g n i f i c a n t l y h i g h e r (P < .01)  amount o f MPG by the  steers  i m p l a n t e d w i t h Z e r a n o l , DES and Synovex-S than the c o n t r o l s t e e r s 11) i s i n agreement w i t h e a r l i e r r e p o r t s on the e f f e c t of compounds  on r a t e of p r o t e i n g a i n (Sharp and Dyer,  Rumsey e t a l . , 1981; Rumsey, 1982).  (Table  these  1971; B y e r s ,  1982b;  The decreased amount of muscle  - 92  -  p r o t e i n g a i n e d by the Z e r a n o l i m p l a n t e d s t e e r s c o u l d be due t o the r e l a t i v e i n c r e a s e i n MPD compared t o DES and Synovex-S i m p l a n t e d steers.  C o n t r a r y t o the t r e n d s o f MPS and MPD observed (namely  i n b o t h MPS & MPD) i n t h i s e x p e r i m e n t , a n d r o g e n i c a n a b o l i c  increase  compounds  have been shown t o d e c r e a s e b o t h MPS and MPD compared t o the u n i m p l a n t e d c o n t r o l a n i m a l s , w i t h the d e c r e a s e i n MPS b e i n g l e s s than the decrease i n the r a t e o f 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 a n a b o l i c compounds on the parameters of muscle p r o t e i n m e t a b o l i s m , namely MPS and MPD c o u l d be b e t t e r e x p l a i n e d 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 ( T a b l e  13).  The e f f i c i e n c y of MPS was found t o be h i g h e r i n s t e e r s i m p l a n t e d w i t h DES and Synovex-S (12%) (8%).  than Z e r a n o l (9%) o r u n i m p l a n t e d c o n t r o l s t e e r s  The h i g h e r r a t e of muscle p r o t e i n gained by the s t e e r s i m p l a n t e d  w i t h DES and Synovex-S than t h a t observed i n Z e r a n o l i m p l a n t e d s t e e r s c o u l d be p a r t l y a t t r i b u t e d t o the lower r a t e s of MPD (as i n d i c a t e d by the h i g h e r 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) c o n c l u d e d  t h a t the i n c r e a s e d muscle growth of the r e l a t i v e l y f a s t growing s t r a i n compared t o t h a t of a slow g a i n i n g hooded s t r a i n e t a l . , 1975) synthesis.  It  rat  (Millward  c o u l d be due t o the i n v o l v e m e n t of l e s s MPD per u n i t i s suggested from the r e s u l t s of the p r e s e n t experiment  t h a t b o t h the r a t e s of MPS and MPD have t o be s i m u l t a n e o u s l y  considered  w h i l e s t u d y i n g the e f f e c t of a n a b o l i c compounds on muscle p r o t e i n m e t a b o l i s m as b o t h a r e i n v o l v e d i n g o v e r n i n g the amount of muscle protein deposited.  - 93 -  CONCLUSION The r e s u l t s of t h i s study demonstrated the u s e f u l n e s s  of  measuring N - 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 , n o n - d e s t r u c t i v e T  method f o r s t u d y i n g muscle p r o t e i n m e t a b o l i s m i n l a r g e a n i m a l s such as beef c a t t l e .  The r a p i d growth r a t e of s t e e r s was accompanied by a r a p i d  r a t e of muscle p r o t e i n t u r n o v e r . The i m p l a n t a t i o n of a n a b o l i c compounds r e s u l t e d i n an i n c r e a s e d amount o f muscle p r o t e i n g a i n e d by the s t e e r s .  A general,  n o n - s i g n i f i c a n t i n c r e a s e i n muscle p r o t e i n d e g r a d a t i o n was observed  in  s t e e r s i m p l a n t e d w i t h a n a b o 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 i g h e r r a t e of muscle p r o t e i n s y n t h e s i s degradation.  than  A n a b o l i c compounds were found t o 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 o f  muscle p r o t e i n gained was h i g h e r i n s t e e r s i m p l a n t e d w i t h DES and Synovex-S than Z e r a n o l .  The d i f f e r e n t i a l e f f e c t s of a n a b o l i c compounds  were c o n c l u d e d t o be due t o the i n v o l v e m e n t of l e s s muscle p r o t e i n d e g r a d a t i o n per u n i t  synthesis.  - 94 -  EXPT. IC COLLAGEN METABOLISM BY T H E URINARY  I N GROWING B E E F STEERS AS A S S E S S E D  HYDROXYPROLINE  E X C R E T I O N AND THE E F F E C T  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 m e t a b o l i s m seem t o be one o f t h e major m e t a b o l i c consequences non-ruminants.  i n ruminants and  A number o f s t u d i e s have demonstrated t h e e f f e c t of  a n a b o l i c compounds (Byers,  o f a n a b o 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 a n i m a l s :  DES  1982b; Rumsey e t a l . , 1981); Synovex-S (Rumsey, 1982); Z e r a n o l  (Sharp and Dyer,  1971).  Even though the g e n e r a l p r o t e i n a n a b o l i c  p r o p e r t i e s o f a n a b o l i c compounds have been e s t a b l i s h e d , o n l y a few e x p e r i m e n t s have been conducted t o 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 t e i n s o f 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 t o e x p l o r e t h e i r e f f e c t on t h e metabolism o f 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 o f t h e mammalian body. Collagen i s a hydroxyproline  (HYPRO) r i c h p r o t e i n , r e p r e s e n t i n g  about 30 per cent o f t h e t o t a l body p r o t e i n s . i n f l u e n c e muscle development 1976)  I t has been shown t o  (De L a Haba e t a l . , 1975; K e t l e y et a l . ,  and has been suggested t o 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  (Robins,  1977).  growth  D u r i n g t h e d e g r a d a t i o n o f c o l l a g e n f i b r i l s , HYPRO i s  r e l e a s e d and t h e q u a n t i t y o f HYPRO i n u r i n e has been c o n s i d e r e d as an i n d e x o f c o l l a g e n metabolism ( Z i f f e t a l . , 1956; K i v i r i k k o , 1 9 7 0 ) . The e x c r e t i o n o f HYPRO i n u r i n e has been r e p o r t e d t o be under t h e i n f l u e n c e  - 95 -  of d i f f e r e n t hormones (Jasin et a l . , 1962;) and several other physiological conditions (See Review by K i v i r i k k o , 1970). This experiment was conducted to study the effect of estrogenic anabolic compounds on collagen metabolism i n vivo i n growing steers. Urinary t o t a l HYPRO (free and peptide bound) excretion has been u t i l i z e d as an in_ vivo index of collagen metabolism. The main objectives of this i n v e s t i g a t i o n were: (a)  to study collagen metabolism at d i f f e r e n t growth stages i n 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 i n EXPT. IA. Two steers from each of the four treatment groups [control Zeranol, D i e t h y l s t i l b e s t r o l (DES) were used.  and Synovex-S ] described i n EXPT.IA  The steers were fed a f i n i s h i n g ration (Table 5) and the  body weight measurements were taken at weekly i n t e r v a l s .  - 96  -  Urine C o l l e l c t i o n Procedure U r i n a r y HYPRO e x c r e t i o n was m o n i t o r e d 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 e e r s over a p e r i o d of 24 h on days 28, 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 o f a n a b o l i c compounds.  The method  o f u r i n e c o l l e c t i o n and s t o r a g e were s i m i l a r t o those d e s c r i b e d Expt.  42,  in  IB.  A n a l y t i c a l Procedure Hydroxyproline (HYPRO) T o t a l HYPRO ( f r e e and p e p t i d e - b o u n d )  c o n c e n t r a t i o n i n the u r i n e  samples was determined a c c o r d i n g to the method d e s c r i b e d 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 h y d r o l y z e d w i t h e q u a l volume o f c o n c e n t r a t e d 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  h y d r o l y z a t e 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 C03.  The pH of the h y d r o l y z a t e was then a d j u s t e d t o 6.0 w i t h 0.1 M  2  citrate-buffer.  The HYPRO i n the a c i d h y d r o l y z a t e was then o x i d i z e d t o  p y r r o l e i n the presence o f i s o p r o p y l a l c o h o l and c h l o r a m i n e - T .  The  reagent m i x t u r e was a l l o w e d t o s t a n d at room temperature f o r 4 minutes and then r e a c t e d w i t h p - d i m e t h y l a m l n o - b e n z a l d e h y d e i n a b o i l i n g water b a t h f o r 2 m i n u t e s .  (Ehrlich's  Reagent)  The reagent m i x t u r e was  then  c o o l e d i m m e d i a t e l y i n an i c e b a t h and k e p t at room temperature f o r 90 minutes.  The f i n a l c o l o r was read a t 575 nm a g a i n s t a reagent  blank.  T o t a l Nitrogen T o t a l n i t r o g e n c o n c e n t r a t i o n i n the u r i n e samples was determined by the K j e l d h a l p r o c e d u r e .  - 97 -  Creatinine C r e a t i n i n e c o n c e n t r a t i o n i n the samples was determined by a c o l o r i m e t r i c procedure as d e s c r i b e d e a r l i e r ( E x p t .  IB).  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 c o n c e n t r a t i o n of HYPRO and the 24 h u r i n e volume. HYPRO e x c r e t i o n per day = HYPRO (mg/ml) x 24 h u r i n e volume  (ml)  (mg/d) D a i l y HYPRO e x c r e t i o n d a t a was expressed as mg/d, mg/d/kg^'^^, HYPRO-N (as % o f t o t a l N i n u r i n e ) .  For the purposes  and  of the l a t t e r  e x p r e s s i o n , n i t r o g e n c o n t e n t o f HYPRO was t a k e n as 10.68 per c e n t . The v a l u e s of d a i l y HYPRO e x c r e t i o n by a l l the s t e e r s on any one of the c o l l e c t i o n dates were averaged and s u b j e c t e d t o l e a s t analysis  of v a r i a n c e u s i n g UBC BMD:10V l i n e a r h y p o t h e s i s  squares  computer  program. RESULTS Urinary HYPRO Excretion at Different Stages of Development U r i n a r y HYPRO e x c r e t i o n a t d i f f e r e n t growth stages presented i n Figures  is  14 and 15.  The HYPRO e x c r e t i o n (mg/d; mg/d/ k g * 0  < .01)  i n steers  7 5  )  i n u r i n e decreased  (P  g r a d u a l l y as the a n i m a l s advanced i n m a t u r i t y and g a i n e d w e i g h t .  U r i n a r y HYPRO e x c r e t i o n decreased u n t i l day 42 (P < .01) and remained a t s i m i l a r l e v e l s on day 56 and 6 3 .  Though a s i m i l a r p a t t e r n was  observed when HPYRO was e x p r e s s e d as HYPRO-N (as a % of t o t a l  nitrogen  i n u r i n e ) t h e r e was a r e d u c t i o n i n u r i n a r y HYPRO e x c r e t i o n even beyond  -  F i g u r e 14.  98  -  U r i n a r y t o t a l h y d r o x y p r o l i n e (HYPRO) e x c r e t e d by s t e e r s on d i f f e r e n t p e r i o d s of the e x p e r i m e n t . Each p o i n t r e p r e s e n t s mean ± SE of v a l u e s o b t a i n e d from e i g h t 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 t o t a l nitrogen) excretion by steers on d i f f e r e n t 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 i g u r e  15).  The r a t i o of H Y P R O : c r e a t i n i n e i n u r i n e on d i f f e r e n t stages development i s p r e s e n t e d i n Table 14.  The r a t i o o f HYPRO:  of  Creatinine  decreased (P < .05) u n t i l day 42 and remained c o n s t a n t d u r i n g the r e s t o f the e x p e r i m e n t a l p e r i o d .  E f f e c t 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 n e by the s t e e r s on d i f f e r e n t times f o l l o w i n g the i m p l a n t a t i o n of a n a b o l i c compound i s p r e s e n t e d i n F i g u r e 16.  The t r e a t m e n t x p e r i o d 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  significant  (P > .05) but a g r a d u a l r e d u c t i o n i n the amount of HYPRO  e x c r e t e d beyond day 28 was observed i n a l l the t r e a t m e n t groups.  In a l l  the p e r i o d s s t u d i e d HYPRO e x c r e t i o n was found to be s l i g h t l y e l e v a t e d i n s t e e r s i m p l a n t e d w i t h DES when compared t o o t h e r  compounds.  When a l l the p e r i o d s (28, 42, 56 & 63 d) were t a k e n 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 i g h e r (P < .05) i n  steers  i m p l a n t e d w i t h DES than Z e r a n o l or Synovex-S or the u n i m p l a n t e d c o n t r o l steers  (Figure 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 Z e r a n o l , Synovex-S o r unimplanted c o n t r o l s t e e r s . The u r i n a r y H Y P R O : c r e a t i n i n e r a t i o was found t o be s i m i l a r among a l l the treatment groups (P > . 0 5 , T a b l e  14).  DISCUSSION H y d r o x y p r o l i n e (HYPRO) e x c r e t i o n i n u r i n e has been used i n t h i s experiment t o m o n i t o r c o l l a g e n t u r n o v e r i n s t e e r s i m p l a n t e d w i t h v a r i o u s anabolic  compounds.  -  T a b l e 14.  101  U r i n a r y h y d r o x y p r o l i n e e x c r e t i o n (mg/g c r e a t i n i n e ) i n s t e e r s on v a r i o u s times f o l l o w i n g the i m p l a n t a t i o n o f a n a b o l i c compounds U r i n a r y HYPRO: days a f t e r  Treatment  -  creatinine  56  42  28  Mean  implantation 63  (± SE)  Control  67.66  42.99  39.56  53.02  50.81 (5.24)  Zeranol  63.22  49.93  58.35  29.29  50.20 (6.76)  DES  85.41  72.48  38.96  57.86  67.21 (7.84)  Synovex-S  70.14  53.11  49.3  39.14  52.92 (5.83)  Mean (± SE)  71.61  a  (7.04)  i,b  Columns w i t h  54.63  b  (4.89)  47.63  b  (5.22)  44.83  3  3  3  b  (5.19)  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  a  (P < .05)  -  102 -  JZ  o ? 101-  CONTROL ZERANOL 1 •••• •••• __ DES SYNOVEX-S _QJ_\  X?  m  %% 44 •4 44  X  8 ^44 O cr JJ 444 444 44 CL 6 44 44 O > 44 i 44 X •\** 44 4 • 44 o >' 4 4 cc <  2  • 44 44 44 4*  o ' «ltt o ,  0  Figure 16.  rl+t<  28  X  X 44 44 44 44 44 4-4 44 44 44 44  o I  o 0  44  a 42  56  DAYS AFTER IMPLANTATION  44 44 44 44 44 44 44 44 44 44 44 44 44 44 44 44 44 44 44 44 44  • • •• • • ••  • 63  0 <  0• 0 o <  Urinary total.hydroxyproline (HYPRO) excretion by steers on d i f f e r e n t days following the implantation of anabolic compounds. Each bar represents mean ± SE of values obtained from two steers.  -  103  -  10 r—  iV. ••••  X  8  O _  $2 >  b  6  ^  DC CvJ  4  X  F i g u r e 17.  fp&SYNOVEX-S  •++ •++ •++ •+• •++ •+• •++ ••+ •++ > •+++ •++ ••+ • « +++ •++ +•+  I  »  0  CONTROL ZERANOL  c  •  >o oc o oc o oc *++ o DC o •••  .•i  E f f e c t of a n a b o l i c compounds on u r i n a r y t o t a l h y d r o x y p r o l i n e e x c r e t i o n by growing beef s t e e r s . Each bar r e p r e s e n t s mean ± SE of v a l u e s o b t a i n e d from two s t e e 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). » B a r s w i t h d i f f e r e n t alphabet d i f f e r (P < . 0 5 ) .  a  t  significantly  - 104 -  V a l i d i t y o f the Method In collagen biosynthesis, proline i s incorporated peptide  chain and i s then hydroxylated  into the  to HYPRO (Peterkofsky and  Udenfriend, 1965) by the protocollalgen-proline-hydroxylase  system.  During the degradation of collagen, HYPRO i s released and i t s excretion r e f l e c t s changes i n the turnover 1970;  Robins, 1977).  Besides,  of collagen i n the body (Kivirikko,  free HYPRO present  i n the b i o l o g i c a l  f l u i d has been demonstrated to originate from the degradation of collagen and not from the direct hydroxylation of proline or p r o l y l - t RNA (Review by K i v i r i k k o , 1970) and has been u t i l i z e d to study collagen metabolism to a large extent i n man (Jasin  et a l . , 1962; K i v i r i k k o ,  1970). Though HYPRO excretion was used as an index, i t s presence i n 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, application of this method.  1972) pose a problem to the  While the N-terminal extension peptides are  released and presumably degraded during normal f i b r i l l o g e n e s i s 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 i n l i v e r was also reported  (Goldstone and Adam, 1965; Efron et a l . , 1968).  Developmental Aspects o f Collagen Metabolism Data i n Figure 14 demonstrate the developmental aspects associated with collagen turnover.  I t tended to decrease as the steers  - 105 -  gained weight as evidenced  by the decrease i n urinary HYPRO. The  developmental changes i n collagen content In the bovine carcass from b i r t h to maturity were presented i n d e t a i l by Boccard (1978).  I t was  reported that the synthesis of collagen decreased, as the a c t i v i t y of prolyloxylase responsible for the hydroxylation f e l l during the ageing process  (Boccard, 1978).  The reduction i n the urinary HYPRO excretion  with advancing maturity observed i n the present study could be interpreted as due to a reduction i n the synthesis of new collagen or due to increased resistance of collagen f i b r i l s to breakdown by collagenolytic enzymes. maturation  K i v i r i k k o (1970) reported that during the  process more and more intermolecular cross-links were formed  and the collagen became increasingly resistant to s o l u b i l i z a t i o n and collagenase attack.  G o l l et_ a l . (1964) reported the presence of more  frequent or strong cross-linkages i n the collagen of mature than young bovine and a decreased  collagen s o l u b i l i t y with advancing maturity.  The developmental changes i n collagen turnover observed i n t h i s study are similar to those reported by Wu et a l . (1981) i n steers. However, i n the l a t t e r study plasma non-protein HYPRO (NPH) was used as an index of collagen turnover.  When the steers were fed a high  concentrate diet, NPH i n plasma increased upto 6 weeks (or t i l l 410 kg i n d i c a t i n g rapid collagen turnover) and decreased 1981).  thereafter (Wu et a l . ,  It i s possible that animals used i n the present study could have  exhibited rapid collagen turnover had they been studied at an e a r l i e r stage of growth.  Besides, the steers used i n this experiment could have  begun to accumulate more fat with continual reduction i n muscle  - 106 -  deposition thus exhibiting reductions i n urinary HYPRO excretion. K i v i r i k k o (1970) suggested that both the rate of collagen synthesis (ki and k ; Figure 18) and degradation ( k , k 2  5  6  and k ) are considerably 7  slower i n older animals i n spite of their greater collagen content of the body. In conclusion, the developmental reduction i n the urinary HYPRO excretion and hence reduction i n collagen turnover  could be attributed  to a reduction i n the t o t a l 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 i n maturity.  Anabolic Compounds and Collagen Turnover The e f f e c t of anabolic compound on collagen metabolism i n growing steers i s shown i n Figure 16 and 17. Among the compounds, DES seem to increase collagen turnover  as indicated by increased  excretion i n urine compared to other treatments.  (Figure 17) HYPRO  In fact, urinary HYPRO  excretion observed i n control and steers implanted with Zeranol and Synovex-S was similar (P > .05).  These d i f f e r e n t i a l actions of anabolic  compounds on collagen metabolism are noteworthy, and since this i s the f i r s t paper on the effect of anabolic on urinary HYPRO excretion, no d i r e c t comparison with the values i n the l i t e r a t u r e could be made. Increased  HYPRO i n urine by the steers Implanted with DES  indicates an increased amount of collagen present turnover.  These observations  and i t s rapid  are comparable to the early reports of  Mcintosh et a l . (1961) on the effect of o r a l l y administered in increasing connective  tissues of lamb skeletal muscle.  stilbestrol Based on  - 107 -  PROLINE K1  Protocollagen  •14 M NaCl k Soluble Collagen k  3  4  k  Less Soluble Collagen  Peptide Hypro in tissues  V  Free Hypro] in Tissues  v11  10  Free Hypro in Plasma  co  2  Urea  14  Peptide y.pro . in Urine H  M  Figure 18.  Insoluble Collagen  K  Pept ide Hypro in Plasma  n  Free Hypro in Urine  A schematic summary of the relationship between urinary hydroxyproline excretion and the metabolism of collagen, to k!5 refers to conversion rate constants.  kl  -  108  -  u n p u b l i s h e d d a t a , they have a l s o i n d i c a t e d i n c r e a s e d c o l l a g e n c o n t e n t of s k e l e t a l muscle a l o n g w i t h m u c o p r o t e i n 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 n c r e a s e i n s k i n c o l l a g e n c o n t e n t was  r e p o r t e d by A n a s t a s s i a d i s  also  e t a l . (1955) from e s t r o g e n t r e a t e d immature  pullets. As most of the a n a b o l i c a c t i o n s of DES i n ruminants through changes i n endogenous p r o t e i n hormones, could hypothesize  are mediated  e s p e c i a l l y GH, one  t h a t the i n c r e a s e d HYPRO e x c r e t i o n i n DES  implanted  s t e e r s may be p a r t l y due t o the i n d i r e c t e f f e c t s of GH, w h i c h has r e p o r t e d to promote c o l l a g e n s y n t h e s i s 1962)  and p o s s i b l y  in_ v i t r o (Daughaday and M a r i z ,  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  collagen synthesis.  But the l a c k of response  i n v a l i d , as the l a t t e r a n a b o l i c compounds  overall  on u r i n a r y HYPRO e x c r e t i o n  due t o Z e r a n o l and Synovex-S i m p l a n t a t i o n s makes the above  endogenous GH s t a t u s  theory  have a l s o been shown t o a l t e r  ( O l s e n et a l . , 1977; r e s u l t s of E x p t .  S i m i l a r (P > .05) H Y P R O : c r e a t i n i n e  been  IIB).  r a t i o ( T a b l e 14) among a l l the  t r e a t m e n t groups suggests f u r t h e r t h a t growth hormone may not be i n v o l v e d i n the d i f f e r e n t i a l responses of a n a b o l i c compounds  on c o l l a g e n  turnover. Though a r a t i o n a l e x p l a n a t i o n 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 t o Z e r a n o l and Synovex-S on c o l l a g e n metabolism c o u l d not be g i v e n , the d a t a p r e s e n t e d e s p e c i a l l y f o r DES, hypothesis  support  the g e n e r a l  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 c o n n e c t i v e  tissue  metabolism. It  i s h y p o t h e s i z e d t h a t the i n c r e a s e d HYPRO e x c r e t i o n i n DES  -  109  -  i m p l a n t e d s t e e r s may be a t t r i b u t e d t o : s y n t h e s i s ( k i and k ; 2  a) i n c r e a s e d r a t e of  F i g u r e 18); b) r e d u c t i o n i n the c o n v e r s i o n  s o l u b l e c o l l a g e n t o l e s s s o l u b l e c o l l a g e n (k3 and k ) ;  c)  n  r a t e of c o l l a g e n d e g r a d a t i o n ( k , n  k^,  i n v o l v e m e n t of a l l the above f a c t o r s . research i n t h i s  collagen  k , 5  kg and k j ) ,  of  increased  and d) the  Data i n d i c a t e a need f o r f u r t h e r  area.  From a p r a c t i c a l view p o i n t , i f h i g h e r HYPRO e x c r e t i o n r a t e t\  is  a s s o c i a t e d w i t h h i g h e r c o l l a g e n c o n t e n t i t would h e l p to e x p l a i n the l o w e r i n g of c a r c a s s 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 e e d i n g .  This  in  l i g h t of the l a c k of s i g n i f i c a n t e f f e c t on c o l l a g e n metabolism due t o Z e r a n o l and Synovex-S i m p l a n t a t i o n 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 d e v e l o p m e n t a l changes a s s o c i a t e d w i t h c o l l a g e n metabolism i n s t e e r s .  Collagen  turnover  d e c r e a s e d as the s t e e r s advanced i n m a t u r i t y and gained weight  as  i n d i c a t e d by the r e d u c t i o n i n the u r i n a r y h y d r o x y p r o l i n e e x c r e t i o n . i s concluded t h a t the r e d u c t i o n i n the u r i n a r y h y d r o x y p r o l i n e w i t h advancing m a t u r i t y of s t e e r s  It  excretion  i s due to a r e d u c t i o n i n the s y n t h e s i s  of new c o l l a g e n f i b r i l s and due t o the i n c r e a s e d 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 t o 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 a n a b o l i c compounds on c o l l a g e n m e t a b o l i s m was a l s o e s t a b l i s h e d i n t h i s s t u d y .  I m p l a n t a t i o n of DES  had  a profound e f f e c t on the i n v i v o c o l l a g e n t u r n o v e r as i n d i c a t e d by the i n c r e a s e d u r i n a r y h y d r o x y p r o l i n e e x c r e t i o n w h i l e the i m p l a n t a t i o n w i t h  - 110 -  Z e r a n o l and Synovex-S had no e f f e c t on c o l l a g e n m e t a b o l i s m . concluded t h a t DES i n c r e a s e s steers.  It  is  the amount o f c o l l a g e n and i t s t u r n o v e r  These a c t i o n s of DES on c o n n e c t i v e t i s s u e m e t a b o l i s m c o u l d be  r e s p o n s i b l e f o r the g e n e r a l l o w e r i n g o f the c a r c a s s q u a l i t y observed other  In  researchers.  by  - Ill -  STUDY I I 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 i n growing beef steers. Attention was focussed on the metabolism of thyroxine, growth hormone and i n s u l i n .  EXPERIMENT IIA:  Effect of anabolic compounds on thyroid hormone status i n growing beef steers. (1)  plasma thyroid hormone levels  (2)  k i n e t i c parameters of thyroxine metabolism.  EXPERIMENT IIB:  Effect of anabolic 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 steers.  EXPERIMENT IIC:  Effect of anabolic compounds on the k i n e t i c parameters of i n s u l i n metabolism i n 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 i n the regulation of growth  and metabolism (Thorngren and Hansson, 1973) i t appears that they might also mediate, at least i n 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 e p i t h e l i a l c e l l s (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 i n the thyroid gland weight and the concentration of plasma protein-bound iodine i n DES treated steers (Trenkle, 1969; Clegg and C a r r o l l , 1956; Trenkle, 1970a).  The effect of DES i n increasing the  fasting metabolic rate of c a t t l e (Rumsey et a l . , 1973; T y r r e l l et a l . , 1975) and sheep (Louw et^ a l . , 1964) strongly suggests the involvement of thyroid hormones i n the estrogen induced growth processess i n farm animals. This study was conducted to investigate the effect of certain estrogenic anabolic compounds (Zeranol, DES and Synovex-S) on the a c t i v i t y of the thyroid gland. levels of thyroxine  Attention was focussed  on the plasma  (T4) and triiodothyronine (T3), on the binding  -  113  -  c a p a c i t y of t h y r o x i n e b i n d i n g g l o b u l i n (as TBG RDA.)  a n <  *  o  n  t  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 o f t h y r o x i n e (as T h y r o x i n e Assessment, FTA) i n the p l a s m a . study the i n v o l v e m e n t of t h y r o i d hormones growth i n  n  e  s  t  a  t  u  s  Free  The major o b j e c t i v e was t o i n the e s t r o g e n mediated  steers.  MATERIALS AND METHODS Animals and Experimental Procedure S i x t e e n H e r e f o r d s t e e r s which were used i n E x p t . IA were used i n t h i s experiment.  The a n a b o l i c compounds,  Synovex-S) t h e i r i m p l a n t a t i o n procedures  (Control, Zeranol,  DES,  and the e x p e r i m e n t a l r a t i o n  used were the same as been d e s c r i b e d i n E x p t .  IA.  B l o o d samples were c o l l e c t e d from i n d i v i d u a l s t e e r s on days 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 n a b o l i c compounds o f j u g u l a r v e i n p u n c t u r e i n h e p a r i n i z e d evacuated tubes tubes, Beckton-Dickinson,  The  were kept i n i c e and were c e n t r i f u g e d i m m e d i a t e l y at 1000 x g;  a f t e r an o v e r n i g h t  by means  (vacutainer  D i c k i n s o n and C o . , New J e r s e y ) .  plasma was c o l l e c t e d and s t o r e d at - 2 0 ° C .  14,  tubes the  B l o o d samples were c o l l e c t e d  fast.  A n a l y t i c a l Procedure The plasma samples were thawed a t 4°C and the c o n c e n t r a t i o n of t h y r o i d hormones was then d e t e r m i n e d . (1)  T o t a l Thyroxine  (T4)  The plasma c o n c e n t r a t i o n o f t o t a l T4 was determined by a s p e c i f i c radioimmunoassay  procedure (Chopra, 1972)  using a  solid-phase  -  radioimmunoassay  114  system (Immuchemical  1-naphthalene-sulfonic  a c i d (ANS)  t o the t h y r o i d b i n d i n g g l o b u l i n  -  Co.);  8-anilino,  was used to d i s r u p t the b i n d i n g of T4 (TBG).  The t e s t sample or T4 s t a n d a r d (0 t o 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 a n t i b o d y coated tubes f o l l o w e d by 125J_-J>4 i  0.01  n  M phosphosaline  b u f f e r c o n t a i n i n g ANS.  v o r t e x mixed and i n c u b a t e d f o r 1 h a t 37°C.  The tubes were  A f t e r the i n c u b a t i o n s t e p ,  t h e s o l u t i o n c o n t a 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 a u t o m a t i c gamma-counter Nuclear  (Model  1185,  Chicago). The c o n c e n t r a t i o n of T4 i n the unknown samples was c a l c u l a t e d  from the s t a n d a r d curve u s i n g the r a p i d procedure d e s c r i b e d by Rodbard et a l . ( 1 9 6 9 ) . (Rodbard,  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  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 c o n c e n t r a t i o n 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 p e r i o d were a n a l y s e d i n the same a s s a y , t o m i n i m i z e assay variations. (2)  Total Triiodothyronine  (T3)  The plasma t o t a l T3 c o n c e n t r a t i o n s were determined by a s p e c i f i c radioimmunoassay  procedure d e s c r i b e d by Chopra e t a l . (1972) u s i n g  s o l i d phase radioimmunoassay  package.  ANS was used t o d i s r u p t  b i n d i n g of T3 to t h y r o i d b i n d i n g g l o b u l i n The t e s t sample o r s t a n d a r d  T  n  o.Ol M p h o s p h o s a l i n e  the  (TBG).  (0-80 ng/100 ml) o r the q u a l i t y  c o n t r o l sample was added t o T3 - a n t i b o d y coated t u b e s . 125j _ 3 £  a  A s o l u t i o n of  b u f f e r c o n t a i n i n g ANS was added and  -  115  -  t h e tubes were i n c u b a t e d f o r 2 h at 37°C.  At the end o f the i n c u b a t i o n  p e r i o d , the s o l u t i o n c o n t a 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 a u t o m a t i c gamma c o u n t e r 1185,  (Model  Nuclear-Chicago). The c a l c u l a t i o n of T3 c o n c e n t r a t i o n i n the unknown samples  the i n t e r - and i n t r a a s s a y  and  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 t o  those d e s c r i b e d f o r T4 - radioimmunoassay.  A l l t h e samples  c o l l e c t e d at  any one time p e r i o d were a n a l y s e d i n the same assay to m i n i m i z e assay variations. (3)  T o t a l T h y r o x i n e B i n d i n g G l o b u l i n (TBG) B i n d i n g  Capacity  The t o t a l TBG b i n d i n g c a p a c i t y was determined by a r a d i o d i s p l a c e m e n t assay (NMS,  1980a).  The t e c h n i q u e measured t h e t o t a l t h y r o x i n e b i n d i n g  globulin  c a p a c i t y of the s p e c i f i c t h y r o x i n e b i n d i n g i n t e r a l p h a g l o b u l i n . excess  A large  of l a b e l l e d ( ^ 5 i _ x4) and u n l a b e l l e d (T4) t h y r o x i n e was added  t o tubes c o n t a i n i n g the t e s t sample or r e f e r e n c e s t a n d a r d s  (high  s t a n d a r d = 20.8 ug/100 m l ; low s t a n d a r d = 10.1 ug/100 ml) or b u f f e r . The tubes were i n c u b a t e d a t room temperature f o r 30 min and the f r e e * 2 5 i _ x4 was s e p a r a t e d from the bound f r a c t i o n by a charcoal-dextran slurry. gamma c o u n t e r (Model 1185, Calculation  The bound f r a c t i o n was counted i n an a u t o m a t i c Nuclear-Chicago).  The b i n d i n g c a p a c i t y o f the t h y r o x i n e b i n d i n g g l o b u l i n i n the unknown samples was c a l c u l a t e d a c c o r d i n g t o the f o r m u l a :  - 116 -  C  ?  M  U N K -  B  A  (RDA) Where: TBG(RDA)  Thyroxine binding globulin capacity by  =  radio-displacement CJ?%NK  •  assay  Bound r a d i o a c t i v i t y i n the unknown tube  ( H i g h standard) Assayed V a l u e  ( L o w standard) Assayed V a l u e  C P M  ( H i g h  CPM  - » (Low std.)  -  / T  CPM  s t d > )  -  ( L o w  A (Assayed value, Low 3  Thyroxine binding globulin opacity was  ^  , . std.)  expressed as ug T4 bound  per 100 ml plasma.  (4)  Free Thyroxine Assessment  (FTA)  The FTA, which i s an index of the free, unbound thyroxine i n the c i r c u l a t i o n was  calculated from the t o t a l T4 concentration i n the  c i r c u l a t i o n and the t o t a l T4 binding capacity, according to the formula:  T4 FTA  where  TBG  (RDA)  FTA  =  Free thyroxine assessment  T4  =  Total T4 concentration (ug/100 ml)  TIH*(RDA)  =  T o t a l thyroxine binding globulin capacity determined  by the radio-displacement  (ug/100 ml)  assay  -  Statistical  117  -  Analysis  The plasma c o n c e n t r a t i o n s of t h y r o i d hormone were averaged  for  each t r e a t m e n t group on a l l the s a m p l i n g p e r i o d s and s u b j e c t e d t o l e a s t squares a n a l y s i s  of v a r i a n c e ( S t e e l and T o r r i e , 1960)  BMD:10V l i n e a r h y p o t h e s i s  computer program.  u s i n g the UBC  The s i n g l e degree of  freedom o r t h o g o n a l c o n t r a s t t e s t ( S t e e l and T o r r i e , 1960) was used t o compare t r e a t m e n t s .  RESULTS Plasma Total Thyroxine (T4) The plasma c o n c e n t r a t i o n 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 n a b o l i c compounds  (Table  15).  T o t a l T4 c o n c e n t r a t i o n i n the plasma was about 23 (P < .05) 18 p e r cent (P = DES,  .08) h i g h e r i n s t e e r s i m p l a n t e d w i t h Synovex-S and  r e s p e c t i v e l y , than i n the u n i m p l a n t e d c o n t r o l s t e e r s .  On the  c o n t r a r y , o n l y a s l i g h t i n c r e a s e i n the plasma T4 c o n c e n t r a t i o n non-significant)  was observed i n s t e e r s i m p l a n t e d w i t h Z e r a n o l  t o 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)  compared t o Z e r a n o 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 i g h e r  (2.2%, compared  i n the plasma  T4 c o n c e n t r a t i o n i n s t e e r s i m p l a n t e d w i t h Synovex-S and DES.  However,  (P <  .05)  c o n c e n t r a t i o n o f plasma T4 was observed i n s t e e r s Implanted  with  Synovex-S.  and  Though the t r e a t m e n t X p e r i o d 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 c o n c e n t r a t i o n on a l l the time p e r i o d s t u d i e d was h i g h e r i n Synovex-S and DES i m p l a n t e d than i n the c o n t r o l s t e e r s .  The  -  T a b l e 15.  -  Plasma t o t a l t h y r o x i n e (T4) c o n c e n t r a t i o n i n s t e e 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 a n a b o l i c compounds  Days A f t e r Treatment  118  14  Implantation 42  28  56  Overall Treatment Mean  SE  J  ug/100 m l Control  4.50  3.65  4.68  4.78  4.40  0.30  Zeranol  5.33  4.26  3.89  4.5  4.50  0.30  DES  5.51  5.01  5.11  5.14  5.19  0.27  Synovex-S  5.53  4.30  4.95  6.84  5.40  0.44  Orthogonal Contrasts  f o r O v e r a l l Treatment Means  Contrasts Control Control Control Zeranol Zeranol DES Control  vs. vs. vs. vs. vs. vs. vs.  P o o l e d S.E.  P Zeranol DES Synovex-S DES Synovex-S Synovex-S Z e r a n o l + DES Synovex-S  N.S 0.08 0.03 N.S 0.04 N.S + N.S  of the mean.  S i n g l e degree of freedom o r t h o g o n a l c o n t r a s t were c a l c u l a t e d u s i n g UBC BMD:10V l i n e a r h y p o t h e s i s computer program.  -  119  -  e f f e c t of Z e r a n o l on the t h y r o i d g l a n d was o n l y m a r g i n a l , w i t h s l i g h t l y i n c r e a s e d response  n o t i c e d 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 c o n c e n t r a t i o n i n the s t e e 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 c o n c e n t r a t i o n 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 compounds  (Table  anabolic  16).  Plasma T3 c o n c e n t r a t i o n was found t o be s i m i l a r (P > .05) i m p l a n t e d and u n i m p l a n t e d c o n t r o l s t e e r s studied.  in  on a l l the time p e r i o d  O v e r - a l l mean plasma c o n c e n t r a t i o n of T3 i n the s t e e r s used i n  t h i s study was found t o be 133.3 ± 6.0 ng/100 m l .  Thyroxine Binding Globulin Capacity (as  T B G R D A  )  The b i n d i n g c a p a c i t y of t h y r o x i n e b i n d i n g g l o b u l i n was d e t e r m i n e d 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 a n a b o l i c compounds r a d i o d i s p l a c e m e n t assay ( T B G values  R D A  ,  Table 17).  O v e r - a l l mean T B G  R D A  of the s t e e r s used i n t h i s experiment was found t o be 15.22 ± .8  ug/100 ml plasma and was i n agreement w i t h the normal v a l u e s u s i n g the r a d i o - d i s p l a c e m e n t assay (NMS, periods  by a  1980a).  reported  When both the time  (days 42 and 56) were t a k e n i n t o a c c o u n t , TBGjyj^ was found t o  be s i g n i f i c a n t l y reduced (P < . 0 0 1 , o r t h o g o n a l comparison f o r c o n t r o l vs a l l the a n a b o l i c t r e a t m e n t s ) i n s t e e r s  implanted w i t h anabolic  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 -  T a b l e 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 o n c e n t r a t i o n i n s t e e 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 o f a n a b o l i c compounds  Treatment  Days A f t e r I m p l a n t a t i o n 14" 42 56  Overall Treatment Mean  SE  ng/100 ml Control  132.7  128.4  150.5  137.1  15.4  Zeranol  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 Contrasts  f o r O v e r a l l Treatment Means  Contrasts Control Control Control Zeranol Zeranol DES  vs. vs. vs. vs. vs. vs.  P Zeranol DES Synovex-S DES Synovex-S Synovex-S  N.S N.S N.S N.S N.S N.S  P o o l e d s t a n d a r d e r r o r of the mean. S i n g l e degree of freedom o r t h o g o n a l c o n t r a s t were c a l c u l a t e d u s i n g BMD:10V l i n e a r h y p o t h e s i s computer program.  - 121 -  Table 17.  Thyroxine binding globulin capacity ( T B G i n steers on d i f f e r e n t days following the implantation of anabolic compounds  Days After  Treatment  Implantation  42  56  RDA  )  Overall Treatment Mean  yg/100 ml . Control  19.41  18.59  19.00  1.36  Zeranol  13.08  12.85  12.97  0.84  DES  17.21.  13.36  15.28  1.40  Synovex-S  16.86  10.39  13.63  1.89  Orthogonal Comparisons for Overall Treatment Means Contrasts Control Control Control Zeranol Zeranol DES Control  vs. vs. vs. vs. vs. vs. vs.  P Zeranol DES Synovex-S DES Synovex-S Synovex-S Zeranol + DES + Synovex-S  0.001 0.02 0.002 N.S N.S N.S 0.001  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.  -  r a t i o o f plasma T4 and T B G  R D A  122 -  (Table 1 8 ) .  O v e r - a l l mean FTA i n the  s t e e r s used i n t h i s experiment was found t o be 0.3281 ± .021 and was i n agreement w i t h the normal v a l u e s o f FTA i n the plasma (NMS, 1980a). When both the time p e r i o d s  (days 42 and 56) were t a k e n i n t o  a c c o u n t , the mean FTA v a l u e was found t o be s i g n i f i c a n t l y h i g h e r  (P <  . 0 5 , o r t h o g o n a l comparison f o r c o n t r o l vs a l l the a n a b o l i c compounds) s t e e r s i m p l a n t e d w i t h a n a b o l i c compounds  than the c o n t r o l s .  in  The FTA was  found t o be 53 (P < . 0 2 ) , 37 (N.S.) and 29 (N.S.) per cent h i g h e r  in  s t e e r s i m p l a n t e d 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 t h e unimplanted c o n t r o l Increased  steers.  FTA v a l u e s  i n the i m p l a n t e d s t e e r s i n d i c a t e an  e l e v a t i o n i n the plasma c o n c e n t r a t i o n o f 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 o f 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 c o n c e n t r a t i o n due t o a n a b o l i c compound i m p l a n t a t i o n s i s p r e s e n t e d i n T a b l e 19.  DISCUSSION  I n t h i s e x p e r i m e n t , the e f f e c t o f e s t r o g e n i c a n a b o 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 o r d e r t o u n d e r s t a n d t h e mechanism of growth induced by a n a b o l i c compounds and e v a l u a t e the i n v o l v e m e n t of t h y r o i d hormones Results  i n such growth  o f t h i s experiment i n d i c a t e an I n c r e a s e  processes.  i n the plasma  t o t a l T4 and f r e e 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  c o n c e n t r a t i o n a r e 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 t a t u s f o l l o w i n g e s t r o g e n a d m i n i s t r a t i o n i n man  -  T a b l e 18.  Mean f r e e t h y r o x i n e assessment (FTA) i n s t e e r s on d i f f e r e n t days f o l l o w i n g t h e i m p l a n t a t i o n o f a n a b o l i c compounds  Days A f t e r Treatment  123 -  Implantation  42  56  Overall Treatment Mean  SE'  Control  0.2498  0.2578  0.2538  0.0241  Zeranol  0.2996  0.3592  0.3294  0.0281  DES  0.3066  0.3908  0.3487  0.0320  Synovex-S  0.2951  0.5119  0.3880  0.0623  Orthogonal  Comparisons f o r O v e r a l l Treatment Means  Contrasts Control Control Control Zeranol Zeranol DES Control  1 o  m  vs. vs. vs. vs. vs. vs. vs.  P Zeranol DES Synovex-S DES Synovex-S Synovex-S Z e r a n o l + DES + Synovex-S  N.S N.S 0.02 N.S N.S N.S 0.03  T o t a l T4 yg/100 m l T B G ^ ug/100 m l  P o o l e d s t a n d a r d e r r o r o f the mean.  - 124 -  T a b l e 19.  Treatment  Summary of changes l n the plasma t h y r o i d hormone concentration i n steers implanted with anabolic compounds  T3  T4  J  TBG-  i  FTA  Control  4.40  137.1  19.00  0.2538  Zeranol  4.50  120.7  12.97  0.3294  DES  5.19  135.0  15.28  0.3487  Synovex-S  5.40  139.2  13.63  0.3880  Orthogonal  Comparisons  Control Control Control Zeranol Zeranol DES Control  Zeranol DES Synovex-S DES Synovex-S Synovex-S Zeranol + DES + Synovex-S  mean mean mean mean  of of of of  vs. vs. vs. vs. vs. vs. vs.  v a l u e s (ug/100 ml) v a l u e s (ng/100 ml) v a l u e s (ug/100 ml) v a l u e s o b t a i n e d on  N.S 0.08 0.03 N.S 0.04 N.S  N.S N.S N.S N.S N.S N.S  0.001 0.02 0.002 N.S N.S N.S  N.S N.S 0.02 N.S N.S N.S  N.S.  N.S.  0.001  0.03  o b t a i n e d on 14, 28, 42 & 56 days o b t a i n e d on 14, 42 and 56 days o b t a i n e d on 42 and 56 days 42 and 56 days  -  (Zaninovich, 1978).  1973)  Increased  125  -  and i n Synovex-S i m p l a n t e d s t e e r s  (Kahl et a l .  plasma T4 c o n c e n t r a t i o n was proposed t o be  responsible  f o r the i n c r e a s e d 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 (Burgess and Lamming, 1960)  steers  and a t r e n d towards i n c r e a s e d T4 s e c r e t i o n  r a t e was r e p o r t e d i n DES-fed lambs ( T r e n k l e , 1969).  Besides,  an  i n c r e a s e i n the h e a r t r a t e and b a s a l m e t a b o l i c r a t e of s t e e r s w i t h DES (Rumsey e t a l . , 1973; T y r r e l l e t a l . , 1975) i n v o l v e m e n t of T4.  t  implanted  s u g g e s t s the  I t would appear t h a t the i n c r e a s e d T4 l e v e l s i n the  Synovex-S i m p l a n t e d s t e e r s used i n t h i s experiment c o u l d have  resulted  i n s i m i l a r changes i n energy m e t a b o l i s m . E l e v a t e d T4 l e v e l observed i n the s t e e r s c o u l d a l s o be due t o a r e d u c t i o n i n T4 d e g r a d a t i o n r a t e and an i n c r e a s e i n TBG b i n d i n g capacity (Zaninovich,  1973).  Thyroxine binding g l o b u l i n c a p a c i t y which  e x e r t s a r a t e 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 m e t a b o l i s m o f t h y r o x i n e (Dowling e t a l . , 1960) was reduced i n b o t h Synovex-S (P < and DES  (N.S.) implanted s t e e r s .  E s t r o g e n a d m i n i s t r a t i o n was  .05)  associated  w i t h i n c r e a s e d b i n d i n g c a p a c i t y of TBG (Sawhney et a l . , 1978; Z a n i n o v i c h , 1973) experiment.  an o b s e r v a t i o n w h i c h was not supported i n  this  The TBG b i n d i n g c a p a c i t y i n t h i s s t u d y , however,  was  measured by a r a d i o - d i s p l a c e m e n t assay and i t s r e d u c t i o n i n the i m p l a n t e d s t e e r s c o u l d 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 , K a h l e t a l . (1978) have r e p o r t e d enhanced b i n d i n g c a p a c i t y of the plasma t h y r o i d hormone b i n d i n g p r o t e i n s i n Synovex-S i m p l a n t e d s t e e r s . It  i s not c l e a r whether the i n c r e a s e d plasma T4 observed i n the  i m p l a n t e d s t e e r s i s a f u n c t i o n of i n c r e a s e d t h y r o i d a l T4 s e c r e t i o n r a t e  - 126  or decreased  -  metabolic clearance from the c i r c u l a t i o n or due to r e l a t i v e  changes i n both the parameters. Increased plasma T4 concentration observed i n the steers, especially with Synovex-S and DES was growth response (Figure 3, Expt. IA).  implanted  similar to the increased  The changes i n 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 i n pigs(Marple et a l . , 1981). The thyroid stimulating effects of estrogenic anabolic compounds, e s p e c i a l l y Synovex-S and DES,  established i n this study are i n  contradiction to that reported for androgenic anabolic compounds (Donaldson et a l . , 1981;  Heitzman et a l . , 1977).  Trenbolone acetate,  either alone or i n combination with e s t r a d i o l has been shown to cause marked reduction (up to 45%) i n the plasma T4 concentration i n wether lambs (Donaldson et a l . , 1981).  The thyroid stimulating effects of  estrogenic and the depressing influence of androgenic anabolic compounds indicate the d i f f e r e n t i a l mechanisms by which they mediate growth i n farm animals.  D i e t h y l s t i l b e s t r o l has been shown to stimulate  thyrotropin (TSH) secretion from the p i t u i t a r y gland both i n i n vivo (Davis et a l . , 1978)  and i n v i t r o ( M i l l e r et a l . , 1977).  The T4 concentration i n the plasma was  not altered by Zeranol  implantation, however, a decreasing trend i n i t s concentration observed beyond day 28 following the implantation. i n plasma T4 l e v e l (Wiggins et^ a l . , 1979)  was  A similar reduction  and a decrease i n the thyroid  - 127  gland a c t i v i t y  -  (Rothenbacher j i t a l _ . , 1975) were r e p o r t e d i n Z e r a n o l  i m p l a n t e d lambs.  However,  t h y r o i d g l a n d weight of Z e r a n o l i m p l a n t e d  lambs was found t o be i n c r e a s e d (Wiggins ^ (Wiggins e t ^ a l . , 1979).  a l . , 1976)  or  decreased  I t was p o s t u l a t e d by Wiggins e t a l . (1979) t h a t  the d e c r e a s e d T4 l e v e l s observed i n Z e r a n o l i m p l a n t e d lambs as  compared  to c o n t r o l s c o u l d lower the b a s a l m e t a b o l i c r a t e and thus c o u l d the p r o p o r t i o n of absorbed n u t r i e n t s a v a i l a b l e f o r g r o w t h .  A trend  towards decreased plasma T4 c o n c e n t r a t i o n i n Z e r a n o l i m p l a n t e d used i n t h i s experiment suggests t h a t p o s s i b i l i t y .  increase  steers  The i m p l a n t a t i o n of  DES on the o t h e r hand seems to i n c r e a s e the b a s a l m e t a b o l i c r a t e i n steers  (Rumsey et a l . , 1973; T y r r e l l e t a l . , 1975).  e x p e r i m e n t , i n c o n j u n c t i o n w i t h the o b s e r v a t i o n s  The r e s u l t s of  of e a r l i e r  workers  ( K a h l et a l . , 1978; Rumsey et a l . , 1973; T y r r e l l e t a l . , 1975; e t a l . , 1979)  this  Wiggins  suggest t h a t the mechanism of a c t i o n of Z e r a n o l , DES,  and  Synovex-S as f a r as t h y r o i d g l a n d a c t i v i t y i s concerned may be different. Plasma T3 l e v e l s i n the i m p l a n t e d and c o n t r o l s t e e r s were s i m i l a r (P > .05)  on a l l the p e r i o d s  s t u d i e d and the r e s u l t s demonstrate  l a c k of i n f l u e n c e on T3 s t a t u s implantations.  It  i n s t e e r s due to a n a b o l i c  non-significant  compound  i s not c l e a r whether T3 i s i n v o l v e d i n the  of growth promotion mediated by the a n a b o l i c compounds.  the  mechanism  Similar  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 i m p l a n t e d s t e e r s was r e p o r t e d by K a h l et a l . (1978) who have concluded t h a t i n the b o v i n e , T4 r a t h e r than T3 might p h y s i o l o g i c a l and m e t a b o l i c a l t e r a t i o n s .  reflect  - 128  -  About one t h i r d o r l e s s of the c i r c u l a t i n g T3, i s of o r i g i n and the r e s t i s d e r i v e d from the e x t r a t h y r o i d a l o f T4 to T3 ( S t e r l i n g and L a z a r u s ,  1977).  thyroidal  monodeiodination  The presence of the  latter  mechanism was r e p o r t e d i n man by Braverman e t a l . ( 1 9 7 0 ) , i n the r a t by Schwartz  et _ a l . ( 1 9 7 1 ) , and i n sheep by F i s h e r e t a l . ( 1 9 7 2 ) .  The  c o n c e n t r a t i o n o f T3 i n the plasma i s t h e r e f o r e a f u n c t i o n o f T4 t o T3 conversion  rate, thyroidal secretion rate, disposal  c o n c e n t r a t i o n of serum b i n d i n g  r a t e , and the  proteins.  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 a n a b o l i c compounds: t h e r e c o u l d be a r e d u c t i o n i n the p e r i p h e r a l c o n v e r s i o n  (a)  of T4 t o T3 i n  the i m p l a n t e d s t e e r s ; enhanced b i n d i n g c a p a c i t y of the t h y r o x i n e g l o b u l i n and reduced f r a c t i o n a l t u r n o v e r 1960; E n g b r i n g and Engstrom,  1959)  binding  r a t e o f T4 (Dowling  £t a l . ,  f o l l o w i n g DES and n a t u r a l  estrogen  a d m i n i s t r a t i o n and t h e i n h i b i t i o n o f d e i o d i n a t i o n of T4 t o T3 i n the i n t a c t c e l l u l a r preparations 1959)  add support  by e s t r o g e n s (Yamazaki and  to t h i s p o s s i b i l i t y ;  Slingerland,  (b) In the i m p l a n t e d s t e e r s  m e t a b o l i s m of T3 i t s e l f c o u l d be r a p i d , namely i t s c o n v e r s i o n 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  to  t h a t are s u s c e p t i b l e t o the  s t i m u l a t i o n of t h y r o i d hormones ( h e a r t , k i d n e y and l i v e r ) have shown t o c o n t a i n enzymes  the  been  f o r the c o n v e r s i o n o f T3 t o t r i i o d o t h y r o a c e t i c  acid i n t h e i r mitochondria (Albright  et_ a l . , 1959)  and t h a t the  latter  m e t a b o l i t e was more a c t i v e than T 3 . 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 ,  r e s e a r c h on the p e r i p h e r a l c o n v e r s i o n f o l l o w i n g a n a b o l i c compound  suggest the need f o r  of T4 t o T3 i n s t e e r s ,  implantation.  further  especially  - 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 t h a t one o f the mechanisms which e s t r o g e n i c a n a b o l i c compounds  evoke growth promotion i n  by  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 e e T4. Implantations  of Synovex-S and DES r e s u l t e d i n i n c r e a s e d growth r a t e and  plasma T4 c o n c e n t r a t i o n .  Only a s l i g h t e l e v a t i o n I n the plasma T4  c o n c e n t r a t i o n was observed due to Z e r a n o 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 t h a t T3 may not be i n v o l v e d i n the growth processes  mediated by the a n a b o l i c compounds  no change i n i t s c o n c e n t r a t i o n was o b s e r v e d .  Further studies  w a r r a n t e d t o i n v e s t i g a t e the e f f e c t of a n a b o l i c compounds on T metabolism i n  as  are 3  steers.  Considering  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  and m e t a b o l i s m of a n i m a l s , i t i s concluded t h a t T4 i s mechanism of a c t i o n of a n a b o l i c  compounds.  growth  i n v o l v e d i n the  -  130 -  EXPERIMENT II A:2 KINETIC PARAMETERS OF THYROXINE METABOLISM IN STEERS IMPLANTED WITH ANABOLIC COMPOUNDS INTRODUCTION Results  of E x p t . I I A :  1 ( s e e page 117) i n d i c a t e d a h i g h e r  plasma  T4 c o n c e n t r a t i o n i n s t e e 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 t h e c o n t r o l s and suggested the i n v o l v e m e n t of T4 i n the growth processes  mediated by a n a b o l i c compounds.  On the o t h e r hand, plasma T4  c o n c e n t r a t i o n was not i n f l u e n c e d by Z e r a n o l i m p l a n t a t i o n s .  Though i t  was concluded t h a t the changes i n t h e c o n c e n t r a t i o n o f plasma T4 i n s t e e r s f o l l o w i n g Synovex-S, DES and Z e r a n o l I m p l a n t a t i o n s  c o u l d be due  t o 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 , t h e exact mechanism f o r such a l t e r a t i o n s c o u l d not be e x p l a i n e d .  A t r e n d towards i n c r e a s e d  thyroxine  s e c r e t i o n 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 had been r e p o r t e d e a r l i e r ( T r e n k l e , 1969).  The c o n c e n t r a t i o n o f T4 i n plasma a t a g i v e n time i s a  f u n c t i o n of s e c r e t i o n r a t e and c l e a r a n c e 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 c o n c e n t r a t i o n s f o l l o w i n g t h e i m p l a n t a t i o n o f a n a b o l i c compounds  c o u l d be due t o 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 , clearance rate or both.  E s t r o g e n a d m i n i s t r a t i o n has been shown t o  decrease d a i l y t h y r o x i n e d e g r a d a t i o n r a t e ( Z a n i n o v i c h , p a r t i c i p a t i o n o f t h y r o i d hormone b i n d i n g g l o b u l i n s been d i s c u s s e d  (Dowling e t a l . , 1960).  1973) and t h e  I n such p r o c e s s e s  has  Thus an experiment on t h e  k i n e t i c parameters o f T4 m e t a b o l i s m 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 t h e i m p l a n t a t i o n of a n a b o l i c compounds  assumes g r e a t i m p o r t a n c e .  This  was t h e r e f o r e conducted t o i n v e s t i g a t e t h e e f f e c t of e s t r o g e n i c  study  -  131  -  a n a b o l i c compounds on the k i n e t i c parmeters of T4 m e t a b o l i s m i n beef  growing  steers.  MATERIALS AND METHODS  Animals: Twenty f o u r H e r e f o r d s t e e r s , w e i g h i n g a p p r o x i m a t e l y 270 k g , were used i n t h i s s t u d y .  The a n i m a l management and the f e e d i n g p r o c e d u r e  were s i m i l a r t o those d e s c r i b e d i n E x p t .  IA.  Anabolic Treatments and Feeding Proceudre: The s t e e r s were a s s i g n e d t o one of the f o l l o w i n g t r e a t m e n t groups (n = 6 per t r e a t m e n t ) : 1.  Control  2.  36 mg i m p l a n t of  DES  3.  36 mg i m p l a n t of  Zeranol  4.  One i m p l a n t of Synovex-S  The method f o r the i m p l a n t a t i o n o f the a n a b o l i c compound has described e a r l i e r (Expt. IA).  been  The s t e e r s were weighed at weekly  i n t e r v a l s and f e d t w i c e 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 2 0 ) .  Water  and s a l t b l o c k s were p r o v i d e d f r e e c h o i c e .  Animal preparation f o r the K i n e t i c Experiments: The s t e e r s were f i t t e d w i t h an i n d w e l l i n g p o l y e t h y l e n e j u g u l a r c a t h e t e r ( P . E . 90, I.D e x p e r i m e n t s began.  = 0.86 mm; O.D = 1 . 2  mm) a day b e f o r e the k i n e t i c  The f r e e end of the c a t h e t e r was taped i n p o s i t i o n  and the c a t h e t e r was f l u s h e d w i t h a 4% sodium c i t r a t e  solution.  -  T a b l e 20.  132 -  Ration Composition  (Expt.  IIA:2)  % of (as  Ingredients  Ration fed)  Steam r o l l e d b a r l e y  79.0  Chopped g r a s s hay  15.0  Soybean meal  3.5  Ground L i m e s t o n e  1.5  Mineral-Vitamin M i x t u r e , 1  1.0  2  S u p p l i e d by B u c k e r f i e l d s L i m i t e d , A b b o t s f o r d , B.C.,  Canada  ^Mineral - vitamin mixture composition: s a l t ( 4 0 . 0 % ) , phosphorus ( 1 0 . 0 % ) , f l u o r i n e ( 0 . 2 % ) , i r o n ( 0 . 4 2 % ) , z i n c ( 0 . 8 % ) , manganese ( 0 . 2 % ) , c o b a l t ( 0 . 0 1 6 % ) , copper ( 0 . 1 5 % ) , v i t a m i n A (700,000 I . U . / k g ) , v i t a m i n D (70,000 I . U . / k g ) .  - 133  -  K i n e t i c Experiments: The k i n e t i c parameters of T4 m e t a b o l i s m were e s t i m a t e d 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 t e e r s from each  t r e a t m e n t group were used a t any one e x p e r i m e n t a l p e r i o d .  The  steers  used i n one e x p e r i m e n t a l p e r i o d were never used i n the subsequent studied.  period  K i n e t i c parameters were o b t a i n e d from e i g h t s t e e r s on each of  the t h r e e p e r i o d s mentioned above.  Experimental Procedure: A s i n g l e b o l u s 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 e t a l . , 1974; Post and M i x n e r ,  1961).  On the day of t h e e x p e r i m e n t , the s t e e r s were weighed, f e d the morning p o r t i o n of the r a t i o n and were p l a c e d i n m e t a b o l i s m c r a t e s w h i c h a l l o w e d some freedom of movement but p r e v e n t e d them from t u r n i n g around.  F o l l o w i n g a p e r i o d of a d j u s t m e n t , two b l o o d samples were  c o l l e c t e d v i a t h e c a t h e t e r t o 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  a n i m a l s 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 c a t h e t e r was i m m e d i a t e l y f l u s h e d w i t h 0.9%  s a l i n e t o make sure t h a t a l l the i n j e c t e d dose of T4 e n t e r  the b l o o d  stream f o r immediate m i x i n g and t o a v o i d the c o n t a m i n a t i o n of  Injected  T4 w i t h the subsequent b l o o d samples t o be c o l l e c t e d through the same catheter.  S e r i a l b l o o d samples were then c o l l e c t e d a t 6, 12, 24,  30,  4 8 , 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 t o m o n i t o r the d i s a p p e a r a n c e 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  w i t h d r a w i n g b l o o d samples, the c a t h e t e r was f l u s h e d w i t h s a l i n e  (0.9%  -  w/v) o r sodium c i t r a t e (4% w / v ) .  134  -  The b l o o d samples were k e p t i n  c o n t a i n i n g h e p a r i n and were c e n t r i f u g e d t o o b t a i n the p l a s m a .  tubes  The  plasma samples were s t o r e d a t - 2 0 ° C .  A n a l y t i c a l Procedure T4  radioimmunoassay:  The c o n c e n t r a t i o n 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 p e r i o d s 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 a s s a y d e s c r i p t i o n , c a l c u l a t i o n o f  T4 c o n c e n t r a t i o n i n the t e s t samples and the d e t e r m i n a t i o n 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 t o t h a t d e s c r i b e d e a r l i e r (T4-Radioimmunoassay,  Expt. IIA:  a n t i b o d y radioimmunoassay  1) w i t h the e x c e p t i o n t h a t a double  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 a u t o m a t i c gamma c o u n t e r 500 C c o u n t i n g system, P a c k a r d I n s t r u m e n t s ,  (Auto-Gamma  I l l i n o i s , U.S.A).  a n t i - r a b b i t gamma g l o b u l i n was used t o p r e c i p i t a t e the f i r s t The i n t e r - and i n t r a a s s a y  The  sheep  antibody.  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 c o n c e n t r a t i o n of 8.0 ug/100 m l . Analysis of K i n e t i c Data The plasma T4 d i l u t i o n curve was a n a l y s e d u s i n g 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 o f t h e l e a s t square e s t i m a t e s , t h e observed d a t a was found t o be b e s t d e s c r i b e d by a s i n g l e e x p o n e n t i a l e q u a t i o n .  Subsequently,  the  k i n e t i c d a t a from a l l t h e e x p e r i m e n t a l s t e e r s were a n a l y s e d 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 d i s a p p e a r a n c e  from the  b l o o d plasma i s d e p i c t e d 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) T4 c o n c e n t r a t i o n at time t  CQ =  =  0 (yg/100 ml)  C  =  T4 c o n c e n t r a t i o n 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 rate constant  (h ) - 1  Calculations 1.  Thyroxine F r a c t i o n a l Turnover Rate (TFTR or k, d e f i n e d as the f r a c t i o n of T4 degraded r e p l a c e d per u n i t time  and  h" ): 1  - 136 -  -  c a l c u l a t e d from the e x p o n e n t i a l e q u a t i o n :  C  =  CQe-  k t  where: k 2.  =  f r a c t i o n a l turnover rate ( h ) . . . Z i l v e r s m i t - 1  (1960)  B i o l o g i c a l H a l f - l i f e (Tl/2, h ) :  .  T 1 / 2  iB_(21  Where: Tl/2 in  3.  biological half-life  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 rate  (h ) - 1  Thyroxine Volume of D i s t r i b u t i o n (TVD, t ) : TVD  JD C  =  P o s t and Mixner (1961) and F r e i n k e l and Lewis (1957)  n  where: TVD  =  volume of d i s t r i b u t i o n  D  =  dose of T4 i n j e c t e d (ug)  CQ =  4.  c o n c e n t r a t i o n of T4 at time z e r o (ug/100 ml)  Metabolic Clearance Rate (MCR, -  (A)  l/d):  d e f i n e d as the volume of b l o o d from which t h y r o x i n e was c o m p l e t e l y and i r r e v e r s i b l y removed i n u n i t t i m e . MCR {llA)  =  TVD x TFTR . . . T a i t and B u r s t e i n  where: TVD  =  t h y r o x i n e volume of d i s t r i b u t i o n (i)  (1964)  -  TFTR  5.  =  137  -  f r a c t i o n a l t u r n o v e r r a t e (d  )  Thyroxine Secretion Rate (TSR, yg/d) TVD x TFTR X B a s a l T4 . . . Post and M i x n e r  TSR (ug/d)  (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 t u r n o v e r r a t e (d  B a s a l T4  =  )  b a s a l T4 c o n c e n t r a t i o n (ug/100 ml)  The k i n e t i c parameters o b t a i n e d from two s t e e r s from each t r e a t m e n t on each of the p e r i o d s s t u d i e d were averaged and e x p r e s s e d mean ± S.E.  The t r e a t m e n t d i f f e r e n c e s were t e s t e d by S t u d e n t ' s  as  t-test  ( S t e e l and T o r r i e , 1960). RESULTS  Plasma Thyroxine Disappearance Plasma T4 c o n c e n t r a t i o n i n s t e e r s i m m e d i a t e l y f o l l o w i n g the b o l u s i n j e c t i o n of L - t h y r o x i n e i s p r e s e n t e d i n F i g u r e 19.  The T4  c o n c e n t r a t i o n i n c r e a s e d 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 a d u a l l y d e c r e a s e d .  The plasma T4 d i s a p p e a r a n c e curve was best  d e s c r i b e d by an one compartment open model and the d a t a o b t a i n e d from all  the s t e e r s were f i t t e d t o 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 o p e , and the z e r o - t i m e i n t e r c e p t of the  - 138 -  100c steer: 3 E  o o  no]  < 100E c/) < 10  steeMO  • observed • predicted  0 15 30 45 60 /b 90 I05 ^ Figure 19.  TIME POST  INJECTION!h)  The d i s a p p e a r a n c e o f t h y r o x i n e from t h e plasma a f t e r a s i n g l e i n t r a v e n o u s i n j e c t i o n o f 12 t o 16 mg o f L - t h y r o x i n e i n s t e e r s . Arrow i n d i c a t e s the time o f T4 i n j e c t i o n . B a s a l T4 c o n c e n t r a t i o n i s i n d i c a t e d by the d o t t e d l i n e s .  -  c o n c e n t r a t i o n vs time curve (C = C°  139  -  The k i n e t i c parameters of T4  m e t a b o l i s m i n s t e e r s o b t a i n e d by the method used i n t h i s experiment  are  presented i n Table 21.  T4 Kinetics:  Effect of Anabolic Compounds  S i n c e 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 o b t a i n e d from s e p a r a t e group of s t e e r s p e r i o d ) , the d a t a o b t a i n e d on each p e r i o d were a n a l y s e d ( T a b l e 22, 23 and  (n = 8 per separately  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 2 2 ) , 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 n c r e a s e d  (P < .05)  in  i m p l a n t e d w i t h Synovex-S compared t o o t h e r t r e a t m e n t g r o u p s . but n o n - s i g n i f i c a n t  steers A slight,  i n c r e a s e 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  i n DES i m p l a n t e d s t e e r s  compared to the u n i m p l a n t e d c o n t r o l s .  noticed  On the  c o n 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 t e e r s i m p l a n t e d w i t h Z e r a n o l was s i m i l a r (P > .05)  t o t h a t of the u n i m p l a n t e d c o n t r o l s .  The T4  f r a c t i o n a l t u r n o v e r r a t e was about 22 per cent lower (P < .05) Synovex-S i m p l a n t e d than the c o n t r o l s t e e r s . was s i m i l a r (P > .05)  in  T4 volume of d i s t r i b u t i o n  i n both the i m p l a n t e d and unimplanted c o n t r o l  g r o u p ; however, i t was s l i g h t l y l a r g e r i n s t e e r s  implanted w i t h Zeranol  than w i t h o t h e r t r e a t m e n t g r o u p s . The MCR of T4 was 20 and 37 per cent lower (P < .05)  i n DES  and  Synovex-S i m p l a n t e d s t e e r s , r e s p e c t i v e l y , than the u n i m p l a n t e d c o n t r o l steers.  On the c o n t r a r y MCR was s l i g h t l y e l e v a t e d (N.S.) due t o Z e r a n o l  implantation.  - 140 -  Table 21.  K i n e t i c parameters of t h y r o x i n e m e t a b o l i s m i n growing beef s t e e r s 1  Mean + S E  K i n e t i c Parameters  41.13 + 1.07  B i o l o g i c a l H a l f - L i f e (h) F r a c t i o n a l Turnover Rate  2  0.41 + 0.01  (d ) - 1  24.20 + 0.68  Volume of D i s t r i b u t i o n ( £ ) M e t a b o l i c C l e a r a n c e Rate Jt/d/kg body w e i g h t  9.97 + 0.40 0.03 + 0.01  J l / d / k g ^ " ^ body weight  0.13 + 0.01  lid  7  835.10 + 42.90  T h y r o x i n e S e c r e t i o n Rate ug/day  2.43 + 0.12  ug/d/kg body weight 0.75 ug/d/kg body w e i g h t  10.87 + 0.52  Mean of v a l u e s o b t a i n e d on 20, 40, 60 days f o l l o w i n g the implantation. Mean ± °SE of v a l u e s o b t a i n e d from 24  steers.  Metabolic Clearance Rate ( M C R ) T h y r o x i n e Secretion Rate T4SR  Thyroxine Fractional Turnover Treatment  1  Control  Thyroxine Volume of  0.75  Biological Half-Life  36.94 ± 1.18  1  DES  39.78 ±0.51  0.42 ±0.01  Zeranol  35.98 ±3.40  0.47 ±0.05  Synovex-S  47.35*» ' ±0.24 t+  +  0.35*' ±0.002  +t  21.74 ±1.00  9.09 ±0.28  0.0331 ±0.002  0.14 ±0.01  837.40 ±33.20  3.07 ±0.42  12.46 ±1.41  26.95 ±4.30  12.77 ±3.20  0.0472 ±0.013  0.19 ±0.05  807.21 ±21.60  2.97 ±0.13  12.06 ±0.47  875.31 ±123.50  3.20 ±0.68  12.24 ±1.79  21.66 ±0.68  *- t **.t* **,t" .11 7.81 0.0257 ±0.01 ±.08 ±-001 , f  , T  ^mean ± 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 s t a t i s t i c a l significance when the calculated t value was compared with tabular t value (sign considered) (Table A.3, Steel.and Torrie, I960).  -  142  -  The TSR was i n c r e a s e d i n a l l the i m p l a n t e d s t e e r s . 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)  a n i m a l v a r i a t i o n observed i n the c o n t r o l group. compounds,  But the  due t o a l a r g e  Among the a n a b o l i c  TSR was s l i g h t l y h i g h e r due t o DES and Synovex-S than Z e r a n o l  implantations. 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 ( T a b l e 2 3 ) , a s i m i l a r t r e n d towards i n c r e a s e d TSR was observed i n Synovex-S i m p l a n t e d s t e e r s . the k i n e t i c p a r a m e t e r s , v i z . , similar  (P > .05)  T 1/2,  All  TFTR, TDS, MCR, TSR, however, were  i n both the i m p l a n t e d and u n i m p l a n t e d 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 l e v a t e d i n a l l the i m p l a n t e d s t e e r s w i t h the e x c e p t i o n o f t h a t i m p l a n t e d w i t h  DES.  On day 60 f o l l o w i n g the I m p l a n t a t i o n ( T a b l e 2 4 ) , 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 i g h e r  (P < .05)  s t e e r s than the c o n t r o l group of s t e e r s .  i n a l l the i m p l a n t e d  There was no change i n T  and TFTR due t o the i m p l a n t a t i o n of a n a b o l i c compounds. mean MCR o f T4 f o r a l l the i m p l a n t e d s t e e r s were h i g h e r steers  and 68 (P < .001)  the  than f o r c o n t r o l  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  The TSR was 43 (P < . 0 5 ) , 51 (P < .05) i n DES,  Although  1/2  (P > 0 . 0 5 ) .  per cent  higher  Z e r a n o l , and Synovex-S i m p l a n t e d s t e e r s , r e s p e c t i v e l y , than the  control steers.  Among the a n a b o l i c compounds, TSR was s l i g h t l y  higher  i n s t e e r s i m p l a n t e d w i t h Synovex-S than DES or Z e r a n o l . Though s e p a r a t e groups of s t e e r s were u t i l i z e d on the d i f f e r e n t periods  s t u d i e d (20, 40 and 60 days a f t e r i m p l a n t a t i o n ) , i n o r d e r t o  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  Table 23. Kinetic parameters of thyroxine (T4) metabolism i n steers on 40 days following the implantation anabolic compounds  Control OJ  0.024 ±.001  DES  45.06 ±3.30  0.373 ±0.027  21.16 ±0.79  7.86 ±0.53  Zeranol  42.09 ±3.33  0.397 ±0.022  24.42 ±2.38  9.63 ±0.18  T  0.0284 ±.0002  1  0.102 ±0.005  733.83 ±49.07  2.22 ±0.10  9.49 ±0.48  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 s t a t i s t i c a l significance when the calculated t value was compared with tabular t value (sign considered) (Table A.3, Steel and Torrie, 1960)  Table 24.  Biological Half-Life  Thyroxine Fractional Turnover  Tl/2 (h)  Rate TFTR, per d  Treatment  Control  Kinetic parameters of thyroxine (T4) metabolism l n steers on 60 days following the Implantation of anabolic compounds Thyroxine Volume of Distribution <*>  Metabolic Clearance Rate (MCR)  t/d/kg (MCRK)  8.81 ± 1.14  0.0243 ±0.002  0.106 ±0.010  10.63 ±1.08  0.0284 ±0.002  0.426 ±0.05  DES  38.64 ±4.67  0.437 ±0.07  24.40* ±0.48  Zeranol  37.2 ±5.84  0.458 ±0.07  **+ 26.04 ±0.29  11.92 ±1.74  Synovex-S  37.97 ±0.31  0.438 ±.004  ** 24.65 ±0.55  10.80 ±0.33  1  20.64 ± 0.29  „..„ 0.75 t/d/kg (MCRM)  i/d (MCR)  39.57 ± 4.57  T  ^  p  ^  ^  p  Mg/d  ug/d/kg  ug/d/kg  633.46 0.28  1.76 ±0.08  0.124 ±0.011  ** 904.49 ±9.67  2.42 ±0.02  10.63 ±0.03  0.0314 ±0.005  0.138 ±0.023  953.78* ±91.9  2.51 ±0.30  11.08 ±1.27  0.0276 ±0.0001  0.122 ±0.001  ***++ 1063.95 ±8.35  'mean ± SE 't**»***p ^  Thyroxine Secretion Rate T4SR  ^ .001, respectively, compared to control.  »tt,p < .05, P < .01, respectively compared to DES. 'Sign indicates s t a t i s t i c a l significance when the calculated t value was compared with tabular t value (sign considered) (Table A.3, Steel and Torrle, 1960).  ±  T T  7.67 ± 0.25  *  2.72 ±0.10  *  **  12.08* ±0.36  -  145  -  hence the changes i n plasma T4 l e v e l s ) , the v a l u e s periods Table  o b t a i n e d on d i f f e r e n t  f o l l o w i n g the i m p l a n t a t i o n were pooled and are p r e s e n t e d  in  25. The TSR (per kg m e t a b o l i c body s i z e ) was found to be e l e v a t e d  -  59% h i g h e r than the c o n t r o l group) i n a l l the i m p l a n t e d  Inspite  of the i n c r e a s e d TSR,  e l e v a t e d (4%)  steers.  plasma T4 c o n c e n t r a t i o n was o n l y  i n Zeranol implanted s t e e r s .  (32  slightly  The MCR on the o t h e r hand,  was decreased due t o DES and Synovex-S i m p l a n t a t i o n and i n c r e a s e d due t o the i m p l a n t a t i o n of Z e r a n o 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, 4 1 , and 4 per cent i n c r e a s e d plasma T4 c o n c e n t r a t i o n i n DES,  Synovex-S and Z e r a n o 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  steers.  The e f f e c t of a n a b o l i c compounds periods  on MCRM and TSRM on a l l the  s t u d i e d i s summarized i n F i g u r e 20 f o r b e t t e r  clarity.  DISCUSSION  Thyroxine K i n e t i c s :  V a l i d i t y o f the Method  The c o n c e n t r a t i o n of T4 i n the b l o o d i s due t o the r e s u l t of  Its  s e c r e t i o n from the t h y r o i d g l a n d , i t s c l e a r a n c e from the c i r c u l a t i o n and i s i n g e n e r a l governed by i t s k i n e t i c p a r a m e t e r s .  I n 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 c h e m i c a l t u r n o v e r method (Post and M i x n e r ,  1961).  the method used i n t h i s e x p e r i m e n t , o t h e r approaches  f o r the  d e t e r m i n a t i o n of T4 k i n e t i c s have a l s o been r e p o r t e d i n the  Apart  from  literature:  s u b s t i t u t i o n method ( P i p e s e t a l . , 1957); p o o l t u r n o v e r method ( I n g b a r  - 146  Table 25.  a  -  E f f e c t o f a n a b o l i c compounds  on plasma t h y r o x i n e  status  Treatment  TSRM  MCRM  Control  8.22  0.13  DES  10.86 (32%)  0.12 (-7.67%)  20.0  Zeranol  11.27 (37%)  0.15 (15.35%)  3.8  Synovex-S  13.12 (59%)  0.12 (-5.8%)  3  Plasma T4 L e v e l (% i n c r e a s e over control)  —  41.0  Mean of v a l u e s o b t a i n e d on 20, 40 and 60 days f o l l o w i n g the implantation.  ^ T h y r o x i n e s e c r e t i o n r a t e per kg m e t a b o l i c body s i z e (ug/d/kg^*^^) v a l u e s i n the p a r e n t h e s i s i n d i c a t e p e r c e n t i n c r e a s e over c o n t r o l . c  M e t a b o l i c c l e a r a n c e r a t e per kg m e t a b o l i c body s i z e ( 1 / d / k g ^ * ^ ) v a l u e s i n the p a r a n t h e s i s i n d i c a t e p e r c e n t i n c r e a s e (+) or decrease over the c o n t r o l .  (-)  - 147 -  250 METABOLIC C L E A R A N C E RATE 200  \  1  150  100  ++ •• •+  o  Sit >C  li  O )C o  +• •• •+  °  •+  ****¥.'  +•  T T«r-M  o  CONTROL SECRETION  20  RATE  ZERANOL DES  1  15  _6_SYNOVEX-S  o  2  t«  10  \  XJ  \  O)  5  o  • K • O )C t• •« <O •• • t )C • O • •1 )C • O • X •• <O ••• iXO • <  T  •« V -->c  20  DAYS AFTER  T  •+ •+ ++ •• •• •+  -  JLIo  TT  + T  •+ •+  ++  •+  ++ •• •+  *•  40  60  IMPLANTATION  Figure 20. Metabolic clearance and secretion rates of thyroxine on days 20, 40 and 60 following the implantation of anabolic compounds i n steers. Each bar represents mean ± SE of values obtained from two steers.  - 148  and F r e i n k e l , 1955).  I n farm a n i m a l s , e s p e c i a l l y i n c a t t l e and sheep,  b o t h the r a d i o t h y r o x i n e (*^I ( u n l a b e l l e d L-Thyroxine)  or 1311 l a b e l l e d ) or the c h e m i c a l  t h y r o x i n e t u r n o v e r methods have been u t i l i z e d  t o study T4 k i n e t i c s ( W i l s o n et_al., Anderson,  -  1977; Yousef and J o h n s o n ,  1971; Post and M i x n e r , 1961; Fox et a l . , 1974).  1967;  The c h e m i c a l  r a t h e r than r a d i o t h y r o x i n e d i s a p p e a r a n c e method was adopted i n  this  s t u d y due t o i t s s i m p l i c i t y and the n o n - d e s t r u c t i v e n a t u r e (as  the  a n i m a l s 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 ) .  Besides  the  k i n e t i c parameters o b t a i n e d u s i n g t h i s method were shown t o be s i m i l a r t o those u s i n g the r a d i o t h y r o x i n e d i s a p p e a r a n c e method (Post and M i x n e r , 1961).  The v a l i d a t i o n o f the c h e m i c a l t h y r o x i n e t u r n o v e r method has  been d i s c u s s e d i n d e t a i l by P o s t and M i x n e r ( 1 9 6 1 ) .  However, Swanson  and M i l l e r (1973) have shown t h a t a b e r r a n t r e s u l t s c o u l d be o b t a i n e d u s i n g the c h e m i c a l t u r n o v e r method. The plasma T4 d i l u t i o n curve was a n a l y s e d m a t h e m a t i c a l l y , t r e a t i n g the system s t u d i e d 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 a t a constant r a t e .  S i m i l a r m a t h e m a t i c a l t r e a t m e n t 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 r e p o r t e d by o t h e r s i n farm a n i m a l s ( P o s t and M i x n e r , and Evans, 1980).  1961; Fox et^ a l . , 1974; Marple eit a l . , 1981;  Ingram  I n a d d i t i o n , the a p p l i c a t i o n of two (Sawhney e t a l . ,  1978)  and m u l t i c o m p a r t m e n t a l ( W i l s o n et_ a l . , 1977; DI S t e f a n o e t a l . ,  1982)  p r o c e d u r e s has a l s o been r e p o r t e d r e c e n t l y . The v a l u e s of T 4 - k i n e t i c parameters o b t a i n e d i n t h i s experiment  were comparable t o those r e p o r t e d f o r c a t t l e ( T a b l e 2 6 ) .  The s l i g h t  T a b l e 26.  K i n e t i c parameters of T4 metabolism i n T h y r o x i n e S e c r e t i o n Rate  Sex  Method  TFTR (d- )  1  (TSR)  2  1  yg/d  yg/d/kg  yg/d/kg  Reference  0 , 7 5  Calves  C.T.  0.40  344  6.83  N.A.  P o s t and M i x n e r ,  1961  Cows  C.T.  0.40  1590  2.89  N.A.  P o s t and M i x n e r ,  1961  Cow  R.T.  0.30  1580  3.16  14.9  Yousef and J o h n s o n ,  Bulls  R.T.  0.23 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 t o 1210  3.24 3.70  Steers  C.T.  0.41  to  835  method used f o r the t h y r o x i n e k i n e t i c s C.T. = Chemical t h y r o x i n e ( L - t h y r o x i n e ) R.T. = R a d i o t h y r o x i n e ( 2  cattle  1 2 5  I  or  1 3 l  I - T4)  turnover turnover  Thyroxine f r a c t i o n a l turnover rate  N.A = not a v a i l a b l e or r e p o r t e d by the workers  2.43  to  10 t o  15.2  10.8  Fox e t a l . , 1974 Expt.  IIA:2  1967  -  150  -  v a r i a t i o n i n the e s t i m a t e s c o u l d have been due t o 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 t e of the a n i m a l s u s e d .  From a d e t a i l e d  s p e c i e s c o m p a r i s o n , Marple e t a l . (1981) have c o n c l u d e d t h a t , TSR e x p r e s s e d p e r kg m e t a b o l i c body weight range  (3.53 p g / d / k g  0 , 7 5  (kg  0 , 7 5  ) was w i t h i n a narrow  i n man t o 20.55 u g / d / k g *  among d i f f e r e n t s p e c i e s .  0  7 5  i n chicken)  The v a l u e s of TSR (10.87 u g / d / k g * ) 0  7 5  o b t a i n e d i n t h i s experiment are s i m i l a r t o those r e p o r t e d f o r beef steers  (Fox et^ a l . , 1974)  Thyroxine K i n e t i c s : Results  and are w i t h i n the i n t e r s p e c i e s  range.  E f f e c t of Estrogenic Anabolic Compounds  of t h i s experiment show c e r t a i n i m p o r t a n t f i n d i n g s  e f f e c t of a n a b o l i c compounds on T4 k i n e t i c s i n s t e e r s .  on the  There appears  to  be an o v e r - a l l i n c r e a s e i n TSR and a decrease i n MCR i n s t e e r s i m p l a n t e d w i t h DES or Synovex-S and an i n c r e a s e i n b o t h the parameters i n Z e r a n o l implanted steers.  The r e s u l t s a l s o i n d i c a t e t h a t d i f f e r e n t i a l p a t t e r n 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 a g a i n s t  Zeranol  i m p l a n t a t i o n and i n g e n e r a l r e v e a l the e f f e c t of e s t r o g e n s  on t h y r o i d  function. Burgess and Lamming (1960) have proposed t h a t I n c r e a s e d TSR c o u l d be r e s p o n s i b l e f o r the i n c r e a s e d growth r a t e f o l l o w i n g e s t r o g e n a d m i n i s t r a t i o n i n farm a n i m a l s .  D i e t h y l s t i l b e s t r o l f e d lambs have been  shown t o e x h i b i t a t r e n d towards i n c r e a s e d TSR ( T r e n k l e , 1969). r e s u l t s o b t a i n e d i n t h i s experiment are thus i n l i n e w i t h the  The  findings  of the e a r l i e r workers and i n d i c a t e t h a t the i n c r e a s e d plasma T4 c o n c e n t r a t i o n s f o l l o w i n g Synovex-S or DES i m p l a n t a t i o n s ( r e s u l t s E x p t . IIA:  of  1) c o u l d be due t o the r e l a t i v e changes i n T4 s e c r e t i o n and  - 151 -  clearance rates.  S i n c e the c l e a r a n c e o f t h y r o i d hormone i s r e l a t e d t o  the s t r e n g t h of the plasma t h y r o i d hormone b i n d i n g p r o t e i n s , o v e r a l l r e d u c t i o n i n MCR 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 s c o u l d be a t t r i b u t e d t o an i n c r e a s e i n TBG b i n d i n g c a p a c i t y .  A significant  i n c r e a s e i n TBG b i n d i n g c a p a c i t y f o l l o w i n g e s t r o g e n t h e r a p y was r e p o r t e d i n rhesus monkeys (Sawhney e t a l . , 1 9 7 8 ) .  F u r t h e r , a r e d u c t i o n i n T4  d e g r a d a t i o n r a t e was observed i n p a t i e n t s t r e a t e d w i t h (Zaninovich,  1973).  estrogens  Serum b i n d i n g o f T4 was a t t r i b u t e d i n p a r t t o t h e  reduced T4 d e g r a d a t i o n f o l l o w i n g e s t r o g e n a d m i n i s t r a t i o n (Robbins and Nelson,  1958; E n g b r i n g and Engstrom,  1959).  The e x t e n t t o which TBG has  p a r t i c i p a t e d i n r e d u c i n g the MCR i n the Synovex-S and DES  implanted  s t e e r s used i n t h i s experiment i s not c l e a r l y u n d e r s t o o d as  simultaneous  measurements  (Expt  of TBG were not c a r r i e d o u t .  1 ) , however, do not support  E a r l i e r findings  IIA:  the above t h e o r y as TBG(RDA) ( T a b l e 17) was  s i g n i f i c a n t l y reduced i n a l l t h e i m p l a n t e d s t e e r s .  B u t , u s i n g T3 r e s i n  uptake as the c r i t e r i o n , K a h l e t a l . (1978) have r e p o r t e d enhanced b i n d i n g c a p a c i t y of the plasma T4 b i n d i n g p r o t e i n i n Synovex-S i m p l a n t e d steers. Results  of t h i s experiment r e v e a l c e r t a i n a s p e c t s on the  mechanism of a c t i o n f o r Z e r a n o l on the t h y r o i d g l a n d and on the p e r i p h e r a l m e t a b o l i s m o f T4.  The TSR,  i n g e n e r a l was s l i g h t l y h i g h e r  Z e r a n o l i m p l a n t e d than the c o n t r o l s t e e r s w i t h s i g n i f i c a n t l y values  higher  (51 p e r c e n t i n c r e a s e over c o n t r o l ) o b t a i n e d on day 60 f o l l o w i n g  i t s implantation.  Thus i n t h i s r e s p e c t the mechanism of a c t i o n o f  Z e r a n o l seems t o be s i m i l a r t o t h a t of Synovex-S and DES,  however  in  -  152  -  Z e r a n o 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 g l a n d compared to the l a t t e r two compounds. thyroid depressive (1975).  The r e s u l t s , t h e r e f o r e , do not support  the  a c t i o n s of Z e r a n o l proposed by Rothanbacher e t a l .  But the mechanism of a c t i o n of Z e r a n o l d i f f e r s from t h a t of  and Synovex-S i n t h a t i t s i m p l a n t a t i o n i n c r e a s e s MCR as w e l l .  DES  Increased  MCR of T4 c o u l d thus be a t t r i b u t e d t o the s i m i l a r T4 c o n c e n t r a t i o n i n both the c o n t r o l and Z e r a n o l i m p l a n t e d s t e e r s  ( E x p t . IIA:  1) i n s p i t e of  the i n c r e a s e d TSR due t o Z e r a n o l i m p l a n t a t i o n . Plasma T4 c o n c e n t r a t i o n i n Z e r a n o l i m p l a n t e d a n i m a l s was found t o be unchanged or s l i g h t l y decreased compared t o the c o n t r o l a n i m a l s (Wiggins e t a l . , 1979)  but the e f f e c t of Z e r a n o 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 n c r e a s e (Wiggins e t a l . 1976)  and a  d e c r e a s e (Wiggins et a l . , 1979)  In  i n i t s weight have been r e p o r t e d .  t h i s study a b a s i s f o r the l a c k of d i f f e r e n c e i n T4 l e v e l between Z e r a n o l i m p l a n t e d and the u n i m p l a n t e d c o n t r o l s t e e r s was f u r t h e r established. CONCLUSION The r e s u l t s of t h i s study r e v e a l e d t h a t the i n c r e a s e d plasma T4 of the s t e e r s i m p l a n t e d w i t h a n a b o l i c compounds s e c r e t i o n r a t e of T4.  i s due to an i n c r e a s e d  Up t o 31-59% i n c r e a s e i n the T4 s e c r e t i o n r a t e  was observed due t o the i m p l a n t a t i o n of a n a b o l i c compounds.  Both DES  and Synovex-S were found t o be more e f f e c t i v e i n i n c r e a s i n g T4 c o n c e n t r a t i o n i n the plasma and i t s s e c r e t i o n r a t e than Z e r a n o l . Implantations  w i t h Z e r a n o l a l s o r e s u l t e d i n an i n c r e a s e d T4 s e c r e t i o n  r a t e but the p a r t i c i p a t i o n of i n c r e a s e d m e t a b o l i c c l e a r a n c e r a t e  - 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 o r no change i n the plasma T4 concentration. It  i s concluded t h a t the i n c r e a s e d T4 s e c r e t i o n r a t e c o u l d be one  of the mechanisms steers.  by which a n a b o l i c compounds  evoke growth response  in  -  154 -  EXPERIMENT I I B E F F E C T OF ANABOLIC COMPOUNDS ON PLASMA L E V E L AND I T S K I N E T I C PARAMETERS  GROWTH  HORMONE  OF METABOLISM I N  GROWING S T E E R S  INTRODUCTION  Changes i n t h e endogenous c i r c u l a t i n g growth hormone (GH) c o n c e n t r a t i o n have been proposed as the major mechanism by w h i c h a n a b o l i c compounds mediate t h e i r m e t a b o l i c and growth p r o p e r t i e s i n ruminants hypotheses  ( B u t t e r y e t a l . , 1978; P r e s t o n , 1975).  These  a r e v e r y a t t r a c t i v e c o n s i d e r i n g the i n c r e a s e d s o m a t i c g r o w t h ,  p r o t e i n a c c r e t i o n and reduced f a t s y n t h e s i s i m p l a n t a t i o n o f a n a b o l i c compounds processes.  promoting  i n ruminants  f o l l o w i n g the  and t h e e f f e c t o f GH on a l l t h e above  However, t h e l a c k o f c o r r e l a t i o n between plasma GH and  growth r a t e i n c a t t l e (Joakimsen and Blom, 1976; K e l l e r e t a l . , 1979; Ohlson et^ a l . , 1981; Purchas et^ a l . , 1970; T r e n k l e , 1977; T r e n k l e and Irvin,  1970; T r e n k l e and T o p e l , 1978) r a i s e s  questions  as t o t h e r e a l  i n t e r r e l a t i o n s h i p between b l o o d GH c o n c e n t r a t i o n and growth r a t e and s u g g e s t s perhaps o t h e r growth f a c t o r s mediated by GH, such as somatomedins,  p l a y an i m p o r t a n t r o l e .  Many o f t h e a n a b o l i c a c t i o n s o f  GH a r e now b e l i e v e d t o be mediated by somatomedins Daughaday, 1976).  (Chochinov and  A r e l a t i o n s h i p between serum somatomedins o r  s o m a t o m e d i n - l i k e a c t i v i t y and growth o f b u l l s 1977), Swine ( L u n d - L a r s e n  ( L u n d - L a r s e n _t_ a l . ,  and Bakke, 1975) and lambs ( O l s e n e t a l . ,  1981; Wangsness e t a l . , 1981) has been r e p o r t e d .  -  155  -  A number o f workers have r e p o r t e d i n c r e a s e d weight of the p i t u i t a r y g l a n d (Burgess and Lamming, 1960; C l e g g  and C o l e ,  1954;  W i g g i n s e t 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 l a n d ( C l e g g and C o l e , 1954)  and plasma GH c o n c e n t r a t i o n  ( B o r g e r et_ a l . , 1973; Donaldson e t a l . , 1981; O l s e n et^ a l . , 1977; W i g g i n s e £ a l . , 1976; T r e n k l e , 1970a; T r e n k l e 1976b) i n t r e a t e d w i t h e s t r o g e n i c a n a b o l i c compounds  (DES,  ruminants  Zeranol, 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 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 ( K a h l e t a l . , 1978) t h y r o x i n e s e c r e t i o n r a t e from the t h y r o i d g l a n d ( r e s u l t s 2).  F u r t h e r the mechanism by w h i c h a n a b o l i c compounds  GH c o n c e n t r a t i o n has not been c l e a r l y u n d e r s t o o d .  of E x p t .  been and IIA:  i n f l u e n c e plasma  Though the  increased  p i t u i t a r y g l a n d weight and number o f a c i d o p h i l s i n the e s t r o g e n t r e a t e d a n i m a l s i n d i r e c t l y i n d i c a t e enhanced s e c r e t o r y a c t i v i t y of the p i t u i t a r y , a b s o l u t e v a l u e s of GH s e c r e t i o n r a t e o r GH k i n e t i c parameters are not known.  S i n c e the plasma GH s t a t u s  r a t e , m e t a b o l i c c l e a r a n c e r a t e and Is  i s a r e s u l t of i t s  i n general  secretion  determined by i t s  k i n e t i c p a r a m e t e r s , s t u d i e s on GH k i n e t i c s i n e s t r o g e n t r e a t e d a n i m a l s w i l l be advantageous anabolic  i n understanding  the mechanism of a c t i o n of  compounds.  T h i s experiment was t h e r e f o r e conducted t o study the k i n e t i c parameters of GH m e t a b o l i s m i n s t e e r s i m p l a n t e d w i t h the a n a b o l i c compounds  estrogenic  i n o r d e r t o 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 :  -  a.  156 -  E s t a b l i s h plasma GH l e v e l s i n s t e e r s i m p l a n t e d w i t h DES, Z e r a n o l and Synovex-S, and  b.  Determine k i n e t i c parameters o f GH m e t a b o l i s m i n s t e e r s implanted w i t h anabolic  compounds.  O b j e c t i v e s (a) and (b) were s t u d i e d i n s e p a r a t e group o f s t e e r s .  MATERIALS AND METHODS  1.  E f f e c t o f Anabolic Compounds on Plasma Growth Hormone Level l n Steers Animals and Experimental Procedure S i x t e e n H e r e f o r d s t e e r s w h i c h were used i n E x p t . IA were u t i l i z e d  i n t h i s experiment t o study t h e e f f e c t of a n a b o l i c compounds GH l e v e l s .  on plasma  The a n a b o l i c compounds used ( Z e r a n o l , DES, S y n o v e x - S ) , t h e i r  i m p l a n t a t i o n procedure and t h e e x p e r i m e n t a l d i e t used were t h e same as described e a r l i e r (Expt.  IA).  B l o o d samples were c o l l e c t e d from i n d i v i d u a l s t e e r s on 14, 28, 42 and 56 days f o l l o w i n g t h e i m p l a n t a t i o n by means o f j u g u l a r v e i n p u n c t u r e i n p r e c h i l l e d h e p a r i n i z e d evacuated tubes ( V a c u t a i n e r Beckton-Dickinson,  D i c k i n s o n and C o . , New J e r s e y ) .  tubes,  B l o o d samples  c o l l e c t e d b e f o r e f e e d i n g the a n i m a l a t 0730 h and a t 1400 h . t a k e n not t o e x c i t e t h e a n i m a l s d u r i n g the sampling p e r i o d . were kept i n an i c e b a t h and were c e n t r i f u g e d at 1000 x g  were  Care was The samples  (Sorvall  superspeed C e n t r i f u g e , Model RC 2B, N o r w a l k , Conn., U.S.A.) t o o b t a i n the plasma which was then s t o r e d a t - 2 0 ° C .  - 157  2.  -  E f f e c t of Anabolic Compounds on the K i n e t i c Parameters of GH Metabolism Animals S i x t e e n H e r e f o r d s t e e r s were used i n t h i s experiment to study  e f f e c t of a n a b o l i c compounds  on GH k i n e t i c s .  the  The a n i m l a s were s e l e c t e d  from a group of 24 s t e e r s t h a t were used i n E x p t . IIA:  2.  The  steers  used i n t h i s study r e p r e s e n t e d a l l the f o u r t r e a t m e n t groups ( c o n t r o l , DES,  Z e r a n o l , Synovex-S) d e s c r i b e d i n E x p t . IIA:  2.  The method of  a n a b o l i c compound i m p l a n t a t i o n , a n i m a l f e e d i n g procedure and the p r e p a r a t i o n of s t e e r s f o r GH k i n e t i c s t u d i e s were d e s c r i b e d i n E x p t . IIA:  2. K i n e t i c parameters were e s t i m a t e d 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 t e e r s per t r e a t m e n t group f o r each p e r i o d  studied).  Experimental Procedure Preparation of GH Solution f o r K i n e t i c Studies NIH-GH-B 18 ( N a t i o n a l P i t u i t a r y Agency, used i n a l l the GH k i n e t i c e x p e r i m e n t s .  B a l t i m o r e , Maryland)  was  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% N a c l and the pH was a d j u s t e d 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 ,  containing  6-8 mg NIH-GH-B18 was s t o r e d f r o z e n i n 5 ml s y r i n g e s a t - 2 0 ° C . syringe  One  c o n t a i n i n g the GH p r e p a r a t i o n was thawed on the day of the  experiment and used as  such.  GH Kinetics A s i n g l e b o l u s i n j e c t i o n , n o n - r a d i o a c t i v e GH k i n e t i c procedure was adopted ( T r e n k l e , 1976a; T r e n k l e , 1977).  -  158  -  On t h e day of the experiment the s t e e r s were weighed, f e d t h e morning p o r t i o n of the r a t i o n and were p l a c e d i n m e t a b o l i s m c r a t e s t h a t a l l o w e d some freedom of movement but p r e v e n t e d the a n i m a l from t u r n i n g around.  The a n i m a l s were a l l o w e d t o r e c o v e r i n the c r a t e f o r an hour  and b l o o d samples were c o l l e c t e d t h r o u g h the c a t h e t e r every 30 min f o r 3.5 h f o r a t o t a l of e i g h t b l o o d samples from each a n i m a l t o a s c e r t a i n b a s a l GH s e c r e t o r y p a t t e r n .  The s t e e 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 b o v i n e GH (NIH-GH-B18; one s y r i n g e as d e s c r i b e d above) and the c a t h e t e r was i m m e d i a t e l y f l u s h e d w i t h 0.9% s a l i n e .  To measure the  d i s a p p e a r a n c e of the i n j e c t e d GH, s e r i a l b l o o d samples were t a k e n a t 5, 10, 15, 20, 2 5 , 30, 35, 40, 4 5 , 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 b l o o d samples were k e p t 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 t o o b t a i n the plasma. s t o r e d a t -70°C t i l l  they were a n a l y z e d .  t r e a t m e n t group were s t u d i e d  3.  The plasma samples were Two s t e e r s r e p r e s e n t i n g a  simultaneously.  A n a l y t i c a l Procedure Plasma GH c o n c e n t r a t i o n 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 ( a n a l y z e d on b o t h 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 a n a l y s e d by a h i g h l y s p e c i f i c double a n t i b o d y radioimmunoassay  procedure.  GH Radioimmunoassay GH c o n c e n t r a t i o n i n the b o v i n e plasma was determined by the radioimmunoassay  method d e s c r i b e d by Niswender e t 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 D a v i s (1972) u s i n g the o v i n e GH radioimmuno-  -  159  -  a s s a y m a t e r i a l s o b t a i n e d from NIAMDD, B e t h e s d a , M a r y l a n d .  Ovine GH  (NIAMDD-oGH-I-1) was i o d i n a t e d by the c h l o r a m i n e - T method  (Greenwood  ejt a l . , 1963)  and was p u r i f i e d as d e s c r i b e d by Purchas et a l . ( 1 9 7 0 ) .  A  d e t a i l e d account of the GH i o d i n a t i o n p r o c e d u r e , and the p u r i f i c a t i o n of 125J_ Q 0  H  i s p r e s e n t e d i n Appendix  Briefly,  II.  the r e a g e n t s were added to d i s p o s a b l e p o l y s t y r e n e  (12 x 75 mm, Amersham C o . , Canada) PBS - 1% BSA (0.01  i n the f o l l o w i n g sequence:  tubes  200 y l of  M phosphate b u f f e r e d s a l i n e , pH 7.0 c o n t a i n i n g 1%  b o v i n e serum a l b u m i n , f r a c t i o n V) or 200 y l t e s t sample or a p p r o p r i a t e amount of oGH s t a n d a r d (0 t o 20 ng NIAMDD-oGH-I-1/tube) and 200 y l of r a b b i t a n t i s e r u m to o v i n e GH ( F i n a l tube d i l u t i o n 1:40000). were t h e n i n c u b a t e d a t 4°C.  Twenty f o u r hours a f t e r the a d d i t i o n of  r a b b i t a n t i s e r u m t o oGH, 100 y l of the Sephadex G-100 ( u s i n g Eppendorf Inc.,  New Y o r k )  The tubes  1 2 5  I - o G H ( p u r i f i e d m a t e r i a l from  Column, 35000 cpm per ml o f PBS-1% BSA) was  r e p e a t e r p i p e t t e , Model 4780, Brlnkmann  dispensed  Instruments,  t o each tube and the i n c u b a t i o n was c o n t i n u e d at 4°C.  Twenty f o u r hours a f t e r the a d d i t i o n of 125T- GH, 100 y l o f 0  goat  a n t i s e r u m t o r a b b i t gamma g l o b u l i n was added t o each tube and the i n c u b a t i o n was c o n t i n u e d at 4°C. ml of PBS (pH 7.0)  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  was added t o each tube and the tubes were c e n t r i f u g e d  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 m i n u t e s .  The s u p e r n a t a n t  c o n t a i n i n g the f r e e 125J._ GH was decanted 0  and the r a d i o a c t i v i t y a s s o c i a t e d w i t h the p r e c i p i t a t e was counted i n an ®  a u t o m a t i c gamma c o u n t e r (Auto-Gamma  500 C Countimg System,  Packard  -  Instruments, duplicate.  I l l i n o i s , U.S.A.).  160  -  A l l a s s a y s were performed i n  B l o o d samples c o l l e c t e d on any one s a m p l i n g p e r i o d or from  any one GH k i n e t i c experiment were a n a l y s e d i n the same a s s a y . The procedure f o r the c a l c u l a t i o n o f GH c o n c e n t r a t i o n i n the plasma samples and f o r the d e t e r m i n a t i o n of i n t e r - and v a r i a t i o n was s i m i l a r t o t h a t d e s c r i b e d f o r the (Expt. IIA:  2).  intraassay  T4-radioimmunoassay  Under the c o n d i t i o n s d e s c r i b e d 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 o f v a r i a t i o n were 3.2% and 9.6%,  respectively,  f o r a sample w i t h a mean c o n c e n t r a t i o n of 16.5 ng/ml.  Analysis of K i n e t i c Data The plasma growth hormone d i s a p p e a r a n c e curve (0 t o 120 min post i n j e c t i o n ) was a n a l y s e d u s i n g 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 e s t i m a t e s the observed d a t a was found t o be b e s t d e s c r i b e d by the b i e x p o n e n t i a l e q u a t i o n :  C  =  A e "  C  =  c o n c e n t r a t i o n o f GH a t time = t  A  =  Y i n t e r c e p t of the f i r s t component of the d i s a p p e a r a n c e  0  * *  Be"  3  1  Where:  curve ( F a s t Phase)  (ng/ml)  a  =  d i s p o s i t i o n c o n s t a n t i n the r a p i d phase  B  =  Y i n t e r c e p t of the second component of the d i s a p p e a r a n c e curve (Slow Phase)  3  =  (min ) - 1  (ng/ml)  d i s p o s i t i o n c o n s t a n t i n the slow phase  (min ) - 1  - 161 -  Model Description The two compartment model describing GH disappearance from the blood plasma i s depicted below:  k  1 2  21  k  Model used to describe GH Metabolism i n Steers where: Cj  ™  Central compartment (represents  the plasma)  C^  •  Peripheral compartment ( s i t e s of GH u t i l i z a t i o n and degradation)  K._  «  the f i r s t - o r d e r rate constant f o r the elimination of GH from the central compartment ( m i n ) -1  K,. 12  -  the f i r s t - o r d e r rate constant for the d i s t r i b u t i o n of GH from the central to the peripheral compartment (min ) -1  *21  •  the f i r s t - o r d e r rate constant for the d i s t r i b u t i o n of GH from the peripheral to central compartment (min ). -1  -  162 -  Calculations: 1.  GH Volume of D i s t r i b u t i o n (£) 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 e q u a t i o n :  A/ot + 2  Volume of d i s t r i b u t i o n  =  D  (A/a  +  B/3  2  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 e t a l . (1969)  2.  Metabolic Clearance rate of GH (MCR, ml. m i n ) : - 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 e q u a t i o n :  MCR ( m l . m i n ) - 1  3.  =  D A  Q  Ap  ° T  g p  Bot  . . . T a i t and B u r s t e i n  (1964)  GH secretion rate or production rate (GH - SR, ng. min *) -  S e c r e t i o n r a t e of GH was c a l c u l a t e d from the m e t a b o l i c c l e a r a n c e r a t e and b a s a l GH c o n c e n t r a t i o n as d e s c r i b e d i n the e q u a t i o n : GH - SR ( n g . m i n ) - 1  =  MCR x b a s a l GH c o n c e n t r a t i o n . . . G u r p i d e and Mann (1970)  4.  H a l f - L i f e of GH T 1/2 ( a )  =  0.693/a  T 1/2 (3)  =  0.693/3  . . . S h i p l e y and C l a r k  (1972)  -  163  -  where: a, the  3 are the r a t e c o n s t a n t s of the r a p i d and slow components  of  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 c o n c e n t r a t i o n s 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 e e r on each sampling p e r i o d ) and were s u b j e c t e d t o analysis  of v a r i a n c e ( S t e e l and T o r r i e , 1960).  by the Newman-Keuls m u l t i p l e range t e s t .  The means were s e p a r a t e d  The hormone  concentrations  were t r a n s f o r m e d t o n a t u r a l l o g a r i t h m s p r i o r t o s t a t i s t i c a l  analysis.  B a s s e t t and Thorburn (1971) r e p o r t e d t h a t l o g a r i t h m i c t r a n s f o r m a t i o n of p r o t e i n hormones i n the plasma conformed more c l o s e l y t o 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 t h a n the a b s o l u t e c o n c e n t r a t i o n .  A similar  t r e a t m e n t o f d a t a was r e p o r t e d by Donaldson e t a l . ( 1 9 8 1 ) .  The  analysis  o f the d a t a u s i n g the a b s o l u t e GH c o n c e n t r a t i o n was a l s o c a r r i e d out and the  r e s u l t s o b t a i n e d from such a n a l y s i s  are p r e s e n t e d i n Appendix  III.  The k i n e t i c parameters were averaged and are p r e s e n t e d as mean ± SE.  The t r e a t m e n t d i f f e r e n c e s were t e s t e d by S t u d e n t ' s  and T o r r i e ,  t-test  (Steel  1960). RESULTS  1.  E f f e c t of Anabolic Compounda on Plasma GH Levels; GH c o n c e n t r a t i o n i n the plasma was e x p r e s s e d as ng  NIAMDD-oGH-I-l/ml.  Mean plasma GH c o n c e n t r a t i o n i n the s t e e r s used  t h i s experiment was 33 ± 7.5 ng/ml (n = 6 4 ) .  U s i n g the  in  radioimmunoassay  -  164 -  p r o c e d u r e d e s c r i b e d i n t h i s e x p e r i m e n t , a GH c o n c e n t r a t i o n o f about 13.9-18.6 ng/ml was o b t a i n e d f o r a b o v i n e plasma p o o l ( f r o m 10 a s s a y s ) . The e f f e c t o f a n a b o l i c compounds  on plasma GH c o n c e n t r a t i o n s on  d i f f e r e n t days f o l l o w i n g t h e i m p l a n t a t i o n i s p r e s e n t e d i n T a b l e 27 see Appendix I I I )  (also  and t h e o v e r - a l l e f f e c t o f t r e a t m e n t s on plasma GH i s  presented i n Figure 21. There was a s i g n i f i c a n t i n c r e a s e i n t h e plasma GH c o n c e n t r a t i o n in  s t e e r s due t o t h e i m p l a n t a t i o n o f a n a b o l i c compounds  C o n t r o l vs a l l t h e a n a b o l i c compounds).  (P < . 0 0 1 ,  When a l l t h e s a m p l i n g  periods  (14, 2 8 , 42 and 56 days) were t a k e n i n t o c o n s i d e r a t i o n , plasma GH c o n c e n t r a t i o n was about 76, 153 and 135 p e r cent h i g h e r i n Z e r a n o l , DES and Synovex-S i m p l a n t e d s t e e r s , r e s p e c t i v e l y , than i n t h e c o n t r o l steers.  S t e e r s i m p l a n t e d w i t h DES and Synovex-S had s l i g h t l y  higher  plasma GH c o n c e n t r a t i o n than those i m p l a n t e d w i t h Z e r a n o l . The t r e a t m e n t x p e r i o d i n t e r a c t i o n was not s i g n i f i c a n t 0.05).  (P >  Plasma GH was m a i n t a i n e d a t a h i g h e r c o n c e n t r a t i o n on a l l t h e  p e r i o d s s t u d i e d i n s t e e r s i m p l a n t e d w i t h DES and Synovex-S t h a n t h e unimplanted c o n t r o l s t e e r s .  On the c o n t r a r y , e l e v a t e d plasma GH i n  Z e r a n o l i m p l a n t e d s t e e r s was observed on day 14 a f t e r t h e i m p l a n t a t i o n and t h e c o n c e n t r a t i o n decreased beyond day 14 t o about 20 ng/ml on day 56. 2.  E f f e c t of Anabolic Compounds on the K i n e t i c Parameters of Growth Hormone Metabolism The d i s a p p e a r a n c e o f GH i n t h e plasma was s t u d i e d a f t e r i n j e c t i n g  the s t e e r s r a p i d l y w i t h 6 mg o f N1H-GH-B18.  A f t e r the i n j e c t i o n , GH  d i s a p p e a r e d from t h e 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 s h i o n over t h e  - 165  T a b l e 27.  -  Plasma GH c o n c e n t r a t i o n s i n s t e e 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 o g e n i c a n a b o l i c compounds 1  Treatment  14  Days A f t e r I m p l a n t a t i o n 42 28 ng  56  Mean  SE'  NIAMDD-oGH-I-l/ml  12.70 ± 0.22  22.98 ± 0.37  23.61 ± 0.24  17.98  0.14  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  Control  14.04 ±0.18  Zeranol  J  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 . t r a n s f o r m e d v a l u e , 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 (P < .05) 4 s t e e r s per t r e a t m e n t .  differ  2  p o o l e d n a t u r a l l o g standard e r r o r .  3  T h e mean of GH c o n c e n t r a t i o n i n 0730h and 1400 h sample from 4 steers.  - 166 -  50 CONTROL ZERANOL •••• •••• ±±± DES >od SYNOVEX-S  40  +• 4+ 44444 44444 ++••4 ••••• +++•• +•••• 44444  ***** ***** ***** ***** *****  30  44444 44444  20  I  •• • •• • •• • •• • •• • •• • •• • •• •  •• • •.V  =; 10  4+++4 •4444 44444 44444 44444 44444 444+4 44444 44444 44444 44444 44444 44444 44444 44444 44444 44444 44444 44444 44444 44444 44444 44444 44444 44444 44444 44444 44444 44444 44444 44444 44444 44444  •• •  •••••  •• • •• •  •• • •• •  0  yzol  Figure 21. E f f e c t of anabolic compounds on plasma GH concentration i n 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). > Bars with d i f f e r e n t alphabet (P < .05).  a  D  differ significantly  -  n e x t 120 min ( F i g u r e 2 2 ) .  167  -  The plasma GH d i s a p p e a r a n c e curve d i s p l a y e d  the presence o f two components:  an i n i t i a l h i g h l y r a p i d phase  lasting  5.0 ± 0.8 m i n , and a slow d i s a p p e a r a n c e phase l a s t i n g 42 ± 3 m i n .  The  v a l u e s o f 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 o b t a i n e d by f i t t i n g t h e d i s a p p e a r a n c e curve t o a two-compartment model, a r e summarized i n T a b l e 28. F r a c t i o n a l r a t e s of GH d i s a p p e a r a n c e from the r a p i d l y t u r n i n g over and s l o w 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 0.017 min  - 1  .  The average volume o f d i s t r i b u t i o n o f GH i n the s t e e r s  used i n t h i s experiment was found t o be 6.1 weight.  and  ± 0.6 per cent of the body  Mean MCR and s e c r e t i o n r a t e s of GH were 21 1/h and 252 pg/h o r  74.5 ml/h/kg and 0.91  ug/h/kg.  The e f f e c t o f a n a b o l i c compounds on the k i n e t i c parameters of GH m e t a b o l i s m 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 p r e s e n t e d i n T a b l e 29 t o 31.  The h a l f - l i f e o f GH i n the r a p i d l y t u r n i n g over compartment  ranged from 3.64  t o 8.02 min i n s t e e r s .  A s i g n i f i c a n t i n c r e a s e i n the  h a l f - l i f e o f GH i n the r a p i d phase was n o t i c e d i n Synovex-S i m p l a n t e d s t e e r s when compared t o the c o n t r o l s t e e r s .  S t e e r s from d i f f e r e n t  t r e a t m e n t groups had s i m i l a r h a l f - l i f e of d i s a p p e a r a n c e of GH i n the s l o w 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 o w l y  turning  o v e r phase ranged from 34 - 49 m i n . The MCR of GH i n s t e e r s ranged from 18.3 - 23.2 1/h ( T a b l e  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 o f GH i n s t e e r s from d i f f e r e n t t r e a t m e n t g r o u p s .  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 i g h e r i n Z e r a n o l (83 vs 73 ml/h/kg)  - 168 -  Figure 22.  The d i s a p p e a r a n c e of GH from the plasma f o l l o w i n g the i n t r a v e n o u s i n j e c t i o n of 6 t o 8 mg of b o v i n e 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 . B a s a l GH c o n c e n t r a t i o n i s i n d i c a t e d by the d o t t e d l i n e .  - 169 -  Table 28.  K i n e t i c parameters of GH metabolism i n growing steers  K i n e t i c Parameters  Mean ± S..E.  1  H a l f - l i f e (min) Fast component (T 1/2 a) Slow component (T 1/2 g)  5.02 41.81  + +  0.78 3.06  F r a c t i o n a l Turnover Rate ( m i n ) Fast component (a) Slow component (3)  0.1674 + 0.0172 +  Volume of d i s t r i b u t i o n (% of body weight)  6.11  +  0.59  20.68 74.47  + +  2.09 6.36  -1  Metabolic Clearance Rate liters/h ml/h/kg body weight ml/h/kg *  body weight  0.0297 0.0013  303.85  + 27.03  252.14 0.91  + 27.72 + 0.09  Secretion Rate Pg/h ug/h/kg  Q  7 5  ug/h/kg *  body weight  ^mean ± S.E. of values obtained from 8 steers  3.70  +  0.37  - 170 -  Table 29.  F i r s t - o r d e r r a t e c o n s t a n t s 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  F i r s t - O r d e r Rate C o n s t a n t s 1 Treatment kl2  k l 2  min Control  0.06800 ± 0 . 0 0 2 1 5  DES  kio  _1  0.05400 ± 0.00896  0.04189 ± 0.00610  0.08762 ± 0.04375  0.05161 ± 0.01463  0.10130 ± 0.07246  Zeranol  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  2  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 t e c o n s t a n t f o r the d i s t r i b u t i o n o f GH from the c e n t r a l t o 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 r a t e c o n s t a n t f o r the d i s t r i b u t i o n o f GH from t h e p e r i p h e r a l t o the c e n t r a l compartment.  kio - F i r s t - o r d e r r a t e c o n s t a n t f o r the e l i m i n a t i o n o f GH from the c e n t r a l compartment. 2  Mean ± s t a n d a r d  error.  *p < .05 compared t o c o n t r o l .  -  T a b l e 30.  171  -  H a l f - l i f e and f r a c t i o n a l d i s a p p e a r a n c e r a t e o f i n j e c t e d GH i n s t e e r s  F a s t Component Treatment  Half-Life (min)  Slow Component  Fractional Turnover Rate (min ) - 1  Half-Life (min)  4.78 ±0.37  1  - 1  (3)  («> Control  Fractional Turnover Rate (min )  0.1460 ±0.011  49.00 ±5.18  0.0143 ± 0.002  DES  3.64 ±1.37  0.2219 ±0.084  41.50 ±2.35  0.0167 ±  Zeranol  3.65 ±1.24  0.2145 ±0.073  34.65 ±4.80  0.0204 ± 0.003  Synovex-S  8.02*" ±0.80  0.0873*" ±0.009  42.07 ±10.19  0.0175 ± 0.004  ^mean ± s t a n d a r d e r r o r *P < .05 compared t o c o n t r o l  0.001  - 172  Table 31.  -  M e t a b o l i c c l e a r a n c e and s e c r e t i o n r a t e s of GH i n s t e e r 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 a n a b o l i c compounds S e c r e t i o n Rate  M e t a b o l i c C l e a r a n c e Rate 1/h  Treatment  Control  ±  18.51 2.07  1  ml/h/kg  ml/h/kg '  72.64 ± 4.30  290.16 ± 20.90  0  7 5  /wi. ° ' Ug/h/kg  ug/h  ug/h/kg  135.01 ± 11.05  0.53 ±0.02  2.12 ±0.09  290.24*" ±50.88  1.04* ±0.08  4.25* ±0.44  DES  18.33 ±8.22  63.95 ±23.40  262.90 ±101.80  Zeranol  22.74 ±4.90  83.30 ±16.90  338.60 ±69.90  299.60* ±14.30  1.10* ±0.07  4.48* ±0.27  Synovex-S  23.15 ±2.50  77.98 ±8.90  323.70 ±36.20  283.72* ±13.60  0.955** ±0.04  3.96* ±0.17  7 5  Mean ± s t a n d a r d e r r o r . P < .05 compared to c o n t r o l . ** P < .01 compared t o c o n t r o l . S i g n 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 v a l u e was compared w i t h t a b u l a r t v a l u e ( s i g n c o n s i d e r e d ) ( T a b l e A . 3 , S t e e l and T o r r i e , 1960).  -  and Synovex-S (78 vs 73 ml/h/kg)  173  -  than i n the c o n t r o l s t e e r s .  A similar  change i n MCR of GH was observed i n s t e e 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 r a t e of GH was c a l c u l a t e d as the product of MCR and b a s a l GH c o n c e n t r a t i o n s .  The s e c r e t i o n r a t e o f GH was h i g h e r i n  i m p l a n t e d w i t h a n a b o l i c compounds  than i n the c o n t r o l s .  C o n t r o l , 135 ug/h vs a l l the a n a b o l i c compounds  (P <  .05,  combined, 291 u g / 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 i n s t e e r s i m p l a n t e d w i t h DES,  steers  higher  Z e r a n o 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 a n a b o l i c compounds were not significant  (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 t e and MCR, b o t h per kg m e t a b o l i c body s i z e , are p r e s e n t e d i n F i g u r e  expressed  23.  DISCUSSION The plasma GH c o n c e n t r a t i o n and the k i n e t i c parameters of GH m e t a b o l i s m i n s t e e r s i m p l a n t e d w i t h a n a b o l i c compounds were s t u d i e d this  in  experiment.  Plasma GH Concentration l n Steers and the E f f e c t 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 h o u r l y samples. i n ruminants  The e p i s o d i c n a t u r e of GH s e c r e t i o n  ( A n f i n s o n e t a l . , 1975; D a v i s e t a l . , 1977a) makes i t v e r y  d i f f i c u l t t o o b t a i n r e p r e s e n t a t i v e samples and the d e t e r m i n a t i o n of GH from s i n g l e samples c o u l d thus be c r i t i c i z e d . analysing  Due t o the d i f f i c u l t y  the l a r g e number of samples g e n e r a t e d i n s t u d i e s as  in  reported  Table 32.  H a l f - l i f e , f r a c t i o n a l d i s a p p e a r a n c e r a t e and m e t a b o l i c c l e a r a n c e r a t e of GH i n s t e e r s on AO days f o l l o w i n g t h e i m p l a n t a t i o n of a n a b o l i c compounds  Slow Component  F a s t Component Treatment  Half-life ( t 1/2 a , min)  Control  4.55 ± 0.22  1  Fractional Turnover Rate ( a , per min)  0.1527 ± 0.007 0.2276" ±0.007  DES  3.05 ±0.10  Zeranol  8.02 ±0.13  Synovex-S  4.44'' ±0.36  **,tt  0.0864 ±0.001 0.1572 ±0.013  *,tt t,+  Fractional Turnover Rate ( 6 , p e r min)  1/h (MCR)  34.44 ±1.30  0.0201 ±0.001  22.51 ±0.39  68.93 ±0.20  292.79 ±1.96  37.9 ±0.93  0.0182 ±0.0004  16.96 ±3.16  51.39 ±8.50  219.00 ±37.52  0.0161 ±0.001  24.30 ±4.00  71.18 ±9.95  305.92 ±44.76  0.0171 ±0.001  23.83 ±3.80  74.08 ±7.14  313.60 ±35.17  Half-life ( t 1/2 0, min)  43.10 ±2.32 40.77 ±3.30  mean ± s t a n d a r d e r r o r . *>**P < . 0 5 , P < .01 r e s p e c t i v e l y , compared to c o n t r o l . " »'l" P < . 0 5 , P < . 0 1 , r e s p e c t i v e l y compared t o DES. r  r  "hp < .05 compared t o Z e r a n o l .  M e t a b o l i c C l e a r a n c e Rat (MCR) ml/h/kg ml/h/kg Body-weight B o d y - w e i g h t (MCRK) (MCRM) 0 , 7 5  -  »  CONTROL  •• ••DO OC •••• •••• x * •• •• • •••• •• •• •  300  200 \  E  -  METABOLIC CLEARANCE RATE  400  \  175  ZERANOL ••••  l&  DES SYNOVEX-S  V.  •• ••  100  V.  • • •  •Pt  r\ .  J  5  Figure 23.  r  y  SECRETION RATE  Metabolic clearance and secretion rates of GH i n steers implanted with anabolic compounds. Each bar represents mean ± SE of values obtained from two steers.  -  h e r e , I t becomes d i f f i c u l t  176  -  t o f o l l o w the s e r i a l b l o o d  sampling  methodology ( D a v i s e t a l . , 1977a; Ohlson e t a l . , 1981) s e c r e t o r y p a t t e r n s of GH.  to o b t a i n  I n s t e a d , b l o o d samples were c o l l e c t e d from  s t e e r s a t 0730 h and 1400 h on each sampling p e r i o d and the average c o n c e n t r a t i o n of GH from these two samples was t a k e n t o r e p r e s e n t GH c o n c e n t r a t i o n .  basal  R e c e n t l y , Donaldson e t 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 t h a t 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 values  o u t l y i n g v a l u e s and the  thus o b t a i n e d were r e p r e s e n t a t i v e of b a s a l GH l e v e l s .  Since a  s i m i l a r procedure w i t h sampling b o t h at morning (0730 h) and a f t e r n o o n (1400 h) f o l l o w e d by the l o g - t r a n s f o r m a t i o n of the mean c o n c e n t r a t i o n s was p e r f o r m e d , the v a l u e s r e p o r t e d i n T a b l e 27 are c o n s i d e r e d t o be r e p r e s e n t a t i v e of b a s a l GH c o n c e n t r a t i o n of the s t e e r s . Mean plasma GH c o n c e n t r a t i o n f o r c a t t l e r e p o r t e d i n e x p e r i m e n t was g r e a t e r than t h a t r e p o r t e d by D a v i s and h i s ( D a v i s e t a l . , 1977a; Ohlson e t a l . , 1981).  this co-workers  Whereas the method of  sample c o l l e c t i o n as d i s c u s s e d above c o u l d have p l a y e d a r o l e , i t  should  be mentioned t h a t the plasma GH c o n c e n t r a t i o n was o b t a i n e d from the radioimmunoassay  u s i n g NIAMDD-oGH-I-1 as the s t a n d a r d .  A large  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 r e p o r t e d i n the literature.  Mean GH v a l u e s o b t a i n e d i n t h i s s t u d y have t o be compared  w i t h the v a l u e s of 3.5 - 5.2 ng/ml i n c a t t l e (Ohlson elt_ a l . , 1981), -  5.7 ng/ml i n wether lambs ( O l s e n e t a l . , 1977),  lambs (Donaldson  e t a l . , 1981),  5-25  ng/ml i n wether  l e s s than 3.07 ng/ml i n lambs  e t a l . , 1976) up to 25 ng/ml i n s t e e r s  (Sharp and Dyer,  3.6  (Wiggins  1971), more than  - 177  -  40 ng/ml i n c a t t l e (Dean e t a l . , 1982)  and 19 - 37 ng/ml i n c a t t l e  ( T r e n k l e , 1970a).  i n the l i t e r a t u r e on plasma GH  These d i s c r e p a n c i e s  l e v e l s i n ruminants suggest t h a t the r e l a t i v e changes i n the c o n c e n t r a t i o n r a t h e r than the a b s o l u t e v a l u e s  s h o u l d be the c r i t e r i o n  f o r comparison between the t r e a t m e n t 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 l e v a t e d i n i m p l a n t e d w i t h a n a b o l i c compounds study support  steers  and the r e s u l t s e s t a b l i s h e d i n  the t h e o r y 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 ( B u t t e r y e t a l . , 1978; P r e s t o n 1975). GH c o n c e n t r a t i o n was s i m i l a r t o the growth response  The plasma  ( F i g u r e 3, E x p t .  o b t a i n e d due t o the i m p l a n t a t i o n of a n a b o l i c compounds used i n study.  These o b s e r v a t i o n s  prolactin, insulin)  p l a y e d a s i g n i f i c a n t r o l e i n enhancing  the growth r a t e of  Sharp and Dyer,  i m p l a n t e d w i t h DES (Wiggins e t a l . 1976) has been r e p o r t e d .  this  c o u l d have steers.  plasma GH c o n c e n t r a t i o n i n s t e e r s i m p l a n t e d w i t h DES  1976b), Z e r a n o l (Borger et a l . , 1973;  1977)  IA)  suggest t h a t GH a l o n g w i t h o t h e r hormonal  f a c t o r s i n the plasma (somatomedins,  Increased  this  1971)  (Trenkle,  and i n lambs  and Z e r a n o l ( O l s e n e t a l . ,  O l s e n et a l . (1977) c o n c l u d e d t h a t a c h r o n i c  exposure t o Z e r a n o l was n e c e s s a r y  to i n c r e a s e the serum c o n c e n t r a t i o n o f  GH and i n s u l i n . The e f f e c t of Synovex-S on plasma GH c o n c e n t r a t i o n 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 experiment.  Whereas no i n f o r m a t i o n  is  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 a n d , i n c r e a s e d growth r a t e ( K a h l et a l . , 1978)  and p r o t e i n r e t e n t i o n (Rumsey, 1982)  following i t s  -  178  -  i m p l a n t a t i o n i n s t e e 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 o f endogenous of a c t i o n .  a n a b o l i c hormones, such as GH and i n s u l i n , i n i t s  mechanism  Thus the i n c r e a s e d plasma GH and growth r a t e ( E x p t . IA)  of  s t e e r s i m p l a n t e d w i t h Synovex-S demonstrated I n t h i s experiment p r o v i d e a b i o c h e m i c a l mechanism o f a c t i o n of Synovex-S i n r u m i n a n t s .  Besides,  i n r u m i n a n t s , Synovex-S seems t o e x e r t a s i g n i f i c a n t i n f l u e n c e on t h e t h y r o i d g l a n d ( K a h l e t a l . , 1978; E x p t . I I A :  1 and 2 ) .  The i n t e r a c t i o n  o f 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 T r e n k l e  (1981).  I n c r e a s e d GH s e c r e t i o n observed i n s t e e 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 Z e r a n o l i s i n g e n e r a l agreement w i t h  t h e e f f e c t of e s t r o g e n i c a n a b o l i c compounds  on the p i t u i t a r y g l a n d  weight (Burgess and Lamming, 1960; C l e g g and C o l e , 1954; Wiggins e t a l . , 1978; W i g g i n s et a l . , 1979).  I n male r a t s a s i n g l e dose o f s t i l b e s t r o l  p r o p i o n a t e has been shown t o i n c r e a s e 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 w e i g h t and serum GH ( L l o y d et a l . , 1971).  Thus the i n c r e a s e d  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 Z e r a n o l , DES and Synovex-S l n s t e e r s c o u l d be due t o the r e s u l t of i t s i n c r e a s e d s e c r e t i o n from the pituitary.  The e x t e n t t o which a n a b o l i c compounds i n f l u e n c e MCR o f GH  i n s t e e r s i s not c l e a r l y known. GH K i n e t i c s The k i n e t i c parameters of GH m e t a b o l i s m i n s t e e r s were d e t e r m i n e d by a s i n g l e , b o l u s i n j e c t i o n k i n e t i c procedure ( u s i n g 6 mg o f N1H-GH-B 1 8 ) .  Though a l a r g e i n j e c t i o n dose was employed i n t h i s  -  179  -  e x p e r i m e n t s t u d i e s i n r a t s (Frohman and B e r n a r d i s , 1970)  and humans  ( T a y l o r e t a l . , 1969) have shown t h a t the m e t a b o l i c c l e a r a n c e o f GH i s independent of the amount o f 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 l e a r a n c e of GH between d i f f e r e n t doses o f GH i n j e c t e d was r e p o r t e d f o r mice ( S i n h a e t a l . , 1979) sheep ( T r e n k l e , 1976a).  and  The dose of GH used i n t h i s experiment was  s i m i l a r t o t h a t used by T r e n k l e (1977) i n s t e e r s .  The m a t h e m a t i c a l  t r e a t m e n t of the plasma GH d i s a p p e a r a n c e c u r v e , however, was d i f f e r e n t from the p r o c e d u r e used by T r e n k l e , (1976a). 1976a) and c a t t l e ( T r e n k l e , 1977),  I n b o t h sheep  (Trenkle,  the t u r n o v e r r a t e of the GH p o o l and  o t h e r k i n e t i c parameters was c a l c u l a t e d from the d a t a o b t a i n e d up t o 40 min post GH i n j e c t i o n .  However, i n t h i s s t u d y , v a l u e s o b t a i n e d up t o  120 min p o s t GH i n j e c t i o n were used t o 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 d i s a p p e a r a n c e curve t o a two-compartment m o d e l .  The  i n j e c t e d GH has been shown t o e q u i l i b r i a t e i n two compartments i n  steers  ( T r e n k l e , 1977) and i n multicompartments i n mice ( S i n h a e t a l . , 1979) man (Bauman, 1979), r a t s (Frohman and B e r n a r d i s , 1970) ( W a l l a c e e t a l . , 1972).  Instead, using  1 2 5  I-GH,  and i n sheep  Yousef e t a l . (1969)  have r e p o r t e d a c o n s t a n t d i s a p p e a r a n c e 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. I n s p i t e o f the d i f f e r e n c e s i n the approaches u s e d , the c a l c u a t e d v a l u e s GH s e c r e t i o n and c l e a r a n c e r a t e s o b t a i n e d i n t h i s experiment were s i m i l a r t o those r e p o r t e d by T r e n k l e (1977) f o r  GH K i n e t i c s :  steers.  E f f e c t of Anabolic Compounds  The r e s u l t s o f t h i s study demonstrate the i n c r e a s e d s e c r e t o r y  -  180  -  a c t i v i t y o f the p i t u i t a r y g l a n d o f s t e e r s i n response  to anabolic  compound i m p l a n t a t i o n s and suggest t h a t the i n c r e a s e d plasma GH c o n c e n t r a t i o n observed i n the e a r l i e r experiment ( F i g u r e 21) and t h a t r e p o r t e d by o t h e r s ( T r e n k l e , 1976b; Borger e t a l . , 1973; O l s e n e t a l . , 1977) gland.  i s i n d e e d due t o an i n c r e a s e d GH s e c r e t i o n from the p i t u i t a r y The MCR o f GH i n the i m p l a n t e d and c o n t r o l s t e e r s was found t o  be s i m i l a r (P > . 0 5 ) .  Thus i t appears  t h a t the i n c r e a s e d plasma GH  observed i n s t e e r s i m p l a n t e d w i t h a n a b o l i c compounds  i s more due t o a  f u n c t i o n o f i t s s e c r e t i o n r a t h e r than c l e a r a n c e from the c i r c u l a t i o n . B e s i d e s a l a c k of d i f f e r e n c e i n the MCR of GH between the i m p l a n t e d and c o n t r o l s t e e 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 ( T a b l e 32) tends c o n f i r m the above h y p o t h e s i s .  to  T r e n k l e (1981) r e c e n t l y c o n c l u d e d t h a t  t h e changes i n the c o n c e n t r a t i o n o f hormone i n the plasma were i n f l u e n c e d more by s e c r e t i o n than by c l e a r a n c e . independent s t u d i e s  F u r t h e r , from two  Sinha and h i s c o - w o r k e r s have demonstrated t h a t the  i n c r e a s e d plasma GH c o n c e n t r a t i o n i n the pregnant mice ( S i n h a e t 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 t e w i t h o u t much change i n MCR  ( S i n h a e t a l . , 1979).  In the r a t s , S t r o s s e r  and M i a l h e (1975) r e p o r t e d  t h a t the MCR i s independent of serum GH c o n c e n t r a t i o n and t h a t 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 gland. the f i n d i n g s  The r e s u l t s o f the p r e s e n t experiment a r e i n l i n e w i t h  of the above works and s u g g e s t , t h a t s e c r e t i o n r a t e o f GH  i s the p r i m a r y k i n e t i c parameter i n f l u e n c e d by a n a b o l i c compounds  in  ruminants. Growth hormone s e c r e t i o n r a t e was s i m i l a r (P > .05) among the  -  a n a b o l i c compounds  181  -  used i n t h i s e x p e r i m e n t , 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 r e p o r t e d e a r l i e r ( F i g u r e Expt. IA).  3,  The k i n e t i c parameters of GH m e t a b o l i s m was o b t a i n e d 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 o f a n a b o l i c compounds.  It  is likely  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 a n a b o 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 a n d . The r e s u l t s a l s o suggest t h a t complex p r o c e s s e s  such as a n i m a l g r o w t h ,  might I n v o l v e the i n t e r a c t i o n o f a number o f hormonal f a c t o r s t h a n the e f f e c t of e i t h e r one a l o n e .  rather  I n sheep, Wagner and Veeinhuzen  (1978) r e p o r t e d t h a t 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 n c r e a s e d 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 Z e r a n o l i m p l a n t e d s t e e r s as observed i n the e a r l i e r experiments 117)  tends to e x p l a i n the decreased growth response  (Expt. IIA,  o b t a i n e d due to  page its  i m p l a n t a t i o n compared t o 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 t e e r s i m p l a n t e d 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 t e 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 s o tends t o 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 s  ug/d/kg0.75  V  )  r  =  0 # 7 1 >  P  = o.05,  n -  thyroxine secretion rate,  8).  CONCLUSION  The i m p l a n t a t i o n o f e s t r o g e n i c a n a b o l i c compounds i n c r e a s e i n the GH c o n c e n t r a t i o n i n s t e e r s .  The growth  r e s u l t e d i n an promoting  p r o p e r t i e s of Synovex-S was a s s o c i a t e d w i t h an i n c r e a s e d plasma GH concentration.  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 a n d .  Implantations  DES and Synovex-S r e s u l t e d i n h i g h e r plasma GH c o n c e n t r a t i o n i n  of  steers  than Z e r a n o l . In the second p a r t , a b a s i s f o r the changes i n t h e plasma GH s t a t u s i n the i m p l a n t e d s t e e r s was e s t a b l i s h e d .  I n s t e e r s , the i n j e c t e d  GH was e l i m i n a t e d i n a m u l t i c o m p a r t m e n t a l 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 t h e disappearance  curve.  The r e s u l t s I n d i c a t e d i n c r e a s e d GH s e c r e t i o n r a t e  i n s t e e r s i m p l a n t e d w i t h a n a b o l i c compounds. i n f l u e n c e d by a n a b o l i c compound i m p l a n t a t i o n s .  The MCR of GH was not In the e a r l y  periods  a f t e r the i m p l a n t a t i o n (20 d a y s ) a l l the a n a b o 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 . It  i s concluded t h a t the e s t r o g e n i c a n a b o 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 t u r n are a s s o c i a t e d w i t h i n c r e a s e d GH  secretion rate with l i t t l e  changes i n the MCR.  - 183 -  EXPERIMENT I I C E F F E C T O F ANABOLIC COMPOUNDS ON T H E K I N E T I C OF I N S U L I N METABOLISM  PARAMETERS  I N GROWING STEERS  INTRODUCTION  I n s u l i n p l a y s an i m p o r t a n t r o l e i n a n i m a l growth and m e t a b o l i s m . Its  e f f e c t on growth r a t e and f e e d c o n v e r s i o n e f f i c i e n c y i n c a t t l e  ( M a r t i n o v i c h and M a r g o l i n , types i n v i t r o ( S t r a u s ,  1975), growth and p r o l i f e r a t i o n o f many  1981), p a r t i t i o n i n g o f 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 S m i t h , 1982) and p r o t e i n s y n t h e s i s have been w e l l documented.  (Manchester, 1972)  The growth promoting p r o p e r t i e s o f  e s t r o g e n i c a n a b o l i c compounds  a r e proposed t o be mediated  changes i n t h e endogenous GH and i n s u l i n c o n c e n t r a t i o n s 1978; P r e s t o n , 1975; T r e n k l e , 1976b). hypothesis,  cell  through  ( B u t t e r y et^ a l _ . ,  I n accordance w i t h t h e proposed  i n c r e a s e d plasma i n s u l i n c o n c e n t r a t i o n s i n a n i m a l s  following  the i m p l a n t a t i o n o f DES ( D a v i s e t a l . , 1970; T r e n k l e , 1970a), Z e r a n o l ( O l s e n et_ a l . , 1977; Sharp and Dyer,  1970; Wangsness e t a l . , 1981) and  e s t r a d i o l (Donaldson e t a l . , 1981) have been r e p o r t e d ; b u t e i t h e r t h e mechanism f o r such changes i n i n s u l i n s t a t u s  or the a l t e r a t i o n i n t h e  k i n e t i c parameters o f i n s u l i n m e t a b o l i s m i n a n i m a l s was n o t c l e a r l y demonstrated.  T h i s experiment was, t h e r e f o r e , conducted t o s t u d y t h e  k i n e t i c parameters o f I n s u l i n m e t a b o l i s m i n s t e e r s i m p l a n t e d w i t h a n a b o l i c compounds  i n o r d e r t o f u r t h e r u n d e r s t a n d t h e mechanism(s) by  w h i c h they a l t e r plasma i n s u l i n s t a t u s  i n steers.  - 184 -  MATERIALS AND METHODS  Animals E i g h t H e r e f o r d s t e e r s were used i n t h i s experiment t o study the e f f e c t of a n a b o l i c compounds metabolism.  The a n i m a l s were s e l e c t e d from a group o f 24 s t e e r s  were used i n E x p t . I I all  on the k i n e t i c parameters of i n s u l i n  A:2.  The s t e e r s used i n t h i s study  that  represented  t h e f o u r t r e a t m e n t groups ( C o n t r o l , Z e r a n o l , DES and Synovex-S)  d e s c r i b e d i n Expt I I A : 2 .  The method of a n a b o l i c compound i m p l a n t a t i o n ,  f e e d i n g procedure and the p r e p a r a t i o n o f a n i m a l s f o r i n s u l i n k i n e t i c s t u d i e s were d e s c r i b e d i n E x p t . I I A :  2.  K i n e t i c parameters were e s t i m a t e d i n s t e e 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 a n a b o l i c  compounds.  Experimental Procedure Preparation of Insulin f o r K i n e t i c Studies: B o v i n e i n s u l i n (Sigma, L o t # 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 a t a c o n c e n t r a t i o n o f 1 mg/ml.  One ml a l i q u o t s o f t h i s s t o c k s o l u t i o n were s t o r e d i n 1 c c  s y r i n g e s at -20°C and were used i n t h e k i n e t i c e x p e r i m e n t s .  K i n e t i c Procedure A s i n g l e b o l u s i n j e c t i o n of n o n - r a d i o a c t i v e i n s u l i n k i n e t i c p r o c e d u r e was used (Madigan and E v a n s , 1973).  On the day o f the  e x p e r i m e n t , t h e s t e e r s were weighed, f e d t h e morning p o r t i o n of the r a t i o n and were p l a c e d i n the metabolism c r a t e s which a l l o w e d some freedom o f movement but p r e v e n t e d t h e s t e e r s  from t u r n i n g a r o u n d .  The  -  185  -  a n i m a l s were a l l o w e d t o remain i n the c r a t e f o r an hour b e f o r e two b l o o d samples  at 10-15 min i n t e r v a l were c o l l e c t e d t o o b t a i n b a s a l  insulin  level.  The s t e e r s were then i n j e c t e d w i t h 1 mg of b o v i n e i n s u l i n  (one  s y r i n g e as d e s c r i b e d 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 t h e t e r was f l u s h e d i m m e d i a t e l y w i t h 0.9% s a l i n e .  In order to  measure the d i s a p p e a r a n c e of i n j e c t e d i n s u l i n s e r i a l b l o o d samples  were  c o l l e c t e d a t 5, 10, 15, 20, 2 5 , 30, 35, 40, 50 and 60 min a f t e r i n j e c t i n g the hormone.  The b l o o d 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 i m m e d i a t e l y t o o b t a i n the plasma.  The plasma was s t o r e d at - 7 0 ° C .  t r e a t m e n t group were s t u d i e d  Two s t e e r s , r e p r e s e n t i n g one  simultaneously.  A n a l y t i c a l Procedure Insulin Plasma i n s u l i n c o n c e n t r a t i o n s were determined by a h i g h l y s p e c i f i c double a n t i b o d y radioimmunoassay  procedure d e s c r i b e d by H a l e s  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 i n g an i n s u l i n radioimmunoassay # IM  package  (Amersham C o . , O a k v i l l e , O n t a r i o , Canada;  Code  78). The r e a g e n t s were added t o d i s p o s a b l e p o l y s t y r e n e 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 o r b o v i n e  o r a p p r o p r i a t e amount of i n s u l i n s t a n d a r d (0 t o 80 yU of insulin/ml)  and 100 y l a l i q u o t of i n s u l i n b i n d i n g reagent  plasma  bovine (guinea-pig  antiserum 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 i n c u b a t e d a t 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 n d i n g r e a g e n t , 100 y l o f ^ ^ I - I n s u l i n was d i s p e n s e d t o each  -  tube.  186  -  The tubes were v o r t e x mixed and i n c u b a t e d a t 2 - 4°C.  After 2 h  of f u r t h e r i n c u b a t i o n , 700 y l o f phosphate b u f f e r was added; the  tubes  were v o r t e x mixed and c e n t r i f u g e d a t 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 J 6 ) . the f r e e  1  J  The s o l u t i o n  containing  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 s o c i a t e d  w i t h the p r e c i p i t a t e was counted i n an a u t o m a t i c gamma c o u n t e r 1185, N u c l e a r - C h i c a g o ) .  (Model  I n s u l i n c o n c e n t r a t i o n i n the plasma and the  a 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 c a l c u l a t e d as d e s c r i b e d i n the T4 radioimmunoassay  (Expt. IIA:  2).  The i n t e r - and  intraassay  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 c o n c e n t r a t i o n o f 15.2  yU/ml.  Analysis of K i n e t i c Data The plasma i n s u l i n d i s a p p e a r a n c e curve (0 t o 60 min) was to an one compartment model. earlier  fitted  The model d e s c r i p t i o n has been p r e s e n t e d  (See T4 K i n e t i c s , E x p t . I I A :  2).  Calculations The c a l c u l a t i o n s of i n s u l i n t u r n o v e r 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 , m e t a b o l i c c l e a r a n c e r a t e and s e c r e t i o n r a t e were c a r r i e d - o u t u s i n g the e q u a t i o n s d e s c r i b e d e a r l i e r (See T4 K i n e t i c s E x p t . IIA:  2).  The k i n e t i c parameters were averaged and e x p r e s s e d as mean ± SE.  The t r e a t m e n t d i f f e r e n c e s were t e s t e d by S t u d e n t ' s  and T o r r i e , 1960).  t-test  (Steel  -  187  -  RESULTS  The d i s a p p e a r a n c e intravenous  of i n s u l i n from the plasma f o l l o w i n g the  i n j e c t i o n i s p r e s e n t e d i n F i g u r e 24.  Immediately  following  the i n j e c t i o n , i n s u l i n c o n c e n t r a t i o n i n the plasma i n c r e a s e d to about 300 - 400 uU/ml and g r a d u a l l y d e c r e a s e d . i n s u l i n metabolism are summarized i n Table  The k i n e t i c parameters 33.  Mean b a s a l c o n c e n t r a t i o n of i n s u l i n of the s t e e r s used i n study was 16.2 uU/ml.  of  this  I n s u l i n was r a p i d l y c l e a r e d i n s t e e r 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 t u r n o v e r r a t e of 0.0505 m i n  - 1  .  The m e t a b o l i c c l e a r a n c e 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 w e i g h t , were 13 ml per min and 208 uU per m i n , r e s p e c t i v e l y . The e f f e c t of a n a b o l i c compounds on the k i n e t i c parameters i n s u l i n m e t a b o l i s m i n s t e e r s i s p r e s e n t e d i n T a b l e 34.  The h a l f - l i f e  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 Z e r a n o l or implantations.  However,  t h e r e was a s i g n i f i c a n t  of  (P < .05)  of  DES reduction i n  the h a l f - l i f e of i n s u l i n In s t e e r s due t o Synovex-S i m p l a n t a t i o n when compared t o the c o n t r o l s t e e r s .  There was a c o r r e s p o n d i n g  increase  the f r a c t i o n a l t u r n o v e r r a t e (11 per cent h i g h e r than c o n t r o l , P < of i n s u l i n i n s t e e r s due t o the i m p l a n t a t i o n of Synovex-S. 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 ,  in .05)  Though not fractional  t u r n o v e r r a t e of i n s u l i n was s l i g h t l y e l e v a t e d i n s t e e r s i m p l a n t e d w i t h Zeranol 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 t e e r s Z e r a n o l was about 20 per cent h i g h e r  (P < .05)  implanted w i t h  than the u n i m p l a n t e d  c o n t r o l s t e e r s and about 25 per cent h i g h e r than the s t e e r s  implanted  - 188 -  F i g u r e 24.  The d i s a p p e a r a n c e of i n s u l i n from the plasma of s t e e r 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 o f b o v i n e 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 . B a s a l i n s u l i n c o n c e n t r a t i o n i s i n d i c a t e d by the d o t t e d l i n e .  -  Table 33.  189  -  K i n e t i c parameters of i n s u l i n m e t a b o l i s m i n  Mean + SE  K i n e t i c Parameters Half-life  13.72  (min)  F r a c t i o n a l Turnover Rate (min Volume of D i s t r i b u t i o n  S e c r e t i o n Rate  +  249.77  1  0.34  0.0505 +  )  - 1  (ml/kg)  M e t a b o l i c C l e a r a n c e Rate  steers  0.001  +  9.16  (MCR)  ml/min/kg body weight  12.69  +  0.55  ml/min/kg  51.62  +  2.40  0  ,  7  body w e i g h t  5  (SR) u U/min/kg body w e i g h t u U/min/kg  0  ,  7  5  body weight  mean ± SE of v a l u e s o b t a i n e d from 8 s t e e r s .  207.73  +  16.87  3.08  +  0.25  Table 34.  Kinetic parametes of Insulin metabolism i n steers Implanted with anabolic compounds Metabolic Clearance Rate _ —  Secretion Rate  (min)  Fractional Turnover Rate min"  Volume of Dlstributlon (ml/kg)  ml/min/kg  14.83 ± 0.001  0.0467 ±.000004  234.99 ±10.9  10.98 ± 0.51  43.84 ±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  *,t 280.07 ±10.01  14.27 ±0.02  57.96 ±0.15  *".t 263.57 ± 4.18  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  *,t 57.06 ±2.69  2.80 ±0.35  14.8 ±0.70  Biological Half-Life Treatment Control  ml/min/kg ' 0  '.tt  75  uU/min/kg  200.44 ± 23.96  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 s t a t i s t i c a l significance when the calculated t value was compared with tabular t-value (sign considered) (Table A.3, Steel and Torrie, 1960).  uU/min/kg  Basal Insulin 0,73  u H/ml  -  w i t h DES.  191  -  Volume o f 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 i m p l a n t a t i o n s when compared t o the c o n t r o l  steers.  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 n c r e a s e d < .05)  i n s t e e r s i m p l a n t e d w i t h Z e r a n o l and Synovex-S when compared t o  the c o n t r o l s t e e r s .  On the c o n t r a r y , when compared t o the c o n t r o l  s t e e r s , MCR was not i n f l u e n c e d by DES i m p l a n t a t i o n . compounds, 0.05)  (P  however, MCR ( m l / m i n / k g  0 , 7 5  )  Among the a n a b o l i c  was s i g n i f i c a n t l y reduced (P <  i n s t e e r s i m p l a n t e d w i t h DES. S e c r e t i o n r a t e of i n s u l i n i n s t e e r s was c a l c u l a t e d as the p r o d u c t  o f MCR and the p r e - i n j e c t i o n b a s a l i n s u l i n c o n c e n t r a t i o n . i m p l a n t e d w i t h a n a b o l i c compounds the u n i m p l a n t e d c o n t r o l s t e e r s .  Steers  had h i g h e r i n s u l i n s e c r e t i o n r a t e t h a n The s e c r e t i o n r a t e (uU/ml/kg)  when  e x p r e s s e d as a percent i n c r e a s e over the c o n t r o l v a l u e was about 74 (P < 0 . 0 5 ) , 42 ( N . S . ) , and 32 ( N . S . ) per cent h i g h e r i n s t e e r s i m p l a n t e d 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 b a s a l  insulin  c o n c e n t r a t i o n was s l i g h t l y h i g h e r i n s t e e r s i m p l a n t e d w i t h Z e r a n o l and DES than the unimplanted c o n t r o l  steers.  The r e l a t i v e changes i n MCR ( m l / m i n / k g (uU/min/kg compounds  0 , 7 5  0 , 7 5  )  and s e c r e t i o n r a t e  ) of i n s u l i n i n s t e e r s i m p l a n t e d w i t h v a r i o u s  are p r e s e n t e d i n F i g u r e  anabolic  25.  DISCUSSION The k i n e t i c parameters of i n s u l i n metabolism i n s t e e r s  implanted  w i t h a n a b o l i c compounds were determined u s i n g a n o n - r a d i o a c t i v e , i n j e c t i o n k i n e t i c procedure.  bolus  -  60 rIC  METABOLIC CLEARANCE RATE  5°;  50 1  \ C  E 40 E 0^  4r  3U ID  \ C  E  D  4  192 -  +••• ••++ ++•• ++++ ++++ ++•• ••+• ••++ •••+ •••• +••• ••++ ****** • *+*+  CONTROL ••  *  ZERANOL  •••tl •••• DES  m  >od  SYNOVEX-S  3 °  :  SECRETION RATE  I 1 ••••  I  TTT+ •+•• ••++ •+•• +++• •••+ •+++ ••++ •+++ +++• ••++ ••+• •++• •••• ••+• •+•• ••+• ++++ •+++ «•••• •••• ••••  t***  O O O O O  o o  F i g u r e 2 5 . M e t a b o l i c c l e a r a n c e and s e c r e t i o n r a t e s o f i n s u l i n i n s t e e r s i m p l a n t e d w i t h a n a b o l i c compounds. Each b a r r e p r e s e n t s mean ± SE o f v a l u e s o b t a i n e d 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 t o  d e t e r m i n e k i n e t i c parameters of i n s u l i n metabolism i n man (Genuth, Orskov and C h r i s t e n s e n , 1969; Sherwin et a l . , 1974; 1975;  1977)  Srivastava et a l . ,  S t e r n e t a l . , 1968), dog ( N a v a l e s i e t a l . 1976), horse  and E v a n s , 1973)  (Madigan  r a t ( W i r t h et^ a l . , 1978) and i n mice ( C r e s t o  has been r e p o r t e d .  1972;  et a l . ,  The plasma i n s u l i n d i s a p p e a r a n c e curve up t o  60 min p o s t - i n j e c t i o n was a n a l y z e d by an one compartment m o d e l .  Both i n  man and i n e x p e r i m e n t a l a n i m a l s , i n s u l i n d i s a p p e a r a n c e i n the plasma has been d e s c r i b e d by one ( C r e s t o e t a l . , 1977), two ( P i e r l u i s s i e t 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;  equations.  Silvers  e t a l . , 1970)  S i n c e 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 a n a b o l i c compounds, attempt was made t o develop i n s u l i n k i n e t i c models i n s t e e r s .  no  However,  when the plasma i n s u l i n d i s a p p e a r a n c e up t o 120 min p o s t - i n j e c t i o n was a n a l y s e d m a t h e m a t i c a l l y (as attempted i n few a n i m a l s —  d a t a not  p r e s e n t e d ) the presence of more than one e x p o n e n t i a l i n the d i s a p p e a r a n c e e q u a t i o n was observed and these o b s e r v a t i o n s  suggest t h a t  i n f u t u r e a m u l t i - c o m p a r t m e n t a l approach should be u n d e r t a k e n t o describe i n s u l i n k i n e t i c s i n steers.  The shape of the plasma i n s u l i n  d i s a p p e a r a n c e up t o 60 min p l o t t e d on a s e m i - l o g paper ( F i g u r e c l o s e l y resembles t h a t r e p o r t e d by (Madigan and Evans, 1973)  24)  i n horses  up t o 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 t e e r s o b t a i n e d i n  this  experiment i s comparable t o t h a t r e p o r t e d by Madigan and Evans (1973) i n  -  194  -  h o r s e s u s i n g a s i n g l e i n j e c t i o n o f equine o r p o r c i n e i n s u l i n ; but was c o n s i d e r a b l y lower than that obtained u s i n g a continuous i n f u s i o n l ^ l l - p o r c i n e i n s u l i n (Madigan and Evans, 1973).  A considerable  s p e c i e s v a r i a t i o n i n the h a l f - l i f e o f i n s u l i n i n the plasma has reported:  4.5 min f o r c h i c k e n (Langslow,  e t a l . , 1977) 1969).  of  been  1976), 10 min f o r r a t s  and between 10 t o 13 min f o r man (Orskov and  (Cresto  Christensen,  The f r a c t i o n a l t u r n o v e r r a t e and the MCR of i n s u l i n c a l c u l a t e d  for steers  ( T a b l e 33) are comparable t o 0.053 t o 0.054 m i n "  1  and  10-13  ml/min/kg, r e s p e c t i v e l y , r e p o r t e d f o r dogs ( P i e r l u i s s i e t a l . , 1978). S i m i l a r v a l u e s of MCR have been r e p o r t e d i n dogs (15 ml/min/kg, et^ a l . , 1974;  10.5 ml/min/kg, N a v a l e s i e £ a l . , 1975).  Sherwin  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 species  r e p o r t e d are of i n t e r e s t c o n s i d e r i n g the l a r g e d i f f e r e n c e s  t h e c a r b o h y d r a t e m e t a b o l i s m between the ruminants and the 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,  in  non-ruminants.  calculated for  s t e e r s i n t h i s experiment i s comparable t o the t u r n o v e r r a t e o r r a t e of d i s a p p e a r a n c e of 290 uU/min/kg ( P i e r l u i s s i et a l . , 1978) represents  ( N a v a l e s i e t a l . , 1976)  r e p o r t e d f o r normal dogs.  and 267  uU/min/kg  The t u r n o v e r r a t e  the amount of i n s u l i n s e c r e t e d by the pancreas per u n i t time  ( N a v a l e s i e t 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 d i s a p p e a r a n c e o f p e r i p h e r a l l y i n t r o d u c e d i n s u l i n and the v a l u e s p r e s e n t e d i n T a b l e 33 may not p r o v i d e  information  about t h e a c t u a l p a n c r e a t i c s e c r e t i o n r a t e as t h e p e r i p h e r a l l y i n t r o d u c e d i n s u l i n does not f o l l o w the endogenous i n s u l i n  secretory  -  pathway.  195  -  I n s u l i n s e c r e t e d by the 3 c e l l s of the pancreas e n t e r s  the  p o r t a l c i r c u l a t i o n p r i o r t o m i x i n g i n the g e n e r a l c i r c u l a t i o n and the l i v e r forms an i m p o r t a n t s i t e f o r i t s d e g r a d a t i o n ( K a p l a n and  Madison,  1959); f o r example i n dogs, about 48 per cent of the s e c r e t e d  insulin  has been shown t o be degraded by the l i v e r ( N a v a l e s i e t a l . , 1976). 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  The into  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 o t h e r a l t e r n a t i v e ( N a v a l e s i e t a l . , 1976).  E f f e c t of Anabolic Compounds The r e s u l t s o f t h i s experiment d e m o n s t r a t e , f o r the f i r s t t i m e , c e r t a i n a s p e c t s of i n s u l i n k i n e t i c s i n s t e e 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 anabolic  compounds.  Increased  I n s u l i n s e c r e t i o n rates i n steers implanted w i t h  Z e r a n o l , DES and Synovex-S demonstrate the e f f e c t o f a n a b o l i c on the p a n c r e a s .  The r e s u l t s support  compounds  the g e n e r a l mechanism of a c t i o n  proposed f o r e s t r o g e n i c a n a b o l i c compounds  i n ruminants  ( B u t t e r y et_ a l . ,  1978; P r e s t o n , 1975), namely the i n v o l v e m e n t 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 c o n c e n t r a t i o n has  been r e p o r t e d i n lambs f o l l o w i n g DES (Wiggins e t a l . , 1976), Z e r a n o l ( O l s e n e t a l . , 1977; Wangsness e t a l . , 1981), e s t r a d i o l (Donaldson al.,  1981)  and i n s t e e r s f o l l o w i n g Z e r a n o l (Sharp and Dyer,  implantations.  et  1970)  On the c o n t r a r y decreased plasma i n s u l i n c o n c e n t r a t i o n  was a l s o r e p o r t e d i n lambs i m p l a n t e d w i t h Z e r a n o l (Wiggins e t a l . , 1976).  These e f f e c t s of e s t r o g e n i c a n a b o 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 t o the i n c r e a s e d i n s u l i n c o n c e n t r a t i o n n o r m a l l y  -  196  -  n o t i c e d i n pregnant women ( S p e l l a c y and G o e t z , 1963)  and the r e s u l t s  t h i s experiment e s t a b l i s h the e f f e c t o f e s t r o g e n i c compounds pancreas.  However,  of  on the  i t i s not apparent i m m e d i a t e l y , whether such an  i n c r e a s e i n i n s u l i n s e c r e t i o n observed i n s t e e r s i m p l a n t a t i o n of e s t r o g e n i c a n a b o l i c compounds  f o l l o w i n g the  i s due t o a d i r e c t e f f e c t  e x e r t e d on the pancreas or due to an i n d i r e c t e f f e c t brought about by t h e changes i n the endogenous hormones  and m e t a b o l i t e s .  Both i n  cattle  and i n sheep, plasma GH was i n c r e a s e d f o l l o w i n g DES ( T r e n k l e , 1976b) and Zeranol implantations  (Borger et a l . , 1973; O l s e n e t a l . , 1977;  Wiggins  e t a l . , 1976). When the e f f e c t of e s t r o g e n s 1965; P r e s t o n , 1975)  on GH s e c r e t i o n ( F r a n t z and R a b k i n ,  and the I n f l u e n c e of GH on i n s u l i n  ( R a b i n o w i t z , 1972) were t a k e n i n t o a c c o u n t , i t appears  secretion t h a t the  i n c r e a s e d 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 o f e s t r o g e n i c a n a b o l i c compounds circulation.  i s a r e s u l t of e l e v a t e d GH i n the  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  secretion  r a t e s observed i n the s t e e r s used i n t h i s experiment f u r t h e r  supports  the above h y p o t h e s i s  secretion  r a t e uU/min/kg;  (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  r = 0.77, P < . 0 5 , n = 8 ) .  F u r t h e r , among the  m e t a b o l i t e s , g l u c o s e and p r o p i o n a t e have been shown to be p o t e n t s t i m u l a t o r s of p a n c r e a 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 e n k l e , 1970b).  A r e l a t i o n s h i p between plasma  glucose  and i n s u l i n s e c r e t i o n thus seems t o e x i s t i n a n i m a l s t r e a t e d w i t h a n a b o l i c compounds.  Increased  plasma g l u c o s e  concentration i n  i m p l a n t e d w i t h DES and Z e r a n o l ( D a v i s e t a l . , 1970;  steers  Sharp and Dyer,  -  1970)  197  -  has a l s o been r e p o r t e d . Increased  s e c r e t i o n r a t e of i n s u l i n i n the i m p l a n t e d s t e e r s  a l s o be due t o the d i r e c t e f f e c t o f a n a b o l i c compounds insulin secretion.  Interestingly,  could  on p a n c r e a t i c  the presence of e s t r o g e n r e c e p t o r s  t h e mammalian pancreas was demonstrated by Sandberg e t a l . ( 1 9 7 3 ) .  in  The  e s t r o g e n i c a c t i v i t y of DES and Z e r a n o l and t h e i r a b i l i t y t o i n t e r a c t w i t h the e s t r o g e n r e c e p t o r s (Dodds e t a l . , 1938; H o s p i t a l e t a l . , 1972; K a t z e n e l l e n b o g e n e t 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 o f a n a b o l i c compounds w i t h the p a n c r e a t i c receptors.  In a d d i t i o n s t e r o i d s  estrogen  such as p r o g e s t e r o n e have been shown t o  i n c r e a s e the a b i l i t y of p a n c r e a t i c ( 5 - c e l l s t o s e c r e t e i n s u l i n (Ashby al.,  1978). Synovex-S seems t o e x e r t s i m i l a r e f f e c t s on i n s u l i n m e t a b o l i s m  when compared t o the o t h e r a n a b o l i c compounds  studied.  Though not  s i g n i f i c a n t , i t s implantation i n steers resulted i n increased  insulin  s e c r e t i o n r a t e when compared t o the c o n t r o l s t e e r s but the r a t e s were s l i g h t l y l o w e r than i n t h o s e Implanted w i t h Z e r a n o l o r DES.  Whereas  the  e f f e c t o f Synovex-S on plasma GH and i n s u l i n has not been documented, i n c r e a s e d GH observed i n Synovex-S i m p l a n t e d s t e e r s IIB)  ( r e s u l t s of Expt.  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 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 Z e r a n o l . r e d u c t i o n i n the i n s u l i n T l / 2  increased  A significant  and an i n c r e a s e i n the f r a c t i o n a l  t u r n o v e r r a t e were a l s o observed due t o Synovex-S i m p l a n t a t i o n . c o n t r a c e p t i v e s w h i c h are s i m i l a r i n t h e i r c h e m i c a l c o m p o s i t i o n t o Synovex-S have been shown t o e x e r t marked a l t e r a t i o n s i n  insulin  Oral  -  metabolism i n p a t i e n t s ( S r i v a s t a v a  198  -  e t a l . , 1975).  B u t , the h a l f - l i f e  of  i n s u l i n was found t o be i n c r e a s e d (5.6 vs 4.4 min) i n p a t i e n t s t r e a t e d w i t h o r a l c o n t r a c e p t i v e s compared t o normal c o n t r o l s . Besides  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 , a n a b o l i c  a l s o seem t o i n f l u e n c e the MCR of i n s u l i n . s t e e r s , MCR was e l e v a t e d (P < .05) s i m i l a r i n DES i m p l a n t e d s t e e r s .  compounds  When compared t o the c o n t r o l  i n Z e r a n o l and Synovex-S and was The mechanism b e h i n d the  increased  i n s u l i n c l e a r a n c e i n Z e r a n o l and Synovex-S i m p l a n t e d s t e e r s i s c l e a r l y understood.  not  But the r e s u l t s i n d i c a t e t h a t the plasma  c o n c e n t r a t i o n 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 l e a r a n c e r a t e ( F i g u r e 25 f o r d e t a i l s ) .  The l i v e r i s  major s i t e of i n s u l i n d e g r a d a t i o n ( K a p l a n and M a d i s o n ,  1959).  Thus t h e  i n c r e a s e d MCR observed i n Z e r a n o l and Synovex-S i m p l a n t e d s t e e r s be a f u n c t i o n of i n c r e a s e d c a t a b o l i c a c t i v i t y of the l i v e r . possible  the  It  could is  t h a t the p e r i p h e r a l s i t e s of i n s u l i n d e g r a d a t i o n a r e more  a c t i v e i n s t e e r s i m p l a n t e d w i t h Z e r a n o l and Synovex-S than the c o n t r o l steers.  Such mechanisms,  however, have been proposed f o r t h e i n c r e a s e d  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 o n t r a c e p t i v e s c o n t a i n i n g e s t r o g e n and p r o g e s t e r o n e  (Srivastava  e t a l . , 1975).  The p a t t e r n 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 a r e not c o n s i s t e n t w i t h the e f f e c t of the compounds steers  ( F i g u r e 3, E x p t . I A ) .  on the growth r a t e of  Among the a n a b o l i c compounds used i n  this  e x p e r i m e n t , Z e r a n o l was found t o 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 t e e r s  than DES o r Synovex-S ( F i g u r e 3 ) .  Yet  its  i m p l a n t a t i o n r e s u l t s i n h i g h e r 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 o b s e r v a t i o n s  suggest t h a t  a n i m a l 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 o f hormonal f a c t o r s r a t h e r t h a n the e f f e c t of a s i n g l e hormone  alone.  CONCLUSION The i m p l a n t a t i o n of a n a b o 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 s e c r e t e d from the p a n c r e a s .  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 status.  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 t e and  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 .  insulin  The p o s s i b i l i t y o f a d i r e c t i n t e r a c t i o n  of a n a b o l i c compounds w i t h the p a n c r e a t i c e s t r o g e n r e c e p t o r s was proposed.  Among t h e a n a b o l i c compounds,  also  implantations with Zeranol  i n f l u e n c e d the p a n c r e a t i c i n s u l i n s e c r e t i o n r a t e t o a g r e a t e r e x t e n t t h a n w i t h DES or Synovex-S.  A n a b o l i c compounds  have a l s o been found t o  i n f l u e n c e the c l e a r a n c e r a t e 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 o t h e r p e r i p h e r a l s i t e s of  insulin  d e g r a d a t i o n have been proposed as p o s s i b l e mechanisms  f o r the i n c r e a s e d  m e t a b o l i c c l e a r a n c e r a t e o f i n s u l i n observed i n s t e e r s i m p l a n t e d w i t h Z e r a n o l and Synovex-S.  I m p l a n t a t i o n o f DES,  seemed t o have  little  i n f l u e n c e on the m e t a b o l i c c l e a r a n c e r a t e o f i n s u l i n . It  i s concluded t h a t 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  o f the mechanisms steers.  by w h i c h a n a b o l i c compounds  one  e l i c i t growth promotion  However, changes i n the m e t a b o l i s m of o t h e r hormones,  in  such as  GH and t h r y r o x i n e might govern the degree of growth promotion a c h i e v e d .  - 200 -  GENERAL D I S C U S S I O N AND  I m p l a n t a t i o n o f a n a b o l i c compounds r a t e of meat p r o d u c i n g r u m i n a n t s .  CONCLUSIONS  r e s u l t s i n i n c r e a s e d growth  The c a r c a s s  from such t r e a t e d a n i m a l s  c o n t a i n s a h i g h e r percentage o f p r o t e i n and a lower percentage of f a t than t h e u n t r e a t e d a n i m a l s . compounds  The b i o c h e m i c a l mechanism by which t h e s e  evoke growth promotion i n domestic a n i m a l s i s not c l e a r l y  understood.  T h i s study was conducted t o i n v e s t i g a t e the m e t a b o l i c and  e n d o c r i n e e f f e c t s of a n a b o l i c compounds  i n ruminants.  From t h e 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 c o n c l u s i o n s were drawn. I n Study I, investigated.  t h e m e t a b o l i c e f f e c t s of a n a b o l i c compounds were  A t t e n t i o n was f o c u s s e d 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 I n growing beef s t e e r s .  The m e t a b o l i c  e f f e c t s a r e summarized i n F i g u r e 2 6 . The r e s u l t s o f EXPT. IA r e v e a l e d t h e growth promoting p r o p e r t i e s of these compounds.  I m p l a n t a t i o n s w i t h DES and Synovex-S were found t o  be more e f f e c t i v e i n promoting the growth of s t e e r s than those w i t h Zeranol.  Implantations  of these a n a b o 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 e d u c t i o n i n t h e plasma c o n c e n t r a t i o n s of n i t r o g e n o u s compounds, acids.  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 n a b o l i c compounds appear t o a l t e r s i g n i f i c a n t l y the n i t r o g e n  m e t a b o l i s m of s t e e r s by i n c r e a s i n g the e f f i c i e n c y o f u t i l i z a t i o n of absorbed n i t r o g e n o u s  compounds.  The d a t a suggest t h a t these  reduce the h e p a t i c p r o d u c t i o n of u r e a and I n c r e a s e amino a c i d s f o r t i s s u e u p t a k e .  compounds  the a v a i l a b i l i t y of  I m p l a n t a t i o n s 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 o g e n m e t a b o l i s m o f s t e e r s than w i t h zeranol. Studies  on muscle p r o t e i n m e t a b o l i s m (EXPT. IB) demonstrated t h e  . PITUITARY  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  2.INCREASE0 CH SECRETION RATE 3.NO CHANCE IM SH METABOLIC CLEARANCE RATE  O _l  o CQ <  EFFECTS OF ANABOLIC COMPOUNDS IN STEERS  UJ  z o o o z  Ui  THYROID (EXPT.UA) 1. INCREASED PLASM TOTAL I FREE T* 2.INCREASED T« SECRETION RATE 3.NO CHANGE IN PLASMA T3  COLLAGEN _ METABOLISM  _ PANCREAS  (EXPT.IC)  1.INCREASED PLASMA INSULIN  1.INCREASED AMOUNT OF CONNECTIVE T1SSUES(DES ONLY)  Figure 26.  (EXPTIIB) 1. INCREASED PLASMA 6H  Summary of the m e t a b o l i c  (EXPT.IIC) 2.INCREASED INSULIN SECRETION RATE  e n d o c r i n e e f f e c t s of a n a b o l i c compounds i n growing beef s t e e r s .  - 202 -  usefulness  of measuring N - m e t h y l h i s t i d i n e i n u r i n e as a T  n o n - d e s t r u c t i v e , i n vivo index of m y o f i b r i l l a r p r o t e i n degradation i n l a r g e a n i m a l s such as c a t t l e .  The r a p i d growth r a t e of s t e e r s was  accompanied by a r a p i d r a t e o f muscle p r o t e i n t u r n o v e r .  Implantations  of a n a b o l i c compounds r e s u l t e d i n an I n c r e a s e d amount of muscle p r o t e i n d e p o s i t e d and among the a n a b o l i c compounds u s e d , DES and Synovex-S were more e f f e c t i v e than Z e r a n o l .  A general, non-significant increase  in  muscle p r o t e i n d e g r a d a t i o n o c c u r r e d i n s t e e r s i m p l a n t e d 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 i m p l a n t e d s t e e r s ,  was f a c i l i t a t e d by a h i g h e r r a t e of muscle p r o t e i n s y n t h e s i s degradation.  however,  than  The a n a b o 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 s y n t h e s i z e d i n the s k e l e t a l m u s c l e .  Implantation  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 y n t h e s i s  than t h a t of Z e r a n o l .  The  d i f f e r e n t i a l e f f e c t s of a n a b o l i c compounds were suggested due to the i n v o l v e m e n t of l e s s muscle p r o t e i n d e g r a d a t i o n per u n i t  synthesis.  H y d r o x y p r o l i n e e x c r e t e d 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 g e n t u r n o v e r as the s t e e r s gained weight and advanced i n m a t u r i t y (EXPT. I C ) .  The r e d u c t i o n i n the u r i n a r y  hydroxyproline  e x c r e t i o n w i t h advancing m a t u r i t y may be due t o a r e d u c t i o n i n the synthesis  of new c o l l a g e n f i b r i l s and the i n c r e a s e d r e s i s t a n c e of the  collagen f i b r i l s  t o the breakdown by the c o l l a g e n o l y t i c enzymes.  I m p l a n t a t i o n w i t h DES caused i n c r e a s e d u r i n a r y h y d r o x y p r o l i n e e x c r e t e d by the s t e e r s , w h i l e Z e r a n o l and Synovex-S had l i t t l e  effect.  These  d i f f e r e n t i a l e f f e c t s of a n a b o l i c compounds on c o l l a g e n metabolism are not c l e a r l y understood a t p r e s e n t , but the r e s u l t s suggest t h a t DES  - 203 -  may i n c r e a s e t h e amount o f c o l l a g e n and i t s t u r n o v e r i n s t e e r s .  These  e f f e c t s o f DES c o u l d be r e s p o n s i b l e f o r t h e g e n e r a l l o w e r i n g o f  carcass  q u a l i t y observed by v a r i o u s I n Study I I ,  researchers.  t h e e f f e c t of a n a b o l i c compounds  on the m e t a b o l i s m  o f 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 e n d o c r i n e  e f f e c t s a r e summarized i n F i g u r e 26. Estrogenic  a n a b o l i c compounds,  e s p e c i a l l y DES and Synovex-S  e x e r t e d a s i g n i f i c a n t e f f e c t on t h e a c t i v i t y of the t h y r o i d (EXPT. I I A : 1 ) .  An i n c r e a s e i n t h e plasma t o t a l and f r e e  gland  thyroxine  c o n c e n t r a t i o n s was observed i n s t e e r s i m p l a n t e d w i t h DES and Synovex-S. Z e r a n o l , on the o t h e r hand, e x e r t e d a d i f f e r e n t mechanism o f a c t i o n as f a r as t h e a c t i v i t y of the t h y r o i d g l a n d i s c o n c e r n e d .  Plasma  c o n c e n t r a t i o n o f t h y r o x i n e 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 t e e r s i m p l a n t e d w i t h Z e r a n o l when compared t o t h e c o n t r o l s . T r i i o d o t h y r o n i n e c o n c e n t r a t i o n i n t h e plasma was not a f f e c t e d by any of the a n a b o l i c compounds;  i t i s s p e c u l a t e d t h a t i t may not be i n v o l v e d i n  the growth process mediated by these compounds. warrants  This  possibility  further investigation.  Studies  on t h e e f f e c t o f a n a b o l i c compounds  (EXPT. I I A : 2 ) on the  k i n e t i c parameters o f t h y r o x i n e m e t a b o l i s m r e v e a l e d an i n c r e a s e and a d e c r e a s e i n t h y r o x i n e s e c r e t i o n and m e t a b o l i c c l e a r a n c e  rates,  r e s p e c t i v e l y , i n s t e e r s i m p l a n t e d w i t h DES and Synovex-S.  Z e r a n o l , on  the c o n 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 l e a r a n c e r a t e s of t h y r o x i n e .  The i n c r e a s e d s e c r e t i o n r a t e of t h y r o x i n e may be i n v o l v e d  i n t h e mechanisms  o f growth mediated by these compounds.  It  is  suggested t h a t Z e r a n o l e x e 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 d e p r e s s i v e et a l . ( 1 9 7 5 ) .  as suggested by  Rothenbacher  I n f a c t , i m p l a n t a t i o n o f Z e r a n o l appeared t o i n c r e a s e  the t h y r o x i n e s e c r e t i o n r a t e and r e s u l t e d I n 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 t h y r o x i n e c o n c e n t r a t i o n due to the p a r t i c i p a t i o n of e l e v a t e d m e t a b o l i c c l e a r a n c e r a t e . k i n e t i c parameters may be b e t t e r I n d i c a t o r s  Measurements  o f plasma hormone  of  status  t h a n the a b s o l u t e c o n c e n t r a t i o n i n the plasma. Estrogenic  a n a b o 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  c o n c e n t r a t i o n 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  this  study i s the d e m o n s t r a t i o n of the e f f e c t of Synovex-S on the p i t u i t a r y gland.  I m p l a n t a t i o n w i t h DES and Synovex-S r e s u l t e d i n h i g h e r plasma GH  c o n c e n t r a t i o n than w i t h Z e r a n o l .  The k i n e t i c parameters of GH  m e t a b o l i s m were determined by a n o n - r a d i o a c t i v e k i n e t i c p r o c e d u r e .  The  r e s u l t s i n d i c a t e d i n c r e a s e d GH s e c r e t i o n r a t e i n s t e e r s i m p l a n t e d w i t h a n a b o l i c compounds.  A l l t h e compounds  were e q u a 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 p e r i o d s 20)  following their implantation.  (day  The m e t a b o l i c c l e a r a n c e r a t e was not  i n f l u e n c e d t o a s i g n i f i c a n t e x t e n t by the a n a b o l i c compounds.  It  is  c o n c l u d e d t h a t e s t r o g e n i c a n a b o l i c compounds mediate growth p r o c e s s e s ruminants  through changes In the endogenous GH s t a t u s ;  in  such changes i n  t u r n are a s s o c i a t e d w i t h an i n c r e a s e d GH s e c r e t i o n r a t e w i t h  little  changes i n the m e t a b o l i c c l e a r a n c e r a t e . The metabolism of i n s u l i n was i n f l u e n c e d to a s i g n i f i c a n t i n steers  i m p l a n t e d w i t h a n a b o l i c compounds  i m p l a n t a t i o n r e s u l t e d i n an I n c r e a s e secretion.  (EXPT. I I C ) .  i n the p a n c r e a t i c  extent  Their insulin  The a l t e r e d plasma i n s u l i n s t a t u s was suggested due to a  - 205 -  r e s u l t of changes i n the c o n c e n t r a t i o n of endogenous GH and m e t a b o l i t e s such as g l u c o s e .  The chances of a d i r e c t i n t e r a c t i o n of the a n a b o l i c  compounds w i t h the p a n c r e a t i c e s t r o g e n r e c e p t o r s were p r o p o s e d . Investigation  This  a l s o 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 t e 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,  Z e r a n o l i n f l u e n c e d the p a n c r e a t i c i n s u l i n s e c r e t i o n t o a g r e a t e r e x t e n t than Synovex-S o r DES, w h i l e the m e t a b o l i c c l e a r a n c e r a t e 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 t e e r s i m p l a n t e d w i t h DES. it  i s concluded t h a t 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 i s one of the  mechanism by which a n a b o l i c compounds ruminants.  From these d a t a  e l i c i t growth promotion  in  However, changes i n the m e t a b o l i s m of o t h e r hormones,  as GH and t h y r o x i n e (and p o s s i b l y  somatomedins  and p r o l a c t i n )  such  might  govern the degree of growth promotion a c h i e v e d . Taken c o l l e c t i v e l y ,  the f i n d 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 t o the s u b j e c t of the mechanism o f a c t i o n o f a n a b o l i c compounds  i n ruminants.  i n d i c a t e t h a t a n a b o l i c compounds  The f i n d i n g s  of t h i s  investigation  i n f l u e n c e the metabolism of the a n i m a l s  f a v o r a b 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  m e t a b o l i s m i n t u r n i s i n f l u e n c e d by the endogenous changes i n the of GH, i n s u l i n and t h y r o x i n e .  status  Thus, the i m p l a n t e d a n i m a l i s i n a  p h y s i o l o g i c a l s t a t e t h a t 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 i n c r e a s e d growth r a t e and p r o t e i n p r o d u c t i o n . r e g a r d a n a b o l i c compounds m e t a b o l i s m i n the It  In  this  seem t o e x e r t a h o m e o r h e t i c c o n t r o l on the  ruminants.  i s suggested t h a t f u t u r e s t u d i e s are needed t o i n v e s t i g a t e  e f f e c t of a n a b o l i c compounds  on a) u r e a c y c l e enzymes and o t h e r  the  enzymes  - 206 -  o f amino a c i d c a t a b o l i s m , b) polyamine m e t a b o l i s m , c) physiology, whole-body  d) p r o l a c t i n m e t a b o l i s m , e) p r o t e i n s y n t h e s i s i n the and i n s e l e c t e d t i s s u e s ,  and f ) i d e n t i f i c a t i o n o f o t h e r serum  growth f a c t o r s t h a t may be i n f l u e n c e d by them. studies,  somatomedin  It  i s hoped t h a t such  i n t h e l i g h t o f knowledge gained from the p r e s e n t  w i l l widen the u n d e r s t a n d i n g compounds.  o f the mechanism o f a c t i o n o f  investigation anabolic  The a p p l i c a t i o n of such i n f o r m a t i o n w i l l , h o p e f u l l y ,  i n improvement  of the growth r a t e of r u m i n a n t s ,  result  as w e l l a s , the amount  and e f f i c i e n c y of meat produced from such a n i m a l s .  - 207  -  LITERATURE CITED A l b r i g h t , E.C., K. 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I l l , D.A. F i s h e r and J . Sack. 1977. System a n a l y s i s and e s t i m a t i o n of key parameters o f t h y r o i d hormone m e t a b o l i s m i n sheep. Ann. Biomed. Eng. 5 : 70-84. W i n i c k , M., J . A . B r a s e l and P. R o s s o . 1972. N u t r i t i o n and c e l l growth. In: N u t r i t i o n and Development [ W i n i c k , M., E d . ] , pp. 4 9 - 9 7 , W i l e y , New Y o r k . W i n i c k , M. and A. N o b l e . 1965. Q u a n t i t a t i v e changes i n DNA, RNA, and p r o t e i n d u r i n g p r e n a t a l and p o s t n a t a l growth i n the r a t . Devel. B i o l . 12 : 451-466.  W i n i c k , M. and A. N o b l e . 1966. C e l l u l a r response i n r a t s m a l n u t r i t i o n at v a r i o u s a g e s . J . N u t r . 89 : 300-306.  during  W i r t h , A . , U. S m i t h , B. N i l s s o n and P. B j o r n t o r p . 1978. I-insulin k i n e t i c s i n p h y s i c a l l y t r a i n e d r a t s . D i a b e t o l o g i a 15 : 281 ( A b s t r . ) . 1 2 5  Wu, J . J . , C.L. K a s t n e r , M.C. Hunt, D.H. K r o p f and D.M. A l l e n . N u t r i t i o n a l e f f e c t s on beef c o l l a g e n c h a r a c t e r i s t i c s and p a l a t a b i l i t y . J . Anim. S c i . 53 : 1256-1261.  1981.  Y a m a z a k i , E. and D.W. S l i n g e r l a n d . 1959. The i n v i t r o m e t a b o l i s m of t h y r o x i n e , t r i i o d o t h y r o n i n e and t h e i r a c e t i c a c i d and p r o p i o n i c a c i d analogues. E n d o c r i n o l o g y 64 : 126-135. Young, J . Z . Oxford.  1950.  The L i f e of the V e r t e b r a t e s .  Clarendon  Press,  Young, V.R. 1970. The r o l e of s k e l e t a l and c a r d i a c muscle i n t h e r e g u l a t i o n of p r o t e i n m e t a b o l i s m . I n : Mammalian P r o t e i n M e t a b o l i s m [Munro, H.N. Ed.] V o l . 4, pp. 585-674, Academic P r e s s , New Y o r k . Young, V.R. 1980. Hormonal c o n t r o l of p r o t e i n m e t a b o l i s m , w i t h p a r t i c u l a r r e f e r e n c e t o body p r o t e i n g a i n . In: Protein Deposition i n A n i m a l s [ B u t t e r y , P . J . and D.B. L i n d s a y , E d s . ] , pp. 167-191, Butterworths, Toronto. Young, V . R . , S.D. A l e x i s , B.S. B a l i g a , H.N. Munro. and W. Muecke. 1972. M e t a b o l i s m of a d m i n s t e r e d 3 - m e t h y l h i s t i d i n e : Lack of muscle t r a n s f e r r i b o n u c l e i c a c i d c h a r g i n g and q u a n t i t a t i v e e x c r e t i o n as 3 - m e t h y l h i s t i d i n e and i t s N - a c e t y l d e r i v a t i v e . J . B i o l . Chem. 247 : 3592-3600.  - 231  -  Young, V.R. and H.N. Munro. 1978. N -methylhistidine ( 3 - m e t h y l h i s t i d i n e ) and muscle p r o t e i n t u r n o v e r : An o v e r v i e w . F e d e r a t i o n P r o c . 37 : 2291-2300. T  Young, V.R. and M.G. P l u s k a l . 1977. Mode of a c t i o n of a n a b o l i c a g e n t s , w i t h s p e c i a l r e f e r e n c e t o s t e r o i d s and s k e l e t a l m u s c l e : A summary review. In: P r o t e i n M e t a b o l i s m and N u t r i t i o n [Tamminga, S. E d . ] , E u r o p . A s s o c . Anim. P r o d . P u b l i c . No: 2 , pp. 14-28, C e n t e r f o r A g r i c . P u b l i c a t i o n and Documentation, Wageningen, H o l l a n d . Young, V . R . , S.C. S t o t h e r s and G. V i l a i r e . 1971. S y n t h e s i s and d e g r a d a t i o n of mixed p r o t e i n s , and c o m p o s i t i o n changes i n s k e l e t a l muscle of m a l n o u r i s h e d and r e f e d r a t s . J . N u t r . 101 : 1379-1390. Y o u s e f , M.K. and H.D. J o h n s o n . 1967. A r a p i d method f o r e s t i m a t i o n o f t h y r o x i n e s e c r e t i o n r a t e of c a t t l e . J . Anim. S c i . 26 : 1108-1112. Y o u s e f , M.K., Y. T a k a h a s h i , W.D. R o b e r t s o n , L . J . M a c h l i n and H.D. Johnson. 1969. E s t i m a t i o n of growth hormone s e c r e t i o n r a t e i n cattle. J . Anim. S c i . 29 : 341-344. Z a n i n o v i c h , A.A. 1973. T h y r o x i n e k i n e t i c s d u r i n g p r o l o n g e d e s t r o g e n a d m i n i s t r a t i o n . J . C l i n . E n d o c r i n o l . Metab. 37 : 949-954. Z i f f , M., A. K i b r i c k , E. Dresner and H . J . G r i b e t z . 1956. E x c r e t i o n of h y d r o x y p r o l i n e i n p a t i e n t s w i t h r h e u m a t i c and n o n - r h e u m a t i c diseases. J . C l i n . I n v e s t . 35 : 579-587. Z i l v e r s m i t , D.B. experiments.  1960. The d e s i g n and a n a l y s i s Amer. J . Med. 29 : 832-848.  of  isotope  - 232 -  APPENDIX I  SOURCES OF MATERIALS SOURCE  CHEMICAL ( i ) Radioactive Chemicals Sodium -  1 2 5  1  1 2 5  I-Iodide  - Thyroxine  Amersham C o r p . , O a k v i l l e , O n t a r i o Canada 1. 2.  N u c l e a r M e d i c a l Systems I n c . New P o r t Beach, CA Immuchem C o r p . , C a r s o n , CA  1 2 5  1  - Triiodothyronine  Immuchem C o r p . , C a r s o n , CA  1 2 5  1  -  Amersham C o r p . , O a k v i l l e , O n t a r i o Canada  1 2 5  1  - Ovine Growth Hormone  Insulin  Prepared i n authors  laboratory  (11) Hormones and Antibody Preparations Bovine Growth Hormone (NIH - GH - B 18)  National Pituitary B a l t i m o r e , MD  Ovine Growth Hormone (NIH - GH - S l l )  (Same as above)  Agency,  Ovine Growth Hormone NIAMDD-oGH-I-1 ( f o r iodination)  NIAMDD, B e t h e s d a , MD  Insulin,  1.  Sigma C h e m i c a l C o . , S t . L o u i s , MO  2.  D r . M.A. R o o t , E l i L i l l y & C o . , G r e e n f i e l d , IN  Bovine  L-Thyroxine  Sigma Chemical C o . , S t . L o u i s , MO  R a b b i t A n t i s e r u m t o oGH  NIAMDD, B e t h e s d a , MD  Rabbit Antiserum to t h y r o x i n e (T4)  1.  N u c l e a r M e d i c a l Systems, CA  2.  Immuchemical C o r p . , CA  Inc.  - 233 Rabbit Antiserum to t r i i o d o t h y r o n i n e  (iii)  (T3)  -  Immuchemlcal C o r p . , CA  Guinea-pig Antiserum to Insulin  Amersham C o r p . , O a k v i l l e , O n t a r i o Canada  Goat A n t i - R a b b i t Immunoglobulin (second a n i t b o d y )  Miles Laboratory, Elkhart,  IN  Other Chemicals l-fluoro-2,  4-dinitrobenzene  Pyridoxal-5'-phosphate Urease (from J a c k Bean) Sigma C h e m i c a l Co, S t . L o u i s , MO  Hydroxyproline 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 a l b u m i n , F r a c t i o n V  Chloramine-T  MCB M a n u f a c t u r i n g C h e m i s t s , NJ  Sodium m e t a b i s u l f i t e  F i s h e r S c i e n t i f i c Co.,  B i o G e l P-60  B i o - R a d L a b o r a t o r i e s , Richmond, CA  Sephadex G 100  Pharmacia F i n e C h e m i c a l s , U p p s a l a , Sweden  lv.  Inc.,  NJ  Catheter Materials P o l y e t h y l e n e C a t h e t e r ( P . E . 90)  C l a y Adams D i v i s i o n of B e c k t o n , D i c k i n s o n C o . , P a r s i p p a n y , NJ  - 234 APPENDIX I I RADIOIODINATION OF GROWTH HORMONE  Materials: 125  -  I, c a r r i e r - f r e e 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 i n 16% sucrose Rinse solution 10 mg/ml KI i n 8% sucrose GH for iodination - aliquots of 2.5 ug NIAMDD-OGH-I-1 per 25 y l 0.01M NaHC03 were dispensed i n iodination tubes (12 x 25 mm) and stored frozen at -70°C u n t i l the day of iodination  Procedure: 1.  Add 24 u l of 0.5M phosphate buffer, pH 7.5 to the iodination v i a l and mix.  2.  Add 0.5 mCi of c a r r i e r - f r e e Iodine-125. Add 15 y l choloramine-T solution. Mix v i a l contents for exactly 15 seconds.  3.  Add 50 u l sodium metabisulfite.  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.  Mix.  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  BSA.  Two peaks r e p r e s e n t i n g  (fractions  IOC  I-oGH ( f r a c t i o n s 4-8)  10-14) were o b t a i n e d .  and f r e e  The l a b e l e d GH from the f i r s t  was p u r i f i e d f u r t h e r on a l a r g e column (1.5 x 44 cm) of Sephadex at 4°C.  The column was e l u t e d w i t h PBS (pH 7.0)  (Peak I I )  and f r e e  125  I  (Peak I I I )  column G-100  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 c o n t a i n i n g 0.5 ml PBS-1% BSA. representing a possible aggregation  I  p r o d u c t (Peak I ) ,  Three  peaks  immunoreactive GH  were o b t a i n e d (Appendix F i g u r e  1).  The f r a c t i o n s from the second peak (from the descending l i m b ) were p o o l e d , s t o r e d a t 4°C and were used i n the radioimmunoassay week of p u r i f i c a t i o n .  on the same  -  236  -  APPENDIX  PEAK I  90  30  40  E L U T I O N  100  SO  V O L U M E ( m l )  IOC  F i g u r e 1.  E l u t i o n of I-oGH d u r i n g 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 a t 4°C. The t h r e e peaks r e p r e s e n t a p o s s i b l e a g g r e g a t i o n p r o d u c t (Peak I ) , immunoreactive I-oGH (Peak I I ) , and f r e e I (Peak I I I ) .  - 237  -  APPENDIX I I I Plasma GH c o n c e n t r a t i o n s i n s t e e 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 o g e n i c a n a b o l i c compounds (Expt. IIB)  Days A f t e r I m p l a n t a t i o n (d) Treatment  14  28 ng  56  Mean  SE  22.98 ±8.9  23.61 ±7.2  17.98  40.28 ±14.9  29.31 ± 7.2  28±77 ±6.3  20.51 ±5.3  29.72  37.59 ±12.5  42.1 ± 7.0  49.65 ±13.9  44.65 ±5.8  43.49  b  45.7 ±25.9  34.92 ±9.8  40.15  b  14.04 ± 2.5  Zeranol DES  39.45 ±8.2  3  42.52 ±9.1  2  NIAMDD-oGH-I-l/ml  12.70 ± 2.5  Control  Synovex-S  42  2.9  a  a b  S t a t i s t i c a l a n a l y s i s based on a b s o l u t e hormone c o n c e n t r a t i o n 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 c o n c e n t r a t i o n i n 0730 h & 1400 h sample from 4 s t e e r s  4.5 4.7 6.8  PUBLICATIONS G o p i n a t h , R., F. E. Newsome and W.D. K i t t s . 1978. P h y t o e s t r o g e n f o r m a t i o n i n c e l l s u s p e n s i o n c u l t u r e s of a l f a l f a . Can. J . Anim. Sci. 58 : 16. Newsome, F. E., R. G o p i n a t h and W.D. K i t t s . 1980. P r a c t i c a l a s p e c t s of f o r a g e p h y t o - e s t r o g e n p r o d u c t i o n . Can. J . Anim. S c i . 60: 558. G o p i n a t h , R. and W.D. K i t t s . 1981. E f f e c t o f a n a b o l i c compounds on plasma l e v e l s of n i t r o g e n o u s compounds and h e p a t i c l e v e l s o f t y r o s i n e a m i n o t r a n s f e r a s e i n growing beef s t e e r s . J . Anim. S c i . 53 ( s u p p l . 1): 494. ( P r o c . West. Sec. Am. Soc. A n i m a l S c i . 32 : 225 - 2 2 8 ) . G o p i n a t h , R. and W.D. K i t t s . 1981. E f f e c t of a n a b o 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 u p p l . 1) : 321. G o p i n a t h , R. and W.D. K i t t s . 1981. N - 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 t u r n o v e r i n beef c a t t l e s t e e r s _in v i v o . The Physiologist 24 : 44. T  G o p i n a t h , R and W.D. K i t t s . 1982. N - 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 t u r n o v e r i n beef c a t t l e s t e e r s ia v i v o : E f f e c t of a n a b o l i c compounds. J . Anim. S c i . 55 ( s u p p l . 1 ) : 2 1 9 . T  G o p i n a t h , R. and W.D. K i t t s . 1982. K i n e t i c parameters of t h y r o x i n e m e t a b o l i s m i n growing beef s t e e r s i m p l a n t e d w i t h a n a b o l i c compounds. J . Anim. S c i . 55 ( s u p p l . 1 ) : 2 1 8 . G o p i n a t h , R. and W.D. K i t t s . 1982. T y r o s i n e f l u x and body p r o t e i n s y n t h e s i s i n growing lambs. J . Anim. S c i . 55 ( s u p p l . 1 ) : 4 2 7 . G o p i n a t h , R. and W.D. K i t t s . 1982. E f f e c t of a n a b o l i c compounds on plasma l e v e l s of f r e e amino a c i d s i n growing beef s t e e r s . Can. J . Anim. S c i . 62 : 1261. G o p i n a t h , R. and W.D. K i t t s . 1982. U r i n a r y h y d r o x y p r o l i n e e x c r e t i o n and c o l l a g e n m e t a b o l i s m i n growing beef s t e e r s and the i n f l u e n c e o f a n a b o l i c compounds. Can. J . Anim. S c i . 62 : 1261. G o p i n a t h , R. and W.D. K i t t s . 1982. Growth, N - 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 t u r n o v e r i n growing beef s t e e r s . Anim. S c i . ( s u b m i t t e d f o r p u b l i c a t i o n ) . T  J.  G o p i n a t h , R. and W.D. K i t t s . 1983. E f f e c t of a n a b o 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 . N u t r . (submitted f o r p u b l i c a t i o n ) .  

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