)V‘ESI.) RETURNING MATERIALS: Place in book drop to LJBRAfiJES remove this checkout from -_ your record. FINES win 7 - be charged if book is returned after the date stamped below. {. MAVEWWN "1 a A"? ,4 3 I 4’C) 3:1-15' .15 q EFFECTS OF'CASTRATION AND ADMINISTRATION OF.ANDROGENS TO CASTRATED MALE PIGS UPON GROWTH AND CARCASS (IIEIEHHION By Donald R. Mulvaney A.DISSERTATION Sutmitted to Michigan State University in partial fulfillment of the requiranents for the degree of DOCTOR OF PHILOSOPHY Department of Animal Science 1984 ABSTRACT EFFECTS OF CASTRATION AND ADMINISTRATION op ANDROGENS TO CASTRATED MALE PIGS UPON CROWTH AND CARCASS COMPOSITION. by Donald Ray Mulvaney Two 5 wk studies involving prepubertal (15 kg) and pubertal (74 kg) pigs were conducted. Pigs at each weight were allotted to the following groups with four pigs in each: I initial slaughter group, boars, castrates and castrates implanted with either a low or high number of silastic capsules filled with either testosterone or dihydrotestosterone. All pigs were fed ad libitum an 18% CP corn soybean diet from weaning to slaughter. At initiation of the study all pigs were given oxytetracycline intravenously to serve as an intravital bone marker. Three pigs of each treatment group were individually penned and feed intake was monitored. At slaughter, right sides of carcasses were dissected into soft tissues and bone. Subcutaneous and perirenal adipose tissue was assessed for fatty acid synthesis, lip0protein lipase and hormone sensitive lipase activities. Several muscles were dissected, weighed and analyzed for composition. Semitendinosus muscle strips were used to assess protein Donald Ray Mulvaney synthesis and degradation rates by an in vitro assay. Feed intake was 14 to 23% lower (P < .05) in boars and 12 to 38% (P- < .05) lower in testosterone implanted pigs compared to castrates and dihydrotestosterone implanted castrates. Gain was unaffected by treatment but feed to gain ratios favored boars and testosterone implanted pigs by 20%, compared to castrates and dihydrotestosterone implanted pigs. Pubertal boars and high testosterone implanted pigs had 1.1 to 1.2-fold more total bone (P < .05) than castrates. Testosterone was more effective (P < .05) in stimulating tibia linear growth than dihydrotestosterone. Androgen administration increased bone thickening compared to castrates. Total carcass fat was reduced (P < .05) in boars and testosterone implanted pigs compared to castrates and dihydrotestosterone implanted pigs. Castration increased (P < .05) fatty acid synthesis and lipiprotein lipase activities, and testosterone but not dihydro- testosterone was effective (P < .05) in lowering these activities expressed on a gram of tissue basis. Hormone sensitive lipase activities were higher (P < .05) in boars and high testosterone implanted pigs relative to castrates. These data support the hypothesis that aromatization of testosterone is required to mediate effects upon fattening. Total muscle mass did not differ (P > .05) between treatments but selected muscles were heavier in boars and androgen treated pigs than castrates. Donald Ray Mulvaney Testosterone was more effective (P < .05) in stimulating fat free semitendinosus muscle growth than dihydrotestosterone. Protein synthesis and degradation rates were higher (P < .05) in boars than castrates. Androgens increased protein synthesis and decreased (P. < 05) degradation rates compared to castrates. ACKNOVIEIXSEMENI’S In retrospect, it seems impossible to acknowledge all those wl'n contributed to the canpletionof this study. To those not mentioned here, I apologize and extend my heartfelt thanks. To all the four-legged creatures who dedicated their lives to the benefit of this project, I am deeply indebted. To Dr. Robert Merkel, I am indebted for his perseveration, confidence and friendship. Without his support and socratic influence, this scion would not have merged. To the members of my committee, Drs. W. G. Bergen, M. G. Hogberg, A. M. Pearson, D. R. Rmsos and H. A. Tucker, I convey my gratitude for their interaction and critique of this manuscript. Tour. RmaldNelson, Iextendmythanks fortheuseof facilities , research aninals and financial retribution through- out my grachlate program. To Dr. Bill Magee, I thank him for his thoughtfulness on numerous occasions and skillful assistance on the statistical analysis of this work. . 'Ib Dora Spooner, to whom I cannot thank enough for the friendship, guidance and technical assistance given throughout my graduate career, I extend my sincerest gratitude. To my comrades, Jan Busbocm, Kris Johnson and Brad Knudson, as well as other graduate students who have moved on, I thank for their friendship and interaction over the years. 'Ib'IUanrtonIespeciallythankforenablingthissmdy tobecatpletedintl'renmmerdesired. Aspecialthankywisexterfledtoallmyfriendswm provided the extra blood and sweat, Darlene Hannenburg, Dave Skjaerlund, Terri Novak and Mary Kraus. ii To my wife Jane , I express my deepest love and gratitude forbeingmybestfriendandtakingaback seatto sciencewhile this project was being perfonted and for canpleting this manu- script. To Shirley Stanke, I convey my love and thanks for her willingness to share in the completion of this manuscript. For my dear parents, Doyle and Stella, my sisters, Joyce, Janice and Judy and brothers, Merle, Verle, Dale, and Gale as well as their families, I am forever grateful for making this endeavor ultimately possible and ardently providing encouragement . iii TABLE OFCON'I'EN'I‘S Page LISTOFTABIES ......................... vii IJSTOFFIGURES ........................ xv”. INl‘KJDUCTICN . . . ........ . ...... . ....... 1 LI'I‘EBA'IUREREVIEV........... .. ........ 6 Intact Male Versus Castrated Male Pigs .......... 5 Growth of Bone, Muscle and Adipose Tissue ......... 11 BoneGrowth............ ...... ...13 Physical Factors . . . . . .......... . 13 EndocrineEffectsonBone..... ....... 19 mscleGrcwth....... ............ .22 Eh'bryonic Proliferation and Differentiation . . . 2 9 Postnatal Myogenic Cell Proliferation . . . . . . 31 Postnatal mscle Protein Accretion ....... 3 4 Factors Influencing Muscle Growth and ProteinTurncver....... ....... 37 Endocrine Factors Influencing Muscle Growth . . . 3 9 Skeletal Muscle Protein Turnover During Growth .................. . 50 Adipose Tissue Development ..... . . ...... . 54 Cellularity............ ....... 54 Imbalance Between Triglyceride Uptake, Synthesis andMobili'zation . . . . . . . . . 60 Uptake of Circulating Fatty Acids . . ..... . 50 Hormonal Control of Lipoprotein lipase . . . . . 53 Lipogenesis...................57 Lipid hbbilization . . . . . . . . ... . . . . . 73 GIAPTERI-GENERALEDGERIMENIALDEBIGNIAI‘DMEIHODS ...... 81 CHAPTER II - RELEASE RATES mm W FILLED SIIASTICCAPSUIESgg Introduction............. ...... ....33 mterialsanduethodsmmm... ..... ..39 InplantationofPilotStudyPigs........... 90 Implantation of Prepubertal and Pubertal Pigs . . . . 90 Catheterization...................91 Blood Collection and Serum Preparation ........ 9 1 Testosterone Determination . ..... . ....... 9 2 iv Results and Discussion ........... - ..... 92 Preliminary Study Results ............ 92 Pilot, Prepubertal and Pubertal Study Results . . 93 CHAPI'ERIII-EFFECI‘SOFCASTRATICNANDAWISTRATIONOF ANDMNSQVBGJYGHMTHANDCARCASSCQ’IPOSITICN. . . .105 Introduction ...................... 105 Materials and Methods ................. 105 Ibsults and Discussion ................. 107 Growth and Feed Efficiency ............ 107 Carcass length, Fat Depth and longissimus ' Muscle Area ................ 113 Carcass Weights and Soft Tissue Composition . . . 119 Carcass Fat-Free Muscle , Fat and Bone ...... 1 2 3 Subcutaneous and Perirenal Adipose Tissue (Imposition ................ 129 Smmary . . . . .................... 14o GiAP'IERIV-EFFECI‘SOFCASTRATIONANDAIIVEENISTRATICNOF ANDKEENS UPCN SKEIEI'AL MUSCLE ACIZREI‘ICN ........ 143 Introduction . . .................... 143 Materials and Methods ................. 145 Ibsults and Discussion ................. 14 5 Muscle Weights . ................. 145 Miscle lengths .................. 154 Muscle Cmposition ................ 157 Surmary ........................ 175 QiAPTERV-EFFTXII‘SOFCASTRATIQJANDAHVIENISTRATICNOF ANDRXEENSUPQ‘IINVITK) PKJI‘EIN SYNI'HE‘SISAND DEGRADATION RATES . . ................. 177 Introduction ...................... 177 Materials and Methods ................. 179 In Vitro Protein Synthesis and Degradation . . . . 179 Results and Discussion . ................ 130 Smmary ........................ 195 CHAPIERVI- EFFECI‘SOFCY-SSTRATICNANDAH’UNISTRATICNOF ANDRIEENSUPQVSKELETALNUSCIEPROI’EINASEACTIVITY. . . 195 Introduction ...................... 19 5 Materials and Metrods ................. 1 9 7 Resilts and Discussion ................. 2 0 o Sumnary ........................ 2 2 1 GiAPIERVII-EFFEXII'SOFCASTRATICNANDAD’ENISTRATICNOF ANDWUPQ‘JADIPCBETISSUEACCREI‘IONANDDIVITK). LIPCIINIC AND LIPCILYTIC ACTIVITIES ........... 222 Introduction ......... . ............ 222 Materials and Metrods ................. 22 3 Fatty Acid Synthesis (FAS) Activities ...... 2 24 Lipoprotein Lipase (LPL) and Hormone Sensitive Lipase (HSL) Assay ........ 2 2 5 'f Results and Discussion . ................. 231 Assay Qatimization . . . . . . . ........... 2 3 1 FAS Activities . . . . . . .............. 2 3 2 LPL Activities . . . ......... . ...... . 242 HSL Activities . ................. . . 2 4 4 W O O C O O C O D O O O C O O O O I O O O O ..... 249 CHAPTER VIII - EFFEIH‘S OF CASTRATION AND AIIVIINISTRATION OF ANDREEENS UPON BONE ACCREI'ION . ..... . . ..... 252 Introduction . . . . . .................. 252 Materials and Methods ................... 253 Ibsults and Discussion ....... . .......... 2555 Bone Weight . . . . ....... . ......... 256 Linear Bone Growth . . . .2 . . .‘ ...... . . . . . 261 Bone Thickness ................. . . . 274 W o c o o o o o o 00000000000 o o ooooo 284 LITERATURE CITED . ................ . . . . . . . 293 APPENDICES ..... . ..................... 286 vi TABLE II-2 II-3 III-l III-2 LIST OF TABLES Endocrine and Localized Factors Affecting Bone . . . . . . . . . . . . . Endocrine Factors Affecting Muscle . . . Experimental Design of Prepubertal and Pubertal Studies . . . . . . . . . Serum Testosterone (Test) Concentrations in Boars, Castrates and Testosterone Implanted Castrates in the Pilot Study Effects of Prepubertal Castration and Administration of Testosterone (TEST) and Dihydrotestosterone (DHT) to Castrated Male Pigs upon Serum Testbsterone Concentrations Effects of Postpubertal Castration and Administration of Testosterone (TEST) or Dihydrotestosterone (DHT) to Castrated Male Pigs upon Serum Testosterone Concentrations . . . . . Effects of Castration and Administra- tion of Testosterone (TEST) to Castrated Male Pigs upon Body Gains, Feed Intake and Feed Efficiency . . . Effects of Prepubertal Castration and Administration of Testosterone (TEST) and Dihydrotestosterone (DHT) to Castrated Male Pigs upon Body ‘ Gain Feed Intake, and Feed Efficiency . . . . . . . . . . . . . . vii Page 23 46 84 95 97 102 108 110 III-3 III-4 III-5 III-6 III-7 III-8 III-9 III-10 Page Effects of Postpubertal Castration and Administration of Testosterone (TEST) and Dihydrotestosterone (DHT) to Castrated Male Pigs upon Body Gains, Feed Intake and Feed Efficiency . . . . . . . . . . . . . . 111 Effects of Castration and Administration of Testosterone (TEST) to Castrated Male Pigs upon Carcass Length, Fat Depth at Tenth Rib and Longissimus Muscle Area . . . . . . . . . . . . . . 115 Effects of Prepubertal Castration and Administration of Testosterone (TEST) or Dihydrotestosterone (DHT) to Castrated Male Pigs upon Carcass Length, Fat Depth at the Tenth Rib and Longissimus Muscle Area . . . . . . . . 115 Effects of Postpubertal Castration and Administration of Testosterone (TEST) or Dihydrotestosterone (DHT) to, Castrated Male Pigs upon Carcass Length, Fat Depth at Tenth Rib and Longissimus Muscle Area . . . . . . . . . . . . . . 117 Effects of Castration and Administration of Testosterone (TEST) to Castrated Male Pigs upon Hot Carcass, Right Side and Soft Tissue Weights and Composition of the Soft Tissues . . . . . . . . . 120 Effects of Prepubertal Castration and Administration of Testosterone (TEST) or Dihydrotestosterone (DHT) to Castrated Male Pigs upon Hot Carcass, Right Side and Soft Tissue Weights and Composition of the Soft Tissues . . . . . . . . . . . 121 Effects of Postpubertal Castration and Administration of Testosterone (TEST) or Dihydrotestosterone (DHT) to Castrated Male Pigs upon Hot Carcass, Right Side and Soft Tissue Weights and Composition of the Soft Tissues . . . . 122 Effects of Castration and Administration of Testosterone (TEST) to Castrated Male Pigs upon Total Carcass FateFree Muscle, Fat and Bone . . . . . . . . . 124 viii III-ll III-12 III-l3 III-14 III-15 III-l6 III-l7 III-18 Effects of Prepubertal Castration and Administration of Testosterone (TEST) or Dihydrotestosterone (DHT) to Castrated Male Pigs upon Total Carcass Fat-Free Muscle, Fat and Bone . . . . Effects of Postpubertal Castration and Administration of Testosterone (TEST) or Dihydrotestosterone (DHT) to Castrated Male Pigs upon Total Carcass Fat-Free Muscle, Fat and Bone . . . . . Effects of Castration and Administration of Testosterone (TEST) to Castrated Male Pigs upon the Percentage of Fat- Free Muscle, Fat or Bone of the Carcass or Live Weight . . . . . . . . . . . . Effects of Prepubertal Castration and Administration of Testosterone (TEST) or Dihydrotestosterone (DHT) to Castrated Male Pigs upon the Percentage of Fat-Free Muscle, Fat or Bone of the Carcass or Live Weight . . Effects of Postpubertal Castration and Administration of Testosterone (TEST) or Dihydrotestosterone (DHT) to Castrated Male Pigs upon the Percentage of Fat-Free Muscle, Fat or Bone of the Carcass or Live Weight . Effects of Prepubertal Castration and Administration of Testosterone (TEST) or Dihydrotestosterone (DHT) to Castrated Male Pigs upon Chemical Composition of Subcutaneous and Perirenal Adipose Tissue . . . . . . . . Effects of Postpubertal Castration and Administration of Testosterone (TEST) or Dihydrotestosterone (DHT) to Castrated Male Pigs upon ChemiCal Composition of Subcutaneous and Perirenal Adipose Tissue . . . . . . . Effects of Prepubertal Castration and Administration of Testosterone (TEST) or Dihydrotestosterone (DHT) to Castrated Male Pigs upon Total Moisture, Ether Extractable Lipid and Protein in Composite Carcass and Perirenal Adipose Tissues . . . . . . . . . . . . . . . . ix Page 125 126 130 131 132 134 135 137 Page III-l9 Effects of Postpubertal Castration and Administration of Testosterone (TEST) or Dihydrotestosterone (DHT) to Castrated Male Pigs upon Total Moisture, Ether Extractable Lipid and Protein in Composite Carcass and Perirenal Adipose Tissue . . . . . . . . . 133 IV-l Effects of Castration and Administration of Testosterone (TEST) to Castrated Male Pigs upon Selected Muscle Weights . . . 145 IV-2 Effects of Prepubertal Castration and -Administration of Testosterone (TEST) or dihydrotestosterone (DHT) to Castrated Male Pigs upon Selected Muscle Weights . . . . . . . . . . . . . . . . . 148 IV-3 Effects of Postpubertal Castration and Administration of Testosterone (TEST) or Dihydrotestosterone (DHT) to Castrated Male Pigs upon Selected Muscle Weights . . . . . . . . . . . . . 149 IV-4 Effects of Prepubertal Castration and Administration of Testosterone (TEST) or Dihydrotestosterone (DHT) to Castrated Male Pigs upon Length of Selected Muscles . . . . . . . . . . . . . 155 IV-S Effects of Postpubertal Castration and Administration of Testosterone (TEST) or Dihydrotestosterone (DHT) to Castrated Male Pigs upon Length of Selected Muscles . . . . . . . . . . . . . 155 IV-6 Effects of Prepubertal Castration and Administration of Testosterone or Dihydrotestosterone to Castrated Male Pigs upon Chemical Composition of Triceps Brachii, Brachialis and Pectineus Muscles . 153 IV-7 Effects of Prepubertal Castration and Administration of Testosterone (TEST) or Dihydrotestosterone (DHT) to Castrated Male Pigs upon Chemical Composition of the Longissimus and Semitendinosus Muscles . . . . . . . . . . 159 IV-8 IV-9 IV-lO IV-ll IV-lZ IV-l3 IV-l4 Page (Effects of Postpubertal Castration and Administration of Testosterone or Dihydrotestosterone to Castrated Male Pigs upon Chemical Composition of Triceps Brachii, Brachialis and Pectineus Muscle . 150 Effects of Postpubertal Castration and Administration of Testosterone (TEST) or Dihydrotestosterone (DHT) to Castrated Male Pigs upon Chemical Compo- sition of Longissimus and Semitendinosus Muscles . . . . . . . . . . . . . . . . . . 151 Effects of Prepubertal Castration and Administration of Testosterone (TEST) or Dihydrotestosterone (DHT) to Castrated Male Pigs upon Total Moisture, Protein and Fat of the Triceps Brachii, Brachialis and Pectineus Muscles . . . . . . . . . . . 153 Effects of Prepubertal Castration and Administration of Testosterone (TEST) or Dihydrotestosterone (DHT) to Castrated to Castrated Male Pigs upon Total Moisture, Protein and Fat of the Longissimus and Semitendinosus Muscles . . . . . . . . . . 154 Effects of Postpubertal Castration and Administration of Testosterone (TEST) or dihydrotestosterone (DHT) to Castrated Male Pigs upon Total Moisture, Protein and Fat of the Triceps Brachii, Brachialis and Pectineus Muscles . . . . . . . . . . 155 Effects of Postpubertal Castration and Administration of Testosterone (TEST) or Dihydrotestosterone (DHT) to Castrated Male Pigs upon Total Moisture, Protein and Fat of the Longissimus and Semiten- dinosus Muscles . . . . . . . . . . . . . . 155 Effects of Prepubertal Castration and Administration of Testosterone (TEST) or Dihydrotestosterone (DHT) to Castrated Male Pigs upon Fat-Free Triceps Brachii, Brachialis, Longissimus, Pectineus and Semitendinosus Muscle Weights . . . . . . . 170 xi Page IV-lS Effects of Postpubertal Castration and Administration of Testosterone (TEST) or Dihydrotestosterone (DHT) to Castrated Male Pigs upon Fat-Free Triceps, Brachialis, Longissimus, Pectineus and Semitendinosus Muscle Weights . . . . . . . . . . . . . . . . . 171 V-l Effect of Prepubertal Castration and Administration of Testosterone (TEST) or Dihydrotestosterone (DHT) to Castrated Male Pigs upon Red and White Semitendi- nosis Muscle Protein Synthesis and Degradation Rates Measured in Vitro‘ . . 134 V-2 Effects of Pubertal Castration and Administration of Testosterone (TEST).or Dihydrotestosterone (DHT) to Castrated Male Pigs upon Red and White Semitendi- nosis Muscle Protein Synthesis and Degradation Rates Measured in Vitro . . 135 V-3 Effects of Prepubertal Castration and Administration of Testosterone (TEST) or Dihydrotestosterone (DHT) to Castrated Male Pigs upon Protein Synthesis and Degradation Rates Measured in Vitro Averaged for the Red and White Portions of the Semitendinosus Muscle . . . . . . . . . . . . . . . . . 139 V-4 Effects of Pubertal Castration and Administration of Testosterone (TEST) or Dihydrotestosterone (DHT) to Castrated Male Pigs upon Protein Synthesis and Degradation Rate Measured in Vitro Averaged for the Red and White Portions of the Semitendinosus Muscle . . . . . . . . . 190 V-S Effects of Castration and Androgen Admini- stration to Castrated Male Pigs upon in Vitro Muscle Protein Synthesis (PSR) and Degradation (PDR) Rates of Prepubertal and Pubertal Male Pigs .. . . . . . . . . . 191 VI-l Effects of Prepubertal Castration and Administration of Testosterone (TEST) or Dihydrotestosterone (DHT) to Castrated Male Pigs upon Acidic, Neutral and Alkaline Proteinase Activity of the Red Portion of the Semitendinosus Muscle . . . . . . . . 204 xii VI-2 VI-3 VI-4 VI-7 VI-8 Page Effects of Prepubertal Castration and Administration of Testosterone (TEST) and Dihydrotestosterone (DHT) to Castrated Male Pigs upon Acidic, Neutral and Alkaline Proteinase Activity of the White Portion of the Semitendinosus Muscle . . . . . . . . . . . . . . . . . 206 Effects of Pubertal Castration and Administration of Testosterone (TEST) or Dihydrotestosterone (DHT) to Castrated Male Pigs upon Acidic, Neutral and Alkaline Proteinase Activity of the Red Portion of the Semitendinosus Muscle‘ . . 203 Effects of Pubertal Castration and Administration of Testosterone (TEST) and Dihydrotestosterone (DHT) to Castrated Male Pigs upon Acidic, Neutral and Alkaline Proteinase Activity of the White Portion of the Semitendinosus Muscle . . 209 EffeCts of Prepubertal Castration and Administration of Testosterone (TEST) and Dihydrotestosterone (DHT) to Castrated Male Pigs upon Composite Proteinase Activity in the Red and White Portions of the Semitendinosus Muscle . . . . . . . . 211 Effects of Pubertal Castration and Admini- stration of Testosterone (TEST) and Dihydro- testosterone (DHT) to Castrated Male Pigs upon Composite Proteinase Activity in the Red and White Portions of the Semitendi- nosus Muscle . . . . . . . . . . . . . . 213 Effects of Castration and Administration of Testosterone (TEST) and Dihydrotestosterone (DHT) to Castrated Prepubertal and Pubertal Male Pigs upon Composite Proteinase Activity of the Semitendinosus Muscle . . 214 Effects of Prepubertal Castration and Administration of Testosterone (TEST) and Dihydrotestosterone (DHT) to Castrated Pigs upon the Percentage of Total Proteinase Activity in Acidic, Neutral and Alkaline Fraction of the Red and White Portion of the Semitendinosus Muscle . . . . . . . 216 xiii Page VI-9 Effects of Pubertal Castration and Administration of Testosterone (TEST) and Dihydrotestosterone (DHT) to Castrated Pigs upon the Percentage of Total Proteinase Activities in the Acidic Neutral or Alkaline Fractions of the Red and White Portions of the Semi- tendinosus Muscle> . . . . . . . . . . . . 217 VII-l Effects of Prepubertal Castration and Administration of Testosterone (TEST) to Castrated Male Pigs upon subcutaneous and Perirenal Adipose Tissue Fatty Acid Synthesis and LipOprotein Lipase Activity . . . . . . . . . . . . . . . 235 VII-2 Effects of Prepubertal and Pubertal Castration and Administration of Testosterone (TEST) or Dihydrotestos- terone (DHT) to Castrated Male Pigs upon Subcutaneous and Perirenal Adipose Tissue Fatty Acid Synthesis . . . . . . 239 VII-3 Effects of Prepubertal and Pubertal Castration and Administration of Testos- terone (TEST) or Dihydrotestosterone (DHT) to Castrated Male Pigs upon Subcutaneous and Perirenal Adipose Tissue Lipoprotein Lipase Activity . . . . . . . . . . . . . 243 VII-4 EffeCts of Prepubertal and Pubertal Castration and Administration of Testos- terone (TEST) or Dihydrotestosterone (DHT) to Castrated Male Pigs upon Subcutaneous and Perirenal Adipose Tissue Hormone Sensitive Lipase Activity . . . . . . . 245 VIII-l Effects of Castration and Administration of Testosterone (TEST) to Castrated Male Pigs upon Selected Bone Weights Removed from the Right Side of the Carcass . . . . . . 257 VIII-2 Effects of Prepubertal Castration and Administration of Testosterone (TEST) and Dihydrotestosterone (DHT) to Castrated Male Pigs upon Total Bone, Scapula, Radius-Ulna, Tibia-Fibula, Humerus and Femur Weights Removed from the Right Side of the Carcass . . . . . . . . . . . 253 xiv VIII-3 VIII-4 VIII-5 VIII-6 VIII-7 VIII-8 VIII-9' Page Effects of Pubertal Castration and Administration of Testosterone (TEST) and Dihydrotestosterone (DHT) to Castrated Male Pigs upon Total Bone, Scapula, Radius-Ulna, Tibia—Fibula, Humerus and Femur Weights Removed from the Right Side of the Carcass . . . 259 _Effects of Castration and Administration of Testosterone (TEST) to Castrated Male Pigs upon Selected Bone Lengths Removed from the Right Side of the Carcass . . . . . . . . . . . . . . . . 252 Effects of Prepubertal Castration and Administration of Testosterone (TEST) and Dihydrotestosterone (DHT) to Castrated Male Pigs upon Scapula, Radius, Ulna, Tibia, Fibula, Humerus and Femur Length . . . . . . . . . . . . 253 Effects of Pubertal Castration and Administration of Testosterone (TEST) and Dihydrotestosterone (DHT) to Castrated Male Pigs upon Scapula, Radius, Ulna, Tibia, Fibula, Humerus and Femur Length . . . . . . . . . . . . 254 Effects of Prepubertal Castration and Administration of Testosterone (TEST) and Dihydrotestosterone (DHT) to Castrated Male Pigs upon Diaphysis Length, Proximal and Distal End Growth Measurements and Proximal and Distal End Epiphyseal Plate Widths of the Radius . . . . . . . . . . . . . . . . 257 Effects of Pubertal Castration of Test- osterone (TEST) and Dihydrotestosterone (DHT) to Castrated Male Pigs upon Diaphysis Length, Proximal and Distal and Growth Measurements and Proximal and Distal End Epiphyseal Plate Widths of the Radius . . . . . . . . . . . . . 253 Effects of Prepubertal Castration and Administration of Testosterone (TEST) and Dihydrotestosterone (DHT) to Castrated Male Pigs upon Diaphysis Length, Proximal and Distal End Growth Measurements and Proximal and Distal End Epiphyseal Plate Widths of the Tibia . . . . . . . . . . . . . . . 271 XV VIII-10 VIII-ll VIII-12 VIII-l3 VIII-l4 VIII-15 VIII-16 _— Page Effects of Pubertal Castration and Administration of Testosterone (TEST) or Dihydrotestosterone (DHT) to Castrated Male Pigs upon Diaphysis, Length, Proximal and Distal End Growth Measurements and Proximal and Distal End Epiphyseal Plate Widths of the Tibia -. . . . . . . . . . . . . . . . . 272 Effects of Prepubertal Castration and Administration of Testosterone (TEST) and Dihydrotestosterone (DHT) to Castrated Male Pigs upon Ratios of Weight to Length of the Scapula, Radius- Ulna, Tibia-Fibula, Humerus and Femur . 276 Effects of Postpubertal Castration and Administration of Testosterone (TEST) and Dihydrotestosterone (DHT) to Castrated Male Pigs upon Ratios of Weight to Length of the Scapula, Radius- Ulna, Tibia-Fibular, Humerus and Femur 277 Effects of Prepubertal Castration and Administration of Testosterone (TEST) or Dihydrotestosterone (DHT) to Castrated Male Pigs upon Thickness Measurements on the Proximal and Distal Ends of the Humerus and Femur. . . . . . . . . . . . 278 Effects of Postpubertal Castration and Administration of Testosterone (TEST) or Dihydrotestosterone (DHT) to Castrated Male Pigs upon Thickness Measurements of the Proximal and Distal Ends of the Humerus and Femur . . . . . . . . . . . . 279 Effects of Prepubertal Castration and Administration of Testosterone (TEST) or Dihydrotestosterone (DHT) to Castrated Male Pigs upon Average Proximal and Distal End Epiphyseal Plate Widths and Tetra- cycline Marked Growth Measurements of the Tibia and Radius and Diaphysis Wall Thick- ness of the Humerus and Femur . . . . . . 231 Effects of Postpubertal Castration and Administration of Testosterone (TEST) or Dihydrotestosterone (DHT) to Castrated Male Pigs upon Pooled Proximal and Distal End Epiphyseal Plate Widths and Tetra- cycline Marked Growth Measurements of the Tibia and Radius and Diaphysis Wall Thick- ness of the Humerus and Femur . . . . . . 232 xvi Figure l.) II-2. II-3. III-l. IV-l 0 LIST OF FIGURES Page Idealized growth curves of muscle and adipose tissue . . . . . . . . . . . . . . . 2 Illustration of expected differences in growth rate, feed efficiency, percentage lean, fat and bone between intact males and castrates . . . . . . . . . . . . . . . 5 Illustration that both cell proliferation and hypertrophy are important to growth . . 12 Developmental changes in fractional protein synthesis and degradation rates . . 52 Silastic implant surface area per kilogram body weight required to elevate serum testosterone for tubes filled with testos— terone, dihydrotestosterone or testosterone propionate . . . . . . . . . . . . . . . . . 94 Serum testosterone concentrations in high (CH) and low (CL) testosterone (TEST) and dihydrotestosterone (DHT) implanted pigs and two boars from the prepubertal study . .100 Serum testosterone concentrations in high (CH) and low (CL) testosterone (TEST) and dihydrotestosterone (DHT) implanted pigs and one boar from the pubertal study . . . .103 Fat-free muscle and fat in carcasses of boars and androgen implanted castrates as a percentage of castrates . . . . . . . .128 Effects of prepubertal castration and administration of androgens to castrated male pigs upon triceps brachii, brachialis, longissimus, pectineus and semitendinosus muscle accretion . . . . . . . . . . . . .150 ' xvii Figure Page IV-2. Effects of pubertal castration and administration of androgens to castrated male pigs upon triceps brachii, brachialis, longissimus, pectineus and semitendinosus muscle accretion . . . . . . . . . . . . . 152 IV-3. Intramuscular fat accretion in the triceps brachii, brachialis, pectineus, longis- simus and semitendinosus muscles of boars, androgen implanted castrates and limit-fed castrates of the prepubertal study as a percentage of castrates . . . . 157 IV-4. Intramuscular fat accretion in the triceps brachii, brachialis, pectineus, longis- simus and semitendinosus muscles of boars, androgens implanted castrates and limit-fed castrates of the pubertal study as a percentage of castrates . . . . . . . 153 IV-S. Fat-free triceps brachii, brachialis, longissimus, platineus and semitendinosus' muscle accretion of boars, androgen implanted castrates and limit-fed , castrates of the prepubertal study as a percentage of castrates . . . . . . . . . 172 IV-6. Fat-free triceps brachii, brachialis, longissimus, pectineus and semitendinosus muscle accretion of boars, androgen implanted castrates and limit-fed castrates of the pubertal study as a percentage of castrates . . . . . . . . . 173 V-l. Time course incorporation of label into protein and specific radioactivity of intracellular pool . . . . . . . . . . . . 9131 V-2. The linear time release of tyrosine from muscle strips . . . . . . . . . . . . 132 VI-l. pH dependence of proteolytic activity of muscle extracts from fasted and fed pigs . . . . . . . . . . . . . . . . 201 xviii Figure V1-2 . VII-1. VII-2. Page Effects of incubation time and ration of extract to hemoglobin sub- strate (v/v) upon proteinase activity at pH 3.8 . . . . . . . . . . . . 202 Effects of incubation time and amount of enzyme on LPL activity . . . . . . . . 233 Effects of incubation time and amount of enzyme on HSL activity . . . . . . . . 234 xix INTROD UCT ION The ultimate goal of modern animal agriculture is to maximize production of lean tissue and minimize waste. Concerns about efficiency of production of desired products has never been keener. Minimal improvements in efficiency can be expected from traditional nutritional and genetic approaches. A clearer understanding of the mechanisms and their regulation of proliferation and differentiation of muscle, bone and adipose tissues and their interrelationships during growth is needed. Several key questions pertaining to growth need answers before the growth process can be modulated more precisely. Some of these questions are: .(1) What controls hyperplasia of myoblasts, adipoblasts and osteoblasts in early growth?; (2) What are the triggers that arrest cell division and differentiation?; (3) What role does hyperplasia have in fully differentiated tissues?; (4) What are the controls for protein and fat accretion?; (5) Can we manipulate hyperplasia and/or hypertrOphy in vitro and in vivo? and (6) How does the partitioning of nutrients vary during growth? (Mersmann, 1982). Growth of domestic animals can be characterized by sigmoidal curves as shown in figure 1. This graph shows three phases of growth: (1) a brief lag period ,for GROWTH UNITS AGE UNITS FIGJRE l. Idealized growth curves of muscle (A and B) and adipose tissue (C and D). Three phases of growth are represented: l) a brief lag phase, 2) an acceleration phase and 3) a deceleration phase. If curves are shifted frcm A to B and C to D, more efficient lean production would be obtained. (modified after Bergen, 1974) . synthesis of nuclear material or ultimate cellular machinery, (2) a steep portion representing and acceleration phase and period of most rapid growth and (3) a deceleration phase or period of reduced growth rate. The broken lines represent fat rates and illustrate that the most rapid deposition of fat occurs during the deceleration phase of growth (Bergen, 1974). Since the most efficient lean gains are made during the acceleration phase, it is desirable to lengthen the duration of this phase or in essence shift curve A to curve B. Likewise, if the curves for fat deposition could be shifted from C to D a postponement of the rapid accretion of fat would occur. “These types) of growth manipulation have been successfully achieved through selection for late -maturing or large. mature size animals (Trenkle and Marple, 1983). The search for growth stimulants or exogenous agents that achieve the aforementioned shifting is of much interest (Heitzman, 1980). However, through castration of male animals, the curves are in essence shifted in the opposite direction of that described. In other words, muscle deposition is effectively decreased and fat deposition is increased by castration. Figure 2 illustrates expected differences of bulls, rams and boars relative to castrated males (Field, 1971, Galbraith and TOpps, 1981: Seideman et al., 1982). Variations in these expected differences between intact males and castrates arise from genetic, nutritional and managerial (time of castration) differences between studies. While the intact male of the three Species shown in figure 2 have advantages in growth rate and lean, the largest commonality appears to be in feed efficiency and percentage fat. If ablation of the testis results in reduced muscle and increased fat deposition, then testosterone becomes a prime candidate for playing a significant role in regulating protein and fat metabolism. To effectively enhance protein deposition rates, testosterone would have to alter protein synthesis and/or protein degradation rates to result in greater net synthesis of protein. Likewise, to reduce fat accretion, testosterone may be involved in reducing lipogenesis and/or uptake and reesterification of circulating lipids and/or increased lipolysis and mobilization of lipid components. It was the primary objective of this investigation to examine the effects of castration and administration of testosterone and dihydrotestosterone to castrated male pigs upon changes in body composition. Muscle, bone and adipose tissue accretion, skeletal muscle protein synthesis and degradation rates, muscle proteolytic activity and adipose tissue lipogenic and lipolytic activities were also studied. These studies are hOpefully the first phase of subsequent investigations which will ascertain mechanisms of action of testosterone upon muscle, bone and adipose tissue. EXPECTED DIFFERENCE OF BULLSIB), RAMSIO) AND BOARSIP) COMPARED TO CASTRATES. FIGURE 2 . BOP ‘X: BONE. .“3- lllll 0- " llllll o 3 |IIIIIIIIIIIIIIIIIIIIIIIIIIIIII » m '0 lllllllllllllll % LEAN FEED EFFICIENCY 0- ‘3‘ lllllll c ."3 IIIIlllllllllll|l||lllllllllllllllllllllllllll m 5 IlllllllllllllllllllllllllllllIIIIllllllllllllllllll ‘ <3 (9 C) 'Q 0| <3 (9 CI 1' CM NPPPPF BONHHSdJIO BOVINSOHBd O- GROWTH RATE 0 I Ranges in the literature Illustration of expected differences in growth rate, feed efficiency, percentage lean, fat and bone -between intact males‘and castrates. % FAT 2 I 2 32 12 LITERATURE REVIEW Intact Male Versus Castrated Male Pigs The merits of intact male pigs were reviewed by Walstra and Kroeske (1968), Wismer-Pedersen (1968), Martin (1969), Turton (1969), Field (1971), Galbraith and TOpps (1931) and Seideman et a1. (1982). Considering the traits of major economic significance, intact male_*p19s present many M MM “‘———. van—n..- advantages over the castrated male. The ensuing discussion 4 lion” I"- _ y ..n. w_ . _ _ n--.“ H- _. Will_ primarily address growth rate, feed efficiency and Lhédy composition differences between boars, and castrates. The largest disadvantage of commercially rearing and marketing boars for meat is that of objectional meat odors and boar taint. The latter is believed to be caused by 5m - androst-lS-en-B-one deposited in the fat of boars (Patterson, 1968). Measures to reduce the incidence of boar taint are currently being investigated throughout the world (Conference Report, 1981 Symposium of Boar Taint, Zeist, Netherlands). Assuming the boar taint problem will eventually be solved,the growth advantages of boars can be eXploited and perhaps greater interest in the mechanisms of testosterone action upon growth in pigs will be stimulated. The literature to date involving comparisonsi of boars to castrated males has been complicated by variations in breed, age and~ weight at castration or ,1 A.) 7 ’ o’ \l {319“ / slaughter and the level of dietary protein fed and the mode of feeding (Kay and Houseman, 1975; Fuller, 1980). V ‘h‘tr’mnl ~ ' ' --— . -.-. Boars generally grow slightly (0 to 10%) Afaster than e castrated counterpart when feed intake is restricted -bu§wga§trates grow faster (0 to 10%) when fed ad_ libitum (Kay and Houseman, 1975). jf Cenerally, there is a sex x m... “$.10!“ .0“ #21,. protein interaction such that boars grow faster than castrates at higher _dietary protein concentrations. Another confounding factor in comparisons of boars and castrated males has been the ”observation that castrates have_1arger appetites which increases -the daily food and energy intake (Pay and Davies, 1973). The effect of castration of boars upon growth rate is unique since it is less than the 10 to 20% growth rate depression observed for Hmfl‘w—P‘ " castrated cattle and sheep (Prescott and Lamming, 1964). ’ While a number of investigations have found no differences“ .‘in grgwth rates between boars and castrates (Winters et "—'——~. al., 1942: Kroeske, 1963; Prescott and Lamming, 1964; Hines, 1966; Omtvedt and Jesse, 1968; Hetzer and Miller, $5,,ow- H.“ “1'... ,1: m. an ‘ and Bowland,1972))§other reports have shown - _ wTWTWW-fl 1972; ewell“ that boars gr ow faster than castrates (Bratzler et al., 4 ’fl’ 1954; Blair and English, 1965; Burgess et al., 1966: Siers, 1975; Campbell and King, 1982; (figgégfland Riley, 198 . i Recent reports comparing growth rates of boars and castrates“ have shown that growth rate varies A during different- phases of growth. Campbell and King (1982) 11"“- wwv “‘1‘ observed no difference in average daily gain (ADG) between boars and castrates during a 20 to 45 kg live weight growth period when fed a 17% CP diet. \ When dietary protein was increased . to 21% and restricted fed, ADC and feed efficiency of boars was improved by 14% 'over those of castrates. ’\\When fed ad libitum, differences between boars and castrates during this 20 to 45 kg live weight growth period were small but slightly favored the boar. \Similar trends were observed during the 45 to 70 kg live weight .____—---#— I 1,- HM lbw-“‘1 growth periodwmwaE—even more dramatic 53% advantage in ADC /' .- 7" cfiuxM‘M 635 reported by Wood and Riley (1982). Pay and Davies (1973) showed no differences in ADC when boars were compared to castrates. The latter investigators also observed that castrates had a higher (6 to 11%) voluntary fodd intake than boars from 22 to 55, 55 to 90 and 22 to 90 kgi weight intervals. These lower intakes in boars occurred for 16, 18 and 20% CP diets. Campbell and King (1982) found the differences in performance and food intake to be most apparent in boars and castrates weighing more than 45 kg. T9333“ latter studies indicated greater potential for utilization of higher protein diets in boars relative to castrates_fland that higher protein diets are needed.for full “expression of this added growth potential. Even though Pay and Davies (1973) showed no difference in ADC between boars and castrates over a 22 to 90 kg live weight growth period, 'there' was a "trend for boars to have more favorable ADG \‘pfl- responsesmgthan castrates per 1% increases in dietary “mg. protein concentrations above 16%, CP and up to 20% crude ,_u. -. protein (3.5% increases in ADC for boars and 2.5% increases g.- in~ ADG for castrates). Since boars voluntarily conSume --- q-_.¢_... -.- - ..—.— "' t/ less feed than castrates, the studies of Campbell and King (1982) become significant. These authors limit-fed boars and castrates equal amounts of isocaloric diets containing 17, 21 and 23% crude proteinL/I/final weights were not ent but backfat thicknesses were 10 to 20% lower and issectable fat in the ham was approximately 15% less in 1 IE“ 2*" ' .4 " ‘M‘u—‘xwé—‘uu 4/_gflwamwur. gs relative to castrates.;¥Th€se data indicate tha a . a r ‘H‘M o c o a mechanism other than reduced feed 1ntake 15 involved 1n \xreduced fatness of boars. \\ “a... In a 139531.. breed, the Pietrain, no differences in nitrogen (N) retention were observed before 60 kg live weight but by 80 kg, boars had 18% greater N retention relative to castrated males (Rerat, 1976). Piatowski and Jung (1966) found N retention to be 28% higher for 30 to 100 kg live weight boars relative to castrated males. These differences in N retention indicate greater capacity for protein synthesis in boars relative to castrates and may partially eXplain the_ increased protein requirements. Boars also “havefl approximately 10% higher heat production than castrates (Fuller et al., 1980). Since boars consume approximately 15% less feed/d than castrates and ADC is equal or higher, boars are superior (10 to 20%) in feed 10 efficiency (Kay and Houseman, 1975). While feed efficiency advantages exceeding 10 to 20% for boars over castrates have been reported (Rerat, 1976) similar efficiencies have been observed in other studies (Charette, 1961; Hines, 1966; Turton, 1969; Pay and Davies, 1973; Siers, 1975; Wood and Riley, 1982). The efficiency of ”converting _d1gestible ener in mu§g1eflhhas fibeenfireported to be_approximate1y 22% higher 19_boars than“ castrated malesfi_(Cahill, 1960; -"‘--.._._, Staun, 1963; Walstra, 1969; Newell and Bowland, 1972.) While boars gain at equal to or faster rates and require less feed to achieve these gains than castrates, boars also are superior in leaness (Carroll et al., 1963; /-\ WM «9...... ‘ ‘MJ‘IM 'l'- ml ‘W (E§a§"afid"fiouseman, 1915). The amount of muscle in boars has _‘W ,4. M been rgpgzrndu.;g_pg_about 3%:highsr (Field, 1971; Wood and K ‘_d/H#f, wwwuoflaw W'V'HW Enser, 1982; ood and Riley, 19 82)) (150 higher (Prescott and Lamming, 1967) and (2032higher (Hansson et al., 1975) than castrates. Bog;§_;engrtedly had 2% (Field, 1971), <39 v/ ' (Wood and Riley, 1982) and 12% (PreScott and Lamming, 1967) greater bone weights than castrates. 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.coneNHHewecHE mcHweHchH cH wcewwomEH en >eE pce cemeHHoo econ ow mocHnIcHwoecoewmo .mewccho econ cH mHmecwcmm cHewowm cemeHHoococ ece cemeHHoo .dzo cemeewocw .nw3owm emeHwaeo mo woweHcmew HeooH .wowoem Ue>HweU emeHwaeOImcU mwowoem H memes u e emmc 29 Embryonic Proliferation and Differentiation. Consider- ation of embryonic muscle cell differentiation is important since this is a time in which muscle cells are programmed (Young and Allen, 1979) and the number of mitotic divisions that occur dictates the number of muscle cells found postnatally -and probably to some degree, the growth potential of 'muscle (Holtzer and Bischoff, 1970; Burleigh, 1980). During embryonic muscle deve10pment, myogenic cells are mononucleated cells with high mitotic activity. MOrphologically, these cells feature cytOplasmic constituents similar to other cell types. Because these cells eventually express muscle cell characteristics upon cloning, they are referred to as presumptive myoblasts (Konigsberg, 1963; Fischman, 1972). In early myogenesis, these presumptive _myoblasts elongate, eventually cease dividing and begin synthesizing muscle specific proteins (Holtzer, 1970). This transition from replicating myogenic cells to muscle fibers results from activation of new genes and suppression of others (Holtzer and Holtzer, 1976; Young and Allen, 1979). After making the transition to this postmitotic or myoblast stage, the cells fuse to form multinucleated cells referred to as myotubes (Okazaki and Holtzer, 1966). It appears that fusion of myoblasts occurs when a critical cell density is reached (Konigsberg, 1971). Fusion of myoblasts 'is characterized by the 30 appearance of muscle specific proteins, namely, myosin, actin, creatine kinase as well as, appearance of cross striations and acetylcholine receptors. In synchrony with these events, DNA replication and nuclear divisions are repressed (Stockdale and Holtzer, 1961; Stockdale, 1970). Subsequent changes that occur are the further accumulation of. muscle proteins and assembly of myofibrils which diSplace the once centrally located nuclei as the nuclei migrate to the periphery of the cell. Cells with these latter characteristics are called muscle fibers or myofibers. Through clonal analysis of myogenic cells in culture, myogenesis appears to be governed by intrinsic, preprogrammed, time dependent factors as Opposed to neural extrinsic factors (Konigsberg, 1963). In general, the cue for synthesis of contractile proteins revolves around two theories. Patterson and Strohman (1972) indicated the critical event is associated with cell fusion; whereas, Holtzer et a1. (1974) viewed the withdrawal of myoblasts from the cell cycle as the prerequisite for subsequent myogenesis. Furthermore, Holtzer et a1. (1982) indicated that only myogenic cells in G1 have the Option to fuse. Cells in S, G2 or M do not fuse. Buckley and Konigsberg (1973) stated that cells which were capable of fusion but that. do not fuse can reenter the cell cycle. Holtzer et a1. (1982) provided evidence that cells with the Option to 31 fuse, withdraw from the cell cycle as a precondition to fusion and cannot be induced to reenter the cell cycle. Dienstman and Holtzer (1975) prOposed a model of myogenic lineage in which cells undergo a quantal mitosis as a requisite of passage from one cell compartment in the myogenic cell’ lineage to another. Also, the myoblast has the unique capability of fusing and cells in the penultimate compartment do not have the Option to fuse. Accordingly, exogenous agents should not be capable lof inducing presumptive myoblasts to fuse. Konigsberg (1982) theorized that cells undergo a protracted G1 period and that fusion is closely related to the time cells remain in this phase of the cell cycle. The time spent in G1 and protracted G1 is related to changes in the cell environment also caused by cells themselves (Konigsberg, 1982). It is apparent that differentiation revolves around the G1 phase of the cell cycle. In theory, a variety of factors could mediate an effect of shortening the G1 portion of the cell cycle and result in additional passes through the cell cycle and ultimately generate greater potential for subsequent postnatal muscle growth (Allen et al., 1979). Postnatal Myogenic Cell Proliferation. In reviewing work by Winick and Noble (1966), Allen et a1. (1979) determined that 80% or more of the DNA content in rat muscle was accumulated after birth. Other studies indicate 32 similar trends of increased total DNA and RNA and decreased concentrations during growth (Burleigh, 1980). This was shown in rats (Mendes and Waterlow, 1953; Devi et al., 1963; Enesco and Puddy, 1964; Winick and Noble, 1965, 1966; Cheek et al., 1971; Howarth and Baldwin, 1971), pigs (Gordon et al., 1966; Robinson, 1969; Gilbreath and Trout, 1973; Tsai et al., 1973; Hakkarainen, 1975; Powell and Aberle, 1975: Harbison et al., 1976: Corring et al., 1982; Wigmore and Stickland, 1983), chickens (Moss et al., 1964, MOss, 1968a, 1968b; Hentges et al.,.1983), rabbits (Corring et al., 1982) and ruminants (Laflamme et al., 1973; Johns and Bergen, 1976; MOstafavi, 1978). Lash et al. (1957) and Enesco and Puddy (1964) demonstrated that there was no. polyploidy in muscle. MUscle was found to be composed of many closely associated fibers, enveloPed by the endomysium and grouped in bundles by the perimysium. MOst muscle nuclei belong to muscle fibers and the remaining nuclei are associated with the connective tissue. At various stages of develOpment in the rat, Enesco and Puddy (1964) found that muscle fiber nuclei represented about 65%, endomysium nuclei 25% and those in the perimysium 10% Of the total. Cordesse and Nougues (1973) showed that the distribution of nuclei between myofibers and related connective tissue did not vary in rabbits between 30 to 150 d Of age. Connective tissue nuclei represented approximately 35% of the total. As 33 indicated by Rozovski and Winick (1979), estimation of DNA content even if uncorrected for nonmuscule nuclei, prOportionately reflects increased muscle cell nuclei. Through combinations of histometric and chemical techniques, Enesco and Puddy (1964) shOwed that individual muscle fibers- in rats did not. increase in number postnatally while the number of nuclei within myofibers and myofiber size did increase. While generally accepted that myofiber number does not increase significantly after birth (Allen et al., 1979), Gordon et al. (1966) observed increased numbers in rats as late as 90 d of age. Other studies have shown increased myofiber numbers in neonatal rats, mice and humans (Chiakulas and Pauly, 1965; GoldsPink, 1962; Mbntgomery, 1962). .In the pig, muscle fiber numbers remain constant shortly after birth (Staun, 1963, 1972; Davies, 1972; Stickland and Goldspink, 1973; Hegarty et al., 1973; Ezekwe and Martin, 1975). In contrast, Swatland (1976) analyzed transverse histological sections of pig muscle and found increased fiber numbers between 56 and 168 days of age. In explanation of the source of the increased muscle DNA during growth, the electron microsc0py work by Mauro (1961) and the thymidine incorporation studies by Moss and Leblond (1970, 1971) clearly showed that a pOpulation of cells which lies between the plasma membrane and the basement membrane Of myofibers called satellite cells are 34 the source of the new nuclei. Cardasis and COOper (1975) complemented this theory with results which showed a decrease in the total satellite cell pOpulation with age. Kelly (1978) found fewer satellite cells in the extensor digitorum longus muscle than the soleus muscle of both develOping and mature rats and these differences in satellite cell- number were correlated with myofiber nuclei density. The soleus had a greater rate of increase in myofiber nuclei per myofiber than the extensor digitorum longus as shown by an autoradiographic assessment of 3H thymidine incorporation. Salleo et a1. (1980) reported satellite cells were also capable of proliferation and fusion to produce new myofibers in rats.. MOss (1968a, 1968b) and Swatland (1977) indicated a direct relationship existed between myofiber 'diameter and the number of nuclei. As pointed out by Hakkarainen (1975) and Allen et a1. (1979) there appears to be a preprogrammed increase in DNA preceding increases in RNA and protein. As summarized by Allen et a1. (1979) the most rapid increase of DNA occurs .during rapid growth periods. Also, the number of nuclei is directly related to fiber size and the number of nuclei which may limit the quantity of protein in the myofiber. Postnatal Muscle Protein Accretion. Postnatal muscle growth is achieved by increased fiber diameter (girth) and length (GoldSpink, 1980c). The increased fiber size occurs 35 gradually and discontinuously, as Opposed to continuously, (Goldspink, 1962) and is a result of increased myofibrillar protein accretion within the myofiber. (Waterlow et al., 1978). Coldspink (1980c) attributed the increased accretion of protein to increased work loads or demands upon muscle. In addition, Goldspink (1970) indicated that once a particular critical size is achieved, myofibrils split longitudinally. This splitting facilitates develOpment of the sarcotubular system and enables additional proteins to be laid down upon the existing myofilaments. In situations of overloaded muscle where a synergistic muscle was removed, muscle. fibers were also Observed to Split. This splitting was incomplete .and localized at the ends of the muscle (Goldspink, 1980c). The extent and significance of muscle fiber splitting during nOrmal growth is not known (Goldspink, 1980c). It appears that myofilaments are added to the periphery of nascent myofibrils (Fischman, 1972) as labeled amino acids administered in vivo are incorporated and localized at the periphery of the myofibrils (Venable, 1969). . The increase in length of myofibers during postnatal growth is largely due to increased sarcomere number in series along the myofibrils and slightly increased sarcomere length (Stromer et al., 1974; Goldspink, 1980a). In mouse soleus muscle, sarcomere numbers increased from 700 to 2200 in the first three weeks Of age (Goldspink, 36 1980a). There are two alternatives for the mechanism of sarcomere addition: interstitial and serial addition. While it appears some insect muscles involve interstitial addition of sarcomeres (Goldspink, 1980a); Griffin et a1. (1971) demonstrated strong evidence for serial addition in mammalian muscle. After incorporation of labeled amino acids into protein, individual muscle fibers were dissected and autoradiography performed. These data showed most of the radioactivity was incorporated into the ends of the myofiber adjacent to the tendons. Goldspink (1980a) reported that the addition of sarcomeres could be suppressed by as much as 50% by immobilizing the limbs with casts. When the casts were removed, the rate of sarcomere addition was accelerated and achieved control numbers within a few days. When muscles of adult cats or mice were immobilized into lengthened or shortened positions with casts, adaptive responses were observed (Tardieu et al., 1980; Williams and Goldspink, 1973). MOuse soleus muscles immobilized in a lengthened position produced 20 to 30% more sarcomeres relative to normal muscles and when immobilized in a shortened position, a 25% loss Of sarcomeres was observed within a few days. These Observations were also similar when muscles were denervated prior to the immobilization (Goldspink et al., 1974). During postnatal muscle growth, net accumulation of muscle prOtein is a result of the imbalance between protein 37 synthesis and degradation rates (Garlick, 1980). With increasing age, the apparent imbalance narrows, and for the adult results in synthesis rates that are completely counterbalanced by degradation rates (Waterlow et al., 1978). Factors which influence either protein synthesis or degradation rates or both are ultimate determinants of skeletal muscle protein mass. Factors Influencing Muscle Growth and Protein Turnover. For the sake of convenience, the factors influencing muscle growth and protein turnover are classified here as: (1) chemical factors which include hormones and nutrients and (2) mechanical factors whiCh include stretch, physical activity pattern and work induced hypertrOphy. GoldSpink (1980a) immobilized a flexor (muscle, the extensor digitorum longus (EDL), and an extensor muscle, the soleus, of 50 9 growing rats by placing plaster casts on the hind leg ankle such that these muscles, were in a shortened state. The EDL and soleus grew about 10 and 20% less, respectively, relative to controls. In vitro protein synthesis rates measured 2 d after immobilization were 30 and 70% lower for the EDL and soleus, respectively, ‘relative to controls. Corresponding degradation rates were 25 and 50% higher for the EDL and soleus, respectively. The differential protein turnover response between the two muscles was eXplained on the basis of differential activity of these muscles. The soleus is recruited for greater work 38 loads relative to the EDL normally and therefore the response to immobilization of the soleus in the shortened state was more evident. In subsequent experiments, GoldSpink (1980a) observed that the EDL and soleus returned to normal within 7 d after removal of the casts. In fact, in vitro protein synthesis rates of the soleus were '90% higher than controls within 3 d after cast removal. These data indicated that a reversal of atrOphy and loss of sarcomeres as well as compensatory hypertrOphy had occurred. Goldspink (1977) reported Opposite trends when the EDL or soleus were immobilized in a lengthened state. In these experiments, the stretch induced growth was accompanied by increased RNA and DNA. This coupling of increased nuclear material and longitudinal growth enabled changes in the nuclear: sarCOplasmic ratios for more efficient maintenance of cellular processes (Goldspink, 1980a). Goldspink (1977) also Observed a stimulation of protein synthesis with stretch induced growth even when de novo synthesis of RNA and DNA was blocked. These investigators concluded that any increased nuclear proliferation in stretch induced growth resulted as a secondary response to the primary affect of enhanced translation. Laurent et al. (1978) stretched the anterior latissimus dorsi muscle Of chickens with weights and induced muscle hypertrOphy. These enhanced growth responses to stretch appear to be 39 mediated through the muscle cell and are not a consequence of neural effects. Goldspink (1980b) reported that the presence of innervation is not necessary for stretch inducement of sarcomere numbers and enhanced protein synthesis rates. When the EDL was denervated and compared to innervated controls and denervated-immobilized with and without stretch, the denervated and denervatedéimmobilized with stretch had respectively, 32 and 75% higher synthesis rates and 97 and 124% higher degradation rates relative to innervated controls. In preliminary studies, Goldspink (1980a) implanted stainless steel electrodes into the soleus muscle at the time of immobilization with plaster casts. Muscles were stimulated electrically for a few hours a day. Electrical stimulation caused sarcomere loss and prevented addition of sarcomeres when muscles were in an immobilized-shortened position. When muscles were in a lengthened but immobilized position, new sarcomeres were added at the ends Of the muscles. Endocrine Factors Influencing Muscle Growth. Kochakian et a1. (1956) and Kochakian and Tillotson (1957) investigated the effect, of castration upon 48 different muscles of guinea pigs and the effect of twelve androgens upon reversing the slowed growth due to castration. Castration resulted in a 10% reduction in muscle weights relative to noncastrates. Injection of 5.6 mg of 40 testosterone propionate restored muscles of castrates to that of noncastrates. Skeletal muscles of male mice are longer and have larger fiber diameters than females (Rowe and Goldspink, 1969) and the greatest rate of increase in fiber diameter is observed in males. ‘ Grigsby et a1. (1976) found greater incorporation rates of 3H-leucine into myofibrillar protein fractions when intact male rabbits were treated with testosterone implants. Powers and Florini (1975) added teStosterone .to L-6 myoblast cultures and showed direct effects on proliferative activity as indicated by a 25% increased labeling by tritiated thymidine and a decreased G1 time. These effects were not observed with dihydrotestosterone. Allen et al. (1983) measured. a=-actin accumulation in muscle cell culture in response to testosterone or growth hormone addition to the medium. Neither testosterone nor growth hormone was effective in selectively stimulating accumulation of this muscle specific protein. Kohama and Ozawa (1978) injected 5 mg of testosterone prOpionate into chickens three' times a week for 6 wk and measured the effects of trOphic factors generated in plasma on muscle cell cultures. These investigators found a significant increase in trOphic activity from testosterone injected chickens but minimal changes in myoblast multiplication 41 were Observed when triiodothyronine, testosterone or estradiol was added to cultures directly. These trOphic factors found in chicken serum have been shown to be transferrin, a necessary component in serum-free culture media (Kimura et al., 1981). Santidrian et a1. (1982) studied the effects of testosterone upon myofibrillar protein breakdown in rats as assessed by N,I -methylhistidine excretion. Castrated or castrated-adrenalectomized rats were injected daily with .2 or 2 mg of testosterone propionate per 100 g body weight, or .8 or 10 mg/100 g body weight of corticosterone. Normal intact rats grew 24% faster than castrated controls which grew at similar rates Observed for the testosterone treated rats. The sum of the gastrocnemius, tibialis, EDL and soleus did not differ among treatments. However, castrates excreted 23% less total NrI -methylhistidine relative to intact rats, and castrated-adrenalectomized rats given .2 mg testosterone/100 g body weight excreted 26% less total NT -methylhistidine. In these studies, adrenalectomy of castrated rats resulted in depressed growth rates which was slightly reversed by testosterone treatment. Large doses Of testosterone given to adrenalectomized-castrated rats that also received corticosterone did not prevent depression of growth or increase NT -methy1histidine output. Interpretatibn of these data is difficult but it appeared that other factors secreted by the testes may be 42 more closely associated with NT-methylhistidine excretion. Additionally, there appears to be an interaction between testosterone and products of the adrenal gland which altered the responsiveness to the testosterone treatment. Vernon and Buttery (1976, 1978) observed decreased NT -methylhistidine excretion in rats as well as decreased glucocorticoid output by adrenal slices incubated in vitro after animals were treated with trenbolone acetate. Lobley et al. (1983) concluded that the mechanism of action between testosterone and trenbolone acetate may be different. Rogozkin (1979) found a 16% increased 14C-leucine incorporation into myosin and a 16% increased DNA dependent RNA polymerase activity in gastrocnemius muscles with methandrostenolone administration to rats. Florini (1970a) Observed a 50 to 70% enhanced uptake Of labeled amino acids into protein and 60% greater mRNA synthesis after intraperitoneal injection of .1 mg of testosterone prOpionate. It was concluded that no new types Of proteins were synthesized and the effect of testosterone was merely to increase availability of existing DNA templates and enhance translational events. In light of the design of that study (Florini, 1970a) and the elevated concentration of circulating insulin accompanying testosterone treatment Observed by Grigsby et al. (1976), the direct effects of testosterone upon protein synthetic events is still questioned. 43 It has not been definitely shown that any anabolic effects of testosterone upon muscle are mediated by direct action upon muscle via binding with intracellular receptors (Michel and Baulieu, 1980). In androgen sensitive reproductive tissues, testosterone enters the. cell passively or is aided by an active mechanism. Upon entry testosterone is reduced by 5-« reductases to dihydrotestosterone which binds to a cytosolic receptor which is activated and translocated to the cell nucleus. In the nucleus, the testosterone activated receptor complex associates with acceptor sites on the chromation enhancing transcription and ultimately synthesis of protein (Vermeulen, 1982). However, other data indicate a different mode Of action in muscle. There is no strong evidence for the reductase enzymes in muscle (Krieg et al., 1974) nor for the prerequisite transformation of testosterone to dihydrotestosterone (Krieg and Voigt, 1977). Krieg (1976) indicated binding sites of dihydrotestosterone in skeletal muscle were 60 and 7 times lower than in the prostrate or bulbocavernous-levator ani, respectively. Mayer and Rosen (1975, 1977) theorized that the response of muscle to testosterone is due to a blockade of the glucocorticoid pleiotypic catabolic effects and is mediated through competitive inhibition and diSplacement of glucocorticoids from receptors (Parra and Reddy, 1962; Mayer and Rosen, 1975). The counteractive effects of 44 glucocorticoids and androgenic steroids such as testosterone lends support to the above theory and would be biologically. compatible. Clucocorticoids elicited responses of decreased amino acid uptake (Kostyo, 1965), reduced incorporation of labeled amino acids into skeletal muscle protein (Hanoune et al., 1972), decreased RNA (Peters et al., 1970) and DNA (Goldberg and Goldspink, 1975) synthesis and greater protein degradation (Goldberg, 1969). Contrary to the ‘ catabolic effects of glucocorticoids, testosterone has been shown to stimulate both increased amino acid uptake and incorporation of labeled amino acids into skeletal muscle proteins (Arvill, 1967; Breuer and Florini, 1965; Florini, 1970b; Rogozkin, 1975), increased messenger and ribosomal RNA_ synthesis (Kochakian et al., 1964; Rogozkin, 1975) and decreased amino acid and protein degradation (Bullock et al., 1969; Young, 1970). Mainwaring (1979) suggested that testosterone may elicit early translational responses as well as time dependent transcriptional responses; however, evidence for nuclear binding has not been indicated. Bicikova et a1. '(1977) showed that there was rapid transport of radioligands into the nucleus and was. dependent‘ upon cytosolic binding. Dionne et a1. (1979) demonstrated- cytosolic binding but low nuclear binding of testosterone in rat skeletal muscle. These authors also concluded that 45 dihydrotestosterone was metabolized so rapidly that it would not be a regulatory homone in skeletal muscle. Max (1983) found high affinity (.6nM=KD) low capacity (3 fmol/mg protein=Bmax) androgen receptors in mouse quadriceps, tibialis, plantaris, soleus, EDL and gastrocnemius muscles. Receptor characteristics were not different between dystrophic and normal mice. Snochowski et al. (1981) provided evidence for testosterone receptors in porcine skeletal muscle. In addition, the receptor complex had dissociation constants twenty times lower than dexamethasone complexes. These data indicated the presence of two separate receptors for androgens and glucocorticoids which is in contrast to the competitive binding theory of Mayer and Rosen (1975) and data of competitive binding between the two hormones provided by Viru and Korge (1979). Michel and Baulieu (1980) found that testosterone and glucocorticoids bind to separate receptors and that the androgen receptor has characteristics much the same as found in other target tissues. Estradiol also bound (Kd=.2 nM) the androgen receptor but diethylstilbestrol did not. Dionne et a1. (1979) found specific estrogen binding receptors in rat thigh muscle and reported the presence of nuclear estradiol binding proteins in the levator ani muscle. The effects 'of other endocrine factors, nutrients and ' growth factors upon muscle growth have been studied. Selected actions and interactions are given in table 2. 46 Amanv cHHnoez HHmmHV .He we conEeo AmmmHv .He we ceHHc AommHv HcHonm a come nomch .Hm we dweneHoo AeemHv mcwasz Ammch owwmox e nommm AmmmHv mHHm .cOHwewooe cHewowm peocence mmHm 0w :0 mo conewmecHEce O>H> cH .wceEmoHe>ep eHomcE HeEwoc pen mmHm Hewem pewemeeer .coneweMHHowm wo conechewemeo .nwzowm co woewme oc neweOHch mewccho HHeo OHcemo>E ow conHpcm .conepewmep cHewowm co woemme oc_ men wcn mHmenwcxm cHewowm peocpew wow eHnHmcommew mH mm mo xoeq .eHo p mH ow e wcone ewes ewew wewme >Hco mHmenwcwm cHewowm cce wwommceww ch ceweHcEme chHwencocH EmewnceHc owwH> cH cH cechocH mu .conewooe czo eHomcE ceweHcEHwe ewew xommn Ow conewmecHEee m0 .conoccowm cheEoweEoe mcHweHcEme ecoEwon OHnmoww e mcHen mo wcomccewm enw Eowm >Hco wcewwomEH en he: MZOZmOm 3930mm .mzo owcH cOHwewomwoocH ecHeHexnw wo conecheweMMHp HHeo OHcemo>E .mewew conepewmep cce mHmenwcwm cHewowm .wwommceww pHoe ocHEe .nw3owm eHomcE ewe newceeeE memcommem .pemc >checveww ceen e>en mewccho HHeo OHcemo>E cce chHw IencocH eHomcE owwH> cH .meHccwm ocHen wowoem ecHwoocce mo eowcom awowewoem mo coneHne wewme wo mHeEHce HeEwoc ow conewmecHEUe o>H> cH aHHeOmes .O>H> cH pce OwwH> cH cHewowm mo cOHwewooe pce ewccho cH mHHeo wochemowQ mo cOHwewemHHowm me nocm memcommew eHomcE cewoeHem hpcwm ow new: ewe menoeowmme mo mweHwe> < mmmvdommmd Aco a cOEHem HommHv cHem AmmmHv HcHonm HommHv mccheh a mecoHIwOmmwc AmmmHv .He we wewHEHem .mHmenwcam cHewowm peweHcEme mwew an ow econwmnw mo conewmecHEO¢ .conewoxe ecHweHn IHwnweEI z cemeewoep pce nwzowm eHomcE peocpew weowoepHowwne mzozmom QHOfiMmB .coneermeU cHewowm ceocpew pce mHmenwcam cHewowm peocence ow meweHewwoo xwoz owwH> cH Uce O>H> cH wmoz .Eewmmm wwommceww ed: pHoe ocHEe cemeewocw ZHADmZH .Aconcmv conecheweHMHp Uce conewemHHowm me HHez me pe>wemno ewe mwoewwe UHHonece Hewecem .mewccho OHcemo>E ow pence cenz .eHomcE EewnmeHe neweHomH mo mcHewowm owcH HeneH e>HwoeoHpew mo conewomwoocH cemeewocH ZHQMEOEen wee mm .mHmenwchm czo pce nw3owm eHomcE cemeewocH pen mwoecw mcHwewoem mm an3 ewem .eNHm ece nwzowm no mcHanoe cH cechmew eOHE owcH mecem mu ecOEwon mo conwemcH eeccchOUIecOEwom nw3owo N names I m mmdm 48 ANmch meHuwsz AmmmHv HcHwOHm comch meaneHoo AommHv cHem HommHv .He we e3emeEcM AHmch mwmneHow a ocwamz .o HommHv .He we wexez comch .Hm we mwmneHoo .m .mwoewme OHHonece e>en mecHEeHonoeweo .cone>meeU HecOEwon wo mHmenwcam cHewowm pemmewmec mo mewewm cH.wenw cewmemmcm .mHmenwcwm cHewowm ece wwommceww eHoe ocHEe peweHcEme ZnIOH we ecHwnmeche .mwew OHweneHc mo EmewnmeHU an3 mwceEHwemxe owwH> cH mmZHZ¢AomUNB¢U .OHHoneweo ewe menw memoc ann we .mwoemwe wceccemee emoa .mEewmwm ewccho HHeo wow nechqem .mewew conepewmep weweewm e>en mHeEHce peweeww UHOOwaooooch Eowm meHomcE mo chHwencocH owwH> cH mQHOUHBmOUOUDAU .conewomwoocH HeneH e>Hwoe IoHpew cemeewoep e>en comeoch an3 newencocH meHomcE neweHOmH .eHomcE mo mwoemwe mo weeHocc .cHeEem ZOOdUqu 9 mo conHcpe >n.peweHcEme ewez conecheweHMHp HHeo OHcemomz .OHHoneweo ewe memoc ann pce OHHonece ewe memop 30H .nwzowm HeEwoc wOm wwemmeoec mH m6 .emoc ann enw ce>Hm mHeEHce cH weann mom ewe3 mewew cOHwepewmeO wcn mewew mHmenwcam cHewowm neweHcEme econwhnw m: N we a: com weane me>Hm mHeEHce xomam Eowm meHomcE mo chHwencocH owwH> cH ceccchOU I ecOEwom pHowhnB m memes n m emem 49 Ammch .Hd we :stm Ammch mwmneHoe a cceEepom .eHomcE eenowewwmcc cH woc wcn eHomcE penowewwm mecewwHEwewcH cH mewew mHmenwcwm cemeewoec cHoe OHEecemoHer @ce cHoenweEOUcH .eHmenwcam cHewowm cH emeewocH mmm e penceo «mom woc wcn mom .mconencocH enw cH pechocH ewe3 mmwm pce cHoe OHcoeHnoewe cen3 wow Ow om meeeewocH owwH> cH mewew cOHwecewmep cHewowm ZHDZflAQflBmOMA m mummy u 4 meme 50 Skeletal Muscle Protein Turnover During Growth. Skele- tal muscle is comprised largely of proteins and associated water. Total muscle mass represents approximately 60% of the carcass weight and as much as 45% of the live weight of growing pigs (Mulvaney, 1981). The rate of skeletal muscle growth is directly related to the rate of protein deposition. If the rate of skeletal muscle protein deposition could be efficiently enhanced during growth, it follows that there would be increases in weight gain and in the efficiency of lean production. This is * not an unrealistic possibility since only 20 to 30% Of ~the skeletal muscle protein that is synthesized per day is deposited in skeletal muscle of young growing boars' (Mulvaney, 1981). The reason for these low values is that proteins in skeletal muscle and most other tissue proteins are continually being synthesized and degraded, a process called protein turnover. Muscle protein deposition or growth rate is determined by the difference between protein synthesis and degradation rate. In order to achieve greater net synthesis or deposition of muscle protein, three basic avenues theoretically exist: (1) increase the- rate of protein synthesis while keeping degradation the 'same or even lowering degradation, (2) keep synthesis rate normal but reduce degradation rates and (3) reduce both synthesis and degradation rates while reducing degradation rates more to expand the margin of difference between‘ the two processes. 51 In order to simplify calculation and interpretation of protein turnover data, the rates of protein synthesis and degradation can be expressed as fractional rates (Swick, 1982). This refers to the percentage or fraction of the total protein in a muscle that is replaced per day (Garlick, 1980).. Figure 4 is an idealized depiction of how the fractional protein psynthesis rate (FSR, %/d) and fractional protein degradatiOn rate (FDR, %/d) undergo develOpmental changes. The hatched portion of the figure is the fractional growth rate (FGR, %/d). The FGR is the difference between FSR and FBR and in the adult essentially becomes zero.‘ The develOpmental decrease in total muscle turnover is due to a combination of the fall in fractional synthesis and degradation rate and the increased amount of muscle protein during growth (Millward, 1980). Quadricep muscles of 3 wk old rats had FSR over 20%/d .and by 1 yr values were less than 5%/d (Millward et al., 1975). In lambs, the FSR of over 24%/d was observed at 1 wk which fell to 2%/d at 16 wk (Arnal et al., 1976). Mulvaney (1981) observed a develOpmental decline in muscle FSR in pigs at 22 to 45 kg but the extent was dependent upon the individual muscle. The work of Maruyama et al. (1978) illustrated a skeletal muscle FSR decrease from 25 to 8%/d in 1 and 2 wk chicks, respectively. It appears that develOpmental changes in protein turnover occur in all muscle types. The pattern of change 52 RATE, %/day in... I IAGE '1 .YOUNG ADULT FIGURE 4. Developmental changes in fractional protein synthesis and degradation rates. 53 varies between muscles such that the hierarcy of relative protein turnover rates change with age. White and red muscles deve10p such that turnover was highest in white muscle of very young animals but the reverse was true in adults (Arnal et al., 1976; Maruyama et al., 1978). In adult rats, turnover rates of 10 to 12%/d, 8 to 10%/d and 4 to 5%/d have been observed for heart, soleus (red muscle) and quadriceps (white muscle), respectively. Laurent et al. (1978) estimated protein turnover rates in chick anterior latissimus dorsi (slow tonic) to be 3 times higher than in the posterior latissimus dorsi (fast twitch) and 5 times higher than the breast muscle. Even after 1 the fully grown anterior latissimus muscle was doubled in size through stretch induced hypertrOphy for 2 mo the same turnover rate was found but during the early phases of stretch, increases in synthesis and degradation rates were observed (Laurent et al., 1978). Two contentious questions about protein turnover remains: (1) does rapid muscle protein turnover accompany rapid muscle growth? and (2) what determines the rates of protein synthesis and degradation in muscle? As mentioned in the previous section, muscle grows through hypertrOphy of the myofibers and acquisition Of new nuclei from satellite cells. This new nuclear material enables the muscle cell to expand its protein to DNA ratio by as much as 300 to 400% (Millward, 1980). Oxidative 54 muscles were reported to have the lowest DNA unit size or protein to DNA ratio (Millward, 1980). During rapid compensatory muscle growth after nutritional insult in rats (Millward et al., 1975) and stretch induced hypertrOphy (Laurent et al., 1978) the RNA to DNA ratio doubled. Millward et al. (1978) found increased protein degradation rates when growth rates were increased as well as decreased degradation when growth was reduced in rats. Maruyama et a1. (1978) indicated rates of protein degradation to decrease to facilitate growth in young chick muscle. Millward et a1. (1981) compared muscle protein degradation rate to cathepsin D activity during normal growth, growth suppression and catch up growth. Degradation rates were higher during rapid growth and decreased shortly after nutritional deprivation. Cathepsin D activities responded similarly. It is uncertain whether normal growth in muscles occurs by the same mechanisms as induced hypertrOphy. Adipose Tissue DevelOpment Cellularity. DeveIOpment of adipose tissue is accomplished by combinations of cellular hyperplasia and hypertrOphy (the imbalance between synthesis and degradation of lipid) and factors which influence ultimate body fat mediate their effects through one or both of these 55 processes (Anderson, 1972; Leat and Cox, 1980). The contribution of progenitor cell proliferation to fattening during growth and Obesity has been a controversial tOpic (Kirtland and Gurr, 1979). Bergen (1974) illustrated the relationship of protein and (fat accretion during growth. In an idealized portrayal, as the rate of protein deposition decelerates, fat accretion rates are accelerating and at greater rates than protein. The plateau in protein accretion rates occurs because muscle cell numbers are fixed at birth (Staun, 1963) and the maximum muscle cell size is attained during this plateau (Allen, 1976). If adipocyte numbers are also fixed at birth or shortly after and there is a maximum cell size, then fat accretion rates also shOuld plateau. The fact it does not plateau indicates that suprpulations of progenitor cells are reserved for later differentiation and/or lipid filling, or hyperplasia can be induced at some time during growth, or that there is no olimitation on the extent of adipocyte cell hypertrOphy (Allen, 1976). However, Anderson et a1. (1972) and Johnson (1978) indicated adipocytes had maximum cell sizes. Proliferating preadipocytes have not been identified as a separate and distinguishable cell type (Hausman et al., 1980). DevelOpment Of adipose tissue. embryonically and identification of precursor adipocyte cells is particularly enigmatic because adipocytes are nOt morphologically 56 distinguishable until lipid. accumulation occurs (Johnson, 1978). The techniques used for estimating adipose tissue cellularity were reviewed by Gurr and Kirtland (1978). Basically, methods were categorized as those that involve some direct measurements of fat and those that measure DNA content. A pOpular method for estimating adipocyte cell size and numbers involves fixation of cells with osmium tetroxide, eventual separation through specified mesh size screens, the counting of cells of varying sizes and integration of cell distribution (Hirsch and Gallian, 1968). Major disadvantages of this method are that cells less than 20 to 25 um in diameter (which would include precursor cells). are not measured and cells swell with osmium tetroxide which overestimates size (Gurr and Kirtland, 1978). Therefore, in situations of increased adipose cell numbers, it is uncertain whether it is due to lipid filling of already differentiated cells or due to newly differentiated cells derived from a pool of dividing precursor cells (Hausman et al., 1980). Another method described by Gurr and.Kirtland (1978) is based upon incorporation of 3H-thymidine by proliferating progenitor cells and cells of the stromal fractions. With time after injection of the label, the amount of radioactive DNA in the stromal fraction decreases while that in the nonproliferating fraction (mature fat 57 cells) increases. Changes in specific activities in the lipid filled fat cell fraction should be a result of newly derived cells. Another method with potential for identifying precursors of adipocytes hinges upon the demonstration of lipOprotein lipase and monoacylglycerol acyltransferase activity in the stromal vascular fraction of develOping adipose tissue (Hietanen and Greenwood, 1977; Dodds et al., 1976). Identification of “precursor cell types via fluorescent antibody techniques could potentially be used (Hausman et al., 1980). Based upon the size sieving technique described above, adipose tissue is thought to undergo an early period of hyperplasia followed by a longer and overriding period of lipid filling and cell hypertrOphy (Anderson, 1972). However, Johnson (1978) indicated additional hyperplasia occurs during postnatal growth after a critical cell size is reached and this hyperplasia results from recruitment of precursor cells. At this time, it appears the osmium tetroxide method of estimating increased cellularity. by itself offers little understanding or verification about precursor adipose cell proliferation. Klyde and Hirsch (1979a, 1979b) injected 3H-thymidine intraperitoneally .into rats and based on labeling of the stromal and adipocyte fractions, suggested the presence of a proliferating cell type which was an adipocyte precursor. Kirtland and Gurr (1980) used 58 3H-thymidine to assess proliferation in pigs. Rate of proliferation was rapid between 2 and 40 days of age but by d 40 lipid filling was beginning to predominate. Greenwood and Hirsch (1974) attempted to distinguish between lipid filling and new cell synthesis via osmium fixation and incorporation of 3H-thymidine into DNA. In rat epididymal fat pads, most cell proliferation was completed by 5 wk of age and the observed increases in cell numbers at 12 wk were due to lipid filling. Kirtland and Gurr (1979) suggested that in situations of increased cell number the terminology 'increase in observable fat cell numbers' should be used as Opposed to hyperplasia or replication of new fat cells. - Hausman et a1. (1980) reviewed white adipose 'tissue differentitation and develOpment and indicated blood vessels and adipocytes form simultaneously. Through active proliferation, mesenchymal cells result in formation of clusters of spherical baSOphillic cells (blood islands) connected to each other by strands of elongated cells. The peripheral cells of the blood islands and the strands ultimately form the primitive blood vessels. Preadipocytes could potentially be derived from these mesechymal cells of the blood island as identifiable adipocytes first appear in the perivascular regions (Hausman et al., 1980). Potential adipocyte precursor cells were identified as endothelial, perivascular reticulum, macrOphagic fibroblastic and perivascular mesenchymal cells (Hausman et al., 1980). 59 Napolitano (1963) described the characteristics of stem cells of the epididymal fat pad of rats. These cells were spindle shaped (fibroblast like) with four to five protOplasmic extensions and cells adjacent to capillaries changed to a nearly spherical shapes or like the mature adipocytes. Slavin (1979) indicated _ similarities Of presumptive adipocytes and fibroblasts were more coincidence rather than fibroblasts developing into adipocytes. If the cell diversification and differentiation scheme of Holtzer and Holtzer (1976) is eXpanded for all cell types, then it would seem fully differentiated fibroblasts could not serve as presumptive adipoblasts unless fibroblasts were capable Of undergoing a quantal mitosis. Leat and Cox (1980) illustrated the remarkable capacity of the adipocyte to store lipid. Since adipocytes have similarities to a sphere (Curr and Kirtland, 1978) and the volume of a sphere is prOportional to the cube of the 'radius, a 2- and 10-fold increase in diameter results in an 8- and 1000-fold increase in volume. A cell with a diameter of 20 um contains about .004 ug lipid, a 100 um cell contains .45 ug lipid and a 200 um cell could contain 3.8 ug of lipid (Leat and Cox, 1980). The factors affecting the observable fat cell numbers (OFCN) and size of OFCN during postnatal growth include Species, strain, age, depot, nutrition and endocrine factors. 60 Differences in postnatal adipocyte proliferation between species appear to be dependent upon the relative degree of maturity of a given depot of the species at birth. For example, rat subcutaneous adipose tissue is less completely develOped than in the guinea pig and the epididymal depot is much more develOped at birth in the guinea pig at birth relative to the rat (Kirtland and Curr, 1979). The order of maturity of the depots in the meat producing porcine, ovine and bovine species is from earliest to latest develOping is: perirenal, subcutaneous, intermuscular and intramuscular (Allen et al., 1976). Imbalance Between Triglyceride Uptake, Synthesis and MObilization. The rate of fat deposition in adipose tissue hinges upon the following metabolic processes: uptake of circulating fatty acids, fatty acid synthesis and esterification, lipolysis and mobilization of fatty acids' and fatty acid oxidation (Allen et al., 1976). Whether an imbalance favors net storage or net mobilization depends upon multiple interactions of hormones with the adipocyte (Saggerson, 1980). Uptake of Circulating Fatty Acids. LipOprotein lipase (LPL) is an enzyme (or closely related enzymes) responsible for extraction of fatty acids from circulating lipOproteins (Allen et al., 1976; Saggerson, 1980; Cryer, 1981; Quinn et al., 1982). LPL functions as an extracellular lipase by“ 61 catalyzing the hydrolysis Of ester bonds of triacylglycerols releasing nOnesterified fatty acids from the lipOprotein carriers, . These fatty acids enter cells leaving a lipOprotein remnant (Quinn et al., 1982). It is currently believed that LPL is secreted by adipocytes and functions at the luminal surfaces Of capillary endothelial cells (Saggerson, 1980). LPL is inhibited by high salt concentrations, activated by the serum factor apolip0protein C-II, heparin and has an alkaline pH (8.0 to 8.5) Optimum (Quinn et al., 1982). Kompiang et al. (1976) prepared antibodies to purified chicken adipose tissue LPL and after administration of the antibody, blocked LPL hydrolysis of chylomicra and very low density lipOprotein _triglycerides which elevated circulating triglyceride concentrations. In addition to playing a role of triglyceride uptake, LPL appears to direct the uptake of fatty acids to adipose or muscle. LPL activity decreased in adipose tissue and increased in muscle during fasting but refeeding stimulated preferential uptake of triglyceride by adipose tissue (Robinson and Wing, 1970; Nilsson-Ehle, 1981). Lee and Kauffman (1974) measured subcutaneous adipose tissue LPL activities in Duroc and Hampshire pigs during growth from birth to 8 mo of age. On a wet tissue basis, Durocs had 25% higher LPL activities over the 8 mo period than Hampshire pigs. In another study of crossbred pigs, 62 LPL activity expressed on a per gram of tissue basis increased 4-fold from birth to 2 wk of age, remained elevated to 70 d and declined between 70 and 150 d of age (Steffen et al., 1978). Lee and Kauffman (1974) observed a similar 2- to 3-fold increase in LPL ‘activity in pigs between 2 and 24 wk of age. In another study, 'obese Minnesota No. 1 pigs had 4—fold higher subcutaneous LPL activity than lean Hampshire pigs (Weisenburg, 1973). MCNamara and Martin (1982) compared LPL activities in fetal and neonatal pigs from lean (Yorkshire) and obese (Ossabaw) pig lines. Even though fetal body composition was not different, obese fetuses had higher adipose tissue LPL activities than lean fetuses. Etherton and Allen (1980) investigated the importance of plasma derived fatty acids relative to fatty acid synthesis in swine adipose tissue. In this latter study, fatty acid synthesis from glucose and esterification of palmitate in adipose tissue was Observed in 57, 94 and 124 kg pigs. The ratio of palmitate esterified to palmitate that was synthesized from glucose de novo in adipose tissue increased 4- to 5-fold in pigs weighing 57 to 124 kg. LPL is an adaptive enzyme (Allen et al., 1976; Cryer et al., 1981). Haugeback et al. (1974) showed that LPL activity was low or nondetectable in subcutaneous, perirenal and intermuscular adipose tissue from lambs fed a maintenance diet. After switching to an ad libitum diet, 63 LPL activity increased and was highly correlated with increased fat deposition. Activity expressed as nmoles of free fatty acids released per 106 adipocytes was 3-fold higher in subcutaneous compared to perirenal adipose tissue. Di Marco et al. (1981) measured a 37% decrease in LPL activity after 10 d of fasting of Holstein steers as well as 100% (of prefasting levels) overshoot upon refeeding. Fasting has been shown to significantly depress adipose tissue LPL activity in pigs (Enser, 1973; Weisen- burg, 1973; Steffen et al., 1981). Hormonal Control of LipOprotein Lipase. One theory concerning LPL activation revolves around the interaction of insulin and lipolytic agents. Insulin stimulates glucose metabolism which accentuates glycosylation and activiation of lipOprotein lipase. Inhibition of LPL activity by fatty acids and lipolytic agents may involve the interference of the formation of precursors needed for glycosylation by diversion to glyceride-glycerol formation (Fain, 1982). ' Insulin has been shown to be intimately involved and positively correlated with the maintenance of -adipose tissue LPL activity (Cryer et al., 1976; Garfinkel et al., 1976; Saggerson, 1980). In adipose tissue Of alloxan (Robinson and Wing, 1970) and streptozotocin (Ishikawa et al., 1982) induced diabetic rats, LPL activity is lowered. 64 Others have reported low correlations of LPL activity and insulin when data were eXpressed on a gram of tissue basis (Kessler, 1963; Redgrave and Snibson, 1977). Saggerson (1980) indicated that insulin plays a primary role in the secretion of an active extracellular form of lipOprotein lipase. Reichl (1972) reported parallel changes in adipose tissue LPL activity with diurnal changes in plasma insulin in meal fed rats. LPL activity in adipose tissue of starved rats also increased during in vitro incubations with insulin and glucose (Salaman and Robinson, 1966; Wing et al., 1966). Ashby and Robinson (1980) reported that glucocorticoids increased the insulin-dependent LPL activity as well as caused subtle independent increases in LPL activity. High concentrations of circulating cortisol are associated with excessive fat deposition in Cushing's syndrome (Rudman and DiGirolamo, 1971). Obesity may involve some increased sensitivity to glucocorticoids which also facilitate insulin stimulation of fatty acid synthesis. Robinson et al. (1983) reported that the synthesis of LPL is enhanced by insulin but further enhanced in the presence of insulin and dexamethasone. It was speculated that dexamethasone stimulated transcription of mRNA and the effects of insulin was a nonspecific translational effect. Insulin stimulated increases in LPL activity are inhibited or prevented by catecholamines (Robinson et al., 65 1983) adrenocorticotrOphic hormone (Davies et al., 1974), glucagon, thyroid stimulating hormone (Nestel and Austin, 1969; Robinson and Wing, 1970), dibutyryl cyclic AMP, caffeine and methylxanthines (Robinson and Wing, 1970; Ashby et al., 1978). The aforementioned agents may inhibit or inactivate LPL activity simply through activation of intracellular lipolysis which causes an increase in intracellular fatty acids. Wilson et al. (1976) measured LPL activity in adipose tissue from 120 to 180 g in male, castrated male and female rats given estradiol cypionate or testosterone enthanate injections. Male rats given weekly doses (for 3 wk) of 5 ug or 500 ug/doses of estradiol had 74 and 91% decreases in LPL activity, respectively. However, weight gain was also depressed by 65% in treated animals indicative of reduced feed consumption (not reported). Female rats given weekly 20 mg doses of testosterone enthanate (for 3 wk) had 12% lower adipose tissue LPL activity. Intact males had 20% lower LPL ~activity relative to castrated controls and the castrated controls had 7% higher activity than male castrates given weekly injections of 500 ug of testosterone enthanate. These investigators viewed the responSe to estrogen to be similar to starvation even though insulin concentrations were unchanged from controls. Hamosh and Hamosh (1975) observed similar responses of decreased LPL activity with administration of 17 B-estradiol to castrated 66 male and female rats. Wade and Gray (1979) and Steingrimsdottir et al. (1981) prOposed that gonadal hormone treatment caused changes in both feed intake (sex and species dependent) and body weight which were possibly due to direct effects on adipose tissue. In testing this hypothesis, Ramirez (1981) gave estradiol to rats and concluded that the estradiol induced decrease in LPL activity occurred prior to depression in food intake. Gray and Greenwood (1982) investigated the changes in food intake and adipose tissue metabolism at 1, 2, 3, 7 and 14 d after estradiol benzoate was administered to ovariectomized rats. After estradiol treatment (2 ug/d), LPL activity decreased by 30% the first day and a 50 to 70% suppression was maintained for the follOwing 14 d. When progesterone (2 mg/d) was given along with estradiol, the responses were approximately 50% of those observed for estradiol alone. Burch et a1. (1982) implanted 33 kg sheep with Revalor (17.5 ‘mg trenbolone acetate and 2.5 mg 17 B-estradiol). After 60 days, adipose tissue LPL activity only tended to be lower in implanted relative to nonimplanted sheep, whereas fatty acid synthesis rates were 50% lower. Prior et al. (1983) implanted bulls and steers with estradiol diprOpionate. While acetyl coenzyme A carboxylase, ATP citrate lyase, NADP-malate dehydrogenase, aconitate hydratase and NADP-isocitrate dehydrogenase activities were generally half those Observed in steer subcutaneous adipose 67 tissue, estradiol increased lipogenic activities in both bulls and steers. Lipogenesis. Lipogenesis has been defined as the synthesis of fatty acids from glucose and their esterification to form triglycerides (Allen et al., 1976). Regulation of the metabolic pathways involved in lipogenesis centers around rate limiting steps. Control of metabolic pathways is exerted through (1) modulation of catalytic activities of rate limiting .enzymes via allosteric effectors, covalent modification or changes in the synthesis and/or degradation of enzymes and (2) control of rate of passage through cell membranes,' changes in availability of precursors from other tissues and flux of substrates and effectors through metabolic pathways (Newsholme and Start, 1973). A variety of enzymes related to lipogenic activity have been studied. Diffusion of glucose into adipose tissue and subsequent phosphorylation by hexokinase is a key step in lipogenesis (Avruch et al., 1972; Romsos and Leveille, 1974). Glucose is the primary precursor of fatty acids in pig adipose tissue (O'Hea and Leveille, 1968; Martin and Herbein, 1976). Adipose tissue hexokinase activity appears to be adaptive and responds in parallel with changes of plasma glucose and insulin (Romsos and Leveille, 1974). Since fatty “acids are synthesized in the .cytosolic compartment, the production of cytosolic NADPH by reactions 68 catalyzed by glucose-6—phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, NADP-isocitrate dehydrogenase and malic enzyme become important considerations (Allen et al., 1976). In addition, phosphofructose kinase, aldolase, pyruvate kinase, pyruvate dehydrogenase, acetyl CoA carboxylase and fatty acid synthetase are involved in the conversion of glucose to fatty acids (Romsos and Leveille, 1974). Glucose 6-phOSphate dehydrogenase catalyzes the first step in the pentose pathway and activity of this pathway is closely associated with fatty acid synthesis (Kather et al., 1972). The reason for this high correlation is that 50% of the NADPH needed 'for adipose tissue fatty acid synthesis is derived from the pentose-phosphate pathway (O'Hea and Leveille, 1968; Kather et al., 1972). As summarized by Allen et al. (1976), breeds of swine or cattle with low thresholds to fattening and with high lipogenic activities had Ogreater NADPH generating capabilities. As animals grow, this NADPH generating capability increases. The glycolytic enzymes phosphofructose kinase, aldolase and pyruvate kinase are implicated in the regulation of fatty. acid synthesis in adipose tissue (Romsos and Leveille, 1974; Saggerson, 1980). Changes in activity of the pyruvate dehydrogenase enzyme complex are correlated with changes in adipose tissue fatty acid synthesis 69 (Jungas, 1970). However, Allee et al. (1971a) observed malic enzyme activity in pig adipose tissue to increase up to 50 kg live weight but declined during growth afterwards. It was indicated that the activity of this enzyme merely responds to changes in fatty acid synthesis (Romsos et al., 1971). The first committed step in fatty acid synthesis is the formation of malonyl CoA via carboxylation of acetyl CoA and is catalyzed by acetyl CoA carboxylase (Numa et al., 1970; Lane et al., 1974). The second step is catalyzed by a multienzyme complex, fatty acid synthetase and involves the condensation of; acetyl CoA and malonyl CoA in the presence of NADPH to form palmitic acid (Mayes, 1976). The primary pathway of triglyceride synthesis in swine adipose tissue is the glycerol 3-phosphate pathway (Stokes et al., 1975). Glycerol 3-phosphate is sequentially acylated by acyl-CoA fatty acids and glycerOphosphate acyltransferase to result in phosphatidate. This is followed by hydrolysis of phosphatidate by phosphatidate phosphohydrolase to yield diglyceride. Another acylation of the diglyceride by the activated acyl-CoA fatty acids is catalyzed by diglyceride acyltransferase to yield triglycerides (Hems, 1975; Raju and Six, 1975; Stokes et al., 1975; Mayes, 1976; Fallon et al., 1977). Glycerol 3-phosphate can be generated from glycolysis or gluconeogenesis. Fatty acyl-CoA can be synthesized de novo 70 in adipose tissue, from plasma free fatty acids or triglyceride via lipOprotein lipase action or from lipolysis of triglyceride stores in the cell (Steffen et al., 1979). There havei been considerable data generated characterizing lipogenic activities in swine adipose tissue. Hood and Allen (1973) assayed malic enzyme (ME), glucose 6-phosphate dehydrogenase (G-6-PDH) and acetyl CoA carboxylase (CBX) in the middle and outer subcutaneous fat and in the perirenal depots of lean and obese pigs. When data were eXpressed per 105 cells, all the above enzyme activities were . higher in the perirenal depot than subcutaneous and were higher in the Obese line compared to the lean line. The magnitude of the differences varied with live weight. Steele and Frobish (1976) reported higher citrate cleavage enzyme (CCE), G-6-PDH and ME activities in obese Duroc pigs relative to a lean line. In that same study, pigs fed a diet containing 15% lard had suppressed enzyme activities. When meal feeding was _compared to ad libitum feeding no significant effects were observed; however, the lean line of pigs appeared to have slightly lowered enzyme activities. Mersmann et al. (1973a, 1973b) investigated lipogenic activities of neonatal and growing pigs. Around birth and until weaning lipogenic enzymes were generally low. The pentose phosphate dehydrogenase activities increased with 71 age and G-6-PDH doubled at d 60. Malic enzyme and glycerolphosphate dehydrogenase activities increased with age. Citrate cleavage enzyme (CCE), CBX and fatty acid synthetase activities were low before weaning but by d 60, had increased 10-fold. Similar trends were reported by Allee et al. (1971b). Anderson and Kauffman (1973) measured CBX, G-6-PDH, CCE in adipose tissue of growing 6 cells, the activities pigs and when expressed per 10 increased markedly at weaning and again at about 4 to 5 mo of age. Between 5 and 6.5 mo, enzyme activities decreased which indicated some subtle changes in lipolytic and oxidative activities may have occurred. Steele et a1. (1982) indicated' palmitate oxidation decreased with increasing age of pig adipose tissue. Anderson et .al. (1972) reported CBX, CCE, ME and G-6-PDH enzyme activities in the outer, middle and inner subcutaneous backfat layers, intermuscular, mesenteric, perirenal and hindleg subcutaneous adipose tissue. For all enzymes assayed, the subcutaneous adipose tissue of the lower leg was lowest and the perirenal was the highest in activity. In addition, lower enzyme activity was observed in the outer subcutaneous backfat layer relative to the middle layer. Scott et a1. (1981) investigated the effects of age, breed and line (lean versus obese) on lipogenic and lipolytic activities of female swine adipose tissue. At 3, 72 4, 5 or 6 mo of age, obese pigs had larger adipocytes than a lean line. In vitro lipogenic activity expressed as nanomoles of 14C glucose incorporated per 105 cells was 30 to 40% lower at 3, 4 or 5 mo of age but was 64% lower at 6 mo in the selected lean line. Highest lipogenic activity was observed within a_line at 4 mo of age. CBX activity expressed on a cell basis was also dramatically 5-, 8-, 3- and 6-fold lower in lean pigs compared to obese pigs at 3, 4, 5 and 6 mo of age. Allee et al. (1971a, 1972) fed 12 or 24% CP diets containing .1 or 13% corn Oil to pigs and observed a 60 to 50% depression in' vitro incorporation of 14C glucose into fatty acids (per 100 mg of tissue) due to feeding fat and 30 to 40% depression due to higher protein. However, in another trial, the protein effect on lipogenesis was not observed. Steffen et al. (1978) also fed 24 or 12.8% fat diets (corn oil) to young pigs for 3 to 4 wk and another group fed 3% fat were fasted 72 h prior to assaying for lipogenesis. Because of greater observable fat cell numbers in the low fat groups, 14C glucose incorporation rates measured in vitro were not different between fat fed groups when data were expressed on a cell basis. However, starvation depressed fatty acid synthesis rates by 85% relative to nonstarved controls regardless Of method of data eXpression. 73 Martin and Herbein (1976) measured several adipose tissue lipogenic enzymes in vitro in 6 mo old pair fed lean and obese pigs. Activities of G-6-PDH, ME, and CCE (expressed per 105 cells) were higher in the obese than lean pigs. Pyruvate kinase and fatty acid synthetase were similar for both groups. Romsos et al. (1971) showed that alloxan diabetic pigs had reduced lipogenic activity. Restoration of lipid biosynthesis was accomplished after in vivo insulin administration. Insulin added directly to an in vitro lipogenic assay caused minimal changes. These investigators demonstrated that changes in ME and CCE activities are a result of changes in fatty acid synthesis and were not the cause of changes. Kasser et al. (1983) measured rates of 14C palmitate and 3H water incorporation into. fatty acids in adipose tissue slices from intact and decapitated pig fetuses. Decapitation - stimulated lipid deposition and lipogenic activity (3-fold) but palmitate esterification only tended to be higher in the intact group. In vitro pancreatic insulin release was also higher in decapitated fetuses relative to intacts. Lipid MObilization. Degradation of triglycerides occurs through the action of hormone sensitive triglyceride lipase (HSL), the rate limiting step ‘in adipose tissue lipolysis (Khoo and Steinberg, 1975; Siddle and Hales, 74 1975). It appears that total triglyceride degradation can be accomplished since HSL includes di— and monoglyceride lipase activity (Khoo et al., 1976). Fatty acid mobilization has been estimated by measuring release of free fatty acids or glycerol from adipose tissue. Since adipose tissue does not have glycerokinase required for reutilization of glycerol, release of glycerol reflects lipolysis of triglyceride minus the amount of reesterification (Allen et al., 1976; Fain, 1982). Lipid mobilization has been reviewed by Renold and Cahill (1965), Jeanrenaud hand Hepp (1970), Scow and Chernick (1970), Bjorntorp and Ostman (1971), Fain (1973, 1977), Fain et a1. (1978), Jungas (1975), Steinberg (1976), Meisner and Carter (1977), Fredholm (1978) and Hales et al. (1978). Insulin appears to be the only peptide hormone which inhibits triglyceride ‘ breakdown. In addition, prostaglandins of the E series and adenosine inhibit lipolysis. Hormones which activate lipolysis include catecholamines, thyroid hormones, thyrotrOpin, growth hormone, glucocorticoids, glucagon and ACTH (Fain, 1982). Lipolysis in isolated swine adipocytes was stimulated by epinephrine and norepinephrine, adrenocorticotrOpin, dibutyryl cyclic AMP but not by glucagon or cyclic AMP (Mersmann et al., 1976). A current working hypothesis is that the above hormones and factors act through binding to 75 surface membrane receptors, stimulation of adenylate cyclase, activation Of cyclic AMP-dependent protein kinase which phosphorylates the lipase (Steinburg and Khoo, 1977). Catecholamines interact with Bl-adrenergic receptors of adipose tissue to aCtivate adenylate cyclase and to accelerate lipolysis. Binding to receptors inhibits O‘2 adenylate cyclase and receptors increases 0‘1 intracellular calcium for a net effect of lipolytic inhibition (Robinson et al., 1971). There are dramatic species differences in the sensitivity. of adipose tissue to lipolytic hormones (Rudman, 1963) and to presence or absence of an alpha-adrenergic. component suppressing lipolysis (Buens et al., 1981). While lipogenic activity of swine adipose, tissue was affected by amount and type of dietary fat (Allee et al., 1971a) and fasting (Mersmann et al., 1973a; Steffen et al., 1977), few studies of the effects of nutrition upon lipolytic activity have been conducted. Martin et al. (1974) reported that a low energy diet fed to neonatal pigs increased the epinephrine stimulated lipolytic response measured at 6 mo Of age. Mersmann et al. (1975a) showed that high fat diets fed to 1 mo Old pigs imparted greater in vitro lipolytic rates and greater sensitivity to epinephrine compared to low fat diets. Mersmann et al. (1975b) reported that weaning of pigs depressed the 76 lipolytic rate and sensitivity to epinephrine. Younger pigs were more sensitive to a 1 agonists than older pigs (Mersmann et al., 1976). Steffen et al. (1981) fed 1 mo old pigs isocaloric and isonitrogenous diets with either 12.8 or 2.4% fat. Adipose tissue lipolytic rates were higher in groups fed high fat but adenylate cyclase, phosphodiesterase and hormone sensitive lipase activities were unaffected. Fasting for 72 h elevated lipolysis and hormone sensitive lipase activity. Scott et al. (1981) found no differences in unstimulated lipolysis between lean and obese lines of pigs but the activity per 103 cells decreased from 3 to 6 mo of age. Mersmann et al. (1975a) reported a decrease with age in the ratio of fatty acid to glycerol released after epinephrine stimulation of swine adipose tissue. Etherton and Allen (1980) indicated that Older swine may have higher rates of esterification. Metz and Dekker (1981) concluded that there were no differences between Large White and Pietrain in fat mobilization as indicated by plasma free fatty acids. No other hormone may be more important in adipocyte metabolism than insulin. An increased release of fatty acids during fasting may be due to decreased plasma insulin rather than lipolytic hormones. Insulin shifts the adipocyte to lipid storage rather than lipOlysis during changes from the fasted to fed states. Insulin inhibits fatty acid release by inhibiting lipolysis as well as 77 stimulation of reesterification of fatty acid and glycerolphosphate. Insulin plays a role in the uptake of plasma lipids by adipocytes (Fain, 1982). Insulin inhibits adenylate cyclase and activates cyclic AMP phosphodiesterase which antagonize the effects of catecholamines (Loten and Sneyd, '1970; Hepp and Renner, 1972; Kono et al., 1975). Insulin apparently independently inhibits activation of cyclic AMP-dependent protein kinase (Walkenbach et al., 1978). Insulin also activated pyruvate dehydrogenase (Seals and Jarett, 1980), glycogen synthetase (Larner et al., 1978) and acetyl CoA carboxylase (Halestrap and Denton, 1973) by cyclic AMP independent phosphorylation of proteins or activation of phOSphOprotein phosphatases. All the aforementioned effects of insulin appear to be . independent of the enhanced glucose uptake even though the insulin stimulated uptake of glucose imparts increased fatty acid synthesis and reesterification (Fain, 1982). Nilsson et al. (1980) showed that insulin and epinephrine antagonize each other through dephosphorylation (inactivation) and phosphorylation (activation) of hormone sensitive lipase. In Opposition of the antilipolytic effects of insulin are growth hormone and glucocorticoids (Rao and Ramachandran, 1977; Fain, 1978). Fain (1982) indicated that both growth hormone and glucocorticoids exert permissiveness by altering adipocyte sensitivity to 78 activators of lipolysis. Machlin (1972) indicated the lipolytic action of growth hormone may be absent in highly - purified forms. Early work showing lipolytic activity associated with growth hormone needs repeating with recombinant DNA produced growth hormone. Nevertheless, it appears that growth hormone and glucocorticoid exert a much greater in vitro lipolytic response when used in combination (Fain, 1982). Vernon (1982) reported that no stimulation of glycerol release was observed when 1 to 104 ng/ml concentrations of growth hormone were incubated with cultured sheep adipocytes; however, fatty acid synthesis rates were about 50% lower compared to incubations with insulin and no growth hormone indicating that growth hormone acts as an insulin antagonist for fatty acid synthesis effects. A potent inhibitor of lipolysis appears to be the feedback of fatty acids (Rodbell, 1965). When ratios of fatty acid to albumin exceeded 2 to 3, basal and hormone stimulated lipolysis was depressed. Another potential regulation of lipolysis is adenosine since removal of it by use of adenosine deaminase elevated cyclic AMP and caused lipolysis in adipocytes (Fredholm, 1981). Very limited data are available showing specific effects of estrogens or testosterone -upon lipid metabolism. ,Hansen et al. (1980) injected noncastrated 200 9 male and female rats intramuscularly with 1 mg Of either 79 estradiol or progesterone and some male rats with 40 mg of testosterone 3 d prior to procurement of epididymal fat pad adipocytes. Female rats that were ovariectomized 15 d prior to slaughter were injected with 1 mg estradiol or 1 mg progesterone 3 h prior to slaughter. Complications due to estrus resulted in the study so that differences in fatty acid synthesis and epinephrine stimulated lipolysis were similar between intact males and females. However, fatty acid synthesis was higher in proestrus than in estrus or diestrus and lipolytic activity was higher in estrus compared to pro- or diestrus. Female rats injected with estradiol 3 d before slaughter had 5-fold lower fatty acid synthesis rates ’and almost 2-fold greater lipolytic rates 5 cell basis. Progesterone treated rats had on a 10 values similar to control values. Ovariectomized female rats given estradiol before isolation of fat cells had similar magnitude of difference as the previously mentioned experiment compared to ovariectomized controls. Treatment of males with estradiol or testosterone depressed fatty acid synthesis by about 65% and increased lipolysis by about 70%. Leszczynski et al. (1982) reported elevated plasma triglycerides after feeding of .05% estradiol to young chickens. Hervey and Hutchinson (1973) indicated that when testosterone doses greater than 1 mg/d are given to castrated females, lean tissue increased but fat decreased. Laron and Kowadlo- Silbergeld (1964) had 80 demonstrated that after a 10 mg injection of testosterone prOpionate to 100 9 female rats, concentrations of free fatty acids in plasma were 70% higher than control rats. In order to separate depression of feed intake responses from true metabolic responses due to castration, Hansen et al. (1983) used rats with ventromedial hypothalamic (VMH) lesions. Compared to control rats,VMH lesioned ad libitum fed rats had increased fat deposition, increased fatty acid synthesis and LPL activity. When castrated VMH lesioned rats were compared to VMH lesioned controls, weight gain was increased but fatty acid synthesis rates were not .different and lipolysis was significantly lowered by 130%. Aloia and Field (1976) indicated that synthetic androgens, (norethandiolone) decreased the hyperglycemia induced by glucagon administration in rats but testosterone had only slight effects. CHAPTER I GENERAL EXPERIMENTAL DESIGN AND METHODS The overall eXperimental design and general methods will .be described here but are fundamental to all subsequent chapters. The effects of testosterone upon growth and composition in pigs was first studied in a pilot study with 26 pigs. In this study, litters with five or more boars were used as one of four replicates. Each replicate was comprised of the. fOllowing five treatments: boars, castrates and castrates which were implanted with testosterone filled silastic capsules designed to deliver physiologically low (prepubertal or serum concentrations of less than 1 ng/ml), intermediate (approximately 2 to 2.5 ng/ml) or high (pubertal or serum concentrations of 4 to 5 ng/ml) concentrations of testosterone. Specific silastic capsule lengths and serum testosterone concentrations are given in Chapter II. These definitions of prepubertal and pubertal groups and testosterone concentrations will be used throughout subsequent chapters. Each treatment was represented by four pigs and each replicate was comprised of littermates (or two litters of similar dam breeding and same sire mating) randomly assigned to the above five treatments or an initial slaughter group. Only pigs that 81 82 were uniform and were representative Of others within each replicate were used. This pilot study was initiated at the time of castration and implantation of silastic capsules when pigs weighed 38 kg and continued for 3 wk. The pilot study was used only to evaluate growth rates, feed consumption and growth rates of selected muscles, bones and adipose tissue depots. These pigs were also used to assess treatment differences in fatty acid synthesis rates and lipOprOtein lipase activity after 10 d into the study and also at slaughter. Six boars were. slaughtered at the initiation of the pilot study so that changes in gross composition, and muscle, fat and bone accretion rates could be evaluated. There were two major studies identical to each other in overall design that were used to investigate the effects of castration and administration of testosterone (TEST) or dihydrotestosterone (DHT) upon carcass composition, muscle, fat and bone growth, as well as, in vitro protein synthesis and degradation rates, crude muscle proteolytic activity and in vitro adipose tissue lipogenic and lipolytic activities. The first study involved prepubertal pigs (30 kg at initiation) and the second involved pubertal pigs (70 kg at initiation). These studies will be referred to throughout subsequent chapters as prepubertal (PREP) and pubertal (POSTP) studies. For each study, littermate (or two litters with similar dam breeding and same sire mating) 83 boars were randomly assigned to either be slaughtered at initiation of the study or assigned to one of the following seven treatment groups (table I-1): boars, castrates, castrates implanted with TEST or DHT filled Silastic capsules at two delivery rates designed to mimic physiologically low (prepubertal) or high (pubertal) concentration of testosterone, and lastly, castrates limited fed (the same quantities as boars). Treatments not receiving TEST or DHT were sham implanted with empty Silastic capsules. As shown in table I-l, each treatment had four pigs. Each of these two major studies was .continued for 5 wk at which time they were terminated by slaughter Of the pigs. In both studies, castration and implantation were performed at the initiation of the study while pigs were under general and local anesthesia. Initial slaughter groups were used to assess the accretion of muscle, fat and bone over each 5 wk period. It should be noted that initial slaughter animals and limit-fed castrates were used only for assessment of composition, and that tissue specific enzyme assays were not assessed on the pigs. In addition, limit fed castrates in the PREP study were not a part of the original design and were comprised of pigs from dissimilar litters. . All pigs' were weighed and bled by venipuncture at weekly intervals. In addition, two pigs from each treatment were catheterized on d 17 in each study to obtain 84 .xecwm x3 m noee mo cOHweHchH we pewceHmEH pce cewewwmeo ewe3 mmHmn .wpcwm e>Hwoemmew enw mo cOHweHchH we e>He>Hwoemmew .mx we pce vH wHeweEonwmme peanez mmHm Hewwencm pceHewwencmewme H H e e e v e e ow e mmHm mo wenecz anm 304 _ anm 30H mweom . mewewwmeo mewewwmeu maoww mewewwmeo cewceHmEH mewewwmeo pewceHmEH new wewnmceHm ecowewmowmee ecoweweowmewowemnHo wHEHA HeHchH Amcoww wo wceEweewB newMHQDBm Ao exewcH peem pce cHewe m.H OH. o.on H.H m.H exam 0 D O O O : eo mv wemm m m oem m mm m mm Emma O oH.mH mHHH.N m.~Hm H.em e.mm emmswo . . . . . H em me much N m 5mm o mm o em Emma o Hm.mm mmm.~ N.Hmm m.mm m.mm U eH.>H eHH.m m.omm m.mm m.ov m Hmwc AcHeOVOeem Hp\cv mev . mev omcoww H99 I cHeO waneS waneB wceEweewB >HHe9 hpom zoom emewe>¢ Hech HeHchH QHONUZ9HUH999 9999 92¢ 99¢92H 9999 ~92H¢0 N909 2099 mem 9H¢2 99B¢989¢U OB ABm9BV 920999908998 90 ZOHB¢MBmH2HZ9¢ 92¢ 20HB¢999¢U 90 9809999 HIHHH 9H9¢B 109 also depressed by 12 to 18% in the TEST implanted castrates. There were no differences in feed conversion among TEST implanted castrates but -the high implanted (CHTEST) group was 10.5% more efficient than castrates. Corresponding data from the prepubertal (PREP) and pubertal (POSTP) studies are presented in tables III-2 and 3, respectively. In neither of these two studies did IBW differ. Pigs ‘implanted with high dihydrotestosterone (CHDHT), low testosterone (CLTEST) and high testosterone (CHTEST) in the PREP study grew more slowly than boars, castrates, castrates implanted with low dihydrotestosterone (CLDHT) or limit-fed castrates (CLFED). Over the 5 wk duration of the PREP study, boars consumed 23% less feed and were 23% more efficient in conversion of feed to gain than castrates. Testosterone but not DHT caused a feed intake depression. In fact, CHTEST pigs voluntarily consumed 38% less feed than castrates but were not different from boars in feed efficiency. In the POSTP study (table III-3), there were no differences in IBW or FBW between treatments. However, boars tended to be faster growing (10 to 19%) than castrates or CLFED pigs. Dihydrotestosterone implanted pigs grew more slowly than boars or CLTEST pigs (P < .06) and tended to be slower growing than all other treatments. Boars and testosterone implanted pigs consumed 110 .999me WGHMSUm UmmmH MO .HOHHO ©HOUGM¥9HSW$ w .mwceHQEH annnmU .mwceHQEH ewereEwewcH.HU .mwceHmEH 3OHNHU .ewewwmeouu .mweonumo .Amo. vmv wemme mmewOmwemcm wcewemep an3 mcEcHoo cHan3 mceeE wceEweewB n .ponem x3 m we>o exewcH peem pce cHeoe mm.~ mo. m.Hm OH.H mm. @299 HH.HH mma.H em.HHe eHe.oH am.mH ommqo w o o .o O o m ewe mm emm H em one emH hm mo mH 9999 O O O I O O NH mmmm Hm 9mm N eh mHo eOH mm mm HH 9999 U . . . . . m mnvm mm @mm m em cow eHh mm Hm HH emu U . . . . . H nHo mm mwmv N am mmm wmw mm Hm mH 929 U nmm Hm mem n ma mHn mmH ov mm HH 0 9Hv.hH evm.H ww.mon mmw.mm mm.HH m Harv :Hno\omoe onme Harv Herc H99 cHew wanez wnOHez Omcowo >HHeo zoom >pom wceEweewB emewe>< HecH9 HeHchH nee>029HUH999 9999 92¢ .9¥¢BZH 9999 2H¢0 M909 2099 90H9 9H¢2 99B¢989¢0 OB A999v 92099890999809999H9 92¢ A999Bv 920998909998 90 20HB¢999H2H29¢ 92¢ 20HB¢999¢0 H¢899999999 90 9909999 NIHHH 9H9¢B .mceeE mewecvm wmeeH Ho wowwe ewepcemeZNm H c .mwceHQEH annumU .mwceHQEH ewereEwewcHu o .mwceHQEH SOHNHU .mewewwmeouo .mweonumo .Amo.v 90 wemep mmewomwemcm wcewemep an3 mcEcHoo cHan3 mceeE wceEweean .poneQ x3 m we>o exewcH ceem pce cHeoe 111 so. mm.~ a.oH om.m mo.~ ozmm mHH.m e.mm.me mo.mHH Hm.ooH HH.me ommHo. . . . . . a mam m e.mH me e.mo mee mm ooH He me Emma o . . . . . H ooH m e.ome Hm eH Hem om HoH Hm He amma o omm.m rma.Ha on.mHH em.ooH He.me amaze Hme.m m.wmo.Hm we.ooe Hm.aa HH.me amoHo rom H r.omH Hm H.ma Han He HoH em me o w.m~H.m we.me eHHS me.moH mm.He m cHnO\omdm Ao\mc (Herc Harv (mmoowo Amxv cHeU waneZ waneS wceEweew9 Hes HHHeo aeom Heom emewe>< Hech HeHchH 9.69029HOH999 9999 92¢ 9M¢92H 9999 .92H¢0 9909 2099 90H9 9H¢2 999¢999¢0 09 A999v 92099990999909999H9 92¢ A9999v 920999909999 90 20H9¢999H2H29¢ 92¢ 20H9¢999¢0 H¢9999099909 90 9909999 MIHHH 9H9¢9 112 approximately 14% less feed than castrates and about 3 to 16% less feed than the CLDHT and CHDHT groups, respectively. Boars and testosterone implanted pigs had 20% lower feed to gain ratios indicative of more efficient feed conversion. Dihydrotestosterone implanted pigs in the POSTP study were intermediate to boars and castrates for feed to gain ratios but were less efficient than testosterone implanted pigs. In a longitudinal growth study of pigs reported by Walstra (1980) castrates tended to consume more feed per day than either boars or gilts up to 6 mo of age. Maximum growth rates for boars were reached between 4.5 and 6 mo while castrates attained maximum growth rates between 3 and 4.5 mo Of age. In addition, Newell and Bowland (1972) and Walstra (1980) found that boars required approximately 12% less feed per kg gain than castrates when fed ad libitum. Boars voluntarily consumed less feed than castrated male pigs in other reported work (Pay and Davies, 1973; Campbell and King, 1982) but boars normally gain at rates equal to or faster than castrates (Wood and Riley, 1982). Bratzler et al. (1954) observed an 8% higher ADG for testosterone implanted castrates but no difference in feed efficiency relative to castrates. The data in the present study confirm data of Walstra (1980) and Wood and Riley (1982) in that boars grew equally as well as castrates and had enhanced feed efficiencies. The observed decreased 113 feed intake of boars and testosterone implanted pigs but not in dihydrotestosterone implanted pigs relative to castrates suggests a role of testosterone or its aromatized metabolite in regulation of feed intake in pigs. This is supported by tendencies of female pigs to consume less feed than castrates and increase feed intake when they are castrated (Rerat, 1976). The growth data for the C FED pigs of this study are L not consistent with the data of Campbell and King (1982) in which castrates were restricted fed to 82% of ad libitum amounts of castrates. In that study, restriction caused a 10% depression in daily gain. The CLFED pigs of the present study indicated no large restrictions in daily gain relative to ad libitum fed castrates. One possible explanation is that the CLFED pigs in this study were restricted to the voluntary intake of boars but were still consuming adequate amounts of nutrients. A more plausible explanation for the lack of any growth depression is that some of the pigs used in PREP CLFED group were from litters not represented elsewhere in the study and since litter and treatment were used to calculate least squares means the genetic difference in growth potential Of these pigs was detected. Carcass Lengph, Fat Depth and Longissimus Muscle Area. Carcass length (CL), fat depth at the tenth rib (FD) and longissimus muscle area (LMA) are given in tables III-4, 5 114 and 6 for the pilot, PREP and POSTP 'studies respectively. In the pilot study, boars and CLTEST treated pigs had slightly longer (5%) carcasses than castrates. However, neither PREP nor POSTP studies showed any statistical differences between boars and castrates but PREP castrates tended to be longer (nonsignificant) than boars. The trend for boars to be longer than castrates found in the pilot and POSTP studies are consistent with data of Knudson (1983) in which boars were about 3% longer than castrates at 105 kg live weight. Walstra (1980) and Wood and Riley (1982) found no differences in carcass length between boars and castrates. , As indicated by Brannang (1971) the age of castration can influence differences Observed in skeletal growth and that vertebrae lengths were reduced in steers compared to bulls. ’Based upon FD measurements alone in the 3 wk pilot study, castration was without effect. However, there was a trend for low and intermediate testosterone treated pigs to have 8 to 10% lower (nonsignificant) FD than castrates and the CHTEST pigs had 25% lower FD than castrates. In the PREP and POSTP studies, boars had 35 to 43% less subcutaneous fat at the tenth rib. This is consistent with the 45% reduction in backfat at the tenth rib recently reported for boars compared to castrates (Knudson, '1983). Walstra (1980) also reported that boars had over 20% less backfat than castrates. Prepubertal and pubertal 115 TABLE III-4 EFFECTS OF CASTRATION AND ADMINISTRATION OF TESTOSTERONE (TEST) TO CASTRATED MALE PIGS UPON CARCASS LENGTH, FAT DEPTH AT TENTH RIB AND LONGISSIMUS MUSCLE AREAarb Carcass Fat LMAe Treatment Length Depth (cmZ) Groupc (cm) (cm) B 73.50h .92h 21.68 c 69.45g .94h 19.97 CLTEST 73.01h .84h 21.59 CITEST 71.169'h .86h 21.52 CHTEST 72.609'h .709 20.43 SEMf 1.20 .08 - .76 .Initial Slaughter'Group 63.15 .69 13.23 aPilot study data. bTreatment means within columns with different superscripts differ (P‘<.05). cB=boars, C=castrates, C =low implants, CI=intermediate implants, CH=high im lants. dFat depth excludes skin. eLMA-longissimus muscle area. fSEM=standard error of least squares means. 116 TABLE III-5 EFFECTS OF PREPUBERTAL CASTRATION AND ADMINISTRATION OF TESTOSTERONE (TEST) OR DIHYDROTESTOSTERONE (DHT) TO CASTRATED MALE PIGS UPON CARCASS LENGTH, FAT DEPTH AT THE TENTH RIB AND LONGISSIMUS MUSCLE AREAa Carcass Fat d Treatment Length Depth LMA Groupb (cm) (cm) (cm2) B 62.8f’g .51f 17.4f'g c 63.8g .909 17.3f'g CLDHT 62.1f’g .90g 18.5g CHDHT 62.0f'g .71f'g - 15.4f CLTEST . 61.2f .67f 16.3g CHTEST 62.4f'g .64f 18.59 CLFED 63.7g .60f 20.5g SEMe .778 .076 1.002 Initial Slaughter 45.9 .34 7.08 Group aLeast square means within columns with different super- scripts differ (P <.05). ' bB=boars, C=castrates, C =low implants, CH=high implants, C FED=limit-fed castrates. L CFat depth excludes skin. dLMA=longissimus muscle area. eSEM=standard error of least square means. 117 TABLE III-6 EFFECTS OF POSTPUBERTAL CASTRATION AND ADMINISTRATION OF TESTOSTERONE (TEST) OR DIHYDROTESTOSTERONE (DHT) TO CASTRATED MALE PIGS UPON CARCASS LENGTH, FAT DEPTH AT TENTH RIB AND LONGISSIMUS MUSCLE AREAa Carcass Fat d Treatment Length Depth LMA Groupb (cm) (cm) (cmz) B 86.51 1.66f 43.769'h c 85.33 2.579'h 33.51f CLDHT 84.17 1.949%h 38.76f'g'h CHDHT 84.43 1.76f’g’h 36.11f'9 CLTEST 85.34 1.63f 37.79“?"h CHTEST 85.89 1.41f 41.18g CLFED 85.69 2.16h 33.47f SEMe .90 .26 2.13 Initial Slaughter 76.95 1.24 24.57 Group aTreatment means within columns with different super- scripts differ (P‘<.05). bB=boars, C=castrates, C =low implant, CH=high implant, CLFED=limit-fed castr tes. éFat depth does not include skin. dLMA=longissimus muscle area. eSEM=standard error of least squares means. l_l8 testosterone implanted castrates in the present study also had 25 to 29% and 36 to 45% leSS fat at the tenth rib relative to castrates for CLTEST and CHTEST pigs of each study, respectively. Even though CHTEST pigs of each study tended to have less FD than the castrates or C DHT, there was a trend for) dihydrotestosterone L implanted pigs to have greater FD (than boars or testosterone treated pigs. Limit-feeding castrates to the ad libitum consumption rates of boars reduced FD by 33% in PREP pigs (P < .05) and by 16% (P < .05) POSTP pigs relative to castrate values. It appears that restriction of feed intake alone was inadequate in reducing fat depth to the same magnitude as Observed in boars or testosterone implanted pigs. Longissimus muscle area is Often used as an indicator of muscling. Many studies have shown larger LMA for boars compared to castrates (Blair and English, 1965; Pay and Davies, 1973; Siers, 1975). There were no differences in LMA of pigs in the pilot study or in the study with the TEST pigs had 31 H and 23% larger LMA, respectively, than castrates. Limit- PREP pigs. However, POSTP boars and C feeding castrates appeared to inhibit LMA in POSTP but not in PREP pigs. Based upon LMA, no definitive statements can be made about the effects of castration or exogenous test- osterone upon muscling. This may relate to the relative insensitivity of LMA mesurements for detecting small muscle area differences. 119 Carcass Weights and Soft Tissue Composition. Hot car- cass weights (HCW), right side weights (RSW), total soft tissues dissected from the skinless right side (TSTW) and proximate analysis of composite soft tissues are given in tables III-7, 8 and 9 for the pilot, PREP and POSTP studies, respectively. Walstra (1980) indicated that boars (tended to have less combined muscle and fat than castrates. No consistent trends in HCW, RSW or TSTW were Observed in this study except that PREP CHDHT pigs had reduced RSW and TSTW relative to other treatments and that PREP CLFED pigs had heavier carcasses and more TSTW than other treatments. This observation indicates that limit-feeding of high protein diets as used here may not be growth limiting over the 15 to 40 kg growth period. Over a 20 to 45 kg growth period in which intake of a 17% crude protein diet was. limited to 75% of ad libitum castrate values, carcass weights were not different (Campbell and King, 1982). In soft tissues of pigs from the pilot study, boars had 7% higher (1.14% absolute difference) concentrations of protein than castrates, but no differences in percentage moisture or ether extractable lipid between treatments were- detected. In the PREP and POSTP studies, boars had higher percentages of moisture (4 and 10%, respectively) and lower percentages (12 and 26%, reSpectively) of ether extractable lipid than castrates. Prepubertal boars did not differ but 120 .99005 90905U9 #9009” M0 .HOHHU @HMUCMHmflzmm .weem pce cme .Ceen echoxe mwanes mmeoweuw .wane3 eDMMHw wwom HewowN39m9 .wanw3 epHm wanwNme .waneB mmeoweo wonnzum .mwceHQEH annnmU .mwceHQEH ewereEwewcHu U .mwceHmEH SOHNHU .mewewwmeouu .mweonnmw .Hmo.uvmv weHpr mwQHwomwemcm wcewemeC an3 mcEcHoo cHan3 mceeE wceEweewB .hpcwm onHmm MH.mH OH.HH ~m.oo mH.m oo.oH NH.o~ coowo wewnmceHm HeHchH Hm. Hm. we. 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A 0399 cm 0299 99 namm mm mm mm on ¢m «9 mm 9999 U . . . . . . 9 039m vm unamm 99 3mm 99 mm mm mm mm ma no 990 U 0 O O O O 0 1H an «N 03mm 99 2mm mm mm mm «M mm we v9 990 U 0990.9N nov.mH nmm.mm 90.9w mm.~m mm.vw U MmH.o~ ommm.9a unmm.aw mm.m~ Hm.vm vm.mo m Amxv Amxv Amwv amsouo umm :wmuonm musumfloz 03999 ©0399 @0309 unmaummu9 M99D99H9 9909 999 90 20H9H909200 92¢ 9990H93 9399H9 9909 92¢ 9QH9 990H9 ~99¢09¢U 909 2093 90Hm 99¢: D99¢999¢U 09 A990v 92099990999909999H9 90 A9999V 920999909999 90 20H9¢999H2H29¢ 02¢ 20H9¢999¢U 9¢9999399909 90 9909999 mIHHH 999¢9 123 POSTP boars had 12% higher (P < .05) protein in soft tissue relative to castrates. Pubertal CHTEST pigs also tended to have higher percentages of moisture, protein and less lipid than castrates. Soft tissue composition of POSTP pigs presented in table III-9 are higher in fat and lower in moisture than data presented by Fortin et al. (1983) but the trend of higher moisture and less fat in boars compared to castrates coincides between that study and data presented here. The relatively constant percentage protein, increased percentage lipid and decreased moisture observed in a comparison of PREP pigs to POSTP pigs is consistent with the changes in body composition with increasing body weight reported by MCMeekan (1940). Carcass Fat-Free Muscle,iFat and Bone. The method of calculating fat free muscle (FFM) and carcass fat (CF) were described in the methods section of this chapter. Tables III-10, 11 and 12 summarize the FFM, CF and bone totals in the carcasses of the pilot, PREP and POSTP studies, respectively. No difference in FFM, fat or bone were observed in the-3 wk pilot study. However, boars at this early stage (55 to 60 kg) tended to have slightly greater FFM and bone and less CF than castrates. All three testosterone groups tended to have less CF than castrates. Slightly different results were obtained for the PREP and POSTP pigs (tables III-11 and 12). PREP boars were not different from castrates in FFM or bone but they had 15% 124 TABLE III-10 EFFECTS OF CASTRATION AND ADMININISTRATION OF TESTOSTERONE (TEST) TO CASTRATED MALE PIGS UPON TOTAL CARCASS FAT-FREE MUSCLE, FAT AND BONEa Fat Treatment ‘Free c Groupb MuscleC FatC Bone (kg) (53) (kg) B 25.03 5.21 5.39 c 24.56 5.51 ,5.11 CLTEST 25.31 5.29 5.35 CITEST 24.10 4.91 5.26 CHTEST 23.71 4.92 4.97 sand 1.56 .54 .10 ‘initial Slaughter 14.21 2.54 3.66 Group - aPilot study pigs. b =low implants, C =intermediate B=boars, C=castrates, I implants, CH=high i9plants. cFat-free muscle (FFM) and fat derived from soft tissue of right side and percentages of side multiplied by hot carcass weight. dSEM=standard error of least squares means. 125 TABLE III-11 EFFECTS OF PREPUBERTAL CASTRATION AND ADMINISTRATION OF TESTOSTERONE (TEST) OR DIHYDROTESTOSTERONE (DHT) TO CASTRATED MALE PIGS UPON TOTAL CARCASS FAT-FREE MUSCLE, FAT AND BONEa Fat-Prep c c Treatment Muscle . Fat Bone Group (kg) (kg) (kg) B 13.93ef 3.05e 3.9lf c 14.14ef 3.61fg 3.89f CLDHT 14.56f 3.94g 3.61ef CHDHT 12.74e 3.04e 3.22e CLTEST 13.26ef 3.46eg 3.64ef CHTEST 13.31e 3.11ef 3.73f CLFED 16.68g 3.98g 3.40ef SEMd .67 .18 .16 Initial Slaughter Group 4.91 .79 1.69 aTreatment means within columns with different super- scripts differ (P <.05). bB=boars, =castrate, C =low implant, CH=high implant, C FED=lbmit-fed cas rates. L CFat-free muscle (FFM) and fat derived from soft tissue of'right side and percentage of right side multiplied by hot carcass weight. d SEM=standard error of least square means. 126 TABLE III-12 EFFECTS OF POSTPUBERTAL CASTRATION AND ADMINISTRATION OF TESTOSTERONE (TEST) OR DIHYDROTESTOSTERONE (DHT) TO CASTRATED MALE PIGS UPON TOTAL CARCASS FAT‘FREE MUSCLE, FAT AND BONEa Fat-Free Treatment MuscleC FatC BoneC Groupb (kg) (kg) (kg) B ‘ 47.56g 11.94ef 9.43f c 41.68e 15.03g 8.11e CLDHT 42.73ef 13 zafe 8.05e f CHDHT 44.69efg 14.18 9 8.28e CLTEST 46.21fg 14.30fg _ 8.23e CHTEST 42.73ef 9.58e 8.54e CLFED 41.25ef 15 399 8.23e sand 1.65 1.04 .11 Initial Slaughter 29.26 7.15 6.22 Group, aTreatment means within columns with different super- scripts differ (P< .05). bB=boars, C=castrates, C =low implant, C H=high implant, CLFED=limit-fed castrgtes. CFat-free muscle (FFM) and fat derived from soft tissue of right side and percentage of right side multiplied by hot carcass weight. dSEM=standard error of least squares means. 127 less CF. Pubertal boars had 14% more PPM, 21% less CF and 16% more bone than castrates. Others have shown that boars have greater muscle and bone but less fat than castrates at typical market weights (Prescott and Lamming, 1967; Walstra, 1980; Wood and Riley, 1982). With the exception of POSTP CLTEST pigs which had 11% more FFM, no definite effects of testosterone or dihydrotestosterone administration were detected in either the PREP or POSTP study reported here. Limit-feeding PREP castrates had a positive influence upon FFM accretion as these pigs had almost 20% more FFM than boars. Limit-feeding of POSTP castrates did not 'reduce FFM accretion relative to ad libitum fed castrates but did result in 13% less FEM relative to boars. Walstra (1980) reported that the amounts of muscle in carcasses from both boars and castrates were increased when fed 70% of ad libitum. However, part of these observed increases may be attributable to the fact that pigs were older when slaughter weights were attained. Aside from boars in the PREP study, only CHDHT and CHTEST pigs had less CF than castrates and they were similar to boars. In the POSTP study, CHTEST depressed fat accretion by 36% relative to castrates but the other implant groups were intermediate to boars and castrates. Limit-feeding was ineffective in depressing carcass fat Iaccretion relative to ad libitum fed castrates. This 128 u l.— < a: l- m ‘1 U h 03:0. 5 '-----0 III...- a... g k““““ m E o “t IIIIIIIIIIIII “- flush—To '- .o‘ooooooo oo—E‘“ < .6565 "- .. ' ‘ I... g I: O I: I n.. I. z E: 00. PPM 52. O 8 8 Jaqam 0/0 aamo1 °L 59.39.1450: a; OAngIOJ {03 pue de FIGURE III-l. Fat-free muscle and fat in carcasses of boars and androgen implanted castrates as a percentage of castrates. 129 observation substantiates the hypothesis that testosterone alters fat deposition by mechanisms other than feed intake depression (Wade and Gray, 1979). Bone accretion was relatively unaffected by either form of androgen treatment or limit-feeding (further data on bone in Chapter VIII). Tables III-10, 11 and 12 also present the data derived from the initial slaughter pigs. Accretion of FFM, CF or bone can be derived by subtracting the initial values from the treatment means. However, the trends remain the same since this was a subtraction of a constant from all treatments. When the FFM, CF and bone data in tables III-10, 11 and 12 are divided by -HCW or live weight, the percentages of FFM, CF or bone are derived. These percentages for the pilot, PREP and POSTP studies are presented in tables III-13, 14 and 15, respectively. In the pilot study no consistent trends in percentage FFM or bone were detected but castration depressed percentage fat by 6 and 12% on a carcass and live weight basis, respectively. Testosterone also tended to result in lower fat percentages on a carcass and live weight basis. In the PREP and POSTP studies no new trends between treatments were apparent when data were expressed as a percentage of carcass or live weight. Subcutaneous and-Perirenal Adipose Tissue Composition. 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OH.H Hm. mzmm oom.m Hem.ma smmH.Hv ammo.~H nmn.mm moe.mo amuse y . . . . . a saw m mmm m flnmm Ne ave «H mas ma :NN op Emma o mam.a flsHh.mH flaom.¢q mmm.HH som.o~ n-.~m ammeuo m-.m Hemo.va whoe.ee comm.ms :NH.HN mom.om amaze mmo.m Hamm.ms mamom.~¢ nmhm.ma :mhaom nos.oo emaqo moo.m Hmm.¢fi moa.He :mHm.~H an.m~ mom.qo o nmm.m nmm~.HH amm.vv Hamo.ma mmm.na nmm.mm m maom mums ozmm mcom mumm 02mm nmsouo ucmEummHB unmflwz m>flq m0 w ommmoumu m0 w memonz m>Hu mo mmaomao was mo mzom mo 94m .muomoz mmmmueam mo momezmommm one zomo mon was: omeamemao oe Assoc mzommemoammeomomeo mo Ramsey mzomme umoemme mo onemmemHszom ozm onemmemmo 449mmmomemom mo meommmm mHnHHH mamas 133 in the PREP and POSTP studies. The proximate analysis results are presented in tables III-16 and 17 for PREP and POSTP studies, respectively. Subcutaneous adipose tissue from the dorsal neck region of boars had 37 and 35% higher moisture, 10 and 6% lower lipid and 37 and 33% higher protein than PREP and POSTP castrates, respectively. While DHT implanted pigs did not differ from castrates, CLTEST and CHTEST pigs had higher percentages of moisture and protein but lower percentages of lipid relative to castrates in both the PREP and POSTP studies. This observation indicates that lipid filling was being depressed by testosterone treatment but not by dihydrotestosterone. For the earlier develOping. PR ~depot compared to the SQ depot, similar trends were observed. Since lipid filling would have its greatest effect on decreasing percentage moisture the effects of castration and CHTEST are most evident on this aSpect in the PR depot. Lipid filling also would decrease the concentration of protein in the depot but some of this decrease would be offset by increased synthesis of membrane proteins due to cell enlargement. In comparing PREP with POSTP pigs it is apparent that PREP pigs had much less lipid but higher protein and moisture concentrations. 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Amxv on .oAmm _ooz _om oAmm _ooz bosono usmfiumwna ouch AmcmuAHmm ovum mmmoumu mmDmmHB mmOmHnfl..flmzmmHmmm 624 mm40m<0 mBHmomZOU ZH ZHmfiomm QZ< QHmHA mflméfivmmexm mflmfim .mmDBmHOZ AflBOB ZOmD mOHm MAAuemmme mmEAu ucmAm3 AmcmuAamm a0 umm mmmoame omAMAooAmo Amuoao .mmumaummo pmMIuAEAAuommAU .mucmAmEA :mAnumU .mucmAmEA 3oAqu .mwumaummouo .mumonnmn .Amo.v me AGMMAU mumAHomamQSm ucmuwMMAU AHAz mGEsAoo cAnuA3 mammE ucmEumeBm 138 m.A m.mm m.¢ No. OA.A AA. 62mm mm.OA aae.vma am.av em. AVoaNA amamA aaaae amo.AA mAAKN ame.mv am. 6mm.a amom.A amaame ama.AA aAA.oN6 amo.em we. maqv.AA on.N amaaae amm.eA ama.amv mao.Ae Nm. mama.AA amm¢.A amaze a6112 n¢.aNm maN.mm Nm. maaA.AA mam.A amaae a692. amo.AAm amA.Ne am. mom.NA amme.A e aa.vA ama.mem aq.oe om. amvv.a maea.A a Ame Ame Ame last imxe lass aasoue ea euma 602 aa mama was pamsamaaa Oumh HMCGHHHQQ Uflmm mMMOHMU mmammaa amoaaa< Aazmmamaa azm mmAA m :AnuA3 com: .mfimme mQHMSUm Ummwfi W0 HOHHG UHMUCMUWHZNM I) Q .mfimnm :A unmAmz mmoa pamAasA amma n .aeaum quAam o.eAA m.vA v.AmA e.NeA m.omA m.cmA «.mmA uoueaaea mDmOC o NNN m Nmamm.vom amo.mAm mN.mev mm.Aav ae.Aem -amaasmsAsmm AAaomua N SAN m mNamm mme am.Amv amm.mAe mm.mAv aA.N: mamoAHa mflumc m.mm ma.mA m.mmA m.maA A.eaA N.ANA N.maA nAammuaaA meAm unmAm mDmOG a.eeN e.mm v.omv v.Amv muamv o.oA¢ m.amq -Aeamuasmm A.mA A.N N.mN o.eN A.mN N.mN «.eN mAAOEma manomm A.Nm A.m amA.NoA amA.AaA Amm.mm 6A.qm Am.OAA mamaAuoma m.amNA A.aAA m.mAAN m.aNON o.mONN N.aGON e.eNmN mssAmmAmaoA «.ma N.N N.mNA m.MNA e.ONA m.mAA m.mNA mAAmAaomua a.oa m.m A.NAA m.NMA e.vAA m.AAA m.¢NA eruAma meAm ameA a unmAm msoaw ZMm :mAm amusA 30A moumuummo .mumom onmsz AmAuAaA .e UnmmBmOHMS mqumDS QMBUMAWm ZOmD mem mqéz QNBflMBWflU OB ABmMBV mzommBmOEmMB m0 ZOHBdemHZHZD< 92¢ ZOHBdMBmH mqmdfi 147 to respond to testosterone implants. Thus it was decided that these muscles could be used as indicator muscles in the subsequent trials of this study. Gross muscle weights of pigs in the prepubertal (PREP) and pubertal (POSTP) studies, respectively, are listed in Tables IV-2 and 3. Boars from the PREP and POSTP studies, respectively, had the following heavier muscles relative to castrates: TB. (by 17 and 30%), BR (by 24 and 31%, LD (by 19 and 10%, PC (by 20 and 14%), teres major (TM) (by 35 and 22%) and ST (by 19 and 21%). As indicated in Chapter III, neither castration nor supplementation with TEST or DHT affected total carcass FFM accretion in PREP pigs. However, in POSTP implanted pigs, total carcass FFM accretion rates were elevated over castrates by 8.5 to 36%. An initial interpretation is that muscle in PREP pigs were not affected by testosterone. However, both androgens had positive effects upon individual muscle weights in the PREP study. If the muscle weight means of the implanted groups in the PREP study are averaged, implanted pigs had 12, 16, 22 and 11% heavier TB, BR, LD and ST muscles, respectively, than castrates. PC, SA and TM muscles of the implanted pigs in the PREP study did not differ from castrates. In light of the total FFM accretion data (Chapter III), these reSponses appear surprising but indicate that some muscles of the PREP pigs were responsive to both forms of androgen. The. data in figure IV-l .mCMQE mOHMSUm Hmmmfl MO .HOHHQ UHMCGmumflzmm o .mmumaummo omwluAEAAnommAu .mucmAmEA nmAcumU .mucmAQEA 3oAnAU .mmumuummouo .mamonumo .mEmnm sA unmAm3 onmszn .Amo.vmv awMMAo muQAuemamQSm ucwHOMMAp nuA3 mCEdAoe :AQHA3 mamas ucmEummuu mumsvm ummwAm «.ma o.OA o.m m.mA A.Nmm m.m~ m.OhA macaw HousmSMAm AMMHAGH em.m mo.m mm.A 0A.m vo.m¢ mm.A AA.mA pzmm . . . . . . . A mm mvm mum mm m mA woo mm mum momA mam mm mmm Nov 9mm 0 . . . . . . . x mm mam mA mA v MA men we no mva we mm mo com Emma U . . . . . . . A 9. mew mew we mm a mA mm om mm ommA mac Ah mm Nov Emma U 4 . . . . . . . . m 11 wow mmm mmm mm v NA mm mm comm mmMA Acmh me an mom Ban 0 O O O O O O O ‘H mm mom mmmA mm N mA mom mm 50m mmMA 50A on mum mmm Ban 0 mm.mmm mmA.mm N.mA om.mm mm.mmOA mA.mm mh.omv 0 mo mwm mA Am m MA mm Ah mMA AAmA Asm om mmm mmm m msmocAocmuAEmm aoflmz msAaoumm mschuomm msEAmmAmCOA mAAmAnouHm AAnomum UQWOHO momma mQOOAHB useEummHB n onmsz mmBmUHMB MAUmDZ DWEUflAmm ZOmD mOHm WAQS OmeH MAQANB 149 .mrmmfi mmamswm ummmA mo House pamosmumnzmm .mmumavmme omwluAEAAnommAu .mucmAmEA amAnumo .mucmAmEA 3OAHAU ~mwumuummeuu .mumonumw . .mfimum GA unmAm3 onmozn .Amovmv amAMAp mumAaemqusm acmnmmon zqu mcEsAoo cAnuA3 mamms unmaummnu mumzwm mmmAm h.mmv m.mm «.mA m.m0A m.mmm~ m.wmA o.mmm msouw amuaosmam AmAAAaa ma.om m>.~ ev.m mm.w mw.o>A mm.h mm.~n pzmm ma.vmo mm.mm mm.A~ wo.>aA A.mo>m m~.va mm.mmMA ommAU mm.mmm www.mm mv.mm mn.mmA m.mmov mmm.oom mm.mh¢A Emmamo www.mAm mm.mm mmo.¢m mm.maA m.vAAv mo.o- am>.mAmA ammBAU aaA.ANm aA.mv a682 mo.aaA «.emNe am.NNN amm.mAmA amaze mmmv.mnh mo.mm mm.AN mv.~wA .A.mmov mmA.OA~. mm.mm¢A BmQAU www.mAh mo.mm mh.Am amm.mmA m.o>mm mmm.Ao~ mm.mNMA U m>.hom mm.he mm.om mo.omA m.wmov mA.¢mN mw.n~hA . m HOMMZ AAnemam omdoau msmocAosmuAEmm wowwe msAHoumm mawcAueom msEAmmAOCOA mAAMAnomum QOOAHB ucmsummua A onmsz CmBmUHHB MAUmDS Omaumflmm ZOmD mUHm mfimz Dm9¢m8m<0 OB ABEQV mzommBmOBmMBomameD m0 Afimmfiv mzommfi ImOBmMB m0 ZOHBflMBmHZHzafl QZd ZOHBH mqmdfi 150 castrate o.ococoooooo‘ooooooooooooooooooooooooooooo .“-““ 'vv'vv'vvv'vvvvv vvv' <01~01itflhtflibflurt .— - '-'ii'3"-'i if; in" 2 a ' a '0 qugH 51' PC lD TB "" CLDHT v00.“ Wt A..- qusr ........ CHIEST — qreo JOMO‘I (%) “4'34“” 04 0145an 00931330 opsnw FIGURE IV-l. Effects of prepubertal castration and administration of arrirogens to castrated male pigs upon triceps brachii , brachialis, longissimus, pectineus and senitendinosus muscle accretion. 151 represent the difference in respective muscle weights between the initial slaughter group and the implant groups expressed as a percentage of the nonimplanted castrates for comparison among treatments. The magnitude of response varied with muscle but from the figure_ it appears that CHTEST was more effective in stimulating growth in these selected muscles than the other implants. . In the POSTP study (table IV-3), there were fewer significant differences for specific muscles between implant groups and castrates but muscles tended to be heavier in the implanted groups. If the implanted group treatment means are averaged for the TB, BR, LD and ST, it appears that implanted androgens stimulated these muscles by 12, 6, 12 and 13%, respectively, relative to castrates. Individual muscle accretion rates were determined relative to castrates and are illustrated for the 'POSTP- study in figure IV-2. It appeared that high DHT had a greater effect on muscle growth than the other implants. However, in the POSTP study no treatment was as effective for muscle growth as intact boars. The inability to detect differences between TEST or DHT upon muscle growth contrasts with- recent reports which suggest that some androgen effects on skeletal muscle may be mediated by aromatization of testosterone to estradiol (Dionne et al., 1979; Knudsen and Max, 1980; Max, 1981). 152 2 O h 1: a U "2222;" ._ O--- m III-III hhfi—n 000...:0 U 55338 a "" 2‘5"" III. 0 U0 U u I I v ’ : . IUI .“ 8 I- ' a III-III..- s“..- ‘ a III-II-iil . o o o o o o o 8 co v N N P Jan's!” JaMO‘I (‘36) 59401430: 0; simple: uoggaaaao apsnw FIGURE IV-2 . Effects of pubertal castration and administration of androgens to castrated male pigs upon triceps brachii, brachialis, longissimus, pectineus and semitendinosus muscle accretion. 153 It is puzzling to explain why individual muscles were stimulated by androgen but no differences in total carcass fat-free muscle mass were evident in the PREP pigs, except' that certain muscles (some of those measured) are responsive to androgens, whereas most muscles are not. On the other hand, POSTP pigs showed a response to androgen in individual and total carcass total fat-free muscle accretion rates. Brannang (1971) used monozygous twin cattle to show that the presence or absence of the testes selectively affected certain muscles more than others. Castration decreased shoulder muscle weights over 15 to 28% but hind leg muscles by. less than 15% on the average. Walstra (1980) made similar observations for groups of muscles in pigs. Kochakian and Tillotson (1957) observed similar trends (in castrated guinea pigs given exogenous testosterone. Explanation of the diverse responses to androgen observed between muscles could _be related to receptor presence, number and function. Androgen receptors have been identified in porcine rectus femoris 'muscle (Snochowski et al., 1981; Lundstrom et al., 1983). In rat skeletal muscle, androgen and glucocorticoid receptors both interact with DNA (Snochowski et al., 1980). 'It also is uncertain whether TEST or DHT is the active ligand which binds to muscle receptors to elicit reSponses (Gustafsson and Pousette, 1975; Krieg and Voight, 1976; Krieg et al., 1974; Tremblay et al., 1977; Michel and Baulieu, 1980). 154 Strong evidence indicates that Scx-reductase activity is nonexistent in skeletal muscle (Krieg and Voight, 1976; Liao et al., 1976; Michel and Baulieu, 1980). Warrenski and Almon (1983) reported that skeletal muscle has considerable 3 a -hydroxysteroid oxidoreductase activity which converts DHT to androstanediol. Chin and Almon (1980) reported that the number of cholinergic receptor sites in some muscles (extensor digitorum longus) are increased but in others (soleus) no change is observed after castration. The demonstration of variation in response to castration indicates that muscles may vary in the species of receptor proteins made as well as reSponsiveness to other hormones such as glucocorticoids. The data in the present study shows that selected skeletal muscles of pigs were responsive to both TEST and DHT both prepubertally and pubertally. The mechanisms underlying this similarity of action cannot be ascertained from these data. Considering the boar taint problem associated with producing boars for meat, it appears that administration of androgens after castration may be a practical approach worthy of further investigation. Muscle Lengths. Muscle lengths taken before removal of muscles from the right sides of the carcasses from the PREP and POSTP studies are presented in tables IV-4 and 5, respectively. Very few differences existed between 155 .mmumaummo omAIuAEAAanm A .Amo.vmv AOMAAU mumAAOmemsm unmammon nuA3 .mcmmE moumsvm ummwA mo Aouum pumpcmumnzmm U .musmAmEA :mAnum o .mucmAmEA 30A" AU .mmumaumMOIU .mumonu me .mEmum GA unmAw3 mAemsz .0 n mCEdAOO aAnuA3 msmmE ucmfiummuu mumsvm ummmAm ea.NA ee.a mm.a «A.N Nm.m «A.N macaw “magmamAm AmAuAaA em. NA. am. NA. NA. mm. exam 0 O O O O I NH qu NA mAAN m AA A 6AA OA was OA mmv m aaa e . . . . . . a aeN mA ammo a me a mma OA mANA AA wmv a amaa e O I O O O O A aeN NA mamA a ma a amom 0A mew AA mAA OA amaa e O O O O O O m Ame NA mama N Am m mama oA mAAA AA . mNo oA aaa e . . . . . . A mam mA mew m cm OA mam AA mam AA mama oA aaa e mam.eA meA.m GA.cA mAa.oA amAN.oA meN.oA e Ama.mA mAm.m mA.oA mom.oA maaA.AA amm.AA a msmocAocmuAEmm momsAuomm msAaoumm mAAoAnomAm AAnomum AOHME Omsouw mmmeAAB momma usefiumwue nonmsz mmmAUmDS QMBUWAMW m0 mfiwzmq 20m: mUHm HA¢S Qm94m9m40 09 ABva mzommBmOBmmEOmomeQ m0 ABmmev mzommfi ImOBmmB ho ZOHBH mamflfi 156 .mCMOE mUHmSUm UmmmH MO .HOHHO QHMUGMUWHZQM m c A U .mucmAmEA 3OAHAU .mmumaummouo .mumonumo .mmumuummo amquAaAAuama e .muamAasA mmAzu .mEmam :A unmAmz onmsz Q . 43.x: ammmAp mumAAemummsm ucmummAAp nuA3 mGESAoe :AAAA3 mammE ucmfiummau mumsvm ummmAm mm.ON NN.oA me.AA mm.NA Nm.NA mA.NA amoue nonzmzzAm AzAuAaa me. Am. ow. ON. em. av. . exam mzA.NN moa.AA ma.mA mma.mA ov.mA ma.mA azaAe mAA.aN mmN.AA am.mA amaN.AA om.AA Am.mA ammaze mAAN.mN amem.NA ON.mA mAm.mA em.AA aA.AA amzaae maao.aN mam.NA am.mA moa.mA aa.AA me.AA amaze amm.vN ammm.NA me.mA mvo.AA mo.mA mm.mA azaae mzm.AN mam.AA oa.mA mmN.AA NN.AA Am.mA e mma.AN aNANA GA.AA amN.AA mo.mA am.mA a mSmOCAocmuAEmm mschuomm mADAoumm mAAMAnomum AAnomnm .HOHWZ Ommono . mQOOAmw momma ucmfiummne nonmaz mmMAUmDE.QmBUmAmm m0 mBGZMA ZOmD mOHm HAH mAm¢B 157 treatments. However, the TB and ST muscles tended to be longer in boars and androgen treated pigs relative to castrates. Muscle Composition. Since wet muscle weights tended to be heavier in boars and androgen treated pigs relative to castrates, composition of TB, BR, PC, LD and ST muscles was determined. Tables IV-6 and 7 show the percentage moisture, ether extractable fat and protein ‘ (Kjeldahl nitrogen X 6.25) determined on muscles from the PREP pigs and tables IV-8 and 9 present the data for the POSTP pigs. The most common feature of castration was to increase percentage fat in muscle by 40% or more, decrease percentage protein by about 3% and percentage moisture by 1 to 2% in both studies. High testosterone implanted pigs tended to have lower percentages of lipid relative to castrates, and in some cases similar or lower percentages of lipid than the initial slaughter group of pigs which in itself indicates an effect on lipid metabolism of the intramuscular depots. Initial group data of the POSTP study which weighed about 75 kg were similar in muscle composition to the boars in the PREP study which 'weighed about 40 kg. Muscle composition data comparing POSTP boars to castrates are in agreement with data of Newell and Bowland (1972) where muscles from boars had 1 to 2% less fat than castrates. 158 .mCMGE mGHMAAmum #mmmd MO .HOHHG UHMQQMUNHEWW .mwumaummo omAIpAEAAnommAU smucmAQEA nmAnumU .mAGMAmEA 30AnAU .mmumuummono .mumonum .Amo. vmv ummon muQAHemummsm pneumAMAo nuA3 mcEdAoo :Anqu memes usmaummae .umm mum .cAmuonm mum .musuonE wuoz AN.A vo.mA me.mh mn.A mv.mA mo.mh hA.A mo.mA MA.mh .0013 (U macaw amaamamAm AmAuAaA AA. NA. AN. AA. mN. AN. GA. Nm. mN. exam amA.N mam.ON mamOA.za amm.m mA.AA 6mm.ea mmmmN.AameN.ON NA.NN amaze. 6SA mammA.ON mNo.aN maN.N AA.mA AANA.NN mmA.AmmAe.mA NA.NN amzaze mNN.A mammo.ON amAa.ea amm.m ma.mA mameo.aa mmmA.AamNm.mA AA.NN ammaAe mme.N amea.mA mAN.zN aom.m om.zA mamm.aa mem.Am6mm.mA oA.Na azame moe.A mNm.ON Ammm.za aANN NN.AA mazA.aa amoa.AAmmo.oN NA.NA amaze mmmm.N mma.mA amaa.ea AAm.m aa.mA amAm.za amom.A mAANA Ae.aa e maA.A a8.3 szm.aa mom.N am.mA moa.aa Amam. aom.oN Ne.aa a a a. as a a as a a oz eaaoue mschuomm mAAMAnomum AAnomum. AGOEUMOHB mQOOAHB amvomsz mmAUmDS mDMZHBUMA 02¢ mHAdeU¢mm .HHmodmm mmmUHmB m0 ZOHBHmOmSOU AdUHmeU ZOmD mUHm mfldz QMEH mflmH qu¢8 160 .mcmmE mmnmzqm ummmA mo Aonum oumocmumuzmm o .mmumaumme owquAEAAuommAU .mpCMAQEA smAnumU .mucmAmEA 3oAuAU .mmumuummouo .mumonume .Amo.v.mv AmmmAp mumAaemammsm unmammmAp SAAB mcEsAoo :Anqu wanes ucmsummuan .umw mum .sAwquQ mum .musuonE NHOZM mA.AN mm.oa ON.N AA.¢N mA.hb mo.A mA.AN mN.o> macaw “mummzmAm AmAAA:A 0N. mA. NA. Am. mm. Nm. AN. ON. 02mm 0 O 0 O I O O O 0 SH memo A mmhm AN mmN mm nmo m Ammm mA Am we mmNm N ammoo AN ANN mm 0mm U o o o o o . o o o o m mNA A AmoA AN wAm on mmA A mmmm ON mA an mmm A smmA AN Amp mm Emma O we moo.NN mNN.m> mmmA.N AmAN.0N h>.mh mmvo.N ALAmmmAN. wom.mh BmmBAU ammo.N mmmA.AN wmA.mn zoom.N mon.oN mm.mh mmom.N nmNm.AN mom.Ah Emamu mumm.A mumm.AN mam.mn :Nm.m Ammh.mA Am.mh mAA.m AmAn.oN Amm.mb BmQAU mAm.N mom.oN mmA.mm som.m mmA.mA .mm.oh mam.m 0mm.oN mmvm.mh U mmmA.A mmom.AN www.mh mmmm.A ANm.oN hm.mn mam.A nmm.AN www.mm m a 02 a a o: a a o: eaaoae mschuomm mAAMAsemnm AAzomum usmfiummne mameAHB Qmonmsz MAUmDZ mDmZHBUmm 02¢ mHA¢HmU¢mm .HH:U¢mm mmmonB mo ZOHBHmOQEOU A¢UHZHSU 2000 mUHm mA¢Z 0MB¢mBm¢U OB mzommsmOBmMBom0w2H0 mo mzommfi ImOBmMB mo 20HB¢MBmH2H20¢ 02¢ ZOHB¢mBm¢U A¢BmmmDmEmOm ho mBUmmhm mI>H mAm¢B 161 .msmme mmumsvm AmmmA mo Hoaum pamocmumuzmm o .mmumaumme omquAEAAHQAMAU .mAGMA0EA nmAsumU .mucmA0EA zoAqu .mmumuummonu .mumonlmo .AMO. vmv Hmmon mu0Auomum05m ucmummqu nuA3 mCEsAoo :AnuA3 mamas ucmEummaen .umm wum .CAOAOA0 mum .mHDAmAOE muozm bM.A MM.ON m>.Om NO.A NO.AN AM.Mh 050nm AmuamsmHm HMAHHGH ON. AN. ON. ON. MM. AN. 62mm ANA.N mma.mA mamm.ma AAOA.N 6mm.NN om.AN amaae m mam.A ANA.AN moa.ma mmN.A mmNA.mN Am.AN amma e . . . . A OAMOWN OABO.ON AmMA mm mmOA A mum MN OM Am Emma U . . . . . . m mmmm N AmON ON OAOO mm AOAM N mmAA MN MO Am 9:0 0 . . . . . . _ A OAOO N OAOO ON Ame mm AAOm N mam NN OM Ah 8:0 0 mhA.M OAMh.ON OOO.A> Smm.N mMm.NN AM.A> U mth.N OAAO.ON OAMO.O> mmmOh.A mmmm.MN NN.A> m m a o: a a 0: eaaoue msmocAocquEmm maEAmmfimGOA ucmEummHB nmmonmsz mmAUmDZ m0002H0209HZHm 02¢ m02HmmH0200 00 20HBHm00ZOU A¢UH2000 2000 mUHO 04¢: 0MB¢mBm¢U 08 A900V 0200m8009m0900000H0 00 ABmmBV mzommBmOBmmB 00 20HB¢0BmH2H20¢ 02¢ 20HB¢MBm¢U A¢Bmmm009m00 00 m80000m mI>H mAm¢B 162 In order to assess protein and fat deposition in selected muscles, the percentage moisture, fat or protein of each muscle was multiplied by the muscle weight to obtain total moisture, fat or protein in each muscle. These totals are given in tables IV-lo and 11 for the PREP study and tables IV-12 and 13 for the POSTP study. As expected from the percentage composition of the selected muscles, the total moisture, protein and fat content of the muscles was similar to that of the percentage composition. The total ether extractable lipid content of muscles from the initial group of pigs was subtracted from the total lipid of the muscles in the treatment groups, to obtain muscle intramuscular 'lipid accretion. When accretion of all treatments is expressed as a percentage of the castrates, the relative effect of castration and administration of TEST or DHT can be readily visualized. The muscle lipid percentage accretions relative to castrates are presented in figures IV-3 and IVr4 for the PREP and POSTP studies, respectively. Castration had a marked effect on intramuscular lipid accretion in both PREP and POSTP pigs but the effect was more pronounced in the POSTP pigs. As shown in figure IV—3, PREP boars had an average of 34% lower intramuscular lipid accretion relative to castrates. Figure IV-4 indicates that POSTP boars had an average of 48.7% less intramuscular lipid accretion than castrates. It' appeared 163 .mCMOE mmhmgmum #mMOH MO .HOHHO MOHMUGMUmH—me . o .mmumaumme pmquAEAAuommAU .mucmAmeA nOAnumU .mucmA0EA BOAHAU .mmumuummouo .mumonnmo .AMO.v my AOAAAU mu0Auemum0sm unmamwon 3AA3 mGEsAoe :AnuAs mammE ucmEummuan .mEmAO cA OAONDE Hem A00 umm paw .Amv :Amuoa0 .Aozv musuonE Amuoem hN. hO.M AA.MA MA. Na.A NM.ON OO.N AO.AM MN.MMA 050Aw umuamamAm AmAuAaa OA. MM. OA.N AA. OM. NN.A MN. Nh.M AO.NA pzmm O O O O O O O O O A OANM A OAOM MA AmNA OM mmNN N mmOA.MA MOON NM AmMM NOAONN ON AOAO ANM omm U 0 O O O I O O O O m ONO OAOMO NA mmAM NA AwMN N AAON MA ANM MO AOOA O ONO OAA OMM NMA HOME 0 O O O O O O O O O QH OMO A AmNA NA mOh MA OAMM N mmOM MA OAMO MM OAMO h AOAA NO AOAN NNM HOME 0 O O O O O O O O I m OMM A OOM AA mMM MA OMO N AAONN AA AOAN OM ONN O OmON NO mmMM AOM Ban 0 I O O O O O O O O O CH OOOA A OAAA MA mmMA OM OAOA N OANN AA :OAO NM OAMO N OMAN AOA OANM MOA 9mm 0 AOA.A AOAO.AA th.MA AAM.N wAN.NA mM0.0M AAM.N OOA.NN OO>.AMM U OmmhA.A ONA.AA AAM.MM th.A nOON.MA AAMM.NM OMM.A OMO.OOA OAMO.MAA m a a as a a . cm a a 0: oasoue mschuemm mAAMAnomnm m0mOAAB usefiumwus mowomsz mm mmqemmz mamzAaema azm maAAAzeH mqm¢8 164 .mcumfi mmumnvm ummmA mo HOAHO cumpcmumHZAM p .mmumuumme omquAEAAnommAu ~mama-A95 nOAnumU .mucmA0EA 3oAuAU .mmumuummouu .mnmonumo .AMO. vmv HOAAAO mumAuemum0sm ACOAOAAAO nuA3 mcEdAow sAsuA3 mammE ucmEummnan .mEmuO :A onmsE Am0 A00 ppm was .Amv cAmuou0 .Aozv musumAOE Amuoam AN.A OO.AA NA.NO NM.M M0.0M hA.MhN 0soa0 mmummamAm AmAAwmm MN. MN.A NM.> MM.M Om.OA OM.NM osmm AmAA.M AOAO.OA eMN.NNA MONA.>N meh.>ON OOO.ANAA Emmemu ANMA.N mmammN.om mmAm.NON amAm.mN ANA.NMN amm.AAm ammaae, Omah.m OAONM.NA AONM.OON OMN.AA OANN.ONN AOh.MOOA emomu mam.m _ mmON.Am amOA.NON OAAo.mm mamN.AmN AAAm.MNOA azaae OMM.N AONM.MA mMO.MNA OAAO.MM mOAMNN wAM.NMN U mmMO M 3AM.MM ANO.ANN mmhM NN OAOO.M~N AAA AOOA _ m a a o: a . a . 0: camoue m mocApcmuAEwm msEAmmAOGOA usefiumwue mAemsz Am 0000002 00002H020BHZ00 02¢ mDEHmmHGZOA 009 00 B¢0 02¢ 2H08000 ~00090002 A¢BOE 2000 mUHO 04¢: 00B¢mam¢0 09 A900V 02000800900900000H0 00 ABm0Bv 020009 Im09m09 00 20HB¢080H2HSO¢ 02¢ 20HB¢090¢U A¢Bm0000000 00 0800000 .AHI>H 0A0¢B 165 .mmumaummo ©OAIUAEAAH000A0 .mucmAOEA SOA£H00 .mucmAOEA BOAHAU .AMO.UV00 AOAAAO mu0Auomum0sm AGOAOAAAO £AA3 mGEdAOO sAnuAB mammE usmEumwue 600005 00HO0U0 U000H MO .HOHHO Uhflficmumflzmm U .mmumuummeno .mumonumo n .NEMAO CA OAemsE Aw0 A00 AMA can .Amv cAmuou0 .Aozv mnsumAOE Amuoam OM.A AM.MN AN.NN NA.M AN.NN AA.NOA NN.MA MO.NNA NO.AAN asome HmunOSMAM AmAuAcH NM. ON.A AN.M NA. OO.A NN.M NN.M A9.0A OM.AM exam NAmAN.N ANNN.NMANNA.NNA NANN.M mNM.NN mNN.NAA Aon.NM mAM.NNN mMN.NANA amaAe . . . . . . . . . m wNO N AmNN NMAmNN NAA mMN N AmMO AA AmNN AMA who NN m.OO NAM mAM NAAA amma e . . . . . . . . . a A Awaa N AmNN NMAmAA NMA OANA M ANA AA ANN NNA mMa OM ANNA NNM AwNM MAAA amma e OANM.M AmAN.NMAmON.MMA OAMM.M ANN.AA AOM.NMA AmOA.NA AmMO.MNM AmOO.AMAA amaze . . . . . . . . . A OAmMO M AmON MM mOA NNA OMN M AeOM AA AmAN OMA ANO MA mNA NOM NOM AOAA azo o ONN.M mAN.AM mOM.MNA ONM.M AeNN.NM AwMM.MMA AAM.NA mMAMNN oNM.OOOA e AmNN.N AMN.OA AOA.AAA ANA.M ONN.AM OAM.NON mAO.AN AOO NNM ANN.NOMA m a a oz a a as a a 0: eazoue mschuomm mAAMAnomum AAnomum NWOOAHB ACOEAMOHB AWonmsz 000000: 0002H9000 02¢ 0HA¢H00¢00 .HH00¢00 0000H09 009 00 9¢0 02¢ 2H09000 .00090H0: A¢909 2000 00H0 00¢: 009¢090¢0 09 A9000 02000900900900090H0 00 A9009v 020009 I009009 00 20H9¢090H2H:0¢ 02¢ 20H9¢090¢0 A¢9000009000 00 0900000 NAI>H 0A0¢9 166 .mcmme mmHmflAmvm 0.0004” MO .HOHHG @HMUGMUmflzmm U .mmumaumme OOAIAAEAAuommAu_ .NAGMAmaA :OAnumU .mAGMA0EA BoAuAU .mmumuummono .mumonumo .AMO.V my AOAAAO mumAAemam0sm uanmAAAO nuA3 mcasAoo :AnuA3 mammE ucmaummuan .mEmHO GA onmSE “$0 A00 AMA 0cm .Amv :Amuou0 .Aozv musumAOE Amuoem MO.N MA.NO M0.0>M AN.NM AA.NOM ON.MO>A 050AO umuzNaNAN AmAuAaIA. AN.A MA.M NN.MN OM.> MM.MA NN.MNA ozmm NANM.NN mNN.NMA mNN.NNM NANN.NN NN.NMN. MA.NNNN aamAe ONN.AA ONA.MNA OAAO.MMM mNA.OM NN.MMN NN.MNOM ammamu . . . . . . A AAM AN OAAO NNA OAMA MAM OmMO ON ON ANN AN OMOM Emma o OAAO.MN OAOO.MMA OAMA.MNN AAAAM.NO AN.ANN NN.MMAM 93000 O O O O O I ‘H OAMO AN OAOO OOA AmOM hNM nOMO OOA NO NAN OM MAOM 9mm U OMM.MN NVOMJNAA eNO.MMM SANA.NO AA.NNN OM.NNhN U AMA.OA OMN.O>A OAA.ONO OONO.ON O0.0MN NO.9NON m A _ 0 02 m 0 oz e0souu mamocAoamuAEmm mSEAmmAOcoA AcmEumeB Qmonmsz 00A000: 00002H0209H:00 02¢ 00:HO0H020A 009 00 9¢0 02¢ 2H09000 .00090H0: A¢909 2000 00H0 00¢: 009¢090¢0 09 A9000 02000900900900000H0 00 A9009V 020009 I009009 00 20H9¢090H2H:0¢ 02¢ 20H9¢090¢0 A¢9000009000 00 0900000 MAI>H 000¢9 167 2 a 1: “ a U .:;....... '— m . IIII .:::....O o -d -- III F I— "" M gifiafi .O...... F U ---- I I ' ' 3 0 0| . -- - : z . s I I It! : 5‘ .- m m .- II .:.:O. m . ” IIIIII 9.0-..2-2 JanIH Jame-I (0,0) sasnusoa OI SAUDI?! uoIIanao In; .IoInasnuImlul FIGURE IV-3 . Intramuscular fat accretion in the triceps brachii, brachialis, pectineus, longissimus and sanitendinosus muscles of boars, androgen implanted castrates and limit-fed castrates oftheprepubertalsuidyasapercentageof castrates. FIGURE IV-4 . 168 '- “" m IIII. I- E 5 E 0 00.000000000000000 ' o i- I“; a “““‘ .1 I -l _J 0 u U 0 U U I . . I a IIII I ,: .: 6 . ’ . I ' 9 a : U “““ a. I... K m . III-I m “““‘ .— III...- 3 $ 6 5 .. .. s2 ,_ g JanIH .IaM01 (70) 584014503 0; aAIID|aJ uouanao In} Jolnasnwoqu Intramuscular fat accretion in the triceps brachii , brachialis, pectineus , longissimus and semi- tendinosus muscles of boars, androgens implanted castrates and limit-fed castrates of the pubertal study as a percentage of castrates . 169 that both implants levels of DHT were ineffective in preventing intramuscular fat deposition but the high dose of TEST reduced intramuscular fat deposition. This indicates that TEST and DHT differ in their effect upon intramuscular fat deposition and supports the hypothesis that testosterone must be aromatized to estrogen metabolites to mediate effects on fat deposition (Wade and Gray, 1979). However, another alternative hypothesis is that reduction of TEST to DHT is minimal, whereas DHT is rapidly converted to diols (Warrenski and Almon, 1983). Limit-feeding castrates also depressed the castration associated lipid accretion to a level similar _to CHTEST in PREP pigs; however, CHTEST in the POSTP study was more effective than the limitmfed castrates. In order to assess the effects of castration and .TEST or DHT upon fat-free muscle accretion or essentially protein plus water accretion, the quantities of fat in tables IV-10 through 13 were subtracted from the muscle weights presented in tables IV-2 and 3. These fat-free muscle weights (FFMW) are presented in tables IV-14 and 15 for the PREP and POSTP studies, respectively. Fat-free muscle weights of initial pigs were subtracted from the treatment groups of pigs and these net values are expressed as a percentage of the net change incurred by castrates. These relative individual muscle FFM acretion rates are illustrated in figures IV-5 and 6 for the PREP and POSTP 170 .00005 WQHMAAmum “0000.. M0 HOHHG UHMUCMHAWHZWM o .mmummumme owAIuAEAAuommAO .mucmAQEA nOAnumo .mucmA0EA 3oAuAO .mwumuummeno .mumonume .AMO. v00 AOAAAO mumAHomammsm AcmHmAAAp nuAs mcadAoe :AnuA3 mamma usmfiumwuan .mEMAO :A USOAOS onmDE mmHA ummm NN.NA A.NA M.NAN A.NN N.NNA .mmoue . umumNamAN AzAquA MO.N AO.M NN.NA MN.A MM.OA Ozmm . . . . . A OM NMN AOO AM AM MMNA OM OM AmM NNA cum 0 . . . . r m AO MMN Awh AM OA MMAA AA ON ON MMM HOME 0 . . . . . A AmM MMN 0N NM AM MONA Omm NO AwA MNA HOME 0 . . . . . m Ame NMN ma MM AA MNNA 5AM MN AmN MNA sun 0 . . . . . A AmA NMN AwA AM OAN ANMA OAN MN OAN AAM Ban 0 0A.AMN mA.NM mN.ONOA ON.NO wA.NAA U AA.NNN AN ON AN NNNA OAM.NM OAN AMM m mamocApcmAAEwm mschuemm msEAmmAOAOA mAAmAnemum AAnemam cmsouw m0meAA9 unmaummaa mAemsz Am mazeamz mAemOz mzmozAazmaAzzm azm mamzAaeza .mzzammaoZOA .maAAAzemmm .AAmemzm mameAza mmzaIama zoae meAa AAA: amaANaNAe oa Aazae mzozmamoammaozawzaa mo Aammae mzozma Imoamma mo zoAamzamAzAzaA azm zoAaAmamme AAammmOamza mo maezaam VAI>H 040¢9 171 .mcmmE mmAMAOm ummmA A0 Hoaum Unmccmumuzmmc A A U .mmumuummonu .mumonumo .mwumuummo UOAIAAEAAuomm U .mAGMA0EA nOAnumU .mucmA0EA 30AM .AMO.v 00 HOAAAU mu0AHomum0sm AcmAOAAAO 3AA3 mQEdAoo cAsuA3 mammfi ucmeummusn .mEmuO cA unOAO3 onmsfi mmAA ummm A.NNA NN.NNA N.NNNN N.MMA N.oNN amoue umunmzwAm.AzAuAaA NN.NM NN.N . NN.NNA AA.N NN.NA exam mN.ANN ANNNA A.NNNN mNNNA NN.MAMA amaAe AN.AAN ANN.NNA N.NNNN Amo.NNA NN.NMAA aNmaze AN.ANN ANN.NNA N.NMNA AN.AAN Amo.MNAA ammaae AN.NNN AAM.NNA N.NMAA AA.NAN AmNNNAA amaze ANM.AMN mA.NMA A.NMNN AmN.NNN mm.AAAA amaAe NN.NNN ANN.NNA A.NNMN AmNANA mA.oNNA e AN.NAN ANNNA .N.NNNN NN.NMN NN.NNNA m mcmocApcmuAEmm msmsAuomm msEAmmAOAOA NAAMAnomum AA£OMAm Omdono m0meAA9 ucmaummna nmonmsz 0900H03 0A000: 00002H0209H:00 02¢ 0002H9000 .00:HO0H0200 .0HA¢H00¢00 ~0000H09 0000I9¢0 2000 00H0 00¢: 009¢090¢0 09 A9000 020009009009000>0H0 00 A9009v 020009 I009009 00 20H9¢090H2H:0¢ 02¢ 20H9¢090¢0 A¢9000009000 00 0900000 mAI>H 040¢9 172 castrate ST 00000? 5 PC I. D Prepuber to I ' I I I a " " " ' CLDHT "0°“ CHDHT ........ CHEST ClFED i ooooooooooo::::’ a. ----_- I- - o o O O . O ‘9 N 1- 1- 1;»?ng % JaMO'I % samusna o; annals: uouanaa was FIGJRE IV-S . Fat-free triceps brachii , brachialis , longissinus , platineus and semitendimsus muscle accretion of bears, androgen' implanted castrates and limit-fed castrates of the prepubertal study as a percentage of castrates. 173 2 O b ‘5 O u "”.'.'.".L°."." .— 0..- m III...- 0.....‘.: U G. I... - h I....... 2 m U U U U U l.- b o o. I o ' : .gll : z : : I . fl.- ' A I : .“‘ - 2 III-II...- ..'3'.".’3'.' a ---- h III-III...- " 1305!“: 13M01% 53401350: 0; aAquaJ uogsanao wag FIGJRE IV-6 . Fat-free triceps brachii, brachialis, longissimus, pectineus and semitendinosus muscle accretion of boars, androgen implanted castrates and limit-fed castrates of the pubertal study as a percentage of castrates. 174 studies, respectively. It appears that castration prepubertally and pubertally affected FFM accretion for the TB, BR, LD, PC and ST muscles. The effect of castration was much more evident in the POSTP pigs than for PREP pigs. All androgen treated groups had greater FFM weights (except for the PC) than castrates. However, muscles varied in the degree of response. The effect of castration in this study resulted in a depression of FFM accretion in selected muscles. This observation is not consistent with data of Knudson (1983) who reported that 105 kg boars and castrates did not differ in fat-free LD, ST or BR muscles. However, there were tendencies for boars in that study to have 13 and 12% heavier fat-free ST and BR muscles, respectively, than castrates. Muscles have differential growth patterns and some muscles exhibit these patterns at different stages of growth (Richmond and Berg, 1982). Increased growth in the neck and shoulder muscles has been observed for bulls (Brannang, 1971), rams (Lohse, 1973) and boars (Walstra, 1980). Whether the age of castration influences differential muscle growth patterns has not been tested in pigs. Since muscles have individual growth patterns, genetic, nutritional and endocrine influences on muscle growth may be different depending upon the stage of develOpment of a muscle or muscle group at the time of imposing the particular treatment. 175 In the present study, the response to castration was observed (n1 selected mmscles both prepubertally and pubertally. The response to exogenous testosterone on selected muscles has not been previously demonstrated in pigs. However, similar types of responses on muscles have been observed in testosterone treated guinea pigs (Kochakian et al., 1956). Ski contrast, previous studies with testosterone administration to pigs have indicated only minimal responses to overall body composition (Sleeth et al., 1953; Bratzler et al., 1954). Differences in the mode of hormone delivery (injection versus implant) may explain some of the differences in total carcass composition between this study and other. Production of as much lean as is economically possible is important to the swine industry. The boar is clearly one alternative for this goal because of their enhanced muscle deposition over castrates. However, the boar is not compatible with the current marketing scheme in the United States due to boar taint at typical market weights. Alternatively, boars could be marketed at lighter weights or castrates could be implanted with androgens, Summary In the 3 wk study, the triceps brachii and semimembranosus muscles were 1.1- and 1.2-fold heavier than castrates. In both 5 wk studies, boars had heavier triceps 176 brachii, brachialis, longissimus, pectineus, teres major and semitendinosus muscles than castrates. Androgen treatment of prepubertal pigs appeared to be more effective in significantly increasing muscle weights over castrates than in pubertal pigs. Of the muscles examined, only the semitendinosus was significantly increased in weight over castrates by the high testosterone implant. High testosterone implanted pigs as well as boars had less intramuscular fat than castrates. However, the fat-free muscle weights followed similar trends between treatments as wet muscle weights. All androgen treated pigs tended to have heavier fat-free triceps brachii, brachialis, longissimus and semitendenosus weights were significantly heavier. These data suggest that some muscles are more responsive to androgens than others and that testosterone may be more effective in stimulating muscle growth than dihydrotestosterone. 177 CHAPTER V EFFECTS OF CASTRATION AND ADMINISTRATION OF ANDROGEN UPON IN VITRO PROTEIN SYNTHESIS AND DEGRADATION RATES Introduction Practical and efficient means of enhancing skeletal muscle protein accretion in growing meat animals is of considerable interest. To study protein accretion, precise measurements of protein synthesis and degradation rates must be determined. The most apprOpriate methods for measuring skeletal muscle protein synthesis rates are by continuous infusion of radiolabeled amino acids (Waterlow et al., 1978) or a flooding dose (tracer + unlabeled amino' acid) method (McNurlan et al., 1982; Goldspink et al., 1983). For eXperiments with large animal species, these methods can be costly. Recently, in vitro muscle incubation methods (Fulks et al., 1975) have been _employed to study changes in protein synthesis and degradation rates during compensatory (Goldspink et al., 1983) and normal (Hentges et al., 1983) growth situations, as well as, in animals varying in endocrine and nutritional ‘status (Goldberg, 1980). Mbderate to large differences in muscle growth rates are readily detectable by this method. 178 Intact male cattle, sheep and swine have greater muscle accretion rates relative to castrated males (Galbraith and TOppS, 1981). In addition, boars have higher metabolizable energy expenditures for maintenance than castrates (Fuller et al., 1980; Walach-Janiak, et al., 1980). Presence of the testes or administration of exogenous testosterone enhances incorporation of labeled amino acids into skeletal muscle proteins (Grigsby et al., 1976) and growth of skeletal muscles (Kochakian, 1976). Protein deposition is associated with elevated protein turnover (Kielanowski, 1976; Reeds et al., 1980). Administration of exogenous testosterone also mobilizes amino acids (Kochakian, 1976). A synthetic analog of testosterone, trenbolone acetate, apparently decreases both skeletal muscle protein synthesis and degradation rates in female rats but accretion of protein is increased due to a disproportionate depression of degradation rates (Vernon and Buttery, 1976; 1978). Testosterone and trenbolone acetate may mediate their effects by different mechanisms. It was the objective of this study to investigate the effects of castration and administration of testosterone or dihydrotestosterone to castrated prepubertal and pubertal male pigs upon in vitro skeletal muscle protein synthesis and degradation rates. 179 Materials and Methods In Vitro Protein Synthesis and Degradation. Immediately after cessation of bleeding of pigs at slaughter, the left semitendinosus muscle was exposed and strips of muscle Obundles were obtained by blunt dissection. Clamps were constructed from welding 2 Mueller alligator clips to a metal wire to separate the clamps by about 1 cm. The muscle strips were clamped to maintain passive tension and then excised. The muscle strips restrained within the teeth of the clamp weighed approximately 100 mg and were less than 2 mm thick. The method described by Fulks et al. (1975) were used to estimate protein . synthesis and degradation rates. All strips were preincubated in oxygenated Krebs Ringer bicarbonate (KRB) buffer for .5 h. Incubation (2.5 h) in oxygenated KRB buffer (pH 7.4) containing insulin (.10/ml), glucose (10mM), amino acids (5X plasma concentration; Appendix B-1) and U-[14C] L-tyrosine (.2 uCi/ml) was used for measurement of protein synthesis rates. For determination of degradation rates, strips were incubated for 3 h in the same buffer as described above except that no labeled or unlabeled tyrosine was added and cycloheximide (.SmM) was added to inhibit protein synthesis. At the end of the incubation, incubation vials were placed on ice, the muscle strips weighed to the nearest milligram and then placed in corex tubes containing 1 ml of phosphate buffer (Appendix B—2). 180 Muscle strips were homogenized by a Brinkman polytron (setting 7) by giving 3 intermittent 20 sec bursts. The polytron tip was rinsed with 1 ml of phosphate buffer into the homogenate tube to which 500 ul of cold 50% TCA were added. Homogenates were centrifuged at 30,000 g for 20 min and then the supernatants or the intracellular pool was decanted and saved. The precipitates were washed two additional times with 5% TCA and the washings pooled with the original intracellular (IC) fraction. Precipitate and IC fractions were washed 3 times with anhydrous ether to extract TCA. Aliquots of the IC pool for synthesis were counted for radioactivity. Dried protein precipitates from Synthesis incubations were digested in 500 ul NCS tissue solubilizer (Amersham) and counted for radioactivity. Media or the extracellular pool (EC) from degradation incubations were precipated with 50% TCA and handled similarly to the IC pools. Aliquots of the IC pool for synthesis and degradation and EC pools for degradation were analyzed for tyrosine contents by the fluorometric pro- cedure described by Ambrose (1974). Results and Discussion Figure V-l illustrates results of preliminary work for in vitro protein synthesis. Incorporation of radiolabel into protein was linear for the 2.5 h time period routinely used in the experiments. In addition, the intracellular 181 PROTEIN SYNTHESIS IN VITRO INCORPORATION (CPM/MOI INCUBATION TIME (H3 2 3 300 : -----------. ‘- > <.. 5'" 200 I, o j< I no; I 0" 100 I (“'3 I 133‘ Pm: ’ In." I A‘s FIGURE V-l . Time course incorporation of label into protein and specific radioactivity of intracellular pool . 182 PROTEIN DEGRADATION IN VITRO 3 3-2 sxrnAcsLLULAI-I/ E 2.8 I a I I E 2.4 ’ I 3 2.0 ,’ 3 1 6 I ' I i ’I a: 1.2 ’ g I 3 .8 ” o 4 I g - I INTRACELLULAR I .- 1 2 3 4 INCUBATION TIME (H) FIGURE V-2. The linear time release of tyrosine from muscle strips . 183 pool was in equilibrium with the extracellular pool and specific radioactivities were constant (after an initial lag) for the period of incubation. Figure V—2 illustrates the linear time release of tyrosine from muscle strips for over 3 h with essentially no change in the intracellular content of tyrosine. The effects of castration and administration of testosterone (TEST) or dihydrotestosterone (DHT) to castrated male pigs upon in vitro protein synthesis and degradation rates in prepubertal (PREP) and' pubertal (POSTP) pigs are presented in tables V-l and 2, respectively. In the PREP study, castration resulted in a 49 and 61% reduction in in vitro protein synthesis rates for the‘ white and red portions of the semitendinosus (ST) muscle, respectively. Due to differences in synthesis rates between red and white muscle strips, interpretation of hormone administration is complicated. In general, red portion strips had 3 to 5% higher synthesis rates than white strips from castrates and the implanted groups. However, red strips from boars had 34% higher synthesis. rates than white strips. This could possibly mean the presence of the testes and factors other than testosterone differentially affect red muscle more than white muscle fibers. Only the high TEST (CHTEST) implanted pigs had synthesis rates similar to boars, but they had 76% higher rates than castrates for white muscle strips. Although 184 .mcmmE mumsvm ummma mo Houuw onmocmumHZMm .mu:.maQEH noes" Imo .mucmHmEH BOHHAU .mmumuummouu .mumonum .Amo.v my Hummeo mumfluomquSm ucmuwMMflp nuflB mcEsHoo casuaz mammz .£\mHOmDE mE\MflcmE ob Ummmwamn no aflououm once omumuomuoocw mnemouxu mo mmaoeoowm mm ommmwumxm mumam .QU'U om.mm mm.~e om.q oo.m czmm mH~.aeH nom.oma ama.mm muee.ma emmemo umme.o~m mma.mea mmwe.mm umoa.om ammaqo emaa.amm mom.mefi mmom.aa mmmn>.~a amaze mam.mmm mae.omm umme.am mmm.~m amaao mam.fiom _ www.mov mna.m~ mmm.m~ o mma.aam moo.maa nma.me mam.mm m cofluuommpmm cofiuuom open: cofluuom pom cofiuuom‘wuflnz ommflonw . ucmfiummue mumn :oflumpmummp cflmuoum mumu mammnucmm :Hmuoum omeH> zH ammommmz mmeam oneeo mamas 185 .mcmmE mumsvm “mama mo uouum oumocmumnzmm U .mucmHQEH smflnumo .mucmHmEfl 3OHHAU .mmumupmmouu .mumonumo .Amo.v my umwwflp mumfluomummsm ucmummwflv nuflB mcEDHoo canufi3 memo: n .£\maomsE @E\mwoms ou Ummmmawu no cflmuoum oucfl omumnomuoocfl mnemouxu mo mmHOEOOflm mm ommmmhmxm mumom H>.ba mm.vm om.v mm.a Ume . . . , . .m who mom mom mmm wmhv mm mum Hm Emma U C O O O ‘H mono mum ohm mam one mm mma om Emma U . , . . . . m mom Nam . mob mom mam Hm mam mm Ema U . . . . A mom mmm mmmm nmm mmoo wm mam «N 9mm 0 moo.aom mmo.vmm mmo.vm mph.om U mam.mmm . mNH.mmv mmv.om mom.Hm m cofluuom pom cofiuuomxmufinz cofluuom pom :ofluuomxmuflsz ommsouw ucmfiummua mumu GOHHMCmnmmU cfimuoum mumu mammnucam cflmuoum n.momeH>.zH DmmDm mqmfie 186 nonsignificant, other treatments tended to have slightly higher~ protein synthesis rates in the white portion. For the red portion strips, high DHT (CHDHT) and (CHTEST) pigs had 53 and 79% higher synthesis rates, respectively, than castrates but 41 and 31% lower rates, respectively, than boars.- The synthesis rates for the red muscle strips for CHDHT and CHTEST were 19 and 31% higher, respectively, than the corresponding low implant treatment groups. These data indicate a dose response trend for DHT and TEST with TEST also being more effective than DHT in stimulating synthesis rates in red muscle of PREP pigs. Protein degradation rates were not different for either white ‘or red muscle strips from PREP boars or castrates. However, red strips from boars and castrates had 8 and 25% higher degradation rates than white, reSpectively. This trend for higher degradation rates in red relative to white muscle strips was also observed for the androgen treated groups and is consistent with reports by Millward (1980). Administration of TEST in the present studies depressed degradation rates relative to boars and castrates. While degradation in red muscle strips of low DHT (CLDHT) implanted pigs was 2-fold higher than CHTEST pigs, no other differences existed between the implanted groups. Nevertheless, subtle dose response trends toward decreased degradation rates were apparent for both red and white muscle strips. CHDHT implanted PREP pigs had about 20% 187 lower (P < .05) degradation rates than CLDHT pigs and muscle strips from CHTEST pigs had 22 and 33% lower (P < .05) degradation rates for white and ared strips, respectively. For POSTP pigs ‘(table V-2), castrates had 34.5 and 32.6% lower synthesis rates’ than boars in white and red muscle strips, reSpectively. This castration associated reduction is lower than the 49 to 61% reduction shown in the PREP pig muscles. Synthesis rates in red muscle strips were 59 to 64% higher than white muscle strips for boars and castrates, reSpectively. PrOportionately, these differences between red and white strips within a treatment group are .greater than were observed in PREP pigs indicating the age of castration may be influencing these differences. Except for red strips from CHDHT pigs, red muscle strips of androgen implanted pigs tended to have higher (P < .05) synthesis rates than castrates but all implanted groups had lower rates than boars. Except for white muscle strips of C DHT pigs, implanted pigs had L about 45 to 72% higher white muscle strip synthesis rates than castrates. POSTP castrates had 26.6 and 24.5% lower degradation rates than boars for the white and red muscle strips, respectively. For degradation rates of white strips, implanted pigs had lower degradation rates than either castrates or boars. No dose dependency was observed but white strips from CHDHT, CLTEST and CHTEST pigs, 188 were approximately 34.5 and 52% lower than castrates and boars, reSpectively, and red strip synthesis rates were consistently 36% lower than boars. Only red strips from CLDHT and CHTEST pigs were different (25.6 and 30.6% lower, reSpectively) from castrates. Degradation rates appeared to be depressed by androgen treatment. Even though, red and white strips differed (P < .05) in synthesis and degradation rates for boars and castrates, these data were averaged and the averages are presented in tables V-3 and 4 for PREP and POSTP pigs, respectively. The averaged data showed similar trends to those previously discussed for separate red and white muscle strips. In order to compare treatment effects, the data of each treatment group were eXpressed as a percentage of the boar values. The synthesis and degradation rates relative to boars are presented in table V-5. The percentages for synthesis and degradation rates of castrates and implanted pigs were all lower than boars (negative). The lower the 'percentage, the closer the rate was to that of boars. Low implant, PREP pigs were not different from castrates, and high androgen implanted pigs had higher synthesis rates than low implants but they were still lower than boars. A dose response trend appeared to have occurred. Implanting PREP pigs with DHT or TEST decreased degradation rates relative to boars or castrates. In addition, a subtle dose response trend was detectable. 189 .mcmmE mumsvm ummma mo Houum pumocmumuzmm U .musmaaefl cmflnwmo .mucmHQEH BOHHAU .mwumuummonu .mumonumo Ame. vmv umwmwp mumfluomummsm ucmumMMflU Suez mcEsHoo Canvas memo: Q .n\maomse mE\mHomE ou Ummmwamm Ho samuoum owcH wmumuomuoocfl mcflmouhu mo mmHOEoon mm Ummmmumxm mumom am.mm sm.m 62mm mos.wma mem.om emmemo mmv.mmg mmmm.mm emmeqo mmo.mom mumm.ma amaze mom.mmm emmm.am emoao usa.~mv mve.m~ o www.mae :m.mo m mumm :oflumwmumwn cflmuoum mama mammnucmm :HmUOHm UQDOHU HamsmmHB £~MMAUmDE mDmOZHQZMBHme Ema ho mZOHBmOm mBHmZ DZfl 0mm WEB mom Qm0< 0mBH> ZH QmmDmfimz mmedm ZOHBfidewmo 024 mHmmmfizwm ZHWBOmm ZOmD mUHm EA¢Z Dm9¢m9m mqmfia 190 .msmmE mumsvm ummma mo Houum pumwnmvmuzmm c .mucmameflmnmfisumo .mucMHQEH BOHHAU .mwumuummouo .mumonumo ,Amo.v my umMMflo muofluomummsm ucmHQMMHU sues mCESHoo sflaufiz mama: n .£\mHOmSE mE\MHUmE ou ommmmamu no :Hmuoum oucfl omumuomnoocfi deflmouwu wo mmHOEooHQ mm ommmmumxm mamas mh.ma m>.H UEmm mmm.mHm .mmefi.mm emmemo www.mqm ummmqam emmeuo mmH.HeN umme.mm amaze 6mm.oem umoa.om sumac mam.aam moa.n~ o moo.ema . meo.aa m mumu :oflumomummc :flmuoum . mums mflmmnushm :Hmuoum omwouo usmEumwmm n.mmn0m32 mDmOZHQZWEHZHm HEB m0 mZOHBmOm MBHSB 02¢ 0mm mmB mom Omwflmm>¢ OMBH> ZH QmmDm mnmfle 191 .mucmaaau emme gens" mmemo .mucmHasn smug a U .mucmamEH Ema BoHuBmo U .mmumuummouu .mumonumw 30H ”Emma o .mucdesn emu nmflcuemam .mcoumumoummuuemme .mcoumumoummuoupmsflpuemop .oomonm>m mum3 mflmocflpcmuflEmm 0:» mo mcowuuom own can muflsz MOM mmummo .mumon mo mmmucmoumm o no ommmmumxm mum mumon mmcflmmn mpcmEummHu Hmsuon .£\mHOmDE mE\mHUmE on ommnmamu no cflmuoum owcH omumuomuoocfl mcflmouwu GHOEOUHQ mm Ummmmumxm mumon Mow macaw o.ma- «.4HI «.meu m.-- emmemo ~.~au H.mm- ~.Hm- «.mau ammeqo m.mm- o.mHI m.mm- m.mmI emomo o.ma- o.m~- «.mau v.04- anode m.m~- m.mm- m.ma m.oma o IIIIIIIIIIIIIII 0w .mumon 0p m>fiumamu mmummIIIIIIIII o.e~a H.Hv v.mmv m.mo m nmom mmmm nmom mmmm m.omsouo ucmEummHB Hmunmnsm Hmuumnsmmum o.n.mmon mq 2H zomo mon was: ome mamas 192 For POSTP pigs, the averaged red and white muscle strip synthesis and degradation rates are presented in tables V-4 and 5. In general, synthesis rates were only 33% lower than boars. As indicated by lower percentages in table V-S, a dose response trend appeared to have occurred even though only the synthesis rates of CHTEST pigs were different (P < .05) from castrates and similar (P > .05) to boars. In general, these protein synthesis data are consistent with the increased muscle ribosome activity of testosterone treated castrate rats compared to castrated rats observed by Florini (1970). Degradation rates were also 26% lower in POSTP castrates than boars and implanted pigs were lower than either boars or castrates. In contrast to the PREP pigs, no dose reSponses for TEST .or DHT upon protein degradation rates were apparent in this study. In Comparing PREP synthesis rates to POSTP rates, it appeared that synthesis rates were 37 and 5% lower (significance not tested) for POSTP boars and castrates, respectively, relative to their PREP counterparts. Degradation rates were 14 and 30% lower (significance not tested) in POSTP boars and castrates, respectively, relative to their PREP counterparts. The effects of providing tension to in vitro incubated muscles alters responsiveness to nutrients in the incubation media and can increase synthesis and decrease degradation rates (Etlinger et al., 1980). While attempts 193 were made to be consistent in the amount of stretch or tension applied to strips used in this study, variation no doubt occurred in these studies. The degree of stretching alters prostaglandin synthesis in incubated muscles (Smith et al., 1983) and PGFZG and PGE2 have been shown to influence protein turnover in in vitro muscle incubations (Rodemann and Goldberg, 1982). Generally in vitro muscle protein synthesis and degradation rates are obtained by incubation of whole thin muscles. Our exPeriments and those of Hentges et a1. (1983) involved muscles that had been cut. However, based upon preliminary data and inulin space, ‘incubation of strips with the clamps to maintain tension had no adverse effects upon either synthesis or degradation rates. Strips incubated with the clamps had lower degradation rates than free floating muscle strips. It appears that the muscle strip technique used in this study was sensitive enough to detect differences in rates of protein synthesis and degradation. between boars, castrates and androgen implanted castrates. The tendency for higher synthesis and higher degradation rates and therefore higher protein turnover in boars relative to castrates is not inconsistent with higher metabolic rates of boars (Thorbek, 1975) relative to castrates nor for the model of increased protein turnover with increased heat production and greater protein deposition rates 194 (Kielenowski, 1976; Reeds et a1, 1980). Fat-free muscle mass accretion rates were similar between PREP boars and castrates but were higher in boars in the POSTP study (Chapter III). Semitendinosus muscle accretion was highest in the boar in both studies (Chapter IV). Thus, is a definite correlation in this study between ,fat-free semitendinosis muscle accretion rates and both increased protein synthesis and degradation rates as measured in vitro. Since the magnitude of difference of in vitro protein synthesis and degradation rates between boars and castrates was greater for protein synthesis rates, it appears that greater semitendinosus muscle accretion occurred in boars because of higher synthesis rate relative to degradation rate. In fact, if a ratio of synthesis rate to degradation rate is calculated, boars had higher ratios than castrates in both studies. Castration decreased in vitro protein synthesis and degradation rates but synthesis rates were decreased to a greater extent _than degradation. TEST and DHT were shown to partially counteract the castration associated fall in synthesis rates but implants also appeared to decrease degradation rates. These responses need to be verified by in vivo mesurements. However, if these in vitro measurements are a true qualitative reflection of the in vivo action of androgen (either direct or indirect) then it would appear that the mechanism of action of these two 195 androgens is mediated differently than trenbolone acetate which decreases both in vivo synthesis and degradation rates (Vernon and Buttery, 1978). In addition, the differences between boars and androgen implanted castrates in in vitro protein turnover indicate that other factors are probably involved in regulating protein turnover in the boar. Based upon the data from this study, implanting castrated male pigs with androgen may have the potential benefit of increasing the efficiency of muscle protein deposition. Failure to show direct effects of testosterone upon protein turnover in muscle cell cultures (Allen et al., 1983; Ballard and Francis, 1983) suggest that in vivo testosterone effects are apparently mediated via some indirect mechanism. Summary In both prepubertal and pubertal studies, boars had higher in vitro protein synthesis and degradation rates than castrates. This indicates that the greater semitendinosus muscle growth rate may be associated with higher protein turnover rates. Androgens tended to increase protein synthesis rates to castrates. These data suggest that both forms of androgen are mediated by similar mechanisms for affecting muscle protein turnover and would appear to increase protein deposition by increasing protein synthesis. and reducing degradation. 196 CHAPTER VI EFFECTS OF CASTRATION AND ADMINISTRATION OF ANDROGENS UPON SKELETAL MUSCLE PROTEINASE ACTIVITY Introduction Protein accretion rates in skeletal muscle are dependent upon the net difference between synthesis and degradation rates. While the mechanisms of protein synthesis and measurement of protein synthesis rates can be assessed by a variety of methods (Waterlow et al., 1978), skeletal muscle protein degradation is a poorly understood process (Millward et al., 1981). The rate of skeletal muscle protein degradation is higher during periods of rapid muscle growth than in adult animals (Waterlow et al., 1978). Attempts to correlate protein accretion and turnover to catabolic enzymes of lysosomal and nonlysosomal origin have met with limited success. Millward et a1. (1981) illustrated a variety of different growth situations which are correlated to changes in cathepsin D (lysosomal enzyme) activity. Other workers (Mayer et al., 1974, 1980; Noguchi et a1, 1974; Dahlmann et al., 1978, 1980, 1981) have demonstrated a high relationship between protein degradation and activity of alkaline proteinases which are closely associated with the myofibrils. In contrast, 197 McElligott and Bird (1981) reported that these alkaline serine proteinases are of mast cell origin and play a minor role in the degradative process of muscle proteins. However, Dahlmann et al. (1980) and Dahlmann and Reinauer (1981) treated castrated male rats with testosterone and attenuated a castration associated increase in alkaline proteinase activity in skeletal muscles. The objective of this investigation was to observe the effects of castration and testosterone or dihydro- testosterone administration to 'prepubertal and pubertally castrated male pigs upon acidic, neutral and alkaline proteinase activity in crude skeletal muscle homogenates. Materials and Methods The amount of enzymatic hydrolysis of 14C hemoglobin at pH 3.8, 7.0 and 8.5 was used as indicator of proteolytic activity of semitendinosus muscle extracts. Labeling of porcine hemoglobin was prepared according to Roth et al. (1971). Small batches of hemoglobin were labeled and later pooled. Five hundred milligrams of porcine hemoglobin were dissolved in 15 ml of mega pure water and adjusted to pH 6.1 with .1 N NaOH. Fifty microcuries of K14CNO (52 mCi/mmol; Amersham) dissolved in mega pure water were added to the dissolved hemoglobin. The mixture was incubated at 50 C for 2 h and then stored overnight at 4 C. Two mililiters of mega pure water containing 20 umol of 198 cysteine hydrochloride (pH 6.1) were added and the mixture incubated at 37 C for 2 h. The solution was transferred to dialysis tubing (12,000 MW cutoff) and dialyzed for 48 h against 8 to 10 changes of deionized water. At the end of the dialysis, .1 ml was counted in 10 m1 ACS (liquid scintillation cocktail; Amersham) and resulted in 7000 to 10,000 Cpm‘ and after precipitation with 5% TCA, 200 to 500 cpm were observed in the acid soluble fraction. The labeled hemoglobin was stored at -20 C until used. Skeletal muscle was extracted by methods described by Rothig et a1. (1978). Approximately 200 to 300 mg of powdered muscle were transferred into previously weighed, rubber stOppered corex tubes and after equilibration of the sample to room temperature (about 5 min), the quantity of muscle was determined to the nearest .1 mg. Samples were placed on 'ice and 5 m1 of cold .05 M tris-HCL (pH 7.4) buffer containing 1 M KCL and .2% (w/v) Triton x-100 were . added. The mixture was then homogenized with a Brinkman polytron at the 7 setting by giving three 20 sec. bursts. Homogenates were- centrifuged at 3000 X g for 10 min at 4 C and supernatants decanted through a single layer of cheesecloth. Homogenization of the remaining precipitate was repeated as above and after centrifugation, supernatants were pooled. The muscle extracts (supernates) were brought to equal volumes (such that protein concentrations were less than 5 mg/ml) with buffer and 199 stored at -20 C until assayed for proteolytic activity and protein (Lowry et al., 1951). After preliminary testing, a standard incubation system was used for determination of proteolytic activity in the muscle extracts. Routinely, 200 ul of muscle extract, 125 ul of labeled hemoglobin and 200 ul of citrate-phOSphate- borate buffer (appendix B—2) were incubated at 37 C in a shaking water bath for 2 h. The reaction was stOpped by addition of 200 ul of cold 50% (w/v) TCA. Precipitated protein was spun down at approximately 10,000 X g for 10 min. Control mixtures or blanks containing all components except enzyme or muscle extract were incubated in parallel with the assay and then muscle extract was added. The acid soluble supernatants resulting after centrifugation were extracted 4 times with 5 ml of ether to rid the system of TCA and then aliquots were counted in 10 ml of ACS (Amersham) counting scintillant. Powdered muscle extracts from the red and white portions of the semitendinosus muscle were assayed at pH 3.8, 7.0 and 8.5. Choice of these pH environments was based upon preliminary studies which are explained in the results section. Activities were expressed as blank corrected Cpm per milligram protein of muscle extract added. 200 Results and Discussion In preliminary experiments, the pH dependency of proteolytic activity in the crude muscle extracts obtained from a fasted and a normal ad libitum fed castrated male pig was assayed. Figure VI-l shows the pH dependence of proteolytic activity in which similar trends for peak activities at Specific pH values were observed for both the fasted and normal pig. Even though activities in the acidic, neutral and alkaline range were higher for the fasted pig relative to the normal pig, this preliminary experiment was not replicated and definitive statements concerning fasting can not be made. Nevertheless, Rothig et a1. (1978) reported similar results in rats and even a larger relative difference of activity at pH 9.0 between diabetic and normal rats. Based upon the preliminary pH dependency data, the major peaks were detected at pH 3.8, 7.0 and 8.5 and thus these 3 pH values were selected for all subsequent assays. In other preliminary work, 4 ratios of l4C-hemoglobin to crude muscle extract were incubated for .5, 1, 2 and 3.5 h. These data are shown in figure VI-2 and indicated some substrate limitation by 3.5 h when the amount of extract exceeded that of labeled substrate by 2-fold. However, for a 2 h incubation, linear activity was observed at all ratios of substrate to enzyme tested. Thus, all subsequent incubations were conducted for a 2 h period. 201 j 0 fi “ 0 IL O O Fed l0 8 In N N N I~ N N v- ' '- NIanud MIMO‘I _ow .cha 1 FIGURE VI-l. pH dependence of proteolytic activity of muscle extracts from fasted and fed pigs. 202 CPM' Ms" Lowav PROTEIN Time (h) FIGURE VI-Z. Effects of incubation time and ration of extract to hemglobin substrate (v/v) upon proteinase activity at pH 3.8. 203 Acidic, neutral and alkaline proteinase activities were assessed in the red or white portions of the semitendinosus muscle. Table VI-l presents the least square means for prepubertal (PREP) boars, castrates, low dihydro- testosterone (CLDHT), high DHT (CHDHT), low testosterone (CLTEST) and high TEST (CHTEST) group for the proteinase activity in the acidic (AP), neutral (NP), and alkaline (AKP) pH ranges for the red portion of the ST muscle. PREP boars had 10.5 and 9.4% higher (P < .05) AP and NP activities than castrates. No difference (P > .05) in AKP activity was observed for PREP boars and castrates (although boars had 12% lower activity). For the implanted groups, only CLDHT pigs differed ‘from castrates with 13.7% higher (P < .05) AP activity. For neutral proteinase activity in the red muscle extract, PREP implanted pigs tended to have 4 to 14% lower activity than castrates; however, only CHDHT and CLTEST differed (P .< .05) from castrates. Alkaline proteinase activity of the red muscle extract from PREP implanted pigs tended to be lower than castrates and similar to boars which is consistent with work by Dahlmann et a1 (1980). High DHT implanted pigs, CLTEST and CHTEST pigs had 15.8, 23.2 and 21.7% lower (P < .05) AKP activity, respectively, in the red muscle extract than castrates which is in agreement with data of Dahlmann et a1. (1980) for testosterone treated male castrated rats. 204 TABLE VI-l EFFECTS OF PREPUBERTAL CASTRATION AND ADMINISTRATION OF TESTOSTERONE (TEST) OR DIHYDROTESTOSTERONE (DHT) TO CASTRATED MALE PIGS UPON ACIDIC, NEUTRAL AND ALKALINE PROTEINASE ACTIVITY OF THE RED PORTION OF THE SEMITENDINOSUS MUSCLEa Treatmegt Proteinase Fraction Group Acidic Neutral Alkaline B 1897.8e 1031.3f 666.2de c 1699.2d 934.4e 745.8e CIDHT 1932.3e 843.5de 664.2de CHDHT 1844.9de 799.1d 628.0d CLTEST 1672.7d 804.1d 572.3d CHTEST 1674.1d 896.2de 583.9d SEMc 70.68 38.88 33.03 aData reported as CPM per milligram of Lowry protein. b CSEM=standard error of least square means. B=boars, C=castrates, CL=low implants, CH=high implants. 205 Proteinase activity determined on the white portion of the ST muscle of PREP pigs is presented in table VI-2. In general, acidic proteinase activities for white muscle extracts were 17% lower than activities in the red fraction. For the acidic fraction of the white muscle extract, castrates had 24.5% lower activities (P < .05) than boars. Pigs implanted with DHT tended to have higher AP activities than castrates but TEST implanted pigs were similar to castrates. The same trends existed for red or white AP activity in PREP pigs. NP activity in the white portion also followed similar treatment trends to those of the red portion. Activities were about 22% lower for the white portion than the red. High DHT implanted pigs had elevated NP activities relative to boars or castrates (54 and 87%, respectively) but CLtest and CHTEST had about 37% lower NP activities (P < .05) than boars and tended to have lower activities than castrates. Sample mixing of red and white portions does not explain the higher NP activities for CHDHT implanted pigs since the red portion was 30% lower than that observed for the white portion. Castration elevated AKP activities by 74% and as already shown for the red portion of the ST muscle, testosterone at both doses and CLDHT significantly lowered AKP activities. This reduced AKP activity is. consistent with the findings of Dahlman et al. (1980). 206 TABLE VI-2 EFFECTS OF PREPUBERTAL CASTRATION AND ADMINISTRATION OF TESTOSTERONE (TEST) AND DIHYDROTESTOSTERONE (DHT) TO CASTRATED MALE PIGS UPON ACIDIC, NEUTRAL AND ALKALINE PROTEINASE ACTIVITY OF THE WHITE PORTION OF THE SEMITENDINOSUS MUSCLEa Proteinase Fraction Treatme t Group, Acidic Neutral Alkaline B 1721.1e 743.6e 294.2d C 1300.0d 612.0d 512.1e CLDHT 1686.9e 725.2e 301.9d CHDHT 1562.0ée 1146.3f 407.5de d CLTEST 1335.0d 471.2d 284.8 CHTEST 1282.7d 457.6d 310.2d SEMC 93.00 82.04 42.99 aData reported as CPM per milligram of Lowry protein. b CSEM=standard error of least square means. =boars, C=castrates, CL=low implants, CH=high implants. 207 Proteinase activities in the red muscle portion of the ST muscle for pubertal (POSTP) pigs are shown in table VI-3. Castration caused a 49% decrease in AP activities, a 54% increase in NP activities and a 43% increase in AKP activities. Neither TEST nor DHT had any influence upon the AP activities relative to castrates as the implanted groups were similar in activities to those of castrates. However, for NP activities, administration of TEST or DHT resulted in decreased activities relative to castrates and activities were similar to those found in the boar. No dose response trend was observed. AKP of androgen implanted pigs had activities which were similar to boars -and therefore 30 to 48% lower than castrates. Since CHTEST were 25% lower than CLTEST and CHDHT had 17% (P < .05) lower AKP activity than CLDHT, a slight dose reSponse trend was obtained. Table VI-4 presents proteinase activities for the white portion of the ST muscle of POSTP pigs. No large differences existed between activities in the red and white ST muscle portions. In fact, NP activities were about 7% higher in white than red and AKP activity was 34% higher. AP activity was 46% lower in castrates relative to boars while NP and AKP activities were 25 and 31% higher, respectively, in castrates compared to boars. These trends between boars and castrates were similar to those in the red muscle portion. Low DHT or TEST had no effect relative 208 TABLE VI-3 EFFECTS OF PUBERTAL CASTRATION AND ADMINIS- TRATION OF TESTOSTERONE (TEST) OR DIHYDRO- TESTOSTERONE (DHT) TO CASTRATED MALE PIGS UPON ACIDIC, NEUTRAL AND ALKALINE PROTEINASE ACTIVITY OF THE RED PORTION OF THE SEMI- TENDINOSUS MUSCLEa Proteinase Fraction Treatment Groupb Acidic Neutral Alkaline B 2126.8e 500.4d 452.6e C 1078.1d 773.4e 647.6f CLDHT 1184.5d 592.5d 454.6e CHDHT 1097.9d 462.9d 375.7de CLTEST 1172.5d 473.3d 447.2e CHTEST 1069.5d 577.4d 336.2d SEMC 118.2 51.35 29.34 aData reported as CPM per milligram of Lowry protein. b CSEM=standard error of least square means. B=boars, C=castrates, CL=low implants, CH=high implants. 209 TABLE VI-4 EFFECTS OF PUBERTAL CASTRATION AND ADMINIS- TRATION OF TESTOSTERONE (TEST) AND DIHYDRO- TESTOSTERONE (DHT) TO CASTRATED MALE PIGS UPON ACIDIC, NEUTRAL AND ALKALINE PROTEINASE ACTIVITY OF THE ngTE PORTION OF THE SEMI- TENDINOSUS MUSCLE Proteinase Fraction Treatment Groupb Acidic Neutral Alkaline B 1492.0f 570.8d 558.8d C 810.7d 713.5e 730.0f CLDHT 1083.7e 704.8e 611.4e CHDHT 1197.1e 456.2d 547.7d CLTEST 1110.48 723.48 650.0e CHTEST 1164.5e 457.1d 544.4d SEM 61.40 32.14 13.70 aData reported as CPM per milligram of Lowry protein. b CSEM=standard error of least square means. B=boars, C=castrates, CL=low implants, CH=high implants. 210 to castrates for NP activities of the white muscle portion but the CHDHT and CHTEST had activities similar to boars and about 36% lower activities than castrates. Alkaline proteinase activities in the white muscle portion followed the same trend as observed for_ the red portion. Administration of DHT or TEST reduced AKP activities relative to castrates and in a dose dependent manner. A qualitative evaluation of these data indicates that boars and castrates differ in proteinase activities. Since the AP activities were higher and AKP activities lower in boars compared to castrates, suggesting that the rate and type of protein degradation was also different. Since semitendinosus muscles of boars were heavier in boars than castrates (Chapter IV), a relationship may exist between higher AP and lower AKP activities and muscle growth. This could be an anabolic increase in AP activities as observed by Millward et al. (1981). The reductions of AKP in androgen implanted castrates is consistent with the data of boars. The inconsistency for androgen implants to elevate AP activity suggest that other factors besides TEST or DHT probably are influencing proteinase activity in boars. Activities in the acidic, neutral and alkaline proteinase assays were totaled for the red and white muscle portions, respectively. Total proteinase AP activities for PREP pigs are presented in table VI-5. Only CLTEST and CHTEST pigs had significantly lower activities than boars 211 TABLE VI-S EFFECTS OF PREPUBERTAL CASTRATION AND ADMINISTRATION OF TESTOSTERONE (TEST) AND DIHYDROTESTOSTERONE (DHT) TO CASTRATED MALE PIGS UPON COMPOSITE PROTEINASE ACTIVITY IN THE RED AND WHITE PORTIONSCE‘ THE SEMITENDINOSUS MUSCLEa Muscle Portion Treatment Groupb Red White B _ '3595.4f 2758.9ef C 3379.4df 2425.0de CLDHT 3440.1e 2714.0e CHDHT - 3272.0df 3115.9f CLTEST 3049.0d , 2091.0d CHTEST 3154.2de 2050.5d SEM 113.43 A 131.24 aData reported as CPM per milligram of Lowry protein. bB=boars, C=castrates, CL=low implants, CH=high implants. CSEM=standard error of least square means. 212 in the red portion of the ST muscle. Even though boars tended to have elevated activities relative to castrates in both red (6%) and white (14%) portions, these differences_ were not significant. For the total proteinase activity in the white portion, C DHT pigs had 28% higher activity and H CLTEST and CHTEST pigs had 13.8 and 15.5%, respectively, lower (P < .05) activities relative to castrates. Both TEST implanted groups had 25% lower total activity than boars. Table VI-6 presents the total proteinase activity assayed in the red or white portions of the ST muscle of POSTP pigs. Castrates had 18.2% and 15.8% lower total proteinase actiVity in the red and white portions, respectiVely, than boars. In the red portion, implanted pigs tended to have lower total proteinase activity than castrates (P < .05) and significantly lower (25 to 41%) activities than boars. Pigs implanted with high doses of androgen tended to have lower activities than those pigs implanted with low doses. In the white portion, the DHT implanted and CHTEST pigs were not different from castrates. In addition, C TEST implanted pigs had higher L total proteinase activities than castrates and did not differ from boars. A summation of the total proteinase activities in the red and white muscle extracts is presented in table VI-7. These data indicate that boars had 9 and 20% greater 213 TABLE VI-6 EFFECTS OF PUBERTAL CASTRATION AND ADMINISTRA- TION OF TESTOSTERONE (TEST) AND DIHYDROTESTOS- TERONE (DHT) TO CASTRATED MALE PIGS UPON COMPOSITE PROTEINASE ACTIVITY IN THE RED AND WHITE PORTIONS OF THE SEMITENDINOSUS MUSCLEa Muscle Portion Treatment Groupb Red White B 3164.6e 2653.9g C 2587.6d 2233.7de CLDHT 2365.1d 2383.4ef CHDHT 1873.6d 2202.7de CLTEST 2133.4d 2475.6fg CHTEST . 1939.2d 2167.0d SEM 191.81 66.20 aData reported as CPM per milligram of Lowry protein. bB=boars, C=castrates, CL=low implants, CH=high implants. CSEM=standard error of least square means. 214 TABLE VI-7 EFFECTS OF CASTRATION AND ADMINISTRATION OF TESTOSTERONE (TEST) AND DIHYDROTESTOSTERONE (DHT) TO CASTRATED PREPUBERTAL AND PUBERTAL MALE PIGS UPON COMPOSITE PROTEINASE ACTIVITY OF THE SEMITENDINOSUSMUSCLEa Pig Group Treatment Groupb Prepubertal Pubertal B 6354.3f 5701.5e c 5804.4e 4753.3d ef d CLDHT 6154.1 4631.5 CHDHT 6387.9f 4137.6d ‘ CLTEST 5140.0d 4576.8d CHTEST ' 5204.7d 4149.2d SEM 172.8 213.3 aData reported as CPM per milligram of Lowry protein. bB=boars, C=castrates, CL=low implants, CH=high implants. cSEM=standard error of least square means. 215 combined activities than castrates for pigs in the PREP and POSTP studies, respectively. Implanting pigs with DHT prepubertally elevated combined red and white proteinase activities relative to castrates. Implanting with TEST prepubertally depressed the combined red and white portion proteinase activities relative to boars and castrates. Implanting POSTP pigs with DHT or TEST depressed combined proteinase activities relative to boars but the decrease in activities were not significantly different than castrates. These data indicate that androgen implanted pigs had lower proteinase activity than castrates. If the proteinase activity is related to protein turnover, the mechanism of TEST and' DHT appear to be similar to trenbolone acetate. Sinnett-Smith et al. (1983) reported decreased cathepsin D activity in muscles of male castrated and female lambs implanted with trenbolone acetate. The percentage of total proteinase activity assayed at acid, neutral or alkaline pH of the red or white muscle portions for PREP .and POSTP pigs, respectively, are presented in tables VI-8 and 9. In both the PREP and POSTP studies, there appeared to be a trend for DHT and TEST to prevent a shift in proteinase activity from the acidic to the alkaline fraction associated with castration. An analysis of the total proteinase activity in tables VI-B and 9 further indicates that no one group of enzymes would be totally responsible for skeletal muscle protein degradation (Dayton et al. 1981). 216 TABLE VI-8 EFFECTS OF PREPUBERTAL CASTRATION AND ADMINISTRATION OF TESTOSTERONE (TEST) AND DIHYDROTESTOSTERONE (DHT) TO CASTRATED pIGs UPON THE PERCENTAGE OF TOTAL PRO- TEINASE ACTIVITY.IN ACIDIC, NEUTRAL AND ALKALINE FRACTION OF THE RED AND WHITE PORTION OF THE SEMITENDINOSUS MUSCLEa Proteinase Fraction Acidic Neutral Alkaline TreatmeBt Group Red White Red White Red White B 52.8de 62.2e 28.7e 27.1d 18.5d 10.7d C 50.2d 53.8d 27.6e 25.2d 22.1e 20.9e CLDHT .56.4e 62.3e 24.4d 26.6d 19.2d 11.1d CHDHT 56.4w 50.6d 24.4d 36.1e 19.2d 13.3d CLTEST 54.8e 63.7e 26.4de 22.6d 18.8d 13.8d CHTEST 52.9de 61.9e 28.5e 23.0d 18.9d 15.1d SEMC 1.20 2.08 .84 2.10 .65 .47 aData reported as CPM per milligram of Lowry protein. bB=boars, C=castrates, CL=low implants, C =high implants. H CSEM=standard error of least square means. 217 TABLE VI-9 EFFECTS OF PUBERTAL CASTRATION AND ADMINISTRA- TION OF TESTOSTERONE (TEST) AND DIHYDROTES- TERONE (DHT) TO CASTRATED PIGS UPON THE PERCENTAGE OF TOTAL PROTEINASE ACTIVITIES IN THE ACIDIC NEUTRAL OR ALKALINE FRACTIONS OF THE RED AND WHITE PORTIONS OF THE SEMITENDINOSUS MUSCLEa Proteinase Fraction Acidic Neutral Alkaline Treatment Groupb Red White Red White Red White B 69.2f 58.6f 16.0d 20.5d 14.8d 20.9d C 43.0d 35.7d 31.1f 31.6e 25.9g 32.6f CLDHT 53.0e 45.1e 27.0ef 29.4e 19.9f 25.5e CHDHT 55.7e 54.2f 24.8e 20.9d 19.5ef 24.8e CLTEST 56.3e 45.1e 22.2e 28.3e 21.5f 26.6e CHTEST 53.6e 53.1f 29.4ef 21.8d 17.1de 25.1e SEMC 2.27 1.88 1.78 1.84 1.06 .63 aData reported as CPM per milligram of Lowry protein. bB=boars, C=castrates, CL=low implants, CH=high implants. CSEM=standard error of least square means. 218 In summary of the proteinase data, it appears that castration reduced acidic proteinase activity. Neither DHT nor TEST significantly altered activity in the acidic fraction. Castration reduced neutral proteinase activity prepubertally but elevated activity in this fraction postpubertally. TEST and DHT either tended to reduce proteinase aCtivity in this NP fraction relative to boars and castrates or else they had little effect. Castration elevated proteinase activity in the alkaline range and TEST and DHT inhibited the castration associated elevation. Santidrian et al. (1982) found that castration of male rats decreased 3-methylhistidine excretion. In addition, testosterone administration appeared to Idecrease 3-methylhistidine excretion in corticosterone treated rats. This latter observation is consistent with work reported by Dahlmann and Reinauer (1981).. In their work on male rats, castration elevated alkaline proteinase activity by 50% and this elevation was SUppressed back to normal with daily injections of .5 mg of testosterone. They also found that basal alkaline proteinase activity was suppressed by 70% by testosterone administration. Their data plus the data in this chapter suggest that, alkaline proteinase activity probably plays a relatively insignificant role in in vivo protein turnover of boars. In fact, data of MCElligott and Bird (1981) indicated that the alkaline proteinase activities observed by Dahlmann et a1. (1979) 219 were of mast cell origin and are not involved in muscle protein breakdown. Nevertheless, the observation that AKP activity was altered by treatment in this study and elevated in obese mice (Trostler et al., 1982) and their ability to degrade myofibrillar proteins (Sanada et al., 1978; Yasogawa et al., 1978) deserves further attention. On the other ' hand, acidic proteinases are probably responsible for the higher skeletal muscle protein degradation data reported in chapter V for boars compared to castrates. Schwartz and Bird (1977) demonstrated that cathepsins B and D are capable of hydrolyzing actin and myosin. Cathepsins H (Bird and Carter, 1980) and L (Sohar et al., 1979) have also been shown to degrade myosin and possibly other myofibrillar proteins. The decreases in degradation rates reported in Chapter V for the implanted groups do not appear to be reflected by the proteinase assay data presented in this chapter. It is possible that neither the proteinase assay nor the in vitro muscIe strip incubation assay reflect true relative. degradation rate differences between treatments. The in vitro muscle incubation procedure has inherent problems, particularly exaggerated degradation compared to in vivo data (Mulvaney, 1981). The proteinase assay used in these studies provides only a crude measure of degradation because a myofibrillar protein, hemoglobin, - served as (the substrate. Thus, 220 interpretation of these data and their relationship to in vivo muscle protein turnover are limited and should only be considered to provide a relative indication of treatment effects. Since the in vitro muscle incubations were assayed at a neutral pH, the NP fraction may be the fraction to examine further. Neutral proteinases could serve as a nonlysosomal system for nicking proteins which possibly enhances degradation by lysosomal enzymes (Bird et al., 1980). However, these results are inconsistent between the two assays because castration stimulated proteinase activity in the NP fraction of pubertal pigs but the in vitro muscle incubation data indicated that castration was associated with a decrease in degradation. While differences between treatments were detected with both the proteinase assay and the in vitro muscle strip incubations, whether true biologic in vivo effects or differences were measured is questionable. Perhaps the only conclusion to be derived from the proteinase data is that the acidic proteinase activity accounted for most of the activity measured and that boars had the highest activities in this pH range. Therefore, the increased (ST) muscle accretion for boars relative to castrates is correlated with high acidic proteinase activity. The reduced in vitro muscle strip degradation rates in androgen implanted pigs is not reflected by significant decreases in the acidic activity relative to castrates. 221 Summary Based upon proteinase activities measured, it appears that boars had greater proteolytic activity than castrates. In addition, acid proteinase activities accounted for the bulk of proteinase activity assessed. It is suggested that further investigation on the relationship of proteolytic activity and muscle growth should focus on the acidic pro- teinases. 222 CHAPTER VII EFFECTS OF CASTRATION AND ADMINISTRATION OF ANDROGENS UPON ADIPOSE TISSUE ACCRETION AND IN VITRO LIPOGENIC AND LIPOLYTIC ACTIVITIES Introduction The pig has a high capacity for fat deposition (Allen et al., 1976). Knudson (1983) reported that 105 kg castrated male pigs had 25.4% carcass fat, whereas littermate boars had 17.0% carcass fat. Numerous studies of growth and body composition differences between intact male and castrated male pigs have been reported and were summarized by Walstra and Kroeske (1968), Wismer-Pederson (1968), Martin (1969), Turton (1969), Field (1971), Galbraith and TOppS (1981) and Seideman et a1. (1982). The recurring dichotomy between castrated and intact male pigs is in the differences in the extent of fatness. While the ontogeny of lipogenic (Allee et al., 1971; Anderson et al., 1973; Hood and Allen, 1973; .Mersmann et al., 1973) and lipolytic (Mersmann et al., 1976; Steffen et al., 1978) capacity in castrated male pig adipose tissue has been investigated, neither the lipogenic nor lipolytic activity differences between castrated and intact male pig adipose tissue have been characterized. 223 Hansen et al. (1980) observed that testosterone treatment of male rats and estrogen treatment of female rats depressed adipose tissue fatty acid synthesis and increased lipolysis. Wade and Gray (1979) hypothesized that gonadal steroids influenced adiposity by direct actions on adipose tissue. Gray et al. (1979) found that treatment of castrated male rats with testosterone prOpionate depleted cytoplasmic estrogen receptors and reduced lipoprotein lipase activity in epididymal fat pads. These effects were blocked by inhibition of the aromatization of testosterone to estradiol via treatment with androsta-1,4,6-triene-3,17-dione. The objectives of this study were to investigate the effects of castration and administration Of testosterone or its nonaromatizable form, dihydrotestosterone, to castrated males upon adipose tissue fatty acid synthesis rates, lipOprotein lipase and intracellular lipase activity in both prepubertal and pubertal pigs. Materials and Methods At the initiation of each experiment, four to six pigs were slaughtered and were physically dissected as described in Chapter II. Determination of total carcass fat and perirenal fat allowed calculation of carcass fat (CFA) and perirenal fat (PRA) accretion over the duration of the experiment. 224 Fatty Acid Synthesis (FAS) Activities. Immediately after exsanguination, subcutaneous and perirenal adipose tissue samples were obtained as described for LPL and HSL assays for determination of FAS activities. Adipose tissue samples were placed in oxygenated KRB buffer (one-half the usual calcium; see Appendix B-3) at 37 C. Slices (.3 mm thick and weighing about 100 mg) were made on a Stadie Riggs microtome using saline solution as a lubricant. Slices were blotted and weighed on either a Cahn or a roller balance to the nearest milligram. Slices were chOpped with scissors to increase surface area and transferred to 25 ml Erlenmeyer flasks containing 3 ml of assay buffer, gassed with oxygen-carbon dioxide mixture (95:5), were stoppered and the flask plus contents placed in a shaker (80 strokes/min) water bath at 37 C for 2 h. The assay buffer consisted .of oxygenated (95:5; 02:C02) KRB buffer (pH 7.4) (one-half the usual calcium) which included porcine insulin (Sigma; .1 U/ml), glucose (10 mM) and tritiated water (.1 mCi/ml). At the end of the 2 h incubation, slices were removed from the flasks, rinsed 3 times in saline solution and transferred to 50 ml culture tubes containing 10 ml of a KOH:ethanol solution (3:7;, 30% KOH: 95% Ethanol) for saponification. The tubes were heated at 60 C for 2 to 4 h or until the tissue was dissolved and then for an additional 30 min. The samples were then extracted twice with 5 ml of petroleum ether with 225 vortexing for 1 full min. The petroleum ether which contained the nonsaponifiable lipids was aspirated and discarded. Approximately 1.5 to 2 ml (one disposable pipette full) of 12 N HCL were added to the sample tubes to bring the pH to about 2 (checked with pH paper). The HCL converted soaps to fatty acids which were extracted 3 times with the addition of 5 ml aliquots of petroleum ether followed by 1 min of vigorous vortexing. After each extraction, the petroleum ether (tOp layer) was aspirated off and transferred to scintillation vials. The ether was driven off under a stream of air and then 10 ml of .4% Omnifluor (New England Nuclear) ethanol:toluene (1:4) liquid scintillation cocktail were added. Assuming pure water in the incubation media to have a molarity of approximately 55 and after counting aliquots of the media, the specific radioactivity (DPM/mol) of the media was determined. The nanomoles converted to fatty acid (FA) per minute per gram of tissue was calculated as follows: E t w SA where C was the counts per minute of the samples counted, E the efficiency of counting determined from channel ratio of tritium standards and counter external standard, t the time of incubation in minutes, w the weight of the adipose 226 tissue slice in milligrams and SA the specific activity of the media. Lipoprotein Lipase (LPL) and Hormone Sensitive Lipase (HSL) Assay. Immediately after exsanguination, subcutaneous adipose tissue (SDAT) samples from the .middle fat layer were obtained approximately 3 cm lateral to the dorsal median plane in the neck region. Immediately after resection of the SQAT sample, an incision was made caudal to the umbilicus and perirenal adipose tissue (PRAT) samples 'were obtained. Samples were placed in ice cold .9% saline solution and then cut into .3mm slices. LPL was determined from 1 g of adipose tissue slices prepared by homogenization in 3 ml of ice cold buffer which consisted of .25 M sucrose -50 mM Tris-heparin (80 U/ml) (pH 8.5) using a Brinkman polytron (three 20 sec bursts at setting 7). The resultant homogenate was centrifuged at 10,000 x g for 30 min at O C. The resulting infranatant (liquid layer below the solid fat cake) was aspirated and strained through a single layer of cheesecloth. The infranatant fraction was kept on ice until used as the LPL enzyme source. For preparation of the HSL source, adipose tissue was treated similarly except that the homogenization buffer consisted of .25 M sucrose - 50 mM HEPES - 10 mM sodium phosphate - 1 mM EDTA (pH 6.8). Homogenates were centrifuged at 1200 X g for 30 min a O C. The infranatant fraction served as a source of HSL enzyme activity (Steffen et al., 1978). 227 Enzyme activity was assayed as outlined by Bensadoun et al. (1974) with modifications of Steffen et al. (1978). The routine assay system for LPL activity involved the following components in a total volume of 1 m1: .2 mmol of Tris buffer (pH 8.5), 1 mmol of NaCl, 10 umol of CaC12, 20 mg BSA (Sigma, fatty acid free), .1 ml of normal pig serum (pooled from several pigs; see below), 2.5 mg of gum arabic and 13.45 umol of glyceryl tri (14C) oleate (.02 uCi/umol of triolein). Substrate was added to the reaction as a gum arabic-triolein emulsion prepared by sonication. 14C-triolein (Amersham) plus appropriate amounts of cold triolein (Sigma) were gassed with nitrogen for 30 min after the odor of solvents were no longer detected. Gum arabic and mega pure water were added to the triolein mixture and sonicated. Substrate, assay buffer and pig serum were preincubated for 20 min before initiation of the assay. with .1 to .2 ml of enzyme. Reactions were continued for 30 min at 30 C with shaking (80 strokes/min) using 50 ml culture tubes (to provide larger surface area for enzyme-substrate interaction). The reaction was terminated by addition of 6 m1 of Dole's extraction solution isopropanol: heptane: 3N H2SO4 in the ratio of 40:10:1 v/v). After vortexing (30 sec) and standing for 5 min, 5 ml heptane (containing .4 umol of triolein) and .5 ml water were added and the mixture vortexed for 30 sec and the phases allowed to 228 separate overnight (12 to 15 h). Fatty acids were isolated from a 3 to 4 ml aliquot of the heptane phase by a resin method of Kelly (1968) as modified by Baginsky (1981). Ion exchange resin (Sigma, Amberlite, IRA 400, 20 to 50 mesh, hydroxyl charged form) was prepared by suspending resin in 2.5 M NaOH (45:1, w/v). After equilibration for 10 h with occasional stirring, the resin was washed with deionized water (until water tested neutral with pH paper) and resuspended in isopropyl alcohol (.7 ml/g). After 24 h, the isopropanol was replaced with an iSOpropyl alcohol: water (9:1) mixture (.7 ml/g) and the resin equilibrated for 6 _h with occasional stirring.. This last step was repeated twice more before the resin was washed several times with n-heptane until the odor of isopropanol was no longer detected. The resin was then stored in dark brown jars at 4 C until used. The heptane aliquot from the reaction tube was added to 20 ml scintillation vials with 1 g of wet resin (previously washed with 2 m1 heptane containing triolein (Sigma, 40 mg/ml) to decrease nonspecific adsorption of labeled triolein to the resin) and the vials were vortexed for 1 min. Excess solvent was aspirated from the vials and the resin washed 3 times with 5 ml of heptane. One milliliter of NCS solubilizer (Amersham) was added to each vial and the resin-NCS suspension incubated at 60 C for 20 min to diSplace the fatty acids from the resin. Fatty acid 229 radioactivity was determined by a Beckman (Medel LS-3133P) liquid scintillation counter in 10 ml of .4% Omnifluor (New England Nuclear) - toluene scintillation mixture. Counting efficiency was determined by addition of l4C-toluene added to vials containing resin and was generally 76%. Recoveries were determined from a standard solution of 14C-oleic acid bound to albumin which was included in the same assay mixture as the samples except emulsions of unlabeled triolein were used. Recovery was calculated from l4C-oleic acid counts obtained with the the ratio of the standard solution included in the assay and the counts obtained with the same amount of standard solution added directly to counting vials containing resin which had been previously washed with heptane and solubilizer as in the assay. Recovery ranged between 50 and 55%. HSL activity was assayed in a. total volume of 1.ml containing .2 mmol HEPES buffer (pH 6.8), 10 mg of BSA (Sigma, fatty acid free), 2.5 mg of gum arabic and 22.6 umol, of glyceryl tri-14C-oleate (.013 uCi/umol of triolein) as a gum arabic-triolein emulsion (see above). The reaction was started with .1 to .2 m1 of enzyme and continued for 60 min at 30 C with shaking (80 strokes/min). Fatty acids liberated were isolated as described above for the LPL assay. The nanomoles of fatty acid (FA) liberated per milligram infranatant protein were calculated as follows: 230 FA = (C x Fp x R) SAapp where C was the counts per minute obtained for the experimental sample (average of triplicates minus counts per minute of blank tubes, Fp was the factor used to correct the volume of infranatant added to the assay to milligrams protein (Lowry et a1, 1951), R was the percent recovery and SAapp was the apparent specific activity. The apparent ' specific activity compensated for quenching due to the resin and solubilizer and was determined for each bath of resin and triolein preparation. For determination of SAapp, aliquots of a l4C-triolein solution was counted in vials of properly treated resin and expressed as counts per minute per nanomole of fatty acid. Blood was collected from 48 h fasted pigs by venipuncture and serum harvested. The serum was heated for 1‘ h at 50 C in a water bath to inactivate basal lipolytic activity, then centrifuged at 480 X. g to remove particulates and stored in 20 ml aliquots at -20 C until used. The amount of serum needed in the LPL assay to produce maximal LPL activiation was determined by adding increasing amounts of serum directly _to typical assay tubes. Maximal activation for the serum batch used was determined to be .1 m1. 231 Results and Discussion Assay Optimization. In preliminary work involving optimization of the LPL and HSL assays, a variety of assay components and conditions were studied in mice and pig adipose tissue. These included effects of varying concentrations of heparin (in LPL homogenization), salts, BSA, gum arabic and triolein. Maximal LPL activity was observed with 60 to 70 U of heparin/ml of homogenization buffer; 200 U/ml appeared to be inhibitory. Variations of CaCl2 from 0 to 50 mM concentrations resulted in a asymptotic curve with a peak activity around 10 to 20 mM. At least .05 mmol of NaCl were needed but decreased activity was observed at .25 mmol, and at .7 mmol complete inhibition occurred. Both BSA and gum arabic concentrations affected LPL activity. Maximal activity occurred with 20 to 40 mg/ml of BSA which was 60 and 40% higher than that at 0 .and 10 mg/ml of BSA. Gum arabic concentratons of 5 to 20 mg/ml indicated similar activities, whereas concentrations of 2 to 2.5 mg/ml resulted in the greatest activity. The effects of heat treated serum and BSA together were tested such that maximal activity was observed when .1 ml of serum and 20 mg/ml of BSA were used. It should be noted that different batches of serum resulted in variation of activity and therefore sufficient quantities of serum were obtained and pooled to complete all assays with the same batch of 232 serum. The effects of incubation time and amount of enzyme present upon LPL activity is illustrated in figure VII-l. Based upon these results .1 to .2 ml of the enzyme source was used in 30 min incubations. The effect of increasing triolein concentration was tested and indicated that maximal occurred activity at 8 to 10 nM and essentially no change up to 20 nM concentrations. Varying pH from 7.0 to 10.0 indicated maximal activity occurred between pH 8 and 9. Preliminary studies with the HSL assay involved the variation of pH, buffer substrate concentrations and incubation time. When the assay media was adjusted to a pH less than 6.5 (5 to 8.5 tested) or greater than 7.0, less activity was observed. Homogenates were prepared with SOmM HEPES, 50 mM HEPES-10 mM phosphate buffer or 50 mM phosphate buffer alone, and the greatest activity was observed with the combination buffers. In addition, maximal activity was observed at triolein concentrations exceeding 20 nM. Figure VII—2 shows a linear relationship of fatty acids released for up to 60 min at enzyme concen- trations ranging from .05 to .3 ml. FAS Activities. Results of fatty acid synthesis activities from the pigs of the pilot study are presented in table VII-1. FAS activities determined on SQ biopsy samples from the middle backfat layer revealed _ that castration had a definite effect on activities by 10 d 233 3 0032 >6 61 .4; 2040 6080100120 Time(min) 3 (022 X 2 E51 .1 .2 .3 Ehzymehnl)’ FIGURE VII-l. Effects of incubation time and amount of enzyme on LPL activity. 234 3 N O ,4 x2 2 8 l l / 20 40 60 80 100 120 time(min) 3 N 2 O ,_I >4 :1 8. .1 .2 .3 enzyme(ml) FIGURE VII-2. Effects of incubation time and annunt of enzyme on HSL activity. 235 .MCMOE mmhmdumum “mmmfi MO .HOHHQ UHMUGMDmHY—mmm .mucmamefl zmflnnmo .mucmHmEfl TDMHUTEHTDCHHH U .mucmamefl 30Huao .mmumuummono .mnmonum O .Amo.v my umwmwp mumwuomummsm DCTHTMMNU sufl3 mcEsHoo cflnuflz mammE unmEummHBo .£\:Hmuoum >H3OA mo mE\UwumumDHH mpeom >uumw NC ~08: mm Umuuommu unfi>auom wmmmfla cfimuoumomflauqmq n .cNE\TDMMNu mmomflpm mo m\pwom Nuumw ou Umuum>soo onm wo HOEC mm Umuuommu >DN>Huom mammzucwm Uflom >uumwnmHBU€ mmfimHA ZHWBOfiQOmHQ 02¢ memmfizwm DHUd NBBflh mDmmHB mmOmHfld AdzmmHmmm 02¢ mDOmZ¢BDUmDm ZOmD mUHm mqfiz DmfidmfimdU OB ABmMBV MZOmmE ImOBmmB m0 ZOHB memes 236 after castration. FAS activities were 90% higher in castrates relative to boars. Testosterone implants were effective in reducing the increase in FAS activities due to castration. There was also a testosterone dose response effect as indicated by the intermediate implanted '(CITEST) and high implanted (CHTEST) pigs having lower incorporation of 3H-water into fatty acids than either boars or castrates. After 3 wk (at slaughter) the magnitude (61% higher for castrates) of the difference between boars and castrates was less than the biopsy sample 10 d removed before slaughter. Part of this reduced magnitude of difference was due to an 18 to 31% fall in FAS between the two samplings. The decrease in FAS activity between biOpsy and slaughter samplings may be a overshoot response for the biopsied samples which by the time of slaughter was reduced due to readjustments of the endocrine system which were disturbed by castration. For the CITEST and CHTEST pigs, the FAS values were higher at slaughter than at biopsy. Since FAS rates have been shown to be affected by feed intake (O'Hea and Leveille, 1969) part of the depression in FAS observed in this study for boars or testosterone treated castrate relative to castrates may be explained by lower feed intake (Chapter III). FAS activities for perirenal adipose tissue obtained at slaughter are presented in table VII-1. In 'general, the 237 FAS activities expressed on a gram of perirenal adipose tissue basis were 2- to 3-fold higher than those in subcutaneous adipose tissue. Since this was only a 3 wk study, the number of cells per gram of adipose tissue probably was similar between treatments (Anderson, 1972). If this assumption is correct, then the relative differences between ‘treatments reflect real differences in adipocyte lipogenic activity. Boars had 39% lower FAS activities relative to castrates. Testosterone treated castrates had 20 to 32% lower FAS activities relative to castrates. Anderson (1972) indicated that adipose tissue depots differ in their capacity to synthesize fatty acids and triglyceride. In general, lipogenic activity expressed on a gram of tissue basis decreases with age of the pig after about 4 mo of age. The differences in subcutaneous and perirenal FAS and LPL activities do not appear to be correlated with differences in the quantity of the fat accretion for the pilot study (see Chapter III). While there appeared to be definite responses in FAS and LPL activities in both depots due to castration and administration of testosterone to castrated male pigs, the duration of the pilot study (3 wk) was apparently not sufficiently long to enable detection of differenCes in carcass fat. It should be emphasized that the castrates in this study and subsequent studies were left as boars up to the initiation of the study. Another 238 factor in the pilot study is that the pigs weighed less than 40 kg when the experiment was initiated and weighed approximately 56 kg pat the termination, a time which corresponds with increased testosterone secretion (Lapwood and Florcruz, 1978; Colenbrander et al., 1978) in boars. Based upon the results observed in the pilot study, 5 wk experiments were conducted for the prepubertal (less than 50 kg at slaughter) and pubertal (heavier than 70 kg at initiation) studies. Subcutaneous (SQ) and perirenal (PR) adipose tissue FAS activities of the pigs in the prepubertal (PREP); and pubertal (POSTP) studies are presented in table VII-2. The effect of castration was apparent both prepubertally and pubertally. Both PREP and POSTP boars had 63% lower subcutaneous FAS activities than castrates. While there were no differences in SQ FAS activities of DHT implanted castrates, CLDHT and C DHT implanted pigs had 2.4- and H 2.1-fold higher SQ FAS activities than boars. CHDHT implanted pigs also had 20% lower SQ activities than castrates but the activities were .similar to CLTEST implanted pigs. The greatest SQ FAS reSponse to testosterone in PREP pigs was observed for the CHTEST group which was similar to boars with 53% lower SQ FAS activities than castrates. Administration of DHT to POSTP castrated pigs had no effect on diminishing the castration associated increase in 239 .MCMOE mOHMSWm Ummmfl MO .HOHHO UHMUCM¥MHEWW .mucmamefl smasnmo .mucmHQEH 3oHnao .mwumuummonu .mumonnmo .Amo..vmv Hmmmep mumfluomummsm usmummmflp Suez mCEsHoo sflnufl3 mumme DGTEumTHB U Q .GNE\m:mmwu mmomwcm mo m\m©Hom xuumm ou.omuum>coo ONmm mo Hoe: mm pmuuommu >DH>Huom mammnucxm cflom >uummnm¢mm m.m m.H hw.v N.N 62mm . . . . m 0N ma mm ma Th mN OH NH Emma U 84.4N mmo.mH wo.am mm.m~ emmeuo . . . . m an we mN mN ma an mum mN 8:0 0 . . . . . A mm we mm mN mm we now Nm Bmo 0 mm mm mm mN we on Sm mm 0 mm.ma Tv.oa . wo.Nm 0v.ma m mmmmm mamom mmmmm mBB mqmdfi 240 SQ FAS activity. While there were no differences in SQ FAS activities between CLTEST and CHTEST pigs these values were 36 and 45% lower than castrates, respectively, but still 73 and 49% higher than POSTP boars, reSpectively. In general, the SQ FAS activities were 22% lower (significance not tested) in POSTP than in PREP boars and castrates. The same trends for treatment difference in PR FAS were observed in PREP and POSTP pigs. Boars had 54 and 51% lower PR FAS activities than PREP and POSTP castrates, respectively. DHT was ineffective in altering PR FAS activities from that of castrates and DHT implanted castrates had.2- to 2.5-fold higher PR FAS activities than boars. However, testosterone implants reduced the increases in PR FAS activities associated with untreated castrates. Testosterone implanted groups did not differ from each other or boars in either the PREP or POSTP pigs. Nevertheless, C TEST pigs tended to have 26 to 33% lower H PR FAS activities than C TEST pigs, thus indicating a L possible dose response trend. In an attempt to decrease variation in the data due to adipose tissue composition between the treatment groups, the milligrams of protein per gram of adipose tissue (Chapter III) were multiplied by the SQ FAS or PR FAS activities to eXpress the data on a nanamole/mg protein/min basis. While not presented in table VII-2, the eXpression of the data on this basis had no effect on treatment 241 differences. Hood and Allen (1973) discussed the various methods for eXpression of adipose tissue enzyme data. When enzyme data were expressed on a per gram wet tissue basis, enzyme activity decreased with increasing live weight. This is consistent with the data in the present study as POSTP pigs had approximately 22 and 40% lower enzyme activities for the SQ and PR depots, respectively, than PREP pigs. When Hood and Allen (1973) expressed enzyme data on an equal number of adipose cells or soluble protein basis, a general increase in enzyme activity was observed in 28 to 109 kg pigs. However, the greatest differences between live weight groups were observed when their data were expressed on a cell basis. The reduction in FAS activities observed in boars and testosterone implanted male castrates compared to castrates is in agreement with data on rats provided by Hansen et al. (1980). In that study, male rats were injected with 40 mg of testosterone or 1 mg of estradiol 3 d before adipocytes were prepared and assayed for lipogenic activities. Both testosterone and estradiol reduced lipogenic activities. The observation in the present study that DHT was unable to reduce an increased lipogenic activity associated with castration lends support to the theory that aromatization of testosterone to estrogen is required for metabolic effects of testosterone upon adipose tissue (Gray et al., 1979). Burch et al. (1982) reported a 50% decrease in 242 sheep subcutaneous adipose tissue fatty acid synthetase activity but only an 18% decrease in acetyl-CoA carboxylase activity due to implanting with a trenbolone acetate— estradiol combination. LPL Activities. LPL activities for SQ and PR depots in PREP and POSTP pigs are presented in table VII-3. When the data were expressed on a soluble protein basis, PREP boars had 3.4- and POSTP boars had 1.7-fold lower SQ LPL activities than castrates. In neither the PREP nor the POSTP study did DHT affect LPL activity relative to castrates. Even though CLTEST PREP pigs had 47% higher SQ LPL activities than boars the CLTEST pigs had over 40% lower activities than castrates and DHT implanted pigs. C TEST implanted PREP pigs were not different from either. H boars or CLTEST pigs but they had 54% lower SQ LPL activities than castrates. . The magnitude of the difference in LPL activities between POSTP boars and castrates was not as large as in the PREP study. In addition, the CLTEST group of the POSTP study had SQ LPL activities similar to castrates and DHT implanted pigs. However, CHTEST Pigs had 61% lower SQ LPL activities than castrates and 33% lower (nonsignificant) activities than boars. The SQ LPL data reported in this study are slightly higher than the SQ LPL data obtained from pigs as reported” by Steffen. et al. (1978). 243 .msme mmumsvm ummma mo Houum cumccmumuzmm .mucmHQEH smflsumo .mucmamefi BOHHAU .mmumupmmouu .mumonnmo v .Amo.v my “Dumap mumfluomummsm ucmuwmmep sua3 mssdaoo Casua3 mcmmE acmfiummua n .caE\msmmau Tmomaom mo m\moaom wuumm ou omuum>coo o Nm NC ~08: mm emuuommu >ua>auom mammnucwm oaom xuummu mdmm m m.amm «.mmm D.HoH m.ama 82mm 0 O O I m we Ham Do mam mam mam mmo mead Emma o . . . . u mm mmea um ammm we mafia no mama emme o am.mmmm um.moam 8H.GHH~ ae.moam emcee m~.omem mm.ommm mm.mmmm ma.emom emauo me.aao~ no.5mmm ma.~mam . ma.moam o mem.mmam 0o.momH ma.m~m Om.moa m umumm umuom gamma amuom omsono . ucmfiummue Hmuumnsm anuumnsmmnm DaweH>Heo< mmEmHu szeommomHu mommHe mmomHom umzmmmmmm Ozm maomzmesomom zoma mon mums omeamemmo oe Assoc mzommemoemmeomowmmo mo Lemmev mzommemoemme mo oneamemHszo< Ozm onemmemao qmemmmom ozm amemmmsmmmm mo meommmm MIHH> mqm<8 244 In analyzing the PR LPL data in table VII-3, prepubertal boars had 4.6-fold lower PR LPL activities than castrates. In both studies, the testosterone treated pigs had PR LPL activities closest to boars with the CHTEST pigs exhibiting a significant depression (almost 3-fold) in their activities relative to caStrates. These data from this earlier maturing depot are difficult to interpret since boars had higher adipose tissue protein than castrates. If it can be assumed from the adipose tissue protein data (Chapter III) that boars had a greater number of cells per unit tissue than castrates the effects of castration upon LPL activity will become more apparent as well as the effect of testosterone administration. The depressed LPL activity observed in boars and testosterone implanted male castrates compared to. castrates is consistent with data reported by Gray et al. (1979) and Slusser and Wade (1981). In those studies, LPL activities were reduced in male rats and hamsters when animals were treated with testosterone or estradiol.‘ In addition, the SO reduced forms of testosterone such as DHT were unable to reduce LPL activity (Slusser and Wade, 1981). HSL Activities. Opposing the lipogenic activities of fatty acid synthesis and the Uptake of fatty acid via LPL are the intracellular lipases or as assayed in this study, the hormone sensitive lipase (HSL). Basal lipolysis is low in swine adipose tissue (Mersmann et al., 1976) and 245 decreases dramatically after 150 d of age (Steffen et al., 1978). HSL activities in SQ and PR depots of PREP and POSTP pigs are presented in table VII-4. SQ HSL activities of PREP and POSTP boars were 74 and 40% higher, respectively, than castrates. Testosterone implants‘ but not DHT, resulted in greater HSL activities in castrates. SQ HSL activities in DHT implanted pigs were not different from castrates. The HSL data in these studies are similar to those observed in pigs by Scott et al. (1981) and Only slightly lower than those reported by Steffen et al. (1978). The combination of increased FAS and LPL activities and lower HSL activities after castration are consistent with the difference in quantity of total carcass fat of PREP and POSTP boars and castrates (Chapter III). As shown in tables III-10 and 11, PREP boars had 15.5% less and POSTP boars had 21% less total fat (excluding perirenal fat) in their respective carcasses at slaughter than castrates. Both the C TEST and C TEST groups had less (4 to 5% I H less for CLTEST and 14 to 36% less for CHTEST) total carcass fat compared to sham- implanted castrates. Total carcass fat of DHT implanted pigs either was similar to castrates or intermediate to that of the CLTEST pigs and castrates. Even though HSL activities were elevated in boars and CHTEST pigs compared ' to castrates, these 246 .Amo.uvmv wowweo mumfiuomummsm ucmumwmwp sues msadaoo sflnufl3 manna uswEummHB .mUGMHQEH nmflnflmu .mUSMHQEH BOHHAU .mmumuumMOHU .mHMOQHmu .mGMOE OH05wm UmMOH MO HOHHO UHMUCMHmflzmm @ Q .n\:Hmuoum >HBOA me \omgmumnfla mpfiom >uumw HOE: mm pmuuomwn >ua>wuom mmmmfla m>fluflmcmmmMCOEHoslqmmm e.om N.NN H.8H «.ma Osmm . . . . m Arm mad mmm was mum ama m e and Emma o same.HHH umo.ama mmmm.aoa m0e.mma ammeuo . . . . m 08 mm mm NmH mm mm 0e mma are U mumm.Hm mo.sma umo.aa mm.m~H emoao m0m.mm 0e.~am mumm.ooa m~.mma o Ama.maa mm.ma~ 0m.mma mm.-~ m ummmm qmm0m ummmm Amman Omsouo unmaummua Hmuumnsm HMDMTQSMTHN mNBH>HBU< mmémHQ m>HBHmzmm MZOZMOE mammHB mmOmHafl A mqmdfi 247 differences become relatively small when data are expressed on a total adipose tissue mass basis since castrates had more total adipose tissue mass than boars or CHTEST pigs. Testosterone accelerates fat mobilization (Laron and Kowaldo - Silbergeld, 1964). and norepinephrine stimulated lipolysis in rats (Hansen et al., 1980). From the data presented in this chapter, it appears that testosterone depressed FAS activity and the uptake of circulating lipid, and increased the mobilization of fat. These observations are in agreement with the data of Hansen et al. (1980) who reported that both the incorporation of 14C-glucose 'into fatty acids and the lipolytic activity were affected by testosterone administration to male. rats. Based upon the relationship of the adipose tissue enzyme assays of the present study and total quantity of carcass fat between treatments (Chapter III), it appears that DHT has little effect on controlling lipid deposition when compared to boars or the TEST implanted pigs. Even though POSTP castrates implanted with DHT had slightly less carcass fat than castrates theSe data suggest DHT has little or no effect in regulating fat deposition. DHT also was essentially ineffective 'in reducing feed intake, whereas TEST and boars voluntarily consumed less feed than castrates (Chapter III). Based upon the higher total carcass fat content of limit-fed castrates (pair fed controls) relative to boars 248 in this study (Chapter III), the fattening process may be more closely controlled by direct or indirect effects of testosterone upon adipose tissue rather than by a mere food intake response. This observation needs further investigation. These data lend credence to the hypothesis that testosterone affects feed intake and fat metabolism only after having been aromatized to estrogens (Wade and. Gray, 1979). Mendelson et al. (1983) reported that there is significant conversion of androstenedione to estrone and to a lesser extent 17-8 estradiol in adipose tissue, and the conversion may be indiced by glucocorticoids. The basis for this hypothesis is due to a dose dependent 20- to 60-fold increase in the conversion of androstenedione to estrone when'dexamethasone was added to adipose tissue cultures. This is consistent with the anti-insulin effects of glucocorticoids described by Baxter (1976). Massively obese human males have reduced circulating concentrations of testosterone and increased concentrations of estrogens (Kley et al., 1980). Ideal body weights for humans appear to be highly correlated (r=.8) with estrogen concentrations. Hamosh and Hamosh (1975)) and W ilson et al. (1976) found reduced adipose tissue LPL activity in estrogen treated male and female rats. In contrast, Prior et al. (1983) reported increased FAS activities in 17-(3 estradiol implanted steers and bulls relative to nonimplanted steers and bulls when the data were expressed on a gram of tissue basis and only subtle increases when expressed on a cell basis. Lipolytic 249 activities were unaffected by estradiol treatment. However, nonimplanted. bulls had 50% lower acetyl CoA. carboxylase activities than steers. There also was a trend for bulls to have greater basal lypolytic activities rates than steers, and for the estradiol implants to have slightly enhanced lipolytic activity in steers. It is possible the dose used in their study far exceeded that which would be optimal for reducing lipogenic and increasing lipolytic activities. Summary Measurement of fatty acid synthesis, lipoprotein lipase and lipolytic activities in subcutaneous and perirenal adipose tissue yielded data that complement the fat accretion data. In the 3 wk study, boars had 38 to 47% lower fatty- acid synthesis activities than castrates. Testosterone reduced fatty acid synthesis rates by 43 to 74% in the biopsied subcutaneous samples and by 29 to 44% in the slaughter samples when data are compared to castrates. Fatty acid synthesis activities in the perirenal depot of testosterone implanted pigs were reduced by approximately 20 to 32% compared to castrates. Similar trends were observed in both 5 wk studies but it appeared that high dihydro- testosterone reduced subcutaneous fatty acid synthesis activities in prepubertal pigs. This suggest that age may influence the response to androgens. Fatty acid synthesis activities suggest that castration elevated activities by 250 approximately 2-fold. Administration of testosterone but not dihydrotestosterone effectively prevented this castration associated increase in fatty acid synthesis activities. Lipoprotein lipase activities (were approximately 40% lower in boars than castrates in the 3 wk study. Lipoprotein lipase activities were 3.4- and 4.6-fold higher in subcutaneous and perirenal adipose tissue samples, respectively, for prepubertal castrates compared to boars. Activities in. pubertal castrates were 1.7- and 1.2-fold higher than boars for subcutaneous and perirenal samples, respectively. The difference between boars and castrates of ”the '2 studies ‘indicate that castration had .a greater response in prepubertal pigs compared to pubertal pigs. In all 3 «studies, testosterone administration reduced lipoprotein lipase activities compared to the data of castrated pigs. Dihydrotestosterone implants had no effect upon lipoprotein lipase activities when compared to castrates. Magnitudes of difference between treatments were less for lipolytic activities; however, prepubertal. boars had 1.7- and l.3-fold. higher activities in subcutaneous and perirenal depots, respectively, than castrates. Minimal effects due to androgens were observed except that high testosterone tended to elevate lipolytic activites relative to castrates and other implanted groups. 251 These data suggest that castration increases fat deposition in pigs by elevating fatty acid synthesis and lipoprotein lipase activities and reducing hormone sensitive lipase activities. Administration of dihydrotestosterone was ineffective in altering these aspects of adipose tissue metabolism. Administration of testosterone reduced fattening primarily by reducing fatty acid synthesis and lipoprotein lipase activities and subtle increases in hormone sensitive lipase activities. It is suggested that either testosterone acts directly, is aromatized to estrogens or that some antilipogenic factor is potentiated. Administration of testosterone could possibly be used to reduce fat deposition in castrated male pigs. 252 CHAPTER VIII EFFECTS OF CASTRATION AND ADMINISTRATION OF ANDROGENS UPON BONE ACCRETION Introduction There is a definite paucity of knowledge on bone growth in meat animal species. Since male meat animals are generally castrated in this country, testosterone is not a prequisite for longitudinal bone growth. However, testosterone influences bone growth (Short, 1980). Castration of young male rats slows growth (Scow, 1952; Aschkenasy-Lelu and Aschkenasy, 1959) but small doses (.lmg/rat/d) of testosterone prevented this diminished growth (Bergstrand, 1950; Scow, 1952). Larger doses (1mg/rat/d) of testosterone were inhibitory to bone ‘growth (Rubinstein and Solomon, 1941). However, the age of castration of rats may influence the response to testosterone (Werff Ten Bosch, 1977). Testosterone stimulates the physis to undergo cell division and widening during growth (Ogden, 1980). The relationships between muscle growth and bone growth are poorly understood. Intact male cattle, sheep and pigs have greater muscle mass as well as more bone than their castrated counterparts (Galbraith and TOpps, 1981). Percentage bone in carcasses of boars will be about 2% more 253 than in male castrates (Field, 1971). Bones of meat animal Species grow in a coordinated pattern but differential bone growth can occur (Richmond et al., 1979). If differential bone growth can be induced by endogenous or exogenous testosterone and if this induced bone growth is measurable, then our understanding of the relationships of bone growth to muscle growth would be enhanced. In addition, .since bone is an endocrine sensitive tissue, increased understanding of the mechanisms of action of growth promoting agents can be facilitated. The objectives of this study were to assess the effects of castration and administration of testosterone or dihydrotestosterone to castrated male ‘pigs upon bone accretion and linear growth rates of bone in male pigs castrated prepubertally and pubertally. Besides gravimetric and. linear measurements on selected bones, tetracycline was used as an intravital marker to assess subtle differences in longitudinal bone growth. Materials and Methods The scapula, radius-ulna, humerus, femur and tibia-fibula were removed from the right side of each carcass, cleaned of adhering tendon, fat and muscle, weighed to the nearest one-tenth of a gram and then frozen at -20 C until further analysis. The radius-ulna and tibia-fibula were weighed as bone pairs due to fusion of 254 these respective bones. Length measurements of the scapula, ulna and fibula were made to the nearest millimeter at the time of slaughter. A meter stick adapted with a stationary back drOp on one end and a sliding back drop on the other end was used to measure lengths of the _radius, humerus, femur and tibia bones. The radius and tibia were sawed into two longitudinal halves such that a 3 mm thick longitudinal center section could be obtained.- Total length and diaphysis length measurements were made on these center sections. On 1 d and 2 d after the initiation of each of the two studies, 20 mg/kg of oxytetracycline was administered intravenously to all pigs. The tetracycline becomes localized in replicatingcells near the epiphyseal plate and remains in those cells even after ossification (Hansson, et al., 1972). Therefore, when the center sections were placed under ultraviolet light, a fluorescent tetracycline band was detected and marked with a lead pencil for subsequent measurements. The distance from the tetracycline band boundary proximal to the epiphyseal plate was measured to the proximal boundary of the epiphyseal plate under a dissecting microscope equipped with an 8 x eyepiece and calibrated in millimeters. Similarly, the distal end of each of these bones also was measured. These measurements were referred to as either proximal or distal and growth. It should be noted that these measurements do not represent total longitudinal bone growth for 3 255 reasons. Firstly, the tetracycline band was too diffuse on the boundary closest to the marrow cavity to enable precise marking of that boundary. Secondly, the tetracycline band was in some cases as much as 1 cm wide. Thirdly, some longitudinal growth occurred in the metaphyses. Proximal and distal epiphyseal plate widths on both surfaces of the longitudinal center sections were measured in micrometers under the same dissecting microscope at approximately 32 x magnification. Due to variability in plate widths within an epiphyseal plate, ten to twenty individual observations were made on each plate and averaged. Observations which were 2-fold wider than the mean were deleted from the averaged values. After the humerus and femur bones were measured for total length, the mid-length point was determined and the bones were Sawed ,perpendicular to the diaphysis at distances equivalent to 10% of the total length of each side of the mid-length point. The outside and inside boundaries of the diaphysis wall On both the proximal and distal surfaces were traced on acetate paper. A planimeter was then used to measure the outer and inner diaphysis wall circumferences and subtraction of the inner circumference from the outer circumference resulted in an arbitrary diaphysis wall area on both the proximal and distal ends. This wall area measurement was used as an indicator of changes in bone thiCkness. 256 Results and Discussion Bone Weight. The weights of the atlas, first thoracic vertebra, pelvic, scapula, humerus, radius-ulna, tibia-fibula and total bone weight from the right side of the carcasses of the pigs in the pilot study are presented in table VIII-1. There were no significant differences between treatments for any bone weights. However, for most bones weighed, boars and testosterone (TEST) implanted pigs had heavier bones indicating a positive effect of testosterone upon bone growth. For subsequent studies, the scapula, humerus, radius-ulna, femur, tibia-fibula and total bones from the right side of each carcass were removed and weighed. Total bone weight as well as scapula, radius—ulna, tibia-fibula, humerus, and femur weights removed from the right sides of the carcass of PREP and POSTP pigs are presented in tables VIII-2 and 3, respectively. These total bone weight data differ from those presented in Chapter III in that the latter were calculated from the percentage bone in the right side times the hot carcass weight. Splitting errors may have under- or overestimated the total bone weight data presented in tables VIII-2 and 3. Since right side weights did differ between some treatments (Chapter III) it is difficult to interpret total bone weight data from the right side only. However, the trends appear to be similar between total bone in the carcass (Chapter III) and that in the right side 257 .mCMOE OHMDUm “mmwfl MO .HOHHO Uhmvcmumflzmm m .mucmHQEH sownnmo .mucmHQEH wumHUTEumucfinHu .mucmamefl soHqu .mmumuummonu .mumonnmm .mEmHmOHNx CH owuuommu we unmwmz econ Hmuoep .mEmHm CA pmuuomwu mum murmflm3 econ HUDUH>N©GHO .Amo. vmv Hemmer nos 00 mGEsHoo segue: mummE mumsvm ummmqn .wesum uOHHQ x003 Tennem m>.H mo.MNH mo.mma ov.vHH mn.vva oo.Nm ON.moa NN.NA on.ma msouo Amunmsmam anwuflcH .oa. mv.m mm.va m.h mm.m No.b me.m ma.v mN.N MZMm Hm.N mm.oma mN.mON om.mma om.mON hm.mma mN.mmH mo.m¢ mm.NN Emmamu vo.N mv.mma mH.ONN ov.ona mo.oHN mm.oma N>.¢ha NN.Nm mo.mH ammBHU mm.N oN.omH NH.mvN ma.mwa mH.NHN va.oma No.H>H o¢.mm oo.aN Emmequ vm.N mm.voa om.m0N NN.mvH Hm.mma Ho.hva H¢.Nma NN.Ne hm.HN U mh.N mm.mma mm.ovN mm.hma mm.MNN N¢.mma mm.mmH Hm.mv Nm.vN m maom wasnwm usEmm OCH: nonmesm wasmmom oa>amm oeomuone mmHHN omnouo cameos INHQHB Imsflemm umufim ucmEummHB omcom ABmMEV MZOmMBmOBmmB m0 ZOHBdemHZHzfld 02¢ ZOHB02mm mBmUHmz mzom OMBqumm ZOmD mUHm MA mqm<8 258 .mseeE eumsqm unwed mo Houue pumpceumnzmme .mEeHm CH Ueuuomeu musmfiez ecomo A m a .meumuummo pequflEHHanm o .muaeHQEN seen" 0 .mucmamefi 30H" 0 .meumuummouu 4mumonum n .Amo.v my uemmap mumfluomuemsm uceHeMMHn nuHB mGEsHoo CASDNB mcmeE uceEueeHu euesvm unmeqe H.85 a.ao a.om m.om A.Nm o.oom mwouu Heunmsmam HEADAEH mm.a m~.a ea.m me.m aa.~ om.mm 82mm mmH.~oH m.mmH emm.ama mm.~oa m.moH mm.saea Ommqo mea.meH H.maH mew.aaa mm.mOH A.Hoa www.4maa emmemo . . . . . . a mm «mm m HaH mm ANA ems NAA N am See mesa Emma o . . . . . . . m mm Hma . o mma mm «HA ema mom m mom ma amma sea o . . . . . . A ewe emH a NQH awe mNHI emm AHA m Had mm owed emo o ema.amH e.oaH mm.aHH mH.m0H a.~oa memo.emma 0 Bee AAA m 44H am Nam .em mHH N mos new emaa m HSEem msuefism mannflm ecHD masmwOm ecom mwouw (Bends (Homemm Hmuoe ucmsummne oecom mmm02mm mBmOHmz MDZMh 02¢ mDmmZDm .dADmHmI¢HmHB .GZADImDHmfim .musmmom .mzom umeoe zoms mon mq<2 Dmadmem mqmde 259 m m .mSMOE Ohmamum HmmmH MO .HOHHQ UHMVCMUmH—zmm c .mEmum :A ceuuomen munmAe3 esomo A m .meumuumeo CeMIDAEAAHQmm U .mwcmAQEA cmAnu U .muceAQEA BOAHAU .meumuummouu .mumonnmn .Amo. v A: HeMMAp mumAuomuemsm useHeMMAp ADAB muesAoo cAnqu memes DceEueeuu emmswm unmeAn a.Am~ S.Nm~ m.oo~ a.maA o.mmA m.mOAm msODO newsmSEAm AMADAGH mm.NA mm.mA mm.m em.m Am.OA mo.moA exam . . . . . . A eo Nmm ev NAm mem ANN em AmN em mVN eN mmoe 0mm 0 O O O O O O m em omm em Nvm mmm mmm em mmN eN AoN mum Nva Emma 0 O O O O O O A ev mNm em Nmm meN ANN eo omN ev CNN men wmAv Emma o O O O O O O m em Nmm em Nvm meN mwN eN omN meA NNN mem NOAv 9mm 0 . . . . . . A em omm ev mNm em mwN eo NVN em mmN em emov 9mm 0 em.Amm eN.Nvm eN.ONN eN.mmN eN.NmN em.mvow 0 mm oov mo mow mm va mm NmN mm «Am 0A wmmv m “seem mDHeEs: OADQAN chD mAsmmom ecom nmsouo IeAQAB ImsAomm Amuoa useEueeHB oesom mmmeummo WEB m0 MDHm BmUHm mme 20mm Qm>02mm mBmOHmz mDth 024 mDmszm . mqmflfi 260 reported in this chapter. Only the right side data will be discussed in this chapter. In the PREP study, total bone weight from the right side of boars and castrates were not different. Boars and castrates tended to have more bone than implanted pigs with the exception of the high testosterone (CHTEST) group. Limit-fed PREP castrates had the least total bone weight of all treatments. In the POSTP study, boars had 16% more bone in the right side than castrates and tended to have more bone than the androgen implanted groups. The CHTEST pigs were closest to boars in tetal bone weight having only 3.5% less bone in the right side, but all implanted pigs in the POSTP study tended to have slightly more (3 to 4%) bone (P < .05) than castrates. Limit-fed castrates had bone masses similar to castrates. In order to assess the effects of castration and administration of testosterone and dihydrotestosterone to castrated male pigs upon bone accretion, the bone weights of the initial slaughter group of pigs were subtracted from the bone weights of pigs within each treatment group in tables VIII-2 and 3 and the accretion was expressed as a percentage of the castrate bone accretion. These data are presented in figures VIII-1 and 2 for the PREP and POSTP studies, respectively. As indicated in figure VIII-1, representing the PREP study, no ' One bone appears to be more responsive to TEST or DHT than 261 another but it appears that the CHTEST treatment had relative bone accretion rates that were similar to those of boars. It appears that dihydrotestosterone at the high dose had an inhibitory effect on bone accretion relative to castrates. As illustrated in figure VIII-2 for the POSTP pigs, boars far exceeded castrates in bone accretion. When considering the radius-ulna, tibia-fibula, humerus and femur, it appeared that CHTEST was more effective than the other implant groups in stimulating bone accretion relative to castrates. Unlike the PREP study, scapula weights were stimulated to a much greater extent by androgen in POSTP pigs with the CHDHT group showing the greatest response. ‘Limited-fed castrates were similar to or had less bone‘ accretion than castrates during the 5 wk experiment. Linear Bone Growth. The differences in bone weights should be related to changes in bone length or thickness. Lengths of the pelvic bone (pilot study only), scapula, humerus, radius, femur and tibula for the pilot study and the PREP and POSTP studies are presented in tables VIII-4, S and 6, respectively. While boars in the pilot study tended to have slightly longer bones than castrates, only the 5% difference in femur length was significant. No other detectable trends in bone length were evident in this 3 wk study. 262 .mGMOE mOHMDmvm “mmma MO .HOHHO UHMflQMUmHEmm .Amo.v mv nemon mumAnomHeQSm usenemon nuAB mGEdAoo :AnnAB mcmee uceEumeMB .mueueEAuceo :A enumaeA econ AesoA>AonH O‘UO) .musmAQEA nmAnumU .muceAQEA euefleeaneucAnAo .mnceAQEA 3oAnAU .meumnumnouo .mueonnmn .Nosum DOAAQ xeez eennem o.~A m.aA N.a a.mA N.4A o.mA msouo HeunmseAm AEAAAGH mm. NN. mo.A mm. em. we. ezmm m.mA AN.SA a.NA N.aA e.mA «.om emmemo o.mA meA.NA a.MA «.mA m.eA m.om emmemo A.mA ea.SA o.MA «.mA a.oA o.o~ Emmeqo o.mA mm.wA m.AA m.vA o.mA o.ON o o.wA mm.NA o.NA N.mA v.0A N.AN m EAnAB usaem msAemm maneesm mAsmeom OA>Aem nmzouw uneaumena ooesom mmwosmm mmeozmu mzom omeomumw zone mon age: omemmemmo oe .emmev mzommemoemme mo onemmemHszom ozm onemmemmo mo meommmm VIHHH> mqmdfi 263 .msmeE meunsvm umnDA mo Houue pnmccmumnzmmw .mueueEAuceo CA oeunomen mnemaeA enomo A m .meumuumno eeMIuAEAAnomm U .mncmAmEA nmAnn o .mAGMAQEA 30A" AU .meumunmmonu .mnmonu m n .Amo.v NV HewmAe mumAAomnerm uceAeMMAo nuA3 mGEDAoo :AnnA3 names useEAeeAn euesvm nmeeAm mA.AA N.OA a.a m.OA a.N m.N N.OA msouo . AeunmsmAm AHADABA aA. om. mm. mm. mA. mA. mm. 82mm 8 m AA ATA.MA m.AA AOA.AA G.a Ama.a OA.MA OAEAO amm.4A A893 G.AA em.4A N.a AOO.OA AN.AA ammemo www.4A AORMA A.AA AO~.4A m.a Awm.a Amm.MA emNEAo O O O O O O O 3 Amp AA mam AA a CA Ama AA m a we a mm «A sea 0 oa.mA mmN.mA S.AA Ama.mA o 0A Amm.a em.8A emOAO mm.8A Amm.mA a.OA Om.mA m.a 0m.a Amm.mA o AVno.3 ma.mA N.AA Amo.4A N.a e~.0A Am.aA m Asaem msAeEDm mAsnAm OAnAe esAD msApmm EAsmeom . nmsouw useEumeAB Omgom mmeozmA ensue ozm mammzsm mAsmAm «AmAe AZAD mvoem «Aammom zoms moAm AAA: omemmemao oe Aemov mzommemoemmeomowon oza Aemmev mzomme ‘ Imoemme mo zoAEAmemAzAzom oza zerAmemmo Amemmmommmm mo meommmm mIAAA> mAmme 264 .WCMOE mmHfl—umvm UmmmH MO HOHHQ CHMUGMumflzmmU .mueueEAuseo :A oeuuomeu enumceA ecomo .meumnnmeo peMIuAEAAnommAU .musmAQEA nmAnan .mucmAmEA 3OAHAO .meumuummono .mumonumn .AAoQE Aemmwp mumAAomnemsm uneuemmwo nuA3 mCESAoo cAnuAB mceeE useEueeAu euesvm unmeAm A.AA A.AA «.4A O.OA A.AA A.NA N.NA mmouo DOOEOOOAO AOAOAOA ON. AA. AA. AA. AA. AA. AA. exam . . . . . . . A Om om Om AA ON AA OA NA A AA O4 AA Om AA one o . . . . . . . m OON om SON AA one AA . mo AA A AA AON AA Om AA NONE O O O O O O O I 1H Om om OA NA OOA AA OOA AA A AA AOA AA ON AA Emma o O O O O O O O m OA om AOA AA one AA AOo AA A AA O4 AA OA om sea o . . . . . . . A ON om Om NA OOA AA AOOA AA A AA AOA AA ON AA Ana 0 OA.o~ OA.AA OOA.AA AOA.AA ~.AA OA.AA OA.AA V o OA.AN mm.AA OA.OA SON.AA A.AA AN.AA AA.A~ m ADEem wsnefism eAsnAm OAnAB mnAD msAemm eAsmmom msouo. us Enneue oecom mmBUZMA MDSmm DZN mDmWZDm eflquHm .flHmHB .fiZAD 4mDHoflm .mADmfium ZOmD mem MAdz Dmfidmfimfio OB ABEQV mzommEmOBmMBOmQMmHQ QZ< Afimmsv mzomMB ImOBmMB m0 ZOHEfimBmHZHzad QZ¢ ZOHBflMBWflU A MAm¢B 265 For PREP boars only the radius, humerus and femur were significantly longer 6, 5 and 5%, respectively, than castrates (P < .05) and a similar trend (P > .05) existed for the scapula, tibia and fibula. Implanted pigs tended to have longer bones relative to castrates but none of them was significantly longer (P < .05) than castrates except for femur length of the CLDHT grOUp. Postpubertally, boars tended to have longer bones than castrates but only the differences in scapula (5%), radius (4.5%), fibula (3.9%), humerus (7%) and femur (7%), respectively,. were significantly (P < .05) different. Implanted pigs generally were not different from castrates and only the tibia (4.4%) and humerus (5.5%) for the CHTEST pigs relative to castrates were longer (P < .05). Walstra (1980) reported that boars had longer bones than castrates when fed ad libitum. In addition, few differences in bone length between boars and castrates were observed before 4 mo of age. The few detectable differences in linear bone growth in these studies either means that differences between treatments are so subtle that simple gross length measurements were not sensitive enough or -that differences in bone accretion by these treatments is more of a function of density or thickening rather than length. Bone is a metabolically active organ with continual remodeling (Ham and Cormack, 1979) and it is feasible that castration and 266 testosterone administration may affect Specific aSpects of long bones without altering total length. In order to assess the effects of castration and androgen administration upon bone growth, the diaphysis (from diaphyseal aspect of epiphyseal plate on the proximal end of the diphyseal aspect of the epiphyseal plate on the distal end) and proximal and distal epiphyseal plate widths were measured. In addition, tetracycline was used as an intravital fluorescent marker to enable measurement of subtle differences in linear bone growth. The radius diaphysis length, growth determined by tetracycline marking on the proximal and distal ends and the proximal and distal epiphyseal plate widths of the radius for PREP and POSTP studies, respectively are shown in tables VIII-7 and 8. Because the radius was longer in boars than castrates so was the radius diaphysis length (by 3%) in the PREP study (table VIII-7). Even though the CHDHT pigs had essentially the same (P > .05) total radius length, the radius diaphyseal length was 4.3% longer (P < .05) than castrates. The other implant groups had slightly longer (P < .05) radius diaphyseal lengths than castrates. Aside from a 42% (.29 cm) greater distal end growth as measured by tetracycline marking in the CHTEST pigs no other differences in growth were ‘ observed. Nevertheless, castrates tended to have the least growth as determined from the tetracycline marked bene. 267 .mceeE neumsvm unneA no Deane punccmumuzmme .mueueEAAAAE :« ceuuoaeu mama .eunAQ AmenazaAme Ou Nuepcson ecAAo>onuueu EGAN censuses zuzoum use Anuon use AnEAxoum neunAQ AmenanmAme use AnEAxoun 0» use AnunAo :0 euOAQ Aoen>zmAne Eouu oeusnees :uoceA nAmasauAno .meunuunno pequAEAAuouon .muceAQEA :mqsuzo .nuanQEA 3oAuAU .neunuunnouo .munonnm Q a o Amoo V any HUHUdU flUQfiHUQHOQOQ UCQHONHdQ nus“: OCEDHOU EMF—HA; MCQOE HCOEUMOHFQ cw. mm. II II 9.9m QDOAU . ueuzmsnAm AOAOAAA AA. AS. AN. NA. . OA. Ozum. OAA. . AA. AA~.A A~.8A u«A.N sumac OAA. _ AA. OAA.A AA.AA muv.8N ammeeo AOAA. AA. OOA.A AA.oA OOA.AN NOONAO AOAA. AA. qu.N . AA.OA A.A.AN amaze AOAA. AA. u3;. AA.AA OOA.AN NeaAo OAAO. AA. OAA.A A..AA AN.AN 0 OAN. ea. AAA.N AA.OA OO.AN A :OOAS OOOAO :OOAz OOOAO auscuo £03046 suchA nmaouo AmenacmAQm AnemazmAam one can nAnazaeAa useauneus Anunwo AnEAxoum Anunwo AnEAxoum COUCQEOHSQQW: ”com 62:05 0:9 m0 9.0.00“: MESA A<0m>=mnmm 020 AdBmHo 02¢ AdZHXOmm 02¢ mfizmtflmamdmz =B30¢U 02m— A‘Bmmn 024 Adxnxomm .zbozmq mHm>=m=H0 02‘ 79mg: mzomwbmoamflh. ho ZOHPGKBmHZHr—Dd 02¢ ZOHF<¢Fm¢U figmmamflmm LO msmhmu NIHHH> Manda. 268 .menmnumeo UeAInAEAAnnmmAO .musmAQEA nmAnnmo .mHGMAQEA BoAuAU .meueuummono .mueonum «.msmeE menmsvm unmeA mo Honue Unnecmumuzmme .mueueEAAAAE CA oeunomeu euro 6 .eumAm AnemwnmAme on mnmpcson ecAAoaomAueu EOAA pensmmeE nuzonm use AmnmAU can AmEAxoum “eueAQ AeemhnmAme pee AeEAxoum on one AmumAe no eumAQ AnemanQAme EOAA pensmmee numceA mwmwnmmwoo a .Amo.v mv HemmAe mumAHomuemsm ucenewmAU nuws mGESAoo :Anufl3 maeeE uceEumeABe oA. AN. II II A.AA msouo AOOASOOAO AOAAAOA AA.NN AN.AN mm. AA. AA. Osmm . _ . . . . . A AAA AAA EAOA A onA A AA AOA one o . . . . . m SANA OAA :mAA A AAN N AA AOA emme o AAA. EmmN. EAOA.A AAA~.N AN.AOA NAAEAO . . . . . m AAA EmmN AAA m AAAA A AA AAA ewe o AAA. AAA. OAAA.~ AAA.A Ao.AAA NmOAo AAAA. rmAN. AAA.~ AAA~.A AA.AA o OAA. nAN. EAN.A :mAN.A AVOAOA m EOOAS OOOAN EOOAS OOOAA EOSOAO Auzouo AOAOOA nmsouo AeemmnmAQm Aeemwnmflmm Cam cam mAmNnmeAn useEumeAB Hmumflfl HwEHxOHm HmmeQ HMENXOHAH oomnceEensmmez ecom mmDHOmH0 02¢ ABmmBV mzomma ImOBmmB ho ZOHBfiMEmHZHZO¢ 02¢ ZOHB mqmdfi 269 The radii of POSTP pigs (table VIII-8), showed that boars had 5.1% (.5 cm) longer diaphysis lengths than castrates which accounts for most of the .6 cm advantage in total radius length. Implanted groups had slightly longer diaphysis lengths than castrates which correlates well with the slightly longer radii data presented in table VIII-6. No definite trends are apparent from the tetracycline data obtained on the radius from POSTP pigs except that CLTEST and CHTEST pigs had more proximal end growth (P < .05) than boars, castrates or DHT implanted pigs (table VIII-8). In both PREP and POSTP studies, the greatest growth of the radius occurred at the proximal ends. In addition, the ratio of the growth on the distal ends to that occurring on the proximal ends as determined by tetracycline marking was about .6 for PREP pigs and about .4 for POSTP pigs, indicating a shift in growth intensity with increased age of the pig. In general the epiphyseal plate widths of the bones examined were greater for PREP pigs relative to POSTP pigs. While boars of both studies tended to have greater (6 to 10% on proximal ends and 10 to 16% on distal ends for PREP and POSTP studies, reSpectively) plate widths than castrates, none of the differences was significant. This trend is not consistent with the concept of wider epiphyseal plate widths observed in castrated male rats (Silberberg and Silberberg, 1971). No other trends due to 270 androgen treatment were. detectable except that implanted pigs like castrates had narrower plate widths than boars which was not unexpected based upon reported effects of testosterone upon epiphyseal plate widths (Silberberg and Silberberg, 1971). The tibia was selected as the representative long bone from the hindleg for longitudinal cross sectional measurements. However, this selection was made prior to statistical analysis of the bone length measurements. A better choice for these measurements might have been the femur for the hindleg and the humerus for the forelimb since these bones showed greater differences in total length between boars and castrates than the tibia. The tibia diaphysis length, growth measurements on the proximal and distal end, and the proximal and distal end epiphyseal plate widths of PREP and POSTP studies are presented in tables VIII-9 and 10, respectively. No differences in tibia diaphysis length were detected for the PREP study but trends were the same as for total length measurements. In the POSTP study,' CHTEST pigs appeared to haVe greater tibia diaphysis lengths than castrates and CLDHT pigs but not the other treatments. As indicated by the ratio of growth on the distal end to that on the proximal end determined from tetracycline marking, greater growth activity occurred in the tibia for PREP pigs compared with POSTP pigs. No distinct differences were 271 mcwuoaomuumu EOuu consumes :u3ouo 6:0 Hmuudo can AmEAxOHQ .mcmoE mmuosvu unwed mo wowwe fiuuccoumuzumw .uuwumEAHaafi :4 cuuuommu mama .quHQ Anomazmfimm Cu aumccson .uumaa Anomanawam 6:0 HmEAxoua On one Amuuwc no manna Haouazmaau Eouu vmusmnwa nausea mau>nauuou .mounuumoo omuu Iu«Eu~noumau .nucuHmEA smaslzu .uusauasq zodnq U .muuauuauouo .muaonumn .Amo. vm. wouuao mumfiuomuwdsm acoumuuev :uA3 unasdoo :Azuq3.u:mofi acmEummuau AA. 84 II II AA; 9.96 umuzasnwmIHmmuucn «no. no. mm. cm. an. 02mm . . . . . A :mw mam mod ca um“ MA a man cum 0 AAAA. AAA. AA~.AA AAA...“ A.HHA amuaao AAA. AAA.A AAA.AA AAA.AA A.AAA ammaqu O O O O O = umh mam mama ca aumw va a wad fine 0 AAA. AAHA.A AAA.A~ AAAA..A A.AAA azaqo :ca uva. mud on now my a med 0 mac. yam. oucn.c~ ouch.vq o.-~ m cava: mumfim cubes wuuam zusouo :uzouo cameo; msouo Amwmanmeam amwnanmfima new new ufluasaauo u: abacus Acumen Amsfixoum Acumen Headache Dbmuflwc—OHDQGQZ 020m N=mnmm Dzm Q=Ho ozm Aammfiv mzomuh ImOEmmfi ho 20H9¢m9mHZHZD< Q24 ZOHP<¢Bm¢U Adbmmmamumm ho mBUNhkfl mIHHH> fldm<fi 272 .QCOQE mmhflflvm wamfl HO HOHHN UHGCCQfiNflZMMU .uumquAAAAE :A cmuuoamu name 0 .mumAQ AnomasmAdw cu zumccson chAoaouuumu Eouu consumes zu30uw 6:0 AauuAp one AmEAxoannmumAm Am0u>chmm new AmEAxoua 0» 6:0 Anumuc co wumAm Ammu>smumm Eouu consumes sumcmn uuw>nmnAoo .mmumuuuao vmquAEAAnommqo .mucmAQEA :quuzo .uucuAQEA zoAuAU .uououuuuonu .uuwonuma .Amc._vmv umuuAc manquomumazm unmumuuuv :uA3 messAou :«zua3 mamas acmEummuan AA. AA. II II A.AAA macho . umuzmsmAm HmAuAcA AA. AA. AA. AA. AA. mzmm AA. AAAA. AAA.A :AAA.A AN.AAA AAAAQ AA. AAA. AAA.A AAAA.AA :A.AAA Ammezu AA. AAA. .AAA.A A..AA.A AAA~.AAA AAAAAA AA. AAAA. AAA.A AAA.A AAAA.AAA Aznzu AA. AAAA. AAA.A AAAA.A AN.AAA AAAAU AA AAA AAA A AAAA A AA. AAA 0 AA. AAA. AAA.A AAAA.AA :AA.~AA A :uAAz ouAAA zqu: AAAAA zuzouo zuzouc nuAcoA nmsouo Ammm>saAmm AnomazaAmm cam can nwuazmnqo unmEummub Acumqo AmEAxoum Amuon AnEAxoum Iw Omucwfimhfimwwz 0:00 mCHmHP m=9 ho m=EQH3 abddm 44mm>=mHam 02m J=m=HG m0 .Emmfiv mzommb ImOmeb ho ZOHP Mdmdfi 273 visualized between treatments of PREP pigs for tetracycline marked growth measurements; however, there were differences in the POSTP study. Boars had 22% greater growth on the proximal and distal ends of the tibia than castrates. In addition, CLTEST and CHTEST pigs had greater (12 to 19%, respectively) proximal end growth than castrates or DHT implanted pigs. Similar trends occurred for growth at the distal end. These observations are consistent with the effects of testosterone upon bone growth in rats measured by tetracycline as an intravital marker (Jansson et al., 1983). Tibia epiphyseal plate widths were greater in PREP pigs than in POSTP pigs. Small differences existed between treatments .in either the proximal or distal end epiphyseal plate widths for PREP pigs. In POSTP pigs, boars had 21% greater proximal end plate widths and 20% greater (P < .05) distal end plate widths than castrates. Although nonsignificant, there appeared to be a trend for greater. plate widths relative to castrates for the testosterone implant dosages used in this study. Because no definite treatment trends in epiphyseal plate widths or tetracycline marked growth of the radius or tibia were detected from the individual observations, the proximal and distal end measurements were added together. These summed plate widths and texracycline marked growth measurements from the proximal and distal ends are 274 presented in tables VIII-15 and 16 for PREP and POSTP pigs, reSpectively. Based upon these summed data, essentially no differences existed for epiphyseal plate widths of the radius in PREP pigs. The summed tibia epiphyseal plate data, indicate that DHT at both doses and TEST at the higher dose may be decreasing the plate width relative to castrates and definitely relative to boars. The summed tetracycline marked growth measurements indicate that TEST and DHT at the low doses were stimulatory to tibia growth relative to castrates; however, the” CHTEST was more effective in stimulating longitudinal growth than CLTEST for the radius and the CLTEST was more effective than CHTEST in the tibia. Higher microsc0pic magnifications should be used for- epiphyseal plate width determinations and fine tuning of the tetracycline marking method is needed. This would involve more precise marking of 'the fluorescent bands and the use of higher microsc0pic magnification to ensure greater sensitivity of the measurements. The growth trends for both the radius and tibia were similar within each study. The greater growth in the tibia compared to the radius is not in agreement with the hypothesis of MCMeekan (1940) that the bones of the forelimb grow at a faster rate than hindlimb bones. Bone Thickness. Knudson (1983) used the ratio of bone weight to length' as an indicator of bone thickening. The bone thickness estimates for the scapula, radius-ulna, 27S tibia-fibula, humerus and femur bones and are presented in tables VIII-11 and 12 for PREP and POSTP pigs, respectively. In the PREP study, there was no suggestion for bone thickening or increased density in boars and testosterone treated pigs relative to castrates based upon bone weight to length. Only the tibia-fibula of boars had a 16% (P .< .05) higher ratio of weight to length than castrates. For POSTP boars the weight to length ratios were 13.4, 8.8 and 6.7% larger for the scapula, radius-ulna and tibia-fibula bones, reSpectively, and the ratios for the humerus and femur tended to be higher than castrates. This confirms observations of Walstra (1980). Based on this ratio of weight to length indication of density or thickening, no differences occurred due to either form of androgen. ' As described in the methods section of this chapter, another indicator of diaphysis wall thickening was examined. The total circumferential area of the diaphysis wall on the proximal and distal ends of a center section of the humerus and femur are given in tables VIII-13 and 14 for the PREP and POSTP studies, respectively. With this method, greater thickening occurred in boars relative to castrates in both studies. Wall thicknesses were 'also almost 2-fold greater in POSTP than PREP pigs. After comparing the treatment group thickness measurements to the initial values for each study, it is apparent that the 276 .mcme mumswm ummmA Mo Houum Unmccmumuzmm o .mmumuummo omMIuAEAAnommAU .mucmamEA zmflnumu .mucmamaw 3OHHQU .mmumuummonu .mnmonumo .Amo.v.mv AmMMAo mumfluomummsm ucmummwflo cufi3 mGEdAoo cAsuAB momma ucmfiummuan .numcmA econ an Um©A>A© unmflm3 mcomm AA.A AA.A AA.A AA.A AA.A msouo - AAAAAAAAA AAAAAAA om. mm. Nv. mm. Am. osmm AAAA.AA AAA.A AAAA.A AAA.AA AA.A AAAAU AAA.AA AAA.AA AAAA.A AAA.AA AA.A emmemo AAAA.AA AmAA.AA AAA.A AAA.AA AA.A AAAAAU I I O O O m mom OH mmmm m mom m wwam AH on A Ema U . . . . . . 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HmuzmsnAm AMAHACH om. mN. «v. vv. mm.mm mN.vv mzmm mNo.o mmo.o NNm.mA Naov.m v.NomA 5N.OOOA ommqo . . m com o smmm o Aon NA AAm OA N mva Ace vaA Emma U . . . . . . A AnmN 0 man 0 anNm mA «Amm OA m NNVA Ago mBOA Emma U . . . . . . m anA o now u nmvm NA some m m quA mAv woAA 9mm 0 nmNo.o mmo.m NanN¢.¢A mam.N o.mmNA mm.Nom amnqo mmo.m mMA.w mmN.mA flmmA.m N.AmNA flAnm.mAAA U 3mm.o nvo.m A0A.NA nAmv.OA h.momA nm.vMNA m noEmm msumEDm MAQAB msAomm mAnAB msAomm szouw unmfiummue ommmchAna ©£u3ouu ocucAB AAmz Ammm>nmmAQ cmxumz chAo>omnumB mHMAm AmwmhnmAmm mmDme QZd mDmmZDm fine no mmMZMUHEB Aflfiz mHmNmmflHD QZ< mDHafim DZ< mqmdfi 283 had thicker (P < .05) humerus and femur diaphyseal walls than castrates but they were less than the diaphyseal thicknesses of boars. Short (1980) discussed the "double threshold" effect of testosterone upon bone growth. High concentrations initially stimulate bone but may also retard growth, whereas low concentrations are usually stimulatory. The effect of large doses of testosterone on growth suppression may not be on bone per se but mediated through inhibition of pituitary function (Silberberg and Silberberg, 1971). The effects of testosterone on bone growth in normal castrated and gonadohypOpysectomized male rats was reported by Jansson et a1. (1983). Testosterone caused a dose dependent increase in linear bone growth' in castrated rats. However, in growth hormone deficient castrated rats, no increase in bone growth -due to testosterone was observed. In the present study, no consistent dose dependent effects upon linear bone growth were observed for either testosterone or dihydrotestosterone. The effect of castration upon bone accretion was less apparent in PREP pigs than POSTP pigs. In addition, bone accretion was more effectively stimulated by testosterone in POSTP pigs than in PREP pigs. The effects of castration reported “here are less obvious than reported in rats (Spencer et al., 1976) but follow trends reported by Knudson (1983). While the 284 effects of castration or androgen administration upon linear growth. were subtle in this study, it appears that the presence of the testes and testosterone administration (at these doese) stimulate bone lengthening and bone thickening in pigs. These effects on bone growth also coincide with a stimulation of selected muscles, particularly the semintendinosus (Chapter IV). Summary Weights of several bones of pigs in the 3 wk study did not differ due to treatment. In the prepubertal study, high testosterone implanted pigs did not differ from castrates but had more bone than high dihydrotestosterone implanted pigs. This latter difference was apparent in the tibia-fibula and femur bone weights. In the pubertal study, boars had greater total bone and individual bone weights than castrates. There was also a tendency for high testosterone implanted pigs to have more total bone than high dihydrotestosterone implanted pigs. A similar trend was observed for low testosterone compared to low dihydro- testosterone. Boars tended to have longer bones than castrates in all studies. Tibia linear growth appeared to be stimulated by high testosterone in both 5 wek studies. This is supported by total length, diaphysis length and tetracycline marked growth. measurements. Since dihydrotestosterone implanted 285 pigs were similar to castrates in these measurements, a differential effect due to the form of androgen was detected. Androgen administration increased bone thickness compared to castrates but generally not to the same degree as the differences observed between boars and castrates. Testosterone implanted pigs tended to. have thicker bones than dihydrotestosterone implanted pigs. ' Based upon the bone data, it appears that castration decreased bone growth and that androgens can increase bone growth and thickening but testosterone was more effective than dihydrotestosterone. This indicates a difference in the mode‘of action of the two forms of the androgen. In terms of use as growth promotants, testosterone proved to be more suitable than dihydrotestosterone in stimulating linear bone growth. The effects observed in these studies may be different from those observed in a more chronic administration. APPENDICES 286 TABLE A.l. Testosterone Assay PREPARATION 1. Set up assay sheet (160 tube maximum - including standards, standard serum, samples). 2. Number extraction tubes (16, X 100 rrm) for Tracers (TR), standard serum (SS) and blanks. ' 3. Number assay tubes (12 X 75 mm) for standards, zeros, SS and samples . EX‘I‘RACI‘IQ‘I EFFICIENCY 1. Add lOul 3H testosterone (refrigerator 3 - no. 3072) to each of 3 scintillation vials and 3 extraction tubes (TR). Dry with nitrogen. (use Hamilton syringe - clean with before and after). , To the scintillation vials add 5.0 ml ACS cocktail, cap and label "'I'I‘C" (tracer total counts) . Set aside. To tubes add appropriate amount of serum (200ul) fran randan samples to be assayed or a standard serum, allow to equili- brate 30 minutes and proceed with extractions . SAMPLING AND EXTRACI‘ICN 1. Sampling - add 200ul serum to extraction tubes according to the assay sheet. a) Standard senrn - low (200ul) and high (50ul) in triplicate. b) Unknowns - 200ul in duplicate. Extraction - add 10 volumes (2 ml) Benzene: Hexane (1:2) to each tube. a) Vortex all tubes for 30 seconds. b) Freeze in the tubes with a NEOH: dry ice bath. c) Decent the supernatant: 1) TR decant into scintillation vials, add 5.0 ml ALB, cap, label "TR" and set aside. 2) Decant samples and standardserum into 12 X 75 nm tubes. 3) Evaporate the solvent in vacuum oven in the hood. STANDARDCURVES 1. With hamilton syringe, add appropriate annunts to tubes. Do in triplicate: 0, 5, 10, 15, 20, 25, 30, 40, 60, 80, 100 ul _.-— 287 Page 2 — TABLE A.l. Standard curves - continued These are taken from stock testosterone solution (10 ng/ml) 2. Allow to dry in vacuum drying oven. ASSAYPHDCEIIJRE 1. Add 200ul antibody*to all tubes (not background), vortex 2 seconds. 2. Allow to equilibrate 30 min at roan temperature. 3. Add 200ul 3H testosterone to all tubes and to 3 scintillation vials, vortex tubes. To scintillation vials add 5.0 ml ACS, cap and label "100%" or "TC". Set aside. 4. Incubate tubes 12-18 hours at 1-5° (cooler, C). Separation of Bound vs . Free homone (.5% charcoal, 1% dextran) 1. Put stock charcoal solution on magnetic stirrer for about 10 minutes. ‘ 2. Put assay tubes on ice bath for 10-15 minutes. 3. Aliquot enough charcoal for assay into small beaker with a stir bar and place in ice bath on stir plate. 4. Add 0.5 ml charcoal to all tubes with a Cornwall syringe. 5. Vortex and spin 15 min at 3000 rpn. 6. Put carriers into ice bath. 7. Decant the supernatants into scintillation vials and mix with 5.0 ml ACS. ***These above steps must be done quickly and without interruption*** (IIJNI‘ING 1. load counter according to the canputer protocol. 2. Count all tubes for 4 min and record on magnetic tape. *Antibody has cross reactivity with DHT of approximately 55%. 288 Page 3 - TABLE A.l. Computer Protocol: back ground "0" std curves TR: TR . samples - (blanks, High serum, low serum and samples) 100% or TC 289 TABLE B. l Stock amino acid solution Amino Acid gél Alanine .6060 Arginine .3640 Aspartie .0538 Cysteine i .1078 Glutamine .4310 Glycine .5454 Histidine .1482 Isolencine .3570 Leucine .4580 Lysine .5926 Methionine .1280 Phenylalanine .1346 Proline .5792 Valine .3030 Threonine .1548 Serine .2290 Glutamine .4512 TryptOphan .2356 Asparagine .0538 Chloramphenicol* .0054 This mixture was calculated to be 5 X porcine plasma values plus corrected for dilution in KRB solution. *Not an amino acid; added as a preservative. 290 TABLE B. 2 Phosphate buffer Stock solution:> Monobasic Dibasic Sodium phosphate Sodium phosphate (NaH2P04'H20) (NazHPO4'7H20) 27.5 g/l 53.6 g/l Volumes of each plus 100 m1 H 20 to give pH desired: p_H_ 6.8 51 49 7.0 34 61 7.1 33 67 7.2 28 72 7.3 23 77 7.4 19 81 7.6 13 87 291 TABLE B. 3 Citrate - phosphate - borate buffer Primary stock solutions: l. 40 g NaOH per liter 2. 35 ml of 85% H3PO4 per liter 3. 70 g citric acid per liter 4. .1 N HCl Mixture of above*: 100 ml H PO 3 4 100 ml Citric acid 343 ml NaOH 3.54 g Boric acid Bring to 1 liter with water *Take 20 ml of the mixture plus corresponding HCl to arrive at desired pH. For pH 6.7 this requires about 34.6 ml of HCl. From: Teorell, T. and E. Stenhagen. 1938. Ein Universalpuffer furden pH-Bereich 2.0 to 12.0. Biochem. 8. 299:416. 292 TABLE B. 4 Krebs-Ringer bicarbonate buffer Primary stock solutions NaCl 4.50g/100 ml KCL 5.75g/100 ml CaCl2 6.10g/100 ml KH2P04 10.55g/100 ml MgSO4 9.25g/100 ml or MgSO4°7H20 19.10g/100 ml NaHCO3* 65.009/1000 m1 *Gassed with 02:CO2 (95:5) for l h. **On day of use, dilute with 130 ml H 15 min. Volume used to 5 X buffer** make 2 25 ml 1 ml .75 .25 .25 O and gas for ml m1 ml m1 LITERATURE CITED ‘F- LITERATURE CITED Aberle, E. C., T. D. Etherton and C. E. Allen. 1977. Prediction of pork carcass composition using subcutaneous adipose tissue moisture or lipid concentration. J. Anim. Sci. 45:449. Allee, G. L., D. H. Baker and G. A. Leveille. 1971a. Fat utilization and lipogenesis in the young pig. J. Nutr. 101:1415. Allee, G.L., D. Romsos, G. A. Leveille and D. H. Baker. 1971b. Influence of age on in vitro lipid biosynthesis and enzymatic activity in pig adipose tissue. Proc. Soc. Exp. Biol. Med. 137:449. Allee, G. L., D. R. Romsos, G. A. Leveille and D. H. Baker. 1972. Lipogenesis and enzymatic activity in pig adipose tissue as influenced by source of dietary fat. J. Anim. Sci. 35:41. Allen, C. E. 1976. Cellularity of adipose tissue in meat animals. Fed. Proc. 35:2302. Allen, C. E., D. C. Beitz, D. A. Cramer and R. G. Kauffman. 1976. Biology of fat in meat animals. North Central Regional Res. Pub. No. 234. Allen, R. E., K. E. Masak, P. K. McAllister and R. A. Merkel. 1983. Effects of growth hormone, testosterone and serum concentration on actin synthesis in cultured satellite cells. J. Anim. Sci. 56:833. Allen, R. E., R. A. Merkel and R. B. Young. 1979. Cellular aspects of muscle growth: Myogenic cell proliferation. J. Anim. Sci. 49:115. Allrich, R. D., R. K. Christenson, J. J. Ford and D. R. Zimmerman. 1982. Pubertal development of the boar: ~Testosterone, estradiol-17B, cortisol and LH concen- trations before and after castration at various ages. J. Anim. Sci. 55:1139. Alcia, J. F. and R. A. Field. 1976. The effect of androgens and related sustances on carbohydrate metabolism, lipid metabolism and diabetes mellitus. Pharmacol. Therap. 1:241. 293 ._ _ ....__.——A—- 294 Ambrose, J. A. 1974. Fluorometic measurement of tyrosine in serum and plasma. Clin. Chem. 20:505. Anderson, D. B. 1972. The cellular development of adipose tissues. Proc. Recip. Meat Conf. 25:9. Anderson, D. B. and R. G. Kauffman. 1973. Cellular and enzymatic changes in porcine adipose tissue during growth. J. Lipid Res. 14:160-168. Anderson, D. B., R. G. Kauffman and L. L. Kastenschmidt. 1972. Lipogenic enzyme activities and cellularity of porcine adipose tissue from various anatomical locations. J. Lipid Res. 13:593. Arnal, M., M. Ferrara and G. Fanconneau. 1976. Nuclear Techniques in Animal Production and Health. Inter. Atomic Energy Agency Rep. No. 205:393. Arvill, A. 1967. Effects of testosterone on the metabolism of the isolated levator ani muscle of the rat. Acta. Endocrinologica 56 (Suppl. 122):1. Aschkenasy-Lelu, P. and A. Aschkenasy. 1959. Effects of androgens and oestrogens on the metabolism of proteins and the growth of tissues. In: World Review of Nutrition and Dietetics. J. B. LippinCOtt Company, Philadelphia. Ashby, P., D. P. Bennett, I. M. Spencer and D. S. Robinson. 1978. Post-translational regulation of lipoprotein lipase activity in adipose tissue. Biochem. J. 176:865. Ashby, P. and D. S. Robinson. 1980. Effect of insulin, glucocorticoids and adrenaline on the activity of rat adipose tissue lipoprotein lipase. Biochem. J. 188:185. Association of Official Analytical Chemists. 1980. Published by Assoc. of Official Analytical Chemists. (13th Ed.) George Banta Company, Inc., Menasha, Wisconsin. Aurback, G. D., S. J. Marx and A. M. Spiegel. 1981. Parathyroid hormone, calcitonin and the calciferols. In: R. H. Williams (Ed.) Textbook of Endocrinology, Chapter 19. W. B. Saunders Company, Philadelphia. Avruch, J., J. R. Carter and D. B. Martin. 1972. The effect of insulin in the metabolism of adipose tissue. In: R. G. Greep and E. B. Astwood (Eds.) Handbook of Physiology, Section 7, Vol. 1. American Physiological Society, Washington, D.C. 295 Baginsky, M. J. 1981. Measurement of lipoprotein lipase and hepatic triglyceride lipase in human postheparin plasma. Meth. Enzymol. 72 (Part D):325. Ballard, F. J. and G. L. Francis. 1983. Effects of anabolic agents on protein breakdown in L6 myoblasts. Biochem. J. 210:243. Baxter, J. D. 1776. Glucocorticoid hormone action. Pharmac. Ther. B. 2:605. Becker, R. O. 1982. Mechanism of Growth Control. C. C. Thomas-Publisher, Springfield, IL. Beeson, W. M., F. N. Andrews, T. W. Perry and M. Stob. 1955. The effect of orally administered stilbestrol and testosterone on growth and carcass composition of swine. J. Anim. Sci. 14:475. Bensadoun, A., C. Ehnholm, D. Steinberg and W. Brown. 1974. Purification and characterization of lipoprotein lipase from pig adipose tissue, J. Biol. Chem. 249:2220.‘ Bergen, W. G. 1974. Protein synthesis in animal models. J. Anim. Sci. 38:1079. Bergstrand, C. G. 1950. The influence of testosterone propionate on skeletal development in the immature rabbit. Acta Endocrinol. 4:91. Bicikova, M., R. Hampl and L. Starka. 1977. The requirement of cytoplasmic receptors for entry of testosterone into nuclei of rat skeletal muscle. Endocrinologica Experimentalis. 11:271. Bidner, T. D., R. A. Merkel and E. R. Miller. 1972. Effect of a combination of diethylstilbestrol and methyltestosterone on performance, carcass traits, serum and muscle characteristics of pigs. J. Anim. Sci. 35:525. Bird, J. W. C., J. H. Carter, R. E. Triemer, R. M. Brooks and A. M. Spanier. 1980. Proteinases in cardiac and skeletal muscle. Fed. Proc. 39:20. Bird. J. W. C. and J. H. Carter. 1980. Proteolytic enzymes in striated and non-striated muscle. In: K. Wildenthal (Ed.) Degradative Processes in Heart and Skeletal Muscle. pp. 51-85. Elsevier-North Holland Bio Medical Press, New York. Bjorntorp, P. and J. Ostman. 1971. Human adipose tissue dynamics and regulation. Adv. Metab. Disord. 5:277. 296 Blair, R. and P. R. English. 1965. The effect of sex on growth and carcass quality in the bacon pig. J. Anim. Sci. 64:169. Branang, E. 1971. Studies on monozygous cattle twins XXIII. The effect of castration and age of castration on the development of single muscles, bones and special sex characters. Part II. Swedish J. Agr. Res. 1:69. Bratzler, L. J., R. P. Soule, E. R. Reineke and P. Paul. 1954. The effect of testosterone and castration on the ' growth and carcass characteristics of swine. J. Anim. Sci. 13:171. Breuer, C. B. and J. R. Florini. 1965. Amino acid incorporation into protein by cell free systems from rat skeletal muscle. IV. Effects of animal age, androgens and anabolic agents on activity of muscle ribosomes. Biochem. 4:1544. Buckley, P. A. and I. R. Konigsberg. 1973. Myogenic fusion and the duration of the post-mitotic gap (G1). Develop. Biol. 37:193. Bullock, G. R., R. F. Peters and A. M. White. 1969 Changes in the mitochondrial structure and ribosomal activity in muscle as a consequence of the interaction between a glucocorticoid and some anabolic steroids. Biochem. J. 115:47. ‘ Bullock, G. R., A. M. White and J. Worthington. 1968. The effects of cataboliC'and anabolic steroids on amino acid inCorporation by skeletal muscle ribsomes. Biochem. J. 108:417. Burch, L., J. R. Scaife and H. Galbraith. 1982. Effect of anabolic steroids on lipogenic and lipolytic enzymes in sheep tissues. Horm. Metabol. Res. 14:52. Burgess, C., E. R. Lidvall, C. B. Ramsey and J. W. Cole. 1966. Performance and carcass quality of litter-mate boars barrows and gilts fed alike. Tennessee Farm and Home Sci. Prog. Rep. 58:6. _Burkhart, J. M. and J. Jowsey. 1967. Parathyroid and thyroid hormones in the development of immobilization osteoporosis. Endocrinology 81:1053. Burleigh, I. G. 1980. Growth curves in muscle nucleic acid and protein: Problems on interpretation at the level of the muscle cell. In: T.L.J. Lawrence (Ed.) Growth in Animals. pp 101-136. Butterworths, London. 297 Burns, T. W., P. E. Langley, B. E. Terry, D. B. Bylund, B. B. Hoffman, M. D. Tharp, R. J. Lefkowitz, J. A. Pharmacological Garcia-Sainz and J. N. Pain. 1981. characterization of adrenergic receptors in human adipocytes. J. Clin. Invest. 67:467. H. 1949. Biological Actions of Sex Hormones. Burrows, Cambridge University Press, Cambridge. L. E. Kunkle, A. L. Moxon and Cahill, V. R., H. S. Teague, E. A. Rutledge. 1960. Measurement of and ways of affecting sex influenced performance on growing- J. Anim. Sci. 19:1036. finishing swine. The infuence of Campbell, R. G. and R. H. King. 1982. dietary protein and level of feeding on the growth performance and carcass characteristics of entire and castrated male pigs. Anim. Prod. 35:177. The hormonal and local regulation of Endocrine Rev. 4:62. - Canalis, E. 1983. 1975. An analysis of bone formation. Cardasis, C. A. and G. W. Cooper. nuclear number in individual muscle fibers during differentiation and growth: a satellite cell-muscle fiber growth unit. J. Exp. 2001. 191:347. Carroll, M. A., F. Hill and P. B. O'Donovan. effects of castration on pig carcass of pork and bacon Irish J. Agr. Res. 2:177. The effects of sex and age of male 1963. Some weights. Charette, L. A. 1961. at castration on growth and carcass quality of Yorkshire swine. Can.J. Anim. Sci. 41:30. Cheek, D. B., A. B. Holt, D. E. Hill and J. L. Talbert. 1971. Skeletal muscle cell mass and growth: the concept of the deoxyribonucleic acid unit. Pediat. Res. 5:312. A study of Chiakulus, J. J. and J. E. Pauly. 1965. postnatal growth of skeletal muscle in the rat. Rec. 152:55. Chin, H. and R. R. Almon. 1980. castration on the cholinergic receptor population in J. Pharmac. Exp. Ther. 212:553. 1978. Anat. Fiber type effects of skeletal muscle. Colenbrander, B., F. H. deJong and C. J. G. Wensing. Changes in serum testosterone concentrations in the J. Reprod. Pert. 53:377. male pig during development. 298 Cordesse, R. and J. Nougues. 1973. Multiplication et grandissement cellulaires, activite cellulaire du tissu musculaire de lapin au cours de la croissance. I. Aspect descriptif. Annls. Biol. Anim. Biochim. Biophys. 13:535. Corring, T., G. Durand and Y. Henry. 1982. Some aspects of development and nutrition in the monogastric animal during postnatal life. erd. Rev. Nutr. Diet. 39:124. Cryer, A. 1981. Tissue lipoprotein lipase activity and its action in lipoprotein metabolism. Inter. J. Biochem. 13:525. Cryer, A. S., E. Riley, E. R. Williams and D. 8. Robinson. 1976. Effect of mutritional status on rat adipose tissue, muscle and post heparin plasma clearing-factor lipase activities: their relationship to triglyceride fatty acid uptake by fat cells and to plasma insulin concentrations. Clin. Sci. Mol. Med. 50:213. Dahlmann, B. and H. Reinauer. 1978. Purification and some properties of an alkaline proteinase from rat skeletal muscle. Biochem. J. 171:803. ' Dahlmann, B. and H. Reinauer. 1981. Adaptation of muscle alkaline proteinase activity to hormonal alterations. Adv. Physiol. Sci. 24:191. Dahlmann, B., B. Mai and H. Reinauer. 1980. The influence of testosterone on the alkaline proteolytic activity in rat skeletal muscle. Biochim. Biophys. Acta 641:479. Dahlmann, B., C. Schroeter, L. Herbertz and H. Reinauer. 1979. Myofibrillar protein degradation and muscle proteinases in normal and diabetic rats. Biochem. Med. 21:33. Davies, A. S. 1972. Postnatal changes in the histochemical fiber types of porcine skeletal muscle. J. Anat. 113:213. Davies, P., A. Cryer and D. S. Robinson. 1974. Hormonal control of adipose tissue clearing factor lipase activity. FEBS Lett. 45:271. Dayton, W. R., R. A. Lepley and J. V. Schollmeyer. 1982. The role of muscle proteolytic enzymes in degradation of the myofibril. Proc. Recip. Meat Conf. 34:17. Devi, A., M. A. Mukundan, U. Srivastara and N. K. Sarkar. 1963. The effect of age on the variations of deoxyribonucleic acid, ribonucleic acid and total nucleotides in liver, brain and muscle of rat. Exp. Cell Res. 32:242. 299 Dienstman, S. R. and H. Holtzer. 1975. Myogenesis: a cell lineage interpretation. In: J. Reinert and H. Holtzer (Eds.) Results and Problems in Cell Differentiation. pp 3-25. Springer Verlag, New York. DiMarco, N. M., D. C. Beitz and G. B. Whitehurst. 1981. Effect of fasting on free fatty acid, glycerol and cholesterol concentrations in blood plasma and lipoprotein lipase activity in adipoe tissue of cattle. J. Anim. Sci. 52:75. Dionne, F. T., R. L. Lesage, J. Y. Dube and R. R. Tremblay. 1979. Estrogen binding proteins in rat skeletal and perineal muscles: in vitro and in vivo studies. J. Steroid Biochem. 11:1073. Dodds, P. F., M. I. Gurr and D. N. Brindley. 1976. The glycerolphosphate, dihydroxyacetone phOSphate and monoglyceride pathways of glycerolipid synthesis in rat adipose tissue homogenates. Biochem. J. 160:693. Dzuik, P. J. and B. Cook. 1966. Passage of steroids through silicone rubber. Endocrinology 78:208. Elliot, M. K. and V. R. Fowler. 1973. The response of growing, castrated male pigs to various hormones of possible anabolic potential. Proc. Nutr. Soc. 32: 103A-104A. Elliot, M. K. and V. R. Fowler. 1974. The effect of sex hormones given orally on lean tissue anabolism in growing female and castrated male pigs. Proc. Brit. Soc. Anim. Prod. 3:106 (Abstr.). Enser, M. B. 1973. Clearing factor lipase in muscle and adipose tissue of pigs. Biochem. J. 136:381. Enesco, M. and D. Puddy. 1964. Increase in the number of nuclei and weight in skeletal muscle of rats of various ages. Amer. J. Anat. 114:235. Etherton, T. D. and C. E. Allen. 1980. Effects of age and adipocyte size on glucose and palmitate metabolism and oxidation in pigs. J. Anim. Sci. 50:1073. Ethlinger, J. D., T. Kameyama, K. Toner, D. Van der Westhuyzen and K. Matsumoto. 1980. Calcium and stretch-dependent regulation of protein turnover and myofibrillar disassembly in muscle. In: D. Pette (Ed.) Plasticity of muscle. pp 541-557. Walter de Gruyter, New York. - 300 Ezekwe, M. O. and R. J. Martin. 1975. Cellular characteristics of skeletal muscle in selected strains of pigs and mice and the unselected controls. Growth. 39:95. Fain, J. N. 1973. Biochemical aspects of drug and hormone action on adipose tissue. Pharmacol. Rev. 25:67. Fain, J. N. 1977. Cyclic nucleotides in adipose.tissue. In: H. Cramer and J. Schultz (Eds.) Cyclic Nucleotides Mechanisms of Action. pp 207-228. John Wiley and Sons, New York. Fain, J. N. 1980. Hormonal regulation of lipid mobilization from adipose tissue. In: G. Litwack (Ed.) Biochemical Actions of Hormones. Vol. 7. pp 119-204. Academic Press, New York. Fain, J. N. 1982. Regulation of lipid metabolism by cyclic nucleotides. Handbook Exp. Pharm. 58II:89. Fain, J. N., R. E. Shepher, C. C. Malbon and F. J. Moreno. 1978. Hormonal regulation of the breakdown of tri- glyceride. In: J. N. Dietschy (Ed.) Disturbances in Lipids and Lipoprotein Metabolism. pp 213-228. American Physiological Society, Washington, D. C. Fallon, H. J., R. G. Lamb and S. C. Jamdar. 1977. . Phosphatidate phosphohydrolase and the regulation of glycerolipid biosynthesis. Biochem. Soc. Trans. 5:37. Felts, W. J. L. 1959. Transplanation studies of factors in- skeletal organogenesis. I. The subcutaneously implanted immature long bone of the rat and mouse. Am. J. Physiol. Anthropol. 17:201. Field, R. A. 1971. Effect of castration on meat quality and quantity. J. Anim. Sci. 32:849. Fischmann, D. A. 1972. Development of striated muscle. In: G. H. Bourne (Ed.) The Structure and Function of Muscles. Vol. 1 pp 75-178. Academic Press, New York. Florini, J. R. 1970a. The effects of testosterone on gene expression in skeletal muscle. Proc. Meat Ind. Res. Conf. p 1. Florini, J. R., 1970b. The effect of testosterone on qualitative pattern of protein synthesis in skeletal muscle. Biochem. J. 9:909. Fortin, A., D. W. Friend and N. K. Sarkar. 1983. A note on the carcass composition of Yorkshire boars and barrows. Can. J. Anim. Sci. 63:711. 301 Fowler, V. R., C. L. Stockdale, R. I. Smart and R. M. J. Crofts. 1978. Effects of two androgens combined with estrogens on the growth and efficiency of pigs. Anim. Prod. 26:358. Fredholm, B. B. 1978. Local regulation of lipolysis in adipose tissue by fatty acids, prostaglandins and adenosine. Med. Biol. 59:249. Fredholm, B. B. 1981. Adenosine and lipolysis. Int. J. Obesity 5:643. Fulks, R. M., J. B. Li and A. L. Goldberg. 1975. Effects of insulin, glucose and amino acids on protein turnover in rat diaphyragm. J. Biol. Chem. 250:290. Fuller, M. F. _1980. Sex differences in the nutrition and growth of pigs. In: W. Haresign (Ed.) Recent Advances in Animal Nutrition, pp 157-165. Butterworths, Boston. Fuller, M. F., J. G. Gordon and R. Aitken. 1980. Energy and protein utilization by pigs of different sex and genotype. In: L. E. Mount (Ed.) Energy Metabolism. pp 169-174. Butterworths, London. - Galbraith, H. and J. H. Topps.- 1981. Effect of hormones on the growth and body composition of animals. Nutr. Abstr. Rev. 51B:521. Garfinkel, A. S., P. Nilsson-Ehle and M. C. Schotz. 1976} Regulation of lipoprotein lipase-induction by insulin. Biochim. Biophys. Acta 424:264. Garlick, P. J. 1980. Assessment of protein metabolism in the intact animal. In: P. J. Buttery and D. B. Lindsay (Ed.) Protein Deposition in Animals. pp 51-68. Butterworths, Boston. Gay, V. L. and J. T. Kerlan. 1978. Serum LH and FSH following passive immunization against circulating testosterone in the intact male rat and in orchidectomized rats bearing subcutaneous Silastic implants of testosterone. Arch. Androl. 1:239. Gilbreath, R. L. and J. R. Trout. 1973. Effects of early postnatal dietary restrictions and repletion on porcine muscle growth and composition. J. Nutr. 103:1637. Goldberg, A. L. 1969. Protein turnover in skeletal muscle. II. Effects of devervation and cortisone on protein catabolism in skeletal muscle. J. Biol. Chem. 24433223. 302 Goldberg, A. L. 1980. The regulation of protein turnover by endocrine and nutritional factors. In: D. Pette (Ed.) Plasticity of Muscle. pp 469-491. Walter de Gruyter, New York. Goldberg, A. L. and D. F. Goldspink. 1975. The influence of food deprivation and adrenal steroids on DNA synthesis in various mammalian tissues. Amer. J. Physiol. 228-310. Goldspink, G. 1962. Studies on postembryonic growth and development of skeletal muscle. I. Evidence of 2 phases in which striated muscle fibers are able to exist. Proc. Roy Irish Acad. 62B:135. Goldspink, G. 1970. The proliferation of myofibrils during muscle fiber growth. J. Cell. Sci. 6:593. Goldspink, D. F. 1977. The influence of immobilization and stretch on protein turnover of rat skeletal muscle. J. Physiol. 264:267. Goldspink, D. F. 1980a. Physiological factors influencing protein turnover and muscle growth in mammals. In: D. F. Goldspink (Ed.) Development and specialization of Skeletal Muscle. pp 65-90. Cambridge University Press, New York. Goldspink, D. F. 1980b. The influence of contractile activity and the nerve supply on muscle size and protein turnover. In: D. Pette (Ed.). Plasticity of Muscle. pp 525-540. Walter de Gruyter, New York. Goldspink, G. 1980c. Growth of muscle. In: D. F. Goldspink (Ed.) Development and Specialization of Skeletal Muscle. pp 19-36. Cambridge University Press, New York. Goldspink, D. F., P. J. Garlick and M. A. McNurlan. 1983. Protein turnover measured in vivo and in vitro in muscles undergoing compensatory growth and subsequent denervation atrophy. Biochem. J. 210:89. Goldspink, G., C. Tardieu, J. C. Tabary, C. Tabary and G. Tardieu. 1974. Effect of denervation on the adaptation of sarcomere number and muscle extensibility to the functional length of the muscle. J. Physiol. 236:733. Gordon, E. E., K. Kowalski and M. Fritts. 1966. Muscle protein and DNA in rat quadriceps during growth. Amer. J. Physiol. 210:1033. Goss, R. J. 1978. The Physiology of Growth. pp 68-89. Academic Press, Inc., New York, New York. 303 Gray, J. M. and M. R. C. Greenwood. 1982. Time course of effects of ovarian hormones on food intake and metabolism. Amer. J. Physiol. 243:E407. Gray, J. M., A. A. Nunez, L. I. Siegel and G. N. Wade. 1979. Effects of testosterone on body weight and adipose tissue: role of aromatization. Physiol. Behav. 23:465. Greenwood, M. R. C. and J. Hirsch. 1974. Postnatal development of adipocyte cellularity in the normal rat. J. Lipid Res. 15:474. Griffin, G. E., P. E. Williams and G. Goldspink, 1971. Region of longitudinal growth in striated muscle fibers. Nature 232:28. Grigsby, J. S., W. G. Bergen and R. A. Merkel. 1976. The effect of testosterone on skeletal muscle development and protein synthesis in rabbits. Growth 40:303. Gurr, M. I. and J. Kirtland. 1978. Adipose tissue cellularity: a review I. Techniques for studying cellularity. Int. J. Obesity. 2:401-427. Gustafssosn, J. A. and A. Pousette. 1975. Demonstrations and partial characterization of cytosol receptors for testosterone. Biochemistry 14:3094. Hakkarainen, J. 1975. Developmental changes of protein, RNA, DNA, lipid and glycogen in the liver, skeletal muscle and brain of the piglet. A methodological and experimental study with special reference to protein synthesis. Acta Veterinaria Scandanavica Suppl. 59:1. Hales, C. N., J. P. Luzio and K. Siddle. 1978. Hormonal control of adipose tissue lipolysis. Biochem. Soc. Symp. 43:97. Halestrap, A. P. and R. M. Denton. 1973. Insulin and the regulation of adipose tissue acetyl-coenzyme A carboxylase. Biochem. J. 132:509. Hall-Craggs, E. C. B. and C. A. Lawrence. 1969. The effect of epiphysial stapling on growth in length of the rabbits tibia and femus. Br. J. Bone Joint Sur. 51:359. ‘ Halse, J. 1979. Parathyroid function in acromegaly. Cacified Tissue Inter. 27 (Suppl. 58):1. Ham, A. W. and D. H.-Cormack. 1979. Histology. pp 377-462. J. B. Lippincott Company, Philadelphia. 304 Hamosh, M. and P. Hamosh. 1975. The effect of estrogen on the lipoprotein lipase activity of rat adipose tissue. J. Clin. Invest. 55:1132. Hanoune, J., A. M. Cambaut and A. Josiporvicz. 1972. The glucose effect and cortisone action upon rat liver and muscle protein metabolism. Archiv. Biochem. Biophys. 148:180. Hansen, F. M., N. Fahmy and J. H. Nielsen. 1980. The influence of sexual hormones on lipogenesis and lipolysis in rat fat cells. Acta Endocrinol. 95:566. Hansen, F. M.., P. Nielsson, B. E. Hustvedt, P. Nilsson-Ehle, H. Holm and A. Lovo. 1983. The effect of gonadectomy on fat and protein metabolism in VMH lesioned rats. Horm. Metab. Res. 15:84. Hansson, I., K. Lundstrom and B. Malmfors. 1975. Effect of sex and weight on growth, feed efficiency and carcass characteristics of pigs. 2. Carcass characteristics of boars, barrows and gilts slaughtered at four different weights. Swedish J. Agric. Res. 5:69. Hansson, L. I., K. Memander-Sellman, A. Stenstrom and K. G. Thorgren. 1972. Rate of normal longitudinal bone growth in the rat. Calcif. Tiss. Res. 10:238. ,\ . / Harbison, S. A., D. E. Goll, F. C. Parrish, Jr., V. Wang and E. A. Kline. 1976. Muscle growth in two genetically different lines of swine. Growth 40:253. Haugeback, D. D., H. B. Hedrick and J. M. Asplund. 1974. Adipose tissue accumulation and cellularity in growing and fattening lambs. J. Anim. Sci. 39:1016. Hausman, G. J. 1978. Cellular and histological aspects of developing porcine adipose tissue. Proc. Recip. Meat Conf. 31:35. Hausman, G. L. 1982. Adipocyte development in subcutaneous tissues of the young rat. Acta Anat. 112:185-196. Hausman, G. J., D. R. Campion and R. J. Martin. 1980. Search for the adipocyte precursor cell and factors that promote its differentiation. J. Lipid Res. 21:657. Hausman, G. J., D. R. Campion and G. B. Thomas. 1983. Adipose tissue cellularity and histochemistry in fetal swine as affected by genetic selection for high or low backfat. J. Lipid Res. 24:223. 305 Hausman, G. J. and R. L. Richardson. 1983. Cellular and vascular development in immature rat adipose tissue. J. Lipid Res. 24:522. Hegarty, P. V. J., L. C. Gumdlach and C. E. Allen. 1973. Comparative growth of porcine skeletal muscle using an indirect prediction of muscle fiber number. Growth 37:333. Heitzman, R. J. 1980. Manipulation of protein metabolism, with special reference to anabolic agents. In: P. J. Buttery and D. B. Lindsay (Ed.) Protein Deposition in Animals. pp 193-203. Butterworths, Boston. Hems, D. A. 1975. Control of hepatic glyceride synthesis. Proc. Nutr. Soc. 34:225. Hentges, E. J., D. N. Marple, D. A. Roland, Sr., and J. F. Pritchett. 1983. Growth and in vitro protein synthesis in two strains of chicks. J. Anim. Sci. 57:320. Hepp. K. D. and R. Renner. 1972. Insulin action on the adenyl cyclase system: antagonism to activiation of lipolytic hormones. FEBBS Lett. 20:191. Herington, A. C., H. J. Cornell and A. D. Kuffer. 1983. Review: recent advances in the biochemistry and physiology of the insulin like growth factor/ somatomedin family. Int. J. Biochem. 15:1201. Hervey, G. R. and I. Hutchinson. 1973. The effects of * testosterone on body weight and composition in the rat. J. Endocrinol. 57:24. Hetzer, H. O. and R. H. Miller. 1972. Rate of growth as influenced by selection for high and low fatness in swine. J. Anim. Sci. 35:730. Hietanen, E. and M. R. C. Greenwood. 1977. A comparison of lipoprotein lipase activity and adipocyte differentiation in growing male rats. J. Lipid Res. 18:480. - Hines, R. H. 1966. The interaction of restricted feed intake and sex on swine performance and carcass quality. Ph.D. Thesis, Michigan State University, East Lansing. Hirsch, J. and E. Gallian. 1968. Methods for determination of adipose cell size in man and animal. J. Lipid Res. 9:110. 306 Holtrop, M. E. and L. G. Raisz. 1979. Comparison of the effects of 1,25-dihydroxycholecalciferol, prostaglandin E and osteoclast activating factor with parathyroid hgrmone on the ultrastructure of osteoclasts in cultured long bones of fetal rats. Calcif. Tissue Res. 29:201. Holtzer, H. 1970. Myogenesis. In: 0. Schjeide (Ed.) Cell Differentiation, pp 476-503. Van Nostrand Reinhold, New York. Holtzer, H. and R. Bischoff. 1970. Mitosis and myogenesis. In: E. J. Briskey, R. G. Cassens and B. B. Marsh (Eds.). The Physiology and Biochemistry of Muscle as a Food. Vol. 2. pp 29-51. The University of Wisconsin Press, Madison. Holtzer, H. and S. Holtzer. 1976. Lineages, quantal cell cycles and cell diversification. In: N. Muller-Berat (Ed.) Progress in Differentiation Research. pp 3-10. North Holland Publishing Company, Amsterdam. Holtzer, H., N. Rubenstein, S. Dienstman,J. Chi, J. Biehl and Somlye. 1974. Perspective in myogenesis. Biochemie 56:1575. Holtzer, H., G. Yeoh, N. Rubeinstein, J. Chi, S. Fellini and S. Dienstman. 1982. Review of controversial issues in myogenesis. In: W. W. Nichols and D. G. Murphy (Ed.) Regulation of Cell Proliferation and Differentiation. pp 87-104. Plenum Press, New York. Hood, R. L. and C. E. Allen. 1973. Comparative methods for the expression of enzyme data in porcine adipose tissue. Comp. Biochem. Physiol. 443:677. Howarth, R. E. and R. L. Baldwin. 1971. Synthesis and accumulation of protein and nucleic acid in rat gastrocnemius muscles during normal growth, restricted growth and recovery from restricted growth. J. Nutr. 101:477. Isaksson, O. G. P., J. O. Jansson and I. A. M. Gause. 1982. Growth hormone stimulates longitudinal bone growth directly. Science 216:1237. Ishikawa, A., T. Murase, N. Yamada, K. Tanaka, Y. Iwamoto, Y. Akanuma and N. Ohsawa. 1982. Lipoprotein lipase activity in adipose tissue of streptozotocin induced diabetic rats. Endocrinol. Japonica29:379. Jansson, J. O., 8. Eden and O. Isaksson. 1983. Sites of action of testosterone and estradiol on longitudinal bone growth. Amer. J. Physiol. 244:E135. 307' Jeanrenaud, B. and D. Hepp. 1970. Adipose Tissue, Regulation and Metabolic Functions. Thieme Publishing, Stuttgart. Johns, J. T. and W. G. Bergen. 1976. Growth in sheep. Pre and postweaning hormone changes and muscle and liver development. J. Anim. Sci. 43:192. Johnson, P. 1978. Studies on adipose tissue cultures. Proc. Recip. Meat Conf. 31:32. Johnston, E. F., J. H. Zeller and R. L. Hines. 1957. Some effects on swine of orally administered methyl testosterone. J. Anim. Sci. 16:1024 (Abstr). Joss, E. E., K. A. Zuppinger and E. H. Sobel. 1963. Effect of testosterone propionate and methyltestosterone on growth and skeletal maturation in rats. Endocrinology 72:123. Jungas, R. L. 1970. Effect of insulin on fatty acid synthesis from pyruvate, lactate or endogenous sources of adipose tissue: evidence for the hormonal regulation of pyruvate dehydrogenase. Endrocrinology 86:1368. Jungas, R. L. 1975. Metabolic effects on adipose tissue in vitro. In: A Hasselblatt and F. V. Bruchhausen (Eds.) _Insulin Action. pp 371-412. Springer Verlag, New York. Kalu, D. N., C. Hillyard and G. V. Foster. 1972. Effect of glucagon on bone collagen metabolism in the rat. J. Endocrinol. 55:245. Kasser, T. R., G. J. Hausman, D. R. Campion and R. J. Martin. 1983. Lipogenesis and pancreatic insulin release in fetal pigs. J. Anim. Sci. 56:579. Kather, M. L., M. Rivera and K. Brand. 1972. Interrela- tionship and control of glucose metabolism and lipogenesis in isolated fat cells. Effect of the amount of glucose uptake on the-rates of the pentose phosPhate cycle and of fatty acid synthesis. Biochem. J. 128:1089. , Kattesh, H. G., E. T. Kornegay, F. C. Grazdauskas, J. W. Knight and H. R. Thomas, 1979. Peripheral plasma testosterone concentration and sexual behavior in young prenatally stressed boars. Theriogenology 12:289. Kay, M. and R. Houseman. 1975. The influence of sex on meat production. In: D. J. A. Cole and R. A. Lawrie (Eds.) Meat. pp 85-105. Butterworths, London. 308 Kelly, P. J. 1971. The effects of thyroid and parathyroid deficiency on bone remodelling distal to a venous tourniquet. J. Anat. 110:349. Kelly, A. M. 1978. Satellite cells and myofiber growth in the rat soleus and extensor digitorum longus muscles. Develop. Biol. 65:1. Kessler, J. I. 1963. Effects of diabetes and insulin on the activity of myocardial and adipose tissue lipoprotein lipase in rats. J. Clin. Invent. 42:362. Khoo, J. C. and D. Steinberg. 1974. Reversible protein kinase activation of hormone-sensitive lipase from chicken adipose tissue. J. Lipid Res. 15:602. Khoo, J. C. and D. Steinberg. 1975. Hormone sensitive triglyceride lipase from rate adipose tissue. Meth. Enzymol. 35:181. Khoo, J. C., D. Steinberg, J. J. Huang and P. R. Vagelos. 1976. Triglyceride, diglyceride, monoglyceride and cholesterol ester hydrolases in chicken adipose tissue activited by adenosine 3',5'-monophosphate-dependent protein kinase. J. Biol. Chem. 252:3829. Kielanowski, J. 1976. Energy cost of protein deposition. In: D. J. A. Cole, K. N. Boorman, P. J. Buttery, D. Lewis, R. J. Neale and H. Swan (Eds.) Protein Metabolism and Nutrition. PP 307-216. Butterworth, New York. Kimura, I., T. Hasegawa, T. Miura and E. Ozawa. 1981. Proc. Japan Acad. 578:200. Kincl, F. A., G. Benagiano and I. Angee. 1968. Sustained release hormonal preparations. 1. Diffusion of various steroids through polymer membranes. Steroids 11:673. Kirtland, J. and M. I. Gurr. 1979. Adipose tissue cellularity: a review 2. The relationship between cellularity and obesity. Int. J. Obesity 3:15. Kirtland, J. and M. I. Gurr. 1980. Fat cell synthesis in pigs assessed after administration of tritiated thymidine in vivo. J. Agr. Sci. 95:325. Kiser, T. E., R. A. Milvae, H. D. Hafs, W. D. Oxender and T. M. Louis. 1978. Comparison of testosterone and androstenedione secretion in bulls given prostaglandin cmx or luteinizing hormone. J. Anim. Sci. 46:436. 309 Kley, H. K., P. Edelmann and H. L. Kruskemper. 1980. Relationship of plasma sex hormones to different parameters of obesity in male subjects. Metabolism 29:1041. Klyde, B. J. and J. Hirsch. 1979a. Isotopic labeling of DNA in rat adipose tissue: evidence for proliferating cells associated with mature adipocytes. J. Lipid. Res. 20:691. Klyde, B. J. and J. Hirsch. 1979b. Increased cellular proliferation in adipose tissue of adult rats fed a high-fat diet. J. Lipid Res. 20:705. Knudsen, J. F. and S. R. Max. 1980. Aromatization of androgens to estrogens mediates increased activity of glucose 6-phosphate dehydrogenase in rat levator ani muscle. Endocrinology 106:440. Knudson, B. K. 1983. Muscle growth and maturity parameters of boars and barrows. M.S. Thesis, Michigan State University, East Lansing. Kochakian, C. D. 1976. Anabolic-Androgenic Steroids. Springer-Verlag, New York. Kochakian, C. D., J. Hill and G. Costa. 1964. Amino acid composition of the proteins of the muscles and organs of the normal, castrated and testosterone treated guinea pig. Acta Endocrinol. 45:613. Kochakian, C. D. and C. Tillotson. 1957. Influence of several C-19 steroids on the growth of individual muscles of the guinea pig. Endocrinology 60:607. Kochakian, C. D., C. Tillotson, J. Austin, E. Dougherty, V. Haag and R. Coalson. 1956. The effect of castration on the weight and composition of the muscles of the guinea pig. Endocrinology 58:315. ' Kohama, K. and E. Ozawa. 1978. Muscle trophic factor: IV. Testosterone induced increase in muscle trophic factor in chicken serum. Muscle Nerve 1:320. Kompiang, I. P., A. Bensadoun and M. W. W. Yang. 1976. Effect of an antilipoprotein lipase serum on plasma triglyceride removal. J. Lipid Res. 17:498. Kono, T., F. W. Robinson and J. A. Sarver. 1975. Insulin-sensitive phosphodiesterase: its localization hormonal stimulation and oxidative stabilization. J. Biol. Chem. 250:7826. 310 Konigsberg, I. R. 1963. Clonal analysis of myogenesis. Science 140:1273. Konigsberg, I. R. 1971. Diffusion mediated control of myoblast fusion. Develop. Biol. 26:133. Konigsberg, I. R. 1982. The culture environment and its control of myogenesis. In: W. W. Nichols and D. G. Murphy (Ed.) Regulation of Cell Proliferation and Differentiation. pp 105-137. Plenum Press, New York. Kostyo, J. L. 1965. In vitro effects of adrenal steroid hormones on amino acid transport in muscle. Endocrinology 76:604. Kream, B. E., M. Jose, S. Yamada and H. F. DeLuca. 1977. A specific high affinity binding macromolecule for 1,25-dihydroxyvitamin D in foetal bone. Science 197:1086. Krieg, M. 1976. Characterization of the androgen receptor in the skeletal muscle of the rat. Steroids 28:261. Krieg, M., R. Szalay and K. D. Voigt. 1974. Binding and metabolism of testosterone and 5 cx-dihydrotestosterone in bulbocavenosus/levator ani (BcLa) of male rats: in vivo and in vitro studies. Steroid Biochem. 5:453. Krieg, M. and K. D. Voigt. 1976. In vitro binding and metabolism of androgens in various organs: a comparative study. J. Steroid Biochem. 7:1005. Krieg, M. and K. D. Voigt. 1977. Biochemical substrate of androgenic actions at a cellular level in prostrate, bulbocavenosus/levator ani and in skeletal muscle. Acta Endocrinol. 85 (Suppl. 214):43. Kroeske, D. 1963. The castration of male piglets. Vect en Zuivelber. 6:254. Laflamme, L. F., A. Trenkle and D. G. Topel. 1973. Effect of castration or breed type on growth of the longissimus muscle in male cattle. Growth 37:249. Lane, M. D., J. Moss and S. E. Polakis. 1974. Acetyl CoA carboxylase. Curr. T op. Cell. Regulation 8:139. Lapwood, K. R. and S. V. Florcruz. 1978. Luteinizing hormone and testosterone secretory profiles of boars: effects of stage of sexual maturation. Theriogenology 10:293. Larner, J., J. C. Lawrence, R. J. Walkenbach, P. J. Roach, R. J. Hazen and L. C. Huang. 1978. Insulin control of glycogen synthesis. Adv. Cyclic Nucleotide Res. 9:425, 311 Laron, Z. and A. Kowaldo-Silbergeld. 1964. Further evidence for a fat mobilizing effect of androgens. Acta Endocrinol. 45:427. Lash, T. W., H. Holtzer and H. Swift. 1957. Regeneration of mature skeletal muscle. Anat. Rec. 128:679. Laurent, G. J., M. P. Sparrow and D. J. Millward. 1978. Changes in rates of protein synthesis and breakdown during hypertrophy of the anterior of posterior lattisimus muscles. Biochem. J. 176:407. Leat, W. M. F. and R. W. Cox. 1980. Fundamental aspects of adipose tissue growth. In: T. L. J. Lawrnece (Ed.) Growth in Animals. pp 137-174. Butterworths, Boston. Lee, Y. B. and R. G. Kauffman. 1974. Cellular and enzymatic changes with animal growth in porcine intramuscular adipose tissue. J. Anim. Sci. 38:532. Leszczynski, D. E., T. Toda and F. A. Kummerow. 1982. Influence of dietary sex hormones on chick lipid metabolism. Horm. Metab. Res. 14:183. Levy, D. D. 1974. A pulsed electrical stimulation technique for inducing bone growth. Ann. New York Acad. Sci. 238:478. Liao, S., S. C. Hung, J. L. Tymoczki and T. Liang. 1976. Active forms and biodynamics of the androgen receptor in various target tissues. Curr. Topics Molec. Endocrinol. 4:139. Lobley, G. E., A. Walker, A. Connell and H. Galbraith. 1983. The effects of trenbolone acetate on growth rate and carcass composition of young female rabbits. Anim. Prod. 36:111. Lohse, C. L. 1973. The influence of sex on muscle growth in Merino sheep. Growth 37:177. Loten, E. G. and J. G. T. Sneyd. 1970. An effect of insulin on adipose tissue adenosine 3':5'-cyc1ic monophosphate phosphodiesterase. Biochem. J. 120:187.‘ Lowry, O. H., N. J. Rosebrough, A. L. Farr and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265. Lundstrom, K. E. Dahlberg, L. Nyberg, M. Snochowski, N. Standal and L. E. Edqvist. 1983. Glucocorticoid and androgen characteristics in two lines of pigs selected for rate of gain and thickness of backfat. J. Anim. Sci. 56:401. 312 Machlin, L. J. 1972. Hormonal influence of fat deposition. Proc. Recip. Meat Conf. 25:40. Mainwaring, W. I. P. 1979. The androgens. In: C. R. Austin and R. V. Short (Eds.) Mechanism of Hormone Action. pp 117-156. Cambridge University Press, New York. Martin, A. H. 1969. The problem of sex taint in pork in relation to the growth and carcass characteristics of boars and barrows: a review. Can. J. Anim. Sci. 49:1. Martin, L. G., J. W. Clark and T. B. Conner. 1968. Growth hormone secretion enhanced by androgens. J. Clin. Endocrinol. 28:425. Martin, L. G., M. S. Grossman, T. Bu Conner, In I“ Levitsky, J. W. Clark and F. D. Camitta. 1979. Effect of androgen on growth hormone secretion and growth in boys with short stature. Acta Endocrinol. 91:201. Martin, R. J. , M. Ezekive, J. H. Herbein, G. W. Sherritt, J. L. Gobble and J. H. Ziegler. 1974. Effects of neonatal nutritional experiences on growth and development of the pig. J. Anim. Sci. 39-521. Martin, R. J. and J. H. Herbein. 1976. A comparison of the enzyme levels and the in vitro utilization of various substrates for lipogenesis in pair-fed lean and obese pigs. Proc. Soc. Exp. Biol. Med. 151:231. Maruyama, K., M. L. Sunde and R. W. Swick. 1978. Growth and muscle protein turnover in the chick. Biochem. J. 176:573. Mauro, A. 1961. Satellite cell of skeletal muscle fibers. J. Biophys. Biochem. Cytol. 9:493. Max, 8. R. 1981. Effect of estrogen on denervated muscle. J. Neurochem. 36:1077. Max, 8. R. 1983. Cytosolic androgen receptor in skeletal _ muscle from normal and dystrophic mice. J. Steroid Biochem. 18:281. Mayer, M. R., R. Amin and E. Shafrir. 1974. Rat myofibrillar protease: enzyme properties and adaptive changes in conditions of muscle protein degradation. Arch. Biochem. Biophys. 161:20. Mayer, M., R. Amin and E. Shafrir. 1980. Differences in response of proteolytic activity in cardiac, skeletal and diaphragm muscles to hormones and catabolic conditions. Mol. Cell. Endocrinol. 18:49. 313 Mayer, M. and F. Rosen. 1975. Interaction of anabolic steroids with glucocorticoid receptor sites in rat muscle cytosol. Amer. J. Physiol. 229:1381. Mayer, M. and F. Rosen, 1977. Interaction of glucocor- ticoids and androgens with skeletal muscle. Metabolism 26:937. Mayes, P. A. 1976. Control of hepatic triacylglycerol metabolism. Biochem. Soc. Trans. 4:575. McElligott, M. A. and J. W. C. Bird. 1981. Muscle proteolytic enzyme activities in diabetic rats. Amer. J. Physiol. 241:E378. McMaster, J. H. and C. R. Weinert. .1970. Effects of mechanical forces on growing cartilage. Clin. Orthop. Relat. Res. 72:308. McMeekan, C. P. 1940. Growth and development in the pig with special reference to carcass quality characteristics. J. Agr. Sci. 30:276. McNamara, J. P. and R. J. Martin. 1982. Muscle and adipose tissue lipoprotein lipase in fetal and neonatal swine as affected by genetic selection for high or low backfat. J. Anim. Sci. 55:1057. McNurlan, M. A., E. B. Fern and P. J. Garlick. 1982. Failure of leucine to stimulate protein synthesis in vivo. Biochem. J. 204:831. Mendelson, C. R., W. H. Cleland and E. R. Simpson. 1983. Regulation of estrogen formation by human adipose stromal cells in culture. In: A. Anbel, C. H. Hollenberg and D. A. K. Roncari (Eds.) The adipcyte and Obesity: Cellular and Molecular Mechanisms. pp 211-215. Raven Press, New York. Mendes, C. B. and J. C. Waterlow. 1953. The effect of low protein diet and of refeeding on the composition of liver and muscle in the weanling rat. Brit. J. Nutr. 12:74. ' Meisner, H. and J. R. Carter. 1977. Regulation of lipolysis in adipose tissue. Horiz. Biochem. Biophys. 4:91. Merimee, T. J., J. A. Burgess and D. Rabinowitz. 1966. Sex-determined variations in serum insulin and growth hormone response to amino acid stimulation. J. Clin. Endocrinol. Metab. 26:791. " Mersmann, H. J. 1982. Direction and Utility of Growth Biology Research. Proc. Recip. Meat Conf. 35:13. 314 Mersmann, H. J., L. J. Brown, R. M. Beuving and M. C. Arakelian. 1976. Lipolytic activity of swine adipo- cytes. Amer. J. Physiol. 250:1439. Mersmann, H. J., J. M. Houk, G. Phinney, M. C. Underwood and L. J. Brown. 1973a. Lipogenesis by in vitro liver and adipose tissue preparations from neonatal swine. Amer. J. Physiol. 224:1123. Mersmann, H. J., G. Phinney and L. J. Brown. 1975a. Onto- geny of epinephrine-induced lipolysis in adipose tissue from swine (sus domesticus). Gen. Pharmacol. 6:187. ' Mersmann, H. J., G. Phinney, L. J. Grown and M. C. Arakelian. 1975b. Factors influencing the lipolytic response in swine (sus domesticus) adipose tissue. Gen. Pharmacol. 6:193. Mersmann, H. J., M. C. Underwood, L. J. Brown and J. M. Houk. 1973b. Adipose tissue composition and lipogenic capacity in developing swine. Amer. J. Physiol. 224: 1130. Metz, S. H. M. and R. A. Dekker. 1981. The contribution of fat mobilization to the regulation of fat deposi- tion in growing Large White and Pietrain pigs. Anim. Prod. 33:149. Michel, G. and E. E. Baulieu. 1980. Androgen receptor in rat skeletal muscle: characterization and physiolo- gical variations. Endocrinology 107:2088. Millward, D. J. 1980. Protein turnover in skeletal and cardiac muscle during normal growth and hypertrophy. In: K. Wildenthal (Ed.) Degradative Processes in Heart and Skeletal Muscle. pp 161-199. Elsevier/ North Holland Biomedical Press, New York. Millward, D. J., P. C. Bates and S. Rosochacki. 1981. The extent and nature of protein degradation in the tissues during development. Reprod. Nutr. Develop. 21:265. ' Millward, D. J., P. J. Garlick, R. J. c. Stewart, D. o. Nnanyelugo and S. W. Waterlow. 1975. Skeletal muscle growth and protein turnover. Biochem. J. 150:235. Millward, D. J. and J. C. Waterlow. 1978. Effect of nutri- tion on protein turnover in skeletal muscle. Fed. Proc. 37:2283. 315 Montgomery, R. D. 1962. Growth of human striated muscle. Nature 195:194. Moss, F. P. 1968a. The relationship between the dimensions of the fibers and the number of nuclei during normal growth of skeletal muscle in the domestic fowl. Amer. J. Anat. 122:555. Moss, F. P. 1968b. The relationship between the number of nuclei during restricted growth, degrowth, and compen- satory growth of skeletal muscle. Amer. J. Anat. 122:565. Moss, F. P. and C. P. Leblond. 1970. Nature of dividing nuclei in skeletal muscle of growing rats. J. Cell. Biol. 44:459. Moss, F. P. and C. P. Leblond. 1971. Satellite cells as the source of nuclei in muscles of growing rats. Anat. Rec. 170:421. Moss, F. P., R. A. Simmonds and H. W. McNary. 1964. The growth and composition of skeletal muscle in the chicken. II. The relationship between muscle Weight and the number of nuclei. Poul. Sci. 43:1086. Mostafavi, M. S. 1978. The effect of growth rate, sex and age on skeletal muscle and adipose tissue growth and development. Ph.D. Thesis, Michigan State Univer- sity, East Lansing. Mulvaney, D. R. 1981. Protein synthesis, breakdown and accretion rates in skeletal muscle and liver of young growing boars, M.S. Thesis, Michigan State University, East Lansing. Napolitano, L. 1963. The differentiation of white adipose cells: an electron microscope study. J. Cell Biol. 18:663. Nestel, P. J. and W. Austin. 1969. Relationship between _ adipose lipOprotein lipase activity and compounds which affect intracellular lipolysis. Life Sci. 8:157. Newell, J. A. and J. P. Bowland. 1972. Performance, carcass composition, and fat composition of boars, gilts, and barrows fed two levels of protein. Can. J. Anim. Sci. 52:543. Newsholme, E. A. and C. Start. 1973. Regulation in Meta- bolism. John Wiley and Sons, New York. 316 Nilsson, N. O., P. Stralfors, G. Fredrikson and P. Belfrage. 1980. Regulation of adipose tissue lipolysis: effects of noradrenaline and insulin on phosphorylation of hormone sensitive lipase and on lipolysis in intact rat adipocytes. FEBS Lett. 111:125. Nilsson-Ehle, P. 1981. Impaired regulation of adipose tissue lipoprotein lipase in obesity. Int. J. Obesity 5:695. ' Noel, J. F. and E. A. Wright. 1972. The growth of trans- planted mouse vertebrae. Effects of transplantation under the renal capsule and the relationship between the rate of growth of the transplant and the age of the host. J. Embryol. Exp. Morphol. 28:633. Noguchi, T., E. Miyazawa and M. Kametaka. 1974. Protease and protease inhibitor activity in rat skeletal muscle during growth, protein deficiency and fasting. Agr. Biol. Chem. 38:253. Noland, P. R. and M. J._Burris. 1956. The effect or oral administration of methyl testosterone on swine growth and develOpment. J. Anim. Sci. 15:1014. Norman, A. W. 1980. Bone and the vitamin D. endocrine system. In: M. R. Urist (Ed.) Fundamental and Clinical Bone Physiology. pp 242-267. J. B. Kippincott Co., Philadelphia. Numa, S., S. Nakanishi, T. Hashimoto, N. Iritani and T. Okazaki. 1970. Role of acetyl coenzyme a carboxylase in the control of fatty acid synthesis. Vitam. Horm. 28:213. Ogden. J. A. 1980. Chrondo-osseous development and growth. In: M. R. Urist (Ed.) Fundamental and Clinical Bone Physiology. pp 108-171. Lippincott, Philadelphia. O'Hea, E. K. and G. A. Leveille. 1968. Lipid metabolism in isolated adipose tissue of the domestic pig (sus domesticus). Comp. Biochem. Physiol. 26:1081. O'Hea, E. K. and G. A. Leveille. 1969. Influence of fasting and refeeding on lipogenesis and enzymatic activity by pig adipose tissue. J. Nurt. 99:345. Okazaki, K. and H. Holtzer. 1966. Myogenesis: Fusion, myosin synthesis, and the mitotic cycle. Proc. Natl. Acad. Sci. 56:1484. omtvedt, I. T. and E. J. Jesse. 1968. Influence of sex and sire-sex interactions in swine. J. Anim. Sci. 27:285. Owen, M. 1978. Histogenesis of bone cells. Calcif. Tiss. Res. 25:205. 317 Owen, M. 1982. Bone growth at the cellular level: a per- spective. In: Factors and Mechanisms Influencing Bone Growth. pp 19-28. Alan R. Liss Inc., New York. Pain, V. M. 1980. Hormonal regulation of protein metabolism. CRC Crit. Rev. Biochem. 14:85. Parra, F. and W. J. Reddy. 1962. Effect of cortisol and estradiol on tissue binding of testosterone. Amer. J. Physiol. 202:340. Patterson, B. and R. C. Strohman. 1972. Myosin synthesis in cultures of differentiating chicken embryo skeletal muscle. Develop. Biol. 29:113. Patterson, R. L. S. 1968. 5a-Androst-16-en-3-one: compound responsible for taint in boar fat. J. Sci. Food Agric. 19:31. Pay, M. G. and T. E. Davies. 1973. Growth, food conversion and carcass characteristics in castrated and entire male pigs fed three different dietary protein levels. J. Agr. Sci. 81:65. Peters, R. F., M. C. Richardson, M. Small and N. M. White. 1970. Some biochemical effects of triamcinolone acetonide on rat liver and muscle. Biochem. J. 116:349. Petrovic, A. G. 1982. Postnatal growth of bone: a per- spective of current trends, new approaches and innova- tions. In: A. D. Dixon and B. G. Sarnat (Eds.) Factors and Mechanisms Influencing Bone Growth. pp 297-331. Alan R. Liss, Inc., New York, New York. Piatowski, B. and H. Jung. 1966. Der erwerbansatz wachsender schweine verschiedenen geschlechts and vershiederner typrichtung. Arch. Tierz. 9:307. Powell, S. E. and E. D. Aberle. 1975. Cellular growth of' skeletal muscle in swine differing in muscularity. J. Anim. Sci. 40:476. Powers, M. L. and J. R. Florini. 1975. A direct effect of testosterone on muscle cells in tissue culture. Endocrinology 97:1043. Prescott, J. H. D. and G. E. Lamming. 1964. The effects of castration on meat production in cattle, sheep and pigs. J. Agr. Sci. 63:341. 318 Prescott, J. H. D. and G. E. Lamming. 1967. The influence of castration on the growth of male pigs in relation to high levels of dietary protein. Anim. Prod. 9:535. Prior, R. L., S. B. Smith, B. D. Schanbacher and H. H. Mersmann. 1983. Lipid metabolism in finishing bulls and steers implanted with oestradiol-17B-dipropionate. Anim. Prod. 37:81. Pritchard, J. J. 1972. The osteoblast. In: G. H. Bourne (Ed.) The Biochemistry and Physiology of Bone, Vol. 1. pp 21-30. Academic Press, New York. Quinn, D., K. Shirai and R. L. Jackson. 1982. Lipoprotein lipase: Mechanism of action and role in lipoprotein metabolism. Prog. Lipid Res. 22:35. Raisz, L. G., E. M. Canalis, J. W. Dietrich, B. E. Kream and S. C. Gworek. 1978. Hormonal regulation of bone formation. Rec. Prog. Horm. Res. 34:335. Raisz, L. G. and Kream, B. E. 1981. Hormonal control of skeletal growth. Ann. Rev. Physiol. 43:225. Raju, P. K. and D. M. Six. 1975. Glyceride biosynthesis in swine adipose tissue microsomes. Comp. Biochem. Physiol. 51B:17l. Ramirez, I. 1981. Estradiol induced changes in lipoprotein lipase, eating and body weight in rats. Amer. J. Physiol. 240:E533. Rao, A. J. and J. Ramachandran. 1977. Growth hormone and the regulation of lipolysis. In: H. Li (Ed.) Hormonal Proteins and Peptides, Vol. IV. pp 43-60. Academic Press, New York. Redden, D. R. 1970. Chronic acceleration effects on bone development in the chick embryo. Am J. Physiol. 218:310. Redgrave, T. G. and D. A. Snibson. 1977. Clearance of chylomicron triacylglycerol and cholesterol ester from the plasma of streptozotocin-induced diabetic and hypercholesterolemic hypothyroid rats. Metabolism 26:495. Reeds, P. J. 1980. Protein turnover in man. In: M. J. Clemens (Ed.) Biochemistry of Cellular Regulation. Vol. II: Clinical and Scientific Aspects of the Regulation of Metabolism. pp 67-88. CRC Press Inc. Boca Raton. 319 Reichl, D. 1972. Lipoprotein lipase activity in the adipose tissue of rats adapted to controlled feeding schedules. Biochem. J. 128:79. Renold, A. E. and G. F. Cahill. 1965. Adipose Tissue. In: Handbook of Physiology, Sec. 5. American Physiological Soc. Washington, D. C. Rerat, A. 1975. Advances in pig technology. In: D. Lister, D. N. Rhodes, V. R. Fowler and M. 'F. Fuller (Eds. ) Meat Animals Growth and Productivity. pp 403- 445. Plenum Press, New York. Richmond, R. J. and R. T. Berg. 1982. Effects of sex, genotype and nutrition on the relative growth of muscles in the pig. Can. J. Anim. Sci. 62:587. Richmond, R. J., S. D. M. Jones, M. A. Price and R. T. Berg. 1979. Effects of breed and sex on the relative growth and distribution of bone in pigs. Can. J. Anim. Sci. 59:471. Robinson, D. W. 1969. The cellular response of porcine skeletal muscle to prenatal and neonatal nutritional stress. Growth 33:231. Robinson, G. A., R. W. Butcher and E. W. Sutherland. 1971. Cyclic AMP. Academic Press, New York. Robinson, D. S., S. M. Parkin, B. K. Speake and J. A. Little. 1983. Hormonal control of rat adipose tissue lipOprotein lipase activity. In: A. Angel, C. H. Hollenberg and D. A. K. Roncari (Eds.) The Adipocyte and Obesity: Cellular and Molecular Mechanisms. pp 127-136. Raven Press, New York. Robinson, D. S. and D. R. Wing. 1970. Regulation of adipose tissue clearing factor lipase activity. In: B. Jeanrenaud and D. Hepp (Ed.) Hormone and Metabolic Research, Suppl. 2. pp 41-59. Academic Press, New York. Rodemann, H. P. and A. L. Goldberg. 1982. Arachidonic acid, prostaglandin E2 and an influence rates of protein turnover in skeletal and cardiac muscle. J. Biol. Chem. 257:1632. Rogozkin, V. 1975. Anabolic and androgenic effects of methandrostenolone ("Nerobol") during systematic physical activity in rats. Brit. J. Sports Med. 9:65. Rogozkin, V. 1979. Metabolic effects of anabolic steroid on skeletal muscle. Med. Sci. Sports 11:160. 320 Romsos, D. R. and G. A. Leveille. 1974. Effect of diet on activity of enzymes involved in fatty acid and chole- sterol synthesis. Adv. Lipid Res. 12:97. Romsos, D. R., G. A. Leveille and G. L. Allee. 1971. In vitro lipogenesis in adipose tissue from alloxan- diabetic pigs (Sus domesticus). Comp. Biochem. Physiol. 40A:569. Roth, J. A., K. Losty and E. Wierbicki.l97l. Assay of proteolytic enzyme activity using a l4C-labe1ed hemoglobin. Anal. Biochem. 42:214. Rothig, H. J., N. Stiller, B. Dahlmann and H. Reinauer. 1978. Insulin effect on proteolytic activities in rat skeletal muscle. Horm. Metabl. Res. 10:101. Rowe, R. W. D. and G. Goldspink. 1969. Muscle growth in five different muscles in both sexes of mice. 1. Normal mice. J. Anat. 104:519. Rozovski, S. J. and M. Winick. 1979. Nutrition and cellular growth. In: M. Winick (Ed.) Nutrition Pre- and Post- natal Development. pp 61-102. Plenum Press, New York. Rubinstein, H. S. and M. L. Solomon. 1941a. The growth depressing effect of large doses of testosterone propionate in the castrate albino rat. Endocrinology 28:112. Rubinstein, H. S. and M. L. Solomon. 1941b. The growth stimulating effect of small doses of testosterone prOpionate in the castrate albino rat. Endocrinology 28:229. Rudman, D. and M. DiGirolamo. 1971. Effect of adrenal cortical steroids on lipid metabolism. In: N. P. Christy (Ed.) the Human Adrenal Cortex. pp 241-255. Harper and Row, New York. Rudman, D. 1963. The adipokinetic action of polypeptide and amine hormones upon the adipose tissue of various animal species. J. Lipid Res. 4:119. Saggerson, E. D. 1980. Regulation of lipid metabolism in adipose tissue and liver cells. Clinical and Scientific Aspects of the Regulation of Metabolism. 2:207-256. ' Salaman, M. R. and D. S. Robinson. 1966. Clearing factor lipase in adipose tissue, a medium in which the enzyme activity of tissue from starved rats increases in vitro. Biochem. J. 99:640. 321 Salleo, A., G. La Spada, G. Falzea, M. G. Denaro and R. Ciccia Rello. 1980. Activation of satellite cells produced by compensatory hypertrOphy, denervation and neostigmine treatment. Adv. Physiol. Sci. 24:255. Sanada, Y., N. Yasogawa and N. Katunuma. 1978. Crystallization and amino acid composition of a serine protease from rat skeletal muscle. Biochem. Biophys. Res. Commun. 82:108. Santidrian, S., M. Moreyra, H. N. Munro and V. R. Young. 1982. Effect of testosterone on the rate of myofi- brillar protein breakdown in castrated and adrena- lectomized rats measured by urinary excretion of 3- methylhistidine. Metabolism 31:1200. SAS Institute. 1979. SAS User's Guide. SAS Institute Inc. Cary, N. C. Schanbacher, B. D. 1980. Testosterone regulation of lutein- izing hormone and follicle stimulating hormone secretion in young male lambs. J. Anim. Sci. 51:679. Schoenle, E., J. Zapf, R. E. Humbel and E. R. Froesch. 1982. Insulin like growth factor I stimulates growth in hypOphysectomized rats. Nature 296:252. Schwartz, W. N. and J. W. C. Bird. 1977. Degradation of myofibrillar proteins by cathepsins B and D. Biochem. J. 167- 811. Scott, R. A., S. G. Cornelius and H. J. Mersmann. 1981. Effects of age on lipogenesis and lipolysis in lean and obese swine. J. Anim. Sci. 52:505. Scow, R. O. 1952. Effect of testosterone on muscle and other tissues and on carcass composition in hypo- physectomized, thyroidectomized and gonadectomized male rats. Endocrinology 51:42. Scow, R. O. and S. S. Chernick. 1970. Transport and utilization of free fatty acids. In: M. Florkin and E. H. Stotz (Eds.) Comprehensive Biochemistry, Vol. 18. Elsevier/North Holland, New York. Seals, J. R. and L. Jarett. 1980. Activation of pyruvate dehydrogenase by direct addition of insulin to an isolated plasma membrane-mitochondria mixture: evidence for generation of insulins second messenger in a subcellular system. Proc. Natl. Acad. Sci. 77:77. 322 Seideman, S. C., H. R. Cross, R. R. Oltjen and B. D. Schanbacher. 1982. Utilization of the intact male for red meat production: A review. J. Anim. Sci. 55:826. Short, R. V. 1980. The hormonal control of growth at puberty. In. T. L. J. Lawrence (Ed.). Growth in Animals. pp 25-45. Butterworth, London. Siddle, K. and C. N. Hales. 1975. Hormonal control of adipose tissue lipolysis. Proc. Nutr. Soc. 34:233. Siers, D. G. 1975. Live and carcass tracts in individually fed Yorkshire boars, barrows and gilts. J. Anim. Sci. 41:522. Silberberg, M. and R. Silberberg. 1971. Steroid hormones and bone. In: G. H. Bourne (Ed.) The Biochemistry and Physiology of Bone. pp 401-484. Academic Press, New York. , Silberman, M., S. Levitan, U. Kleinhaus and S. Finkelbrand. 1979. Long bone growth during prolonged intermittent corticosteroid treatment and subsequent rehabilita- tion. Cell Tissue Res. 201:51. Simpson, M. E., W. Marx, H. Becks and H. M. Evans. 1944. Effect of testosterone propionate on the body weight and skeletal system of hypophysectomized rats. Synergism with pituitary growth hormone. Endocrinology 35:309. Sinnett-Smith, P. A., N. W. Dumelow, and P. J. Buttery. 1983. Effects of trenbolone acetate and zeronal on protein metabolism in male castrate and female lambs. Brit. J. Nutr. 50:225. Slavin, B. G. 1979. Five structural studies on white adipose tissue differentiation. Anat. Rec. 195:63. Sleeth, R. B., A. M. Pearson, H. D. Wallace, D. H. Kropf and M. Koger. 1953. Effects of injection of testos- terone, estradiol and a combination of the two upon growing-fattening swine. J. Anim. Sci. 12:322. Slusser, W. N. and G. N. Wade. 1981. Testicular effects on food intake, body weight and body composition in male hamsters. Physiol. Behav. 27:637. 323 Smith, R. H., R. M. Palmer and P. J. Reeds. 1983. Protein synthesis in isolated rabbit forelimb muscles. The possible role of metabolites of arachidonic acid in the response to intermittent stretching. Biochem. J. 214:153. Snochowski, M., E. Dahlberg and J. A. Gustafsson. 1980. Characterization and quantification of the androgen and glucocorticoid receptors in cytosol from rat skeletal muscle. European J. Biochem. 111:603. Snochowski, M., K. Jundstrom, E. Dahlberg, H. Petersson and L. E. Edqvist. 1981. Androgen and glucocorticoid receptors in porcine skeletal muscle. J. Anim. Sci. 53:80. Sohar, I. O., F. Takacs, F. Guba, H. Kirschke and P. Bohley. 1979. Degradation of myofibrillar proteins by cathepsins from rat liver lysosomes. In. Protein- stoffwechsel. Part 2. pp 121-125. Martin Luther Press, Halle-Wittenberg. Sommerville, E. M. and M. F. Tarttelin. 1983. Plasma testosterone levels in adult and neonatal female rats bearing testosterone proprionate filled silicone elastomer capsules for varying periods of time. J. Endocrinol. 98:365. Spencer, H., J. A. Friedland and I. Lewin. 1976. Effect of androgens on bone, calcium and phosphorous metabolism. In: C. D. Kochakian (Ed.) Anabolic Androgenic Steroids. pp 419-440. Springer Verlag, New York. Staun, H. 1963. Various factors affecting number and size of muscle fibers in the pig. Acta Agr. Scand. 13:293. Staun, H. 1972. The nutritional and genetic influence on number and size of muscle fibers and their response to carcass quality in pigs. erd. Rev. Anim. Prod. 8:3. Steele, N. C. and L. T. Frobish. 1976. Selected lipogenic enzyme activities of swine adipose tissue as influenced by genetic phenotype, age, feeding frequency and dietary energy source. Growth 40:369. Steele, N. C., R. W. Rosebrough, J. P. McMurtry and L. T. Frobish. 1982. Effects of dietary 1,3-butylene glycol on adipose tissue metabolism from lean and obese swine. J. Anim. Sci. 54:116. 324 Steffen, D. G., M. C. Arakelian, G. Phinney, L. J. Brown and H. J. Mersmann. 1981. Effect of nutritional status on swine adipose tissue lipolytic activities. J. Anim. Sci. 52:1306. Steffen, D. G., L. J. Brown and H. J. Mersmann. 1978. Ontogenic development of swine (sus domesticus) adipose tissue lipases. Comp. Biochem. Physiol. 59B:l95. Steffen, D. G., E. Y. Chai, L. J. Brown and H. J. Mersmann. 1977. Effects of diet on swine glyceride lipid metabolism. J. Nutr. 108:911. Steffen, D. G., G. Phinney, L. J. Brown and H. J. Mersmann. 1979. Ontogeny of glycerolipid biosynthetic enzymes in swine liver and adipose tissue. J. Lipid Res. 20:246. Steinberg, D. 1976. Interconvertible enzymes in adipose tissue regulated by cyclic AMP-dependent protein kinase. Adv. Cyclic Nucleotide Res. 7:157. Steinberg, D. and J. C. Khoo. 1977. Hormone sensitive lipase of adipose tissue. Fed. Proc. 36:1986. Steingrimsdottir, L., J. Brasel and M. R. C. Greenwood. 1981. Hormonal modulation of adipose tissue lipo- protein lipase may alter food intake in rats. Amer. J. Physiol. 239:El62. Stenstrom, A., L. I. Hansson and K. G. Thorngren. 1982. Effect of ovariectomy on longitudinal bone growth in the rat. Anat. Embryol. 164:9. Stickland, N. C. and G. Goldspink. 1973. A possible indicator muscle for the fiber content and growth characteristics of porcine muscle. Anim. Prod. 16:135. Stockdale, F. E. 1970. Changing levels of DNA polymerase activity during the develOpment of skeletal muscle tissue in vivo. Development. Biol. 21:462. Stokes, G. B., L. W. Poteat and S. B. Tove. 1975. Distri- bution of fatty acids incorporated into triacylgly- cerols by microsome-cytosol preparations from adipose tissue. Biochim. Biophys. Acta. 380:245. Stratton, L. G., L. L. Ewing and C. Desjardins. 1973. Efficacy of testosterone-filled polydimethylsi- loxane implants in maintaining plasma testosterone in rabbits. J. Reprod. Fert. 35:235. 325 Stromer, M. H., D. E. Goll, R. B. Young, R. M. Robson and R. C. Parrish, Jr. 1974. Ultrastructural features of skeletal muscle differentiation and development. J. Anim. Sci. 38:1111. Stubbs, D. 1970. Skeletal function and weightlessness. A mechanism for hypogravic skeletal atrophy. Aerospace Med. 41:1126. Swatland, H. J. 1976. Effect of growth and plane of nutrition on apparent muscle fiber numbers in the pig. Growth 40:285. Swatland, H. J. 1977. Accumulation of myofiber nuclei in pigs with normal and arrested development. J. Anim. Sci. 44:759. Swick, R. W. 1982. Growth and protein turnover in animals. CRC Crit. Rev. Fed. Sci. Nutr. 16 (2):1l7. Tardieu, C., J. C. Tabary, G. Tardieu and C. Tabary. 1980. Adaptation of sarcomere numbers to the length imposed on the muscle. Adv. Physiol. Sci. 24:99. Telegdy, G. 1980. The responses of the developing endo- crine system to hormones and drugs. Pharamacal Ther. 10:537. Thorbek, G. 1975. Studies in energy metabolism in grow- ing pigs II. Protein and fat gain in growing pigs fed different feed compounds. Efficiency of utiliza- tion of metabolizable energy for growth. Beretning fra Staten Husdrybrugs forsog No. 424. Thorngren, K. G. and L. I. Hansson. 1974. Bioassay of growth hormone. I. Determination of longitudinal bone growth with tetracycline in hypophysectomized rats. Acta Endocrinol. 75:653. Thrasher, G. W., T. W. Perry, F. N. Andrews, W. M. Beeson and M. Stob. 1959. The effect of estrogenic and androgenic compounds upon growth and carcass compo- sition of swine. J. Anim. Sci. 18:399. Tonna, E. A. 1973. Hormonal influence on skeletal growth and regeneration. In: J. Lobue and A. S. Gordon (Eds.) Humoral Control of Growth and Differentiation. pp_ ' 275-359. Academic Press, New York. Topel, D. G. 1971. Physical and Chemical changes during growth and development of fat and muscular pigs. Proc. Recip. Meat Conf. 24:304. 326 Tremblay, R. R., J. Y. Dube, M. A. Ho-Kim and R. Lesage. 1977. Determination of rat muscles androgen-receptor complexes with methyltrienolone. Steroids 29:185. Trenkle, A. H. and D. N. Marple. 1983. Growth and develop- ment of meat animals. J. Anim. Sci. (Suppl. II) 57:273. Trostler, N., R. Amin and E. Shafrir. 1982. Increased pro- tease activity in muscle of obese-(ob/ob) mice. Inter. J. Obesity 6:557. Tsai, R., R. G. Cassens and M. L. Greaser. 1973. Changes in RNA species during differentiation of fiber types of skeletal muscle. J. Anim. Sci. 36:25. Tulloh, N. M. and B. Romberg. 1963. An effect of gravity on bone development in lambs. Nature 200:438. Turton, J. D. 1969. The effect of castration on meat pro- duction from cattle, sheep and pigs. In D. N. Rhodes (Ed.) Meat Production from Entire Male Animals. pp l-49. J. & A. Churchill LTD., London. Underwood, L. E. and J. J. VanWyk. 1981. Hormones in normal and aberrant growth. In: R. H. Williams (Ed.) Text- book of Endocrinology. pp 1149-1191. W. B. Saunders Co., Philadelphia. Urist, M. R., R. J. Delange and G. A. M. Finerman. 1983. Bone cell differentiation and growth factors. Science 220:680. ‘ VanSickle, D. C. 1982. Control of postnatal bone growth. J. Anim. Sci. (In Press). VanWeerden, E. J. and J. A. Grandadam. 1976. The effect of an anabolic agent on N-deposition, growth and slaughter quality in growing castrated male pigs. In: F. Coultson and F. Korte (Eds.) Environmental Quality and Safety, Suppl. Vol. V. pp 123-130. Georg Thieme Publishers, Stuttgart. Vaughn, J. 1981. The Physiology of Bone. Clarendon Press, Oxford. Venable, J. H. 1969. Turnover of myofibrils in stable and hypertrophing perineal muscles of mature mice. Anat. Rec. 163:279. Vermeulan, A. 1982. The Androgens. In: C. H. Gray and V. H. T. James (Eds.) Hormones in Blood. pp 335-416. Academic Press, London. 327 Vernon, R. G. 1980. Comparative aspects of lipid meta- bolism in monogastric, pre-ruminant and ruminating animals. Biochem. Soc. Trans. 8:291. Vernon, R. G. 1982. Effects of growth hormone on fatty acid synthesis in sheep adipose tissue. Int. J. Biochem. 14:255. Vernon, B. G. and P. J. Buttery. 1976. Protein turnover in rats treated with trienbolone acetate. Brit. J. Nutr. 36:575. Vernon, B. G. and P. J. Buttery. 1978. Protein metabolism of rats treated with trienbolone acetate. Anim. Prod. 26:1. Viru, A. and P. Korge. 1979. Role of anabolic steroids in the hormonal regulation of skeletal muscle adaptation. J. Steroid Biochem. 11:931. Wade, G. N. and J. M. Gray. 1979. Theoretical review: Gonadal effects on food intake and adiposity: a metabolic hypothesis. Physiol. Behav. 22:583. Walach-Janiak, M., M. Kotarbinska and J. Kielanowski. 1980. Energy metabolism in growing boars. In: L. E. Mount (Ed.) Energy Metabolism. pp 157-161. Butterworths, London. Walkenbach, R. J., R. Hazen and J.Larner. 1978. Reversible inhibition of cyclic AMP-dependent protein kinase by insulin. Mol. Cell. Biochem. 19:31. Walstra, P. 1969. Experiments in the Netherlands on the effects of castration of pigs in relation to feeding Kslevel. In: D. N. Rhodes (Ed.) Meat Production From Entire Male Animals. pp 129-141. J. A. Churchill LTD, London. Walstra, P. 1980. Growth and carcass composition from birth to maturity in relation to feeding level and sex in Dutch Landrace pigs. Mededelingen Landbouwhogeschool Wageningen, The Netherlands. Walstra, P. and D. Kroeske. 1968. The effect of castration on meat production in male pigs. World Rev. Anim. Prod. 4:59. Warrenski, J. and R. R. Almon. 1983. Effect of castration on the metabolism of androgens in rat skeletal muscle. Int. J. Biochem. 15:1149. 328 Waterlow, J. C., P. J. Garlick and D.;I.Millward. 1978. Protein Turnover in Mammalian Tissues and in the Whole Body. North-Holland Publishing Company, New York. Weisenburg, C. L. and C. E. Allen. 1973. Adipose tissue metabolism in obese and lean pigs. J. Anim. Sci. 37:293. (Abstr.). WerffTenBosch, J. J. Van Der. 1977. Testosterone as growth stimulant in man. Pharmacol. Ther. 2C:l7. Whitaker, M. D., H. Brown, C. E. Barhart, J. D. Kemp and W. Y. Varney. 1959. Effects of methylandro- stenediol, methyltestosterone and therprotein on growth and carcass characteristics of swine. J. Anim. Sci. 18:1189. White, A. M. 1967. The anticatabolic effects of synthetic anabolic androgenic steroids. In: Proc. Int. Congr. Horm. Steroids pp. 576. Wiedemann, E., E. Schwartz and A. G. Frantz. 1976. Acute and chronic estrogen effeCts upon serum somato- medin activity, growth hormone and prolactin in man. J. Clin. Endocrinol. Metab. 42:942. Wigmore, P. M. C. and N. C. Stickland. 1983. Muscle development in large and small pig fetuses. J. Anat. 137:235. Williams-Ashman, H. G. 1975. Metabolic effects of testi- cular androgens. In: R. O. Greep and E. G. Astwood (Eds.) Handbook of Physiology, Sect. 7, Vol. 5. American Physiological Society, Washington, D. C. Williams, P. E. and G. Goldspink. 1973. The effect of immobilization on the longitudinal growth of striated muscle, J. Anat. 116:45. Wilson, D. E., C. M. Flowers, 8. E. Carlile and K. S. Udall. 1976. Estrogen treatment and gonadal function in the regulation of lipoprotein lipase. Ather- sclerosis 24:491. Wing, D. R., M. R. Salaman and D. S. Robinson. 1966. Clearing factor lipase in adipose tissue. Factors influencing the increase in enzyme activity pro- duced on incubation of tissue from starved rats in vitro. Biochem. J. 99:648.