I .I I I I I IIIIIHIHIH. Dill! THESlS .2000 IllllllllllllllllllllllIlllllllHlllllllllllllllll 293 02048 6399 LIBRARY Michigan State University This is to certify that the dissertation entitled INFLUENCE OF BOVINE SOMATOTROPIN ADMINISTRATION TO HOLSTEIN STEERS ON GROWTH, LIPID METABOLISM, AND CARCASS CHARACTERISTICS presented by Michael L. Schlegel has been accepted towards fulfillment of the requirements for Ph.D. degree in Anlmal Sc1ence W ,/ [I / Major professor Baez/‘3 0’ q 9 MS U i: an Affirmative Action/Equal Opportunity Institution 0-12771 PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE _ DATE DUE 11/00 edCIHMpBS-nu INFLUENCE OF BOVINE SOMATOTROPIN ADMINISTRATION To HOLSTEIN STEERS ON GROWTH, LIPID METABOLISM AND CARCASS CHARACTERISTICS By Michael Lynn Schlegel A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Animal Science 1 999 ABSTRACT INFLUENCE OF BOVINE SOMATOTROPIN ADMINISTRATION TO HOLSTEIN STEERS ON GROWTH, LIPID METABOLISM AND CARCASS CHARACTERISTICS By Michael Lynn Schlegel Two experiments were conducted to study the effectiveness of bovine somatotropin (bST) to alter the growth, carcass characteristics and lipid metabolism of Holstein steers. The first evaluated the effectiveness of long-term (354 d) bST administration to growing-finishing Holstein steers at the beginning, end or during the entire feeding period on lean, skeletal, and carcass measurements. One hundred and sixty-eight Holstein steer calves (185 kg) were blocked into four body weight (BW) groups and randomly allocated to initial harvest (8 steers) or four treatments (10 steers/treatment) within a block. Treatments were control, no bST; bST d O to 182; bST d 183 to harvest; and bST d O to harvest. Doses were 320 mg bST/14-d injection from d O to 112 and 640 mg bST1/14-d injection from d 113 to harvest. Two steers from each block were harvested on d 199 with the remaining steers harvested when block BW averaged 615 kg. The last treatment was administered 31 d before harvest. Administering bST to young, light-weight Holstein steers increased skeletal growth and reduced carcass fat content producing a leaner product. Somatotropin increased noncarcass components without increasing carcass weight. Steers receiving bST during the entire study showed the greatest effects with the majority of lipid reduction occurring during the latter part of the study. Somatotropin increased protein accretion and therefore red meat yield. The second study evaluated the effect of bST administration to Holstein steers on measures of lipogenesis and lipolysis during the latter half of the feeding period when fat deposition was the greatest. Twenty-eight Holstein steers (460 kg) were blocked by weight and randomly assigned to an initial harvest group (8 steers), or to 116 daily treatments of control (no bST, 10 steers) or bST (100 pg bST/kg BW, 10 steers). Three perirectal adipose tissue (AT) biopsies were collected from each steer (5 to 16 d before treatment and 40 to 49, and 103 to 112 d after treatment). Lipogenesis was measured in triplicate in vitro as tritium incorporation into fatty acids, fatty acid synthase activity, and NADP-isocitrate dehydrogenase activity. Lipolysis was measured in triplicate in vitro by glycerol release from AT and in vivo by a seven-dose epinephrine challenge administered intravenously beginning on d -24, 32 and 95. It was clear that somatotropin improved the efficiency of carcass protein accretion and content, while drastically reducing carcass lipid accretion and content. The decrease in lipid accretion was a result of depressed lipogenesis through inhibiting lipogenic enzymes and enhancing lipolysis in the adipocyte. Bovine somatotropin was effective in reducing carcass fat and increasing edible lean in both experiments. Administering bST to young, light-weight steers increased skeletal growth and noncarcass weight, but did not increase the total carcass weight and reduced the quality grade of the carcass. Somatotropin decreased lipogenesis and increased lipolysis in AT of finishing-Holstein steers in positive energy balance. Dedicated to my parents, Kay and G. Sidni Schlegel ACKNOWLEDGMENTS lwould like to thank members of my committee, Dr. Romsos, Dr. Doumit, Dr. Banks, and Dr. Emery for their support, ideas, and perspective to make the project and the results meaningful. I owe a great deal of gratitude to Dr. Bergen and his staff, Patty Weber, Sharon Debar, and Michelle Mater. They allowed the use of the lab, equipment, time, and expertise in finishing this project. Without their help the study could not have been completed. I would like to thank Dr. VanderHaar and Bal Sharma for assistance in analyzing lGF-l concentrations and mRNA abundance. I would like to thank Dr. Tucker and his staff, Larry Chapin, Dr. Paula Gaynor, Dr. Christine Simmons-West for allowing the use of the lab, and assistance with the somatotropin and insulin assays. I would like to thank the personnel of the Meats Lab and the Beef Cattle Teaching and Research Center and their managers Tom Forton and Ken Metz for their assistance and to Bob Burnnet for his lab expertise. I would like to thank Dr. Yokoyama for the use of his lab and plate readen Special thanks go to the many fellow graduate students for their assistance in sample collection at the processing plant, blood collection and analysis. I must thank Dr. Aubrey Schroeder and Lilly Research Labs for their contribution of the somatotropin and technical support. TO the faculty and administration of Delaware Valley College, thank you for your encouragement, support and patience. To Dr. Benson for her mpral support and encouragement. To Dr. Steven Rest, my major professor, thank you for your insight, assistance and encouragement. It has been 11 years since I started my graduate programs at Michigan State, thank you for help and friendship. And finally to my parents, family, and colleagues who were very supportive and asked "Is it done yet?" vi TABLE OF CONTENTS LIST OF TABLES ............................................... ix LIST OF FIGURES ............................................. xiii LIST OF ABBREVIATIONS ....................................... xv INTRODUCTION ................................................ 1 CHAPTER 1: LITERATURE REVIEW ............................... 6 Regulation of endogenous somatotropin secretion ................. 6 Effect of somatotropin on plasma hormones and metabolites ......... 7 Effect of somatotropin on feedlot performance ................... 13 Effect of somatotropin on skeletal growth ....................... 18 Effect of somatotropin on the carcass .......................... 20 Lipogenesis .............................................. 30 Lipolysis ................................................ 36 CHAPTER 2: EFFECTS OF LONG-TERM BOVINE SOMATOTROPIN ADMINISTRATION ON GROWTH, CARCASS CHARACTERISTICS, AND TISSUE WEIGHTS IN GROWING-FINISHING HOLSTEIN STEERS .............. 43 Abstract ................................................. 43 Introduction .............................................. 44 Materials and Methods ..................................... 45 Results ................................................. 56 Discussion .............................................. 78 Implications .............................................. 87 CHAPTER 3: LONG-TERM BOVINE SOMATOTROPIN ADMINISTRATION TO GROWING-FINISHING HOLSTEIN STEERS DECREASED LIPOGENESIS AND INCREASED LIPOLYSIS ......................................... 90 Abstract ................................................. 90 Introduction .............................................. 91 Materials and Methods ..................................... 92 Results ................................................ 105 Discussion .............................................. 115 Implications ............................................. 1 33 CONCLUSION ............................................... 135 vii APPENDICES ................................................. 139 APPENDIX A: Raw data and procedures from chapter 2 (Experiment 809002) ............................................... 140 APPENDIX B: Raw data and procedures from Chapter 3 (Experiment BC9402) ............................................... 205 SAS program to calculate area under the curve of epinephrine Challenge and to calculate a segmented curve to a plateau (Rm response) ......................................... 241 APPENDIX C: Approval letters from the All-University Committee on Animal Use and Care ..................................... 244 LITERATURE CITED ........................................... 247 viii LIST OF TABLES Table 1-1. Summary of recent studies on the effect of somatotropin on beef cattle feedlot performance. ............................................ 14 Table 1-2. Summary of recent studies on the effect of somatotropin on beef cattle carcass characteristics. .......................................... 21 Table 2-1. Guaranteed analysis and ingredients of medicated-supplement starter pellet ........................................................ 46 Table 2-2. Guaranteed analysis and ingredients of pelleted supplement . . . 48 Table 2-3. Effect of bovine somatotropin on body weight, average daily gain, daily dry matter intake and feed efficiency ................................ 59 Table 2-4. Effect of bovine somatotropin on hip height and daily hip height gain .............................................................. 61 Table 2-5. Effect of bovine somatotropin on intermediate-harvest metacarpal bone characteristics ................................................. 62 Table 26 Effect of bovine somatotropin on final-harvest metacarpal bone characteristics ................................................. 63 Table 2-7. Effect of bovine somatotropin on intermediate-harvest carcass characteristics ................................................. 66 Table 2-8. Effect of bovine somatotropin on final-harvest’s carcass characteristics .............................................................. 67 Table 2-9. Effect of bovine somatotropin on intermediate-harvest semitendinosus muscle and internal organ weights ................................. 70 Table 2-10. Effect of bovine somatotropin on final-harvest semitendinosus muscle and internal organ weights ....................................... 71 Table 2-11. Effect of bovine somatotropin on intermediate-harvest carcass composition and tissue accretion rates .............................. 73 Table 2-12. Effect of bovine somatotropin on final-harvest carcass composition and tissue accretion rates ........................................... 75 Table 2-13. Effect of treatment on carcass premium, discounts and value. . . 88 ix Table 3-1. Guaranteed analysis and ingredient content of the pelleted supplement .............................................................. 95 Table 3-2. Effect of bovine somatotropin treatment on feedlot performance . 110 Table 3-3. Effect of bovine somatotropin treatment on carcass characteristics . ............................................................. 1 1 1 Table 3-4. Effect of bovine somatotropin treatment on carcass composition and accretion rates ................................................ 1 13 Table 3-5. Effect of bovine somatotropin on tritium incorporation into fatty acids, fatty acid synthase activity, and isocitrate dehydrogenase activity ........ 114 Table 3-6. Effect of somatotropin treatment on basal and epinephrine-stimulated glycerol release ............................................... 1 16 Table 3-7. Estimates of Rm, and ED50 of nonesterified fatty acids area epinephrine dose-response curves .......................................... 1 18 Table A-1 a. Initial harvest carcass characteristics for Experiment 809002 . 141 Table A-1 b. Initial harvest carcass composition for Experiment 809002 . . . 141 Table A-2. Record of removal of steers from Experiment 809002 and disposal . ............................................................. 142 Table A-3. Steer weight for Experiment 809002 ...................... 147 Table A-4a. Weekly pen dry matter intake for pens 1 to 4 for Experiment 809002 ............................................................. 168 Table A-4b. Weekly pen dry matter intake for pens 5 to 8 for Experiment BC9002 ............................................................. 1 71 Table A-4c. Weekly pen dry matter intake for pens 9 to 12 for Experiment BC9002 ............................................................. 174 Table A-4d. Weekly pen dry matter intake for pens 13 to 16 for Experiment 809002 ............................................................. 177 Table A-5. Carcass characteristics of steers from Experiment 809002 . . . . 179 Table A-6. Ninth-tenth-eleventh rib composition from Experiment 809002 . 183 Table A-7. Organ and muscle weights from Experiment 809002 ......... 187 X Table A-8. Steer hip height for Experiment 809002 ................... 191 Table A-9. Metacarpal bone characteristics for Experiment 809002 ...... 195 Table A-10. Serum IGF-l concentrations (nglml) following initial and subsequent control or bST treatments, and following the last control or bST treatment for Experiment 809002 ............................................ 202 Table A-11. Serum lGF-I concentrations following the final control or bST treatment, and liver IGF-l mRNA abundance for Experiment BC9002 ...... 203 Table A-12. Steer equations predicting carcass composition using the 9-10-11 rib section (Hankins and Howe, 1946) ................................. 204 Table 8-1. Time line of activities for Experiment 809402 ............... 206 Table 8-2. Steer weights for Experiment 809402 ..................... 209 Table 8-3. Daily dry matter intake of steers from Experiment 809402 ..... 212 Table 8-4. Carcass characteristics of steers from Experiment 809402 . . . . 213 Table 8-5. Ninth-tenth-eleventh rib composition from Experiment 809402 . 215 Table 8-6. Plasma glucose concentrations (mmol/ml) of steers for Experiment 809402 ...................................................... 216 Table 8-7. Plasma glycerol concentrations (pmol/ml) of steers for Experiment 809402 ...................................................... 217 Table 8-8. Plasma nonesterified fatty acid concentrations (mEq/L) of steers for Experiment 809402 ............................................ 218 Table 8-9. Plasma insulin concentrations (nglml) of steers for Experiment 809402 ............................................................. 219 Table 8-10. Plasma lGF-l concentrations (nglml) of steers for Experiment 809402 ............................................................. 220 Table 8-11. Plasma bovine somatotropin concentrations (nglml) for steers from day 94 of Experiment 809402 ........................................ 21 xi Table 8-12. Tritium incorporation into fatty acids, fatty acid synthase activity, NADP-isocitrate dehydrogenase activity, and enzyme assay soluble protein content for fat biopsies from Experiment 809402 ............................ 223 Table 8-13. Basal and epinephrine-stimulated glycerol release (nmol glycerol released-2h'1-100 mg tissue") for fat biopsies from Experiment 809402 . . . 224 Table B-14. Nonesterified fatty acid response (MEq/L) to seven epinephrine doses (pg/kg 8W) during three different weeks for Experiment 809402 ......... 225 Table 8-15. Plasma glycerol response (pmol/L) to a 1.6 pg epinephrine /kg BW dose during the 15th week of treatment for Experiment 809402 .......... 240 xii LIST OF FIGURES Figure 2-1. Lateral and medial measurements of sectioned metacarpals, and total cross sectional area (T CA), marrow cavity area (MCA) and bone area ..... 52 Figure 2-2. Effect of treatment, control (0) or bST (I) on serum IGF-I concentrations after initial treatment on d 0 and subsequent treatments as indicated by arrows. “Means with unlike letters differ (P < .01). .................. 57 Figure 2-3. Effect of treatment, 0-0 (0), C-bST (e), bST-C (III), or bST-bST (I), on serum IGF-l concentrations after final treatment 31 d prior to harvest as indicated by the arrow. "’Means with unlike letters differ (P < .05). ................ 57 Figure 2-4. Top panel -Effect of treatment on liver lGF-I mRNA abundance from Block 1 final harvest steers. Bottom panel - Serum lGF-l concentrations 7 to 28 d following the last treatment in 0-0 (0), C-bST (o), bST-C (El), or bST-bST (I) Block 1 steers. “Means with unlike superscripts differ (P < .05) ......... 77 Figure 3-1. Schedule of epinephrine challenges and adipose tissue biopsies 94 Figure 3-2 -Plasma bovine somatotropin concentrations over an 11.5-h period in Holstein steers treated with either a daily injection of sodium phosphate (0) or bovine somatotropin (I). *bST steers differ from control steers (P < .05). . . . 106 Figure 3-3. Plasma lGF-1 concentrations versus days of treatment in Holstein steers treated with either a daily injection of sodium phosphate (0) or bovine somatotropin (I). *bST steers differ from control steers (P < .05). “*bST steers differ from control steers (P < .001) ................................ 106 Figure 3-4. Plasma NEFA concentrations versus days of treatment in Holstein steers treated with either a daily injection of sodium phosphate (0) or bovine somatotropin (I). **bST steers differ from control steers (P < .01). "*bST steers differ from control steers (P < .001) .......................... 107 Figure 3-5. Plasma glycerol concentrations versus days of treatment in Holstein steers treated with either a daily injection of sodium phosphate (0) or bovine somatotropin (I). **bST steers differ from control steers (P < .01). “*bST steers differ from control steers (P < .001 ). ................................ 107 Figure 3-6. Plasma glucose concentrations versus days of treatment in Holstein steers treated with either a daily injection of sodium phosphate (0) or bovine somatotropin (I). *bST steers differ from control steers (P < .05). **bST steers differ from control steers (P < .01) ................................. 109 xiii Figure 3-7. Plasma insulin concentrations versus days of treatment in Holstein steers treated with either a daily injection of sodium phosphate (0) or bovine somatotropin (I). *bST steers differ from control steers (P < .05). **bST steers differ from control steers (P < .01). **bST steers differ from control steers (P < .001) ........................................................ 109 Figure 3-8. Response in plasma concentrations of NEFA to varying doses of epinephrine during 3 weeks prior to treatment (panel a) and after 6 (panel b) and 15 weeks (panel c) of control (0) and bST (I) treatment. Analyses of individual animal curves are in Table 3-7 .................................... 117 Figure 3-9. Response in plasma glycerol concentrations to a 1.6 pg/kg BW epinephrine infusion during the 15th week of treatment with either control (0) or bST (I). Area under the response curve (0 to 20 min. after infusion) was calculated using data points -30 to 0 and 120 to 130 min. relative to infusion as the base line. Control area under the curve = 604 :t 100 pM-min and bST area under the curve = 648 :l: 94 pM-min ..................................... 119 xiv LIST OF ABBREVIATIONS A00 ........................................ acetyl 00A carbcxylase ACS .......................................... acetyl CoA synthase ADG ............................................ average daily gain AT ................................................. adipose tissue AUF ............................................. Animal Use Form 8W .................................................. body weight BAR ........................................ beta-adrenergic receptor bST ............................................ bovine somatotropin BRSV .............................. Bovine Respiratory Syncytial Virus BVD .......................................... Bovine Viral Diarrhea C ........................................................ control °C ................................................. degree Celsius cc ............................................... cubic centimeters cDNA ............................. chromosomal decxyribcnucleic acid cm ................................................... centimeter cm2 ............................................. square centimeters CcA ................................................. Ccenzyme A CP ................................................. crude protein 0PM ............................................. counts per minute d ......................................................... day(s) DES ............................................. diethyl stilbestrol DM .................................................... dry matter DMI ......................................... daily dry matter intake DP ............................................... dressing percent DPM ...................................... disintegrations per minute E ............................................ efficiency of detection ED50 .................................. one half of maximum response EDTA ................................. ethylenediaminetetraacetic acid EEL ........................................... ether-extractable lipid FA ..................................................... fatty acids FAS ............................................ fatty acid synthase 9 .......................................................... gram Gi .............................................. inhibitory G protein G ............................................ stimulatory G protein GDP ........................................ guanosine diphosphate GLM .......................................... general linear models GHRF ............................... grcvvth hormone releasing factor GDP ........................................ guanosine diphosphate GTP ........................................ guanosine triphcsphate h .......................................................... hour 3H ........................................................ tritium HCW ........................................... hot carcass weight XV HSL ....................................... hormone-sensitive lipase IBR .................................. Infectious Bovine Rhinotracheitis ICD ................................. NADP - isocitrate dehydrogenase IGF-I ...................................... insulin-like growth factor- I i.m. .............................................. intramuscular(ly) INAD ................................ Investigaticnal New Animal Drug IU .............................................. International Units kg ...................................................... kilogram km ..................................................... kilometer KPH ......................................... kidney-pelvic-heart fat KRBB ................................ Krebs-Ringer—bicarbonate buffer L ........................................................... liter lb ...................................................... pound(s) m ........................................................ meter rrI2 ................................................. square meters M ......................................................... molar MCA ............................ marrow cavity area of metacarpal bone pCi .................................................... micrccurie ME ........................................... metabolizable energy mEq ............................................... milliequivalent pg .................................................... microgram mg ..................................................... milligram min ...................................................... minute MJ .................................................... millijoules pl ...................................................... microliter ml ....................................................... milliliter pM .................................................... micromolar mm .................................................... millimeter mM .................................................... millimolar mRNA ................................... messenger ribonucleic acid NADPH ........................... niCctinamide dinucleotide-phcsphate NEFA ........................................ nonesterified fatty acid NCW .......................................... non-carcass weight ng ..................................................... nanogram nm .................................................... nanometer oST ............................................ cvine somatotropin P ..................................................... probability pg ..................................................... picogram pH ..................... the negative logarithm of the hydrogen ion activity Pl3 ........................................... Parainfluenza 3-Virus PIA ..................................... Ne-phenylisoproyladenosine Pl-PLC ........................... phosphotidylinositol phospholipase 0 PM .................................................. Pscas major pST .......................................... porcine somatotropin rbST ................................ recombinant bovine somatotropin REA .................................................. ribeye area xvi RF ................................................. Rectus femcris RIA ............................................. radioimmunoassay Rm ........................................... maximum response RNA .............................................. ribonucleic acid 8 ........................................................ second SA ................................................. specific activity s.c. .............................................. subcutaneous(ly) SE ................................................ standard error SED .................................. standard error of the difference SEM ..................................... standard error of the means SRIF ............... somatostatin (somatotropin releasing inhibitory factor) SS ................................................. Supraspinatus ST ............................... somatotropin (a.k.a. growth hormone) STMUS ..................................... Semitendinosus muscle t ............................................. . ............. time T8 ................................................. Triceps brachii TBA ............................................ trenbolone acetate TCA ....................... total cross-sectional area of metacarpal bone wt ........................................................ weight INTRODUCTION After nearly 25 years (1974 to 1995) of selecting for lean, growth-type cattle, camss characteristics have changed (NCBA, 1995). Carcass weight (HCW) and ribeye area (REA) have increased 10 and 8.5%, respectively, with backfat and kidney-pelvic heart (KPH) fat decreasing 24 and 30%, respectively (NCBA, 1995). With these changes, yield grades have improved but marbling score and the number of carcasses grading USDA choice or higher have decreased. It has been estimated that it costs the beef industry $2.4 billion per year to put excess fat on cattle and another $2 billion per year for the retail food industry to trim the excess fat from beef (H.D. Ritchie, personal communication). Carcass backfat has decreased from 1.6 cm (.62 in) to 1.2 cm (.47 in; NCBA, 1995). To meet consumer preference most packers and retailers trim carcasses to a fat trim of .64 cm (.25 in) or less (H.D. Ritchie, personal communication). Therefore, there is a need for leaner carcasses. Second generation growth promotants (e.g.. zeranol, and estradiol) have been shown to increase (P < .05) ADG (9.8%), HCW (7.5%), and REA (6.8%) over non-implanted Holstein steers (Apple et al., 1991). Trenbolone acetate (TBA), a synthetic androgen with 50 times the potency of testosterone (Preston, 1987), does not increase ADG to the same extent as the estrogenic-growth promotants. The benefit of TBA is in combination with the estrogenic implants and increases (P < .05) the ADG of Holstein steers up to 14.8%, HCW by 9.6% and REA by 16.5% over non-implanted steers (Apple et al., 1991 ). Reducing the waste fat of beef carcasses is a major industry goal. 1 Estrogenic implants or the combination of estrogen and TBA have increased growth of Holstein steers, but the study by Apple et al. (1991) demonstrated no significant effect on carcass fat thickness, KPH fat, or marbling score (Apple et al., 1991). Although in crossbred beef steers which received an estradiol benzoate- progesterone implant, KPH fat, marbling score and the number of steers grading choice or higher decreased compared to non-implanted control steers (Preston et aI,1995) Recently, interest in another growth promotant, somatotropin (ST), to alter growth and carcass composition has increased. The primary goal has been to increase circulating serum levels of ST to increase milk production, increase muscle mass or increase the proteinzfat ratio of the final product (Ethertcn and Kensinger, 1984; Ethertcn and Smith, 1991). When exogenous ST was administered to pigs, ADG increased 10 to 20%, feed efficiency improved 15 to 30%, lipid accretion decreased 20 to 80% with a 20 to 50% increase in protein deposition (Ethertcn and Smith, 1991). Responses varied in cattle administered ST. In cattle, ADG may increase 6 to 24% (McBride and Moseley, 1991). The most pronounced effect of ST treatment is the decrease in carcass fat (McBride and Moseley, 1991). Moseley et al. (1992) showed a linear decrease in carcass fat of steers given increasing levels of ST administration (0, 33, 100 300 pg/kg BW per day). The most dramatic decease in carcass fat (55% less than control steers) was from steers treated daily with 300 pg ST/kg BW. Early et al. (1990a) did not observe a decrease in backfat thickness but the lean to fat ratio was increased . Even though bST increases growth and decreases carcass fat in cattle, Early et al. (1990a) found that bST had no effect on HCW, but observed an increase (P < .05) in non-carcass weight and lower (P < .05) dressing percent (DP) as compared to control steers. The bST steers had 19.9% greater (P < .10) digesta weight. Furthermore, 75% of the nonlcarcass weight was due to greater gut fill which accounted for 64% of the greater live weight. Early et al. (1990a) hypothesized that by giving younger cattle ST over a longer period might increase not only non-carcass tissue, but the more economically important, carcass tissue. A review article by Ethertcn et al. (1993), proposed that the decrease in adipose tissue growth from pST-treated pigs was the result of a decrease in lipogenesis rather than an increase in lipolysis. The review further stated that "metabolic effects of ST in adipose tissue of cattle and pigs are a function of energy balance . . . (and when) . . . animals are in'positive energy balance, ST decreases lipid synthesis whereas the effects of lipolysis are modest at best." Both Mikel et al. (1993) and Dunshea et al. (1992a) demonstrated increased non-esterified fatty acid (NEFA) concentrations in plasma of growing pigs administered ST. Evidence exists that plasma NEFA concentrations are elevated in ST treated animals, thus indicating an increase in lipolytic capacity (Eisemann et al., 1986). In a 266 d study, beef heifers treated with ST had plasma NEFA concentrations 26 to 46% greater than control heifers (Schwarz et al., 1993). A greater increase in lipolytic capacity of adipose tissue of bST-treated steers was shown by Boisclair et al. (1989) and Peters (1986), who demonstrated a 43 and 86% increase in NEFA response to an epinephrine challenge following 15 and 28 d of ST treatment, respectively. Few studies have been completed investigating the effect of long-term bST administration on lipolysis in finishing steers. With the increased use of biotechnology, researchers are able to elucidate and define the mechanisms of cell action. With an understanding of the basic mechanisms of cellular lipid metabolism, new methods of modulating this system can be evaluated. Somatotropin modulates lipid metabolism by decreasing carcass fat and lipid accretion. Evidence suggests that ST uncouples insulin from its secondary messengers (Roupas, et al., 1991), producing insulin resistance (Walton et al., 1987), therefore, reducing lipogenesis and enhancing lipolysis. The first series of objectives were to test a hypothesis by Early et al. (1990a), that administering ST to younger animals for a longer period might increase growth of carcass as well as noncarcass tissue. The second set of objectives were to further elucidate the role of ST in modulating lipid metabolism of the growing- finishing Holstein steer. Therefore, the objectives of this dissertation were: 1) To evaluate the effectiveness of long-term ST administration to growing-finishing Holstein steers at the beginning, end or during the entire feeding period on: a) feedlot performance b) skeletal growth 0) lean and adipose tissue accretion and body composition d) carcass trait Characteristics e) internal organ growth f) serum lGF-1 concentration 9) and, liver IGF-1 mRNA abundance 4 2) To determine the effect of long-term ST administration to finishing Holstein steers on: a) lipogenesis as measured by in vitro tritium incorporation into fatty acids, fatty acid synthase activity and isocitrate dehydrogenase activity b) lipolysis as measured by in vitro basal and epinephrine stimulated glycerol release into the media, and by in vivo NEFA and glycerol dose response to epinephrine Chaflenges c) and, changes in lipogenesis and lipolysis during the finishing period Chapter one will review the current literature regarding the use of bovine somatotropin to improve growth, reduce carcass lipid content and modulate lipid metabolism. Chapter two will outline the methodology, results and discussion of the experiment which addresses objective one. Objective two will be discussed in chapter 3, and both experiments will be reviewed in the conclusion. Raw data, detailed methodology, and animal use approval letters are contained in the appendices. Chapter 1 LITERATURE REVIEW Regulation of endogenous somatotropin secretion Somatotropin (ST) is a single polypeptide chain of 191 amino acids (chchick and Cioffi, 1991) stored and secreted by acidophile cells of the anterior pituitary gland (Bennet and Whitehead, 1983). Regulation of ST is under both positive and negative control as well as feedback inhibition. The hypothalamus produces a ST releasing factor (growth hormone releasing factor, GHRF) and an inhibitor of ST release (somatostatin, SRIF). Growth hormone releasing factor and SRIF are neurally regulated by serotonin, dopamine and catecholamines. The hypothalamus is under negative feedback control by ST and other factors of this system. Plouzek and Trenkle (1991 a) determined that overall plasma ST decreases with age (5 - 15 months) in beef cattle. This was due to a decrease in ST baseline, amplitude of secretory periods, the number of ST spikes, and the amplitude of ST spikes. They further determined that the reason ST decreases with age may be due to decreased responsiveness to GHRF. Additionally, Plouzek and Trenkle (1991a) found that bulls have greater overall plasma ST concentrations, greater baseline concentrations, greater amplitude of secretory periods, and greater amplitude of ST spikes than steers. The number of secretory periods and spikes in a 12-h period were similar between bulls and steers (Plouzek and Trenkle, 1991a). This may explain, in part, why bulls gain faster and are leaner than steers. Unlike humans, plasma ST concentrations do not increase during puberty in bulls, but decreases from birth to maturity (Plouzek and Trenkle, 1991a; McAndrews et al., 1993). Somatotropin acts directly through binding receptors on precursor bone, muscle, and adipose cells causing their proliferation, or indirectly by binding hepatocytes causing production andrelease of insulin-like growth factor-I (lGF-l) which acts on peripheral tissue to stimulate growth (Kopchick and Cioffi, 1991) Although ST plays a major role in partitioning nutrients for support of physiological and developmental processes (Bauman et al., 1982), many functions are indirect and are medicated by lGF-l (Campion and Novakofski, 1990). Even though Plouzek and Trenkle (1991a) demonstrated a decrease in plasma ST concentration with age in beef cattle, plasma IGF-l concentrations increased more than three-fold in bulls and steers from 5 to 12 mo of age (Plouzek and Trenkle, 1991b). Additionally, bulls had greater plasma lGF-I concentrations than steers after 5 mo of age. Plouzek and Trenkle (1991b) suggest that lGF-I in cattle appears to be controlled by many regulator hormones including ST along with the sex hormones, insulin and thyroid hormones. Effect of exogenous somatotropin on plasma hormones and metabolites. Somatotropin. Boisclair et al. (1994) demonstrated that within 1 h after exogenous bovine ST (bST) administration (29.2 IU bST/d), plasma bST increased to its peak above the control and then decreased in the remaining 12 h. Eisemann et al. (1986a) and Enright et al. (1990) saw plasma bST increase to its peak within 2 h after exogenous administration with 40 pg bST/kg BW and 120 pg bST/kg BW, respectively. Although, mean and baseline plasma bST concentrations increased with bST administration, Enright et al. (1990) determined that the number and amplitude of endogenous bST pulses decreased. Roeder et al. (1994) observed over a two- fold increase in serum bST baseline with administering 160 mg bST/wk, and observed a decrease in peak amplitude and a trend towards a decrease in the frequency of bST spikes. The decrease in frequency of bST spikes and amplitudes observed by Roeder et al. (1994) were not evident in steers receiving bST in a study by Moseley et al. (1982). Moseley et al. (1982) did observe an increase in baseline serum bST concentration and an increase in the number of bST spikes, but amplitude of the spikes was not changed. One reason for the discrepancy maybe in the mode of administration. Moseley et al. (1982) evaluated three patterns of administration; infusion, pulse (six pulses per day), or a combination. When all bST patterns were compared to the control, the number of spikes were increased (7.5 vs. 11.2 for the control and bST treatment, respectively), but only the pulse method increased the number of spikes per day as compared with infusion (13.3 vs. 9.8 spikes per 24 h, respectively) and the combination was intermediate (10.4 spikes per 24 h). Preston et al. (1995) demonstrated a quadratic response in serum bST concentration with exogenous bST administration of 0, 80, and 160 mg bST/wk. But bST concentrations were lower for steers receiving bST than control steers. This could be explained by differences in the procedures utilized. Blood samples were collected prior to the weekly bST implant, therefore, blood was collected when the implant was depleted and combined with the fact that exogenous bST reduces endogenous secretions, could explain why bST-treated animals had reduced serum bST concentrations. Insulin-like growth factor-I. Previous research (Enright et al., 1990; Dalke et al., 1992; Moseley et al., 1992; Boisclair et al., 1994; Roeder et al., 1994; Preston et al., 1995) has demonstrated that bST administration increased blood lGF-I concentrations 13 to 150% in cattle. In growing lambs, plasma lGF-l concentration increased after one week of treatment and increased three-fold after ten weeks of ST administration (Pell et al., 1990). Both Dalke et al. (1992) and Moseley et al. (1992) observed linear increases in serum lGF-l concentration with increasing doses of bST (0 to 160 mg bST/wk and 0 to 300 pg bST/kg 8W, respectively). The increase in serum IGF-I concentrations occurs rapidly after bST administration. Roeder et al. (1994) observed a 7.8% increase in serum lGF-l concentration 8 h after a 160 mglwk dose was administered to steers and the lGF-l concentration continued to increase to 35.8% over controls at 32 h post- administration, with the elevated concentrations maintained for 88 h after the initial implantation of bST. The liver is the main source of IGF-I in the blood (Pell, 1997). Grant et al. (1991), Coleman et al. (1994) and Ramsay et al. (1995) demonstrated a 100 to 300% increase in liver lGF-I mRNA with ST administration to pigs. Mathews et al. (1986) demonstrated an increase in liver mRNA within 2.5 h after mice were injected intraperitoneally with human ST. The increase in lGF-l mRNA abundance is transient. In both the liver (Mathews et al., 1986) and gastrocnemius muscle (lsgaard et al., 1989), lGF-l mRNA abundance decreased after reaching its peak within 12 h after a single ST injection in mice. Ramsay et al. (1995) demonstrated in pST-treated pigs, that lGF-I mRNA quantity increased 4 h after a single injection of pST, remained elevated for 12 h and began to decrease by 20 h after treatment. Grant et al. (1991) showed a 270 to 300% increase in liver lGF-l mRNA in pigs 24 h following the last pST injection of a 24-d treatment period. Insulin. Somatotropin induced increase in plasma insulin has been observed in cattle (60 and 114%; Eisemann et al., 1986a; Boisclair et al., 1994, respectively), sheep (81 and 150%; Johnsson et al., 1985; Poll et al., 1990; respectively), and pigs (502 and 514%; Dunshea et al., 1992a; Hansen et al., 1997b; respectively). Schwarz et al. (1993) demonstrated a ST-dose-dependent increase in plasma insulin concentration of 15 to 84% and 37 to 118% in heifers with doses of 62 and 126 pg STIkg BW, respectively. The drastic increase observed in pigs, described above, occurred 7 (Dunshea et al., 1992a) and 28 d (Hansen et al., 1997b) following the start of pST treatment. There is evidence that ST decreased insulin sensitivity. Hart et al. (1984) observed a reduction in the insulin-induced decrease of plasma glucose during an insulin tolerance test and Roupas et al. (1991) suggested that insulin uncouples from its second messengers. Nonesten'fied fatty acids and glycerol. Eisemann et al. (1986a) observed a chronic elevation in plasma nonesterified fatty acids (NEFA) in growing beef heifers with bST administration, but Enright et al. (1990) and Peters (1986) observed no increase in plasma NEFA of growing steers. Pell et al. (1986) demonstrated plasma NEFA levels were unaffected by ST administration to growing lambs. Dunshea et al. (1992a) demonstrated an increase in plasma NEFA of barrows within 7 h of the 10 first pST injection and the response increased on the second and seventh day of treatment. It has been suggested by Boisclair et al. (1997) that the increase in plasma NEFA concentration with bST treatment of animals in positive energy balance is a result of mild disturbances or stress during blood collection. This was suggested because bST causes an increase in B—adrenergic receptor (BAR) number in rat adipocyte membranes (Watt et al., 1990), therefore, these animals respond to catecholamines to a greater extent than non-treated animals. Houseknecht et al. (1995) determined that bST increased the maximum binding of a BAR agonist to the BAR without a change in the binding affinity. Boisclair et al. (1997) cautions that proper protocol is required to prevent inadvertent responses which may suggest lipolytic activity. Eisemann et al. (1986a) demonstrated both an increase in irreversible loss and oxidation of NEFA with bST treatment in heifers. Their data further suggested that NEFA are used as an energy source and perhaps this spares oxidation of other metabolites (e.g. amino acids) during bST treatment. Plasma glycerol concentrations were increased 31% in sheep (Doris et al., 1996) and 79% in barrows (Dunshea et al., 1992a) after receiving ST. This may be the result of triacylglycerol breakdown as suggested by Pell et al. (1990). Sechen et al. (1990) stated that because adipose tissue has very low glycerol kinase activity, alteration in plasma glycerol concentration relates to lipolysis whereas Changes in NEFA concentration reflects mobilization (i.e., the difference between lipolysis and NEFA re-esterification). A consistent increase in blood NEFA and glycerol concentrations has been shown to occur in animals that are in 11 negative energy balance. Plasma NEFA and glycerol concentrations increased 743% and 46%, respectively, in steers under a prolonged fast (Rule et al., 1985). Sechen et al. (1990) saw a 372% increase in plasma NEFA and 119% increase in plasma glycerol concentration in bST-treated lactating dairy cattle. Additionally, Peters et al. (1986) observed a 63% increase in basal-plasma NEFA with feed restriction. Glucose. Plasma glucose was increased 5 and 11%, respectively, in bST- treated growing cattle (Boisclair et al., 1994) and in ST-treated lambs (Pell et al., 1990). Dunshea et al. (1992a) observed an increase in plasma glucose within 2 h after an initial injection of porcine ST (pST) to barrows. Early et al. (1990a) and Enright et al. (1990) in steers and Sechen et al. (1990) in lactating dairy cattle, did not observe an increase in plasma glucose with ST administration. Pell et al. (1990) stated that the increase in plasma glucose could be due to an increase rate of gluconeogenesis, or a decrease in peripheral glucose utilization. Boisclair et al. (1994) demonstrated a decrease in hindlimb glucose uptake in growing steers administered bST. Additionally, Hart et al. (1984) demonstrated that bST had diabetogenic activity and reduced insulin’s depression of plasma glucose during an insulin tolerance test and suggested that bST decreased glucose uptake by body tissues as seen by Dunshea et al. (1992c) in pST-treated pigs. Pell et al. (1990) suggests they have unpublished data which demonstrates an increase in gluconeogenic potential by hepatocytes isolated from ST-treated lambs and therefore, glucose production may be increased, but would require an adequate supply of gluconeogenic precursors, glycerol being one. 12 Effect of somatotropin on feedlot performance Growth. Additional evidence since the study by Evans and Simpson (1931) has shown a growth response in rats due to chronic ST administration. Administering ST to farm animals is of interest because as animals grow older, the proportion of fat in weight gain increases and there is a need to produce meat with less fat (Ethertcn and Smith, 1991). When administered exogenously, ST increased ADG 10 to 20% in pigs (Ethertcn and Smith, 1991) and 19 to 24% in sheep (Wise et al., 1988; and Beermann et al., 1990a, respectively). Excellent tabular summaries of the effect of ST on beef cattle feedlot performance can be found in McBride and Moseley (1991) and Moseley et al. (1992) which summarize data from heifers, steers, and bulls from 1959 to 1990. The following review will primarily focus on the effects ST has on steers. A summary of recent research on the affects of ST on beef cattle feedlot performance is presented in Table 1-1. The range in growth (ADG, kg) response to ST as compared to controls varies from -6.3 to +26%. Three studies have looked at ST dose response on feedlot performance (Dalke et al., 1992; Moseley et al, 1992; and Preston et al., 1995). Moseley et al. (1992) demonstrated ST increased ADG in crossbred steers up to a daily dose of 33 pg Sleg BW, and decreased ADG with doses of 66 to 300 pg Sleg BW with the 300 pg dose having the greatest reduction in gain. Observations by Dalke et al. (1992) and Preston et al. (1995) showed similar results in bST-treated cattle. In sheep, ovine ST (oST) increase lamb weight gain 19 to 24% (Beermann et al., 1990b; Wise et al., 1988), although early work by Muir et al. (1983) did not show 13 Table 1-1. Summary of recent studies on the effect of somatotropin on beef cattle feedlot performance Duration Reference ADG, DMI, kg ' Feed (animals used) wt, kg dose, of kg " efficiency, pg/kg treatment, g/kg a 8W d Dalke et al., 378 0 28 1.70 10.5 ° 161 ° 1992 1’ (steers) -.10 12 28 -1.2 -5.7 +4.9 0 24 28 +1.1 -4.0 +6.0 0 49 28 +5.3 -70 +138 Moseley et al., 392 0 131 1.14 8.9 ° 165 d 1992, Exp. 1 (steers) 0 33 121 +7.9 1 -5.8 +137 1 0 100 131 —7.0 1 -130 1 +7.3 +01 1 300 151 1 -37.7 1 -17.4 1 -26.2 1 Moseley etal., 417 0 132 1.10d 8.7° 126d 1992, Exp. 2 (stem) -.01 8.25 133 -.01 -6.9 t +5.9 0 16.5 121 +9.0 .34 +144 1 0 33 121 +10.8’r 57* +1781 0 66 135 -3.6 -12.6 1 +107 1 Preston eta|., 379 0 84/119' 144° 8.1 ° 177° 1995° (steers) -.01 25 84/119 +3.5 -37 +8.8 0 51 84/119 +4.9 4.9 +102 Rathmacher at 357 0 140 1.44 8.26 171 al., 1998 9 (steers) +1.4 49.8 140 -.6 +2.7 -29 Rumsey et al., 182 0 56 1.37 5.40 249 1996 (steers) +.01 100 56 +244“ +23 T +232“ Schwarz et al., 286 0 257 .91 7.40 122.8 " 1993 , (heifers) O 62 239 +8.5 -3.9 +12.9 +73 124 232 10.6 1 +5 +10.0 14 Table 1-1. (cont’d) ‘Control values are given in actual units, bST doses are percent change from controls; a difference does not indicate a statistical significance. bbST dose was given as 40, 80, and 160 mglwk. °Significant linear response (P < .10) with increasing bST dose. dSignificant quadratic response (P < .10) with increasing bST dose. ‘A 2 x 3 factorial experiment was conducted; with or without an estradiol benzoate progesterone-trenbolone acetate and bST doses of 0, 80, 160 mglwk. 'Duration of treatment was determined by initial body-weight block. 1A 2 x 2 factorial experiment was conducted; with or without 120 mg trenbolone acetate and 24 mg estradiol-17B and with or without 160 mg bST/wk. ~ hEnergy consumption, MJ of MElkg of gain was decreased (P < .05) 11.7% with treatment of 62 pg Sleg BW. ‘Different from control (P < .20). 1Different from control (P < .10). “Different from control (P < .01 ). 15 an improvement in ADG of oST-treated lambs. Dry matter intake. In the reviews by McBride and Moseley (1991) and Moseley et al. (1992), the effect of ST on dry matter intake (DMI) varies greatly. Swine treated with ST show a marked depression in DMI (Bonneau, 1991). Based on the review by McBride and Moseley (1991) and Table 1-1, when ST is given to cattle with initial weights less than 260 kg, DMI is increased and when BW is over 260 kg, DMI is decreased. There are exceptions, for example, Fabry et al. (1987) and Rathmacher et al. (1996) demonstrated a 1.5% and 2.7% increase in DMI in 439 kg heifers and 357 kg steers, respectively. Schwarz et al, (1993) did not observe an effect on DMI of heifers administered ST with a beginning weight of 286 kg. Groenewegen et al. (1990) observed a 24% increase in DMI of ST-treated bull calves and also noted an increase in digestive tract volume. The reticulo-rumen, small intestine and large intestine weights were 24, 28 and 19% greater, respectively, in ST-treated bull calves (45 kg BW, Groenewegen et al., 1990). This is also supported by Early et al. (1990b) who observed a 2% increase in DMI with ST treatment in steers (231 kg BW) and an increase in gut fill. In the three dose response studies summarized in Table 1-1, DMI decreased linearly with increasing daily doses of bST from 3.4 to 17.4%. Cattle in those studies weighed greater than 370 kg. One plausible reason for the discrepancy in ruminants is suggested by the quote from McBride and Moseley (1991). “A hypothesis to explain this depression in DMI, might be that the abundance of nutrients from mobilized fat stores cannot be utilized by growing tissues at a rate fast enough to reduce Circulating nutrient concentrations, therefore that animal responds by reducing DMI and bringing body to homeorhesis. On the other hand, in young growing cattle and sheep treated with somatotropin, it would be expected that the protein and energy requirement 16 cannot be met by mobilization of energy reserves, therefore DMI may be increased to help support the stimulated metabolic response to somatotropin.” Feed Efficiency (gain/feed). In cattle and sheep, the most consistent ST response is improved feed efficiency (McBride and Moseley, 1991). Studies by Enright et al. (1990) demonstrated a 4% increase in feed efficiency over controls, although in the same study steers receiving an estradiol implant exhibited a 8% increase in feed efficiency. Moseley et al. (1992) reported an improvement in feed efficiency of 14 and 7.3% when daily treatments of 33 and 100 pg bST/kg BW were administered to steers, respectively. When the daily bST dosage was increased to 300 pg bST/kg 8W, feed efficiency was reduced 26.2% as compared to control steers (Table 1-1). The other two dose response studies, Dalke et al. (1992) and Preston et al. (1996) demonstrated similar results. The effect of ST treatment on feed efficiency in sheep is consistent with that of cattle. Feed efficiency was shown to increase 8 to 24% in oST-treated lambs (Muir et al., 1983; Wise et al., 1988; Beermann et al., 1990b). The above findings are supported by studies by Early et al. (1990a) in which ST-treated steers exhibited a 12.2% increase in the efficiency of metabolizable energy (ME) intake used for gain as compared to control steers. Early et al. (1990a) showed that 75% of the noncarcass weight (NCW) of bST-treated steers was due to greater gut fill, while rumen and intestine weights were numerically 4.8% greater, than control steers. From this data, one can infer that bST-treated animals may have a greater capacity to digest and absorb nutrients. This concept is supported by a study by Moseley et al. (1982) which showed a 5% increase in dry 17 matter digestibility and a 7% increase in nitrogen digestibility in bST-treated steers. These findings are corroborated by Lapierre et al. (1992) which demonstrated a 2% increase in dry matter (DM), energy , nitrogen, and organic matter digestibility with GHRF administration. Wray-Cohen et al. (1991) found a 3 to 4% increase in apparent nitrogen digestibility in pST-treated pigs. These researchers (Wray— Cohen et al., 1991) hypothesized that the increase was due to decreased feed intake. In other studies DM digestibility of bST-treated steers (Eisemann et al., 1986b) and N digestibility of ST-treated lambs (Pell et al., 1990) were similar to control animals. Effect of somatotropin on skeletal growth Somatotropin is considered to be the main regulator of long-bone growth (Scheven and Hamilton, 1991) and has direct effects on cartilage and indirect effects on cartilage through lGF-l (Slootweg, 1993). Both ST and lGF-l increased fetal and neonatal longitudinal metatarsal bone growth in vitro, with the response arrested with the addition of lGF-I monoclonal antibody (Scheven and Hamilton, 1991). Because anti-lGF-I antibody decreased bone growth, this suggests that ST may work through locally produced lGF-I (Scheven and Hamilton, 1991). Martinez et al. (1991) demonstrated that Systemic injection of ST caused an increase in bone—protein synthesis in the tibia of normal female rats without an increase in lGF-l concentrations, again, suggesting that the actions of ST have a direct effect on bone or mediated by autocrine or paracrine mechanism, independently. Early et al. (1990b) demonstrated that ST administration to steers increased 18 humerus length 4.5% and rate of growth in length 45%, and femur Circumference growth by 18.7%. In steers which were treated with bST from 231 to 384 kg, the increase in bone growth with bST is possible because the proximal epiphysis of cattle ossifies when cattle are 18 to 24 months of age (Emara, 1937, Sisson, 1953). Johnsson et al. (1985) observed an increase in bone circumference of the femur and humerus of ST-treated lambs, and Butler-Hogg and Johnsson (1987) demonstrated an increase in bone weight in ST-treated lambs. Although ST stimulates growth when given exogenously, research by Greiner (1993) showed that endogenous bST and lGF-I concentrations may not reflect differences in height. For example, unselected Hereford steers had an average frame score of 1.6 which was significantly lower than the average frame score of 5.3 for Herefords selected for growth. Although frame scores were different, plasma bST and IGF-I concentrations were greater in the unselected Herefords (3.70 and 880.5 nglml, respectively) than in Herefords selected for growth (3.31 and 795.3 nglml, respectively). In human medicine, the focus of ST treatment is to prevent osteoporosis in the elderly (Slootweg, 1993). Johnsson et al. (1985) did not find a change in bone density of ST-treated lambs. Hardt et al. (1995) in a study evaluating the relationship between estrogenic implants, ST, lGF-l and skeletal characteristics did observe an increase in circulating ST concentrations with implantation of estradiol- benzoate progesterone and tended to see an increase in the breaking load of metacarpals of implanted steers which suggest an increase bone density. Hardt et al. (1995) also observed a decrease in metacarpal length with the estrogenic 19 implant due to the fact the estrogens inhibit long bone growth. Effect of somatotropin on the carcass Carcass Characteristics. A review of recent studies on the effect of ST administration on beef cattle carcass characteristics is summarized in Table 1-2. In the two studies by Moseley et al. (1992), hot carcass weight (HCW) decreased with increasing ST doses. This effect was evident in studies by Dalke et al. (1992) and Preston et al. (1995). Conversely, HCW increased in the study by Rumsey et al. (1996). Carcass weight was increased 7.4% in prepubertal heifers given ST for 15 wk in a study by Vestergaard et al. (1995), but not in an earlier study (Vestergaard et al., 1993). The difference in the response may be due to dose, Moseley et al. (1992) used the highest doses and Rumsey et al. (1995) used the lightest-weight cattle. In the summary by McBride and Moseley (1991), research demonstrated there was a positive response in carcass tissue accretion with ST administration, although, not necessarily statistically significant. There was also an increase in weights of non-carcass tissues (McBride and Moseley, 1991). Early et al. (1990a) and Johnsson et al. (1985) observed significant increases in non- carcass weight (NCW) in ST-treated steers and lambs, respectively. Early et al. (1990b) attributed 75% of the increase in NCW to an increase weight of the gastro-intestinal tract. Early et al. (1990a) theorized that by administering bST to younger, light-weight cattle, the carcass component could be increased to a greater extent than the non-carcass components. If final weight does not Change, but HCW decreases and NCW increases, changes in dressing 20 Table 1-2. Summary of recent studies on the effect of somatotropin on beef cattle carcass characteristics‘ Reference Daily ST HCW, Dressing Back Ribeye Marbling Yield dose, kg percent fat, areab score I" grade b pg/kg mm b cm 2' 8W Dalke, et al., 0 331 60.1 14.0 ° 80.6 510 ° 3.2 ° 1992 1' 12 0 +1.2 -2.1 +3.6 -39 -94 24 +1.5 +2 -1.4 +2.5 +9.8 -6.3 49 +1.8 +2 -19.3 +4.1 -13.7 -15.6 Moseley et 0 334 ° 62.7 ° 19.4 °-' 52.7f NG *1 NG al., 1992, 1, 1 1 1 Exp. 1 33 -2.1 -2.2 -9.3 +7.4 NG NG 100 -2.7 1 -2.2 1 -28.4 1 +7.2 1 NG NG 300 -9.0 1 -6.4 1 -62.4 1 +6.6 1 NG NG Moseley et 0 356 ° 63.2 ‘ 23.2 ° 71.8 °-' NG NG al., 1992, 1 , T 1 Exp. 2 8.25 -1.1 -.9 -6.0 +4.1 NG NG 16.5 -1.4 1 -1.1 1 -6.5 1 +7.8 1 NC NG 33 -.8 1 -.9 1 4.7 1 +8.8 1 NG N6 66 -3.1 1 -2.1 1 -22.8 1 +7.0 1 NG NG Preston et 0 329 63.1 11.2 ° 86.1 490 ° 2.4 ° al., 1995 1' 25 +9 +3.2 -8.0 +2.7 -4.1 -8.3 51 +6 -.5 -15.2 +1.7 —6.1 -125 Rathmacher 0 353 62.5 6.6 83.5 NG 2.5 et al., 1996' 49.5 -.4 0 -17.3 -1.9 NG -4.o Rumsey et 0 147 N6 NG NG NG NG al., 1996 T 100 +5.5 N6 N6 NG NG NG Schwarz et 0 287 57.7 NG NG NG NG aL,1993 62 +1.8 +1.2 NG NG NG NG 124 +7 +7 N6 N6 N6 N6 21 Table 1-2. (cont’d) 'Duration of dose and initial body weight is presented in Table 1-1. bControl values are given in actual units, bST doses are percent. change from controls; a difference does not indicate a statistical significance. ‘400 = slight, 500 = small, 600 = modest. ‘ bST dose was given as 40, 80, and 160 mglwk. ‘ Significant linear response (P < .10) with increasing bST dose. ' Significant quadratic response (P < .10) with increasing bST dose. “Not given in tabular form or not determined. '1A 2 x 3 factorial experiment was conducted, with or without an estradiol benzoate progesterone-trenbolone acetate and bST doses of 0, 80, 160 mglwk. iA 2 x 2 factorial experiment was conducted; with or without 120 mg trenbolone acetate and 24 mg estradiol-17B and with or without 160 mg bST/wk. 1Different from control (P < .20). *Different from control (P < .10). 22 percent (DP) would occur. Although, Dalke et al. (1992) and Preston et al. (1995) did not see a change in DP with increasing ST doses, both Moseley et al. (1992) and Early et al. (1990a) observed a decrease when an increasing ST dose was administered. The greatest effect of ST treatment on carcass characteristics is the decrease in carcass fat (McBride and Moseley, 1991; Table 1-2). Dalke et al. (1992), Moseley et al. (1992), and Preston et al. (1995) showed a linear decrease in carwss backfat of steers with increasing doses of ST administration. The most dramatic was a 62.4% decrease in backfat with a daily ST dose of 300 pg/kg 8W. Peters (1986) observed a 24% reduction in backfat with bST administration following 29 d of treatment, but Early et al. (1990a) did not see a difference in backfat of steers administered ST for 112 d. Additionally, Enright et al. (1990) and Preston et al. (1995) demonstrated a decrease in kidney-pelvic-heart (KPH) fat with bST administration. In regards to marbling score, intramuscular fat content, Preston et al. (1995), Dalke et al. (1992), and Schwarz et al. (1993) observed a dose-dependent decrease in marbling score with increasing concentrations of ST (see Table 1-2 for data from Preston et al., 1995 and Dalke et al., 1992). The study by Schwarz et al. (1993) determined a 13 to 28% reduction in marbling with 62 and 124 pg ST/kg BW, respectively, with controls having a marbling score of 3.83 on a scale of 1 to 5 where 1 is poor fat and 5 is extremely fat. In conjunction with the decrease in marbling observed by Schwarz et al. (1993), they also determined that the percentage of intramuscular fat was reduced (P < .05) 46.6% with a daily dose of 23 124 pg ST/kg BW. The decrease in marbling score was translated into a decrease number of animals achieving the USDA choice quality grade (Dalke et al., 1992; Moseley et al., 1992; Preston et al., 1995), although, Early et al. (1990a) did not see a change in Canadian carcass grade with bST administration. There was a trend towards increased ribeye area (REA) with ST administration (Table 1-2). A significant linear or quadratic response in REA due to ST was only evident in the studies by Moseley et al. (1992). The greatest response in REA was observed with a daily ST dose of 33 pg/kg BW (Table 1-2). With a decrease in carcass backfat and KPH fat, and an increase in REA, there were improvements in carcass yield grade (Dalke et al, 1992; and Preston et al, 1995; Table 1-2). Moseley et al. (1992) did not demonstrate an effect on carcass yield grade with increasing dose of ST, but as mentioned previously, did observe an decrease in backfat and an increase in REA. Individual muscles and internal organs. Butler-Hogg and Johnsson (1982) observed greater muscle weight throughout the carcasses of ewe lambs treated with bST. In regards to muscle groups (primal cuts of the carcass), Early et al. (1990b) observed an increase in the flank and shank primals, and hypothesized that the flank increased to accommodate the larger gastro-intestinal tract in bST animals. Elsasser et al. (1998) observed an increase in the Rectus femoris (RF), Triceps brachii (TB), and Supraspinatus (SS) muscles with bST or in combination with 20 mg 17-B estradiol benzoate plus 200 mg progesterone. The Semitendinosus (STMUS) and Psoas major (PM) were less responsive to the hormonal modifiers which may be related to the functions of the muscle. Elsasser 24 et al., (1998) theorized the differences in response of muscles to ST treatment may be due to 1) the muscles are already increasing in size to their maximum potential given the nutrients available and 2) in relation to function, 9.9. the RF , TB and SS are involved in locomotion and therefore may respond in conjunction with long bone growth as compared to the STMUS and PM which are involved with hindlimb flexor and vertebral posture, respectively. Early et al. (1990b) did not see a significant affect of bST on the STMUS, but the Vastus lateralis muscle exhibited a 42% increase in growth. Also, Eisemann et al. (1989) did not observe an increase in STMUS weight with bST, but did observe a 11% increase in Longissimus weight but not relative to empty BW. Brameld et al. (1996) observed an increase in lGF-l mRNA in STMUS of pigs but not the Longissimus muscle after 7 d of bST administration, but neither muscle increased in weight. Beermann et al. (1990a) did Show an increase in STMUS with pST administration up to 120 pg pST/kg 8W which was due to individual muscle fiber hypertrophy. As discussed previously, NCW was increased with long-term bST administration. Part of this increase would be increases in internal organs. Moseley et al. (1992), Early et al. (1990b) and Rumsey et al. (1996), reported increases in liver and kidney weights in bST-treated steers and Schwarz et al. (1993) in bST-treated heifers. Rumsey et al. (1996) observed an increase in spleen and heart weight, but Early et al. (1990b) did not. Early et al. (1990b) did however observe an increase in lung and trachea weight with bST treatment. 25 Carcass composition and accretion rates. A consistent effect of ST administration to growing animals is increased nitrogen retention (Eisemann et al., 1986b). In the study by Eisemann et al. (1986b), they reported a 500% increase in nitrogen retained by ST-treated heifers. The increase was due to a 11% decrease in urinary nitrogen loss. These findings are supported by a 16% increase in nitrogen retention in steers (Moseley et al., 1982), and a 19% increase in nitrogen retention in lambs (Pell et al., 1990) receiving ST. Wallace and Bassett (1966) believed that the increase in nitrogen retention shown with ST administration is dependent on the associated increase in plasma insulin. The increase in plasma insulin would drive more nutrients (e.g. amino acids and glucose) into cells fueling the anabolic effects. Early et al. (1990C) observed a greater whole-body protein accretion with bST administration, but this was due to greater noncarcass protein accretion rather than in the carcass. Protein accretion was increased 22% with bST administration in Angus-Hereford crossbred steers (Rumsey et al., 1996). Rumsey et al. (1996) demonstrated a 23.5% decrease in lipid accretion after 56 d of bST administration in light-weight cattle (182 kg initial weight, and 266 kg average final weight.) and 100 pg bST/kg BW dose. Boisclair et al. (1994) demonstrated a greater protein accretion in the hindlimb of bST-treated steers. In conjunction with the decrease in fat, the percent protein and water increased linearly with increasing levels of bST administration (Moseley et al., 1992). Early et al. (1990b) did not see a significant decrease in total steer carcass fat, but the total lean to fat ratio was increased with ST administration. A similar 26 response was exhibited in lambs treated with ST (Pell et al., 1990). Treated lambs had carcasses with lower fat content, increased protein, and an increased lean to fat ratio (Pell et al., 1990). Product quality. Growth modifiers may increase ADG, improve feed efficiency, increase protein and decrease fat, but if the product produced is not acceptable to the public, the point is mute. Moseley et al. (1992) saw a 17 to 95% reduction in the percent of carcasses grading choice. The 95% reduction was seen in steers receiving 300 pg bST/kg BW and they did not reach market weight. At 100 pg bST/kg BW, 70% fewer carcasses graded choice. Preston et al. (1995) documented a linear decrease in marbling score (6%), and percent carcasses grading USDA choice (58%) with increased bST dosages. Few studies have evaluated the effect of ST in cattle on the resulting tenderness of the product. In young, light-weight (223 kg at harvest) prepubertal- Friesian heifers, treated with bST for 15 weeks, lean and fat color was improved and closer to the Danish ideal (Vestergaard et al., 1995). Additionally, there was a tendency for decreased intramuscular fat percentages with bST treatment, but bST did not affect tendemess (Vestergaard et al., 1995). Vestergaard et al. (1993) observed no effect of bST treatment on Longissimus dorsi muscle pigmentation, meat color, shear force or cooking loss. They did observe a decrease in intramuscular fat. Additionally, a trained-taste panel found no differences between loins prepared as steaks from control or bST-treated heifers (Vestergaard et al., 1993) Allen and Enright (1989), as reported by Vestergaard et al., (1993), reported 27 taste-panel scores for tenderness and overall acceptability to be slightly lower for bST-treated animals as compared to controls. An absence of difference in panel acceptability is consistent in the lack of affect of bST-treatment on collagen content and solubility (Vestergaard et al., 1993), and glycogen content of the meat (Vestergaard et al., 1995). Bovine ST administered for eight weeks to East Friesland, Oxford, and Texel sheep did not affect meat quality in regards to ultimate pH, lightness, hue, or saturation in the loin joint (Sinnett-Smith et al., 1989). In barrows, pST administered for 18 wk reduced sensory-panel scores for juiciness of loin chops and overall tenderness (Klindt et al., 1995). There was a linear decrease in juiciness of loin chops from boars as length of pST treatment increased. Loin Chops from boars receiving pST for 18 weeks had lower boar taint intensity than control boars. Somatotropin does not always show an affect on tenderness, but when there is an effect, it is negative (Klindt, 1995). Goodband et al. (1990), Solomon et al. (1988), and Beermann et al. (1990a) observed an increase in shear force values of pork chops with pST treatment. Hagen et al. (1991) and Goodband et al. (1990) demonstrated a decrease in tenderness scores assigned by a sensory panel to meat from pST-treated pigs. A consumer study demonstrated that consumers showed no difference in preference for loin roasts from control or pST-treated pigs, but consumers did prefer ham roasts from control versus pST-treated pigs (Prusca et al., 1993). Combination of ST and other anabolic agents. When ST is combined with another anabolic agents such as estradiol (Enright et al., 1990), TBA plus estradiol 28 (Preston et al., 1992; Rathmacher et al., 1996), or a B-agonist (Maltin et al., 1990; Hansen et al., 1997b); the combined affect is additive for specific traits. The use of estradiol and ST in feedlot steers increased ADG 12.8% and 19.3% over only ST-treatment and control steers, respectively. Also, the addition of estradiol to ST- treated steers improved feed efficiency 10% and 14.5% over only ST-treated and control steers, respectively (Enright et al., 1990). Combining ST and TBA plus estradiol did not increase feedlot performance or alter carcass characteristics as compared to these compounds given separately (Rathmacher et al., 1996). In the study by Rathmacher et al. (1996), implanting cattle with TBA plus estradiol improved feedlot performance (ADG and feed efficiency), HCW, and REA more than administration of bST. Administering bST did alter carcass composition by increasing the weight of muscle and decreasing the fat when combined with TBA plus estradiol. Preston et al. (1995) demonstrated an additive response between ST and TBA plus estradiol on ADG and feed efficiency. Although alone, the steroid implant increased ADG and feed efficiency to a greater extent than ST alone. In veal calves, the use of the B-agonist, clenbuterol, and ST improved feed efficiency over clenbuterol-treated calves, but only tended to improve feed efficiency over ST-treated calves. Average daily gain was not significantly different between the clenbuterol, ST plus clenbuterol, and ST treatments; although, clenbuterol plus ST tended to increase ADG 6.6% over the growth promotants individually (Maltin et al., 1990). Hansen et al. (1997b) showed additive effects of pST and the B-agonist, salbutamol, given to pigs as evidenced by increased feed 29 efficiency, carcass protein and water accretion, longissimus muscle area, and STMUS weight. Lipogenesis Adipose tissue is responsible for more than 90% of the fatty acid (FA) synthesis in the ruminant and the majority of FA synthesis in the pig and guinea-pig (Vernon, 1980). This differs from man and birds where the liver is the main site of FA synthesis and the rat, mouse, and rabbit in which both the liver and AT synthesize substantial amounts of FA (Vernon, 1980). Acetate is the primary carbon source for FA synthesis in ruminants unlike rats, where glucose is the primary precursor. In mature ruminants, most glucose is derived from gluconeogenesis rather than derived from carbohydrate digestion and absorption in the small intestine as in monogastric animals (Vernon, 1980; Fahey, 1988). Therefore, glucose is needed to be spared for basal metabolism (Vernon, 1980). Lactate, pyruvate, propionate, methylmalonate, butyrate, and B-hydroxybutyrate are used to some extent for FA synthesis (Vernon, 1980). The use of propionate leads to odd—chained fatty acids and methylmalonate produces branched-Chain FA (Vernon, 1980). The three critical (or control) enzymes in the pathway of FA synthesis are acetyl CoA synthase (ACS), acetyl CoA carboxylase (ACC) and fatty acid synthase (FAS; Vernon, 1980). The process of FA synthesis (palmitic acid, 16 carbons) requires 8 acetyl CoA and 14 molecules of nicotinamide dinucleotide-phosphate (NADPH; Vernon, 1980). The primary source of NADPH is from the pentose- 30 phosphate pathway. NADP-isocitrate dehydrogenase (ICD) provides the additional NADPH needed for FA synthesis above what can be produced from the pentose- phosphate pathway (Vernon, 1980). Regulation of lipogenesis. Lipogenesis is regulated by both plasma metabolites and hormones (Vernon, 1980). Increased plasma acetate and glucose will increase FA synthesis. Increased plasma glucose is rarely observed in ruminants (Vernon, 1980). The increase in dietary FA reduces FA synthesis in the adipocyte of the ruminant (Vernon, 1980). AcetyI-CoA carboxylase is regulated by both phosphorylation/ dephosphorylation and by changes in enzyme concentration (Wolf, 1996). Fatty acid synthase is only regulated by a change in enzyme concentration (Wolf, 1996). The change in enzyme concentration was found to occur via the action on the rate of mRNA transcription. Although in a summary by Girard et al. (1994), they suggest that the increase in FAS mRNA may be due to post-translational stabilization rather than only an increase in transcription. Vernon (1980) states that changes in NADPH production is determined by the rate of FA synthesis. Total fat content of steers increases as the age at harvest increases (Koch et al., 1979; Cianzio et al., 1985; Loy et al., 1988). Associated with the increase in quantity of adipose tissue is the decrease in the number of adipocytes per gram of tissue (Cianzio et al., 1985). Pothoven et al. (1975) observed a 62% decrease in in vitro lipogenesis as crossbred steers increased in 8W from 363 to 505 kg. The decrease in lipogenesis as BW increases is associated with an increase in adipocyte size and fewer cells per unit weight of tissue (Hood and Allen, 1973). 31 Effect of somatotropin on lipogenesis. Somatotropin has been found to decrease FA synthesis in adipose tissue (AT) treated in vitro (Vernon, 1982; Sinnett-Smith and Woolliams, 1989) and Walton and Ethertcn (1986) demonstrated that ST can antagonize insulin’s stimulation of lipogenesis in cultured porcine AT. Kramer et al. (1993) observed a 35% decrease in FA synthesis, as measured by tritium incorporation into FA, and a 56% decrease in malic enzyme activity in pigs treated with pST for 24 d. Just as with ICD, malic enzyme generates NADPH to be used in FA synthesis (Zubay, 1988) Glucose transport. Harris et al. (1993) observed an 86% reduction in glucose incorporated in FA after barrows were treated 11 d with pST and reduced FAS and ICD activity (67 and 31%, respectively). Because AT biopsies are in a net degradative state a soon as they are collected, in vitro measurements of lipogenesis may be underestimates (Kramer et al., 1993). Dunshea et al. (1992c) utilized an in vivo method to determine glucose utilization rates for lipogenesis and the effect which pST had on them. Rates of glucose incorporated into lipids were reduced 76%. Specifically, glucose incorporated into triglyceride-FA was reduced 78% as compared to glucose incorporated into triglyceride—glycerol which was reduced 66%. Donkin et al. (1996) found the mRNA abundance of GLUT 4, the major insulin regulated glucose transporter in adipocytes, was not decreased with pST administration, but Kilgour et al. (1995) determined that altering plasma ST concentration in the rat caused the GLUT 4 protein to translocate from the intracellular pool to the plasma membrane when ST was decreased. 32 Insulin and insulin resistance. Blood insulin concentrations increase in ruminants that are treated with ST (Davis et al., 1969; Wagner and Veenhuizen, 1978; Hart et al., 1984; Eisemann et al., 1986a). If insulin stimulates lipid accretion, ST must be blocking insulin’s affect by making the adipocytes insulin resistant. Insulin resistance is the subnormal biologic response to a specific insulin level (Moller and Flier, 1991). Insulin resistance is observed in genetically Obese mice following ST administration (Roupas et al., 1991). Studies by Walton et al. (1987) support the results of Roupas et al. (1991). Walton et al. (1987) used dose response curves to demonstrate that pST decreases the sensitivity of AT explants to insulin. Both the basal and maximally- stimulated conversion of glucose to lipid were reduced 50% and the insulin concentration which produced the half-maximum response was increased 10 fold. In addition, Walton et al. (1987) demonstrated that not only had the adipose tissue from ST-treated animals become insulin resistant, but lGF-I resistant as well. An experiment by Vernon et al. (1991a) can in part explain how lipogenesis is decreased in insulin-resistant AT. Vernon et al. (1991a) determined the effect of insulin, dexamethasone (a synthetic glucocorticoid) and ST on FA synthesis and ACC activity of AT from wethers. Insulin and dexamethasone acted synergistically to increase FA synthesis, but this affect was blocked by ST. Although ST clearly decreased insulin’s effect of FA synthesis, the total activity of A00 was unchanged. The proportion of A00 in the active state correlated to the effects seen in FA synthesis. Insulin increased activation of A00 whereas the addition of dexamethasone did not. 33 Somatotropin did not inhibit ACC activation after 24 h of incubation, but completely blocked the effect of insulin and dexamethasone following a 48 h incubation. The mechanism for ST blockage of insulin’s action is unclear, but it is not due to a decrease in insulin binding or insulin receptor kinase activity as determined by Magri et al. (1990) in pig adipocytes. These findings suggest that a post-receptor mediator of insulin action may be affected (Roupas et al., 1991; Vernon et al., 1991; Magri et al., 1990). GTP-binding proteins. One way ST may induce insulin resistance is to uncouple the insulin receptor from its signal transduction GTP-binding protein (G protein). The G proteins are heterotrimeric proteins comprised of or, B, y subunits (Simon et al., 1991). The action of ligand binding to a receptor activates the G protein which induces the exchange of guanosine diphosphate (GDP) bound to the or subunit for guanosine triphcsphate (GTP). The binding of GTP causes the or subunit to dissociate from the By heterodimer. The 01 subunit-GTP complex and By heterodimer can interact with effector proteins that are second messengers. Examples of second messengers are phosphodiesterase, phospholipase C, adenylate cyclase, phospholipase A2, and ion channels. Each second messenger is stimulated by a stimulatory Gs protein and inhibited by an inhibitory Gi protein (Simon et al., 1991). Work done by Roupas et al. (1991) demonstrated that ST antagonized the phosphotidylinositol phospholipase C (Pl-PLC) stimulation by insulin. Although PI-PLC activity was unchanged, the results suggested ST interfered with the mechanism of signaling. As stated above, G proteins require GTP to become activated. Roupas et al. (1991) demonstrated the activation of PI- 34 PLC by the addition of GTP[vS], a hydrolyzable GTP analog, to the membranes from saline-treated mice. When GTP[vS] was incubated with membranes of adipocytes from ST-treated mice, Pl-PLC was not stimulated. This finding suggests ST interferes with Gi protein activation of PI-PLC by insulin. What happens to the G, protein of the Pl-PLC pathway is unknown. In addition, ST effects on the G proteins of the adenylate cyclase pathway are unclear. Change in hormone activity and amount. Vernon et al. (1 991 a) determined in culture, ST decreased the activation of A00 of AT from wethers. Magri et al. (1990) demonstrated a decrease in the lipogenic enzyme activities of FAS, glucose- 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and malic enzyme in AT of barrows treated for 7 d with pST. Additionally, Magri et al. (1990) determined that glucose transport into adipocytes was reduced 62% with pST administration, but insulin binding to the adipocyte nor the insulin receptor’s tyrosine kinase activity were affected. Roupas et al. (1991) discovered that ST interferes with the signal transmission of a G protein between the insulin receptor and PI-PLC, thereby, even though insulin binding is not inhibited (Magri et al., 1990) its signal is being interrupted. Borland et al. (1994) determined that a short- lived protein may also be needed for ST to inhibit lipogenesis in sheep AT. This was determined after actinomycin D, an inhibitor of protein synthesis, blocked ST depression of lipogenesis in vitro. Ornithine carboxylase activity was shown to increase during ST treatment and coincided with the decrease in lipogenesis (Borland et al., 1994). Additionally, a polyamine may be needed for the full ST affect (Borland et al., 1994). 35 In addition to decreasing enzyme activation and interfering with signal transduction, ST alters A00 (Liu et al, 1994) and FAS (Mildner and Clarke, 1991; Harris et al., 1993; Donkin et al., 1996) synthesis. Liu et al. (1994) observed a 40% decrease in ACC mRNA in pST-treated barrows. Similarly, Harris et al (1993) demonstrated a 74% decrease in FAS mRNA with pST treatment for 11 d and Donkin et al. (1996) determined that FAS mRNA decreased in a linear fashion in porcine adipocytes with increased dosage of pST. No change in ICD activity was observed in lactating ewes (Vernon et al., 1991a) and lactating cows (Lanna et al., 1995), although FAS activity decreased. In a similar fashion, lngle et al. (1973) observed a decrease in in vitro lipogenesis, as measured by acetate incorporation into FA, with significant reduction in ACS and A00 with fasting; but did not show a significant decrease in ICD although, numerically activity decreased 15 to 30%. Additionally, lngle et al. (1972) observed a tendency in ruminant AT for the dehydrogenase to have greater activity when rates of FA synthesis were greater. Lipolysis Lipolysis is the mobilization of AT lipids for use by other tissues of the body and requires the hydrolytic cleavage of triacylglycerols to glycerol and free FA (Vernon, 1980). Hormone-sensitive lipase (HSL) catalyzes the rate limiting step of lipolysis in adipocytes (Cordle et al., 1986), and hydrolyzes triacylglycerol to diacylglycerol and free fatty acids. The last step is the conversion of 2- monoacylglycerol to free fatty acids and glycerol and is catalyzed by 36 monoacylglycerol lipase (Belfrage, 1985). Reversible phosphorylation controls the activity of HSL. Activation of HSL results from phosphorylation catalyzed by cyclic AMP-dependent protein kinase (Cordle et al., 1986) and deactivated by dephosphorylation with protein phosphatase 2C (Sztalryd and Kraemer, 1994). Egan et al. (1992) demonstrated that the lipolytic stimulation of adipocytes caused the translocation of HSL from the cytosol to the lipid droplet resulting in an increase in lipolysis. Catecholamines such as epinephrine and norepinephrine result in an almost immediate increase in plasma NEFA with a corresponding increase in plasma glycerol concentration. These compounds interact with the adipocytes BAR to increase the rate of lipolysis. Catecholamine stimulation of lipolysis can be decreased with both or- and B-adrenergic blocking agents (Vernon, 1980). Norepinephrine can be increased by stimulation of the sympathetic nervous system due to handling, injecting experimental animals, cold exposure, or hypoxia, and therefore, increase lipolysis. Insulin is the primary antilipolytic hormone in ruminants and causes a reduction in plasma NEFA and glycerol concentration within minutes after being administered (Vernon, 1980). Insulin not only decreases the rate of lipolysis, but also decreases the rate of FA release from AT by stimulating glucose uptake and FA re-esterification. The actions of insulin on lipolysis may act through a low-Km phosphodiesterase or by inhibiting adenylate cyclase activity (Vernon, 1980). Glucose decreases plasma glycerol concentration and glycerol entry rates in sheep when administered intravenously. This effect is primarily due to an increase in 37 insulin (Vernon, 1980). Unlike insulin and glucose, glucagon is a mild lipolytic stimulator (Vernon, 1980). Pothoven et al. (1975) found no correlation between basal and stimulated lipolysis. Rule et al, (1992) observed a 134% increase in basal glycerol release per mg protein with increased 8W of Holstein steers, from 277 to 528 kg. Because the main control of HSL activity is by phosphorylation/dephosphcrylation (Vernon, 1980), an increase in basal glycerol release without an increase in epinephrine- stimulated lipolysis suggests a change in enzyme activity without a Change in enzyme concentration. The same quantity of enzyme when maximally stimulated would have similar glycerol release rates. Greater lipolytic rates with changes in BW were observed in AT from perirenal (309%), omental (200%), and intermuscular (111%) depots than inner or outer backfat (Rule et al., 1992) without a change in stimulated lipolysis. Pothoven et al. (1975) observed an increase in basal glycerol release as crossbred steers increased in weight from 363 to 505 kg, but a decrease in epinephrine-stimulated lipolysis. Effect of somatotropin on lipolysis Hart et al. (1984) did not find an increased lipolytic activity in AT incubated with bST as measured by glycerol release per 4 big of tissue. Doris et al. (1996) found that basal glycerol release in AT was increased 59% in vitro with in vivo ST administration to sheep for 7 d. The AT from ST-treated sheep also responded to a greater extent to the B-agonist, isoproterenol, than AT from control sheep. Additionally, AT from ST-treated sheep responded less to the antilipolytic agent, N6- 38 phenylisoproyladenosine (PIA), an adenosine analog, than control AT (Doris et al., 1996). Doris et al. (1996) did not find an increase in glycerol response to a catecholamine with ST administration. An increase in glycerol release was not observed when sheep (Vernon, 1982; Hart et al., 1984) and swine (Walton and Ethertcn, 1986) AT were treated in vitro with ST. Additionally, Kramer et al. (1993) and Sinnett-Smith and Woolliams (1989) did not observe an increase in glycerol release from AT of pigs and sheep treated with ST in vivo. Dunshea et al. (1992a) suggested that the increase in lipolytic activity may in part be related to decreased insulin sensitivity, which would decrease the inhibition of lipolysis. Increased lipolytic activity may also be the result of a heightened sensitivity or responsiveness to lipolytic stimuli as suggested by Boisclair et al. (1997) and discussed previously. Through calculating grams of triglyceride mobilized per day, Dunshea et al. (1992b) determined a nearly 50% increase in lipid mobilization with pST (56 to 109 g of triglyceride mobilized per day). Although when compared to the reduction in lipid accretion ( > 200 gld), enhanced lipolysis would constitute approximately 25% of the reduction in daily lipid accretion, the balance being reduced lipogenesis. Peters (1986) observed an increase in plasma NEFA response area to a Single epinephrine challenge with ST administration, although Peters (1986) saw a greater response with feed restriction than with ST treatment. Likewise, Sechen et al. (1990) found that lactating cows receiving bST had greater response in circulating levels of NEFA at all doses of epinephrine, and an increase in the maximum response of plasma glycerol from epinephrine challenges. As discussed 39 earlier, glycerol response relates to lipolysis, whereas, NEFA alterations reflect mobilization (Sechen et al., 1990). Lanna et al. (1995) did not observe a increase in basal glycerol release in vitro, but an increase in HSL activity of AT was observed with bST administration to lactating cows (Lanna et al., 1995 and Liesman et al., 1995). Beta and alpha-2 adrenergic system. The second possible mechanism ST enhances lipolysis of AT is to increase the number of B-receptors as compared to or2 receptors. Beta receptors are mediated through Gs proteins and stimulate cyclic AMP through the adenylate cyclase pathway (Levitzki, 1988) and would stimulate lipolysis. Alpha-2 receptors inhibit cyclic AMP and therefore inhibit lipolysis (Levitzki, 1988). Watt et al. (1991) showed that the binding of B-receptors increased as compared to 012 receptors when sheep AT was incubated with ST. Roupas et al. (1991) suggests that if ST inhibits the Gi protein, then the Gs protein axis would allow greater activation and subsequent stimulation of lipolysis. Doris et al. (1994) demonstrated in rats that ST alters the amount of Gi2 proteins in rat AT, which inhibits the cyclic AMP signaling systems and inhibits HSL. Decreased suppression of HSL allows greater stimulation of the enzyme producing increased lipolysis. Doris et al. (1996) did not find altered quantities of the Gi protein, but found that ST alters the cyclic AMP-based signaling systems of AT by several mechanisms. The first, by increasing the maximum rate of B-adrenergic- stimulated lipolysis, due to an increase in the number of BAR; and the second by decreasing the inhibition of antilipolytic agents. 40 Inhibition of antilipolytic agents. Lipolysis might be enhanced by decreasing the response of AT to antilipolytic agents such as adenosine and proStaglandin E1 or the local production of the antilipolytic agent, prostaglandin E2 by the adipocyte (Doris et al., 1996). Adenosine’s actions on AT mimic those of insulin and include increased glucose uptake, inhibition of lipolysis and enhanced lipoprotein lipase activity (Carey, 1995). Extracellular adenosine in AT could originate as adenine nucleotides from purinergic neurons, as adenosine transported out of endothelial cells and adipocytes, or adenine nucleotides transported out of adipocytes (Carey, 1995). Doris et al. (1996) demonstrated that AT from bST-treated sheep responded less (a higher rate of lipolysis) to the adenosine analog PIA than control sheep. As with PIA, lipolysis in AT from bST-treated sheep was inhibited by prostaglandin E1 (PGE) to a lesser extent than AT from control sheep. (Doris et al., 1996). Additionally, PGE2 concentrations were lower in bST-treated sheep (Doris et al., 1996). Adipocytes release arachidonic acid when stimulated with catecholamines which can be used to synthesize PGE2 by the stromal-vascular cells of AT (Richelsen, 1992). Although there was an associated increase in plasma glycerol with the decrease in PGEZ, the cause and effect is not clear (Doris et al., 1996). It is clear that ST is a potent modifier of growth when given to feedlot cattle. In some circumstances ST increases ADG, decreases DMI and improves feed efficiency. Research has shown that ST can alter the composition of the carcass by decreasing the lipid content and increasing protein. Early et al., (1990a) suggested that ST should be given early in the feeding period and for a longer 41 period of time to observe its full potential and to reap the benefits of increased red meat yield. Previous research has demonstrated the effect ST has on lipid metabolism. The majority of the decrease is through a decrease in lipogenesis but enhancement of lipolysis is possible. The question remains, what portion of the reduced lipid accretion can be attributed to lipolysis and at what point does it occur. The two studies which follow will try to answer these questions. 42 Chapter 2 USE OF GROWTH PROMOTING SUBSTANCES FOR INCREASED SKELETAL AND LEAN TISSUE GROWTH OF HOLSTEIN STEERS1 Abstract A study was conducted to evaluate the effectiveness of long-term bovine somatotropin (bST) administration to growing-finishing Holstein steers at the beginning, and or during the entire feeding period on lean, skeletal, and carcass measurements. One hundred-sixty Holstein steer calves (185 kg) were blocked into four weight groups to determine the long-term (354 d) effect. Steers were randomly allocated to four treatments (10 steers/treatment) within a block. Treatments were control, no bST (0-0); bST d 0 to 182 (bST-0); bST d 183 to harvest (C-bST); and bST cl 0 to harvest (bST-bST). Steers received a s.c. injection of bST or placebo at 14-d intervals. DOses were 320 mg bST/injection from d 0 to 112 and 640 mg bST/injection from d 113 to harvest. The last treatment was administered 31 d before harvest Steers received a 14% CP diet from d 0 to 182 and 11.5% CF from d 183 to harvest consisting of dry, whole-shelled corn and a pelleted protein- mineral supplement Steers were harvested when 8W per block averaged 615 kg. During the first 182 d, bST-0 and bST-bST steers were heavier, with greater (P < .05) ADG, feed efficiency, hip height and hip height gain as compared with 0-0 and C-bST steers. Steers receiving bST during the first part of the study had lower (P < .10) carcass fat measurements but similar protein composition than control steers. From d 183 to harvest, cattle on all treatments had similar ADG, but C-bST steers 1This study was funded in part by Lilly Research Laboratories, Greenfield, IN and the Michigan Agricultural Experiment Station 43 had reduced (P < .05) daily DMI and greater feed efficiency than bST-C steers. Steers receiving bST (C-bST, bST-C, and bST-bST) had greater (P < .05) hip height and skeletal growfl'I than 0-0 steers. Noncarcass weight was increased and dressing percent reduced (P < .05) in C-bST and bST-bST steers as compared with C-0 and bST-C steers. Measurements of carcass fatness including quality grade was reduced (P < .05) the greatest in bST-bST steers as compared to 0-0 while bST-C and C-bST carcasses were intermediate. Steers receiving bST had greater (P < .05) carcass bone, protein and water composition and greater (P < .05) protein accretion than 0-0 steers. Bovine somatotropin was effective in reducing carcass fat and increasing edible lean. Administering bST to young, light-weight steers increased skeletal growth and noncarcass weight, but did not increase the total carcass weight and reduced the quality of the carcass. Key words: Somatotropin, Steers, Lean, Skeletal growth, Carcass measurements Introduction A greater awareness and demands by the consumer for healthier beef products has prompted the beef industry to wage a "war on fat". Emphasis on producing lean beef which maintains consumer acceptance is a key objective. Identification of production strategies which accomplish this goal has led researchers to investigate exogenous growth promotants such as somatotropin (ST). Research has documented ST ability to stimulate growth and reduce carcass lipid content in pigs (Krick et al. 1993), lambs (Butler-Hogg and Johnsson, 1987), 44 and mttle (Early et al., 1990a; Moseley et al., 1992; Preston et al., 1995; Rumsey et al., 1996). Previous results in beef cattle administered bovine ST (bST) have demonstrated greater increases in noncarcass weight than lean and skeletal tissue (Early et al., 1990a). Additionally, as cattle on high-concentrate diets increase in weight and age, carcass lipid mass increases at a greater rate than carcass protein (Owens et al., 1995). To alter carcass lipid and protein content, administration of bST to younger animals during peak periods of lean and skeletal tissue growth may increase the effectiveness of the exogenous hormone on economically important tissues. Therefore, a study was designed to evaluate the effectiveness of long-term bST administration to growing-finishing Holstein steers at the beginning, end or during the entire feeding period on lean, skeletal and organ growth; carcass characteristics and composition; serum lGF-l concentrations; and liver lGF-l mRNA abundance. Materials and Methods Animal Management. This study was completed under the approval of the Michigan State University All University Committee on Animal Use and Care (AUF # 10l90-308-02) and under an INAD (6673-0004 - Bovine Somatotropin) issued to Michigan State University. Sixty-four to 71 d prior to the start of the study, 243 Holstein steers (116 kg) were received from Indiana and Wisconsin at the Beef Cattle Teaching and Research Center at East Lansing, MI. Upon arrival, steers were placed in partially-covered pens (4.3 m x 11.9 m, 10 to 12 steers/pen) and fed 1.36 kg of dry, whole-shelled com, .91 kg medicated-pelleted supplement (Table 45 Table 2-1 - Guaranteed analysis and ingredients of medicated-supplement starter pellet‘” Nutrient Guaranteed analysis Crude protein, minimum 25.0% Crude fat, minimum 1.0% Crude fiber, maximum 9.0% Calcium, minimum 1.5% Calcium, maximum 2.5% Phosphorus, minimum 1.0% Salt (NaCl), minimum 2.0% Salt (NaCl), maximum 3.0% Vitamin A, minimum 110,000 lU/kg Vitamin D3, minimum 55,000 lU/kg Vitamin E, minimum 220 lU/kg 'lngredients: Processed grain by-products, soybean meal, meat and bone meal, linseed meal, sunflower meal, dehydrated alfalfa meal, ethoxyquin (a preservative), molasses products, monocalcium phosphate, dicalcium phosphate, calcium carbonate, sodium bicarbonate, salt, vitamin A acetate, D-activated animal sterol (source of vitamin D3), vitamin E supplement, riboflavin supplement, calcium pantothenate, niacin supplement, choline chloride, vitamin B12 supplement, ethylenediamine dihydriodide, menadione dimethylpyrimidinol bisulfite (source of vitamin K activity), manganous oxide, iron sulfate, copper sulfate, cobalt carbonate, magnesium oxide, zinc oxide, potassium chloride, and sodium selenite. ”Contains 2.2 g oxytetracyclinel kg of supplement. °Rebound® supplement, Kent Feeds, lnc., Muscatine, IA. 46 2-1), and had ad libitum access to second cutting alfalfa hay and water. Steers were ear-tagged, vaccinated against IBR, PI3, BVD, BRSV, and were mass medicated with 10 cc of an antibiotic mixture (225 ml long-acting penicillin, 225 ml of spectinomycin, and 50 ml of vitamin B complex). Animals with rectal temperatures of 40 °C or higher were treated for two additional days with the described antibiotic mixture. Two weeks after arrival, steers received a booster vaccination against IBR, Pl3, BVD, BRSV, and initial vaccinations against Hemophilus somnus and Clostn'dial organisms. Steers were mass medicated with oxytetracycline (10 cclsteer, 100 mg/ml oxytetracycline), checked for growth promotant implants, dehorned and castrated as needed . Thirty-four days prior to the initiation of the study, the medicated supplement was removed from the diet, and replaced by a 50% CP pellet (Table 2-2), and steers were given a 2 ml injection of Vitamin E and Selenium (Mu-SE®, Schering- Plough Animal Health, Kenilworth, NJ). The steers were fed a 14% CP diet consisting of 86% dry, whole-shelled corn and 14% pelleted supplement until the initiation of the study. One hundred and sixty eight steers (185 kg) were selected from the initial 243 animals based on breed characteristics, ADG, and health history. Eight steers were used as an initial harvest group to determine initial tissue weights (Table A- 1a) and body composition (Table A-1 b). The remaining 160 steers were divided into four weight blocks and randomly assigned to one of four treatments (10 steers-treatment"oblock"). The four treatments were: 47 Table 2-2. Guaranteed analysis and ingredients of pelleted supplementa Nutrient Guaranteed analysis Crude protein, minimum” 50.0% Crude fat, minimum .4% Crude fiber, maximum 4.0% Active drug ingredient Content Monensin sodium 330 mglkg Tylosin phosphate ~ 132 mglkg 'lngredients: soybean meal, calcium carbonate, urea, flash dried blood meal, corn distillers dried grains with solubles, salt, vitamin A acetate, D-activated animal sterol (source of vitamin D3), vitamin E supplement, ethylenediamine dihydriodide, manganous oxide, ferrous sulfate, copper sulfate, cobalt carbonate, zinc oxide, and sodium selenite. t’lncludes no more than 10.0% equivalent crude protein from non-protein nitrogen. 48 1) Control: no bST, d 0 to harvest (3-0 2) Control: d 0 to 182, bST: d 183 to harvest C-bST 3) bST: d 0 to 182, Control: d 183 to harvest bST-C 4) bST: d 0 to harvest bST-bST Steers were housed in partially covered pens (4.3 m x 11.9 m) with ad libitum access to feed and water. The diet consisted of dry, whole-shelled corn and a pelleted supplement (Table 2-2); and formulated to meet or exceed NRC (1984) requirements. During the first 182 d, steers received a 14% CP diet and a 11.5% CP diet from d 183 to harvest, based on component analyses. Two steers from each pen (32 steers total, 16 control, 16 bST treated) were randomly selected for harvest when treatments were Changed (intermediate harvest). These steers received their last treatment injection on d 168 and remained with their treatment group until d 199 when steers were transported to a commercial-harvest facility for processing. In order to address the objective of strategic timing of bST administration, 64 steers remained on their initial treatment after d 182, or were switched to the opposite treatment to provide the four different treatment groups as previously described. Steers were harvested when weight blocks were predicted to reach 615 kg and a 31 d withdrawal period from last treatment was observed. Blocks were harvested on d 322, 350, 364, and 377, with final weights of611, 609, 602, and 619 kg, respectively. The INAD required a 31 d withdrawal period from the last bST dose before cattle went to harvest. If a steer gained (ADG) less than 50% of its pen-mates for two consecutive weight periods (28 d), the steer was removed from the study. Thirty steers were removed from the study. These included: 22 for gaining 50 % less than their pen 49 mates, 3 for respiratory problems, 2 died, 1 was lame, 1 was not completely castrated, and 1 was incorrectly treated prior to harvest (see Appendix A-2). Preparation and administration of treatments. Bovine somatotropin (Lilly Research Laboratories, Greenfield, IN) and control treatments were preloaded in 10 cc syringes and frozen till used. The carrier agent was white wax. One hour before administration, the required number of bST and control treatments were thawed at room temperature. Steers received a s.c. bST or control (placebo) injection (16 gauge needle) at 14-d intervals beginning on d 0. From d 1 to 42, injections were administered in the mid-thoracic region just caudal to the shoulder, on alternating sides. From d 43 to harvest, treatments were given s.c. in the perirectal area. Dosages of bST administered were 320 mg bST/injection (22.9 mg/d, lot # 48631) from d 0 to 112 and 640 mg bST/injection (45.7 mg/d, lot # 48632) from d 113 to harvest. The change in dosage after 112 d were as directed by the sponsor and helped maintain the bST close relative to BW. Control steers received a similar volume of carrier agent as the bST treatment steers. Treatments were administered between 0730 and 1100. Feed/of performance. Initial, intermediate (d 182), and final weights were calculated as the average of weights taken on two consecutive days. Interval weights were determined every 14 d. Orts were weighed weekly to determine daily DMI. Feed efficiency was calculated as ADG (9) divided by daily DMI (kg/d) for a specific time period. Skeletal growth. Skeletal growth was assessed through hip-height measurements (Altitude Stick, NASCO, Fort Atkinson, WI) on d 0, and 1 (averaged 50 for initial hip height), 181, 183 (averaged for d 182 hip height), and on two consecutive days prior to harvest (averaged for final hip height). Skeletal growth (cm/d) was calculated for each period (d 0 to 182, d 183 to harvest, and d 0 to harvest) of the study. At harvest, the right front limb was separated from the carcass proximal to the carpo-metacarpal joint (knee), placed in a labeled-plastic bag and returned to the Michigan State University Meat Laboratory for dissection. The hide, tendons, ligaments, muscle, and fat were removed from the bones, and the metacarpal bone was separated from the carpus and phalanges. The metacarpus was frozen for future analyses. Third metacarpal bone length was determined and the bones were sectioned at their midpoint and measurements obtained according to Coble et al. (1971 ). Medial and lateral width and depth (Figure 2-1), and Circumference of the bone section were determined. Total cross-sectional area (T CA) and marrow cavity area (MCA) were determined using a compensating polar planimeter (Keuffel and Esser 00., Germany). Bone area was determined by difference (TCA - MCA). Bone volume was determined by water displacement and used to calculate bone density (glcc). Carcass measurements. At harvest, hot carcass (HCW), lungs, liver, spleen, heart, kidney, and semitendinosus muscle (STMUS) were weighed. Samples of the liver were collected from the last weight block (Block 1, lowest initial BW) to be harvested, frozen in liquid N for mRNA analysis and stored at -80 °C until analysis. After a 24—h, post-harvest chill; ribeye area (REA), 12th-rib backfat, kidney-pelvic- heart fat (KPH), marbling score, and USDA quality grade were determined. 51 Lateral Depth .) Medial Depth Medial \Mdth 6 Lateral WIdth .\\ \\ \ \ \ \ \\ \\\\\\\\\\\\\\\\\\\ t . \ \ \: - s \\ k‘ \~. \ \ \‘\ \ \ ////// TCA MCA Bone area Figure 2-1. Lateral and medial measurements of sectioned metacarpals, and total cross sectional area (TCA), marrow cavity area (MCA) and bone area. 52 Noncarcass weight (NCW), dressing percent (DP) and yield grade were calculated. Noncarcass weight was calculated as the difference between the final harvest weight and HCW. The 9th through 13th rib sections were removed from the left side of the carcass for compositional determination and transported to the Michigan State University Meats Laboratory. The 9-10-11 rib sections were separated from the larger rib portion (Hankins and Howe, 1946), weighed and the soft tissue was separated from bone. The bones and soft tissue from each rib were weighed. The soft tissue was ground three times (once coarsely, and twice finely) with mixing in between each grind to ensure uniformity. Approximately 500 g of sample was collected and placed into a Whirlpak bag (NASCO, Fort Atkinson, WI) and stored at -30 °C. The frozen sample was homogenized with liquid N in an industrial Waring blender. Tissue samples were analyzed for DM, CP and ether-extractable lipid (EEL). Tissue sample DM was determined in triplicate by placing 2 g of homogenized tissue in a desiccated-labeled aluminum pan (5 cm diameter) and dried for 48 h in a 55 °C oven. Tissue moisture was determined by difference. Ether-extractable lipid was determined in triplicate using samples previously dried. A cotton ball was placed in the center of each aluminum pan and the sides folded inward to prevent loss of sample and the cotton ball. The dried samples were placed in a Soxhalet apparatus and extracted with petroleum ether for 24 h. Ether- extractable lipid was calculated as the loss of weight during ether extraction. Crude protein was analyzed in duplicate for total-Kjeldahl N (CP = N * 6.25, AOAC, 1984) using a Technicon auto-analyzer system (Bran + Luebbe Inc., 53 Tanytovm, NY). Percent carcass bone, lipid, protein and moisture were calculated based on equations by Hankins and Howe (1946, Table A-10). Accretion rates of lipid and protein were determined from equations developed by Anderson et al. (1988) using initial harvest data and average treatment intermediate harvest data. Blood collection and Serum lGF-I assay. Blood samples were collected on d 0, 7, 14, 21, 28, and 35 at the beginning of the study and prior to final harvest 7, 14,21, and 28 d following the last treatment. To reduce influence of ST spikes on IGF-l concentrations, the evening prior to blood collection, steers were removed from feed for 16 h, and the following morning, fed .5 h prior to having blood collected by jugular venipuncture. Blocks (4 pens) were staggered every 1 h with feeding started at 0700. Blood was collected in a 10 ml, sterile blood-collection tube (Vacutainer Brand, Becton Dickinson and 00., Rutherford, NJ). Blood samples were allowed to coagulate at room temperature for .5 h and then stored at 4 °C overnight. Serum was harvested after centrifugation (959 x g, Beckman J2-21 refrigerated centrifuge, Beckman Instruments Inc. Palo Alto, CA) for 15 min at 5 °C and stored in 12 x 75 mm polypropylene tubes (Sarstedt, Newton, NC) at -20 °C until analyzed. Serum IGF-l concentrations were measured on 2 steers from each pen by radioimmunoassay (Shame et al., 1994) after removal of insulin-like growth factor binding proteins by formic acid-ethanol extraction (Bruce et al., 1991; Sharma etaL,1994) Liver IGF-l mRNA abundance. Liver lGF-I mRNA abundance was determined by Northem-blot hybridization as described by Sharma et al. (1994). Total liver RNA was isolated from 1 g of tissue using the RNA STAT-601M reagents 54 and procedure (Tel-Test “8”, Inc. Friendswood, TX). Concentration of single- stranded RNA was determined spectrophotometrically at a wavelength of 260 nm. Samples of total RNA (20 pg) were electrophoresed on agarose-formaldehyde gels, and the separated RNA bands were electrophoretically transferred to nylon membranes. Membranes containing liver RNA were hybridized with a 32P-labeled human lGF-I cDNA (Bell et al., 1984), and the abundance of mRNA for lGF-l was quantified by densitometric scanning of the resulting band. Liver mRNA abundance was expressed as arbitrary densitometric units. Statistical analysis. The study was designed as a completely randomized block. Feedlot performance (BW, ADG, DMI, and feed efficiency) was analyzed with the GLM procedure of SAS (1994) with block and treatment in the model and the pen as the experimental unit. Means for traits evaluating performance from d 0 to 182 were separated using a contrast statement to determine the difference between control (0-0 and C-bST) and bST (bST-C and bST-bST) treatments during the first part of the study. Performance trait means from d 183 to harvest, were separated using a Tukey’s test for all pair-wise comparisons and a contrast statement to determine the difference between control (0-0 and bST-C) and bST (C-bST and bST-bST) treatments during the last part of the study. Hip height, hip-height gain, metacarpal bone characteristics, carcass characteristics, carcass composition and tissue weights were analyzed with the GLM procedure of SAS (1994) with block and treatment in the model and the animal as the experimental unit. Least square treatment means were calculated and separated using the probability of difference function. Differences between control 55 and bST treatments for each part of the study were evaluated with contrast statements as before. Serum lGF-l concentrations were analyzed using the univariate repeated- measures analysis of SAS (1994) with block and treatment in the model and using the animal as the experimental unit. Treatment means within a day were separated by procedure outlined in Gill (1987). In the case of serum lGF-I concentrations from the last weight block to be harvested, concentrations were analyzed with only treatment in the model. Liver lGF-l mRNA abundance was analyzed with the GLM procedure of SAS (1994) with treatment in the model and least square means separated with the probability of difference function. One control steer, from the intermediate harvest group, was removed from the study because it received an incorrect treatment three days prior to harvest. All individual data was omitted. As discussed previously, an additional 30 steers were removed from the study and individual data omitted (see Table A-2). Raw data is provided in Tables A-3 to A-11. Results Serum IGF-I Concentrations. Steers receiving bST had 151% greater (P < .01) serum IGF-l concentrations from d 7 through d 35 of the study as compared to control steers (Figure 2-2). Following the final treatment injection, 31 d prior to harvest, C-bST and bST-bST steers had 298 and 223 % greater (P < .05) serum lGF-I concentrations 7 and 14 d following the last treatment, respectively, than C-0 56 1000 _ SED=40.1 g 800 c a a a —,-~ 600 a a 1‘5 -' 400 .. +V° 5 //IT 6 b o o 200 ¢ b ¢ ¢ (0 0 I T I I I I I 0 7 14 21 28 35 Days after initial bST treatment and bST-C steers (Figure 2-3). Twenty-one days following the last treatment, bST- Figure 2-2. Effect of treatment, control (0) or bST (I), on serum lGF-l concentrations after initial treatment on d 0 and subsequent treatments as indicated by arrows. “Means with unlike letters differ (P < .01). 1400 SED=91.9 3' E 1200 U) a 1000 It; 800 E 600 E 400 r3 200 I Days after final bST treatment Figure 2-3. Effect of treatment, C-0 (0), C-bST (o), bST-C (El), or bST-bST (I), on serum lGF-l concentrations after final treatment 31 d prior to harvest as indicated by the arrow. “Means with unlike letters differ (P < .05). 57 bST steers had 163 % greater (P < .05) serum lGF-l concentrations than C-C, bST- C, and C-bST steers. All steers had similar serum IGF-l concentrations 28 d following the last treatment injection. Feedlot performance. Steers receiving bST during the first 182 d of the study (bST-C and bST-bST) had 5% greater BW (P < .01) than control steers (0-0 and C-bST, Table 2-3). However, at the end of the experiment, steers had similar BW. Average daily gain during the first 182 d was 8.9% greater (P < .01) in steers receiving bST (bST-C and bST-bST) than control steers (0-0 and C-bST). Treatment had no effect on ADG during the latter part of the study or overall. Dry matter intake was similar among treatments for the first 182 d of the study. During the latter part of the study, C-bST steers had 9.4, 11.6, and 9.7% lower (P < .05) DMI compared with C-C, bST-C and bST-bST steers, respectively. Overall, C-bST steers had 7% lower (P < .01) DMI than 0-0 and bST-C steers. Steers receiving bST during the latter part of the study (C-bST and bST-bST) had lower DMI (P < .05) during that same period and over the entire study. Feed efficiency was 9% greater (P < .01) in bST treated steers (bST-C and bST-bST) during the first 182 d as compared to control steers (0-0 and C-bST). Steers receiving bST only during the latter part of the study (C-bST) had 25.6% greater (P < .05) feed efficiency during the latter part of the study and 6.8 % greater feed efficiency overall than bST-C steers. Steers receiving bST during the latter phase (C-bST and bST-bST) of the study had greater (P < .01) feed efficiency during the latter part of the study and over the entire study than C-0 and bST-C steers. 58 Table 2-3. Effect of bovine somatotropin on body weight, average daily gain, daily dry matter intake and feed efficiency Treatments Probability Trait C-C C-bST bST-C bST-bST SEM Model Contrast No. of 4 4 4 4 pens Body weight, kg Initial 185 185 185 185 1.5 .0001 .81 ' d 183 419 419 440 439 4.8 .0002 .002 a Final 599 608 611 622 6.3 .21 .13b Average daily gain, kg d 0 to d 1.29 1.29 1.41 1.40 .03 .08 .004 a 182 d 183 to 1.06 1.10 1.00 1.06 .03 .07 .11 1 Final d 0 to 1.17 1.20 1.21 1.24 .02 .14 .21 1’ Final Daily dry matter intake, kg d 0 to d 6.79 6.52 6.61 6.68 .09 .0005 .93 1 182 d 183 to 8.29‘" 7.51 " 8.50 ‘" 8.32 ‘1 .15 .01 .01 1’ Final d 0 to 7.54w 7.02 " 7.56 ‘” 750"" .11 .01 .03 1’ Final Feed efficiency, 9 gain/kg DMI d 0 to d 190 198 214 209 3.4 .0006 .001 a 182 d183 to 128'“ 1471’ 117" 128““ 4.6 .01 .0091’ Final d 0 to 156 "’ 1711 160‘“ 165 "V 3.0 .01 .007 1’ Final 'C-0 and C-bST versus bST-C and bST-bST. I’C-C and bST-C versus C-bST and bST-bST. “Means with unlike superscript letters differ (P < .05). 59 Skeletal growth. On d 182, hip height was increased 1.2% (P < .01) in steers receiving bST during the first part of the study (bST-C and bST-bST) as compared to control steers (0-0 and C-bST, Table 2-4). Steers receiving bST during the entire study (bST-bST) had 3.3, 1.4, and 2.2% greater (P < .05) hip heights at harvest than C-C, C-bST, and bST-C, respectively. Steers receiving bST during only one part of the study, C-bST and bST-C, had 1.8 and 1.1% greater (P < .05) hip heights at harvest than 0-0 steers, respectively. At harvest, steers receiving bST during the latter part of the study (C-bST and bST-bST) had 8.8% greater (P < .01) hip heights than control steers (C-0 and bST-C). Daily hip height gain was increased (P < .01) 8% in steers receiving bST during the first 182 d (bST-C and bST-bST) than control steers (0-0 and C-bST). Steers receiving bST during the latter part of the study (C-bST and bST-bST) had 66% greater (P < .01) daily hip height gain from d 182 to harvest than 0-0 and bST-C steers. Overall (d 0 to harvest), bST-bST steers had 15, 7, and 10% greater (P < .05) daily hip height gain than 0-0, C-bST and bST-C steers, respectively. Steers receiving bST during one part of the study (C-bST and bST-C) had 8 and 5% greater (P < .05) daily hip height gain than C-C steers. Intermediate-harvest metacarpal bone characteristics were similar for all treatments (Table 2-5). Steers receiving bST throughout the entire experiment had greater (P < .05) lateral bone depth than bST-C steers at harvest. Bone area tended to be 4% greater (P < .10) in bST-bST steers than C-C steers. Bones of 0-0 steers were 3% denser (P < .05) than C-bST steers. (Table 2-6). At harvest, steers receiving bST during the latter part of the study (C-bST and bST-bST) 60 .30. v 108:6 28am. 35233 95:: 53> 38.22; Hmnfimn can .590 0.329, oihwn new 0.0.. 0.09-th new 0-...09 329 .590 new 00.. .mcmoE Boson ammo... o 58. 68. E. s 8. 3. x 8. S. x 3. S. s 2.. as“. - o o a 38. 58. B. s :- 8. x em. 8. s we. 8. x em. as“. - a2 a a 88. 38. mo. of no. 3.. 8. em. 8. on. «8. o 6 EE .58 £99. a: son a 88. 8o. R. s N. a: o. x mam. o. a 32 e. s 32 as“. 3 5o. :5. m. 32 m. on? m. «N? m. tum. we. a a 5.. 38. e. 32 e. 32 e. 32 e. 4.8. o a so .299. 9... 8288 .882 mm 56-56 mm 0.5.. mm 590 mm 0.0 go: £52.26 8558: .Emm 306ch 3% new £99.. 9: :0 53:22.8 0:33 00 “norm TN ofimh 61 Table 2-5. Effect of bovine somatotropin on intermediate-harvest metacarpal bone characteristics ‘ Treatments Probbility Control SE bST SE Model Contrast” Trait (C-C, C-bST) (bST-C, bST-bST) Length, cm 21.8 .1 21.8 .14 .28 .89 Medial width, mm 18.6 .6 18.9 .6 .63 .77 Lateral width, mm 35.1 .6 36.0 .6 .28 .30 Medial depth, mm 14.0 .4 13.7 .4 .54 .67 Lateral depth, mm 26.1 .5 25.7 .4 .59 .55 Circumference, 108.5 1 .5 1 10.1 1 .5 .22 .45 mm TCA, cm 1° 8.3 .3 8.3 .3 .63 .94 MCA, cm 2-1' 2.9 .2 2.9 .2 .90 .90 Bone area, cm 2 5.4 .1 5.4 .1 .32 .75 TCA/MCA 2.9 .1 2.9 .1 .98 .99 Density, glcc 2.0 .02 2.0 .02 .98 .58 'Least square means. bProbability of difference: control versus bST. cTotal cross-sectional area of metacarpal bone. clMarrow cavity area. 62 N. 38. No. .2 as: No. .9 was No. a N3 No. 3 8.. 85 .2228 r9 3. F. on F. 3 a. N...” a. N...” 5.228. NF. 8. a. .3 C. see a. see a. .. to N :8 see 88 8. 8. e. on a. N a. E” .. aN ea :6 .52 8. 8. N. we N. no N. ea N. 3 4N :6 doe 8. em. 3 me: o. No: a. No: a. we: EE .ooeeseaoeo 8. 8. n. :08 n. x 2N a. s,oeN N. SEN EE .5666 .223 9.. N. n. 4.: a. 5.2 n. 3; m. 92 EE .5666 .682 SN. 3.. e. 4.8 e. we.” e. New 4. one as .59; .293 am. 8. e. 2: e. 2: e. 4.2 e. 2: SE .56.; .6622 me. 8. NF. ENN Ne. BNN :. mmNN :. SNN :6 .523 3.8580 .882 mm 5956 mm 0-5.. mm 56-0 mm 0.0 to: £32.90 £5.53; 82.988920 econ .maemofloe “momemc: co cfiomeEom oc_>on Co Bdrm .0.N 2an 63 .30. v 10.8% Bozo. 3.5933 95:: 53> mcmosrs .6 F. v n: Leta. Bozo. 5:04:33 95:: £5, mcmozé 62m 338 3952.3 .ocon 69899: Co noun 6553888 .988 #09..th pcm 0.09-0 389 0909 cam 0-0 60:995.. 00 2:538} .mcmoE ocmzcm “one... APEOOV .miN 03m... tended to have greater lateral bone width (P < .10) lateral bone depth (P < .10), bone circumference (P < .01), and TCA (P < .10) than control steers (C-0 and bST- C). Carcass characteristics, composition and organ weights. After 182 d, harvest weight, HCW, NCW, DP, measures of REA or backfat were similar among treatments as determined in the intermediate harvest group (d 199, Table 2-7). Steers receiving bST for 182 d had 30% lower (P < .05) KPH, 10% lower (P < .10) marbling score, 6% lower (P < .05) USDA quality grade, and 12% lower (P < .05) calculated yield grade than control steers. At final harvest, C-bST and bST-bST had 5.5% greater (P < .01) NCW, and 3% lower (P < .01) DP (Table 2-8). Steers receiving bST only during the latter part of the study (C-bST) tended to have greater carcass (P < .10) REA expressed as cmzlkg HCW than bST-C steers at the final harvest. Backfat thickness at final harvest was 24% lower (P < .05) in bST-bST carcass than 0-0 and bST-C carcasses. Steers receiving bST during the latter part of the study (C-bST and bST-bST) had 40% less KPH than C-0 and bST-C carcasses. Marbling score decreased (P < .05) 21, 19, and 31% in C-bST, bST-C, and bST-bST carcasses, respectively, as compared to 0-0 carcasses. Steers receiving bST throughout the study (bST-bST) had 13% lower (P < .05) marbling scores than steers receiving bST during only one part of the study (C-bST and bST-C). Quality grade was decreased (P < .05) 7, 7 and 12% in C-bST, bST-C, and bST-bST carcasses, respectively, as compared to C-0 carcasses. Steers receiving bST throughout the study (bST-bST) had 5% lower (P < .05) quality grade than C-bST and bST-C. 65 Table 2-7. Effect of bovine somatotropin on intermediate-harvest carcass characteristics "1 a Treatments Probability Control SE bST SE Model Contrast ° Trait (C-C, C-bST) (bST-C, bST-bST) Number of 15 16 steers Harvest wt., kg 439 5 448 5 .0001 .20 HCW, kg 247 4 251 3 .0006 .45 NCW, kg 192 3 197 3 .001 .15 DP, % 56.3 .4 55.9 .4 .57 .50 Ribeye area, 64.9 1.3 66.4 1.2 .84 .41 cm2 Ribeye area, .263 .005 .266 .004 .03 .73 cmzlkg HCW Backfat, mm 3.4 .3 3.0 .3 .3 .37 KPH, % 3.4 .16 2.4 .15 .004 .001 Marbling 461 1 8 41 3 17 .26 06 score“ Quality grade' 16.9 .3 15.9 .3 .21 .04 % Choice 26.7 12.5 % Select 60.0 43.8 % Standard 13.3 43.8 Calculated 2.4 .08 2.1 .08 .04 .02 yield grade ‘Least square means. bd 199 of study, steers received last treatment on d 168. 1'Probability of 0-0 and C-bST versus bST-C and bST-bST. 1‘400 = slight, 500 = small. '15 = high standard, 16 = low select, 17 = high select. 66 25.0 283 8. 58. a. .mN a. .. as a. me a. son 88.8.8 3 o 3 o 9886 as N8 oeN e8 3 828 3... RR oNN 4.8 o.8 865 s o o o see 286 s 8. 38. N. 3.2 N. s.c.: N. in: N. 1.2 e688 £80 8. S8. 8 I Be 2 x 8m 2 x 2m 2 s so soon 8:26.: 8. 38. F. x 3 a. s we a. x eN a. en.» 3.. in: B. 8. No. a 8.8 E. :86 8. .2 88 3. :88 55 586.8 26: 95 :6 8. m a. .8. ._._ m N. +8. .. 8N. e8. EN. :8. e. a .N. {mm No. 8. 3 QB 3 at 3 n8 .3 N8 N :8 {mm 8. E8. 5. 8.3 5. 2.8 8. 2.8 3. .38 as .60 :8. 88. es 3 N8 no .. 8N m...” s8N me XSN 9. .302 R. 8. we con 3. N8 3 m8 3 8N 9. .26: N 8 .8 8 ease .6 62 38880 .882 mm 538 mm 0-5.. mm Boo mm 0.0 8:. $582.... 8888: amozmtofimemco mmmocmo “mozmclficc co E958: Co Dorm .miw 2an 67 .60. v n: 00:6 80:0. 5:00.003 05E: 53> 0:00.25? .8 _.. v n: .050 2020. 090080030 95:: 53> acme—2.... 006:0 008020 .I- 0.. 00.050 30. u 0.. 000.00 :9: u t. .0200 30. 0 0: 99685 u 02 .888. u 08 .__oEa .i- 08 .088 u 8s. 0.8-98 ocn 56-0 .9, 0-56 98 0-0 ”enscoo Co 38828.. .wcmmE Ohm—40m «mel—a 6.880 .3 some 68 Calculated yield grade of C-bST and bST-bST carcasses were 16.7% lower (P < .05) than C-0 and bST-C carcasses. Semitendinosus muscle, liver, lung, and spleen weights did not differ between treatments of the intermediate-harvest group (Table 2-9). Steers receiving bST during the first part of the study had smaller hearts relative to BW than control steers, but bST steers tended to have larger (P < .10) kidney weights on an absolute (9.2%) and relative (7.1%) basis than control steers. At the conclusion of the study, C-bST and bST-bST steers had 6.9% greater (P < .05) STMUS than C-C steers (Table 2-10). Relative to BW, C-bST steers had 5.2 % greater (P < .05) STMUS than 0-0 steers. Relative to HCW, bST-C and bST-bST steers had 7.8 and 6.3% greater (P < .05) STMUS than 0-0 steers. Liver weights of bST-bST steers were 7.5% greater (P < .05) than C-C steers. Steers receiving bST throughout the entire study (bST-bST) had 16.3, 12.5, and 11.1% greater (P < .05) spleen weight than 0-0, C-bST, and bST-C steers, respectively. When expressed on a carcass weight basis, bST-bST steers had 11.4 and 9.6% greater spleen weights than 0-0 and C-bST steers. The bST-bST steers had 23, 10.4, and 12.6% greater (P < .05) kidney weight than 0-0 and C-bST and bST-C steers. Absolute kidney weights of C-bST steers were 11.3% greater (P < .05) than 0-0 steers. Relative to BW, bST-bST steers had 18.9 and 9.6% greater (P < .05) kidney weight than 0-0 and bST-C steers. Carcass bone content of the intermediate-harvest group was similar among treatments (Table 2-11). Lipid content of bST carcasses were 11% less 69 Table 2-9. Effect of bovine somatotropin on intermediate-harvest semitendinosus muscle and internal organ weights"b Treatments Probability Basis Control SE bST SE Model Contrast ° (C-C, C-bST) (bST-C, bST-bST) Semitendinosus muscle wt. 9 1610 44 1626 42 .16 .79 glkg 8W 3.66 .08 3.63 .08 .28 .75 glkg 6.51 .13 6.48 .12 .31 .89 HCW Liver wt. 9 5918 1 89 6034 182 .86 .66 glkg 8W 13.54 .45 1 3.49 .44 .28 .94 Lung wt 9 6000 179 5775 173 .80 .37 glkg 8W 13.73 .40 12.92 .39 .08 .16 Spleen wt. 9 929 45 101 1 43 .36 .20 glkg 8W 2.11 .10 2.26 .09 .64 .31 Heart wt. 9 1653 .03 1550 45 .12 .13 glkg 8W 3.78 .10 3.46 .10 .08 .03 lfidney wt. 9 1 1 72 35 1280 35 .28 .04 glkg 8W 2.67 .08 2.86 .08 .16 .08 " Least square means. 1’0 199 of study, steers received last treatment on d 168. 1for contrast Control versus bST. 70 2. :. B. a mod 8. s 2: 8. x 3.. 8. x 43 26 9.3 3. 8. 2. a £3 3 x3: 3 x mm: 8 x 82 a is .cooaw 8. R. cm. 8. 3 mm. 5.3 mu. 3.3 mm. 8. 5 3m 9% 3.. B. «3 82 3 83 N: 83 4: 82 a is .95.. cm. a. R. S. : a. 2. : SN. 3.. t «N. 6.: 3m 9.3 2. mm. X: :8: a. 59...: mm. .28: E. 788 m .E .32.. m8. 8. NP. 32.8 E. .s 68 NF. :38 Q. 33 26: 9% 1. t. B. :8...” 8. sum...” 3. 38.4 3. x8.” 25 9.3 3. 8. mm :0ch 3 .288 E :23 5 Bow a is .282: 88:55:68 .. $228 .282 mm 55-5.. mm 0-5.. mm 590 mm 0.0 $8 2.532“. 355.8: _. 35995 50.6 .mEEE new 2838 33:65:83 69.242: co EmEHmo: Co .035 .0 TN 2an 71 .80. v n: .wtfi 82.0. Etomcmazm 93:: 53> 33.22; Fwnfimn ucm hwnd 389 9.59 ocm 0-0 “mmzcoo ho b___nmno.nr .mcmmE 990m “moo... N8. 88. B. a 8d 8. x o 3 8. .2 EN 8. x SN 26 98 :8. 38. we :83 3 a. N8. 3 x 5.2 we 28 a .8 $83. 8. 8. 8. 3m 8. 8.8 8. 8.» 8. 8.” 26 98 8. 8. 3.. «Na om 85 8 £8 8 88 a is £81 a $2.80 .082 mm .538 mm 0-2.8 mm 590 mm 0.0 88 3:52.05 mace—Ewe; 8.38v .03 928 72 Table 2-11. Effect of bovine somatotropin on intermediate-harvest carcass composition and tissue accretion rates'-b Treatments Probability Trait Control SE bST SE Model Contrast ° (C-C, C-bST) (bST-C, bST-bST) Carcass composition, % Bone 16.7 .3 17.4 .3 .41 .12 Protein 14.2 .2 14.3 .2 .71 .69 Lipid 21.2 .8 18.7 .8 .20 .03 Water 48.8 .5 50.1 .5 .14 .07 Protein accretion rates, g/d d 0 to 199 85.7 3.7 90.6 3.5 .65 .34 Lipid accretion rates, g/d d 0to199 220.9 10.3 193.8 9.9 .17 .07 'Least square means. bd 199 of study, steers received last treatment on d 168. °Probabi|ity of contrast Control versus bST. 73 (P < .05) and water content 4% greater (P < .10) than control carcasses. Carcass protein accretion rates were similar among treatments, but lipid accretion of bST carcasses from d 0 to 199 tended to be 11% lower (P < .10) than in control steers. Steers receiving bST throughout the entire experiment had the greatest increase (P < .05) in carcass bone, protein, and water composition and the greatest decrease (P < .05) in carcass lipid relative to CC carcasses (Table 2-12). Steers which received bST during only one part of the study (C-bST and bST-C) had greater (P < .05) carcass bone, protein, and water; and lower (P < .05) lipid content than C-C steers, but not to the extent of the bST-bST treatment. Steers receiving bST only during the latter part of the study had greater bone and less lipid than bST-C steers. Steers receiving bST during the latter part of the study (C-bST and bST-bST) had 18.6 and 14.3% greater (P < .05) protein accretion from d 199 to harvest than C-C steers. Overall, bST-bST had the greatest protein accretion, followed by bST- C steers, C-bST steers and C-C steers. Lipid accretion was reduced (P < .05) 39.8% for steers receiving bST during the latter portion of the study as compared to C-C and bST-C steers from d 199 to harvest. Overall, bST-bST had the lowest lipid accretion (P < .05) followed by C-bST, bST-C, and C-C steers. Abundance of IGF-I mRNA in the liver was similar for all treatments following final harvest of Block 1 steers (Figure 2-4, top panel). Serum lGF-l concentrations were 310% greater (P < .05) in C-bST and bST-bST steers 7 d after final treatment as compare to C-C and bST-C steers (Figure 2-4, bottom panel). Fourteen days following final treatment, bST-bST and C-bST steers had 268% and 74 .329. _.oo. 58. mo ”moor mo xmoNN No QooN mo zoomN 0. o o ”.322 Foo. Fooo. 53 Limo? v.3. aoNoN N.3 xmoow v.3. aoNom 0. mm: u 2o .35.. 5:9on 2a... .3sz moo. mooo. 7N zoom fiN Sooo FN onm FN Boo 0. o u 0.83me moo. moo. Nd zNooF we Soda o.v 18.5.. I. Loo 0. mm? o 2o .36. cozmbom 520$ roo. Pooo. v. 303 v. xvsv v. xmov v. 3.3 563 5o. ..ooo. m. N foN m. xmdN m. >o.NN m. .5me Eu... Foo. 58. r. 3 5: F. x N: r. 3.3. F. .99 529.”. Foo. Fooo. N. :99 N. 11.0.. N. 1.9 N. No.3 9.0m .x. 62.6868 $850 8.8.580 .322 mm .52-th mm 0...? mm .590 mm 0-0 :9... 5:59.95. 3:958; .36. coombom comm: ncm 3:62.88 3850 6822.3: co .cmEumo: .o .025 .N..-N flow... 75 .8. v 1.55.8 92.0. 5.8833 95:: 5.2, 28.5.8.3 .co.._manoo mm? o 238.8 2 new: 28> «.029 .5589. .3ch ofifimEEE. momLm> 300 d). The lack of response in ADG during the latter part of the study may be a function of dietary CP content. Rumsey et al. (1996), Early et al. (1990a), Moseley et al. (1992), and Preston et al. (1995) used diets containing 21, 16.7, 16.5 and 13.9% CF, respectively. These values are considerably higher than the 11.5% used by the author during the latter part of this study. The CP content used by the author was sufficient to allow .85 to 1.37 kg of ADG based on the low and high treatment DMI, respectively (NRC, 1984). The compounding effect of reduced intakes associated with bST is discussed later. 79 The effect of bST on DMI is not consistent among previous studies. Rumsey et al. (1996) observed an increase in DMI with bST administration, Preston et al. (1995) and Moseley et al. (1992) reported a decrease with increasing levels of bST, and Early et al. (1990a) saw no effect. McBride and Moseley (1991) suggest that in animals which may have deposited fat, the administration of bST increases the amount of circulating nutrients which can not be fully utilized by the animal, therefore, the animal reduces its DMI to compensate. Conversely, animals which are growing rapidly and have little body fat may need to increase intake to satisfy nutrient demand as a result of bST administration. Rumsey et al. (1996) started with light-weight cattle (182 kg), whereas, Preston et al. (1995) and Moseley et al. (1992) used cattle that were heavier (378 kg and 393 kg, respectively), and Early et al. (1990a) used cattle which were intermediate (229 kg) in size. In the present study, DMI intake was not affected by bST treatment during the initial 182 d of the study. The largest decrease in intake was observed in C-bST steers which only received bST during the latter part of the study. These steers had greater body fat than the bST-bST and bST-C steers after 182 d of bST administration, and therefore, would fit the theory suggested by McBride and Moseley (1991). As discussed previously, the lower CP concentration of the diet and reduced DMI associated with bST treatment may have prevented expression of increased gain with bST administration. Research in pigs have shown that the supplementation of additional lysine and CP allows greater bST-mediated gains (Krick et al., 1993). Additionally, there is evidence that cattle implanted with anabolics have greater potential for growth if fed diets higher in CF content (Bartle 80 and Preston, 1994). Either by increasing ADG, decreasing DMI, or both, feed efficiency increases with bST administration. The present study demonstrated an increase in efficiency during the latter part of the study which is in agreement with Preston et al. (1995), Rumsey et al. (1996), and Early et al. (1990a). Moseley et al. (1991) saw an increase in feed efficiency with increasing bST concentration up to 100 pg bST/kg BW. At 300 pg bST/kg BW, ADG was reduced so drastically in relation to DMI, that efficiency decreased. Skeletal and bone growth. Although hip height increased 5 cm (3.3%) from C-C to bST-bST steers, increased length of the metacarpal (front limb bone) was not apparent. Loy et al. (1988) also observed an increase in hip height in estrogenic-implanted steers over controls steers without a corresponding increase in metacarpal length. Early et al. ( 1990b) observed a 1.3 cm increase in humerus length in steers administered bST, but not in the length of the femur, tibia or radius. The question then arises whether there was differential bone growth rates between the front and rear limbs; do non-significant increases in length occur in multiple bones with an additive effect, or do changes in the angle of joints occur to increase hip height? Bovine ST administration increased lateral width, lateral depth, circumference, and bone surface area of the metacarpal bone; and the percent bone in the carcass. This is in agreement with research in ewe lambs receiving bST (Johnsson et al., 1985). In contrast to the present study, Johnsson et al. (1985) did not see a change in bone density. Johnsson et al. ( 1985) observed an 81 increase in bone circumference of the femur and humerus. Daily growth in circumference was also observed in the femur of bST-treated steers (Early et al., 1990b). In the current study, increased skeletal growth was evident after 182 d of bST administration by greater hip height, although no other measure were affected at that time. The proximal epiphysis of cattle ossifies when cattle are 18-24 months of age (Emara, 1937 and Sisson, 1953 as referenced by Grant et al., 1972). Therefore, bST should have been able to stimulate long-bone growth throughout the study. This is supported by the fact that the bST-bST steers had the greatest hip height for the entire experiment, bST-C and C-bST steers were intermediate, and C-C has the shortest hip height. There was no difference in daily hip height gain between C-bST and bST-bST during the latter part of the study, suggesting that bST continued to have the ability to stimulate long-bone growth. It is obvious that long- bone growth decreased by two-thirds from the first part of the study to the second, which is in agreement with the sigmoidal growth curve of meat animals in which bone matures first (Boggs and Merkel, 1993). Using data from both the intermediate and final harvest groups, hip height was positively correlated with metacarpal length (r = .53, P < .01), lateral width (r .50, P < .01), lateral depth ( r = .51, P < .01), TCA (r = .53, P < .01), and MCA (r .23, P < .01). Metacarpal length was negatively correlated with bone density (r = -.28 P < .002), positively correlated with lateral width (r = .28 P < .002) lateral depth (r = .32 P < .001), bone circumference (r = .32 P < .001), TCA (r = .36, P < .001), and bone area (r = .38, P < .001). 82 Carcass measurements. Moseley et al. (1992) observed a significant linear decrease in HCW with increasing amount of bST administration (0 to 300 pg bST/kg BW). Preston et al. (1995) found no affect of bST on HCW, but Rumsey et al. (1996) found an increase in HCW with bST treatment. Moseley et al. (1992) and Early et al. (1990a) found a decrease in DP with bST administration. The present study did not see an affect on HCW but did see a decrease in DP. The cause for the decrease in DP was a result of an increase in NCW at final harvest. Early et al. (1990b) attributed 75% of the increase in NCW to an increased weight of the gastrointestinal tract (GIT), but this was not evident in Rumsey et al. (1996) data and was not measured in the present study. Therefore most of the weight gained as a result of bST treatment would end up in the offal bin and not on the rail. Early et al. (1990a) theorized that by administering bST to younger, light- weight cattle, the carcass component could be increased to a greater extent than the noncarcass components. The present study started with cattle that were 45 kg lighter and used a higher dose of bST throughout the study than Early et al. (1990a). Not only did the present study not see an increase in the carcass component as compared to NCW, but NCW was increased to a greater extent than that observed by Early et al. (1990a) which may be the result of harvesting Holstein steers 226 kg heavier than the Herefords used by Early et al. (1990a). Ribeye area was only greater in C-bST steers as compared with bST-C steers, and there was not difference between control and bST-treated steers. Preston et al (1995) and Peters (1986) did not see an increase in REA with bST administration, but Moseley et al (1996) observed a 4 to 8% increase with bST 83 doses of 8.25 to 300 pg bST/kg BW. As discussed, bST was a potent modifier of carcass fat. After 182 d of treatment, bST reduced KPH fat percent, marbling score, USDA quality grade, and the number of carcasses grading USDA choice. At the conclusion of the study, backfat was also reduced. Peters (1986) observed a 24% reduction in backfat with bST administration following 29 d of treatment. Moseley et al. (1992) saw a 9 to 62% reduction in backfat and a 17 to 95% reduction in the percent of carcasses grading USDA choice. The 95% reduction was in steers receiving 300 pg bST/kg BW which failed to reach market weight. At 100 pg bST/kg BW, 70% fewer carcasses graded USDA choice. Preston et al. (1995) documented a linear decrease in backfat (15%), KPH fat (28%), marbling score (6%), and percent USDA choice (58%) with daily treatments of 0, 25 and 51 pg bST/kg BW. However, Early et al. (1990a) did not see a difference in backfat or Canadian carcass grade in bST- treated cattle. As fat decreased, red meat yield increased in the present study as evidenced by a reduction in yield grade. This agrees with Preston et al. (1995) in which bST reduced yield grade scores by 12.5%. In this experiment, long-term bST administration increased the weight of STMUS both in absolute and relative terms, but did not increase REA, as discussed. Early et al. (1990b) and Eisemann et al. (1989) did not see a significant effect of bST on the STMUS. Early et al. (1990b) demonstrated that bST increased vastus Iateralis daily muscle growth 42%. Eisemann et al. (1989) observe an 11% increase in longissimus weight but not relative to empty BW. In regards to muscle groups (primal cuts of the carcass), Early et al. (1990b) observed an increase in the 84 flank and shank primals, and hypothesized that the flank increased to accommodate the larger GIT in bST animals. Additionally, Butler-Hogg and Johnsson (1982) observed greater muscle weight in ewe lambs treated with bST. Beermann et al. (1990) showed an increase in STMUS with porcine ST administration up to 120 pg pST/kg BW which was due to individual muscle fiber hypertrophy. Noncarcass weight was increased with long-term bST administration. A portion of this increase occurred in the internal organs. In this study, the liver, spleen, and kidney were increased in absolute values and the spleen and kidney increased in relative value with bST administration. This agrees with Moseley et al. (1992), Early et al. (1990b) and Rumsey et al. (1996), who reported increased liver and kidney weights with bST treatment. Rumsey et al. (1996) observed an increase in spleen and heart weight, but Early et al. (1990b) did not. Early et al. (1990b), however, observed an increase in lung and trachea weight with bST administration. Early et al. (1990c) observed a greater whole-body protein accretion with bST administration, but this was due to greater noncarcass protein accretion and not that of the carcass. In another study, protein accretion was increased 22% with bST administration in Angus-Hereford crossbred steers (Rumsey et al., 1996). The lower dietary CP in the present study may not have allowed the greatest nutrient repartioning to take place and limited protein gain and total ADG as discussed. Somatotropin decreased lipid accretion to a greater extent when lipid accumulation was the greatest (12.5% from d 0 to 199 and 43% from d 199 to harvest). Rumsey et al. (1996) demonstrated a 23.5% decrease in lipid accretion 85 after 56 d of bST administration in light-weight cattle (182 kg initial weight, and 266 kg average final weight.) with a 100 pg bST/kg BW dose. Even though bST does decrease lipid accretion, it does not prevent it entirely. Liver lGF-I mRNA concentration. No differences were found in liver lGF-l mRNA at harvest. Insulin-like growth factor-I mRNA abundance has been shown to increase in the porcine liver (Grant et al. 1991; Coleman et al., 1994; Ramsay et al., 1995), adipose tissue (Coleman et al., 1994; Ramsay et al., 1995), and to a smaller extent in the vastus Iateralis muscle (Ramsay et al., 1995) of pigs following pST treatment. Brameld et al. (1996) observed an increase in lGF-l mRNA in STMUS of pST-treated pigs, whereas Ramsay et al., (1995) found no response. Both Coleman et al. (1994) and Ramsay et al. (1995) observed no increase in lGF-I mRNA in the longissimus muscle of pST-treated pigs. Additionally, Johnson et al. (1998) demonstrated a 150% increase in liver lGF-I mRNA in wether lambs 24 days after implantation of a combined trenbolone acetate-estradiol implant with a concomitant increase in blood lGF-l concentrations. In the present study, serum lGF-l concentrations were similar to control steers 28 d post-administration of bST and would explain the lack of difference in mRNA abundance. The abundance of IGF-I mRNA decreases rapidly after reaching its peak stimulation by ST. Matthews et al. (1986) and lsgaard et al. (1989) demonstrated the rise to peak quantities and a return to pretreatment values of lGF-I mRNA abundance in the liver and gastrocnemius muscle, respectively, within 12 h after a single ST injection in mice. Carcass Value. On an economic basis, the goal of using bST is to produce greater quantities of red meat without sacrificing the quality of the product which 86 would make it unacceptable to the consumer. Using an average grid pricing system (USDA, 1998) based on quality grade, yield grade and HCW; Table 2-13 demonstrates the premiums and discounts using average treatment values. All carcasses fell within the acceptable HCW range (250 to 431 kg, 550 to 950 lb). Although C-bST and bST-bST carcasses had lower yield grades and therefore greater cutability, the added value of the increased lean did not compensate for the reduced quality grade, and would be valued at $41.64 less than C-C carcasses. Carcasses from bST-C steers are not only discounted for quality grade, but also are discounted for yield grade and, therefore, had $50.26 lower carcass value than C-C carcasses. Some of the loss in carcass value could be offset by the lowered DMI and reduced feed cost, but handling charges (every two weeks) or cost of the product would need to be accounted for as well. A leaner carcass that contains an acceptable quality is difficult to produce especially when marbling is the last fat to be deposited and its during this period that bST has its greatest negative influence on fat accretion. Before the use of bST is looked at seriously, the premium between yield grades 2 and 3 needs to be increased to be adequately rewarded for production of lean meat. Implications Administering bovine somatotropin to young, light-weight Holstein steers did increase skeletal growth and reduce carcass fat content, thereby producing a leaner product. Somatotropin increased weight of noncarcass components 87 Table 2-13. Effect of treatment on carcass premium, discounts, and value“-b Treatments Criteria c-c C-bST bST-C bST-bST Carcass Value Adjustments, $lkg Quality grade6 0 -.14 -.14 -.14 Yield grade ‘ -.002 .022 -.002 .022 Total -.002 -.1 18 -.142 -.1 18 Carcass Value, $- Deviation from base ‘ -.72 -42.36 -50.98 -42.36 ‘Based on the National carcass premiums and discounts for harvest steers and heifers (USDA Market News, June 29, 1998). l’USDA choice base. cAdjustments for quality grade: Select ($-.14lkg). “Adjustments for yield grade: 2.5 - 3.0 ($+ .022/kg), 3.0 - 3.5 ($- .002/kg). °Based on total carcass value adjustments x 359 kg average carcass weight and represents the deviation from the value of a USDA choice carcass. 88 without increasing carcass weight. Steers receiving somatotropin during the entire study showed the greatest effects with the majority of lipid reduction occurring during the latter part of the study. Somatotropin did increase protein accretion and therefore red meat yield, but not to the extent that would compensate, economically, for the loss in carcass quality. Additionally, the use of bovine somatotropin would not be practical until a longer delivery mechanism is developed. 89 Chapter 3 LONG-TERM BOVINE SOMATOTROPIN ADMINISTRATION TO GROWING- FINISHING HOLSTEIN STEERS DECREASED LIPOGENESIS AND INCREASED LIPOLYSIS"2 Abstract The objective of this study was to determine whether long-term bovine somatotropin (bST) administration to Holstein steers would alter measures of lipogenesis and lipolysis in growing-finishing Holstein steers. Twenty-eight Holstein steers (460 kg) were blocked by weight (2 replicates lblock) and randomly assigned to initial harvest (8 steers), control (no bST, C; 10 steers) or bST (100 pg/kg BW, 10 steers) treatment. Treatments were administered daily via s.c. injection during the 115 d study. Steers were individually penned indoors (3.9 m’) with ad libitum access to water and a diet containing whole-shelled corn, soybean meal, and a pelleted supplement. Blood was collected via jugular venipuncture on d 0, 3, 17, 30, 52, and 87 and analyzed for plasma lGF-l,,insulin, glucose, glycerol and NEFA. Three perirectal adipose tissue (AT) biopsies were collected from each steer, one during each of the periods 5 to 16 d prior to treatment, 40 to 49 d, and 103 to 112 d of the trial. Lipogenesis was measured in triplicate in vitro as tritium incorporation into fatty acids, fatty acid synthase activity, and NADP-isocitrate dehydrogenase activity. Lipolysis was measured in triplicate in vitro by glycerol release into the ‘ The author would like to acknowledge the contribution of bovine somatotropin to the study by Lilly Research Laboratories, Greenfield, IN. 2 The author would like to thank Patty Weber, Sharon Debar, and Michelle Mater for their laboratory assistance and expertise. 90 medium with and without epinephrine stimulation. In vivo lipolytic potential was determined by dose response-epinephrine challenges administered intravenously beginning on d -24, 32 and 95, with a single epinephrine concentration (0, .1, .2, .4, .8, 1.2, and 1.6 pglkg BW) administered each day for seven consecutive days. Plasma NEFA responses were measured over a 20 min period after the epinephrine challenge. Bovine ST chronically increased (P < .05) plasma glucose, glycerol, IGF-l, insulin, and NEFA concentrations. Lipogenesis, measured as tritium incorporation and fatty acid synthase activity in AT, was reduced (P < .10) after 40 d of treatment in the bST group. Basal glycerol release was increased (P < .05) after 103 d of bST treatment. Maximum response to the epinephrine challenge was increased in bST steers during challenges after 6 and 15 weeks of treatment. Based on this study, bST treatment decreased lipogenesis and increased lipolysis in AT of growing-finishing Holstein steers in positive energy balance. Key Words: Steers, Somatotropin, Lipogenesis, Lipolysis Introduction The initial experiment conducted by this author (Chapter 2) demonstrated a decrease in carcass lipid, backfat, kidney-pelvic-heart (KPH) fat, marbling score, USDA quality grade, and calculated yield grade of bovine somatotropin (bST)- treated steers. Treatment with bST reduced daily lipid accretion 12% during the first 182 d of treatment, and 20 to 29% the last 172 d of the study as compared to controls. Fat accretion increased 37% in control steers as time on feed increased whereas fat accretion decreased 18% for steers receiving bST. Clearly, bST had 91 a significant effect on the lipid deposition of steers, especially during the period when fat accretion was accelerated. The decrease in carcass lipid can be accomplished through decreased lipogenesis or increased lipolysis. Somatotropin treatment has been shown to reduce fatty acid synthesis (Vernon, 1982; Walton and Ethertcn, 1986; Sinnett- Smith and Woolliams, 1989), fatty acid synthase activity (FAS, Harris et al., 1993; Kramer et al., 1993), and fatty acid sythase mRNA protein (Harris et al., 1993). In more recent studies, ST has been shown to increase lipolysis through increased number of B-adrenergic receptors (Watt et al., 1990) and the reduced inhibitory effects of adenosine and prostaglandin E1 and E2 (Doris et al., 1996). While most studies have concentrated on the ruminant at maintenance or lactation, this study will evaluate the influence of bST on growing animals in positive energy balance. Specifically, the objective of this experiment was to determine the effects of long- terrn bST administration to Holstein steers on lipogenesis and lipolysis throughout the growing-finishing period. Materials and Methods Animal management. This project was approved by the Michigan State University All-University Committee on Animal Use and Care (AUF# 06/94-141-01) and conducted under an INAD (6673-C004 - Bovine somatotropin) issued to Michigan State University. Twenty-eight Holstein steers (460 kg) were individually identified; vaccinated against IBR, Fla, and Clostn'dial organisms; and implanted with estradiol benzoate-progesterone (Implus, The Upjohn Co., Kalamazoo, MI). 92 Steers were selected from a larger group of 40 steers to provide experimental units that were uniform in BW, frame size and body condition. The steers were blocked by weight and randomly assigned to 1 of 3 groups; initial harvest (8 steers); placebo-treated (control, 10 steers), or bST-treated (10 steers). The initial harvest group was used to determine initial carcass characteristics and composition. The remaining 20 steers were housed in an environmentally controlled room (12 h light: 12 h dark) in individual slotted-floor pens (2.0 m x 1.8 m, 3.6 m2) with ad libitum access to feed and water. Steers were adapted to the environment and fed a whole-shelled corn and pellet diet for 10 d prior to initiation of the study (Figure 3-1). On a DM basis, the diet contained 86.3% dry, whole-shelled corn; 12.4% protein-mineral pellet (Table 3—1); and 1.3% soybean meal. The diet was formulated to contain 14% CF (13.4% actual CP) and met NRC (1984) requirements for minerals and vitamins. Steers were fed twice daily, 60% at 0800 and 40% at 1500. Sufficient feed was offered to allow 5% to remain. Orts were weighed daily. Steers were removed from their pens weekly, weighed, had blood collected, and turned into a common outdoor dirt exercise lot for two hours. To assess in vivo lipolysis, epinephrine challenges were conducted on d -24 to -18, d 32 to 38, and d 95 to 101 (Figure 3-1). In vitro measures of lipogenesis and lipolysis were performed on adipose tissue (AT) from biopsies collected between d -16 to -5, d 40 to 49, and d 103 to 112 of the study. Details of the epinephrine challenge and AT biopsies are discussed later. One control steer was 93 Day of study .5 O 100 110 120 _> _> —.> D :> 11% d -36 to —26: adaptation period d -24 to -:18 1st series of epinephrine challenges (I -16 to -5: 1st series of biopsies d 0 Treatments began d 32 to 38: 2nd series of epinephrine challenges d 40 to 49: 2nd series of biopsies d 95 to 101: 3rd series of epinephrine challenges cl 103 to 112: 3rd series of biopsies d 115 last Treatment d 117: Slaughter Figure 3-1 - Schedule of epinephrine challenges and adipose tissue biopsies. 94 Table 3-1. Guaranteed analysis and ingredient content of the pelleted supplement'I Nutrient Guaranteed analysis Crude protein, minimumb 50.0% Crude fat, minimum .4% Calcium, minimum 5.0% Calcium, maximum 6.0% Phosphorus, minimum 1.0% Salt (NaCl), minimum 2.0% Salt (NaCl), maximum 2.5% Potassium, minimum 2.0% Vitamin A, minimum _ 22000 IU/kg Active Drug Ingredient _ Content Monensin Sodium 220 mglkg Tylosin phosphate 66 mglkg I'lngredients: soybean meal, calcium carbonate, urea, flash dried blood meal, corn distillers dried grains with solubles, salt, vitamin A acetate, D-activated animal sterol (source of vitamin D3), vitamin E supplement, calcium iodate, manganous oxide, ferrous sulfate, copper sulfate, cobalt carbonate, zinc oxide, sodium selenite. t’Does not included more than 18.5% equivalent CP from non-protein nitrogen. 95 removed from the study on d 17 due to lameness. Treatments. A daily placebo or bST injection (100 pglkg BW, Lilly Research Laboratories, Greenfield, IN) were administered subcutaneously in the mid-thoracic region just caudal to the shoulder between 0800 and 0900. The treatment was divided between two injection sites each day and alternated daily between the left and right side of the animal. Every two days, crystalline bST (Somidobove, Elanco Products Co., Indianapolis, IN, 92% potent, Lot # 014BM3) was dissolved in .01 M sodium phosphate buffer and adjusted to pH 7 at a concentration of 1 mg bST/ml. Control steers received .1 cc of .01 M sodium phosphate buffer [kg BW. Dosages of treatments were adjusted weekly based on BW and continued through d 115 (116 treatments). Treatment concentration remained constant and volume of treatment varied to deliver the required dose. Bovine somatotropin absorption time course. Prior to the third series of epinephrine challenges (d 94), the rate at which bST was absorbed from a s.c. injection into the peripheral circulation was determined. The animals were fitted with an indwelling jugular catheter on the previous day. Blood samples were collected every 30 min beginning at 0630 and continued to 1800 for a total of 24 samples. Steers received treatments between 0730 and 0800 and were fed at 0930 and 1500. The blood was collected in a 10 cc syringe and dispensed into a 7 ml, sterile blood-collection tubes containing 10.5 mg EDTA and .014 mg potassium sorbate (Vacutainer Brand, Becton Dickinson and Co., Rutherford, NJ) to prevent coagulation. The filled tube was inverted several times and chilled in an ice water bath until centrifugation. Plasma was separated by centrifugation at 959 x g 96 (Beckman J2-21 refrigerated centrifuge, Beckman Instruments Inc. Palo Alto, CA) for 20 min at 4 OC and stored in 12 x 75 mm polypropylene tubes (Sarstedt, Newton, NC) at -20 °C until analyzed. Plasma bST concentration was determined with a double antibody radioimmunoassay (RIA, Gaynor et al., 1995). Plasma metabolite and hormone analyses. Blood samples were collected on d 0, 3, 10, 17, 24, 30, 52, 59, 73, and 87 by jugular venipuncture into 7 ml, sterile blood-collection tubes containing 10.5 mg EDTA and .014 mg potassium sorbate (Vacutainer Brand, Becton Dickinson and Co., Rutherford, NJ); and processed and stored as described earlier. Plasma insulin-like growth factor-I (IGF-l) was measured by RIA (Sharma et al., 1994) after removal of insulin-like growth factor binding proteins by formic acid-ethanol extraction (Bruce et al., 1991; Sharma et al., 1994). Plasma nonesterified fatty acid (NEFA) concentrations were analyzed with an enzymatic kit (WAKO Chemicals, Dallas, TX) using a 96-well, micro-plate as described by Johnson and Peters (1993) and used the procedures of McCutcheon and Bauman (1986) to dilute reagents. Plasma glycerol concentrations were determined with a enzymatic kit (Procedure No. 337, Sigma Chemicals, St. Louis, M0) by placing 20 pl of sample into 1 ml of triglyceride (GPO-Trinder) blank reagent. Concentrations of plasma glucose were assayed by a enzymatic kit (Procedure 510, Sigma Chemicals, St. Louis, MO). Plasma insulin was analyzed using an lmmuChemTM Coated Tube 1"5| RIA kit (ICN Pharmaceuticals, Inc, Costa Mesa, CA). Animal performance. Animal performance was characterized by body weight (BW), average daily gain (ADG), daily dry matter intake (DMI) and feed efficiency. 97 Initial and final weights were determined from the average of weights taken on two consecutive days (d -1 and 0, and d 115 and 116). Feed intake was determined daily and averaged across the periods d 0 to 30 and d 31 to 115. Samples of all feed ingredients were collected daily, composited weekly, and analyzed for dry matter (DM, 48 h in a 55 °C oven) and crude protein (CP, total-Kjeldahl N, CP = N * 6.25, Technicon auto-analyzer system, Bran + Luebbe lnc., Tarrytown, NY). Feed efficiency was calculated as grams of gain divided by kilograms of DMI over the same intervals as daily DMI. Carcass characteristics, composition, and accretion rates. The initial harvest group and the 20 treated steers were harvested at a commercial packing plant. At the time of harvest, liver weight and hot carcass weight (HCW) were determined. After a 24-h, post-harvest chill; ribeye area (REA), 12th-rib backfat, KPH fat, marbling score, and USDA quality grade were determined. Non-carcass weight (NCW), dressing percent (DP) and yield grade (Boggs and Merkel, 1984) were calculated. Non-carcass weight was calculated as the difference between final weight and HCW. The 9th through 13th ribs section was removed from the left side of each carcass for compositional determination and transported to the Michigan State University Meats Laboratory. The 9-10-11 rib portion was separated from the larger rib section (Hankins and Howe, 1946), weighed and the soft tissue was separated from bone. The bones and soft tissue from each rib were weighed. The soft tissue was ground three times (once coarsely, and twice finely) with mixing in between each grind to ensure uniformity. Approximately 500 g of sample was collected and 98 placed into a Whirlpak bag (NASCO, Fort Atkinson, WI) and stored at —30 OC. The frozen sample was homogenized with liquid N in an industrial Waring blender. The samples were analyzed for DM, CP and ether-extractable lipid (EEL). Tissue ‘sample DM was determined in duplicate by placing 2 g of homogenized tissue in a desiccated-labeled aluminum pan and dried for 48 h in a 55 °C oven. Tissue moisture was determined by difference. Crude protein was analyzed in duplicate for total-Kjeldahl N (CP = N * 6.25, AOAC, 1984) using a Technicon auto-analyzer system (Bran + Luebbe lnc., Tarrytown, NY). Ether- extractable lipid was determined in triplicate by placing 2 g of sample in a desiccated-weighed and numbered #1 filter paper circle (Whatman Inc., Fairfield, NJ). The filter paper was folded to prevent loss of sample, secured with paper clips, and dried for 48 h in a 55 C’C oven. The dried samples were placed in a Soxhalet apparatus and extracted with petroleum ether for 24 h. Ether-extractable lipid was calculated as the amount of dry weight lost after ether extraction. Percent carcass bone, lipid, protein and moisture were calculated based on equations by Hankins and Howe (1946). Accretion rates of lipid and protein were determined from equations developed by Anderson et al. (1988). Adipose tissue biopsy procedure. Adipose tissue biopsies were collected from steers within a 10 d period with 4 steers (1 block) biopsied every second day. Steers were moved from their pens prior to the moming feeding and treatment, and placed in individual stalls. The left or right perirectal area was clipped and scrubbed clean. Under the direction of a veterinarian, the animal was given epidural anesthesia (2% Lidocaine hydrochloride, - without epinephrine) to effect. 99 Using sterile technique and instruments, an incision was made over the sacral-siatic ligament. Using forceps and blunt scissors, 3 to 5 g of AT was excised. The excised AT was divided between Krebs-Ringer-bicarbonate buffer (KRBB, pH 7.4), or .25 M sucrose solution (pH 7.4) each maintained at 37 °C for transport to the laboratory. The subcutaneous tissues of the biopsy site were sutured with #2 chromic gut and the skin was closed with #3 monofilament suture. Steers received an i.m. injection of penicillin G procaine (22,000 units! kg BW, Anthony Products Co., Arcadia CA) in the neck and were monitored closely to prevent complications. One bST steer (#184) developed an infected biopsy site after the second biopsy. The site was flushed with a warm Betadine“D solution for three days (twice a day), then with warm water, six days, until the wound closed. The wound healed and the steer finished the study. In vitro lipogenesis. In vitro lipogenesis was assessed as the amount of 3H incorporated into fatty acids (FA, Mulvaney, 1984). Triplicate samples (100 mg) from each biopsy sample were sliced with a microtome and placed in 25 ml Erlenmeyer. flasks containing oxygenated- KRBB with 100 pCi 3HZO/ml and incubated for 2 h at 37 °C. Following incubation, the tissue slices were rinsed three times with saline solution and placed in 50 ml culture tubes containing 10 ml of a KOH:ethanol solution (3:7 vol/vol; 30% KOH: 95% ethanol) for digestion and saponification overnight in a 60 °C water bath. Lipids were extracted from each sample with 5 ml of petroleum ether and vortexed for 1 min. The petroleum ether which contained the nonsaponifiable lipids was aspirated and discarded. This 100 extraction procedure was repeated to ensure complete separation of the lipid fraction. The remaining soaps were converted to FA with 1.5 ml of 12 N HCl. Fatty acids were extracted three times with the addition of 5 ml petroleum ether followed by 1 min of vortexing. After each extraction, the petroleum ether, top layer, was aspirated off and transferred to a scintillation vial. The ether was evaporated under a hood overnight and 10 ml of a non-aqueous scintillation fluid (ScintiVerse®, Fisher Scientific, Pittsburgh, PA) was added. Radioactivity was determined in a liquid-scintillation counter (efficiency for counting 3H = 65.6%). Based on equations of Mulvaney (1984), the assumption that pure water in the incubation media has a molarity of 55, and the specific radioactivity (DPM/mol) of the media; the nanomoles of 3H converted to FA per minute per gram of tissue could be calculated from: FA = CPM x (11E) x (1/t) x (1000/wt) x (1ISA) where CPM = the counts/minute of the samples E = the efficiency of the liquid scintillation counter counting tritium t = time of incubation in minutes wt = weight of the AT slice in milligrams SA = specific activity of the media In vitro lipolysis. Glycerol release into the media in the absence and presence of epinephrine was used as a measure of in vitro lipolysis. Six AT slices (100 mg) cut as previously described were placed into 25 ml Erlenmeyer flasks containing 3 ml of KRBB media (prepared as before with the addition of 30 mg/ml of essentially fatty acid free bovine serum albumin; A-6003, Sigma Chemical Co., St. Louis, MO). Three of the flasks contained 25 pM epinephrine-HCI (E-4642, Sigma Chemical Co., St. Louis, MO). The slices were incubated for 2 h in a 101 shaking water bath at 37 °C. After incubation, the KRBB was removed by aspiration and transferred into glass test tubes (12 x 75 mm) and chilled on ice until assayed in triplicate for glycerol with the glycerol enzymatic food analysis kit (No. 148270, Boehringer Mannheim Biochemicals, Indianapolis, IN). The reagents were diluted 1:3 with Milli-Q water (Dickerson, 1990) and the change in NADH was read at 340 nm (Varian® Cary 2200 UV-VIS spectrophotometer, Sugarland, TX). Enzyme activity. Five hundred milligrams of AT were homogenized (Polytron, Brinkrnann Instruments, Westbury, NY) in 5 ml of .25 M sucrose solution (37 °C) in a 15 ml Corex tube for 10 s bursts and centrifuged at 20,000 x g (Sorvall RC-5, DuPont Instruments, Newtown, CT) at 4 °C for 20 min. The supernatant was removed and placed in 12 x 75 mm glass test tubes for enzyme activity measurements. Triplicate analyses were performed for each AT sample. Fatty acid synthase (EC 2.3.1.85) activity (Muesing and Porter, 1975) was measured as the conversion of acetyl CoA and malonyl CoA to palmitate utilizing NADPH. The disappearance of NADPH over time was measured as the change in absorbance over time at 340 nm and 30 °C (Varian® Cary 3 UV-VlS spectrophotometer, Sugarland, TX). NADP-isocitrate dehydrogenase (ICD, EC 1.1.1.42) activity (Plaut, 1962) was measured as the conversion of isocitrate to or-ketoglutarate producing NADPH. The appearance of NADPH over time was measured as the change in absorbance over time measured at 340 nm and 30 °C. Following both enzyme assays, the tissue supernatant was frozen until soluble protein content was determined by folin- phenol procedure (Lowry et al., 1951). Enzyme activity was expressed as units/mg 102 protein. Epinephrine challenges. Steers were fitted with an indwelling jugular cannula on the day before initiation of each series of epinephrine challenges. Epinephrine challenges ( 0, .1, .2, .4, .8, 1.2, and 1.6 pg! kg BW) were administered over seven days, with one dose concentration administered per day (Sechen et al., 1990). Epinephrine HCI (1 mg/ml, The Butler Co., Columbus OH) was diluted with sterile saline (.15 M NaCl) to a concentration of 100 pglml. Epinephrine challenges (2-5 ml) were injected via the jugular catheter immediately followed by 4 ml of sterile saline. Epinephrine challenges were administered to nine steers at 0930 and the other ten at 1030. The latter group was treated and fed an hour later on days challenges were administered. Blood samples were withdrawn from cannulas at - 30, -15, -10, -5, 0, 2.5, 5, 10, 15, 20, 30, 45, and 120, 125, and 130 min post- epinephrine infusion (starting at 1000 and 1100 for each group of 10 steers, respectively) into a 10 cc syringe. Collected whole blood was dispensed into a 7 ml, sterile blood-collection tube containing 10.5 mg EDTA and .014 mg potassium sorbate (Vacutainer Brand, Becton Dickinson and Co., Rutherford, NJ) to prevent coagulation. The filled tube was inverted several times and chilled in an ice-water bath until centrifugation. Plasma was separated by centrifugation at 959 x g (Beckman J2-21 refrigerated centrifuge, Beckman Instruments Inc., Palo Alto, CA) for 20 min at 4°C and stored in 12 x 75 mm polypropylene tubes (Sarstedt, Newton, NC) at -20 °C until analyzed for NEFA, glycerol and glucose concentrations. Plasma NEFA concentrations were assayed as previously outlined on all samples. Plasma glycerol and glucose concentrations were analyzed on samples from the 103 third series of challenges using the 1.6 pg epinephrine] kg BW dose. Non-esterified fatty acid and glycerol responses to the epinephrine challenge were calculated as area under the response curve from 0 to 20 min post-challenge (SAS program provided in Figure B-1). Response areas were corrected for differences in base-line concentration (the average of samples collected -30, -15, -10 , -5, 0, 120, 125, and 130 min relative to the challenge). The NEFA response area for each dose was used to construct a dose-response curve for each animal utilizing the non-linear regression procedure of SAS (1994). A segmental curve was fitted to each set of challenges comprised of a quadratic curve to a plateau (maximum response, Rm, Figure B-1). When developing curves, the response of the 0 pglkg BW was set at 0. If a plateau was not reached, within the range of epinephrine dosages, the responses for the 1.2 and 1.6 pglkg BW dose (6 curves) or the .8 to 1.6 pglkg BW doses (4 curves) were averaged and the curve recalculated. The epinephrine dose which gave half of Rm, the EDSO, was calculated using the quadratic portion of the curve. Of the 19 steers completing the study, one steer did not have the first two series of epinephrine challenges completed due to problems with the catheter. Statistical analysis. This study was designed as a completely randomized block. Animal performance was analyzed using the GLM procedure of SAS (1994) with treatment and block in the model as class variables. Least square means were calculated and separated with probability of difference function. Plasma hormones and metabolites were analyzed using the univariate repeated-measures analysis of SAS (1994) with treatment and block in the model as class variables and least 104 square means, treatment and day within treatment, separated by a procedure outlined by Gill (1987). Tritium incorporation, enzyme activity, and glycerol release were analyzed using the mixed procedure of SAS (Littell et al., 1996) with the spatial power law covariance structure to account for the unequally spaced repeated measures. The epinephrine challenge maximum response, Rm, and ED50 were analyzed using the mixed procedure of SAS (Littell et al., 1996) with an autoregressive covariance structure assuming equally spaced repeated measures. Measures of lipogenesis and lipolysis utilized treatment and block in the model as class variables, whereas Rm, and EDSO utilized treatment and replicate in the model as class variables. Results Bovine somatotmpin time course and blood metabolites. Plasma bST concentrations began to increase (P < .05) above baseline within .5 h after the s.c. injection of bST and remained elevated 10 h when blood collection stopped (Figure 3-2). Somatotropin concentrations peaked 1.5 h after bST administration. Prior to treatment, bST concentrations were similar between treatments for at least 1.5 h. Prior to the start of treatment, bST steers had 34% greater (P < .05) plasma lGF-l concentration than control steers (Figure 3-3). Three days following initial bST treatment and continuing throughout the entire study, bST steers had 112 to 157% greater plasma lGF-l concentration than control steers. Steers receiving bST averaged 88% greater (P < .01) plasma NEFA concentration and 45% greater (P < .01) plasma glycerol concentration from d 3 to 87 than control steers (Figure 3-4 105 Plasma bST. nglml 700 800 900 1000 1100 1200 1300 4.— Treatment 1500 1600 1700 1800 Hour Figure 3-2 - Plasma bovine somatotropin concentrations over an 11.5-h period in Holstein steers treated with either a daily injection of sodium phosphate (0) or bovine somatotropin (I). ‘bST steers differ from control steers (P < .05). Plasma lGF-1, ngImI W SED=64 o t-l-i-slfifi-l 010 20 30 4o 50 60 7o 80 90 Days of treatment Figure 3-3 - Plasma lGF-l concentrations versus days of treatment in Holstein steers treated with either a daily injection of sodium phosphate (0) or bovine somatotropin (I). *bST steers differ from control steers (P < .05). “*bST steers differ from control steers (P < .001). 106 250 *** E zoo .- <' in" 150 Z 0 A“ u 10 = ' E 3 E 5° SED=21 0 I . i - i - Jr - i - i w i - i8 i - i o 10 20 so 40 so so 70 so so Days of treatment Figure 3-4 - Plasma NEFA concentrations versus days of treatment in Holstein steers treated with either a daily injection of sodium phosphate (0) or bovine somatotropin (I). **bST steers differ from control steers (P < .01). “*bST steers differ from control steers (P < .001). N O #11:!!! 60 In: *1": am: / 50 Plasma glycerol, uM N O l I A O I I SED = 5.0 o L I l 1 L l l l I I I fl I I I I I I I I I I I I T I I 010 20 30 40 50 60 70 80 90 Days of treatment Figure 3-5 - Plasma glycerol concentrations versus days of treatment in Holstein steers treated with either a daily injection of sodium phosphate (0) or bovine somatotropin (I). **bST steers differ from control steers (P < .01). “*bST steers differ from control steers (P < .001). 107 and 3-5, respectively). On average, bST steers had 11% greater (P < .05) plasma glucose concentration from d 3 to 87 than control steers (Figure 3-6). From d 17 to 87, bST steers had 665% greater (P < .05) plasma insulin concentration than control steers (Figure 3-7). Feedlot performance. Steers receiving bST tended to have 3% greater (P < .10) live weight than control steers after 30 d of treatment (Table 3-2). Any advantage gained in the first 30 d was not maintained throughout the entire study. The greater BW after 30 d of treatment was due to bST steers having 43% greater (P < .05) ADG during this period, but as with BW, no improvement in ADG was evident from d 31 to d 115. Dry matter intake was similar for treatments during the first 30 d of the study, but during the remaining 85 d, DMI tended to be 9% lower (P < .10) in bST steers than controls. Over the entire study, treatments had similar daily DMI and protein intake. Feed efficiency of bST steers was 47% greater (P < .01) during the initial 30 d of the study and 19% greater (P < .05) over the entire study. Carcass characteristics, composition and accretion. Steers had similar HCW, but steers receiving bST tended to have 6% greater (P < .10) NOW and 2% lower DP (P < .10) than control steers (Table 3-3). There was no difference between treatments in liver weight. Ribeye area, as expressed on a HCW basis was 9% larger (P < .05) in bST steers than control steers. Measures of carcass fatness were reduced with bST administration. Bovine somatotropin decreased backfat 28% (P < .05), KPH fat percentage 50% (P < .001), marbling score 21% (P < .001), and USDA quality grade 9% (P < .001). Administration of bST decreased 108 ** I II 0 ll: 0 0 1110 I ILJLI Plasma glucose, mM O A N u «h 0| 0: Days of treatment Figure 3-6 - Plasma glucose concentrations versus days of treatment in Holstein steers treated with either a daily injection of sodium phosphate (0) or bovine somatotropin (I). *bST steers differ from control steers (P < .05). **bST steers differ from control steers (P < .01). 25 E **III ”I, «a a 20 ...-. C * * =§ 15 * In E 10 E III 5 , SED=5.1 2 - - a f v .. 0 l I i I i r i I4} I i I I , 4' r + 1 1I 0102030405060708090 Days of treatment Figure 3-7 - Plasma insulin concentrations versus days of treatment in Holstein steers treated with either a daily injection of sodium phosphate (0) or bovine somatotropin (I). *bST steers differ from control steers (P < .05). **bST steers differ from control steers (P < .01). ”*bST steers differ from control steers (P < .001). 109 Table 3-2. Effect of bovine somatotropin on feedlot performance“ Treatments Probability Tran Control SE bST SE Model Contrast b No. of steers 9 10 Body weight, kg Day 0 460 3.9 460 3.6 .91 .97 Day 30 502 6.6 519 6.2 .20 .08 Day 115 594 11.3 608 10.6 .38 .37 Average daily gain, kg DaysOto 30 1.38 .17 1.97 .16 .15 .03 Days 31 to 115 1.09 .08 1.05 .07 .60 .74 Days 0 to 115 1.15 .08 1.28 .08 .35 .28 Dry matter intake, kg/d Days 0 to 30 9.81 .36 9.52 .34 .29 .57 Days 31 to 115 9.78 .34 8.88 .32 .19 .07 Days 0 to 115 9.79 .33 9.05 .31 .21 .12 Protein intake, g/d DaysOto115 1312 44 1214 41 .21 .12 Feed efficiency, 9 gain/kg DMI Days 0 to 30 140 15 206 14 .09 .007 Days 31 to115 110 5 116 5 I .35 .47 Days 0to115 118 5 140 5 .07 .01 'Least square means. I’Probability of difference: Control versus bST. 110 Table 3-3. Effect of bovine somatotropin on carcass characteristics‘I Treatments Probability Chem-3.13m Control SE bST SE Model Contrast” Hot carcass wt., 355 7.1 355 6.6 .45 .98 k9 Noncarcass wt., 240 5.8 254 5.4 .36 .10 K9 Dressing 59.7 .51 58.3 .48 .51 .07 percent Liver wt, 9 8634 228 9029 213 .1 1 .22 % of BW 1.45 .03 1.48 .03 .32 .43 Ribeye area, cm 2 77.1 2.7 83.7 2.7 .66 .11 cm 21kg How .216 .006 .236 .006 .20 .03 Backfat, mm 6.1 .58 4.4 .54 .29 .04 KPH, % 2.2 .06 1.1 .06 .0001 .001 Marbling score c 560 12.3 445 1 1.5 .0003 .001 Quality grade " 18.1 .19 16.4 .18 .0008 .001 % Choice 78 0 % Select 22 100 Calculated yield 2.7 .14 2.0 .13 .03 .002 _grade aLeast square means. bProbability of difference for Control versus bST. ‘400 = slight, 500 = small. ‘19 = Choice°, 18 = Choice' 17 = Select *, 16 = Select'. 111 (P < .01) calculated yield grade 26% and therefore increased red meat yield. Carcass composition was altered significantly with bST administration (Table 3-4). Steers administered bST had 11% greater (P < .001) bone, 9% greater (P < .001) protein, and 10% greater (P < .001) water composition of the carcass and 38% greater protein accretion than control steers. Lipid composition of the carcass was decreased 32% (P < .001) with 70% lower (P < .001) lipid accretion in bST steers than control steers. Efficiency of protein accretion was increased (P < .001) 50% with bST administration. Lipogenesis. Measures of in vitro lipogenesis, tritium incorporation, and enzyme activity are presented in Table 3-5. Tritium incorporation into AT was decreased (P < .05) by 54 and 56% for control and bST steers, respectively, from pretreatment through 103 d of the study. Tritium incorporation was decreased 45% (P < .05) in AT from bST steers than control steers during the second biopsy period, 40 d of treatment, but treatments were similar during the last biopsy period. Similarly, FAS and ICD decreased (P < .10) throughout the study in AT from control and bST steers. Fatty acid synthase activity tended to be lower (P < .10) in AT of bST steers than controls during the second biopsy period. lsocitrate dehydrogenase activity was similar between treatments throughout the study, but activity in AT of bST steers decreased sooner than controls. The correlations between tritium incorporation and FAS and ICD activity were r = .41 (P = .002) and r = .40 (P < .001), respectively. The correlation between FAS and ICD was r = .77 (P < .001). 112 Table 3-4. Effect of bovine somatotropin on carcass composition and accretion rates' Treatments Probability Variable Control SE bST SE Model Contrast b Carcass composition, % Bone 15.0 .3 16.6 .2 .09 .001 Protein 13.2 .2 14.4 .2 .007 .001 Lipid 25.6 .7 17.3 .7 .0001 .001 Water 47.1 .5 52.0 .4 .0002 .001 Carcass accretion rates, gld Protein 92 8 127 8 .09 .01 Lipid 355 29 106 27 .0009 .001 Efficiency of protein accretion Day 0 to 115 ° 70 6 105 5 .02 .001 ‘Least square means. I’Probability of difference for Control versus bST. 6daily 9 protein accretion/ kg daily dietary protein intake. 113 Table 3-5. Effect of bovine somatotropin on tritium incorporation into fatty acids, fatty acid synthase activity, and isocitrate dehydrogenase activity‘il Period Control bST nmol 3H converted to fatty acid-min"t100 mg “b d-16to-5 3.44x 3.15x d 40 to 49 2.50 *7 1.37 y.- d103to112 1.59’ 1.39" Fatty acid synthase activity, units-mg protein' 1" d -16to -5 10.96j 13.29- d40 to 49 6.83 k 3.12vT d 10316112 7.62“ 457 . NADP-lsocitrate dehydrogenase activity, units-mg protein "d d ~16 to -5 281.6 " 284.5 " d 40 to 49 210.5"y 153.8y d103to112 160.9y 135.8y ‘Least square means. bSED = .47. °SED = 2.25. ‘SED = 41.1. j"‘Means within columns and response variable with unlike superscripts differ (P < .10). x”Means within columns and response variable with unlike superscripts differ (P < .05). TDiffers from Control within period (P < .10). *Differs from Control within period (P < .05). 114 Lipolysis. Basal-glycerol release (Table 3-6) was increased (P < .05) 42% in AT from bST steers after 103 d of treatment as compared to AT from control steers. Epinephrine-stimulated glycerol release was similar between treatments at all biopsy periods. Nonesterified fatty acid response to an epinephrine challenge was used as an in vivo measure of lipolysis. Response curves for NEFA are presented in Figure 3-8 with descriptive statistics in Table 3-7. The maximum NEFA response (Rm) to an epinephrine challenge was 84 and 40% greater (P < .01) in bST steers during the 6‘“ and 15‘h week of treatments as compared to control steers. Within bST steers, the Rm (P < .05) was 76 and 65% greater than pretreatment values during the 6th and 15‘" week of treatment, respectively. The ED50 dose was higher (P < .05) in bST steers prior to the start of the study, but during treatment, the ED50 dose was similar between treatments at both series of challenges. The plasma glycerol response to a 1.6 pg epinephrine/kg BW response (Figure 3-9) was similar between treatments. The correlation between basal glycerol release and Rm was r = .32 (P = .02). There was not a significant correlation between basal glycerol release in vitro nor Rm, with the area under the glycerol response curve. Discussion Bovine somatotropin time course and blood metabolites. Plasma ST concentrations were similar between controls and bST treatment for 1.5 h prior to each daily treatment. Peak plasma ST concentrations occurred within 2 h after the s.c. injection. This is a similar pattern to Eisemann et al. (1986a), Enright et al. 115 Table 3-6. Effect of somatotropin treatment on basal and epinephrine- stimulated glycerol releasea Period Control bST Basal, nmol glycerol released- 2h"-100 mg tissue "b d -16 to -5 73.5 93.5 d 40 to 49 95.4 105.5 d103to112 83.4 118.5.- Stimulated, nmol glycerol released- 2h’1-100 mg tissue “° d -16 to -5 251.4 284.1 d 40 to 49 261.1 274.9 d 103 to 112 265.2 232.2 'Least square means. I’SED = 16.5. °SED = 29.6. *Differs from Control within period (P < .05). 116 NEFA area,pmoI-mlnoL'1 0 l I I I I 0 0.4 0.8 1 .2 1 .6 Epinephrine dose, pglkg BW I I I I Figure 3-8 - Response in plasma concentrations of NEFA to varying doses of epinephrine during 3 weeks prior to treatment (panel a) and after 6 (panel b) and 15 weeks (panel 0) of control (0) or bST (I) treatment. Analyses of individual animal curves are in Table 3-7. 117 Table 3-7. Estimates of Rm and ED50 of nonesterified fatty acids area epinephrine dose-response curves“ Week Control bST Rm, pmol~min-L'"’ -3 1094 1246" 6 1192 2196 y... 15 1465 2056 V” EDSO, pg epinephrine-kg BW"° -3 .16 .27 * 6 .19 .24 15 .19 .22 ‘Least square means. bSED = 206. cSED = .05. ”-V‘Means within columns and response variable with unlike superscripts differ (P < .05). *Differs from Control within week (P < .05). **Differs from Control within week (P < .01). ”*Differs from Control within week (P < .001). 118 1111 11111 1111111111111111111 Till[TITIIIIITTIITTTIIIIIIIIITI -30 0 30 60 90 120 Minutes relative to epinephrine infusion Plasma glycerol, pM 8 D Figure 3-9 - Response in plasma glycerol concentrations to a 1.6 pglkg BW epinephrine infusion (arrow) during the 15th week of treatment with either control (0) or bST (I). Area under the response curve (0 to 20 min. after infusion) was calculated using data points -30 to 0 and 120 to 130 min. relative to infusion as the base line. Control area under the curve = 604 :I: 100 pM-min and bST area under the curve = 648 3: 94 pM-min. 119 (1990) and Boisclair et al. (1994) with 29.2 IU bST/d, 40 pg bST/kg BW and 120 pg bST/kg BW, respectively. Enright et al. (1990) and Boisclair et al. (1994) observed a peak in plasma ST concentrations 1 h after administration with a gradual decline to baseline by 12 h. In the present study, elevated plasma ST concentrations persisted for 11.5 h after injection. Similar to the first study (Chapter 2), and consistent with previous research (Moseley et al., 1992; Roeder et al., 1994; Preston et al., 1995; and Enright et al., 1990) bST administration increased plasma lGF-l concentrations. In growing lambs, plasma lGF-l concentration increased after one week of treatment and increased three-fold after ten weeks of ST administration (Pell et al., 1990). Both plasma NEFA and glycerol concentrations increased dramatically in the present study. This is in agreement with Eisemann et al. (1986) who observed a chronic elevation in plasma NEFA in growing beef heifers, but Enright et al. (1990) and Peters (1986) observed no plasma NEFA increase in growing steers, and Pell et al. (1990) demonstrated no effect in growing lambs treated with ST. Dunshea et al. (1992a) demonstrated an increase in plasma NEFA and glycerol of barrows within 7 and 10 to 12 h, respectively, of the first porcine ST (pST) injection and the response increased on the second and seventh day of treatment. In agreement with the present study, Doris et al. (1996) observed a 31% increase in plasma glycerol concentration growing sheep treated with ST for 7 d. It is suggested by Boisclair et al. (1997) that the increase in plasma NEFA concentation with bST treatment of animals in positive energy balance is a result of mild disturbances or stress during blood collection. This is suggested because 120 bST causes an increase in B-adrenergic receptor (BAR) number in rat adipocyte membranes (Watt et al., 1990) and therefore respond to catecholamines to a greater extent than non-treated animals. Houseknecht et al. (1995) determined that bST increased the maximum binding of a BAR agonist to the BAR without a change in the binding affinity. Boisclair et al. (1997) cautions that proper protocol is required to prevent inadvertent responses which may suggest lipolytic activity. It is true that the animals in the current study were disturbed during blood collection. They were removed from their pens, taken outside, worked through a typical beef cattle-handling facility and placed into a squeeze chute for jugular venipuncture. Eisemann et al. (1986) demonstrated both an increase in irreversible loss and oxidation of NEFA with bST treatment in heifers. The data further suggested that NEFA are used as an energy source and perhaps this spares oxidation of other metabolites (e.g. amino acids) during bST treatment. It could also be theorized that the adipocytes are stimulated continuously due to the increased adrenergic receptors causing elevated plasma NEFA concentrations. Without measures of stress (e.g. cortisol) one can not determine if the animal experienced a mild disturbance. Plasma cortisol was not different in the study by Peters (1986), but NEFA concentrations were not elevated either. Plasma NEFA and glycerol concentrations increase 743 and 46%, respectively, in steers under a prolonged fast (Rule et al., 1985). This is significantly greater then the increase In plasma NEFA observed in the present study. These changes were in response to decreased plasma glucose, insulin, and ST concentrations and were used for homeostatic control of plasma glucose, 121 utilizing glycerol for gluconeogenesis (Rule et al., 1985). In the present study where plasma glucose concentrations were elevated, glycerol would not be needed for gluconeogenesis, but may be elevated due to increased lipolysis. This is in agreement with Sechen et al. (1990), where a 372% increase in plasma NEFA and 119% increased plasma glycerol concentration in bST-treated lactating dairy cattle were observed. Additionally, Peters et al. (1986) observed a 63% increase in basal plasma NEFA with feed restriction. Plasma glucose was chronically increased from d 3 and thereafter in bST- treated steers. This increase in plasma glucose is supported by Boisclair et al. (1994) in bST-treated growing cattle and Pell et al. (1990) in ST-treated lambs where increases of 5 and 11% were observed, respectively. Dunshea et al. (1992a) observed an increase in plasma glucose and insulin within 2 and 4 h, respectively, after an initial injection of pST to barrows. Early et al. (1990a) and Enright et al. (1990) in steers, and Sechen et al. (1990) in lactating dairy cattle, did not observe an increase in plasma glucose with ST administration. Pell et al. (1990) stated, that the increase in plasma glucose could be due to an increased rate of gluconeogenesis, or a decrease in peripheral glucose utilization. Boisclair et al. (1994) demonstrated a decrease in hindlimb glucose uptake in growing steers administered bST. Additionally, Hart et al. (1984) demonstrated that bST had diabetogenic activity and decreased the insulin depression in plasma glucose during an insulin tolerance test and suggested that bST decreased glucose uptake by body tissues. Glucose uptake was decreased in pigs treated with pST (Dunshea etal,1992c) 122 There is evidence that ST decreases insulin sensitivity. Hart et al. (1984) observed a reduction in the insulin-induced decrease of plasma glucose during an insulin tolerance test and Roupas et al. (1991) suggested that insulin is uncoupled from its second messengers in the presence of ST. If insulin sensitivity is reduced, that would explain the increase in plasma insulin observed in this study. Somatotropin induced increase in plasma insulin has been observed by Wallace and Bassett (1966), Johnsson et al. (1985), Eisemann et al. (1986), Pell et al. (1990) and Boisclair et al. (1994) in ruminants and by Hansen et al. (1997a) in pigs. Pell et al. (1990) suggests they have unpublished data which demonstrates an increase in gluconeogenic potential by hepatocytes isolated from ST-treated lambs and therefore, glucose production may be increased, but would require an adequate supply of gluconeogenic precursors, glycerol being one. Plasma glycerol concentrations were increased in the present study and Doris et al. (1996) in growing animals and may be the result of triacyglycerol breakdown as suggested by Pell et al. (1990). Wallace and Bassett (1966) believed that the increase in nitrogen retention with ST administration is dependent on the associated increase in plasma insulin. The increase in plasma insulin would drive more nutrients (AA, glucose) into cells and stimulate anabolism. Feedlot performance. Just as in the previous study, bST increased ADG and BW early during treatment, but the increase was not maintained throughout the entire study. Early et al. (1990a) reported an increase in ADG during the first 8 d of a 112-d study, but Rumsey et al. (1996) and Preston et al. (1995) demonstrated increased ADG throughout their 56 and 84 to 119-d studies, respectively. In the 123 current study, the dietary CP concentration was increased to 13.4% as compared to the 11.5% in the previous study (Chapter 2). There was not a sustained increase in ADG of bST steers with the increased dietary CP. Daily gains exhibited by both treatments in this study are similar to the previous study (Chapter 2), although adequate undegraded intake protein (518 gld and 482 gld for the control and bST- steers, respectively) was available to achieve ADG of 2.02 and 1.73 kg, respectively (NRC, 1996). Based on the NRC (1996) diet evaluator, energy would limit ADG to 1.68 and 1.5 kg for the control and bST-steers, respectively. Dry matter intake was reduced in bST-treated steers. This is in agreement with Dalke et al. (1992), Moseley et al. (1992) and Preston et al. (1995) who reported a decrease in DMI of steers with increasing levels of bST administration. Early et al. (1990a) observed no effect of bST on intake and Rumsey et al. (1996) demonstrated an increase in DMI with bST treatment. The decrease in DMI suggests that as an animal mobilizes lipids and energy due to bST, these nutrients are directed away from lipogenesis, the animal compensates for the extra circulating nutrients by reducing its intake (McBride and Moseley, 1991). The effect may be dependent on BW and lipid content of the animal. In light-weight cattle which have less body-fat reserves, such as those used by Rumsey et al. (1996), the increased growth observed may stimulate intakes to supply the increased energy needs. Animals that have been on feed for some time (Preston et al., 1995; Moseley et al., 1992) have added body-fat reserves and the action of bST to enhance lipolysis increases circulating blood nutrients thereby decreasing DMI. Although, ADG did not increase significantly and DMI decreased marginally, 124 feed efficiency increased. This is supported Early et al. (1990a), Moseley et al. (1992), Preston et al. (1995), and Rumsey et al. (1996) who all observed increased feed efficiency with bST administration. Carcass measurements. In the present study, HCW was not influenced by bST treatment. Hot carcass weight has been shown to decrease linearly with increasing bST doses from 0 to 300 pg bST/kg BW (Moseley et al., 1992), although Rumsey et al. (1996) observed a 5.5% increase in HCW with bST administration. The increase in NCW and the decrease in DP observed in the present study is consistent with previous research. The increase in NOW is supported by Early et al. (1990a) and the decrease in DP by Early et al. (1990a) and Moseley et al. (1992), but Preston et al. (1995) did not observe an effect of bST on DP. Early et al., ( 1990a) estimated that the increase in NCW was primarily due to an increase in the GIT, but this was not observed in the study by Rumsey et al. (1996). Ribeye area when expressed on a HCW basis did increase in the present study and was similar to the response observed in the previous experiment (Table 2-8) in steers receiving bST during the latter part of the study as compared to control steers. This increase in REA is supported by Moseley et al. (1992) which observed a 4 to 8.7% linear and quadratic increase in REA with increasing bST doses (8.25, 16.5, 33, and 66 pg bST/kg BW), and a 7.2 and 6.6% increase in REA at doses of 100 and 300 pg bST/kg BW. An increase in REA with bST administration was not observed by Peters (1986) or Preston et al. (1995). The increase in REA is consistent with the increased protein composition and accretion of bST-treated steers. Rumsey et al. (1996) demonstrated a 22% increase in 125 carcass protein accretion with bST administration in young steers and Early et al. (1990c) determined whole-body protein accretion was increased with bST administration by not of the carcass. Eisemann et al., (1989) demonstrated increased protein synthesis of the biceps femoris, longissimus dorsi, gastrocnemius and triceps brachii muscles. The increase in efficiency of protein accretion estimated in the present study is in agreement with Eisemann et al. (1986) who demonstrated a marked improvement in the efficiency of protein deposition (a decrease in the grams of protein synthesized l grams of protein deposited), and a 5.7% increase in whole-body protein synthesis. Additionally, these results would be consistent with the findings that ST reduces blood urea nitrogen suggesting reduced amino acid degradation and increased nitrogen retention and protein deposition (Wallace and Bassett, 1966; Eisemann et al., 1986b, and Sinnett-Smith et al., 1989) All measures of carcass lipid content (backfat, KPH, marbling score, USDA quality grade, lipid composition and accretion rates) were decreased with bST administration. Peters (1986) observed a 24% reduction in backfat with bST administration following 29 d of treatment. Moseley et al. (1992) reported a 9 to 62% reduction in backfat and a 17 to 95% reduction in the percent of carcasses grading USDA Choice. The 95% reduction was in steers receiving 300 pg bST/kg BW, growth rate was reduced and these steers did not reach market weight. At 100 pg bST/kg BW, 70% fewer carcasses graded USDA Choice (Moseley et al., 1992). Preston et al. (1995) documented a linear decrease in backfat (15%), KPH fat (28%), marbling score (6%), and percent grading USDA choice (58%) with 126 increased bST dosage. Early et al. (1990a) did not see a difference in backfat or Canadian carcass grade. As fat decreased, the percentage of lean tissue increased in the present study as shown by reduced yield grade. This agrees with Preston et al. (1995) in which bST reduced yield grade by 12.5%. In the present study, carcass lipid composition and accretion were decreased to a greater extent in bST-treated steers than in steers receiving bST only during the latter part of the previous experiment (Chapter 2, 18 vs. 32%, and 39 vs. 70% reductions, respectively). The average bST dose was different between the two studies and may explain these differences. In the current study, 100 pg bST/kg BW was administered, but in the previous study a set quantity per 14 d was given and averaged 89 pg bST l kg BW for steers only receiving bST during the latter part of the study. This finding would be consistent with bST dose-response studies (Dalke et al., 1992 and Moseley et al., 1992). Total carcass lipid was decreased in steers treated with bST (Peters, 1986) and Rumsey et al. (1996) demonstrated a 23.5% decrease in lipid accretion after 56 d of bST administration. Early et al. (1990b) did show a statistically-significant reduction in carcass fat or lipid accretion with bST treatment, although, numerically this values decreased 11.5% and 11%, respectively. Lipogenesis. All three measures of lipogenesis demonstrated a decrease over the period of the study. Pothoven et al. (1975) observed a 62% decrease in in vitro lipogenesis as crossbred steers increased in SW from 363 to 505 kg. The decrease in lipogenesis as BW increases is associated with an increase in adipocyte size and fewer cells per unit weight of tissue (Hood and Allen, 1973). 127 In the present study, after 40 d of treatment, both tritium incorporation into FA and FAS activity were reduced in bST-treated steers as compared to control steers. Somatotropin has been found to decrease fatty acid synthesis in AT treated in vitro with ST (Vernon, 1982; Sinnett-Smith and Woolliams, 1989). Walton and Ethertcn (1986) demonstrated that ST can antagonize insulin’s stimulation of lipogenesis in cultured porcine AT. Kramer et al. (1993) observed a 35% decrease in fatty acid synthesis, as measured by tritium incorporation into FA, and a 56% decrease in malic enzyme activity in pigs treated with pST for 24 d. Additionally, Harris et al. (1993) reported a correlation between FAS and lipogenesis was r = .45, which is similar to the correlation between FAS activity and measures of lipogenesis in the present study. Harris et al. (1993) observed an 86% reduction in glucose incorporated in FA after barrows were treated 11 d and reduced FAS and ICD activities of 67 and 31 %, respectively. Because AT enters a net degradative state after biopsy, in vitro measurements of lipogenesis may be underestimates (Kramer et al., 1993). Dunshea et al. (1992c) utilized an in vivo method to determine glucose utilization rates for lipogenesis and the concurrent effects of pST. Rates of glucose incorporated into lipids was reduced 76%. Specifically, glucose incorporated into triglyceride-FA was reduced 78% as compared to glucose incorporated into triglyceride-glycerol which was reduced 66%. Vernon et al. (1991 a) determined that ST decreased the activation of acetyl- CoA carboxylase (ACC) when AT from wethers was treated with ST in vitro. Magri et al. (1990) demonstrated a decrease in the lipogenic enzyme activities of FAS, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and 128 malic enzyme in AT of barrows treated for seven days with pST. Additionally, Magri et al. (1990) determined that glucose transport into adipocytes was reduced 62% with pST administration, but insulin binding to the adipocyte nor the insulin receptor’s tyrosine kinase activity was affected. Donkin et al. (1996) found GLUT 4 mRNA abundance, the major insulin regulated glucose transporter in adipocytes, was not decreased with pST administration, but Kilgour et al. (1995) determined that a decreased in plasma ST concentration in the rat caused the GLUT 4 protein to translocate from the intracellular pool to the plasma membrane. Roupas et al. (1991) discovered that ST interferes with the signal transmission of a G protein between the insulin receptor and phosphoinositol- phospholipase C, thereby, even though insulin binding is not inhibited (Magri et al., 1990) its signal is being interrupted. Borland et al. (1994) determined that ashort- lived protein may also be needed for ST to inhibit lipogenesis in sheep AT. This was determined after actinomycin D, a protein synthesis inhibitor, blocked ST depression of lipogenesis in vitro. Ornithine carboxylase activity was shown to increase during ST treatment and coincided with the decrease in lipogenesis (Borland et al., 1994). Additionally, a polyamine may be needed for maximal ST expression (Borland et al., 1994). In addition to decreasing enzyme activation and interfering with signal transduction, Harris et al. (1993) and Donkin et al. (1996) have shown that ST can alter enzyme synthesis. Harris et al. (1993) demonstrated a 74% decrease in FAS mRNA with pST treatment for 11 d and Donkin et al. (1996) determined that FAS mRNA decreased in a linear fashion in porcine adipocytes with increased dosages of pST. 129 In the present study, ICD was numerically lower (16 to 26%) in AT of bST- treated steers as compared to control. In support of these findings, similar ICD activity was observed in lactating ewes (Vernon et al., 1991a) and lactating cows (Lanna et al., 1995). In a similar fashion, Ingle et al. (1973) observed a decreased in vitro lipogenesis, as measured by acetate incorporation into FA, and a significant reduction in acetyl CoA synthase (ACS) and ACC with fasting; but did not show a significant decrease in ICD (although, numerically activity decreased 15 to 30%). Additionally, Ingle et al. (1972) observed a tendency in ruminant AT for the dehydrogenase to have greater activity when rates of FA synthesis were greater. Lipolysis. Basal-glycerol release was increased in AT from bST steers after 103 d of treatment. Stimulated-glycerol release was not affected by treatment or time on feed. This agrees with Jones and Marchello (1983) who demonstrated that basal and epinephrine-stimulated lipolysis did not change with the length of time on feed from 166 to 214 d. Pothoven et al. (1975) found no correlation between basal and stimulated lipolysis which appears to be the same as in the present study. Rule et al. (1992) observed a 134% increase in basal glycerol release per mg protein with increased BW of Holstein steers, from 277 to 528 kg. Greater lipolytic rates with changes in BW were observed in AT from perirenal (309%), omental (200%), and intermuscular (111%) depots than inner or outer backfat (Rule et al., 1992) without a change in stimulated lipolysis. Pothoven et al. (1975) observed an increase in basal-glycerol release as crossbred steers increased in weight from 363 to 505 kg, but a decrease in epinephrine-stimulated lipolysis. An increase in basal- glycerol release without an increase in epinephrine-stimulated lipolysis would 130 suggest a change in enzyme activity without a change in enzyme quantity (Vernon, 1980). Egan et al. (1992) demonstrated that the lipolytic stimulation of adipocytes caused the translocation of hormone-sensitive lipase (HSL) from the cytosol to the lipid droplet resulting in an increase in lipolysis. Hart et al. (1984) found similar lipolytic activity of AT incubated with rbST as measured by glycerol release. Doris et al. (1996) found basal-glycerol release in AT was increased 59% in vitro in sheep given ST for seven days. The AT from ST- treated sheep also responded to a greater extend to the B-agonist, isoproterenol, than AT from control sheep. Additionally, AT from ST-treated sheep responded less to the anti-lipolytic agent, NG-phenylisoproyladenosine, an adenosine analog, than control AT (Doris et al., 1996). Like in the present study, Doris et al. (1996) did not find an increase in glycerol response to a catecholamine with ST administration. Vemon (1982), Hart et al. (1984), and Walton and Ethertcn (1986) did not observe an increase in glycerol release when sheep and swine AT, respectively, were treated in vitro with ST. Additionally, Kramer et al. (1993) and Sinnett-Smith and Woolliams (1989) observed similar glycerol release in AT from pigs and sheep treated with ST in vivo. Dunshea et al. (1992a) suggested that the increase in lipolytic activity may in part be related to decreased insulin sensitivity, which would decrease the inhibition of lipolysis. Increased lipolytic activity may also be the result of a heightened sensitivity or responsiveness to lipolytic stimuli as suggested by Boisclair et al. (1997). Through calculations, grams of triglyceride mobilized per day, Dunshea et al. (1992b) determined a 50% increase in lipid mobilization with 131 pST (56 to 109 g of triglyceride mobilized per day). Although when compared to the reduction in lipid accretion ( > 200 gld), the increase in lipolysis would constitute approximately 25% of the reduction in daily lipid synthesis, the balance being reduced lipogenesis. Measures of in vivo lipolytic activity were increased in bST steers after 40 d of treatment as seen by the increased response to the epinephrine challenge. This is in agreement with Peters (1986) who observed an increase in plasma NEFA response area to a single epinephrine challenge with ST administration. Although, Peters (1986) saw a greater response with feed restriction than with ST treatment. Likewise, Sechen et al. (1990), found that lactating cows receiving bST had greater response in circulating levels of NEFA at all doses of epinephrine. In the current study, plasma glycerol did not increase in response to the highest dose of epinephrine. In contrast, Sechen et al., (1990) observed an increased maximum response in plasma glycerol from epinephrine challenges. Adipose tissue has very low glycerol kinase activity, so alterations in glycerol response relates to lipolysis, whereas, NEFA alterations reflect mobilization (ie. the difference between lipolysis and NEFA re-esterification, Sechen et al., 1990). The EDso for the NEFA response in the present study and in Sechen et al., (1990) was not altered by bST. Sechen et al. (1990) suggests that the absence of any changes in E050 make it unlikely that the elevated glycerol response was due to changes in epinephrine clearance rate or the number of epinephrine receptors, and further suggest that a change in a post-receptor mechanism enhanced lipolysis. Lanna et al. (1995) did not observe an increase in basal-glycerol release in vitro, but an increase in HSL activity of AT 132 was observed with bST administration to lactating cows (Lanna et al., 1995 and Liesman et al., 1995). Roupas et al. (1991) suggested that if ST inhibits a G inhibitory protein (G,), then the G stimulatory protein axis may allow greater activation and subsequent stimulation of lipolysis. Doris et al. (1994) demonstrated in rats that ST alters the amount of Gi2 proteins in rat AT, which inhibit the CAMP-signaling systems which inhibit HSL. Less repression of HSL will allow greater stimulation of the enzymes involved in lipolysis. Although in a follow-up study in sheep, Doris et al. (1996) did not find altered quantities of the Gi protein. Based on work by Doris et al. (1996) it can be hypothesized that ST alters the CAMP-based signaling systems of AT by several mechanisms. The first, by increasing the maximum rate of B-adrenergic- stimulated lipolysis, due to an increase in the number of BAR. Secondly, decrease the response of AT to lipolytic agents such as adenosine and prostaglandin E,, and lastly, decreasing the production of the anti-lipolytic agent prostaglandin E2 by the adipocyte. These factors would explain why Hansen et al. (1997b) observed an additive effect in the reduction of carcass lipid content when both pST and the B- agonist, salbutamol, were administered together. Implications Bovine somatotropin is a potent modifier of growth in cattle that alters the composition of beef. It is clear that somatotropin increases carcass protein accretion, and efficiency, while drastically reducing carcass lipid accretion and content. The majority of the reduction in lipid accretion is a result of depressed lipogenesis through decreased glucose utilization and decreased activity and 133 quantity of acetyl CoA carboxylase and fatty acid synthase. In conjunction, lipolysis is enhanced through suppression of antilipolytic factors such as adenosine, insulin, and prostaglandins. Although, the mechanism in which somatotropin stimulates lipolysis in growing animals is less clear. 134 CONCLUSION Based on the results from the previous discussed studies, the following conclusions can be drawn. Both studies showed somatotropin (ST) decreased dry matter intake (DMI) later in the feeding program and improved feed efficiency. Both affects would be beneficial to the economic feasibility of using ST. Although, due to the decrease in DMI, increased nutrient density may be necessary to obtain the full growth potential of ST. The increase in dietary CP content from 11.5% in Chapter 2 to 13.5% in Chapter 3 did not increase daily gains of either the control or ST-treated steers. Estimates of undegradeable intake protein supplied, would have allowed gains to be higher than observed. Houseknecht et al. (1992) suggest that due to limitations in amino acids supplied from microbial and ruminal escape protein in young-growing cattle, amino acid supply may limit nitrogen retention response to ST. Before ST becomes commercially available, a better delivery system with a longer payout period needs to be developed to reduce the frequent injections used in this study. The author understands that a subdermal pump is in development that would allow longer treatment periods and the pump could be removed at harvest. Contrary to the assumptions by Early et al. (1990a), giving ST to light-weight steers, for a longer period, did not increase the carcass components in relation to the noncarcass component of the animal. Noncarcass weight (NCW) was greater in ST-treated steers as compared to control steers in both studies, although, steers receiving ST only during the first part of the feeding program (bST-C, Chapter 2) 135 had similar NCW as control steers at harvest. Red meat yield was improved in both studies with the administration of ST. Somatotropin increased ribeye area, carcass protein composition, protein accretion and improved yield grade. As expected, ST reduced measures of carcass lipid content (backfat, kidney-pelvic-heart fat, marbling score, carcass lipid composition, and lipid accretion). Unfortunately, on an economic basis, the reduction in carcass value due to the decreased USDA quality grade was greater than the premiums associated with increasing red meat yield and decreasing yield grade. At the start of this research there was a drive to decrease the fat content of beefto compete with the healthy image of poultry. Additionally, there has been a drive to increase the economic efficiency of bringing beef to the table. As mentioned in the Introduction, it costs about $2 billion to remove the fat from beef carcasses that producers spent $2.4 billion to put on (HD. Ritchie, personal communication). The reduction in external fat would be a clear example where ST would be beneficial to the beef industry. The resurgence of steak houses in the US. has poised another problem. Although the need to reduce external fat is apparent, consumers still demand a quality steak which requires a carcass to grade USDA choice or higher. Therefore, a genetic or a pharmacological method needs to be developed to minimize external fat but maintain marbling in the muscle. It does not appear that ST is the answer for this production segment. Somatotropin would be useful to produce cattle that, overall, have a reduced amount of carcass fat with increased red meat yield. Consistent with previous research, the decrease in lipid accretion was 136 primarily due to a decrease in lipogenesis with some added benefit from increased lipolysis. As discussed previously, ST administration decreases the activation state of lipogenic enzymes (Vernon et al., 1990a), decreases enzyme activity (Chapter 3), and reduces enzyme quantity (Harris et al., 1993; Donkin et al., 1996) contributing to the decrease in lipid accretion. Adipose tissue samples collected from the author’s study are being used by another colleague to look at changes in lipogenic enzyme mRNA abundance. In the author’s study, there was a problem with limited adipose tissue during the third biopsy. Using the initial-harvest steers to get a baseline value would have been possible and would have eliminated one biopsy per animal. Consequently, the first and third biopsy would hot have been taken from the same location. Although, using the initial-harvest steers to establish the base line would have eliminated the opportunity for each animal to serve as its own control The present study did observe an increase in lipolysis with ST administration as measured by in vitro glycerol release and in vivo with a series of epinephrine challenges. Consistent with the understanding of the control of hormone-sensitive lipase (HSL), the quantity of the enzyme did not change as evidence by the lack of response in the values for epinephrine-stimulated glycerol release. Therefore, the change in lipolysis would be due to change in HSL activity. A phosphorylation site on HSL has been identified for the B-adrenergic agonist, isoproterenol, and showed the associated increase in lipolysis (Anthonsen et al., 1998). No such site has yet to be demonstrated for ST, and would be an area for future research. Also, Egan et al. (1992) demonstrated that lipolytic stimulation of adipocytes caused HSL to 137 translocate from the cytosol to the lipid droplet increasing lipolysis. This has not been shown, to date, with ST. Further research must be aware of the problems associated with excitability of the animals and the possible rise in plasma NEFA and glycerol due to excitability as compared with the effects of ST. In the present study, the author may have been overly ambitious to conduct the epinephrine challenges on ten steers at one time. By reducing the number of animals, the human participants would not need to be as hurried in the collection and possibly reduce the potential excitability of the steers, and improve the results. Regarding both lipogenesis and lipolysis, changes in the number of cells per gram of adipose tissue over time and with ST administration should have been documented. This would have allowed the in vitro measures of lipid metabolism to be expressed on a per cell and a per gram of tissue basis. As with any new technology, its acceptance by the consumer is required. Researchers and producers must be aware of effects the new technology has on tenderness, palatability and shelf-life of the resulting product. For example, the introduction of the callipyge gene in sheep, although it adds significant quantities of red meat to the carcass, the carcass is less tender and is unacceptable to the consumer. Somatotropin can fit into a beef production system that produces lean red meat from decreased lipogenesis and increased lipolysis, but further evaluation of the end product must be completed. 138 APPENDICES 139 APPENDIX A: Raw data and procedures from Chapter 2 (Experiment 809002) 140 M! 3.9 34 EN EN .3... 8.2 38 8.8 6.8 New mop «.2. o.vm o.vN om? ms? «Nu omn msm «NF vow Nov o.m.. mNm F. N oéN fit mom omn mom F. C omF mo? NNP N.vm o.o.. m. N mow Nox- nmn hoe r.N.. No to. o.Nw vvm SN 65 m5. Non QE- v.om mo E- N? mop odm vow NmN v.5 Non ooh mom No no 0.3. Q: :m m.oN Nov Nov N. E omn va m. 3 n N.mF m.9 odm m.oN _..oN of. N. K mm» N. 3 v. 3 m e\e 6.0.. axe 6.9. 29809.6 23.09.36 9. 6st 9. . Locum axe 6.99m .x. :25; axe 6:8 .95 £6 6.22.. .x. .863 606 .o .x. com .86... 6:2. .80... 5%.” 88.60 6:8: cow 2% £91 Nooo0m EoEanxm .0. 5:63:50 mmmofio .mmamz _m.._c_ .n T< flow... o. m m.mm N... o.mN RN mom Nmm omo mwv 30o five vo.. NON o.mm v.ov N. F m.mN mom No v ..m mno Fvv ONvm N.m_. om? vow _..vm m.Nm N. F FNN ooo voN Pom moo mmv mono Nor mow om .. fivm odm N. v v.oN voo vom 00v moor Km mNom mm? mNN No v.ov o. _.v N. P m.mN 5m oom on oom ovm vvoo s.c.. mm? vn fivm r. m N. .. v.mN mow mvm Nom moo Nmo onm N .o v _.o.. no finm mmm N. .. m.mm mow Nvm ovm No.. oNv omnm five NON n m Km N.mm N. F mmN oom mnv vmv mom Ev mmmm o.vw 0 N m 8.... :3 85 .8? EE .5. .5 a .5, 26:5 29¢ 53 a a a 9. 9_ .5, .62 low. fits mono-.8 .oI xomm mama mzwocficogEmw o :25 6:3. res two... :5. cacao J; .2,... J? on... «moan... .620 NOOGOm acmEtmaxm .05. mozmtmuowhmfio mmmohmo ummamfi _NEC— .mrt< GEN-P 141 Table A-2. Record of removal of steers from Experiment 809002 and disposal Steer no. Date removed Reason Disposal Date 1 3/16/92 Harvest #1 Killed 3/16/92 2 4/1 3/92 Harvest #2 Killed 4/1 3/92 4 7/21/91 Below 50% ADG Killed 3/16/92 5 3/16/92 Harvest #1 Killed 3/16/92 6 4/23/91 Initial Kill Killed 4/23/91 7 4/30/91 Initial kill Killed 4/30/91 8 11/11/91 Intermediate Harvest Killed 11/11/91 9 5/1 1/92 Harvest #4 Killed 5/1 1I92 10 11/11l91 Intermediate Harvest Killed 1 1/1 1/91 1 1 11/11/91 Intermediate Harvest Killed 11/11/91 12 5/1 1/92 Harvest #4 Killed 5/1 1/92 13 3/16/92 Harvest #1 Killed 3/16/92 14 11/11/91 Intermediate Harvest Killed 1 1/1 1/91 16 4/27/92 Harvest #3 Killed 4/‘27/92 1 7 4/1 3192 Harvest #2 Killed 4/13/92 18 4/27/92 Harvest #3 Killed 4/‘27/92 19 8/31/91 Below 50% ADG Killed 11/11/91 20 3/16/92 Harvest #1 Killed 3/16/92 21 3/16/92 Harvest #1 Killed 3/16/92 25 5/1 1/92 Harvest #4 Killed 5/1 1/92 27 11/11/91 Intermediate Harvest Killed 11/11/91 29 4/27/92 Harvest #3 Killed 4/27/92 31 4/27/92 Harvest #3 Killed 4/27/92 32 11/1 1l91 Intermediate Harvest Killed 11/11/91 34 4/1 3/92 Harvest #2 Killed 4/1 3/92 37 4/27/92 Harvest #3 Killed 4/27/92 38 4/1 3/92 Harvest #2 Killed 4/13/92 39 1 1/9/91 Injected before harvest Killed 3/16/92 40 1 1/1 1l91 Intermediate Harvest Killed 1 1/1 1l91 42 3/16/92 Harvest #1 Killed 3/16/92 43 4/27/92 Harvest #3 Killed 427/92 44 4/1 3/92 Harvest #2 Killed 4/1 3/92 45 11/1 1l91 Intermediate Harvest Killed 1 1/1 1/91 46 3/16/92 Harvest #1 Killed 3/16/92 47 4/27/92 Harvest #3 Killed 4/27/92 49 5/25/91 Below 50% ADG Killed 11/1 1/91 50 3/16/92 Harvest #1 Killed 3/16/92 54 3/16/92 Harvest #1 Killed 3/16/92 55 5/1 1192 Harvest #4 Killed 5/1 1/92 56 10/26/91 Below 50% ADG Killed 4/13/92 57 4/27/92 Harvest #3 Killed 4/27/92 58 4/27/92 Harvest #3 Killed 4/27/92 142 Table A-2. (cont’d) Steer no. Date removed Reason Dismsal Date 59 511 1192 Harvest #4 Killed 5/1 1192 60 11111191 Intermediate Harvest Killed 1 1/11191 62 411 3192 Harvest #2 Killed 411 3192 63 511 1192 Harvest #4 Killed 511 1192 65 411 3192 Harvest #2 Killed 4/1 3192 67 4123191 Initial kill Killed 4/23/91 70 411 3192 Harvest #2 Killed 4/1 3192 72 1 1111191 Intermediate Harvest Killed 1 111 1191 73 4/1 7192 Below 50% ADG Killed 4127/92 74 4123191 Initial kill Killed 4123/91 75 4127192 Harvest #3 Killed 4127/92 76 4127192 Harvest #3 Killed 4127192 77 311 6192 Harvest #1 Killed 3116192 78 11/1 1191 Intermediate Harvest Killed 1 1/1 1/91 79 1 0121 191 Died Necropsy/lncinerated 80 3116/92 Harvest #1 Killed 3/1 6192 83 3116192 Harvest #1 Killed 3/1 6192 84 10121 191 Died Necropsy/Incinerated 85 1 111 1/91 Intermediate Harvest Killed 1 111 1191 86 511 1192 Harvest #4 Killed 511 1192 87 4/1 3192 Harvest #2 Killed 411 3192 88 10126191 Below 50% ADG Killed 3116192 89 5/1 1192 Harvest #4 Killed 5/1 1192 90 4/1 3192 Harvest #2 Killed 4/13/92 91 3129192 Below 50% ADG Killed 5/11192 92 4130/91 Initial kill Killed 4130/91 94 12121191 Below 50% ADG Killed 411 3192 95 11111191 Intermediate Harvest Killed 1 1/1 1191 97 4127/92 Harvest #3 Killed 4127/92 98 1 1/1 1191 Intermediate Harvest Killed 1 1/1 1191 99 1 0/1 5191 Respiratory problems Killed 3/1 6192 100 1 1/1 1/91 Intermediate Harvest Killed 1 1/1 1191 101 4113192 Harvest #2 Killed 4113192 102 311 6192 Harvest #1 Killed 3116192 103 5/1 1192 Harvest #4 Killed 5/1 1192 104 5/1 1192 Harvest #4 Killed 5/1 1192 105 8131/91 Below 50% ADG Killed 1 1/1 1191 106 11111191 Intermediate Harvest Killed 1 111 1191 107 4127192 Harvest #3 Killed 4127/92 108 4127192 Harvest #3 Killed 4127/92 109 3116192 Harvest #1 Killed 3/1 6192 1 10 1 1/1 1191 Intermediate Harvest Killed 1 1/1 1191 143 Table A-2. (cont’d) = Steer no. Date removed Reason Disposal Date 1 1 1 10/14191 Respiratory problems Killed 411 3192 1 12 4127192 Harvest #3 Killed 4127192 1 14 6122/91 Below 50% ADG Died 711/91 1 15 4/1 3192 Harvest #2 Killed 411 3192 1 16 4/1 3192 Harvest #2 Killed 4113192 1 17 3/1 6192 Harvest #1 Killed 3/1 6192 118 11/1 1191 Intermediate Harvest Killed 11111191 1 19 4/1 3192 Harvest #2 Killed 4/1 3192 120 11111191 Intermediate Harvest Killed 11111191 121 4127/92 Harvest #3 Killed 4/27/92 122 3/1 6192 Harvest #1 Killed 3/1 6192 123 2117192 Below 50% ADG Killed 3/1 6192 124 1 1123/91 Below 50% ADG Killed 3/1 6192 127 8131191 One testicle/Not castrated Killed 1 1/1 1191 128 11111191 Intermediate Harvest Killed 1 1/1 1191 129 1111 1191 Intermediate Harvest Killed 11/1 1191 130 411 3192 Harvest #2 Killed 4/1 3192 133 5/1 1192 Harvest #4 Killed 5/1 1192 135 8131/91 Below 50% ADG Killed 1 1/1 1191 1 36 4127192 Harvest #3 Killed 4127192 137 715191 Below 50% ADG Killed 1 1/1 1191 1 39 411 3192 Harvest #2 Killed 4113192 140 813191 Below 50% ADG Killed 1 1/1 1191 141 1 119191 Below 50% ADG Killed 311 6192 142 511 1192 Harvest #4 Killed 5/1 1/92 143 3/1 6192 Harvest #1 Killed 3/1 6192 144 411 3192 Harvest #2 Killed 4/1 3192 147 4113192 Harvest #2 Killed 4113/92 148 4126191 Poor health Killed 1 1/1 1191 150 4123191 Initial Kill Killed 4123/91 151 5111/92 Harvest #4 Killed 5/1 1192 153 11111191 Intermediate Harvest Killed 1 111 1191 155 511 1192 Harvest #4 Killed 5/1 1192 157 11/1 1191 Intermediate Harvest Killed 1 1/11/91 157 4/1 3192 Harvest #2 Killed 411 3192 158 511 1192 Harvest #4 Killed 5/1 1192 159 411 3192 Harvest #2 Killed 4/1 3192 161 4127192 Harvest #3 Killed 4127192 163 4127192 Harvest #3 Killed 4127192 164 4130191 Initial kill Killed 4130/91 165 511 1192 Harvest #4 Killed 5/1 1192 166 4127192 Harvest #3 Killed 4/27/92 144 Table A-2. (cont’d) Steer no. Date removed Reason Disposal Date 170 4127192 Harvest #3 Killed 4127192 171 11/11/91 Intermediate Harvest Killed 11/1 1191 172 11111191 lnterrnediate Harvest Killed 11111191 173 1 1/1 1191 Intermediate Harvest Killed 1 1 /1 1191 180 5/1 1192 Harvest #4 Killed 5/1 1/92 1 83 3/1 6192 Harvest #1 Killed 3/1 6192 184 3/1 6192 Harvest #1 Killed 3/1 6192 187 11/11/91 Intermediate Harvest Killed 11111191 1 92 3/1 6192 Harvest #1 Killed 3/1 6192 194 511 1192 Harvest #4 Killed 511 1192 1 95 4/1 3192 Harvest #2 Killed 4/1 3192 1 96 411 3192 Harvest #2 Killed 4/1 3192 1 97 3/1 6192 Harvest #1 Killed 3116/92 1 98 311 6192 Harvest #1 Killed 3/1 6192 199 5/1 1/92 Harvest #4 Killed 511 1192 201 11111191 Intermediate Harvest Killed 1111 1/91 202 4130191 Initial kill Killed 4130/91 203 5125/91 Below 50% ADG Died 6/4/91 205 4113/92 Harvest #2 Killed 4/1 3192 206 5125191 Below 50% ADG Killed 11/11/91 208 6122191 Below 50% ADG Killed 3/1 6192 210 5125191 Below 50% ADG Died 718/91 211 11111191 Intermediate Harvest Killed 11111191 212 4127192 Harvest #3 Killed 4127192 213 11111191 Intermediate Harvest Killed 11111191 215 3/1 6192 Harvest #1 Killed 3/1 6192 216 314192 Severe Inflammation of R. foot Killed 511 1192 217 3/1 6192 Harvest #1 Killed 3/1 6192 21 8 4/1 3192 Harvest #2 Killed 4/1 3192 221 5/1 1/92 Harvest #4 Killed 5/1 1192 222 3/1 6192 Harvest #1 Killed 3/1 6192 225 311 6192 Harvest #1 Killed 3116/92 227 11/1 1191 Intermediate Harvest Killed 1 1/1 1/91 228 4/1 3192 Harvest #2 Killed 4/1 3192 231 1119191 Below 50% ADG Killed 4/1 3192 232 3116/92 Harvest #1 Killed 3116/92 234 511 1192 Harvest #4 Killed 511 1/92 236 4/27192 Harvest #3 Killed 4127192 237 111 8192 Below 50% ADG Killed 4127192 238 3116192 Harvest #1 Killed 311 6192 239 4127192 Harvest #3 Killed 4127192 241 3/1 6192 Harvest #1 Killed 3/1 6192 145 Table A-2. (cont’d) Steer no. Date removed Reason Disposal Date 243 411 3192 Harvest #2 Killed 4/1 3192 146 oon ono ooo oNe oNe non oon oon oNn oen oon 5..-5.. e N o Nn oon ono o no one oee n we oNe o .e ooe onn oeN 5.3 n n n en oon ooo ooo one ooe nen onn oon oNn oon oNN 0-5.. N F 2 Nn oon onn ooo ono oNe oz. oNe one ooe onn oNN 0-5.. N N N on non ooo ooo oee ooe onn onn onn oon oNn oNN 0-0 F N n oN ooo ooo one one oNn nen oon oen oNn oon oNN 0.5.. N n : NN ono ono oNe ooe oon oon oen oon oNn oon onN 0-0 F F on oN oon onn oon ono oNo one one one one oNn oNN 0-5.. N e e. .N onN onn oon oNo no . nNe oNe oNe ooe oNn onN 0.5.. N e e. R ooo oNe oNe ooe oon nNn onn oNn oon oNN oNN 5.3 n P N. on oen ooo oeo ooo one :e 2e ooe oon oon onN 0-5.. N N N no onn oon ono oNo one one one one ooe onn oeN 595.. e n N No on ono So oNe oNe nNn oNn onn oon oon oNN 0-0 F N n no ono oeo one one one oon oon onn oNn oNn ooN 0.5.. N N N en oon onN onn oon ono noe one ooo one onn oNN 0.5.. N e e. n. one ooe ooe oNn onn oon oon oon onN oeN onN 0-0 F F on N. ono ono one one oee non oon oon onn oNn onN 0-0 F N n : oon onn oon ono So oNe one oNe one onn onN 595.. e e no on non oNe one oon oen nen oen oen oNn oon oeN 595.. e F o o o .n ono oNo oNe one nnn oon onn oNn onn ooN 5..-0 n N n n ooN oNn oon ooo oNo nne one one one oon onN 0-0 V e n. o oeo oen So oNe oee oNe one one oNe oon own 595.. e n N e oen oon ooo oNo one nee oee oee one onn onN 0-0 F n n N ooN oon oNn ono ooo oNe oNe one one oNn onN 0-0 F e n. n n. :3 n. J; 2 J; n. J; n. J; n. J; n. J; n. 5.; n. J; 2 J; n. _..3 .2. 2 one no .60 Ne N8 nN .8 e. nnn. 3...... N... .n... no a. _no. :0 3.5.2.. 858nm 5.58: .85 one .85 FonoRo _.onch PoRoNoo roNvNNmo _.oBtmo oomom>< FoRNNv wonNNv FoNoEv FoNNNNm _.ng Noonon Egonoxm 5. Eon; new .9. 2.5 147 ono ooo one oee ooe oNn oNn onn oon an oeN 590 n N n nN onn oNo ooo oNe one non oon ooe oNn oon onN 595.. e N o oN oeo ooo ooe oNe oNn oon oon oon onn onN oNN 0-0 F F 8 2 oon ono one one oNn nen oen oon oNn onN oFN 0-0 F F S NN oon onn oNo ooo oNe nNe oNe one oFe onn ooN 0-0 F n F 2 oNn oNo oNo oNe ooe noe ooe oFe nnn onn onN 590 n n n on non ono one one ooe non oon oon oen onN oNN 590 n F NF nn onn oon ooo one one oNe oNe oNe oon oen ooN 0-0 F n F No oon oeo oNe ooe ooe . nnn oon oNn oNn onn onN 595.. e N n on ono 8... one one onn oen oen oen an onN oFN 590 n F NF no oNn ono no one onn nnn oon onn oNn an oeN 0-5.. N N N no oNn oNo ono one oFe non ooe nnn onn onN oNN 0.5.. N N N No oFN onn an oNo one one one oee oNe onn oeN 0.5.. N n e no 8.... oNo one oFe onn an an an onN ooN oNN 0-5.. N F FF no onN onn an oen oNo noe one one one oNn nnN 0-5.. N e 3 en oNN onn oon ooo ooo one one one oee oen onN 590 n e 2 oo oen oon nnn oon onn onn oNn onN 0-0 F N n oe onn ooo oeo one oee ooe ooe ooe onn onN onN 595.. e N o Ne ooN oNn oon ono one nee oee one one oen oNN 0-5.. N e 3 ne ooN oon oon oeo one nee ooe oee one oon onN 0-0 F n F oe onn an ono ooo one one oNe oee oFe onn ooN 0.5.. N n e ee ono oeo one oee oFe ooe ooe ooe oNn oon oeN 0-0 F N n ne onn oon oon oeo ooo nne one one oee oon onN 0-0 F e nF Ne oNn oNo one one ooe nNn onn oNn oon oon onN 595.. e N o oe oNn oen ono oNo oon nne oNe one one oon onN 590 n e oF on non oNn one ooo ooe nNe oNe oNe oFe oNn ooN 590 n n n on n. J; n. :3 n. é... o. :3 o. J; ... .5 n. _..z n. :3 n. J; 2 ....s 2...; .2. E nnn. no nnn. Ne .60 nN >8 2 one 3...... N... _..5 .5 F... as no 35...... 2.283. .558: .85 one .85 3.8.3 FQFNno 5.8.8 FQeNoo FnaFoo none)... FQNN... 3.er Sate FnNQn FenNN 6....8. .n-< mane 148 oon ono ono one one oon oon oon onn oNn oNN 590 n N n NoF oon oon ooo ono oFo one oNe one oee oon onN 590 n e 2 SF oon oNn an ono oFo nNe one one one oNn onN 0-0F e 8 ooF ooo oeo oNe oee ooe onn onn oNn oen oNn oeN 0-0F F S eo. oon ooo one one onn nen oon oen oNn onN ooN 0-5.. N F FF noF oNN oeN oNn an oNo oNo oNo oFo ooo ooe oon 590 n e oF NoF ooN, onn oNo ono one oFe oFe oFe onn an onN 590n n n FoF oon an ooo oFo oee noe one one oNn an onN 0-5.. N N N ooF oon ooo ooo ooe ooe non onn oon oNn oon oeN 595.. e F n no oon an ono ooo one noe oFe ooe onn an oFN 0-0F N n no ooo ooo oFo one oFe nNn oNn oNn oen oNN oeN 0-5.. N N N No oon ooo ono one ooe oNe oNe oNe ooe oNn onN 0-5.. N n e on ooo one oee one onn nen oen oon onn oNn ooN 590 n F NF en oen ooo oen ooo oee oFe oFe ooe oon oen oeN 590 n N n Fn oon oNn ooo one oee noe ooe oFe oon oon oNN 595.. e n N on oNo one one ooe onn oen oen oen onn oon oeN 595.. e F n on one oee onn onn oNn nnN oon onN onN onN ooN 0-5.. N F FF nn oNN oon ooo ooo oNe one one oee oFe onn oeN 595.. e n N No ono oFo one oNe oNn onn onn oon onn oon oeN 0.0F F S on oen an ooo oNo oNe nee oee nee oNe oen onN 0-0F n F on ono oeo oNe one oFe oon oon onn oNn oen onN 595.. e N o en oon oon onn ono ono nNe oNe oNe one oon oNN 595.. e e 2 nn oon onn oNo oFo oNe nee oee ooe ooe oNn oNN 595.. e e 2 on So one one one oen nNn oNn oNn an onN oeN 0.5.. N F FF 2 oNo ooo one one oFe nnn onn oNn oon oon onN 590 n F NF 2 oon onn oon oeo one one one ooe one onn onN 595.. e e 2 NN n. ....s n. ....s n. :3 n. J; n. a...) 21.; n. J; n. a...) n. ....s 2 _..z n. J; .2. E .30 no.8 Ne 30 8 .30 3 30 3...... N... 3... .5 F... 3... ..o 35...... 9.5.93”. .5533 .85 5.. 3.5 Foxmotb 9onng _.oRoNoo _.oNvNNmo FoNotmo oomoo>< FoRNNv PonNNv _.oNotv FoNNNB wok-”NR 8.38. .9... 2.3» 149 onn oon ooo one oee nFe ooe oNe oon oNn oeN 595.. e n N onF ono oeo oNo ooo ooe oNe oFe oNe ooe onn ooN 0-5.. N n e NnF onn ooo oFo one oFe oNn oNn oNn oon onN onN 0-0 F N n on. oeo So one oee ooe oNn oNn onn oen oNn onN 0-0 F F 8 onF oNo oNo one oFe oen an an an oon onN onN 0-5.. N F FF nnF oon oon ooo So one one one one oFe onn onN 590 n n n onF oNN nnn oon oeo ooo one one one one oon onN 595.. e e 2 nNF onn oNn ono oFo oee oNe oNe one one onn oeN 590 n n n nNF oNN oNn onn ono oFo nNe oNe oNe ooe oon oon 595.. e e S NNF oon oeo oNo one oNe ‘ onn oon onn an ooN 590 n N n eNF oon ono ooo one oNe nNn oNn oNn oen an onN 0.5.. N N N nNF oon onn onn ono oNo one one one one oNn onN 0-0 F e 2 NNF ooo ono one one oFe onn onn onn onn oNn onN 595.. e N o FNF oFN onn oNn ono ono nNe oNe oNe ooe oNn onN 0-0 F e 2 oNF onn ono ono ooo one nee oee oee oFe oNn onN 0-0 F n F nFF oNn oon ono one one non ooe oon onn oon ooN 590 n N n nFF onn oen ooo ono ooo nne one oNe oee oen onN 595.. e e n. NFF oNn onn ono oNo one oee oee oee oFe onn oeN 590 n n n nFF onn an ooo one oee one one one oNe oon onN 590 n n n oFF one oFo oNe oon nnn oNn onn oen an ooN 0-5.. N F FF eFF ono ono one one oee oon oon oon oNn an onN 595.. e N o NFF ooo one one onn oon an an an onN oNN oNN 595.. e F n FFF ooo So one oNe oNn oon oon oon onn onN oeN 595.. e F n oFF onN oon onn oNo ono one one oNe one oNn onN 0-5.. N e 3 SF onn oNn ooo oNo one oFe oNe oFe ooe oNn oeN 595.. e N o n8 e _..z n. J; n. :3 n. J; n. ....s n. J; o. :3 n. ....s n. ....s 24...... n. J; .2. E .30 no .30 Ne >8 8 .30 3 30 3...... N... 3.... .5 Fe 3.... ..o 35...... 9.3381 .558: v.85 5.. .85 «oNn—95o FQFNBO SEES woNvNNmo PoBCmo oou.o>< _.oRNNv 5‘va woxotv woNNNNm FoBNNN cue-.00. .o-< oEm.- 150 151 onn oNn ono ooo ooo nee oee one one onn oNN 595.. e e 2 NnF oeN ooN onn ono ooo oNe oNe oNe ooe oNn oon 0-5.. N e 2 NnF onn onn ono oeo ooo oNe oNe oNe oee oNn onN 590 n e oF en. o.N ooN onn oNo ono nNe one oNe ooe nnn onN 0-0 F e nF nn. oNn oNo oNo oNe o.e onn onn oNn onn on onN 595.. e F n onF ono oeo ooo ooe onn noe onn oFe onn oon oNN 595.. e n N nNF an ooo ooo oNe onn nnn oNn onn oon onN ooN 595.. e F n NNF oNo oee oNe ooe oon nFn o.n oNn oon onN oeN 590 n F NF FN. oen oNn ooo one oee noe oFe ooe oFe oon oeN 590 n N n oNF oen oon ooo ooo one nFe oNe ooe ooe oNn ooN 590 n N n nnF ono ooo ooo one onn . oNn oNn oNn onn onN oNN 0-5.. N F FF onF oen oon ono ooo oee ooe ooe ooe oNn an oNN 590 n N n nnF onn ono ono ooo one onn onn onn onn onN oNN 0-0 F N n FnF oNn onn ono ono oNe nee oee oee one oen ooN 0-5.. N n e noF ono oeo oNe one onn oen oen oen an an oeN 590 n F NF noF onn oon oon oeo ooo noe oee one nee onn oNN 0-5.. N e 3 NoF ooo oNo one one onn non oon onn oen onN onN 595.. e F n no. oNn ono oNo one oee o.e oFe oNe onn onn onN 0.0 F n F eoF onn oNn ono oNo one oee one oee oNe oNn onN 590 n n n noF ono ono So one onn onn onn oon onn ooN onN 0-5.. N F FF .oF onn ono ooo ooo one 8.. oNe oFe oFe oen onN 0-0 F n F NeF onn o.n ono ooo ooe nNe oNe one onn onn onN 590 n n n eeF ooN onn an oNo ono oNe oNe oNe oNe onn onN 590 n e 2 ne. ooo one oNe onn onn nFn on on oNn onN onN 590 n F NF Ne. oNo ono one oee o.e onn onn onn oNn onn onN 0-0 F N n Fe. onn an ono ooo oFo oNe one oNe oNe oon onN 0-5.. N e e. oe. ... ...... ... ...; ... ...; ... ...... ... ...... ......z ......z .. ...... ... ...... ... ...... ... ...... .2. 2 ..no no .8 Ne .30 nN .8 e. ..no 3...... we 3... ..o 2.. 3... ..o 33...... 2.55.. .555: ......n 5.. 5....n FonoRo ooN—NEG wot-90° 3330 33:8 ooooo>< rot-N? FonNNv FoNoEv _.oNNNNm PoNoNNN 6.38. .n-< 2.3. onn oon ono ono o.o noe one one one oon oon 0-0 F e 2 NnN onn onn oNo oNo one oee oee oee o.e oNn oNN 0-0 F n . FnN onn onn oNo no one n.e oFe oFe onn oen oNN 595.. e n N nNN oen an ono one one nne one oNe oee oNn onN 0-0 F e n. NNN oNN onn ono ono one one one one oNe onn ooN 595.. e e 2 oNN oNn oen oon oNo one noe one one oee oon onN 0-5.. N e 3 NNN oon oeo one one onn nen oon oen onn oNn onN 0-0 F F 2 FNN onn oon ono o Fo one one oNe one one onn onN 595.. e n N nFN oNn oon an ono ooo oNe oNe oNe ooe onn an 595.. e e n. NFN oFN oon ono oNo oNe one oNe one one oon onN 595.. e n N nFN onn onn ono ono ooo , ooe one one one onn onN 590 n e 2 oFN oNn oen ono ono ooe oee one oee Se oen oNN 595.. e n N nFN ono ono So one onn nnn onn onn onn o.n onN 0-0 F N n NFN oFN oon ono ooo oee n.e oFe oFe onn oon onN 0-5.. N n e FFN onn onn onn onn onn onn oen oNN 590 n N n oFN oNe ooe one one onn onn onn onn oNn oNN 595.. e N o noN an an n.n an oNn o.n oNn oeN 595.. e F n noN onn oNn oNo oFo ooe oNe oFe oNe onn oen onN 0-5.. N n e ooN o.e one nNe one oNe ooe onN onN 0-0 . n . noN oeo oFo oee ooe oen nFn o.n an oon onN onN 0-0 . . 2 EN an ono ooo ooe onn non onn oon oen oNN 0.0 F . oF nnF onn onn ono ono ooo one one one ooe oon onN 0-0 . e 2 nn. ooN oNn an ono oNo one one one one onn oNN 590 n e oF Nn. oon oon oNo oFo one n.e ooe one onn oen oNN 0-5.. N n e nnF oNn oen ono oNo oNe one oNe one oFe oen onN 0.5.. N n e on. oeo ooo oee oFe oNn nnn oNn oen o.n oon onN 590 n F N. enF ... ...; ... ...; ... ...; ... ...... ... ...; ... ...... ...ns. ... ...... ... ...; ... ...; ... ...... .2. 2 .30 no .8 Ne 3.. 8 ..no 2 ..no 3...... N... 3... ..o ... 3... ..o 35...... 9.55m .555: .85 5... .55 wonoR-o PQPNBc wot-QR. _.oNvNNmo _.oBSmo ooo5>< FoRNNv vonNNv woNoEv _.oNNNNm _.oNoNNN ......8. .9... 2.3. 152 onn ono oNo oNe oNe ooe ooe oFe ooe oen onN 0-5.. N n e neN onn oNn an ono oNo one one one one oon onN 590 n e o. FeN oNn ono ono one one one ooe onn onn oen onN 0-5.. N N N nnN oNN onn onn ooo o.o one one oNe one oNn oNN 590 n e o. nnN ono ooo one one onn nNn oNn oNn onn oen onN 590 n F N. NnN oon oon ooo ooo ooe onn ooe onn onn onn onN 0-5.. N N N nnN oeo oNo oNe oNe onn oon oon oon oen an onN 595.. e F n enN ... ...; ... ...; ... ...; ... ...... ... ...; ... ...... ... ...; ... ...; ... ...; ... ...; ... ...; .2. 2 .30 5...... Ne .30 nN .30 e. ..no 3...... N33... ..o .3355 35...... 9.53.3”. .555... ......n 5.. .55 .233 5.58 3.8.8 .n.eN.oo 55:8 33.9.... Fn.NN.e .2er Fonze .n.NN.n Fn.nN.N 3.58. .n-< 2.3. 153 mmo oom omo mvm mmo mNo oon mvn m :- mnm ovm o em on o For 93 mmo omo omo mNo mom mmo m: mNN ooh omm no moo omo mvo mvm mvm mNm ooh mmn oNN mom mmo ovm vm o _.o o no ooo moo ooo o _.o omo m 5 oon ooh mom mvm mNm Nm oNo _. mmo moo omo o _.o _. oNo omo ooo ohm mmo mos mvh _.m mvo oNo ooo ooo oom mum mvm oon o:- mos ooo omo oN mvo mNo ovo moo moo m _.o mom o 5 mos omn o E mmo o 5 RN ono mvo m.Nom mom ooo mmo ovm mt. ooh mom mmo oNo mN omo w omo m.Nmo omo mmo mmo ooo omo mNm mos ooh mvn ..N moo _. m o F ooo mmo moo oNo omo moo mmo mNm mos mm» on . o _.m mom mom ovm o — mvo F omo omo mmo mmo mNo mom mmo mom mmh o E mom m P mmo? omo? moo ooo.- ooo mmo m o ooo mmo ooh ovh m _.N t oho mNo m.Noo moo ooo ooo mom mo» mvh o K mmm mvm 0.. moo moo ooo mNmm mom omo omo oNo o: mNh ooo omo omo v_. ooo F o: _. m.NoP _. moo r o F P F mmo_. omow moo omo mmo ooo omo or mom mox- mNms ooN. mmn mNN omo mmo omo mom omm mNm N— ovo ovo omo mNmm omo mmm mmm m 3 mm» m2. mom mum omm F P mmo_. mmo.. mvow mNmoF mNoe ovoF moo F mmo omo omo m _.m o...» mos o.- oon onn onN onN oon onN ooN oFN oNn oNn ono ooo n oen oen onn oon onn oon onn onn oon onN onN ooN oNn n 8.. ono. oFoF onn oNoF oFo. onn oNn oon onn o.n oNN o ono e oeo. onn o.Nen oen oen o.n oNn oen onN onN ooN oNn N oNo. oFo. oNnn onn onn onn onn an oen oNn onN ooN F ......s 5...... .22.: ......z ......z ......z ......z ......2 ......z ......s ......z ......z 2...... ......z .8 oFN ..no ......z 5.50 No.30 52.8 5.30 5.30 5.30 3.50 5.30 N530 no 3n. 5.8 .55 ..oNNNNZ. 5029. 23.85.25 539 : oowoo>< ..oRNNoP PonNNoF Po: (or _.oRQoo _.oxm Coo PoNono Fontmo _.oNNoBo 53 (no 3.58. .n-< 2.3. 154 ooo mwo mN Fo moo ooo ono o¢o ooo ooh m FN. ooo mvo on moo F moo ovo ooo omo m F.o moo omo ooh omm oon mno mu moo ooo omo oNo ovo m Fo o2. o: moo ooo oNo com on mmo mmo moo m Fo m Fo m Fo o Fo moo ooo mun omx. o Fm moo Nu m Fo F omo mKNo ooo mNo ooo moo ooo moo mon mos mom on m Fo mno omo mvo mmo mNo o Fo mom on» mos ooo ooo mo ooo mho moo ooo o Fo moo omo o Fo mmn oon ooo mvo no mvo ovo ooo mno moo moo ooo mom oNN oon moo oNo No ooo ooo moo oNo m Fo mmo moo mmo oNo mun ooh ooo ovo oo ooo ooo mmo moo moo ovo mun obs mun moo ooo oNo om o Fo F mho ooo oNo ovo ooo ooo o Fo mun own ooo moo om o Fo F moo ooo mNo mmo ooo ooo o Fo mun on... mow moo hm mNoo ooo moo ooo F o Fo F moo oNo moo mNo mus om mmo mVo m.N Fo moo oNo omo mNo mom mvn mob omo moo mm mom F on F F oNF F o F F F on F F moo F moo F o Fo F moo moo ooo ooh Vm mv F F mmo F ooo F mmo F moo F moo ooo m Fo moo mvo o Fo moh om ov mvo F ooo F mNoo mmo ono oNo o Fo oho oNo mom oNn ooo he ooo F oNo F mKoo ooo moo omo ooo oho m Fo oon ovn mob ow o Fo F oNo F ooo F m.Noo ooo moo mho o Fo ooo ooo ooo on» omh mv mvo F moo omo oFo omo ooo moo moo moo mt. mos o F» 3 moo o Fo ooo moo moo omo ooo ooh ooh ooh ono m Fo ow moo F ooo F moo moo moo mmo mNo moo ooo moo ooh mmh NV ooo omo ovo vao ovo mmo moo mso oNo mob mm... mos moo ov ooo ooo m.~oo moo ooo moo moo moo ohm mow mm» oon on 2...; 5E: 56:: 2...; a}; 2...; 2...; 2...; 2...; 9...; 2...; 2...; 2...; 2...; .2. 228 2...; 828 828 828 828 3:8 318 318 8:8 «:28 8:5 35985 Euggoza; 258,525 332: 8223 32.3. .232 5E2 .258 .238 .288 56:8 .238 55:8 AFESV .m-< 2an 155 156 8.8. 8.8. 8.8. 888 888 888 888 888 88 888 88. 8.. 88. 88. 88. 8... 8. 88. 8. 888 88 8.88 888 88 888 8.8 88. 8.. 88 8.8 888 ..8. 888. 888. 8.88 8.8 888. 88 888 8.8 888 88. 8.. 8.8 88. 88.. 88.. 88.. 88.. 8.. 888. 888. 8.8 888 888 88 88 88. 88.. 888. 888 888 888 888 8.8 888 888 8.8 88. 88. .8. 888 88 888 8.88 88 8.8 88 88 888 8. 88. 8.. 888 88. 888 88. 88. 8.. 88. 8.8 888 88 8.8. 888. 888. 8.888 888 888 888 888 88 8.8 88. 88. 888 88 8.8. 8.8 8.88 8.8 888 888 888 88. 88. 8... 88 88 .8 888 888 888 888, 8.8 888 88 8.8 88 888 8.. 8. 888 88 88 8.8 8.. 8.. 88. 8... 8.. 8.8 888 888 888 888 ..8 88. 888 8.88 888 888 888 888 8.8 888 88. 888 888 .8 888. 8.8. 88 888 888 88 888 888 888 88. 8. 8. 88 888 888 8...8 8.8 888 8.8 888 88. 88. 88. 88 88 88 8.88. 88. 8... 88. 8. 88. 8.8 888 888 888 88 88.. 88.. 8.8. 888. 88. 888. 8.8 8.8 888 888 888 8.. .8 88 8.8 8.8 88 8.8 888 8.8 8. 88. 8.8 888 8.8 8 888. 8.8. 888 88 8.8 88 888 88 88 88. 8. 88. 88 8 8. 8. 8.. 88. 888 88 888 8 8... 88. 8.888. 888. 888. 8.8. 8.8 88 8.8 88 88. 8.. 88 88.. 8.. 8.888. 888. 888. 888. 8.8 888 88 888 88. 8. 88 88. 88. 88. 888 888 8.8 88 8. 888 88 88 888 88 88 888 88. 88. 88. 88 8.8 8.8 8. 88.. 8.8. 8.8. 8.8. 88. 88 88 888 88 888 88. 8... .. 2...; .82.... .88.... 8...? 2...; 8...? 8...? 2...; 2...; 8...? 8...? 83; 8...? 8...? .88 8.8.80 8...? 88.8.88 88.83 88280 .8188 88:88 88:80 83.88 88:80 8.180 88.80 88.8988 Foam: F «8028.. 2850.525 Fo.oo. FF oomB>< FoRQoF Fo\mN\oF 3. F :2 FoRQoo 3% Foo Fo\oo.oo Fo\o F\oo FoNQoo Fo\o FRo 6.8888 .8-< 8.88. nnF ooo ovo oNo o Fo ooo ooo omo moo moo mt. oNN. moo on F mom ohm mom oom mo F mNo F mmo ovo ooo omo moo mvo m Fo omn o Fm ooo m Fo on F mmo F ooo mNmo mvo ooo moo mmo mvo ooo mt. oen ooh on F oN F F mN F F mN F F mNno F oho F mmo F o Fo F ooo m Fo moo mVo mos own oN F moo mvo moo m Fo o Fo oNo ooo omo moo mus o: mos m FF oNF omo ooo com um F moo ooo mKoo ooo mso moo m Fo mun omh oNn moo omo VN F moo F oeo m.N Fo moo oNo mmo moo mob ooh oNn ono omo om F moo ovo mNoo moo ooo ooo moo moo mom mun ooh mvs NN F moo mvo o Fo ooo oNo ooo oNo mvn mmh ooh mmo m Fo FN F mmo F ooo F moo F m.~vo F m3 F ovo F ovo F moo oNo omo ooo m Fo mt. oN F omo m Fo ooo ooo ooo oVo mNo mos m¢n m F» moo omo o F F ooo omo mmo ooo ooo ooo ooo moo ooo oNN m: m Fm mho o F F oo F F omo F mNo F m Fo F moo F mmo mNo moo mvo mob mt. mm» b F F mno F oNo F mKno oNo moo moo o Fo moo mom oon mvs o Fn o F F mvo F ooo mKoo moo ovo moo omo oeo o Fo oou omh mos m F F «F F ooo moo mKNo oNo moo ooo mmo moo on» m E. ooo oNo N F F moo moo mom mun ooo ooo o Fo F F F mmo ooo ooo vao ovo mvo o Fo moo own mos ooo mvo o Fo o F F 88.. 8... 888. 8.8. 88. 88. 888. 8.8 88 888 888 88. 88. 88. 8.8. 888 888 888 888 888 888 8.8 88. 8. 88. 88. 888 88 888 88 88 8.8 888 88 8.. 88. 888 888 .8. 8...? .83... .88.... 9...; 8...? 8...? 2.8; 2...; 8...? 8...... 8...? 8...? 8...? 8...? .88 8.8.88 818; 88.8.8 88.83 88.88 .8180 88.88 .8288 81.80 88.88 8.18 88.88 8.89828 Foam. F F .8028: 285952... FQoQ FF mom.m>< FoRQoF FQonF FQ F :3 FoRQoo Fo\o too FQoQoo Fo.o too FoNQoo Foxo FRo 6.8888 8-1.8.88. 157 158 888. 888. 8.8. 888. 8.8. 888 888 888 888 8.8 88. 88. 88. 88. . 888. 888. 888. 8.8. 888. 888 8.8 888 888 888 8.. 88. 88. 888. 8...8 8.8 888 88 888 8.8 88. 88. 88. 888 88. 888 888 888 8.88 888 888 888 888 888 88. 888 8.8 8.8 8.. 888 888 888 8.8 888 888 888 888 8.8 8.. 88. 88. 888 8.. 888 888 888 8.888 888 888 888 8.. 88. 888 8.8 888 888 ... 888. 888 8.8 8.8 8.8 8.8 888 888 88. 8.. 88. 88. 8.. 888. 888 8.8 888 8.8 8.8 888 8.8 888 8.. 88. 888 88. 888 888 8.888 8.8 88 888 88. 88. 88. 888 8.8 888 88. 888. 888 888 888 88 888 8.8 888 888 88. 88. 88. 88. 888. 8.8. 8.888 8.8 888 888 888 8.8 888 88. 88. 8.8 .8. 88. 888. 8.8 8.8 8.8 888 888 888 88. 8.. 88. 88. 88. 888 88 8...8 888 888 888 88. 8.. 88. 8.8 888 888 88. 888. 888. 888. 888 888 888. 888 88 888 888 888 88. 8. .8. 888 888 888 88 888 8.8 88. 8... 88. 8.8 888 888 88. 888. 888 8.8 8.8 8.8 88 88 888 88. 88. 88. 888 ..8. 888 888 888 8.8 888 888 88 888 88 88. 8.. 88. 88. 88. 888. 888 888 888 8.8 888 888 8.8 88. 88. 8.8 888 .8. 8.8. 888 888 888 888 888 888 888 888 88. 888 888 .... 8.8. 888. 8.8 888 8.8 888 8.8 888 8.8 88. 8... 88. 3.. 888. 888. 888 888 888 8.8 888 888 88 888 88. 8... 8... 8.. 88. 8.... 8.. 88. 88. 8.8 888 8.8 888 888 8.8 8... 88 888 888 888 8.8 88. 8.. 8.8 8.8 888 888 .... 888 888 88. 88. 888. 8.8 888 888 888 888 888 8.. 8. 88. 8.8 88. 2...; .83... .88.... 8...8; 8...8; 8...? 2...; 2...; 8...; 2...; 8...... 8...? 8...; 8...; .88 8.8.88 83; 88188 88.88 88.88 .8288 88.888 8.88 83.8 88.88 8...8 88 .88 8.89828 FoNQF F .8828: 238.53:— Fo\oo\FF oom8m>< FoRQoF Fo\mN\oF 3. F :9 FoRQoo 58 too Fo.oo\oo Fo\o too FoNoBo Fo\o FRo 6.8888 .8-< 8.88,. o FN F mo F F moo F moo F . moo F mmo F o Fo F moo mmo ovo moo mom mNN o F F F omo F moo F moo m Fo F ono ovo m Fo moo o Fo ooh mNn NNN ooo F ooo meo ovo mvo moo mno mvo mom mom ooo ooo FNN ooo F oNo F m.N Fo F moo F oNo F moo ooo ooo o Fo mun mos moo o FN mo F F ooo F mNNo F ooo F mNo F moo F ooo o Fo mNo ooo o2. mvn n FN mo F F m Fo F moo F ooo F o Fo F moo moo o Fo moo mmo mom mmn o FN mmo F ooo ooo moo moo moo omo moo on... mos o?» oon m FN moo moo ooo F mNoo ooo moo mmo o Fo omo ooo mow omn m FM o FN ooo moo ovo moo mvo moo mun omn o F» ooo ooo ooo N FN mmo F ooo F mmo F ooo F mvo F mmo F ovo F ooo m¢o ooo ovo ooo oon F FN o FN ooN ooN o Fo F ooo oNo m Fo mNo moo mmo mos oon oi. oi moo mom ooN moo ooo moo mNoo ooo moo m Fo mos mmn m Fm ooo omo ooo FoN ooo mmo mKoo moo ooo ooo moo m Fo mom m F5 oNn ooo oo F on F F moo F m... Fo F oNo F m Fo F moo oNo m Fo omo ooo mom men oo F mm F F mmo F mNNo F ooo F mmo F moo mvo moo mvo ooo onn mos no F oNo F ooo vao mvo omo m Fo mmo ooo ooo mom ooh ooo oo F mvo F ooo F moo ooo ooo ovo moo moo moo ooh mos mNn mo F ooo mvo moo mos m Fo ooo own mm. ooo moo ooo mom vo F oo F F mo F F mNo F F mo F F o F F F mmo F ooo mmo ooo ooo moo omn No F m F F F oNF F mN F F moo F moo F mo F F mmo F ooo F moo ooo mno oNo ooh no F 8...? 8:5. .88.... 2...: 2...; 8...; 8...; 2...: 8...8; 8...; 8...... 8...? 8...? 2.83.88 8.8.88 8...? 8...8 8828 88.8.8 .828 88.888 .8288 83.88 88.88 8...8 88.88 8.89828 FoNQF F .8828: 23.80.53... Fo.oo. FF mo90>< FoRQoF Fo\mN.oF Fo\ F FBF FoRQoo Fo\o too Fo.oo.oo Fo\o too FoNQoo Foo FRo 6.8888 8.8. 8.88. 159 o F F F mno F mNoo F mNo F ovo F ooo moo ooo oNo ooh ovu ooo oVN oooF oooF mooF mooF mooF mmo oNo moo mmo oFo oon mvn F¢N mmo moo oFo moo mFo ooo moo mFo mus ooh moo moo ooN oo F F ono F mNoo F ooo F moo F oNo F mmo ooo moo moo mos mun ooN moo mvo mNoo moo o Fo ooo mmo m Fo omn o FN ooo ooo noN oooF ooo mNo oNo ooo ooo mmo moo ooh oon mNn ooo ooN ooo moo ovo moo moo oNo mom mos moo omo mNo mom vow mNoF mmo oNo oNo oNo ooo moo ooo mom mvn mob moo Now mmo mNVo moo omo moo ooo ooo ooN mow o3. mow FoN oFFF ovoF oFoF mFoF mooF moo mFo ooo omo ooh mmn mVn oNN oNo ovo moo moo moo m Fo mno mvo ooo mt mmn ooh ooo NNN 160 2...: .83... .88.... 8...; 8...; 8...8; 8...8; 8...? 8...8; 8...8; 8...8; 2...; 2...; 2...: .88 8.8.80 8...8; 8.80 88288 88.88 .8188 88.88 8...8 82.88 88.83 8...8 8.80 8.80.8.8 8.88.3822 2868.528. .888... 888.82 .8888. .8888. .8228. 5.888 88.88 .88888 88:88 .8888 88.8.8 88.8888 .8-< 8.88. th F mom F mom F omo omN F mvm F mo F F mm F F oo F F omo F ovo F moo F mho oo 8.888. 888. 888. ..88 88. 8.8. 888. 888. 888. 888. 8.... 888. 88.. 88. .8 88.. 88.. 88.. 888 88.. 88.. 8... 88.. 8.8. 8.8. 888. 8.8 8.8 ..8 . 88 888. 888. 8.8. ..88 888. 888. 888. 888. 88.. 8.... 88.. 88.. 888. 888. .8 8.88. 888. 888. 888 8.8. 888. 88. 888. 8... 8.... 8... 8.8. 8.8. 888 88 .8 8.8.8. 888. 888. ..8 888. 888. 8.8. 8.8. 888. 8... 88.. 8... 8.8. 888. 888. 88 8.8. 888. 888. 888 8.8. 88.. 88.. 88.. 888. 888. 888. .8 8.8. 888. 888. 888 888. 888. 8.8. 88.. 88.. 8... 88. 88 8. 888. 888. 888. .88 888. 8.8. 888. 8.8. 88.. 88.. 88.. 88.. 888. 888. 8. 8.888. 888. 888. 888 88. 888. 888. 8.8. 888. 888. 88.. 88.. 88.. .. 8...8. 888. 8.8. 888 888. 888. 888. 888. 8... 88.. 888. 888. 8.8. 888 8. ... 8..... 88... 88... 888 888. 8.8. 888. 8.8. 888. 8.8. 8.8. 8. 88.. 88.. 88.. ..8 88.. 88.. 888. 888. 888. 888. 888 888 888 888 888 8. .. 8. 888. 888. 888. ..8 888. 888. 888. 88.. 88.. 888. 888. 8.8. 888 888 888 8 8 888. 8.8. 888. 888 888. 8.8. 888. 888. 88.. 8.... 88.. 8 8 8.888. 88. 88... 888 8.8. 8.8. 888. 888. 888. 8... 88.. 888. 88. 8 88.. 8... 88.. 888 8.8. 888. 88.. 8... 88.. 888. 888. . .8., .8... 8. ...; 8. ...; 8. ...; 8. ...; 8. ...; 8. ...; 8. ...z 8. ...; 8. ....s 8. ...; 8. ...; .88 < 8.. .... ..8 .88 .88 888 .88. 888 .88 888 .88 888 .88 888 .88 888 .88 888 .88 888 .88 888 .88 ..88 .80 .828 8.8.; .88.“. 8.88 88.88.88 88.8 ..3 88..8.88 88.8 ..88 88.88.88 88.....88 88..8..8 88.....8 8.88.8 8.88. 8.8.8. .8888. .8-... 8.88. 161 mNoo F ooo F moo F woo ooo F mNo F ooNF mNNF m FN F mo F F o¢F F moo F mmo F m Fo F on 162 8.8.8. 8.8. 8.8. 888 8.8. 8.8. 888. .888. 8.8. 88.. 88.. 888. 88. 888. 8. 888. 88.. 88.. 88.. 888. 888. 888. 888 888 888 8. 8. 888. 888. 888. 888 888. 888. 888. 888. 888. 88.. 88.. 888. 88. 8. 888. 8.8. 888. 888 88.. 88.. 88.. 8... 8.8. 888. 888. 888 88 88 888. 8.8. 888. ..8 888. 888. 888. 8.8. 888. 88.. 88.. 88.. 888. 888. 888. 88 8.88. 888. 888. 888 8.8. 888. 8... 88.. 88.. 888. 888. 8.8. 8.8. 88 88 8.888. 888. 888. ..8 888. 8.8. 888. 888. 888. 888. 8.8. 88.. 88.. 888. 8.8. 88 888. 888. 888. 888 888. 888. 888. 88. 88.. 88.. 88.. 88.. 88. 888. 88 888. 888. 888. 888 888. 88. 888. 888. 8.. 88.. 88.. 888. 88. 888. .8 88 8.888. 8.8. 888. ..8 888. 88. 888. 888. 888. 88.. 88.. 88.. 8.8. 888. 888 88 8.888. 888. 8.8. 888 888. 888. 888. 888. 888. 888. 888. 88 8.888. 8.8. 888. 888 888. 888. 888. 888. 888. 88.. 88.. 88 88 8.8.8. 888. 888. 888 888. 888. 888. 888. 888. 888. 888. 88.. 8. .. 888. .8 888. 888. 88. 888 888. 888. 888. 88.. 8... 8... 888. 88 88 8.88. 888. 888. 888 88. 888. 8.8. 8.8. 888. 88.. 88.. 8. .. 888. 88 8.88. 888. 888. 888 8.8. 888. 888. 888. 88.. 88.. 8. .. 88. 888. 8.8. 88 8.8. 888. 888. 888 888. 888. 888. 888. 888. 88.. 88.. 88 88 88 . .8.... 8. ..8... 8. ....s 8. ...; 8. ..8... 8. ...z 8. ..8... 8. ..8... 8. ....s 8. ....s 8. ..8... 8. ..8... .8.. 8.2 8... .... 88 888 .88 888 .88 888 .88 888 .88 888 .88. 888 .88 888 .88 888 .88 888 .88 888 .80 888 .88 .828 ... ....s .88.". 8.88. 88.88.88 88.8 ..88 88..8.88 88.8 ..88 88.88.88 88.8 ..88 8.8.5 88.....8 8.88.8 .8.88.8. 8.88.8. .8888. .8-... 8.88. ooF moF meo F mvo F omo F hno mNo F ooo F ooN F oNN F oNNF oo F F mv F F m F FF moo F oNo F oooF vo F mhv F mmv F mov F 85o ooo F ooo F mmo F omo F moo F mom F mNN F m FN F om F F o F FF oo FF oo F ooF. F mhv F mom F NNo oov F out F Not F ooo F o Fo F mum F ooN F No F oovF oooF oovF omo oovF m FvF mooF mmoF mFoF ohNF ooNF moFF omFF FoF ooF mNNN F moN F ooN F voo omN F moNF ooN F mv F F oo F F mm F F ooo F ooo F ono F moo F no ooo ooo vo oooF ovoF mooF oFoF omNF oVNF moFF oFFF mooF Fo 8.8. 8.8. 888. 888 888. 888. 8.8. 888. 888. 88.. 88.. 8... 888. 88 888. 888. 888. ..8 8.8. 888. 888. 888. 888. 8... 88.. 88.. 8.8. 8.8. 8.8 88 88 8.88. 888. 8.8. 888 8.8. 888. 8.8. 888. 88. 888. 888. 88.. 88.. .8 888. 888. 888. ..8 888. 88. 888. 888. 88.. 88.. 88.. 88.. 888. 888. 8.8 8 88 88 888. 888. 8.8. 888 8.8. 888. 888. 8.8. 888. 8.8. 88.. 88 8.888. 888. 888. 888 888. 888. 888. 888. 8.8. 888. 8.8. 88 8. 8. 8.888. 888. 888. 888 888. 888. 888. 8.8. 888. 88.. 8... .. .8)... 8.. .... .8... 8. ....s 8. ....s 8. ....s 8. ..8... 8. ..8... 8. ...... 8. ..8... 8. ....s 8. ...; 8. ...z 8. ..8... .8.. ..8 888 .88 88 .88 888 ..8 888 .88 888 .80 888 .88. 888 .88 888 .80 888 .88. 888 .8 888 .80 .828 n. ....s .85“. 88.30 NQVQE No8 Evo NoRQoo No.o too No.oN.No No? {No No. Fo.Fo No.8 F. Fo NQoQFo Fo.oN..NF FQooNF 6.888. .8-... 8.88. 163 hoF 8.888. 8.8. 888. 888 888. 888. 888. 888. 888. 8.8. 88.. 8... 888. 888. 88. 88. 8.88. 888. 888. ..8 8.8. 8.8. 8.8. 8.8. 888. 8... 88.. 88.. 888. 888. 8.8. 88. 888. 8.8. 888. 888 888. 888. 888. 888. 888. 888. 88.. 88.. 888. 88. 88. 88. .8. 88. 88.. 88.. 88.. 888. 888. 888. 88. 88.. 88.. 88.. 888 8... 88.. 88.. 8.8. 888. 8.8. 888 88. 888. 888. 888. 888 888. 888. 8.8. 888. 8.8. 8... 88.. 888. 888. 888. .8. 88. 888. 888. 888. 888 888. 88.. 8... 88.. 8... 88. 888. 888. 8.8 8.. 8.. 888. 888. 888. 888 888. 888. 888. 8.8. 888. 8... 88.. ... 8.888. 888. 888. 888 8.8. 888. 888. 888. 8.8. 888. 8... 88.. 88.. 8.. 888. 888. 888. 888 888. 888. 888. 888. 888. 8... 88.. 8... 8.8. 8.. 8.. 888. 8.8. 888. 888 888. 888. 88. 888. 888. 888. 88.. 8... 8.8. 888. 8.. ... 8.. 8.8.8. 888. 888. 888 888. 8.8. 888. 8.8. 888. 888. 88.. 88. 8.888. 8.8. 888. 888 8.8. 888. 888. 888. 888. 8.8. 88.. 88.. 8... 88.. 88. 888. 888. 8.8. 888 888. 8.8. 888. 8.8. 88.. 88.. 888. 888. 888. 8.8 .8. .8.... 8. ..8... 8. ..8... 8. ..8... 8. ....s 8. ...z 8. ..8... 8. ...; 8. ...; 8. ...z 8. ...; 8. ...; .8.. .83. 88 ... ..8 888 .80 888 .88 888 .88 888 .88 888 .88 888 .88 888 .88 888 ..8 888 .88 888 ..8 888 .88 .828 a. ....s _mcE 88>an. NQvao Noxo :3 NoRQoo No8 too NQoQNo No? (No NQFQFo No.5 F. Fo NQoo. Fo FQoQNF FQooNF 6.888. .8-.. 8.88. 164 oomw owNw 0N2 Nmm come mwmp mme mm: mv: 3 _.F mmo? v3 888: 888: 888. ~88 888: 888. 888. 888: 88.: 82:: 88:: 88: 82 82 82 28 82 8: 82 88: 2: 82 82 82 82 8: 8: 82 2. N2 :2 82 22 82 :8 82 82 22 82 82 82 8: 8: 8: 22 82 82 82 88. v8 8: 22 82 82 82 8: .8: 8: 82 82 8: 82 22 82 28 82 82 82 82 82 2: 8: 2: 22 82 82 82 8.82 22 82 ..8 82 82 22 22 82 82 8: 8: 82 88. 82 8.82 82 82 :8 2: 8: 8: 88: 82 22 82 2: 2: 8: :2 8.82 82 82 88 82 82 22 22 82 8: 8: 8: 82 82 82 82 82 28 82 82 82 82 82 2: 8: 82 82 88 88 82 22 8.82 82 22 28 22 82 82 8: 8: 8: 8: 22 82 22 88 82 82 82 82 88 82 22 82 82 82 8: 8: 22 82 82 m2 8: 82 82 28 2: 82 22 82 82 82 8: 8: 2: 22 82 2 8.82 82 82 88 82 82 82 .82 2: 82 82 82 82 2: 8.82 82 22 88 22 82 82 82 82 22 .8: 8: 8: 8: 82 82 82 N8 82 22 82 22 8: 8: 8: 8: 82 82 82 28 82 82 2: 8: 8: 82 82 88 88 88 28 2.: 2: 82 82 82 82 88 82 82 82 82 82 8: 8: 8: 8: 82 .8..» 8. ....s 8. ....s 8. ...; 8. ....s 8. ....s 8. ...z 8. ..E 8. ...; 8. ....s 8. ...; 8. ...; .8.. .82 2. x... ..8 .8 ..8 88 ..8 88 ..8 N8 ..8. 88 ..8 v8 ..8 88 80 888 ..8 m8 ..8. 88 ..8 88 >88. .88 8. ..8; .88.“. 28 8.8.3 8.2.3 8288 88 :8 8888 88:8 8238 8.2.8 882:. 8.8.2 8822 $.88. .m-< 8.882 165 NNN 166 8.28: 88: 22 N8 82 88: 2: .82 88: 82 82 88 8.82 82 82 N8 82 82 8:: 8:: 8:: 8:: 2: N8 88: 88: 28: 28 88: 82 82 22 82 82 82 2: 2: 82 82 :8 82 82 22 88 22 22 82 82 8:: 8:: 2: 22 82 2m 8282 22 82 N8 82 82 82 82 22 2: 8: 28 888: 888: 888: 88:: 888: 88:: 88:: 8:8 8.22 82 82 N8 82 82 8:: 8: 8:: 22 82 2m 8 :N 82 82 22 88 82 8: : 8 :: 8: : 82 82 22 88 88 88 28 :8 2m 88 88 8.82 82 82 88 22 82 22 82 8:: 8:: 82 82 82 m8 m8 :8 .828: 82 82 28 82 82 22 22 82 8:: 8:: 8:: 22 82 88 8: 8.22 22 82 88 82 82 82 82 22 82: 8:: 8: 8.82 82 82 N8 82 82 82 82 8:: 2:: 8:: 8: 22 22 22 88 82 82 82 82 8:: 8:: 8:: 22 .82 8: 22 22 82 88 82 82 22 22 82 .8: 8:: 8:: 82 8: 82 8:: 22 28 8: : 8 : : 8: : 8:: 82 82 88 88 88 8 :8 88 8: 8.2: 82 88: N8 88: 82 82 22 82 82 82 8: 8: .8: 8. ...; 8. ...; 8. ..E 8. ...; 8. ....s 8. ...; 8. ...; 8. ...; 8. ...; 8. ....s 8. ..8... .2. .82 8 s... 88 :8 >88. 88 ..8. 88 80 N8 ..8 88 >80 88 ..8 88 ..8. 88 ..8. N8 ..8 88 >8 «8 ..8 .88 8. ..8... .88.“. 28 8888 8.2.3 8.8.8 8.2.8 8.88 8.28 8.828 8.2.8 8.8.8 882 8.8.2 $.88. .m-< 8.8m: 2: 28: 88: 88 88: 8: 22 82 82 82 22 2:: 8:: .88 82 22 82 N8 82 82 88: 22 8:: 8:: 8:: :8 .828: 82 82 v8 82 82 22 2:: 8: 8:: 8:: 82 82 88 88 82 82 88: N8 82 82 22 82 82 8:: 82: 88 82 82 82 28 8.8. 828: 82 22 :8 82 82 82 22 82 88: 8:: 8:: 82 22 88 82 82 22 28 82 22 82 82 2:: 8:: 8:: 22 82 82 88 88 8.22 22 22 m8 2:: 8:: 8:: 8:: 82 82 82 N8 :8 8.82 82 82 88 8: 82 82 88: 82 82 82 8:: 8:: 8w .2 .8.... 8. ...; 8. ...; 8. ..E 8. ....s 8. ....s 8. ...; 8. ....s 8. ....s 8. ....s 8. ...; 8. ...; .88 < 8 :8. 88 ...8 ..8 88 ..8 8m ..8 N8 ..8 88 ..8 28 >80 88 .83 88 :8 «8 >8 ..8 .88 88 ..8. .88 8.. :25 .55“. 9mm. NQfiflvo N29 :3 NQNNBO NQm {no NQwQNo Na; (No NQPQS NmR : 5 ~99: ro SEEN: 3899 \l! I| I! 3.88. .m-< 8.88» 167 8.2 8.2 8 8:: 8.8 8.28 8 2:: 8.8: 8.28.:. 8 :::: 8.8 8.28.8 8 2:: 88 8:28:88 2.2 «..8 8 :2: .8 8.28.8 8: 8:: 8.2 8.28.8 8 :8: 8.8 8.22. 8 8:: 88 8:28:88 8.28 2 828.8 8.2: 8 828.2 8.28 2 828.8 8.28.8 2 828.8 8:28:88 82 2 8 82 88 8.28 8 82 82 8.28.8 8 8:: 8.8 8.28.8 :8 82 88 8:28:88 :2 8.8 8 82 8.8 8.2:..2 8 82 8.2 8.28 8 8:: 8.8 2.28.8 8 8: 8.8 8:28:88 8...: o 2 88 8.8 8.28 8 82 8.8: 8.28 8 22 8.8 8.28.8 8 22 88 8:28:88 8.2 88:3 82 8.8 :8: :8 8 82 8.8: 828.8 3 82 8.8 8.2323 82 8.8 8:28:88 8.2 8.82.8 8: 8.8 8.2.228 8:: 8.2 828.8 8 3: 8.8 8.28.888 8:: 88 2:32:88 8.2 E28: 22 8.8 8.28: 2: :8: 8.2 8.2 :.2::.2 8:: 8.8 8.28.8:8: 22 8.8 2:28:88 8.2 «.8 8 8:: 8.8 828.8 8: 8:: 8.2 8.28.: 8 «8 8.8 8.2:..8 8 22 88 8:98.88 8.2 :8 8: 8:: 8.8 8.22.8 3: 8:: 8.2 8.28.3. 8: 8: 8.8 82:2. 8 8:: 8.8 82888 8.2 ......8 8 88 8.8 8.28.8 8 82 8.8: 8.28.2 8 82 8.8 8.22.88 8 88 8.8 8288.8 2.2 8.8 8: 22 8.8 8.28.8 8: :2: 8.2 82:2 8: 82 82 8.28.8 8: 8:: 8.8 33288 2.2 2.8 8 :8 8.8 8.28.8 2: 82 82 8.22.2 2: 82 8.8: 8.2:.8 :2 :8 8.8 22:28.8 8.2 8 :2 :2: 8.2 8.28 8: 8:: 8.8: 8.2:. 8: :2: 8.2 8.28.8 2: 82 8.8 22888 8.2 :8 8.8: :8 82 8.28.2 2: :8: Q2 8.28.8 2: 82 8.2 8.28.8 :2 88 8.8 838%.. 8.28.2 8.8 «8 8.2 8.2:..2 8: 38 8.8: 8.28.: ::: 28 8.2 5.28.: 8: :8 8.8 82888 2.2 «..8 «.2: 8:: 82 8.85.8 2: 82 ed: 8.38.8 8.82 32 82 8.28.88 8: 88 8.8 83888 2.3 2 2: 22 8.2 3.28 8: 88 8.2 8.28 2: 888 8.8: 8.28.8 8: 88 8.8 2.2888 2.2 88:2: «8 8.2 8:328: 38 8.2 2:88 2: 38 8.8: 2.28.83: m8 :8 8288.8 8.: m8 8: «8 8.2 8.2:..2 8: 88 8.8: 8.288 8: :8 8.8: 828228: 88 8.2 8288.8 2.2 8.222: 82 82 8:28:82 88 8.2 2:328: 88 8.2 828.83: 22 8.2 892.88 oz «2 2: 82 8.202 «2 8: 28 8.202 «2 8: 88 8.2002 ..mz 8:: 82 8.2 22288 8.2 2.8 2: 82 8.2 8.8 «.8 8: 88 8.8: 8.8 :8 8: 88 8.2 2.8 8.8 3: :8 8.2 22:88 8. 8. 8. 8. ......o 8. 8. 8. 28.8.28. 8. 8. 8. 98.8.30 8. 8. 8. 288.38.. .8. 8. 8. 2828 8...8 .95 82.88. €88.82 8.8 .95 82.88. 8.88.82 8.8 .95 8...... 8.8.8.82 ....8 .95 82.88. 8.88.82 ... con. 8 con. 8 con. : con. 8.88. Noomom «CO—.5503 50h V Ow _. mcma 50.. 0x35 .0305 EU Ema 2:00)) .NVI< 03Gb. 168 2.2 8 8 2.. .8 8.2.. 8 m8 .... 8.28 8 2.. .8 8.2...8 :m 8: .... m8 2 :8 :2. 8.2 8.2 8 :8 .8 :288 8 ..8: ..8 8.88: 8 N8 .8 8.2828 :8 .... 88 2.8 ..8 8.2 o 8 8.. .8 8.28 8 82 .... 8.8 8. 8 :8 o... 8.8 ..8 8 88 .8 ..8 2 m8 ..8 8:: 8 8 88 .8 8.22 2 :2: ..8 ::m ...: 8 ..8 .8 8.8 8:8 8 :8 .8 88 22.. ..8 8.2 8...: 8.8 ..8 .8 8.28... 8 ..8 .... 2.288 8 ..8 :8 8.2 8.88 ...:m 8.. .8 2m 2 ..8 ..8 8.8 ::: 2 2: .8 8.2.. 8.8 :8: ..8 2.2: 8.8 82 ...: 8:8. ..8 8.8 :8 ...... 8.. 2 8.8 ..8 8.2 o 88 8:8 .8 2.2.. 8.8 2.. o... 2.8.. 8.8 82 ...: 8.2.... 8...8 8:8 .... :8 2 m8 ..8 8.8 m2 8 8..: o... 2.282 N: 8: .... 2:82. 8:: :::: ...: 8:38 ..8 ..8 .8 882888.. 8.2 .. 88 ..2 .8 :...2.. 8...8 ..2 .... 82:2 ..8 22 ...: 8.28.” 2.8 8: .8 :8 2 :8 ..8 8.8 8 8 8.. .8 8.28 8 2:: ..8 8.8 8 8 2: ...: 8.8 82:8 :8 .8 ..8 2 88 ..8 8.8 N 8 ..8: .8 828.8 8 82 o... 828.8. 8.8 882 ...: 8.2.... :8 :8 .8 28 2:8 ..8 8:: 8..:8 8 :8 .8 8.22. 8 22 o... 8.2.. 8 82...: 882.88 8 ..8 .8 8m 2 ..8 ..8 8.8 8 8 :8 .... 2.28.2 .... 82 ..8 8.28 :8 22...: 8.8 8.8 8 88 o... ..8 282.8 8.2 .. 8 88 .8 8.28 :8 88: ..8 8.22 8 88 ...: 8.2.... 8 «,8 o... «8 2 88m ..8 8.2 o ..8 8:8 ...... 8.28 8.8 ..8 o... 2.2.. 8 ..8 ...: 882...... :8 8: .8 8m 2 ..8 ..8 8.2 o 8 ..8 .8 8.28 8 8:: ..8 2.28... :8 8: ...: 8.2.... .88 :2 .8 ..8 2 N8 ..8 8.2 :: 8.8 88 .8 82.. 8.8 :8 .... 2.28. 8.8 :8 ...: 8.2.8 88 8.. .8 :8 2 m8 ..8 8.8 ..8 8 8.. .8 2.28 8 28 .8 8.8.. 8 ..8: ...: 8.882 8 28 .8 88 2222. 8.2 o .88 :8 .8 8.22 8.8 ..8: o... 8:88 8 8:: ...: 8.88.8 8 88 .8 ::~ 2:28 8.8 8.8 8 88 .8 828.8 8 22 o... 8.2...8 8 22...: 2.2.88 8 :8 .8 2m 2888:. 8.2 8 8 2.. .8 8.22 8 82 2. 2.28 8 8.. 8.: 8.2...8 :: 82 8..: 88. 282.8 8.2 8.8 8.8 :82 ..8 82 «8:8: 2:: ...2 828.8 8.8 2: .8 82 02:2 82: .8 8: 2 8:88 82 8.2 8.8 82 :8 8.2.. 8:: 22 ...2 8.288 2 :8: .8 8.2.... 8 ::2 .8 8:28:88 8.2 «.8: m8 ::2 .8 828.8 2 :8: ...2 8.2.88 8 82 o... 8.28.... 2 8:: .8 8:22:88 82 .88 2 8:: .... 888.8 2 :2: ...2 2.22 8: 8:: .... 228.8 8: 8: .8 2: 2 82:8 ... ... ... ... ...).o ... ... ... 28:8 ...).o ... ... ... 28.» ..zo ... ... ... 2828 ...)... ... ... ... 282m ....8 .95 8...... 28.0.02 ....8 .95 ......on. 5.8352 ....8 .95 228...... 5.8362 ....8 .95 ......on. 28.8.2 8 5n. m 5n. N 8n. : 8n. 82.8 6.38.- 218 28: 169 2mm; 35 ..8 :20 9: mm 83 mm; 333 Emomfim 22 9: ho mom.m>m m5 .xomncgmz, mEmflE .nmEEQmu 62° .8289 .oz,d 208-662,. 3.9 o mm an» ad Eda Nan we 39 ed no.9 0 mm m3. o6 no.9 od mm m: o6 own 2 3m >3 2 n. a. a. ..sa 2 e 2 233.30 a. e a. £83 ...20 e a a. 23$ ...20 e a. e «3% 2.8 .95 .28 .58362 2.8 .95 J23". 5.8362 38 .m2, 5.2. .58352 £8 .25 5.8 .58st ¢ ..8 n 5a N Sn. F 5a neon. 368* 91 293 170 2.2. 3. 82 3 8.288 8 22 3. 2.2.8 8 ...2 3 2.2.... 8 88 3 2.28.5. 8.2.. 8 ..8. 3 2.28.. 8 88. 3. 2.23 8 2.. 3 8.28.8 2 ..8 3 8.28.5. 8.2.. 8 8.. 3 2.28... 8 8.. 3. 8.2.. o. ..8 3 8.23 8 .8 3 8.28.5. 8...8 8 8.. 3 8.28... 8 8.. 3. 828.8 8 8.. 3 8.28.8 8 82 3 8.2825. 8.28 8. 82 3 828.8 8 .2. 3. .223 ... ..8 3 ....28 8 88 3 .2225. 8.2.. N. 88 3 8.2.8 8 82 3. 828.2 8 22 3 8.238 8 82 3 2228.5. 8.2... 8 82 3 .228 8 2.. 3. 8..... 8 8.. 3 8.28 .. .8 3 8.2.25. 8.28.8 o. .8. 3 8.28.2 ... m8 3. 8.2.8 8 .8. 3 8.222.... 88 3 8.28.5. 8.22 8 .2. 3 22.2.8 8 8.. 3. 8.28 8 2.. 3 8.288 8 82. 3 2.22.5. 8.22.28. 8.. 3 8.8.8.8. 82 3. 8.2.88 8. 8.. 3 8.2328. 82 3 2.28.5. 8.2 ..2 8 .8. 3 828.8 8. 2... 3. 8.2.8 8 8.. 3 8.2 ..8 8 .8. 3 8.285. 8.28... 8 .8. 3 8.23.. 8. 2... 3. 8.2 ..n 8 .8. 3 8.2.... 8 82 3 8285. 8.28.. 8 m8 3 8.28.2 8 82 3. 8.2.8 8 m8 3 8.28.8 8 88 3 8 285. 8.28.2 2. 82 3 8.2..8 8. 2... 3. 8.28.... 2. 82 3 8.2.. .2 88 3 8 285. 8.2.8. 8. ..8 3 8.2.... .2 .8 3. 2.2.8 8 m8 3 8.28.8 8 .8 3 .. 2 ..5. 8.28 2. 82 3 8.28 8. 2... 3. 8.28 ... 82 3 8.2 .. 8. 22 3 o. 285. 2.2.. 8 8. 3 2.28,. 8. .8 3. 2.23 8 .8 3 8.23 8 8. ..8 82.85. 8.28.2 .8 88 3 8..... 8 m8 3. 8.2.8 8 N8 3 8.2.2 8. ..8 3. 8285. 8.23 .2 88 3 8.22.8 2. 88 3. 8.28.. 8 m8 3 8.2...8 8. ..8 3. 8285. 8.2.. .2 8o 3 8.28 .2 88 3. 8...8 8 8. 3 ...2o 8 .2 3. 8285. 2.2..8 8. 8. ..8 8...8 8. 8. 3. ......3. 8 2.. ..8 8.2328. 8. 3. 8285. 8.8 o .8 .8 3. .3... ... 8. 3. 8.8 8.8 8. m8 3. 8.2.. 8. 8. 3. 8285. 8.3.88.2 88 3. 8.2.8.2. ... 3. 2.2....28. 2. 3. 8.8 32.2 8.. 3. .8225. oz «.2 8. 8. 3. oz «2 8. 28 3. oz «2 .2 8. 3. 0oz 82 2. 2. 3. 2285. 8.8 8.8 8. 8.. 3. 8.88.8.2. ... 3. «..8 8.2.8. ..8 3. 2.8 8.8 .8 N8 3. .2.5. 2 2 2 2 220 2 2 2 28.... ...20 2 2 2 28.... ...20 2 2 2 28.3.20 .2 2 2 .82» .28 .25 8:8 28.0.02 28 .25 8:8 .58... .oz 28 .25 22.8 288.02 28 .95 8:8 288.02 8 8.. . ..8 o 8n. m 8n. 88.. moomom .cmEtmaxm .2 m 2 m .8ch .2 9.2.: Esz Eu :3 2.825 .91.. m_nm._. 171 8.2 ..8 2. .8 3 ...2. 8 8.. .... 2.28 8 ... 3 2.2.... .. 8. 3 ..8 2.8 5. 8.2 ......8 8. 3 8.23 8 8.. .... 2.23 8 8. 3 2.2.... 8. ... 3 ..8 2.8 5. 8... .. 8 8.. 3 8...8. .. ...2. .... 8.2... 8 8.. 3 8.2.. .... 2.. 3 8. 2885. ....8 2 8 .8 3 8...8 8 ..8 .... 2.2. 8 .8 3 8.2. 8 .8 3 ..8 38 5. 2.2.. 8 ..8 3 88.8.88 8 8.. .... ...2. 8 ..8 3 2.... m... 8 .8 3 .8 22.. .8 8... 228.8 .8 3 82...... 8 .8 .... 8.28 8.8 .8 3 8.8... 8.8 ..8 3 2... 28. 5. .... .. ...... .8 3 ...2. 8 ..8 .... 8...8 8.8 .8 3 8..... 3. .2 3 ..8 2.8 5. 8... 2.. 3. ... 3 8.28... 8.8 m8 ... 8..... 3. ... 3 8.28... ...... ..8 3 .8 28.5. ....2 8 8.8 .8 3 ......m 8 823 8.8.. 8 ..8 3 8.2.. 8.8 ..8 ...... 8. 28.5. ...2 .. 8 2.. 3 ...2. 8.... ...... 3 8......... 8.8 ..8 3 82.. ..8 .8 3 8.2.85. ....2 .. 8 ..8 3 2.28.8 .. 8.. 3 2.3.8 .... ...... 3 2.28.8 m8 8.. 3 8.2.85. ...2 m2 3. 8.. 3 8.2.. ... 8.. 3 8.22 8 2.. 3 8..... ... 8.. ...... 2. 2.85. 8.28 8 8.. 3 8.2... 8 2.. 3 8...8 8 ..8 3 2.28 8 8.. 3 8.28.5. ...2 . .. 8. 3 ...2. 8 82 3 8.... 8 8 2.. 3 8.2.... .. 8. 3 ..8 2 8. 5. 8.2 . .. ..2 3 8.2. 8 88 3 2.2.. m... ... 3 8.2.. ...... 2. 3 8.28.5. 8... .. 8 .8 3 8.2.. 8 22 3 .....2. .. 8. 3 ...2... .. ... 3 ...... 28.5. 8.2 .. .. ... 3 ...2. 8 82 3 8.23 8 ..8 3 8.2.. .. ... 3 8.28.5. 2.2 3 m. 8. 3 8.2. 8 ...... 3 8.2.. 3. 88 3 8.23 m... ... 3 .8 28.5. 8.2 .. .. .8 3 8.2.. 8 .2. 3 8.2.. m. 8. 3 8...... .. 8. 3 ... 22. 5. 8.2 8.8 m8 .8 3 8.2.. 8 .8 3 8.28 8 .8 3 8.2.. .. 8.. 3 2.22.5. .....2 .... ... ..8 3 2.28 ... .... 3 8.28... .... .2 3 8.28 ... 8.. 3 2.2.85. 2... m... 8 ..8 8.. 8.2.. .. .... 3 8.3.8 8 ..8 8.. 8...... 8 2.. 3 8.28.5. 8.2.8 .28 8.. 3 22.2.2 2.. ...... ....2 ...28... .... 3 8.2.... 8 .8 3 8.28.5. 8.2 ..8 .... .8. 3 8.2.... m... 8.. ...2 8.2.22 8... 8.. 3 8.23. 8 .8 3 8.28.5. 8... 8 2 .2. 3 82...... 8 8... ...... 8.2 ..8 ..8 .8. 3 8.2.8 8 8.. 3 8.22.5. 2 2 2 2 22.. 2 2 2 288...)... 2 2 2 28.230 2 2 2 28.3.20 2 2 2 28.. ..8 .2.. 8:8 28.902 .28 .25 8:8 28.8.02 ...... .2.. 8...... 28.0.02 28 ...2. 8:... 28.902 .. ...... . 8n. o 8.. ... ..8 8...... .....82 .2}. 28. 172 ...33 25 .2 :20 85 mm 03: mm; 8232. E0023 0.2 85 ..o 383.... 85 132353 9.88.... .352:an .02. .3888 .02.. 288882.. aim. 0.8. cm mm. 9m 3.9. cm moo .... 3.8.8.8 mm mg 0.0 mad. 3... mm «mm od .3 28...... >2. 8..». K 8. mo. o.m 3.8.9. 8 .3 .... 8.8.3 mm .8 od 3.38 cm .88 ad .3 ... .3 >2. 2 2 2 2 22.. 2 2 2 2828 ...20 2 2 2 2828 220 2 2 2 282.. 220 2 2 2 28.8 2.... .82. .2.... 8.8.0.02 2.... .82. .8:... .58882 ..8 .82, 8...... .Eoo.o...z 28 .82. .2.... 28.0.82 8 8n. . 8n. 8 8n. 8 8n. .2.... .....co... .2}. 28. 173 2.2.... Q8 ..8 Q8 8.2m... Q8 .8 Q8 8.2.8 Q8 .8 Q8 8...8 Q3 88 Q8 2.281% .338 Qm. 88 Q8 8.2.... Q2 28 Q8 8.288 Qo. .8 Q8 8....Q2 Q.. 88 Q8 8.281% 8.38.3.3. 82 Q8 2.23. Q8 82 Q8 2.2 ..8 Q8 82 Q8 8.2.... Q0. ..8 Q8 8.281% 8.23 Q.. 88 Q8 8.3.8 Qo. 88 Q8 .82.... Q8 22 Q8 8....Q2 Q... 82 Q8 8.281% 22»... Q8 8.» Q8 8.23... Q8 88. Q8 8.288 Qo. 88 Q8 8....Q8 Q.. 88 Q8 ....2...1% 8...8 Qm. «8 Q8 ...2~.8 Q8 2.. Q8 2....Q8 Q8 8.. Q8 8......2 Qm. ..8 Q8 3.281% .v.¢.Q.~ o... 8.8 ..8 8.23 Q8. .8. Q8 8.20.8 Q8 2.. ..8 2.20.8 Q8 8.8 Q8 8.281% 8.28. Qe. 88 Q2 8.28.8 Q... 88 Q8 8.28.2 Q8. .8. Q2 8.2Q8 Qo. 88 Q8 8.281% 8.338 Q8 82 Q2 2.28.8 Q8 8.8 Q... 2.20.8 Q8 2.. Q2 2.2Q8 Q8 .8. Q8 2.221% 2.2 ...2Q8 8.. Q2 22283.8 :2 Q8 8.2Q8 Q8. 8.. Q2 8.3.8.98. 8.. Q8 2.281% .Q2Q8 Q8 88 Q2 8.22....” Q8 28 Q8 .22 ..8 Q8. 22 Q2 .~.2Q8 Q8 82 Q8 8.281% 8.288 Q8 82 Q2 8...... Q8 88 Q8 8....Qw Q8 .8. Q2 8.28. Q8 88 Q8 8 281% 8.28.8 Q8 88 Q2 8.28.8 Q8 88 Q8 2.238 Q8 .8. Q2 8....Q2. Q8 88 Q8 8 281% 8.33. Q... 82 Q2 8.28 Q8 28 Q8 2.2.8 Q8. 8.. Q2 8.2.2.2. Q8. 88 Q8 828.1% 2.2 Q2 Q8. .8 Q2 2.2.8 Q.» 88 Q8 2838 Q8. 88 Q2 8.2...8 Q8 8.» Q8 .. 2 ..1% 2.2 Qo Q8 8.» Q2 82...: Q8 88 Q8 8.2.8. Q... ..2 Q2 8.28.8 Q8 8.» Q8 o. 281% 8.2 Q8 Q8. 2.... Q2 8.3... Q8 8. Q8 .Q2...~ Q8. 28 Q2 8...~..m Q8 8. Q8 82.1% 8... Q2 Q8 88 Q2 2.28.8 Q8 8. Q8 .Q2Q.m Q8. o8 Q2 .m...Qo Q8 8. Q8 8 281% 8.2 Q2. Q3. 8.» Q2 8...8... Q8 8. «.8 8.2m..~ Q8. «.8 Q2 8.23. Q8 ..8 Q8 2.281% 8.2 Qo Q8 8. Q2 8.2Q2. Q8. «8 Q2 8...Qo Q8 ..8 Q2 8......» Q8 8. Q8 2.281% 8... «83.8. 8. Q2 2.....8 Q8. 8. Q2 2.3.... Q8. 8. Q2 2...Q8.Q8 8. ..8 82.81% 28 Q8 Q8. 88 Q2 8.2 ..2.Q2. 2.. Q2 8.8 ..8 Q2 88 Q2 8.8 Q8 Q8 28 Q2 8285. 8... Q8 Q8. .8 Q2 8.» ..8 Q8 88 Q2 8.8 ..8.Q8. 88 Q2 8.» Q8 Q8 .8 Q2 .~ 281% oz m2 Q8 .8 Q2 oz «2 Q8 8.83.2 oz .mz Q8 28 Q2 ooz ...2 Q8. e8 Q2 221% ..8 3.3.8 8... Q2 8.8 ..8 Q8. 88 Q2 2.8 88 Q8 28 Q2 2.8 8.83.88 .8 Q2 .21% 2 2 2 2 ...20 2 2 2 288 .So 2 2 2 28.3.20 2 2 2 288.30 .2 2 2 288 28 .95 22.8 28352 28 82, 22.8 .283 521.8 8.5 22.8 28862 2.5 .25 22.8 28862 2 ..8 .. :8 2:8 8 :8 8:8 Noomom EmEtmaxm .0. N. o. m 2.3 .2 39.2 .288 Eu :3 12925 .ov.< 2an 174 2.23 3.. ..8 3 223 Q... .8 3 8.23 Q8 .8 Q. 8.23.3 Q2. 28 3 ..8 231% 3...... Q8 88 3 .338 Q... 8. 3 8.33.8 Q8 ... Q. .3838 Q8 ... 3 m3 2 .8 1% 38.... Q8 .2 3 8.2Q8 Q8 8. 3 .Q..Q.. Q8 28 Q. 2238 Q8 3. 3 88 281% 2.3.8 Q2. 88 3 8.23.. Q8 8. 3 2.3.2 Q8 .8 Q. 2.3... Q2. 88 3 8.. 281% 8.3... Q8 88 3 2.23 Q8 38 3 2.83.3 Q8 88. Q. .8838 Q8 8. 3 88 221% 8.3.2 Q2. .8 3 .338 Q8 8. 3 8.3.8 Q8 88 Q. 8.23.8 Q3 23 3 2.. 281% 8.3.... Q3 88 3 .Q8 Q8 Q8 .2 3 .338 Q8 82 Q. 88 Q8 Q2. 8. 3 83 2 88 1% .Q.3.. Q8 .8 3 ...23 Q8 8. 3 ...23 Q8 8.. Q. .33.... Q3 8. 3 .8 281% 2.2.3. Q3 88 3 ....83 Q8 ... 3 2.23 Q3 82 Q. 88 Q23... ..8 3 88 28.1% 8.23. Q8 83 3 8.23 Q8 .8 Q3 2.23 Q8 .8. Q. 3.23 Q8 28 3 882.81% 8.238 Q8 88 3 8.23838 8. 3 8.23.3 Q8 2.. Q. 8.8 3.23.8 8. 3 38 2 3.81% 8.3.83.2. 88 3 8.23 Q8 8. 3 8.23.8 Q8 28 Q. ...23 Q8 .8 3 28 2 .812. 8.3.83.8 .2 3 8.8.2... Q8 2.. 3 8.23.2. Q8 ..8 Q. 8.23.8 Q3 8. 3 88 28.1% 8.3.8 Q8 8. 3 8.23.2 Q8 3. 3 .338 Q8. .8 Q. ....2Q8 Q8 88 3 m8 281% 8.23 Q8 88 3 8.3.3 Q8 .8 3 8.23 Q8 28 Q. 8.38 Q8 38 3 88 2 81% 2.23 Q8 .8 3 8.23 Q8 ..8 3 8.33.3 Q8 8.. Q. 8.23 Q8 83 3 8. 28.1% 8.33.. Q3 ... Q. 8.23... 3.. 2. 3 8.3.3 Q8 88 Q. 8.3.8 Q8 38 3 88 281% 8.23 3.. 8. Q. .333 Q2. 8. 3 8.3.3 Q8 88 Q. 8.38 Q... 88 Q3 .8 28.1% .33.. Q8 ..8 Q. 8.... Q8 Q8 8. 3 283... Q8 28 Q. 8.23... Q3 3. 3 ..8 221% 2.8 3 Q8 .8 Q. 8.23.. Q8 88 3 8.3.. Q8 8.. Q. 8.23.8 Q8 88 3 2.2.8.2. 8.3.3.8 8.8 .... Q. 8.23. Q3 8. 3 ...23 Q2. .2 Q. .33... Q... ..o 3 2.281% 8.23.8 Q... 8. Q. 8.3.8 Q8 2. 3 8.23.8 Q8 2... Q. 8.33.838 2o 3 88 2 .21% 8.2.... Q3. ..8 3 8... ..8 Q... ..8 Q. 8.2.8 Q8 82 3 8.2.8 Q8 .8 Q. 8.281% 8.3.3.8 Q8 88 3 8.2Q. Q8 .8 ... 8.2.... Q.. 22 3 8.23.8 Q8 .8 Q. 8.281% 8.2.8 Q8 82 3 2238 Q8 2.8 3 8.3.2 3. .2. 3 2.3.83.8 88 Q. 8.221% 2 2 2 2 ...20 2 2 2 28.3.20 2 2 2 28... ...20 2 2 2 28.3.30 2 2 2 28.... .80 .2.. 8:8 28.0.02 .80 .2.. .28 28.052 .80 .2., 8:8 28.202 .80 .2.. 22.8 28.902 2 =8 .. 8n. 28... 8 ..8 32.8 6.82 .2128. 175 .3889. .02.. .232. 8.5 .2 =20 2.. mm 88: 882. 8x32, .3023 9.... m... .0 83.53 2.. £39298; 9.7.8:: .umEEQmo .ozo ...2. 20.8 So... 202.382... 2888.25. 2.3 mdm odm m... 3.. 3.90% odm w: Qm 8.30.». 98.. 35 ad mm.m.m.m 93 mm. 3.. mum 0:33.. 8.83.8 8.83 cm. 3 8.2.3. 8.3 3.8 3. 2.28.88 8.88 3.8 3 8.8...83 Q8 82. 3 ..8 281% 3.9 ..mm 3.3 mmm O... 88.9 ...n c.33 mam o.m m...m.m.~ adv mo. od ondpmd 9.3 mmo Qm 30m 03mm 3.. 3.8.8.838 83. 3 8.23.88 8.83 8. 3 8.8.8.838. 88. Q. 8.3.233 3.8 3. 88 2 881% a. 2 2 a. ......o 2 2 2 2828.38. 2 2 2 2828...)... 2 2 2 2828.88. 2 2 2 8.828 280 .95 3:9". .EoOSdZ >=mD .m>> .2238. £303.02 >=mo .m>> 3:85 6.60.062 280 .m>> £2.98. £503.02 NF 38. _.F 3a 6.2.8. .213 8.8. econ. m 3n. 888 176 829° 98 988 98 88:8 98 98:98 8.898 98 98:98 8.898 98 98898 8:28:28 829° 98 98:98 8.898 98 988 98 8.29.. 98 98:98 829* 98 98:98 8:28:28 8898 98 98898 8.898 98 98898 8.298 98 93:98 8.298 98 98:98 8:28.18 829° 98 98:98 889° 98 98:98 8.89o 98 98898 8.28. 98 98:9... 83.828 8.298 98 98:98 8.898 98 988:98 8.898 98 988 98 8.898 98 98898 8:23.28 8.898 98 9:8: 98 8.898 98 98898 8.890 98 98:98 8.892 98 98898 8:08.85” 8.83 98 98::8 8.898 98 92.898 8.898 98 98:98 8.898 98 98::8 «82888:. 8.898 98 98898 3.898 98 98898 8.28.8 98 «.8898 «989: 98 98:98 8:38.18 88” 8: 8: 98 :9298 8: 88 98 8.8988: :8: 98 8.29:. ~88 88 98 8:28:28 8.8988: «8.: 98 8.2983: 88 98 8898::8 88 98 8.898 98: 88 98 8:2828 8.8:: 8 8: 98 8.898 8 88 98 8.898 8 88 :8 :928 8: 38 98 88288:. 8.89:: 8: 88 98 8.8: 8 8:: 98 8.898 3: 8:: 98 8.298 8: :8: 98 8288a 8.898 8: 88 98 8.89:: 8: 88 98 8.898 8 88 98 8.89:. 8: 8: 98 8288:. 3.898 8.: 88 98 8.298 8: 88 98 89298 8: 88 98 88° 8: 88 98 8288.: 8.89:. 8: 8: 98 8.89m 8: 2.: 98 8.8:.8 8: 88 98 8.898 8: 8.: 98 82:28 898° 8: 8: 98 8.88 :8 8.: 98 8.88 .8 8.: 98 :988 8: :8: 98 82318 8.898 8: 88 98 8.898 8: 88 98 8988.8 8: 88 98 8.898 8: 3.: 98 82838 8.898 ::: 88 98 8.898 88 88 98 :9892. «8 8.: 98 8.88 8: 88 98 828E. 8.8g 8: 2.: 98 2.8a 8: 8: 98 8.8:.8 2: 88 98 8.898 8: 88 98 82818 2.8g 8: 88 98 8.898 8: 88 98 8.898 8: 88 98 8.88. 8: 88 98 8.285. 8.898 8: 88 98 8:98 8: «8 98 8.898 8: 88 98 8.898 8: to 98 8 28:8 8.8: 8: 88 98 8.89: 8: 98 98 89:98 8: :8 98 8.898. 8: .8 98 8288.. 89:98:88 88 98 8.898: :8 N8 98 8.89838 :8 98 8:98:88 88 98 828E. oz mz N8 88 98 oz «2 8: .8 98 oz m2 :8 88 98 0oz a_mz 8: 98 98 82:8 2: 8 8: :8 98 8:98 «8 88 98 8.89888 88 98 8:98 :8 :8 98 8228 a. a. 2 2 .__2o 2 e 2 8% .__2o 2 2 n. 9871.5 e e e 28855 .2 e 2 8% 8o .95 8:8 .58 .662 2:5 .95 8:8 .58 362 £8 .95 8...8 .58 5.02 8o .95 8:8 .58 .052 8 8a 8 8a 8:8 8 8a 88m moomom EmEtmoxw .2 m: 2 m: 8.3 .8 9.8:. 5sz Eu omo >333 .214. flow... 177 gem; m2. .2 :20 9: me new: me; $33 Emomfim 92 m5 *0 3905 05 £03599, 0:828 .vmc_E.23 ooze 33.89 82.. 282925. «we o.o o.ew owev o.> Fww o.o o.ew own:~ o.e «we o.o o.ew ow? o.> ewe o.o o.ew o.wmv ow owe 2 ewe >3 ow.vw he: o. we o.oww. o.> wo._‘w ewe. o.oe o.o>wp o.e ov. Pw we: o.o> ooe: o.> eeep e.we owe o.eeo o.> wwe 2 ewe >3 Peep o.o ewe o.owe o.> emf o.e? o.oe o.omo o.e oeo? o.eo flee o.eeow o.> oeep o.e: o.em o.wee o.> own 2 ooe >3 weew e.e> e.o> o.mwww o.> 8.: moe m.o> o.oBF o.e meow F. For nee oweow o.> 8.3 N F; ode o.eoo o.> eoe 2 woe >3 neéw o.oe o.w> o.ew: o.> 3.3 93. owe o.eee o.e ever o.e mew oie o.» 3.9 o.e o.em o.eme o.> 3e 2 mow >3 no.3 o.w eée o. _.oo o.> 3.: o.o o.oe o.e>ov o.e Sow o.e e.>e o.emow ow 3.ow o.oe eee o.eBP o.> vow 8 eew >3 Fe._.w m.me m. E o.o:w o.> no.2 mom o.ee o.oeoF o.e eod; o.o o.oe o. 3» ow ooe? o.ww mée o. Poor o.> new 2 Few >3 3.9 o.o o.oe o.eeo o.> no.2 mop owe o.vwow o.e ewow o.o o.ve o.veop o.> wz... mw o.em o.ooo o.> oew 2 3w >3 meow o.o o.we o.evop o.> 5.9. o.e o.oe o.ooo o.e ems? me o.>m o.evo ow owow mom o.ee o.eeow ow 2w 2 new >3 mw. Fw o.e owe o.ep S o.> ever e.vw o.ee ow? S o.e w>.ow Noe o.ee oéoov o.> ew.ow e.o> oée o. to? ow eew 2 oew >3 whew o.e o.ee o.eeo o.> vwer o.e mew o.e; o.e mew.‘ mow owe o.oeo o.> who? o9~ o.oe o.ooow ow mew 2 new >3 mwsw o.o oém o.eoo o.> on: o.o o. E o.ene o.e 8.9 o.o m5. o.em> o.> 5.: o.e o.ee o.evo ow wow 2 evw >3 ever o.o o.we o.ewow o.> m5; o.w o.em o.>ee o.e ew.ep o.o nne oweo o.> ow: o.o oée o.owo o.> new 2 mew >3 Que? mo o.ee o.emo o.> 5.? mo owe o.:m o.e o.o? o.e? o.oe o.eoow o.> wee. e; o.—m o.>ee ow eew 2 wow >3 3.9 o4 o5. o.>oe o.> over o.o o5. o.>oe o.e 3.ow mew o.ee o.e>ow o.> emew o.ee o.oe o.weo ow Few 2 eww >3 3. w o. w o.ee o3 3 o.> ooe? o.ee o.ee o.ooow o.e we.ow o.o o.ee o.eeop ow >e.ow ed o.ee o.ooop ow vww 8 er >3 over ed men o.emo o.> ooe? o.o mom o. 3e o.e meow o.ow o. re o.owop ow eoow we: o.ee o.ew: ow 5w 8 :w >3 ooe? o.o o. 5 o. 3e o.> oeo. o.o o.ee o.oeo o.e wfiow m. w o.ee o.eeop ow Fee. o.oe o.we o.eeow ow 2w 2 vow >3 ewew mow o.oe o.m>ow as 3.3 eww o.oe o.eeo o.e mt: o.oe o.ee o.oeop as ete. o.om owe o. Em as now 2 new >3 3.2 o.o men o. 3w? o.o ooe? w.>w o. _L. o.ew: o.o. mfiow Nw o.ve owe? o.o 8.9 Q: o.e> o.ew: o.o em? 2 23 >3 ewe? o.e o..ee osoww o.o owe? eNF own oiww o.o. 3w: o.e mi. o.oe: o.o 3.: >4 oi. oéowr o.o me? 2 me? >3 5.3 tee o. _‘e o. Few. o.o 3.9 wee o.e> 3.wa 92 3.9 mee o.o> oi: o.o owew ode o.o> o.oo: o.o New 2 e: >3 9. a. a. a. ...20 2 e e 28m. ...20 2 e a. 92%. ..sa 2 a. 2 £83 ...20 a e e 90% >zen. .m>> ..o__on_ .500 3 .02 2:5 .m>> 3:8 .500 he .oz 230 .m>> .5?“ 630 he .oz 2.8 .m>> Join. .Eoo .0 62 er 5a or com 35a 2 com Eton. E83 El 293 178 Table A-5. Carcass characteristics of steers from Experiment 809002 Stee Date Slaughter HCW, REA, Backfat, Marbling Qual' Calculated no. harvested wt., Kg kg cm 2 mm KPH, % score ' gade yield Ede ° 1 03/16/92 535 313.1 62.6 2.45 2.0 690 19 2.67 2 04/13/92 632 367.8 78.4 8.58 3.0 540 18 3.17 5 03/16/92 599 336.0 71.0 6.13 2.5 630 19 2.92 8 11/11/91 424 246.7 58.1 3.68 3.5 460 17 2.75 9 05/1 1I92 581 336.9 74.2 3.68 1.5 480 17 2.32 10 11/11/91 479 269.0 71.0 2.45 2.5 430 16 1.97 11 11/11/91 426 234.6 67.1 2.45 2.5 430 16 1.88 12 05/11/92 522 316.3 74.8 9.80 3.5 950 22 3.14 13 03/16/92 653 375.4 81.3 14.70 2.5 540 18 3.61 14 11/11/91 411 226.4 63.2 2.45 2.0 310 14 1.90 16 04/27/92 598 361.0 85.8 4.90 3.0 510 18 2.37 17 04/13/92 627 353.7 81.6 3.06 2.0 380 15 2.13 1 8 04I27/92 587 357.3 80.6 2.45 2.5 460 17 2.24 20 03/16/92 596 347.3 83.5 7.96 3.0 540 18 2.68 21 03/16/92 562 309.1 67.1 6.13 3.0 570 18 2.99 25 05/11/92 623 371.8 69.7 6.74 3.5 880 21 3.55 27 11/1 1/91 424 235.4 67.7 2.45 3.0 460 17 1 .95 29 04/27/92 589 352.5 80.6 4.90 2.0 480 17 2.35 31 04/27/92 590 352.5 64.5 4.90 3.0 500 18 3.35 32 11/11/91 413 232.0 60.6 2.45 2.0 420 16 2.07 34 04/13/92 540 324.6 74.8 9.80 2.5 520 18 3.01 37 04/27/92 600 353.6 76.1 2.45 1 .5 440 16 2.24 38 04/13/92 578 335.2 95.5 3.68 1 .5 390 15 1 .25 40 11/11/91 433 245.8 69.0 2.45 2.5 340 15 1.87 42 03/16/92 616 381.9 91.6 3.68 2.5 770 20 2.03 43 04/27/92 602 358.0 77.4 4.90 3.5 530 18 2.86 44 04/13/92 613 364.9 83.5 4.90 3.5 550 18 2.61 45 11/11/91 460 254.7 60.6 3.68 3.5 500 18 2.69 46 03/16/92 572 349.3 78.0 6.13 3.0 440 16 2.78 47 04f27/92 641 387.3 89.0 4.90 2.0 440 16 2.23 50 03/16/92 650 377.4 79.7 8.58 2.0 490 1 7 2.98 54 03/16/92 663 389.0 79.0 10.41 2.5 530 18 3.40 55 05/1 1192 627 374.8 80.6 3.68 3.0 550 18 2.62 57 04/27/92 587 357.0 79.7 7.35 3.5 620 19 2.99 58 04/27/92 587 360.9 68.4 5.39 3.0 460 17 3.28 59 05/11/92 654 382.8 86.4 7.96 2.0 510 18 2.63 60 11/1 1191 440 234.9 56.8 3.68 2.0 350 15 2.42 62 04/13/92 561 328.0 82.6 9.80 3.5 550 18 2.85 63 05/11/92 651 367.2 77.4 7.35 2.5 510 18 2.99 65 04/13/92 556 316.9 82.6 2.45 2.0 520 18 1.71 70 04/13/92 608 356.0 78.0 4.90 3.5 610 19 2.81 179 Table A-5. (cont’d) Marbling Quality Calculated ee ‘ ‘0... ‘ harvest ch, REA, —Bar.:k1a no. harvested wt., lgg kg cm 2 mm KPH, % score flade yield grade 72 11l11l91 433 244.9 67.1 2.45 2.0 410 16 1.86 75 04l27/92 595 342.8 76.8 3.68 2.0 420 16 2.34 76 04I27l92 61 8 357.4 76.8 2.45 2.0 500 1 8 2.34 77 03/16I92 627 364.2 72.2 4.29 3.0 500 1 8 3.00 78 11/11/91 400 227.4 59.3 4.90 4.5 560 18 2.85 80 03/16I92 645 356.0 73.2 4.90 1 .5 480 20 2.65 83 03116192 617 370.7 86.1 4.90 2.0 450 17 2.23 85 11/11191 457 265.7 71.0 6.13 3.5 610 20 2.52 86 05/11/92 615 358.0 80.0 6.13 3.0 560 18 2.76 87 04/13/92 638 385.4 91 .6 4.90 1 .5 460 17 1 .98 89 05/11/92 599 340.1 75.5 9.19 1.5 460 17 2.84 90 04/1 3/92 596 343.4 74.2 3.68 2.0 430 16 2.47 95 11/11/91 449 250.9 62.6 2.45 3.5 510 18 2.44 97 04/27/92 579 343.9 74.8 3.68 3.0 500 18 2.64 98 1 1/11/91 456 245.6 67.1 2.45 3.0 440 16 2.07 100 1 1l11/91 450 250.7 72.2 3.68 3.0 520 18 1.98 101 04/13/92 635 382.3 86.1 10.41 2.0 420 16 2.89 102 03/16/92 676 392.2 78.7 6.13 3.5 620 19 3.21 103 05/11l92 669 395.3 78.7 6.74 3.0 550 18 3.20 104 05/11/92 616 372.3 69.0 11.64 5.0 570 18 4.38 106 11/1 1191 458 251.0 59.3 4.90 3.0 530 18 2.75 107 04/‘27/92 592 336.6 65.1 4.41 1 .5 470 17 2.84 108 04/27/92 627 375.9 79.3 2.94 1 .5 430 16 2.31 109 03l16/92 623 370.7 78.7 10.41 3.5 730 20 3.46 110 11/11/91 398 222.9 67.7 2.45 2.5 390 15 1.74 1 12 04/27/92 628 366.2 80.6 4.41 2.0 600 19 2.42 115 04/13l92 615 366.7 87.1 3.68 2.0 540 18 2.03 1 16 04/13I92 654 387.4 94.2 4.90 1 .5 520 18 1 .87 117 03/16/92 612 340.7 66.4 4.29 1.0 510 18 2.70 118 11/11I91 442 247.0 67.7 2.45 3.0 450 17 2.05 119 04/13/92 565 334.8 73.5 6.13 4.0 930 22 3.08 120 1 1/1 1l91 489 289.2 68.4 3.68 4.5 460 17 2.79 121 04/27/92 587 350.1 89.7 7.35 2.0 360 15 2.13 122 0311 6/92 526 343.5 66.4 4.90 3.5 740 20 3.28 128 11I11l91 426 250.2 66.4 4.90 3.5 450 17 2.49 129 11/11/91 509 281.1 65.8 2.45 2.5 430 16 2.33 130 04l13192 628 358.3 87.7 6.13 1 .5 560 18 2.07 133 05/11/92 616 372.0 85.1 6.74 2.5 500 18 2.58 136 04/27/92 618 364.8 81.3 4.90 3.5 630 19 2.72 139 04/13/92 626 374.7 72.2 7.1 1 2.5 430 16 3.28 142 05/11/92 603 341.1 72.2 2.45 1.5 430 16 2.32 143 03/16l92 610 363.6 75.1 9.19 1.5 490 17 3.06 144 04/13/92 593 354.8 91.6 4.29 2.5 510 18 1 .87 180 Date Harvest Table A-5. (cont’d) Steer HCW. REA, Backfat, Marbling Quality Calculated no. harvested wt., lgg kg cm 2 mm KPHL% score grade yield mde 147 04/13I92 589 357.3 74.5 6.74 3.5 760 20 3.18 151 05/11/92 649 391.9 84.5 7.96 3.0 440 16 3.00 153 11/11191 448 243.1 67.1 2.94 3.0 360 15 2.09 154 04l13l92 617 370.1 80.6 6.74 3.0 550 18 2.89 155 05/1 1/92 591 332.5 82.6 5.51 2.0 450 17 2.15 157 11l11/91 466 260.8 66.4 4.90 2.0 460 17 2.27 158 05/11/92 612 357.3 68.4 7.35 1.5 450 17 3.15 159 04I13/92 613 372.2 80.6 5.51 4.0 560 18 2.98 161 04l27l92 679 415.0 90.3 11.52 4.0 640 19 3.47 163 04/27/92 582 337.8 77.4 3.68 2.0 480 17 2.26 165 05/1 1l92 599 351 .0 74.2 6.74 2.5 510 18 2.95 166 04l27l92 633 387.7 89.7 6.86 2.5 630 19 2.50 170 04l27l92 574 330.1 76.8 5.39 1 .5 520 18 2.31 171 11/11/91 401 225.2 66.4 1.23 3.0 450 17 1.80 172 11/11I91 443 254.7 74.8 3.68 2.5 440 16 1.78 173 11/11/91 420 232.3 57.4 3.68 2.0 340 15 2.36 180 05/1 1l92 708 403.2 82.6 13.48 1 .5 430 16 3.46 183 03l16l92 628 367.6 79.0 7.35 4.5 730 20 3.31 184 03/16l92 590 344.4 81 .3 7.35 1 .5 550 18 2.40 187 11/11/91 507 278.5 69.7 2.45 1.5 390 15 1.91 192 03/16/92 641 375.5 62.6 6.74 1 .5 640 19 3.53 194 05/11/92 547 316.7 67.7 3.06 2.0 610 19 2.51 195 04/13/92 624 376.1 71 .0 9.19 3.5 490 17 3.77 196 04/13192 594 359.6 85.8 4.90 4.5 570 18 2.66 197 03/16/92 613 354.8 78.7 3.06 2.5 730 20 2.38 198 03/16/92 625 382.9 77.4 9.19 3.0 680 19 3.41 199 05/11/92 616 359.5 81 .9 2.45 2.5 520 18 2.20 201 11/11/91 407 221.8 58.1 2.45 3.5 310 14 2.42 205 04/13/92 589 346.5 73.9 7.35 3.0 500 18 3.09 211 11/11/91 480 283.1 69.0 4.90 2.5 390 15 2.43 212 04/27/92 553 341.1 76.8 7.35 3.5 580 18 3.00 213 11/11/91 451 254.7 68.4 1.84 2.0 440 16 1.82 215 03/16/92 577 342.4 82.6 4.29 2.5 570 1 8 2.21 217 03l16l92 602 352.9 81.3 4.90 1.0 420 16 2.12 218 04/13l92 572 333.8 80.3 4.29 2.0 410 16 2.15 221 05/1 1l92 662 405.6 87.1 4.90 4.0 820 21 2.88 222 03/16/92 584 344.8 77.4 4.90 3.0 770 20 2.65 225 03/16/92 679 388.1 108.0 4.90 1 .5 510 18 1 .19 227 11/11/91 422 241.4 68.4 2.45 4.0 480 17 2.17 228 04/13192 682 374.7 71 .3 2.45 2.5 440 16 2.85 232 03/16/92 550 337.8 83.9 14.09 3.0 640 19 3.21 234 05/1 1l92 615 339.7 68.4 4.29 2.0 420 16 2.79 236 04l27l92 616 362.6 83.9 2.45 3.5 450 17 2.33 181 Table A-5. (cont’d) Steer Date Harvest HCW, REA, Backfat, Marbling Quality Calculated no. harvested wt., kg kg cm 2 mm KPH, % score grjde yield grade 238 03l16/92 628 378.4 71 .6 8.58 1 .5 470 1 7 3.29 239 04/27/92 561 331.3 79.3 7.35 2.5 450 17 2.59 241 03/16192 603 345.6 87.1 4.90 2.0 540 18 1 .98 243 04/13/92 669 388.8 75.5 6.13 2.5 560 18 3.14 “Marbling scorez300 = traces, 400 = slight, 500 = small, 600 = modest, 700 = moderate, 800 = slightly abundant, and 900 = moderately abundant. bUSDA quality gradez14 = low standard, 15 = high standard, 16 = low select, 17 = high select, 18 = low choice, 19 = average choice, 20 = high choice, 21 = low prime, 22 = average prime. cCalculated yield grade equation2YG = 2.5 + (2.5 * backfat) + (.0038 * HCW) + (.2 * KPH) - (.32 * REA). 182 Table A-6. Ninth-tenth-eleventh rib composition from Experiment 809002 Steer 9-10-1 1 bone Soft tissue, Bone in Soft tissue m "'b wt.» 9 wt» 9 9 "'b. % Water, % EEL, % CP, % 1 4515.1 762.3 3752.8 16.88 45.57 40.72 12.11 2 5231.4 764.8 4466.6 14.62 45.41 40.53 14.01 5 5150.3 967.3 4183.0 18.78 50.86 31.95 14.98 8 3846.5 687.8 3158.7 17.88 47.64 37.34 12.64 9 4816.6 1034.3 3782.3 21.47 55.42 25.62 16.46 10 3495.3 718.8 2776.5 20.56 56.43 26.25 15.84 11 3471.2 658.0 2813.2 18.96 52.22 30.83 15.85 12 4809.6 683.1 4126.5 14.20 45.70 39.56 13.89 13 5242.0 912.6 4329.4 17.41 49.73 34.14 14.61 14 3404.6 705.9 2698.7 20.73 58.70 20.98 17.93 16 5368.5 836.5 4532.0 15.58 50.52 32.78 16.05 17 5499.3 1195.6 4303.7 21.74 57.22 24.80 17.22 18 4905.4 878.3 4027.1 17.90 54.23 27.90 16.91 20 4941.5 940.5 4001.0 19.03 48.93 35.20 14.83 21 4120.3 818.7 3301.6 19.87 47.20 36.55 14.23 25 5450.8 782.3 4668.5 14.35 41.39 45.57 12.07 27 3314.7 696.3 2618.4 21.01 57.55 23.86 16.67 29 5108.2 977.7 4130.5 19.14 49.03 34.93 13.10 31 4698.9 913.1 3785.8 19.43 51.80 31.55 15.46 - 32 3288.5 658.4 2630.1 20.02 57.63 24.33 16.17 34 5357.0 1082.7 4274.3 20.21 48.91 37.12 13.12 37 4965.8 1035.7 3930.1 20.86 57.04 24.60 17.76 38 4774.8 807.2 3967.6 16.91 54.39 28.74 16.03 40 3560.8 696.6 2864.2 19.56 59.18 19.69 17.13 42 5195.8 894.0 4301 .8 17.21 49.00 35.63 13.86 43 5354.8 916.5 4438.3 17.12 43.01 44.73 12.11 44 4979.1 805.0 4174.1 16.17 52.14 31.07 15.93 45 3625.1 702.9 292.2 19.39 51.00 31.71 14.49 46 4808.5 846.6 3961.9 17.61 50.28 31.94 15.30 47 6135.5 1012.6 5122.9 16.50 56.36 24.42 17.49 50 5477.3 1025.2 4452.1 18.72 49.04 34.70 14.91 54 5484.9 1029.5 4455.4 18.77 45.21 40.54 13.68 55 5417.2 891.1 4526.1 16.45 50.09 31.93 14.90 57 5003.5 753.9 4249.6 15.07 46.73 38.26 14.15 58 5452.2 799.5 4652.7 14.66 45.30 39.66 14.46 59 5893.0 984.1 4908.9 16.70 50.06 34.55 14.82 60 3056.8 735.4 2321.4 24.06 49.58 35.09 13.85 62 5011.4 834.8 4176.6 16.66 49.34 34.63 14.01 63 5370.4 814.7 4555.7 15.17 52.91 30.71 15.63 65 5210.6 884.0 4326.6 16.97 52.15 32.23 14.70 70 5519.5 937.7 4581 .8 16.99 45.71 40.22 14.11 183 Table A-6. (cont’d) Steer 9-10-11 bone Soft tissue, Bone in Soft tissue no. rib wt. 9 wt. 9 9 rib. % Water, % EEL, % cp, % 72 3864.3 809.9 3054.4 20.96 60.79 20.1 1 17.28 75 5103.7 1068.6 4035.1 20.94 55.57 26.20 16.35 76 5002.8 1025.3 3977.5 20.49 54.99 28.42 15.10 77 5234.9 1115.7 4119.2 21.31 51.95 31.25 15.73 78 3308.5 572.2 2736.3 17.29 48.49 35.98 13.86 80 5018.1 1105.6 3912.5 2.03 52.70 28.64 15.76 83 5953.1 1224.8 4728.3 20.57 56.79 26.15 16.84 85 3911 .4 652.3 3259.1 16.68 47.73 36.82 13.33 86 5063.6 696.5 4367.1 13.76 46.02 38.87 13.03 87 5153.1 996.4 4156.7 19.34 52.84 31.28 15.08 89 4981 .1 984.5 3996.6 19.76 54.41 27.20 16.69 90 4893.6 879.0 4014.6 17.96 52.64 30.59 15.82 95 3403.8 702.6 2701.2 20.64 51.80 31 .81 14.47 97 5031 .1 936.9 4094.2 18.62 49.73 34.58 13.94 98 3595.0 751 .4 2843.6 20.90 54.98 26.55 17.14 1 00 3673.4 663.8 3009.6 1 8.07 55.44 26.69 14.32 101 5789.4 1 156.2 4633.2 19.97 51 .30 32.52 16.00 102 5647.9 994.0 4653.9 17.60 44.33 41 .64 13.05 103 5422.1 1044 4378.1 19.25 48.25 35.62 14.11 104 5551.5 917.1 4634.4 16.52 42.11 44.49 11.85 106 3381.7 712.0 2669.7 21.05 50.38 33.19 14.92 107 4757.8 1010.7 3747.1 21 .24 56.63 24.38 18.67 108 5406.8 1017.1 4389.7 18.81 59.01 21.60 17.86 109 5929.4 1262.3 4667.1 21 .29 40.75 45.68 12.13 110 3144.1 704.1 2440.0 2.39 55.19 28.01 15.03 1 12 5064.6 855.8 4208.8 16.90 52.91 30.41 16.36 115 5587.0 987.4 4599.6 17.67 52.36 29.38 15.43 116 5341.0 931.3 4409.7 17.44 51.01 33.17 15.69 117 4775.6 1022.5 3753.1 21.41 51.32 32.36 15.12 118 3685.8 683.5 3002.3 18.54 53.51 28.89 15.88 119 4221.6 818.3 3403.3 19.38 42.09 44.70 12.47 120 3781.0 743.1 3037.9 19.65 55.15 27.67 15.10 121 5444.3 1033.2 4411.1 18.98 55.37 25.77 17.62 122 4015.4 780.1 3235.3 19.43 50.46 34.14 14.09 128 3288.6 646.6 2642.0 19.66 48.80 35.19 16.32 129 3818.6 786.5 3032.1 20.60 55.42 28.00 14.44 130 5545.5 995.0 4550.5 17.94 54.05 29.18 16.20 133 5125.3 893.7 4231.6 17.44 50.12 32.43 15.31 136 4791 .5 760.4 4031 .1 15.87 50.45 32.69 15.65 139 5069.9 1108.9 3961.0 21.87 51.02 33.17 14.76 142 4624.8 989 3635.8 21 .38 57.57 24.09 17.81 143 5301.3 1017.3 4284.0 19.19 50.52 33.57 15.15 184 Table A-6. (cont’d) m E = Steer 9-10—1 1 bone Sofl tissue, Bone in Soft tissue "0- rib Wt» 9 Wt. 9 9 lib. % Water, % EEL, % CP, % 144 5098.5 990.0 4108.5 19.42 47.17 38.60 16.21 147 5127.9 791.6 4336.3 15.44 46.56 38.70 13.81 151 5590.6 865.8 4724.8 15.49 52.74 29.20 16.60 153 3360.9 788.4 2572.5 23.46 57.67 22.95 17.44 154 5414.3 925.5 4488.8 17.09 43.21 43.59 12.41 155 5066.9 950.7 4116.2 18.76 57.53 23.59 17.57 157 3860.0 854.7 3005.3 2.14 55.43 26.96 14.49 158 4974.0 875.1 4098.9 17.59 50.68 32.86 14.30 159 5624.9 963.3 4661.6 17.13 47.93 37.22 14.71 161 6060.4 860.8 5199.6 14.20 45.43 40.18 13.94 163 4717.0 955.3 3761.7 20.25 53.01 28.85 16.82 165 5564.6 827.2 4737.4 14.87 49.00 33.51 16.56 166 5884.9 976.4 4908.5 16.59 50.53 32.71 15.27 170 5366.1 972.5 4393.6 18.12 50.72 31.44 15.32 171 3348.9 675.9 2673.0 20.18 58.38 21 .92 16.92 172 3474.7 602.0 2872.7 17.33 54.05 28.44 15.44 173 3309.7 865.3 2444.4 26.14 57.31 24.55 15.87 180 6323.7 1119.1 5204.6 17.70 49.47 35.08 13.94 183 5250.9 774.9 4476.0 14.76 44.52 41 .50 12.82 184 5203.0 1050.0 4153.0 20.18 50.62 33.65 14.03 187 3711.8 721.0 2990.8 19.42 58.41 22.87 16.93 192 5432.4 1064.6 4367.8 19.60 44.69 40.67 1 3.42 194 4429.7 777.1 3652.6 17.54 53.08 28.40 14.94 1 95 5492.4 872.3 4620.1 15.88 55.46 27.25 1 3.72 196 5349.7 820.6 4529.1 15.34 46.88 38.51 14.92 197 5191.5 1101.5 4090.0 21.22 54.04 28.12 16.32 198 5732.9 843.3 4889.6 14.71 41.94 44.87 11 .70 199 5895.1 1026.8 4868.3 17.42 50.78 32.67 15.54 201 3087.2 600.1 2487.1 19.44 56.22 25.47 15.85 205 5471.1 1043.0 4428.1 19.06 48.00 36.42 14.21 211 4399.6 846.3 3553.3 19.24 56.51 24.71 16.88 212 4907.8 915.3 3992.5 18.65 46.92 37.68 13.39 213 3415.5 729.7 2685.8 21.36 57.22 23.89 16.41 215 4991.8 897.3 4094.5 17.98 48.77 35.62 13.51 217 5354.7 1254.8 4099.9 23.43 54.73 27.38 16.70 218 5101.1 946.6 4154.5 18.56 54.33 28.04 17.02 221 6024.4 984.9 5039.5 16.35 49.53 34.54 14.82 222 5472.4 945.3 4527.1 17.27 49.35 34.36 13.81 225 6098.0 1 121.3 4976.7 18.39 59.09 21 .79 18.10 227 3510.9 728.8 2782.1 20.76 53.10 30.57 13.67 228 5191.0 1071.2 4119.8 20.64 54.12 28.84 16.38 232 4706.9 731 .4 3975.5 15.54 43.99 41 .87 12.83 185 Table A-6. (cont’d) Steer 9-10-1 1 bone Soft tissue, Bone in Soft tissue 00- rib wt. 9 Wk 9 9 "'b. % Water, % EEL, % cp, % 234 4404.2 880.3 3523.9 19.99 57.53 24.91 17.29 236 5277.0 921.1 4355.9 17.45 53.35 28.30 16.94 238 5930.3 1072.7 4857.6 18.09 49.74 33.97 14.23 239 4526.6 744.9 3781.7 16.46 54.16 28.01 16.51 241 5296.9 981.0 4315.9 18.52 51.80 32.83 14.75 243 5311.8 948.1 4363.7 17.85 46.01 40.10 14.04 186 Table A-7. Organ and muscle weights from Experiment BC9002 r Ste e r Liver Spleen Kidney Heart Lung ST muscle no. vangl “@197 vnugLi “4,9 vans; mm,g___ 1 8377 1414 1264 2290 6282 1958 2 7041 1273 1032 1880 7930 2658 5 8186 1173 1214 2159 7473 2587 8 5492 818 1096 1703 5420 1327 9 6350 968 1723 2298 9142 2600 10 6247 978 ND ‘ 1652 5293 1852 11 5832 863 1013 1604 6115 1427 12 6159 945 ND 1758 7322 1922 13 7991 1132 1514 1876 7866 2576 14 5218 1104 1100 1502 5075 1413 16 6914 1241 973 2108 6441 2615 17 8127 1223 1286 2160 7358 2183 18 7759 1250 1277 2144 7167 2566 20 6809 1364 1286 1834 6573 2620 21 5955 1136 1800 2053 7126 2037 25 6536 1132 1341 1515 4191 2602 27 5930 1100 1352 1423 5509 1755 29 6432 895 1068 1821 7502 2294 31 6477 1268 1605 2396 7645 2279 32 5528 710 1054 1403 5374 1557 34 5550 1300 918 1916 7394 2067 37 7750 1236 1555 2247 8528 2490 38 7559 1218 1177 1960 7055 2314 40 6519 986 1403 1684 5556 1725 42 7132 1150 1409 1757 7932 2745 43 5764 918 1077 2318 5698 2394 44 7305 1177 1209 2312 7089 2477 45 5929 978 1208 1605 5780 1621 46 6736 1036 1386 1790 6412 2308 47 9032 1036 1300 2032 7488 2616 50 7186 1414 1873 2085 7396 1839 54 7159 1286 1541 2315 7790 2567 55 6391 1241 1327 2100 6928 2442 57 6627 868 1005 2731 7313 2249 58 7232 ND' 1059 2025 6069 2527 59 7800 1173 1368 2032 8424 2694 60 5906 1052 1342 1782 7067 1261 62 5959 1073 895 1879 6160 1886 63 7150 991 1395 1963 7896 2470 65 6205 1277 1200 2056 6161 2123 70 7318 1050 1100 2015 7775 2176 187 Table A-7. (cont’d) Steer Liver Spleen Kidney Heart Lung ST muscle no. vngjl “419 “mug m¢,g unugL44, mm,g 72 5764 1026 1184 1593 6186 1809 75 6873 1405 1350 1940 6121 2171 76 7032 955 1195 2030 7150 2259 77 7068 1336 1909 1960 7943 2236 78 8673 788 1354 1572 5525 1462 80 7468 1545 1564 1291 N0 2675 83 7159 1114 1545 2036 6950 2795 85 6075 850 1308 1527 5582 1719 86 5973 1009 1027 2184 8575 1934 87 7618 1668 1441 2105 7983 2132 89 7195 1023 1700 2002 7412 2143 90 7700 1405 1318 1897 6298 2278 95 5330 994 1040 1692 5948 1432 97 7605 927 1209 2235 7042 2362 98 5580 1170 1146 1544 5497 1617 100 6646 980 1219 1812 6902 1675 101 7586 832 1659 2042 6396 2610 102 6527 1164 1355 2130 8037 2572 103 7205 1182 1314 2144 8788 2534 104 6309 955 1227 1950 8140 2603 106 5318 748 1266 1621 6405 1540 107 6032 1341 1559 1937 7099 2753 108 7709 1286 1409 2177 7410 2557 109 6950 1077 1273 2158 7440 2430 110 5892 794 1309 1224 6567 1510 112 7600 1377 1377 2302 7492 2460 115 7055 955 1000 2035 6508 2475 116 7877 1705 1018 1731 6719 2756 117 7455 1041 1405 1946 6348 2366 118 5878 1028 1310 1720 6583 1644 119 6559 1164 1141 1812 7368 1850 120 6555 922 1240 1945 6117 2030 121 7173 1459 1232 1896 6683 2163 122 6486 932 1255 1728 5670 1971 128 5810 1146 1058 1904 5591 1630 129 7130 1094 1390 1549 5476 1716 130 8473 1241 1364 2166 6681 2681 133 6582 909 1241 2110 7380 2327 136 8218 1200 1495 2441 7367 2376 139 7086 1018 1405 1922 6196 2257 142 8682 864 1745 2040 9096 2709 143 6459 1277 1359 2184 7678 2570 144 7123 1141 1641 1990 6946 2315 188 Table A-7. (cont’d) Ste 9 r Liver Spleen Kidney Heart Lung ST muscle no. wmég vnugLi “Rug Vang; “mug, vw1;L__ 147 7245 914 1200 2103 6197 2103 151 6691 1477 1273 2150 7696 2757 153 5776 1154 1270 1489 6188 1782 154 6582 1027 1027 1663 7265 2258 155 5977 1377 1127 1900 6509 2330 157 5981 1151 1083 1834 5255 1753 158 8714 891 1536 1873 9712 2638 159 7741 1027 1205 1585 8307 2050 161 8673 1082 1309 2593 7470 2260 163 7973 1395 1405 2016 6020 2562 165 6991 1145 1095 1947 6732 2224 166 7459 1218 1395 2301 6600 2566 170 7809 686 1041 2544 7860 2571 171 5158 724 1040 1669 7008 1580 172 6143 932 1410 1438 4936 1605 173 6509 1214 1355 974 4306 1460 180 7473 1295 1618 2218 6622 2665 183 7368 1091 1386 2105 7043 2000 184 6550 1059 1209 2023 7200 2358 187 6530 1454 1348 1665 6831 1836 192 7518 1055 1618 2100 7130 2640 194 7068 836 1186 2428 7162 1908 195 7468 977 1509 2035 7293 2389 196 6500 1136 1086 2098 6741 2216 197 7745 1291 1395 1953 7400 2352 198 7327 995 1523 2036 7718 2350 199 5632 1455 1195 2191 7235 2495 201 5586 981 980 1706 6037 1402 205 7300 1205 1236 2201 7225 2266 211 5396 716 1372 1733 6769 1867 212 6077 868 1064 1931 7254 2307 213 5631 924 1410 1436 5538 1596 215 5923 1227 1109 1934 6762 2252 217 6882 909 1318 1830 6130 2734 218 7073 1345 1259 2190 6632 2213 221 9100 1164 1686 1836 7610 2784 222 7445 1141 1295 1935 7095 2345 225 8677 1709 1859 2368 7655 3018 227 5186 706 1092 1506 5941 1435 228 8618 1618 1995 2975 7526 2751 232 5891 1368 1191 1979 7729 2121 234 7305 1295 1455 3048 11193 2207 236 8509 1036 1541 2316 7641 2487 189 Table A-7. (cont’d) Steer Liver Spleen Kidney Heart Lung ST muscle no. wt.,L wt., g wt., L wt.,g wt.,g wt., g_ 238 5914 1295 1436 2135 6696 2787 239 7795 1 391 1 705 2080 6750 1 956 241 5818 982 1486 2102 6944 2254 243 7945 1277 1082 2050 8052 2829 'Not determined. 190 Table A-8. Steer hip height for Experiment BC9002 04l25/91 04/26191 Average 08/30/91 10l25/91 10/27/91 Average Final hip ht., in Steer Initial #1 Initial #2 Initial Day 112 Day 181 Day 183 Day 182 #1 #2 Avg. no. hip ht., in hip ht., in hip ht., in hip htfl in hip htfl in hip ht., in hip ht, in 1 44.13 44.00 44.06 50.25 52.13 52.88 52.50 55.00 55.69 55.35 2 44.31 43.75 44.03 50.13 52.88 53.25 53.06 57.13 56.44 56.79 4 44.00 42.69 43.34 5 44.38 44.44 44.41 50.50 52.88 54.63 53.75 58.13 57.44 57.79 8 43.38 43.00 43.19 48.13 52.00 51 .75 51 .88 9 42.25 43.25 42.75 48.94 52.38 52.75 52.56 57.25 56.38 56.82 10 46.31 47.00 46.66 52.50 55.00 56.06 55.53 1 1 43.25 44.75 44.00 49.38 52.38 53.13 52.75 12 41.88 42.19 42.03 48.56 49.88 50.69 50.28 54.88 54.81 54.85 13 44.19 43.19 43.69 50.13 52.81 53.56 53.19 55.38 55.00 55.19 14 42.63 42.13 42.38 47.50 50.38 50.25 50.31 16 42.63 42.13 42.38 48.31 52.38 52.56 52.47 56.13 55.44 55.79 17 42.56 43.44 43.00 50.38 53.50 53.63 53.56 59.13 58.50 58.82 18 42.94 43.81 43.38 50.19 52.25 53.75 53.00 57.81 57.50 57.66 19 40.50 42.13 41.31 47.50 20 44.19 43.94 44.06 49.50 52.50 53.31 52.91 56.69 55.81 56.25 21 43.25 43.50 43.38 48.63 50.38 51 .38 50.88 53.50 53.63 53.57 25 43.13 43.13 43.13 49.69 51.81 52.63 52.22 56.06 56.06 56.06 27 41 .31 38.88 40.09 47.69 50.06 50.38 50.22 29 42.81 43.25 43.03 48.88 51.88 52.56 52.22 56.63 56.81 56.72 31 44.56 45.13 44.84 50.56 54.00 54.63 54.31 58.50 58.88 58.69 32 42.44 42.88 42.66 49.00 51 .38 52.50 51 .94 34 43.88 45.06 44.47 50.06 53.75 52.69 53.22 57.75 57.13 57.44 37 42.88 43.06 42.97 48.44 51.50 52.13 51.81 57.06 56.94 57.00 38 42.56 43.81 43.19 48.13 51.06 52.88 51.97 56.81 56.75 56.78 39 43.00 43.81 43.41 49.00 52.00 51 .63 51 .81 40 42.50 42.25 42.38 49.19 51 .50 53.06 52.28 42 44.88 44.19 44.53 51.50 52.75 53.44 53.09 56.75 56.94 56.85 43 43.13 43.13 43.13 48.56 51.75 52.50 52.13 55.44 56.00 55.72 44 43.50 42.63 43.06 49.19 51.38 52.63 52.00 56.88 56.06 56.47 45 43.94 43.88 43.91 49.56 51.19 51.75 51.47 46 44.44 43.38 43.91 49.88 52.56 52.38 52.47 56.75 55.50 56.13 47 42.56 43.19 42.88 49.06 51.88 52.25 52.06 58.25 58.19 58.22 49 43.50 43.63 43.56 50 44.50 43.69 44.09 51.13 53.25 53.25 53.25 56.81 57.00 56.91 54 43.38 44.44 43.91 50.44 53.00 54.00 53.50 56.81 56.19 56.50 55 4125 42.19 41.72 48.50 52.00 53.88 52.94 57.81 57.38 57.60 56 42.75 42.56 42.66 49.13 52.38 52.25 52.31 57 42.19 42.00 42.09 47.50 51.50 51.50 51.50 55.44 53.75 54.60 58 43.38 44.63 44.00 49.25 52.50 53.25 52.88 56.88 57.00 56.94 191 04I25/91 04/26/91 Table A-8. (cont’d) Average 08/30/91 10l25l91 10727l91 Average Day 112 Day 181 Day 183 Day 182 #1 hip ht., in hip ht., in hip ht., in hip ht., in hip ht., in hip ht., in hip ht., in Final hip htLin #2 Avg. Steer Initial #1 Initial #2 Initial no. 59 40.00 40.13 40.06 60 42.50 41.00 41.75 62 41 .94 40.94 41 .44 63 41.38 40.81 41.09 65 42.25 41 .69 41 .97 70 45.00 44.25 44.63 72 41 .00 40.50 40.75 73 42.44 42.25 42.34 75 43.88 42.25 43.06 76 42.75 42.13 42.44 77 43.31 43.13 43.22 78 41.25 41.94 41.59 79 39.88 39.69 39.78 80 44.75 44.44 44.59 83 43.44 44.00 43.72 84 45.25 43.94 44.59 85 42.63 42.19 42.41 86 42.00 42.56 42.28 87 42.75 43.00 42.88 88 42.50 40.63 41 .56 89 41.81 41.75 41.78 90 43.56 43.44 43.50 91 43.56 44.63 44.09 94 42.38 42.56 42.47 95 42.00 42.63 42.31 97 41.50 41.06 41.28 98 43.88 43.38 43.63 99 41 .63 42.63 42.13 100 43.88 43.88 43.88 101 44.13 43.38 43.75 102 45.94 45.44 45.69 103 41 .88 41.56 41.72 104 41 .88 42.19 42.03 105 44.63 45.44 45.03 106 44.63 43.81 44.22 107 43.50 42.69 43.09 108 43.38 42.75 43.06 109 44.13 44.50 44.31 110 41.75 43.19 42.47 111 40.50 41.13 40.81 47.25 48.94 47.75 47.56 46.94 50.19 50.00 47.31 49.06 47.56 49.50 49.75 47.50 51 .50 50.25 50.00 49.00 49.00 50.63 47.00 49.25 50.19 48.06 48 .63 49.06 47.38 48.06 49.56 49.69 49.44 51 .38 50.31 48.06 52.50 50.00 49.06 49.13 51 .00 48.06 49.00 192 49.25 52.75 51 .00 50.88 49.75 53.13 52.88 51 .13 52.75 50.50 52.88 51 .38 54.81 53.38 51 .00 51 .00 53.25 49.88 52.25 52.75 51 .00 52.50 53.00 51 .38 52.00 52.00 51 .50 53.00 52.50 51 .19 52.50 53.38 52.75 53.00 51 .94 50.56 53.13 49.19 50.56 49.38 53.25 52.00 51 .56 53.88 51 .00 52.50 51 .63 55.69 52.94 51.25 52.00 53.88 50.13 53.13 53.44 52.00 52.25 52.44 53.13 52.50 52.38 50.50 53.50 53.81 51 .25 52.94 53.25 52.50 54.31 51 .56 49.91 55.13 55.63 55.38 52.94 50.09 53.25 53.50 53.38 50.72 54.06 54.50 54.28 49.56 53.75 52.88 53.32 53.19 56.56 57.88 57.22 52.44 51.34 53.31 57.50 57.75 57.63 50.75 56.75 56.75 56.75 52.69 54.38 55.50 54.94 51 .50 55.25 58.88 59.44 59.16 53.16 56.31 57.31 56.81 51.13 51.50 55.81 56.25 56.03 53.56 58.75 59.25 59.00 50.00 52.69 58.50 57.38 57.94 53.09 57.56 58.13 57.85 51 .50 52.38 52.72 52.25 55.06 55.50 55.28 52.25 52.19 51.00 57.56 57.94 57.75 53.25 56.94 58.00 57.47 53.16 58.25 57.38 57.82 51.22 55.81 55.50 55.66 52.72 53.31 59.88 59.00 59.44 52.63 59.44 60.25 59.85 53.66 56.69 56.25 56.47 51.75 Table A-8. (cont’d) 04/25/91 04/26/91 Average 08/30l91 10l25l91 10/27/91 Average Final hip ht.,in Steer Initial #1 Initial #2 Initial Day 112 Day 181 Day 183 Day 182 #1 #2 Avg. no. hip ht., in hip ht., in hip ht., in hip ht., in hip ht., in hip ht., in hip ht., in 112 42.81 42.94 42.88 48.69 52.00 52.94 52.47 58.06 57.81 57.94 114 41.44 40.94 41.19 115 43.44 43.50 43.47 47.56 51.25 50.56 50.91 55.94 55.38 55.66 116 44.63 44.69 44.66 50.25 54.38 53.56 53.97 59.13 59.06 59.10 117 44.88 46.00 45.44 52.00 54.50 55.00 54.75 58.81 59.50 59.16 118 42.50 42.56 42.53 49.81 51 .88 51.31 51.59 119 43.75 43.44 43.59 49.50 53.44 52.81 53.13 56.06 56.50 56.28 120 44.50 44.75 44.63 52.75 53.00 54.56 53.78 121 41 .63 41.56 41.59 48.50 51 .25 52.06 51.66 57.50 56.50 57.00 122 43.50 43.38 43.44 49.13 50.50 51.50 51.00 53.06 53.06 53.06 123 42.19 43.13 42.66 49.06 52.94 53.25 53.09 124 43.00 42.19 42.59 48.13 52.88 51 .50 52.19 127 43.69 42.69 43.19 50.00 128 44.63 44.00 44.31 50.00 52.75 53.25 53.00 129 44.38 43.75 44.06 49.75 52.88 53.75 53.31 130 43.94 43.81 43.88 48.50 50.50 51 .06 50.78 55.50 56.75 56.13 133 42.50 42.13 42.31 49.75 52.56 52.69 52.63 56.19 56.75 56.47 135 42.44 43.00 42.72 47.63 136 43.25 43.31 43.28 48.75 53.75 53.50 53.63 57.44 57.63 57.54 137 43.63 41 .88 42.75 139 42.50 44.31 43.41 49.88 52.50 52.25 52.38 57.19 56.94 57.07 140 44.94 46.88 45.91 141 42.38 43.50 42.94 46.19 51.00 51.50 51.25 142 42.63 40.44 41.53 47.25 49.50 49.81 49.66 56.00 56.50 56.25 143 46.25 45.19 45.72 51.00 53.25 54.44 53.84 59.25 58.38 58.82 144 41.63 41 .44 41.53 47.88 50.50 52.13 51.31 56.25 55.88 56.07 147 43.94 45.00 44.47 51.38 53.75 53.50 53.63 57.06 57.75 57.41 151 42.00 41.69 41.84 50.19 51.19 53.50 52.34 58.00 57.38 57.69 153 44.25 44.63 44.44 48.69 52.06 52.69 52.38 154 42.88 43.44 43.16 49.25 52.50 53.00 52.75 55.50 55.88 55.69 155 42.13 41 .69 41.91 48.56 52.25 51.88 52.06 58.44 57.50 57.97 157 45.88 46.44 46.16 52.88 55.50 55.94 55.72 158 41.38 40.38 40.88 49.00 52.75 52.25 52.50 57.50 53.25 55.38 159 43.63 43.13 43.38 49.19 52.50 52.50 52.50 56.38 55.88 56.13 161 42.44 41.00 41 .63 48.19 52.38 52.88 52.63 55.63 55.63 55.63 163 43.25 43.13 43.19 48.13 53.25 53.06 53.16 58.38 58.44 58.41 165 42.94 42.31 42.63 48.19 51.75 52.56 52.16 55.31 55.00 55.16 166 44.50 43.94 44.22 49.75 52.88 53.13 53.00 59.00 59.00 59.00 170 43.00 43.38 43.19 49.56 52.50 52.50 52.50 57.00 56.63 56.82 171 41.94 42.31 42.13 48.19 52.00 52.06 52.03 193 Table A-8. (cont’d) 04l25l91 04l26/91 Average 08/30/91 10l25l91 10/27/91 Average Final hip ht., in Steer Initial #1 Initial #2 Initial Day 112 Day 181 Day 183 Day 182 #1 #2 Avg. no. hip ht., in hip ht., in hip ht., in hip ht., in hip ht., in hip ht., in hip ht., in 172 40.50 41 .00 40.75 45.00 50.25 50.06 50.16 173 43.13 42.56 42.84 49.38 53.00 53.50 53.25 180 41.38 42.63 42.00 48.69 51.94 52.88 52.41 58.19 57.63 57.91 183 43.38 43.88 43.63 49.25 51.00 52.50 51 .75 54.56 54.81 54.69 184 45.13 43.63 44.38 49.94 52.00 52.38 52.19 56.25 56.63 56.44 187 42.63 43.19 42.91 49.75 52.81 53.50 53.16 192 44.13 43.31 43.72 50.50 52.88 54.44 53.66 57.63 58.06 57.85 194 41.88 42.00 41 .94 47.44 51.00 52.00 51.50 56.06 56.50 56.28 195 43.13 44.00 43.56 48.94 52.38 53.88 53.13 57.13 57.50 57.32 196 43.19 42.31 42.75 48.19 51.25 ' 52.19 51.72 55.13 54.69 54.91 197 44.25 43.94 44.09 50.75 52.94 53.44 53.19 56.50 57.00 56.75 198 44.00 43.88 43.94 48.69 51.00 50.00 50.50 54.06 54.00 54.03 199 43.00 42.94 42.97 45.75 52.56 52.25 52.41 57.13 56.75 56.94 201 41.25 41 .69 41.47 48.63 51.38 51.69 51.53 203 42.00 43.06 42.53 205 43.63 43.13 43.38 49.88 52.75 54.00 53.38 57.56 57.25 57.41 206 42.88 40.88 41 .88 208 43.06 42.69 42.88 210 43.13 42.75 42.94 211 43.81 44.00 43.91 50.13 53.50 55.00 54.25 212 42.75 42.19 42.47 47.25 52.00 51.81 51.91 54.38 54.56 54.47 213 43.81 43.88 43.84 49.81 53.50 53.88 53.69 215 44.94 43.00 43.97 50.81 52.00 52.31 52.16 56.56 56.75 56.66 216 43.44 42.38 42.91 50.50 53.00 53.75 53.38 217 43.69 43.13 43.41 48.25 52.00 52.25 52.13 54.13 55.38 54.76 218 42.25 42.50 42.38 49.00 52.00 51 .00 51 .50 56.63 56.50 56.57 221 43.75 40.88 42.31 50.00 52.88 53.50 53.19 57.00 57.88 57.44 222 44.25 44.00 44.13 50.63 52.88 53.50 53.19 56.50 56.06 56.28 225 43.75 44.69 44.22 51.00 52.88 54.00 53.44 57.63 58.00 57.82 227 42.50 42.88 42.69 47.88 50.50 50.50 50.50 228 42.50 43.50 43.00 49.44 52.50 51.63 52.06 57.31 57.31 57.31 231 43.75 44.13 43.94 51 .50 53.60 53.38 53.49 232 43.31 43.88 43.59 48.94 50.06 51 .50 50.78 53.13 54.00 53.57 234 42.94 43.31 43.13 49.19 52.25 53.63 52.94 58.00 57.81 57.91 236 41 .38 41 .88 41 .63 48.50 51.50 51.44 51.47 56.75 56.75 56.75 237 43.50 44.50 44.00 51 .00 54.00 54.25 54.13 238 44.44 44.06 44.25 50.00 51 .75 53.13 52.44 56.00 56.88 56.44 239 41.94 42.69 42.31 47.31 51 .38 51.25 51 .31 54.50 54.63 54.57 241 45.00 44.38 44.69 51 .63 53.38 53.75 53.56 57.69 56.63 57.16 243 44.75 43.75 44.25 49.44 53.00 54.88 53.94 59.13 58.25 58.69 194 med vmd wed em; 5.? ted o.e: 9.: oeeé weed End 8.? eve. we: wweede wdw we evd evd evd ew... ow; swé o.o: moo; >9; {o.o >e>d Pow «e.ww 3: oevvém e.ww we ned and end weé wed ow; odor e3... one; mwed eeed em; omd— oowé ewpede new we omd ovd omd evé ev... e1. o.e: em: oomé eoed eeud oo.w o_..ow mp: oovvde o.ww ow eed eed emd on; oo.w em; o.e: enoé nee; eved oood Pow end? we: eewowe :w >w evd 3d 3d ové ové otw o.e: eeo... eomé need weud «ed ee._.w ow: ooeede flew mw wed wed oed eeé 3.? em; o.ww? we: need oeed mmed oo.w eodp ow: >wew.ee e.ww Fw wed 3d ovd we... on; we; ed: e3; o3... {ed wend ve... 3.2. cm: ooeeée o.ww ow med ovd ovd em; oo.w em; o.ewp mo: oveé owed mend oo.w oeéw mt... weomév e.ww er end end e>d eeé ooé eeé o.eew oewé ewe; ee>d 5o; oeé wodw we: ovepdv e.ww 5 3d evd med we; evé ové e5... ow: one; mned eesd ofiw efiew om: eopode flow or vehemeoE 85.20230 =e .o: .333 «a so 3 evd med evd 3: med em; o.e: Feoé eevé enmd eFed oo.w eedp we: Refine e.ww e9 wed wed wed o: ow; e: o.mo_. eeoé ewe; eoed eeed Rd 3.3 eooé veo_..ee e.ww w_. med wed med oo.« o: B; odo 3o; owe... voed wved wow at! we: Kfidw e.ww 2 o.e: 32:32: 8538930 =e .o: .80st on 50 or 3d 3d ovd 3.? med 3.? o.e: wt; eon; enmd Pwnd t4 ew.ww or: eeomde o.ew o wvd ovd 3d d: e: ow; meow ewoé ooe; weed weed no; we.3 cw: eowedw o.ww e wed wed oed wed eed med oNe eeed mm: oeed eefio we; med. _.w: Pewtt. o.ow > ewd nwd mwd wed wed wed oNe >eed ev: eoed deed mi. 3.2. 9: oeopdp v.3 e med med med mmé mm... mm; o._.w_. on: ooeé e>ed wend vow 3.2. we: voohow e.ww m svd evd >vd we... etp ow; o.e: ooo; eoeé weed eond ovw eodp on: diode o.ww w wvd evd ovd me; he; weé o.o: oo: eevé oeed mwnd oeé 3.3 mew... eooede e.ww P . . EE . c_ . c_ . s . E E0 E0 .... so u: Eo. o o>< w¢ 3 o>< we E .5326 33 533 563 £33 . ...th .om::_o> deflow. Swede ESL—cued?»— 32m w E .33 Bate-2 «5 .38 .83. .923 .3522 woooom EoEtooxm Loo 85:23.30 econ $93922 d-< mfimh 195 196 3d nmd Rd mud mud Ed o.ww 03d mnoé movd mvod do; sod cm: 32.: mdp 3. mvd mvd med nmé um; um; mi; 3: N9; Rod 3:. 3.? 8.3 mm: 83.3 firm Nu ovd dvd dvd mmé mm; cm; dd: do: mum; onmd Sid SN 5.? ~34 moods...” oNN on mud ”Nd owd mud and cod nNn End ..8; mdvd _.omd m5. 5.0.. cm: nomad? Nd? no mvd 3d ~¢d mm; mm; 3.? dd: 3: 3m; mumd Ekd no.9 2.3 mm: 33.3 mém mm and and and on; 5... ma... odor 50.. own; wed domd mm; 3.3 o3... mvmmdn FNN no 3d 3d mvd on; 3.? on; mam: 39. 0mm... dumd domd mod no.2 or: 33.8 ......N mm mvd mvd omd NNF cmé one 0K3 5o; NNvé mnmd End 3... 8.9 up: 83.8 o.ww 8 dvd and dvd 3.? 3... NY? mi: or: 5mm; vad dmud mm; and. Fed; 9oz." o.ww mm mod mmd mad 5.? mwé Nd... dd: mwdé omvé onvd wand 3.. 3.3 oom; «mid» nNN on 3d 3d Red on; 3.? mm; dd: mm: 03.? mdmd and 8.. 3.9 5: 58.3 nNN hm omd mmd Ed mm... 3.9 mm; md: ms: at: NEd opod oné smdw and; 2&me nNN mm mvd dvd ovd Fm; mm; and dd: 2:... com; ommd «cod oo.w 3.2. 3: 83.3 ddw vm mvd mvd mvd and cm; mm... mémp cm: omo; nmmd wand 34 mmdw mu: mddev tum on and omd and so; we; mmé o.wwp 5: mmo; o—md Nde 5.? mmdm nod; 53d» 93 2. mad mnd mmd and an; on; 3...: d2: 03.? Nvmd Sod 34 3.3 cm: Eaton vdm ov and and nmd o: o: m: 93.. odd; o9... ~_.md «and odd 8.: 5: 83.8 nNN me mvd nvd Nvd mm; 3... 3.? d4: no: onmé mmmd and SN 3.? 3d; wommdm Bk 3 and omd dmd cm; I: 3.? o.ww? so: mow; Smd 3nd SN mod? mg; 33.8 nNN mv vmd vmd 3d mm; mm; mmé ddmp 8582: magmtouofluzfificamozgfi 50 «v mvd ncd 3d mm; mm; vué 0.3? So... m3: mnmd mid mdN 2mm. mow; mmpmdn NFN ow wvd std mvd 9; we; 34 o.ww? 3: com; Bod 02.0 8.? mad? 8: mmomdm vdm an and dmd mmd 5.. nmé om; o.ww? oFN; 30.? mmmd mood mmé «e.ww 3: 88.9. o.ww mm . . EE . E .5 . E .5 503 :8 5.59.2 art» E9598. .o: 92 at K u>< 0* Pt ..EREO finou 5&3 finou 53> .mgmcoo .mmSB) doaoow :ouomw 3ansz 33m N 5 62a Betas. ~ 5 do..." .38. .823 .332 6.203 .m-< osfl. med mvd ovd 3.? 2; 93 md: Nv: dmmé mvmd Sod SN cod? mode 33.? o.ww do? 2d end end an; an; on; o.ow? wow... mam: oood. m3d mm; 8.3 mm: «30.3. 03 SP wwd mvd ovd mm; cm... mm; ode mmo; N9; mmmd End 3.. 8.3 an: odomdm SN 8.. and mvd Ed and 3: mad m6: 3: «$4 momd mdmd do; mod? 8: 089mm mdm vow dmd wmd cod K; mm... as; msmw mmwé omo; mmod Sod R... 2.6m do: ddmodv 5mm mow mmd mmd Ed Pm; mm; om... md: 3: Sum; vmmd ddod mm; 9.9 8: new?” tum Now 3d 8d mmd No... 5.? No.9 o.wmw mm: «mm; Smd owed am; 3d? a: mmmmdm mNN 2: 5d de mmd do; 3... mo... 9:: Edd mom; Ed mdnd «dd 3.3 do: smnndm NFN ooF omd dvd 3.0 mm; mm; 3...? od: o3; Bvé mumd 3nd oo.w 8.2, 3: 33.3 nNN dd nmd and mmd mm; vmé dvé o.ww? m»: mum; Edd omod mm; msdw mm: moat; firm 3 Nvd mvd 3d om; oNF om... man: mmdé dmvé Imd Sod noN 3.9 3: @9de de mm 3d .....d and 3d 3d 8d odd mpmd mpmé dpmd did do... of: mp: Povwdp mdN Nd mvd ovd ovd :4 34 3.; o.ww? mm: 264 End 3nd 8; nmdm mm: Nemndv nNN om Ed 3d 8d vmé mm; vmé odmr 8532: motorcaoeazflfidoc..womemSo mm nvd dvd mvd cm; mmé mm; mdww mm: mm»... dvmd mmdd EN mmdm mo: “NNN; nNN so sad mad and an; and um; dd: mad; dam... Emd omnd 8.? odd? who; 98d» nNN on 3d Nvd ovd ow; R... «N. QB? mud; mot? momd domd SN 3.: 8..... dmmmdm NFN mo dmd dmd mvd mm; 3... mm; mémr 3: odd; :wd mwdd SN mwdm we: Nmnodv nNN mm and and dvd 3.. an; nvé dd: do: 93.? wmmd omnd mod mod? mmo; opvmdn v.3 do mvd med mvd mm; FNF cm; 0.2: So; 3m... dmmd «mud odd «_..mw om: newton o.ww on 3d hvd mvd of? 33 we; dd: 3532: magmtofiemcflfido: .fimzafm 50 R ovd 36 dvd mm; and mm; dd: who; Ev; Svd wood 3... 8.9 mm: «893 o.ww on and and and £1 med 3.? md: mm: _.omé Ed «and mm; mddm mm: dammdv _..NN ms .9 .9 EE . u_ . 5 . 5 . 5 M503 :8 5.59.0. arts $0.595. .o: < at I“ < «a r: ..E:2_o 5 2. £33 .32. 523 $5.80 .092; donoow 5:03 3983;. 505 mu... 62a Betas. :. .35 .30... .223 .3322 N €203 .m-< 2an 197 198 vmd vmd mad 3; El 3.? md: 9.: Em; momd mid oo.w vmdm do: dmmwdv Ndm Fm? mmd mmd mad and 3d and mdm m3... 8: Nvmd mvnd mm; NI; 8: mmmmdw odm omF mmd vmd nmd mmé nmé mm... mNNw 9m; 3m; odmd mdmd mod 8.9 5:. «Nada mNN t; dmd Pmd med mmé mm; mm; md: 3: NE; dwmd 85d 5.? oo.ww on: mimdv o.ww v3 omd 3d de vmé mm... mm; dd: mm: mwmé wmmd mtd mm... mvdm ~34 mvwmdm nNN m3 and dvd mad 3; av... mvé dd: 3N? 5m; mnmd vmsd mm; Nada 3d... 98de nNN N: mvd 3d med mm... mmé oo.w mdmw on: mmo; mmmd nmmd omé mmdw m5... @903 mém m9 and and umd mm; mm; of? dd: do: m8; mmmd mmsd mod 8.9 mom... ..mmmdm mNN m9 mwd mvd med om; om; omé dd: dad; oomé mmmd mmmd mm; vwém m8; donmdm 5.3 mm. and 5d ovd 3.? ové :3 as: mm: 3.0.. 3md wmmd 3N Fmdm m3; mvmmdv mdm on? and and nmd NNF em; mm; dd? 8: vomé nvmd NKd Nod no.5 mN: mmmmém m...~ map mvd mvd mvd mm... No; and dd: mmo; F3: Nomd ovmd mmé 3.9 8: mPEdm o.ww wmw ovd ovd ovd 3... 3.? 2... cm: om: cum; ddmd mmmd mod no.3 3: 38.5 dém NNF mvd mvd mvd mmé vmé om... o.ww? mom; mum; mdmd mmmd mmé dmNm mm: 38.3. mém FNF mcd 9d mvd mm; mm; 34 dd: mmo; mmvé mvmd mmnd Nd; mmsr amp... mBde tum our mmd mmd mmd mvé cm; 5‘... md: 3: omm... mEd m3... mmé mmdw mp: 33mm o.ww m: mvd dvd mvd Na; vmé omé QBF omo; mam; mmmd mmud mod «amp 8..... Sand...“ v.3 m2. dcd and dvd Nvé ové 3; md: mm: dome 3md doud moé mud? omo; vonvdm o.ww n: dmd mcd dmd mm; No.9 no; o.ww. mm: on»; Smd omnd 8N 3.3 mm: 93.9 mdm m: nmd ...md mmd oo.w mm... mm; o.ww? no: mmo; mnmd dmmd mm; Sim cm: PENN? NFN m: omd omd dmd mm; om... vmé dd: on: cam; mdmd mmud 3.? Edm oFN... ..nmmdw tum N: and and and a: 9... m: 9de mmo; m3; owmd dzd dd; 5.9 mm: 33.8 mém o: mvd 3d uvd mvé mvé mvé dd: mm: ommé oumd mEd Rd 5.9 dd: 83.9... o.ww mow .9, .9 EE . u. . c. . m. . c. nEek. Eu 5.595. mi? 59595. d: < 9 ..u < g E» ..E:2_o 5 at 52.5 5 on £23 .2350 .2920) 5.83 5.33 69850.2 .35 c. .35 2.2.6.2 5.35 .53... .823 3.8.2 N N 6.68. .m-< 2an 199 med med med mm... mm; 5.? dd: d5; m3; opmd 55d 84 dmdp om: Pmmndm NNN c2 and Fed Rd and deé one o.e: an: mum; mdmd dend ed.N 8.3 mmo; ..eNddm méN mm— dmd omd med and mm; and dd: dad; 5m... ..6d NEd on; ew.ww mm: eFNrdm NdN e3 Fwd de dmd and mm; mm... o.ww? 8N4 Rm... ddmd mend 3.9 nmd~ do: Podmde o.wN N9 dmd med dmd mm; 84 e.ww mN: dud; mNmé wood wood 8N Ed? eddé 38.5 FNN so? mad end mmd me... Ne; me... o.o: 8: now; dmmd ..mnd do; RAN mm: emhdée dNN e3 dmd dod dmd nmé hm; one oNNF 3: duo; mood ddmd dd; NudN mN: medm d.NN m3 ..ed Fed Fed Ne; Ne; Ne; as: mN: one; dmmd Bad ewe emdN on: 32.3 m.NN do? med med med oNé mN... mm... deP omo; ope... mend oeud S; 3.? d9... memedm NFN m: 5d and de odd 8; oo.w mdg mood and; weed mNod mdN N93. 3: medeN ndN N: mod mod 3d an... an; and 0on oom... 85F Sud mod; 3.? NNNN mN: NNmmde QFN E; mud and and 9.4 med hm; d8: on: mam... owed mNud ed.N RAN 3: omneee NNN d2. eed med med eeé Ne; me; dd: Pm: mmo; mwmd 85d Be 5.8 8: ommsde déN mm? and and ded and um; and dd: 9.: wemé demd eeud and om...N mm: 83.8 oNN mow wed med wed add add odd odd dmdd mmNé meod Edd 5.? ed mm: Nddddp mdp e2 med med med eeé me; me; o.e: dN: woe; mde eowd ed; 8.3 mm: odmndm ndN m2. med Fed eed om; om; cm; as: omF... mum; mde mend 9N 5.9 odd; somedn m.FN ..ow de mmd 5d 84 um; emé dd: 3: wow; dmmd Sud ed.N 3.8 on: 2.3.3 e.NN amp Ed ..md cod and mm; 84 md: N93 304 emmd odmd on; 3.8 dd: 33.3 ER on? mmd dmd omd ee... ..eé we; dd: 2.: mmeé emmd hand dd; 2..: dew... mmpmdm th BF eed eed eed mm; Nm... mm... dd: 3: mum; mwmd mid ed.N dde dud; 38.; SN mm; and de NNd Rd N... N; ode mmo; Noe... mmed muod mm; 8.9 dd: 85.8 e.NN em? Ned med oed 5N; 5N4 5N4 dd: mod; hoe; mumd mmud mod dew we: ..enwdm NNN nne . . EE . c. . c. . c. . 5 Son :8 5. cm a: Eu. 5 .oc a>< Ni 2. a>< Nu _.n ..E:o...0 fidmv 523 :32. £93 .mgmcwn. .mPEo) dofimm. cofidw .aEwWwdaoeh. .ooum 5 .83 26:32 5 62a .80... .223 .932 N N 6.38. .m-< 28p 200 med med med me... med med mm: om..d mum... mmed dmnd No.N omdN dmdd emmmde m.NN mmN mmd mmd emd mmd mmd _.md dd: ..m..d nmmd mmmd homd 8N mNdd eddd mmemNm NNN 0mm bed med med eed med med 9m... mhdd mmmd semd moud mmd mmdN eddd mnomdm «.mN emN hed med med emd mmd emd ON: mmo... mom... dumd eemd de mndd ded oom—..em QNN NmN mmd mmd mmd de add mNd d.Nd.. omo... Nmmd dmmd dmmd ooN mEmd mt... Nmmedm mmN oNN mmd emd mmd ....d N..d mdd m.ed.. mhmd ooe... dmed 08d mm... owed m3... mommdN edN NNN med Fed med mm... mm... hmd mm: ooo... dmed ommd mnnd emd medd dud... domomm m.NN mNN _.md de omd de de de dd: omdd Nem... hmed mhmd mad ded eed... mNbde d.NN NNN Ned eed ded hmd hmd smd 9de omdd eemd Nmed eend Nod medN mNdd meom.ee N.mN ..NN dmd dmd med _.ed Ned oed 9m... Nod... momd mdod mmmd mm... thd dddd mNNomm m.NN mdN mmd mmd mmd ..ed oed ded me: mm..d odmd oemd dend NON mmd.. ON..d mmmodm m.NN 5N mmd emd mmd med Ned med mddd omod ded mdmd edmd ..md mN.m.. mud... Nmeomm m.NN mdN odd mmd nod om... and cm... ode word mmmd mood dmmd mad 5.: om..d Nmem.mm mdN mdN med med med ..ed oed Ned om: ee..d demd m..md edud eo.N NN.m.. mt... mmmNdm NNN NdN omd omd med mmd oed nmd me: eddd mom... Nmod omnd and 9.0.. dN..d thdem NNN :N ded ded ded dmd dmd dmd dd: mmdd 5mm... oomd mmmd om... omNN 2N... dmumme e.ww mow mNd eNd mNd mud mud mud omo ommd mood coed dwmd and emd mmdd mNmNKd add NON dmd de bed mNd mNd mNd mddd mmmd dded mdmd mmhd mm... mde 59.... mNNedm ddw ..dN med Ned med med Ned med dd: mmdd .Lmd emmd mehd de ..mNN medd mmomde d.mN add mmd dmd mmd mod mod mod c.0Nd Nde mend mde wmmd em... nmNN mhdd mddeee oNN mad de omd mmd dmd omd dmd oNNd odd... mEd mwmd mde mod dedd ded mddem ...NN 50.. . . EE . c. . c. . c. . c. Eon Eu 5. co m: Eu. 5 .oc d>< me ..e d>< g ..u ..E:o..0 5%.. 52; Snow 5!; $22.00 $5.0) powwow. conodw 39%.?“ 32$ a. .33 26.5.2 ~ 5 62m .80... .883 .532 N 3E8. .m-< mafl wed med med dmd omd mmd mde mtd mmmd ddmd mmnd mm... Nmmd mmod eeemdm m.mN meN mmd emd mmd dmd mmd de dde emNd mend dmmd mnmd mmd NNmN 2.: ..mdNee NNN FeN med med med de... ded Ned om: md..d nmmd mhmd ddmd ed.N ee.m.. mmd... eemmdm d.NN mmN :. c. c. c. . .. . . .m .m EE . a . . a . E2m Eu c. 598. m .2. E0 595. o: >< N.“ we >< Nu I. .8385 5 mm 55.3 5 mm 55.2. ...”..wcoo 692.5 .8503 comomw 398822 .35 N 5 68¢ 26:82 N a. do; .83 .823 .8522 8.28. .m-< 223 201 Table A-10. Serum lGF-l concentrations (nglml) following initial and subsequent control or bST treatments, and following the last control or bST treatment for Experiment 809002 ‘ Steer Days after initial treatment Days after last treatment "°' 0 7 14 21 28 35 -7 -14 -21 -28 13 467 710 NC b 928 950 776 500 500 536 523 17 378 497 442 660 570 660 1244 1094 648 628 18 333 608 394 622 565 666 407 510 465 428 37 252 378 483 480 519 537 1091 922 698 478 42 452 426 284 444 368 422 509 499 559 544 44 325 496 441 442 491 568 439 450 395 398 46 351 687 515 478 443 652 421 394 455 463 62 318 350 450 375 380 428 496 523 486 447 77 404 575 447 958 479 670 1661 900 510 498 86 227 323 288 322 256 348 282 267 296 304 87 391 629 NC 663 729 523 1 103 1 103 729 676 89 235 360 328 370 296 447 1305 1295 817 462 97 369 579 488 424 420 576 457 539 440 426 101 291 395 424 417 430 325 1307 954 487 442 102 282 396 358 398 349 286 1263 712 260 250 103 158 319 301 503 489 551 205 265 278 233 121 303 394 406 557 347 605 1129 785 504 355 130 303 442 392 495 436 458 1426 1044 617 506 136 330 283 343 370 450 430 427 515 448 517 142 101 141 257 344 256 336 1184 512 360 397 154 292 263 419 425 358 384 462 475 467 484 158 252 226 326 275 341 337 1308 979 453 363 163 353 366 460 455 476 485 1052 999 508 321 165 360 782 819 738 492 863 452 485 402 421 166 293 398 273 286 344 277 1118 1062 651 474 180 284 388 340 460 306 430 1299 967 697 480 183 329 396 427 364 336 506 328 307 305 297 195 405 619 546 704 545 728 529 507 525 507 199 283 275 359 360 377 421 440 400 446 448 212 284 346 308 461 417' 455 448 409 457 394 225 654 848 1002 816 802 865 1244 1367 1069 614 238 284 358 621 434 328 419 1503 994 849 491 and 28. Blood was collected prior to treatment. bNot collected. ‘Two steers were sampled per pen. 202 Steers were treatment on day 0, 14, Table A—1 1. Serum lGF-l concentrations following the final control or bST treatment, and liver lGF-l mRNA abundance for Experiment 309002 ' Steer Days following last treatment, lGF-l, ng/ml Liver lGF-l mRNA no. abundance, '7 '14 '21 ’28 densitometric units 9 593 753 819 617 111516 12 386 340 382 384 143915 25 251 383 298 314 88813 55 322 374 372 289 67567 59 987 1037 697 785 103413 63 475 396 710 457 62957 86 282 267 296 304 56249 89 1305 1295 817 462 96360 103 205 265 278 233 62135 104 225 312 183 224 104637 133 407 348 454 374 106711 142 1184 512 360 397 47797 151 424 450 434 383 70336 155 1086 848 423 499 98677 158 1308 979 453 363 100200 165 452 485 402 421 105150 180 1299 967 697 480 96664 194 1293 603 437 380 105541 199 440 400 446 448 111729 221 406 399 340 342 69999 234 1216 1058 462 373 107842 “Steers from Block 1, the last group to be harvested. 203 Table A-12. Steer equations predicting carcass composition using the 9—10- 11 rib section (Hankins and Howe, 1946) Carcass moisture, % = 16.83 + .75 x Rib % H20 Carcass fat, % = 3.49 + .74 x Rib % EEL Carcass protein, % = 6.19 + .65 x Rib % CP Carcass bone, % = 5.52 + .57 x % Bone in rib 204 APPENDIX B: Raw data and procedures from Chapter 3 (Experiment 309402) 205 Table B-1. Time line of activities for Experiment BC9402 Day of Date Activity study 3-3-94 360 Holstein steers received from 3-9-94 Wisconsin 3-1 8-94 4-21-94 Steers adapted to a forage diet 5-6-94 Steers implanted with lmplus implant Steers adapted to a concentrate diet -36 9-27-94 Steers weighed and sorted into Adaptation metabolism room pens period d -36 to d -26 -25 10-8-94 Jugular catherization -24 10-9-94 1 3' day of 1 3‘ series of epinephrine 1 " epinephrine challenges challenge series -18 10-15-94 7" day of 1 3‘ series of epinephrine d '24 to '18 chaflenges -16 10-17-94 1 " day of adipose tissue biopsies 1 " adipose . . . . tissue biopsy nd -14 10-19-94 2 day of adipose tissue biops1es series -9 10-24-94 3" day of adipose tissue biopsies d -15 t0 -5 -7 10-26-94 4'h day of adipose tissue biopsies —5 10-28-94 5"1 day of adipose tissue biopsies -1 1 1-1-94 1 " weight taken for initial weight Initial weight 0 1 1-2-94 1 " treatment injection and 2 “" weight taken for initial weight 3 11-5-94 Steers weighed, blood collected 10 11-12-94 Steers weighed, blood collected 17 1 1-19—94 Steers weighed, blood collected 24 11-26—94 Steers weighed, blood collected 206 Date Table B-1. (cont’d) Activity Day of study 30 12-2-94 Steers weighed, blood collected 31 12-3-94 Jugular catherization 32 12-4-94 1 " day of 2 "‘ series of epinephrine 2 "‘ epinephrine challenges challenge series 38 12-10-94 7‘" day of 2 "‘ series of epinephrine d 32 to 38 chaHenges 39 12-1 1-94 Steers weighed 40 12-12-94 1 " day of adipose tissue biopsies 2 "" adipose 42 12-14-94 2 "“ day of adipose tissue biopsies :::: biopsy 44 12-16-94 3" day of adipose tissue biopsies d 40 to 49 45 12-17-94 Steers weighed 47 12-19-94 4th day of adipose tissue biopsies 49 12-21-94 5m day of adipose tissue biopsies 52 12-24-94 Steers weighed, blood collected 59 12-31-94 Steers weighed, blood collected 68 1-9-95 Steers weighed 73 1-14-95 Steers weighed, blood collected 80 1 -21-95 Steers weighed 87 1-28-95 Steers weighed, blood collected 92 2-2-95 Steers weighed 93 2-3-95 Jugular catherization 94 2-4-95 bST absorption time course 95 2-5-95 1 " day of epinephrine challenges 3" series of epinephrine 101 2-11-95 7th day of epinephrine challenges °ha"°"9°s d 95 to 101 207 Table B-1. (cont’d) Day of Date Activity study 102 2-12-95 Steers weighed 103 2-13-95 1 " day of adipose tissue biopsies 3" adipose . . . . tissue biopsy rid 105 2-15-95 2 day of adipose tissue blOpSleS series d 103 to 107 2-17-95 3" day of adipose tissue biopsies 1 12 110 2-20-95 4th day of adipose tissue biopsies 112 2-22-95 5‘" day of adipose tissue biopsies 115 2-25-95 Last treatment injection, 1 " weight Final weight taken for final weight 1 16 2-26-95 2 '" weight taken for final weight 117 2-27-95 Steers transported to Ada Beef for slaughter 208 209 82 2.2 22 82 82 8.. .88 m8 ..8 8.. 5.. z 8 P .8 82 ..8. 82 82 82 m8 38 ..8 m8 88 .280 z 8 m 8... 82 82 ..8 ..8 m8 ..8 .88 m8 ..8 o 8 .228 m 2. e h 8 82 82 22 22 82 m 8 8.88 28 28 .2.. .228 z 2. N «8 88 m8 8.. 82 ..8 8.. .38 ..8 o 8 28 .228 z 8 v 8. 82 82 82 22 82 ..8 988 m 8 88 o 8 5.. w 8 m .2 82 82 88 ..8 82 ..8 .88 m8 ..8 m8 5.. w 8 P ...2 8: 2: 82 82 82 28 8...8 ..8 m8 m8 5.. z 8 m .2 82 82 2.2 82 82 ..8 .8 8 o 8 88 8.. 5.. z 3. e 82 82 22 82 82 88 88 2.8 m8 m8 8.. 5.. z 8 n 2. 82 82 22 ..8 2.2 ..8 .38 ..8 m8 2.. .880 m 8 m 2. 2.2 82 22 82 82 ..8 .88 m 8 o 8 8.. .228 m 2. m e: 82 82 2.2 2.2 82 ... 8 o.o 8 ..8 88 ..8 .228 m 8 m 8 82 82 22 22 22 88 ...28 ..8 28 8.. 5.. 2 8 N 8 82 .82 82 82 82 88 8.8.. 8» 8.. 8.. .228 w 8 F 8 82 ..8 88 22 e8 88 8.. 8 88 m 8 m 8 388 z 8 m 8 8.2 2.2 82 82 82 ..8 Q8.. ..8 8.. m8 5.. m 8 N 5 82 2.2 82 ..8 82 ..8 m... 8 ... 8 88 8.. 5.. w .... e 8 82 88 82 82 m8 8.. 38 ..8 m8 2.. 688 z S P 8 82 82 22 82 m8 8.. 38 .28 88 m 8 5.. m 8 m 2 .. ...2, 2.. .. ...; 2.. ... ...2. 2.. ._ ...; 2,. ... ...2, 2,. ._ ...2. 2,. .. ...; 9.. ... ...2 e2. ..3; 2.. e ...2 e... 88:: 8.8.: 3.8 88...: 8:2: 882 .885 8.8.8 8.8.8 88 :8 8.8 .2. ..8... .2. .5558... 3.23m zen. 2902. 5.5 3.160 m+ >80 om8o>< o+ >80 ..- .60 mm. 50 om8e>< mm- >80 mm. in. 888 .8888 .o. 28.8; .88 N-.. 8.8; ommw CNN? mmNF oFNF mmvw moFF mmwp corp mvpp ovww omFF corp non omNF cmww ome onN? mmmw mpmw ONNF mow? mhpw mvrp omFF omFF oFFF NNN mnrp oomw moFF mow? cow? omww moww mwww mwpw mmoF chow mob? mvow NFN mvmw onN? mNNF mPNP omFF omFF onpw mvww mwpw corp ONPF mow? mac? NON map osmw onN? mom? ONNF camp mare mnpw mmww mwww mNFF cow? mmoF mmoF haw mam? mmNP mvmw mom? +mrNF mom? comp mnww mcpw mnww mva corp chow war ovvw vaw cove owcw camp m¢mw own? omnw mwmw omNP comp ommw coww nmw comp ommp mrnw mam? ome mnmw GVNF CNN? mo—w omrw omFF meF cow? mm? mPNF mPNF mmww omFF map? mnww mwww omFF omow mow? omo? Chow or? oomw mwmw omnw ownw camp camp mvmw ONNP mmpw mopw mmpw omrw mow? www ommw mmNP onNP mme cme oFNw new? car? map? omww Orr? omo? whow VP? ommw mwmw camp own? mum? omNF ommw ONNP mmpw mnww mow? owww omcw mm mmNF mam? mow? mnmw omNP ome oFN? mwwr mva warp mvww our? over as mmNP ovmp ONNF mmNF oFN? mFNF moww omFF corp omFF oowF ocww cmov mo mNNF ONNF omFF omFF omFF mvvv mmww corp omow mvow mmoF omow mm mnmw omNF mmww omNF mme ommw oFNF oowr our? mvww mvwv meF mow? mm ova mme ommw mNNF mNNw corp oo.w moFF ovww our? more oopw mm mom? cur? our? our? ourp omFF mop? ONFF who? mmoF omow mvow omow mm cmNF mnwp onN? mPNP ONNF mom? mare cow? carp omww oo.w onow mp e ...2. oz. 2 ...; 9.. ... ...2. 2.. .. ..8 2.. e ...2 9,. .. ...2. 2.. e ...z 9.. 29.; 2.. ... ...2. 2.. 2...: 2.. ... ...; ...... a. ...2. 2,: ... ..8 a... 8:28.. 888.. 8:82.. 88:3 83:8 88:8 82:2 88:2 88:2 822 8822 88:: 88:: .2. 82+ 80 81.8 3+ ..8 8+ ..8 21.8 818 81.8 8+ ..8 .2; .8 82:2 81.8 81.8 21.8 .88 5.83 .8-... 28» 210 Table B-2. (cont’d) Steer Day +105 Day +108 Day +115 Day +116 Average In 02/14/95 02/18/95 02/25/95 02/26/95 off-Uial live wt., lb live wt., lb live wt., lb live wt., lb live wt.le 15 1260 1285 1290 1300 1295 33 1220 1260 1270 1265 53 1270 1290 1320 1335 1328 57 1275 1275 1285 1280 58 1205 1210 1235 1245 1240 69 1285 1330 1325 1328 73 1325 1360 1370 1365 92 1355 1380 1385 1383 114 1265 1300 1315 1308 116 1390 1415 1420 1418 118 1200 1220 1225 1220 1223 133 1390 1415 1420 1418 157 1480 1525 1535 1530 184 1310 1355 1360 1358 187 1285 1300 1310 1305 199 202 1280 1300 1310 1305 217 1200 1220 1220 1220 222 1295 1320 1335 1328 307 1230 1260 1305 1315 1310 211 Table B-3. Daily dry matter intake of steers from Experiment B09402 Steer Total feed intake, lb DM "0- d-36t0-1 d0t030 d31t0115 d0t0115 15 615.65 590.26 1614.80 2205.07 33 674.55 566.44 1708.57 2275.02 53 722.18 561.75 1576.85 2138.60 57 778.43 663.92 1584.80 2248.72 58 629.01 602.06 1783.32 2385.38 69 638.72 641.61 1874.90 2516.51 73 673.73 663.24 1768.96 2432.19 92 726.03 718.28 21 19.77 2838.05 1 14 705.41 653.77 1808.31 2462.08 1 16 689.07 691 .70 2062.96 2754.66 1 18 675.25 582.81 1501 .87 2084.68 133 728.89 653.68 1923.33 2577.01 157 763.03 778.88 2023.52 2802.40 184 661 .99 609.39 1696.45 2305.85 187 667.12 545.80 1576.99 2122.79 202 675.49 651 .40 1824.33 2475.72 217 634.70 534.50 1466.81 2001.31 222 727.19 819.78 2102.30 2922.08 307 694.67 636.14 1530.93 2167.07 212 213 88 2 88 82 8.8 8.22 88 2 8.2 82 82 82 .288 28 8.8 2 88 82 8 8.22 82. 8 8.28 82 22 82 .228 N8 28 88 2.2 8 8.2 852...... 82 2.8 2 28 82 8.2 8.2 82. N 8.2 82 22 82 5.. 22 8.2 2 28 22 8.2 8.2 2v 2 8.8 82 82 82 5.. 82 2.8 2 88 2.8 8.2 8.2 8.. 2 8.8 82 82 82 5.. 22 8.2 2 88 88 8.2 8.2 8.. 8 2.8 22.2 82.2 22.2 5.. 82 28.2 2 28 85 88 8.2 82. 2 8.2 82 82 82 5.. 22 8.8 2 28 88 8.8 8.2 8... 2 28.8 22.2 82.2 22.2 .2258 22 8.8 2 88 8:. 8.8 8.22 88 8 8.2 82 22 82 .288 2.22 8.8 2 88 28 8.8 8.22 88 2 2.82 82 82 82 .8850 8 8.2 2 28 28 8.2 8.2.2 82. 2 28.2 82 82 82 5.. 2. 2.8 2 88 22 ..8 8.2 m8 8 8.2 82 82 82 .8850 8 2.8 2 28 82 8.8 8.2 88 2 8.2 82 2.2 82 .2250 8 8.8 2 28 28 8.2 2.22 82. 8 8.8 82 82 82 5.. 2.. 88 2 88 8.2 8.2 8.22 ...2. 8 28.2 82 82 82 5.. 8 8.8 2 88 82 88 8.22 88 8 8.2 82 82 82 .228 8 28.2 2 28 82 8.2 2.2 m2 8 8.2 82 82 82 5.. 2 22.2 2 28 88 8.8 8.2 88 8 82 82 882 .8.... 82 8.2 2 28 8.. 8.2 8.2 82. 8 88 88 88 .8.... 82 8.2 2 88 88 8.8 8.22 24 o 88 88 88 .8.... 8 8.2 2 28 2.... 88 2.2 82. 8 88 88 82 .8.... 8 22.8 2 28 28 8.8 88 88 8 88 88 88 .8.... 28 28.8 2 88 88 8.8 2.22 82. 8 82 22 82 .8.... 8 8.2 2 88 28 8.8 8.2 82. 2 82 82 82 .8.... 8 8.2 2 28 88 8.2 8.2 82. 8 82 82 82 .8.... 2 2.28.6 22> 52.85 .2.. $62.2 8.2.. .288 .285 2...; J>m u 3 68.9.8 .52 u 3 .8285 :92. u t .2885 26. u 9. 85.8.25 no... u m2 ..8nt 3.53.8 <09... .8280... u oom .=mEm u oom .2598 u oov .8882 n com .885 053522.. 8.8558me 085590.53 .__mEm 502 o. 952 u N 225588 85 2QO 888% .0 88858838 __mEm >5> 03. .0 8:0 22.0 n 2 .5888QO o: n o .8on 882.8 .82... mm... 9 2.6 {k m2 mt: m3 0 3.2 33 99 mo? .52. En an...” t. «to no» o.~ mw. 2 2 mov o mmdm mm? mm? om? _ozcoo NNN u 8285 5.83 o 285 .2.. .2... .2.? N .2.. a 8on a 28m e ...2, .3 N... 2% .o: .280 >525 22x25 2 .2201 (mm a8.5.20.5. 52.. .82.. m. ...2. .oEdalmfi 2252588.. 505 6.2.8.- 218 8.8.2 214 Table B-5. Ninth-tenth-eleventh rib composition from Experiment BC9402 Steer 9-10-11 Bone wt., Soft Bone Soft Tissue no. rib wt., 9 9 tissue wt., 9 in n'b, % H20, % EEL, % CP, % 10 3750.0 750.5 2999.5 20.01 56.19 26.23 15.09 25 3809.6 646.7 3162.9 16.98 55.30 27.91 14.61 27 4081.7 781.0 3300.7 19.13 52.19 31.80 13.67 47 3531.2 664.8 2866.4 18.83 53.51 29.72 14.10 60 3933.8 690.6 3243.2 17.56 59.31 22.12 15.36 68 3719.4 716.6 3002.8 19.27 58.36 24.03 14.72 138 3587.9 700.2 2887.7 19.52 55.71 26.36 15.05 179 3972.8 718.9 3253.9 18.10 60.61 20.42 15.95 15 5384.7 1035.9 4338.1 19.24 58.72 21.73 16.80 33 5588.0 860.1 4719.8 15.39 48.83 36.10 12.70 53 5728.1 1093.3 4652.5 19.09 53.01 30.08 13.83 57 5136.6 1043.3 4085.9 20.31 58.20 22.94 16.37 58 5329.5 961.1 4356.9 18.03 45.89 40.02 11.53 69 6524.0 1073.6 5440.1 16.46 51.16 32.18 13.18 73 6029.7 1 127.4 4940.6 18.70 57.34 25.09 15.20 92 6266.6 966.6 5279.6 15.42 46.39 38.79 12.51 114 5317.5 918.0 4385.4 17.26 49.78 33.80 13.35 116 6289.0 866.1 5374.9 13.77 47.38 37.24 13.35 118 4904.1 1019.6 3886.3 20.79 61.54 18.39 17.11 133 5985.1 1174.0 4805.7 19.62 58.56 22.76 15.55 157 5738.1 1 161 .3 4576.8 20.24 53.44 29.42 14.22 184 5351.1 1023.4 4301.2 19.13 62.35 18.13 16.64 187 5481.9 1062.8 4403.6 19.39 59.88 21.53 15.20 202 5332.0 1097.7 4257.0 20.59 51.65 31.68 14.68 217 5345.6 895.5 4456.7 16.75 48.77 35.85 12.97 222 5449.7 881.3 4587.8 16.17 48.37 35.41 13.06 307 5459.2 1006.3 4456.7 18.43 58.81 21.97 15.75 215 Table 86 Plasma glucose concentrations (mmollL) of steers for Experiment BC9402 Steer no. Days relative to first treatment 0 3 17 30 52 87 15 4.74 4.89 5.13 5.25 5.27 4.98 33 5.03 5.10 5.02 4.74 4.94 4.86 53 4.87 5.43 5.26 5.42 5.22 5.35 57 5.32 5.68 6.02 5.42 5.73 5.30 58 4.73 4.57 4.63 4.45 4.89 4.51 69 4.92 4.60 4.63 4.60 4.69 4.59 73 5.62 6.43 6.86 6.70 5.61 6.38 92 5.51 5.26 5.56 5.72 4.88 4.97 1 14 4.92 5.04 4.55 4.85 4.65 4.61 116 4.89 5.14 5.31 5.33 4.92 5.01 118 5.17 5.43 6.03 5.49 5.20 5.10 133 5.20 6.21 5.42 5.96 5.71 5.66 157 5.00 5.48 6.39 5.65 6.87 4.86 184 4.46 4.78 5.02 5.07 4.88 4.57 187 4.80 5.13 5.64 5.88 5.11 4.91 202 5.71 5.92 6.12 5.53 5.69 5.54 217 4.93 4.89 4.98 4.92 4.49 4.29 222 5.61 4.90 5.27 5.21 5.05 4.65 307 4.71 5.53 5.31 5.46 5.24 4.89 216 Table B-7. Plasma glycerol concentrations (pmol/L) of steers for Experiment BC9402 Steer Days relative to first treatment "0- 0 3 17 30 52 87 15 55.39 58.85 76.16 48.47 58.85 62.32 33 44.14 47.08 26.48 29.43 35.31 44.14 53 52.31 76.84 67.03 49.05 65.39 60.49 57 32.93 49.39 69.14 60.91 62.56 77.37 58 51 .50 53.34 38.62 44.14 34.94 38.62 69 48.83 34.88 26.16 40.1 1 36.62 36.62 73 45.20 59.32 56.50 36.73 22.60 73.45 92 36.35 38.08 36.35 34.62 25.97 38.08 114 34.57 27.99 16.46 19.76 14.82 37.86 1 16 71 .50 64.52 41.85 36.62 43.60 45.34 118 44.14 58.85 49.66 45.98 57.01 58.85 133 53.67 48.02 53.67 64.98 50.85 67.80 157 42.38 50.85 42.38 59.32 53.67 62.15 184 48.83 59.29 52.31 41.85 45.34 59.29 187 73.24 78.47 62.78 55.80 57.55 47.08 202 45.20 31 .08 28.25 28.25 22.60 39.55 217 39.24 34.33 34.33 29.43 42.51 39.24 222 50.85 42.38 39.55 39.55 28.25 45.20 307 35.31 50.03 47.08 47.08 44.14 41 .20 217 Table B-8. Plasma nonesterified fatty acid concentrations (mmollL) of steers for Experiment B09402 Dag relative to first treatment Steer "0- 0 3 10 17 24 30 52 59 73 87 33 0.196 0.288 0.094 0.070 0.097 0.090 0.080 0.101 0.085 0.124 58 0.119 0.1 14 0.087 0.074 0.082 0.193 0.069 0.077 0.092 0.089 69 0.121 0.092 0.059 0.079 0.072 0.099 0.092 0.109 0.137 0.101 92 0.082 0.102 0.106 0.124 0.057 0.097 0.069 0.065 0.067 0.090 114 0.112 0.084 0.060 0.052 0.082 0.097 0.067 0.075 0.087 0.119 116 0.176 0.139 0.121 0.095 0.106 0.084 0.102 0.106 0.112 0.117 202 0.131 0.092 0.131 0.062 0.082 0.173 0.062 0.097 0.094 0.109 217 0.092 0.080 0.074 0.080 0.122 0.104 0.095 0.132 0.136 0.099 222 0.114 0.111 0.079 0.095 0.107 0.129 0.089 0.112 0.111 0.126 15 0.116 0.216 0.194 0.193 0.173 0.151 0.147 0.129 0.228 0.276 53 0.099 0.296 0.126 0.220 0.300 0.223 0.204 0.189 0.171 0.240 57 0.104 0.296 0.246 0.278 0.280 0.278 0.223 0.256 0.235 0.347 73 0.101 0.193 0.102 0.136 0.119 0.097 0.169 0.127 0.142 0.188 118 0.109 0.198 0.116 0.147 0.188 0.119 0.233 0.147 0.142 0.191 133 0.127 0.196 0.265 0.132 0.219 0.174 0.154 0.221 0.188 0.149 157 0.092 0.151 0.117 0.119 0.119 0.203 0.137 0.201 0.104 0.226 184 0.067 0.201 0.196 0.147 0.166 0.164 0.161 0.231 0.144 0.162 187 0.173 0.256 0.238 0.265 0.196 0.240 0.176 0.194 0.204 0.174 307 0.072 0.183 0.147 0.139 0.169 0.152 0.178 0.147 0.196 0.159 218 Table B-9. Plasma insulin concentrations (nglml) of steers for Experiment 309402 "0- 0 3 10 17 24 30 g 59 73 87 114 116 118 133 157 184 187 202 217 307 1.708 2.150 2.818 3.531 1.317 2.21 1.651 3.315 1.968 2.683 3.622 1 .747 8.697 23.901 1.210 1.70 6.854 1.265 0.820 1 .193 2.685 11.046 1 .942 2.285 2.64 2.007 2.444 2.135 7.673 2.568 10.794 9.715 5.93 4.535 3.000 1 .129 2.404 14.008 aNot determined 4.281 1 .807 18.285 16.513 2.008 3.44 6.246 2.384 1 .777 2.204 1 0.628 3.036 23.766 6.51 4.490 3.041 2.193 6.520 9.550 4.807 5.216 9.503 39.435 1 .902 2.55 1 5.482 1.764 1.303 2.817 1 5.472 8.509 34.517 3.67 4.669 3.876 1 .870 9.270 1 1 .669 4.424 2.223 8.991 73.469 2.197 2.86 14.706 4.238 1 .008 2.980 6.986 32.663 5.35 5.453 1 .051 6.447 23.551 1 .320 1 .52 14.188 2.525 1 .015 4.538 36.529 48.331 8.102 17.371 11.20 7.877 10.157 2.957 1.925 5.917 23.089 219 1.535 1.468 6.992 9.019 9.702 2.779 16.095 37.635 2.395 2.74 3.680 2.109 1.338 3.876 29.607 1 3.754 12.699 41 .543 7.34 7.053 4.423 1 .655 8.496 9.222 2.503 11.365 ND ' 4.568 1.74 10.513 2.214 0.762 3.481 ND ND 8.68 11.422 3.054 1 .957 6.586 10.468 11.905 8.477 11 .482 4.674 11.182 36.059 66.853 3.084 2.00 1 0.238 3.178 1 .292 4.879 54.292 10.688 36.838 7.11 7.456 3.193 1.471 4.396 9.274 2.402 14.035 3.603 1 .80 19.451 2.480 1 .125 8.743 51 .697 17.904 12.669 4.39 6.076 2.840 1.411 2.709 8.069 Table B-10. Plasma lGF-I concentrations (nglml) of steers for Experiment 809402 Steer 3 Days relative to first treatment "0- 0 3 10 17 24 30 52 59 73 87 15 580 1169 1220 1187 1205 1104 1206 1224 1227 1076 33 585 581 482 491 476 464 458 491 534 459 53 573 954 1 108 1103 904 1075 1 158 1 198 1072 1083 57 779 1426 1258 1674 1615 1677 1550 1558 1 551 1514 58 523 512 474 529 539 509 546 502 482 479 69 549 51 8 538 572 538 546 546 455 477 474 73 861 1368 1 367 1 568 1 367 1468 1481 1458 1 585 1 598 92 543 471 400 462 435 480 388 394 390 366 1 14 522 589 543 541 519 413 506 529 459 433 1 16 529 487 581 550 522 576 532 541 516 514 118 758 1094 1137 1081 1293 1142 1218 1084 1087 1142 133 618 1337 1308 1354 1551 1459 1388 1380 1565 1673 157 802 1240 1141 1238 1276 1158 1078 1160 1050 1116 184 673 1034 1104 1116 1154 1317 1175 1035 1183 1184 1 87 1 077 1 103 1 149 1092 1 178 629 908 902 1 1 36 988 202 803 809 684 750 746 669 669 697 595 631 21 7 519 542 499 520 450 433 421 366 416 395 222 555 524 564 601 608 498 572 529 533 524 307 758 1086 1103 1111 1193 1162 1104 1046 1015 1019 220 82.5an .2058 2 26 888:8 mmEEam oz non 8...8 888 888 22.8 28.2 22.8 288 888 2.88 828 88 8.4.8 888 888 22.8 .82 -~ 8~.~ 28.2 .2288 8.4.8 888 2.88 .82 8.4.8 288 2.88 888 2.88 22.8 888 888 888 2~ 88 ~88 82.8 ~28 228 82.2 88.2 28.2 228 288 22.2 2.88 2.8.2 288 288 888 ~8 222.2 88.2 82.8 88.8 82.8 28.28 228.8 88.8 88.28 28.8 88.: 88.8 28.22. 28.2. 2.28.2. 8...8 22 28.8 22.8 82.8 ~88 2.8.8 ~88 8...8 2282 ~22 88.22 88.22 22.8 ~88 82.8 2.88 28.2 2.2 88.2 28.2 882 28.2.2 28.2 22.2 -2.2~ 28.2 88.2 ~22 842 88.2.2 888 82..~ 228 2.28 22 82.8 28.2 88.2 88.8 28.8 88.22 28.8 82.8 88.8 88.8 82.8 24.8 228 288 2.88 28.2 22 .82 828 888 228 888 888 8~.2 888 828 22.2 2.88 2.88 888 288 888 888 22 2.88 888 ~88 888 288 8~.~ 22.2 88.2 8.4.8 228 288 82.8 288 888 8.8 888 22 2.88 288 ..8... ~88 88 ~88 88.2 888 288 82.2 82.2 £82 888 828 22.2 288 8 28.22 22.2 88.2 88.8 8~8~ 82.8 28.8 .88 88.2. 88.2. 228.8 28.28 88.8 82.22 28.2. 828 2 2.88 888 8.4.8 288 288 ~28 ~88 28.2 888 88.2 88 ~88 288 288 2.88 888 8 832 28.22 28.22 ~22 88.2 88.2 88.2 ~28 88.8 28.8 288 2.2.2 282 888 .88 ~28 5 82.2 ~82 88.2 88.2 28.2 22.2 28.- 2.28.8 88.8 88.8 88.8 28.8 2.2.2.2 2.8.2. 828 2.88 8 28.2 888 828 288 88 288 82.8 88.2 8.8 888 888 82.8 2.~.2 82.2 88~ 2.2 8 88.8 88.8. 2.2.28 88.8 82.8 88.8 28.8 88.22 2.882 28.22 88.2 22.2. 28.2 888 8~.:. 88.2 2 82 82 882 88 882 822 822 82 82 88 88 88 88 82 82 88 .2. >m|u2|oaoz . 52w NOVmom uC®E_._mwa *0 Vm >NU E0: mgmwum ..Ow 2::va wcozmbcmocoo EQOhOum—tom 6:302 mEmmF— . _+ rum 039—. 221 Table B-11. (cont’d) Steer no. 1430 15 46.268 33 53 57 69 73 92 114 116 118 157 184 187 202 217 222 307 No samples collected due to catheter problems 2.1 13 1 1 .689 9.837 1 .197 10.935 1 .361 0.433 1 .091 76.531 10.923 19.588 10.990 0.537 1 .697 0.487 1500 38.220 1 .657 12.920 13.967 0.457 12.209 1 .102 0.485 0.920 67.950 10.668 19.848 12.951 0.527 0.706 0.479 1530 29.633 0.910 14.000 14.183 0.414 10.849 0.485 0.559 0.998 66.803 13.641 11.508 12.957 0.569 0.547 0.705 Hogr of @v 1600 31.061 1.015 14.630 13.944 0.514 14.724 0.733 0.513 0.664 65.446 15.196 13.586 12.146 0.632 0.584 0.488 1630 ND 2.780 10.540 15.223 1700 28.461 1.610 10.546 11.678 ND 0.555 1 1.208 0.660 0.434 0.474 64.817 12.120 16.451 9.275 0.853 0.425 0.640 12.735 0.793 0.570 0.649 59.381 11.064 15.324 9.008 0.810 0.590 0.463 1730 30.768 1 .531 8.723 1 1 .816 0.594 8.878 0.806 0.536 1 .1 1 1 55.476 1 1 .022 13.353 9.027 2.573 0.631 0.480 1800 28.983 0.822 11 .245 11.967 0.601 9.652 1 .229 0.424 0.566 58.631 11.931 9.847 10.984 0.744 0.487 0.562 . 3Not determined 222 8628 E0: 8889 828 62.8880 82. 223 mm.m nNn mmé mods R. E: ovdom mod. 8...... m ...NF 2N mad nNn 3m 36 oo.w cud 3.»... 3.3m 8.8m oNn mmd no.9 med 3.? 3N NNN mod 3.» Ed and? mod: 3...»? oo.w nNN N: am; wad mad x. 3 mms mod. vmé 2.3 3.»: otmww 2.6 m3. 8.0 cu... ~88 ~...m Now 02 02 oz 02 oz ew.ww? oz 02 «o.o oz _. 02 Ed m9 :6 02 .38 K. ..NP and: NmNNP cod mo... 5.0 «N. «o.o SN .38 was Sum oNn «9mm 3N: NodaN NNN 33 3. 3 3.8 vmd Inn 38 NV.” wad «Va and? 8.03 and? 3.8 x. 3 5. E n 3 mmN and um? mvd ..~.o de when under 38 ..n 36 mo; «How 3N mmd on.” m? ~86 mvé and $69 3.3.. 3.3m and n F... otmm and 8.8 mod n: mtm and mvé 959 8.0 K 3m: oo.ww 3.6 3.8 mod 3m «Na 0: mod mad. «he 3.3.. omSNN 38mm $8 9.0 8.3 and 3.8 3.8. v: 36 an.» 3d 3.2: and? «ZEN mvé mwd 3.2 3N and o tn 3 mod tum 96 o fimwm 8.3m ohmmm on. E. oo.w In? mm. 8 No. 8 mm. 8 Q. 8N and 8N 36$ EN ..m «0.5% ..mdw 3.2. ow: aw; 2.... 88m 8 t.” 36 mad 3.3m m 88mm afimnm 5.3 mm. ..8 3.9 3.8 3+ mm...” mm nod mm.» 3.: 3.58 3.89 3d S cm. P mod mm.» mv. v we. .. «in. 5 mmd cud 3.3 m 33 8.22. ew.ww on. P 0: «WV 3.... mo. 8 oo.w mm 5.9 de 96 3.3 8.38 madam oné mmé om.o.. 8...8 wed 8N mm mm. P mod on. w modem 8wa wmdov nnn mad 9.» mad 36 ..nd 3 8 .6828 ~ 888.8 P 888.8 8 8888.8 ~ .8828 F .6828 8 88.8 ~ 888.8 . 88.8 8 .6828 ~ 88.8 8 >828 s.c.oadEEE: p. ozmmn 9:8 78- ...E. .0: 3mm: 99: £53 38:30:82.3 522.. 953...: (n. 2 8:268 o I .05: 82w 6.28.. 8...—..8 Egfiofldog .508 9.85:? How Emu. £88098... Esau... Novmom €88.8me So: 8.305 8. .2 .9250 5205 833.08 .688 mE>~cm new 5.26m 88:392.ch m.m:.oom_do89 .mBom 38m. 2:. 5.8.0900... 83...; .NTm 2an Table B-13. Basal and epinephrine-stimulated glycerol release (nmol glycerol released-2h "-100 mg tissue ") for fat biopsies from Experiment BC9402 Steer no. Basal Epinephrine stimulated Biopsy 1 Biopsy 2 Biopsy 3 Biopsy 1 Biopsy 2 Biopsy 3 15 93.8 50.8 105.5 240.3 292.3 255.0 33 127.8 109.3 87.7 416.0 223.0 294.6 53 23.0 127.7 54.5 283.8 235.2 325.2 57 51.8 187.5 151.4 ND ' 331.5 288.9 58 63.1 65.1 68.5 224.0 411.5 260.8 69 106.3 34.9 99.9 295.0 143.0 316.7 73 86.3 149.1 203.7 277.7 319.2 325.9 92 51.0 69.3 54.5 355.5 340.9 244.0 114 75.3 115.1 75.0 199.8 308.7 265.8 116 14.5 67.9 58.6 167.1 187.3 235.5 118 51.1 105.7 162.1 192.0 338.5 295.8 133 83.2 151.4 103.3 340.8 284.6 213.0 157 232.0 58.9 132.9 199.1 293.5 268.0 184 74.6 105.6 47.5 273.2 312.6 330.7 187 104.2 32.9 139.9 333.8 131.0 293.0 199 83.8 ND ND 245.4 ND ND 202 99.6 52.7 84.5 229.5 281 .7 352.5 217 53.2 178.7 101.2 256.9 314.2 235.9 222 90.7 82.3 102.4 206.7 137.1 179.0 307 135.5 95.2 84.6 359.4 210.4 226.9 'Not determined, steer no. 199 removed from study 224 mood oood NNod mvod vood oood oN Pd mood o3d mood oood mood god vd oo o- oood ovod osod mood omod oood oood wmod . oz Nood omod o3d omod Nd oo o- oood omod Nvod ovod Nmod omod NNod wood _.mod oood wood omod onod rd oo o- Fmod omod mood Nmod omod omod Pmod omod Nvod oood hood oz omod oo o- E. Pd oo Pd or rd Nm Pd on —d ode N No omNd ood Nod omod omod oood m4 me m? or Pd mood No Pd no Pd mu Pd VoNd NoNd cord wood oood Nood oood ood N. F mp mp _.Nwd 3 rd oo ...o oNod om Pd oo Pd ¢o rd oo Pd mod omod vmod Nmod hmod o.o mp m— mo Pd oood o w Pd oood m _. ...o mm Pd vo Fd vvpd Nmod Food omod hood nod vd mw mF oNNd _.VNd ooNd oo Pd mtd mPNd oNNd NNFd ooNd Np Pd «Cd oo Pd _..o Nd m_. m— oo Fd oo _..o mm _.d No Pd or _..o oo Pd Nord mm rd omod omod Nood oood oood Pd m? m« 02 oz om Pd oood oood Nood 02 oz onod oz 02 oz Nmod m w m v 02 oz 02 02 oz 02 02 oz 02 02 oz 02 oz oé mw o _.vpd oN_.d oo Pd oo rd ooNd omod oood ooNd NNFd op Fd oo Pd oo Pd ::d N. _. m_. o o No om Pd ov_.d oNNd ooNd moNd o _.Nd or _..o oood oo Pd oood di no Pd o.o o No Pd NNPd mo Pd No Pd NVNd oz FoNd NVNd omod hood mood No Pd om Pd vd m_. m mNFd No Pd oNPd mo Pd oo Pd oo Pd oo Pd mo Pd oood hood mood oood mood Nd m_. m o No NPNd ooNd mid on «d o PNd ooNd moNd Novd mo Pd oo pd vo Pd FoNd Pd m_. m mood oood oovd Nood mood PNod mNNd RNd oNd NNd oNd mood nod m_. m ovod vmod oood mood oN _..o ooNd oo Pd o P Pd omod omod omod vmod shod oé m F o- mood _.3d Nmod ohod oood Nood mN Pd omod omod Eod omod vood omod N. P mw o- Rod oo Pd oood ov _..o ooNd oNNd voNd R Pd nod oood mood wood wood o.o m P o- Nmod omod omod eNFd o.. _..o oowd om Pd omod oood ohod Sod oo rd omod vd m_. o- 02 oz 02 02 02 oz 02 oz 02 02 02 02 a 02 Nd mw o- N3d omod Food Nnod shod oood oood o F Pd omod hood Rod oood oood Pd m P o- _.ood omod omod wood omod mood god Nnod Nnod mnod mod oood wood o mw o- oo_. mN_. oNP oN m _. o P m m.N o m- o T m 7 oo- . moon .2. xoo>> 35:96 055353 2: 2 026.2 835.2 ..ow 88w oczao A>>m 9.33 momoo mEancao co>om 9 3:955 owcoomo. Eom >sz ooztoumocoz «Yo o.omh Novoom Eoetooxm 5.. 9.33 .5on 925 225 mad oood hood omod wood 02 mo pd vvod Nood omod Ncod mmod _.ood Pd om m mmod Food Nvod ovod mood vmod Nmod vmod ovod omod mvod Nhod ovod om m Nvod mood ovod §.o no _.d wwwd PN Pd mo Pd oood omod ovod _.mod omod mé om o- _.Nod oNod mood oood god or _.d Nd _.d onod oNod oNod oNod wood oNod N. _. om o- mood oood Sod Nvod oood o F Pd de vo _..o oood wood oood vood oVod o.o om o- oeod oNod vvod hmod vuod oood ond omod Vood odd Sod ovod mood vd om o- mood Nvod oood ovod 33d vsod 2. rd ohod «Nod oVod oood oood oood Nd om o- mvod oood _.vod Nmod Nmod Nood god Nood vood omod mood ocod o _.od Pd om o! oNod oNod Nood mmod oood oood omod 3rd oNod oood oNod omod oood om o- Nood oood NVod oood «N _..o Nm Pd ovpd oood _.vod omod oood oood Vmod mé oo m _. ovod vmod Neod mmod omod m w Pd ovwd 02 OZ omod Nmod Nvod vmod N. F oo m _. mood Nvod nmod oood shod oN pd mv _..o Nm _..o ovod mmod vmod oood mmod o.o oo m _. Nvod oood NVod hmod Nhod No Pd 0.. ...o oo _..o mmod Nmod omod Fvod ovod vd oo m _. mNod oNod ovod mood ohod oood oood Nhod OZ Nmod ovod ovod Nmod Nd oo m F wvod mood oVod Nvod oood mood Vood _.ood mad omod mood hmod vmod _..o oo m w mvod oood Nvod omod mmod Vmod mood mmod ovod ohod odd oood o3d oo m r god Nood oNod ovod mhod N _..o on wd omod Nood mmod oood oood oood o. _. oo o god omod omod hood Nwod mmod oood mood oNod mNod _.Nod 02 hood N. F oo o vood Yvod mvod wvod {o.o wo pd 03d mo Pd god Nood wood odd _.mod o.o oo o Vood OZ mad mood :od 3 Pd No _..o vsod mvod hood Fvod :od N3d to oo o mvod Nood N3d oood Nvod mmod omod ovod ovod ovod ovod oood mvod Nd oo o :od N3d ovod hood oood oood mmod oood NVod odd oood oood mmod _..o oo o oood Nood Nood mood ovod Nwod oood ovod oNod Nood mood mood hood oo 0 odd Pmod _.vod Nvod oood oo _..o oo Pd oo _..o ovod oood ovod oood oood oé oo o- ovod odd oood vmod oood om Pd mo pd Nood ovod Pmod wood 02 oood N. v oo o- _.Vod mvod ovod mvod mm _..o hood ....Pd vsod omod oood o _.od 02 mood o.o oo o- oo_. me oNF oN m _. o F m m.N o m- o P- m T oo- - coco .oc xoo>> |¢mcm=m£o mate—QQEQG 05 Du wgflmo omen-ES. .am ..mOum A353 .34“ 2%» 226 227 oNPd wm...o wtd oo..d Nm...o oowd Nm...o word oood oood mwod oood NNPd 0.? mm 0 Food mmNd mmNd ..wwd med ond ..wwd oNNd no Pd NNNd mde ode No «d N. F hm m thd woNd ooNd NmNd oood mood m Pwd oood o ..Nd mNNd woNd woNd wo .. .o o.o Nm 0 omwd cord ooNd ww_.d nowd oNNd _.mNd mood moNd oood oood oood de wd km 0 oood ..o ...o wood mp rd oood oo ...o oo ...o ow rd wood omod omod Nood mmod Nd mm m N.._.d ..mwd Nowd o:d wwpd ..o_.d oopd on..d mood oo...o o..wd ohod mmod Pd nm 0 owod oood ow rd ..mod wNod Pmod Fmod onod mmod omod omod oood mhod mm o mmod owod oood Nmod m.. ...o 0.. rd so ...o mood oood Nood owod mwod oood 0... km o- ohod mood oood omod Nmod oo Pd N «d Rod NNod oood owod mNod mwod N. _. mm o- wmod oood wmod Nmod oood oN rd NN..d Fo rd oood Nood NNod omod shod o.o m o- mwod Nwod mwod ..mod mmod omod NN ...o omod NNod owod oNod Nood omod wd mm o- mmod oood omod oo ...o mo rd NmNd m _.d o v ...o ohod wood mmod ohod owod Nd mm o- mmod nmod ..wod oood mwod wmod omod oood mood ..wod wood oood omod ...o hm o- ohod oood mmod wmod omod mwod Nmod Nmod Fwod owod Nmod Nmod mwod mm o- omod omod omod oN Pd NoNd NNNd oo rd oo Pd omod wwod mmod wwod omod mé om m _. oo Pd Nood hood oo ...o om..d N ..Nd oo ...o mm ...o Nood Nmod oood wood mood N... om mw ..o...o NNNd m..N.o NNNd mwod omNd mNNd om...o ..o...o mowd wood mood Nood o.o om m.. oood wwod oood ..mod owod FNFd m.. ...o N rd omod owod oNod wood wood wd om m.. oood omod oNod mmod oood Food 0.. ...o oood oNod oood Nood oNod oNod Nd om mp shod shod oood shod NNod oood oo ...o oo ...o Fwod oood ..wod mmod mood ...o om m P oood o .. ...o mmod owod mwod omod owod Fmod mood owod oNod mwod Nood om m .. owod omod oNod wood wood ow ...o oood onod oood omod mwod omod mmod m... om o ..ood Nwod omod oood _.wpd oNNd oo..d oood owod wmod omod shod 02 N. F om m omod ..mod omod wood mN ...o ..NNd ooNd NN Pd oo ...o o .. rd omod Kod ohod o.o om m hood OZ oood omod oo ...o mw rd ow Pd oood oood ..mod owod oood oood wd om m oood mood omod o F ...o Nood oood oood wN _..o Nwod Nmod wmod mmod mwod Nd om o oo.. mN.. oNF oN m .. o .. m m.N o m- o ..- m ..- oo- o omoo .oc xoo>> 35:20 octzomcao o... o. 9:32 8.35.2 ..om 82m 6.38. i-m. 2an ..oNd No..d No...o std NrNd otd NNFd oowd ..w..d oo...o oNFd Nood ww...o o.o om mp omod owod owod oood oo wd Nood ow ...o ohod owod oood mmod oood Nmod wd om m r oood omod Nood omod oood mood oood oo Pd mood mood owod owod mwod Nd om mp Nmod mwod owod owod mhod Food oo ...o ..ood wmod Nmod oood owod nmod ...o om mp Nood wood oood mood omod oood mood owod wmod omod Nmod Nmod mmod om m.. omod oood oood omod mN ...o —o Pd wN ...o ohod owod oood mwod ..mod omod o. F om o omod omod Nood mood oNPd ..o Pd oo Pd oo Pd owod wwod NNod nood hood N... om o oood oood oood No rd wN Pd No ...o ow ...o omod wmod Nwod owod oood mwod o.o om o oood owod o Pod omod omod w.. ...o PN..d oo Pd oood oood nwod o ..od Nood wd om o Nwod oood oood omod oood oood ..ood omod omod wwod omod owod Nwod Nd om o {o.o omod Nwod Nwod wood oo ...o No vd omod hood Nood mood oood mood ...o om o omod omod Nood hood Nwod ..mod owod Nood swod Nood mood Nwod omod om o owod omod ood mmod oN ...o o..d oowd oood owod ..mod oo ...o mod omod o. F om o- oN ...o oo ...o t ...o Nood mo...o o...o on ...o oood omod mod mo ...o Nood {o.o N... om o- omod oN ...o mod omod oood om ...o mowd ..o Pd {o.o hood hood hood ood o.o om o- oood mood omod omod oood oo ...o prd onod mod nood hood oood omod wd om o- omod mmod hood wmod wood oood no ...o oood wwod wood oood wwod ood Nd om o- hood oNod hood owod mod hood ood ohod wwod Nood oood oood mmod ...o om o- omod NP ...o mmod wmod hmod owod mmod owod owod hood wood owod {o.o om o- oo ...o hood Nood mo ...o oNNd mood wood ms ...o oood omod owod Nwod mo Pd o. .. hm m _. Nood oowd omwd oo wd woNd wNNd NmNd mo ...o omod mood owod omod oo Pd N. .. mm m .. omod shod Nnod omod oood so ...o ooNd oo ...o NNod Nood NNod mood wood o.o hm m .. N.. ...o oo...o oood mood owwd o5 ...o moNd oo rd wood oood oood omod omod wd hm mp oo ...o otd mu Pd mood oN..d oo...o on ...o oo ...o omod omod mood omod wmod Nd mm m.. o..w.o Nowd mood oNNd oNNd ooNd omNd Nde No..d hm Pd mm ...o om ...o ..N ...o ...o Nm m.. N Pod Nood ooNd ..N ...o oo ...o No Pd Nood oo ...o oood oood ohod wwod wood o no m.. oow mNF oNr oN m .. o F m m.N o m.. o w- m ..- oo- o coco .o: xoo>> oucmzmco QCESQoEn—o 05 on mggo. mQSE—Z .am home” 6.28. .....m .2an 228 oood oood wood ohod oood wood Nood mo pd owod oood owod wwod :od Nd on o- mo ...o oo Pd onod mood shod ..Nod oood oood owod Nwod oood Nmod mood Pd on o- omod oood oood oN..d ompd Nwwd FNPd oNPd Nowd Nood oowd oo...o oowd on o- owod oood oood mood oo vd 2. ...o oo ...o oood oNod oood oood owod owod o. _. oo m P oood NNod Nood ohod wo Pd NNNd oNNd Nood Nood oood NNod owod Nood N. P oo m .. ..wod mwod owod oood No...o Ford o..N.o oowd mwod wwod mwod owod ..wod o.o oo mw oood oood mwod owod oood wo ...o Nowd wN...o OZ wwod oood mood omod wd oo m e mmod omod owod wo ...o oood ow rd oo ...o NNwd mwod Nmod Nmod wmod omod Nd oo m _. omod omod Nmod ohod oood mood wood wood Nwod owod omod owod Nwod Pd oo m P owod oood msod owod mood owod owod omod _.ood owod ..wod oood Nood oo m r oood oNod oood owod w.. Pd ow...o No Pd mp vd oood owod wwod Nmod ..wod o. w oo o owod Nmod omod oood oo Pd mo Pd om ...o wood ..wod mwod omod owod oood N... oo o mood owod owod mood oo ...o oo ...o ow...o Nood owod owod mwod oood omod o.o oo o owod owod omod oood mood no ...o oo...o owvd Nood Nood oood omod Nood wd oo o 02 02 02 02 02 02 02 02 02 02 02 02 OZ Nd oo o ..ood ..ood owod ..ood oood oood omod oood wood Nwod omod owod Nwod ...o oo o owod owod oood Pm Pd owod oood mood mood NNod oood oood OZ omod oo o owod mwod wmod mood ..o Pd oo ...o ..o ...o oood ..ood wwod Nwod Nwod owod o. .. oo o- hmod hmod shod mood on ...o o «Nd o FNd Nood owod oood wwod Nood oood N. w oo o- mwod wmod ..mod omod o _. pd wk pd on ...o oood owod omod owod mwod owod o.o oo o- oo Pd oood oo «d o de oNNd omNd ode FoNd oN ...o oo Pd oo ...o NN rd wm wd wd oo o- Nmod Nwod oood wood wN Pd Nm ...o wm ...o oood owod Nmod omod owod mwod Nd oo o- oood oood omod Nmod ...wod omod omod oood oood oood oood owod Nwod ...o oo o- owod owod oood Nmod owod omod mwod Nmod NNod oood oood OZ omod oo o- owod mmod owod oood oo ...o ..w pd on ...o owwd wwod mood oood mwod Nmod o. .. om m .. hood smod mood Nood oo ...o noNd ..N rd wood mood mood mood wmod omod N... om m _. ooF va oN.. oN m w o F m m.N o m- o P- m T. oo- . omoo .o: xoo>> G§__m£0 @ctcflmcam 05 Ow claw—0.. mmSEE .am howuw EEBV i-m. .033 229 230 888 888 888 888 888 888 888 888 888 888 888 888 888 8 ~8 8 888 888 888 888 .88 8...8 88.8 888 888 2.88 ..88 888 888 8.. ~8 8- 888 888 888 888 888 8.8 88.8 888 888 888 888 888 88 ~.. ~8 8- 888 888 .88 888 888 888 8.8 88.8 888 888 .88 888 ~88 88 ~8 8- 888 888 888 888 888 8.8 888 8...8 .88 888 8.8 888 88 88 ~8 8- 888 ..88 888 888 888 .8.8 8...8 888 888 ~88 8.88 ~88 888 ~8 ~8 8- 888 :88 8.88 888 888 888 888 888 888 ~88 .88 ~88 8.88 .8 ~8 8- 888 888 8.88 .88 888 ..88 888 888 88 888 .88 888 888 ~8 8- 888 ~88 888 888 88.8 .88 888 8:8 888 ~88 888 ..88 ~88 8.. 8 8. 888 888 .88 888 88.8 88.8 v.~8 88.8 8.88 ~88 888 .88 .88 ~.. 8 8. 88.8 88.8 ...-.8 88 888 8~8 888 8.8 .~.8 ~..8 888 88.8 888 88 8 8. ..88 888 .888 8...8 88.8 8~8 23 88.8 888.8 8.8 .88 8...8 888 88 8 8. 888 888 8.8 8...8 8.8 88.8 88.8 8.8 888 888 888 ..88 888 ~8 8 8. ...8 8.8 8.8 8...8 88.8 88.8 8.8 8.8 8.8 888 888 8.8 oz .8 8 8. 888 888 888 ~¢~8 888 88.8 8.8 888 888 888 ..88 388 ..88 8 8. 888 ~88 8.8 22 8-8 888 .88 8-8 388 8.8 3.8 8.8 8888 8.. 8 8 8.8 88.8 .88 888 888 .88 ~88 8888 8.88 8~8 ....~8 8...8 8...8 ~.. 8 8 88.8 8.~8 888 888 888 8888 ~..~8 88.8 ...8 88.8 88.8 ...-..8 oz 88 8 8 2.88 .88 888 8.8 83 888 -~8 8.8 888 888 8888 888 888 88 8 8 888 .888 888 ~...8 2.8 88.8 3~8 -~8 .88 8...8 8.8 888 888 ~8 8 8 ..88 8.8 ....8 88.8 88.8 88.8 88.8 8...8 888 ~88 8.8 888 .~.8 .8 8 8 oz oz oz oz oz oz oz oz oz oz oz oz oz 8 8 888 888 888 ....8 8.8 83 88.8 8.8 888 ~88 888 888 888 8.. 8 8- ~88 888 888 .~.8 8.8 v-8 8.8 88.8 888 .88 888 888 888 ~.. 8 8- .88 .88 «88 8.8 8.8 .88 88.8 888 888 888 888 888 .88 88 8 8- ~88 8.88 888 8.88 .88 .888 8...8 8.8 888 3.88 8.88 ~88 888 ..8 8 8- 88. 8. 8. 8 8. 8. 8 8.~ 8 8- 8.- 8.- 88- .88.. .8 3.82, omF—2350 oatnnmcao Q5 Ow ogflmh mot-.52 .Em bomuw .888. ...-8 288 mood Nood Nood oood Nood oN Pd wood god mood hood omod owod mood N. P w P .. o omod omod omod omod mood NP Pd oNPd oo ...o wwod wwod wwod oood Nood o.o w.._. o wwod mNod mood Fwod hood wood ..o «d oood oNod oood mNod owod mood wd w: o o ...o mo ...o Nood Nood oood oood mo ...o oood Nood oood omod omod wmod Nd w: o owod owod wwod Nood oood Nood Nood omod wwod omod owod ..wod ..wod ...o w: o owod omod owod owod owod owod hood Nood owod omod wwod wmod omod o ww .. o oood oood oood oood Nnod ..ood oood oood NNod oood o ..o-o owod omod o. w w.. .. o- mwod Nwod wmod wmod Food oood o F ...o mood wood mwod mwod omod oood N. .. w.._. o- mwod mwod OZ mmod ..ood NNod wN ...o wmod oood Nwod owod mmod ..wod o.o w: o- oood oood Nood oood wmod oood Nood mmod oood wood oood oood mwod wd ww F o- omod oood oood Nwod ..wod Nwod shod oood wood Nwod wood oood oood Nd w.. r o- \Lod Nwod owod mood omod oood oood oood oood oood oood oood oood ...o w.. r o- wood oood Nmod wmod Nmod omod mwod oNod wwod owod oood omod wmod o w.. .. o- wood Nood omod ow Pd wo ...o oo Pd No ...o Nood omod Nmod Nmod oood shod o. F No m .. oNod oood wood oood ow rd on vd ow Pd oo rd wood oood oood oood Nood N... No m .. mhod :od oood oood oN ...o mN Pd no ...o wp Fd nod Nod Nhod Nood oood o.o No m .. omod Nood hood Nood oowd omwd om wd oowd wood omod mhod nod ..Nod wd No me omod Nood wN...o PNPd o.. ...o ow..d Nowd wwpd OZ omod ood oood :od Nd No m. omod ..ood oood mood No rd oN ...o oN ...o oood Nmod oood Rod Food Nood ...o No m.. Nhod Nwod omod Nood w..-o.o ohod ohod wood mwod wood nod onod owod o No m _. Nood ohod oood mood oo Pd oo ...o ow Pd ow rd omod god omod wwod .Lod o. F No o Nowd Ford oood oo.d oN_..o mmrd oFNd Nopd wmod oood wo..o word oo..o N.. No o oood oood owod hood m. ..o NN..o woPd oN.d mood mood NN..o oowd oo...o o.o No o omod oood oood oood o.. ..o Po ..o NN Pd mood oood oood Nood oood oood wd No o Kod oood onod oood o. ..o 02 om ..o E ..o oood mood oood mood Nood Nd No o Food omod onod oood oood Nood Nood o.. ..o mood oood oood ood oood ..o No o oo. mN. oN. oN m 8 o F m m.N o m- o T m F- on. _. oood .oc xoo>> ooco_.m..o 23853 2.. o. 9.5.8 8.35.2 ..om 82o .888. ...-8 838» 231 mood wmod mmod ..ood o r ...o ..m ...o oo ...o oo Pd omod omod omod oood ohod wd o: m . oood omod omod mood mood oood onod omod oood Pwod owod ..wod Nwod Nd o... m F oood OZ oood oo ...o o ...o prd no ...o om rd wood oood \Lod oo ...o Nood ...o o... m .. rood Nwod mwod ..wod omod mwod Nwod omod Nwod omod wmod wood wood o: m _. owod onod omod oNod Nood oNFd No Pd oo...o Pwod omod owod Nood oood o. w o: o 02 DZ 02 02 02 02 02 02 02 02 02 02 02 N... o.... o mwod oood owod omod oo ...o oo Pd om Pd oo rd hood omod wwod Nood owod o.o o.... o omod mo ...o owod owod ..ood oood oood no ...o owod owod mood omod mwod wd o: o owod _.wod wmod wood oood ..ood o.. ...o oo ...o omod omod mmod owod oood Nd o.... o omod Nood owod owod oood OZ omod mnod oood mwod mwod omod mwod pd o..? o oood Nood oood owod ..ood wood ..Sd o.. ...o hood owod mmod owod OZ o2. o hood hood oood oood Nood om ...o oN Pd oood Nwod omod omod oood owod o. .. o .. .. o- oood wmod Nood wwod mhod oN Pd oo ...o oN_..o oood owod Nood omod oood N... or F o- {o.o omod owod shod onod Food ond Nhod oood oood oNod omod owod o.o o.... o- wwod Nood oood omod owod oN_..o oN Pd Nood oood owod owod Nwod owod wd o.. .. o- Nwod oood mwod Nwod omod wmod oood omod Nood oood owod oood oNod Nd o: o- omod oood oood mood oood Nwod owod Nood oNod mood Nood mwod wwod «d o.... o- Pood ..ood Nwod wmod oood oood omod ..mod owod omod mmod wmod wwod o: o- ..ood omod oood oowd o FNd ode oN_..o wood omod oood hood omod oood o... w.... m.. owod oood wood owod ..ood mo ...o om Pd shod owod oNod NNod oood wwod N. w w w .. m r Nood mNod oNod mood oo...o wwwd oo Pd x... _.d wmod oood oNod owod owod o.o w: m.. wood owod oood owod oood hood Nw pd wN...o ooo-o oood omod mwod owod wd w: m w Nwod wood oood Nwod omod oood mood oNod wood oood owod oood owod Nd w .. w m w oood oood wwod ..wod omod Nood oood mood oood owod ..wod ..wod owod ...o w: m F owod wwod omod Nmod omod hood Nood omod omod oood omod omod Nood w_... m _. oood owod omod owod mo vd m .. Pd ..Nod owod mood owod oood oood oood o. .. w: o oo.. mN.. oN.. oN m w o F m m.N o m- o T m 7 oo- o oooo .o: xoo>> 095:2". octcoocio o... o. o>oo_o. 8.35.2 ..om 89o .8888. «.-8 838 232 233 omod omod oood owod oood oood .ood oood oood owod owod omod mmod ..o oo. o- omod .wod omod owod wwod owod .wod .mod oood owod omod owod omod on. o- omNd ooNd m Pod moNd .Nod oood oood oNNd ..oNd woNd oo ..o o FNd oowd o. _. o: m. oo.d NoNd omwd . .o ..o ooNd woNd oo ..o oo ..o ....d o w ..o Nood wood NNPd N.. o: m. oo.d ow.d oo ..o .oNd Nood oood .oNd mNNd oo.d oo ..o oN..o oo ..o oN..o o.o o... m. Nw..o oo ..o woNd wwod wo..o .oo.d oood oowd mood oood ..ood .ood oood wd o... m. oNNd oo ..o oo.d ww ...o ww...o mood .oNd wo..o Nood oood wmod wmod oood Nd o: m. oood oowd oo..o Nood owod _.w..o oo..o oo ..o wood oood mood wood mood ...o o: m. oSd ow..d oo..o om.d ow..o wm..d o..—d oN..o NN..o 83d oood oN..o o..d o: m. oo..o oo ..o o. ..o oowd wde ooNd oood omNd om ..o mo ..o ..N.d oN_..o o. ..o o... o.. o wood oo...o .ood o. ..o wo ..o o No ooNd mN.d oood oood owod omod wood N; o... o oood oood wo ..o ww..o o .Nd ode ooNd No ..o wood omod oood oood wood o.o o... o oo.d oo...o wo ..o mo ..o wp ..o ..Nd oNNd ww..o oo.d wood oood wood Nood wd o.. o oo ..o o. ..o Nopd wood .m..o Nowd .o ..o No ..o oood oood Food oood mood Nd o: o wood oood oN.d oood om ..o oo ..o oo..o oN..o Nood owod omod wmod oood ..o o: o w....o mo..o oood oN...o oo...o oo...o oo..o oo.d mo.d oo.d mwod FNPd ooto o: o wood omod mod oood o. ..o mo.d _.opd wood omod wwod .o..d omod oood o. . o.. o- oood omod wwod owod Nood w.d mood oo ..o oood omod o. ..o Nood oood N. _. o.. o- oood oood omod Nood .o ..o oo Pd om ..o oood ood ood o. ..o oood oood o.o o: o- Nood wood o F ..o ood oood o. ..o oo..o o. ..o ..ood wood m. ..o oood oood wd o: o- _.mod mmod omod omod oood or d wo ..o oo ..o wood .mod oood wood oood Nd o... o- wwod oood omod wwod owod ood Nw..o wood .mod oood wood owod oood ..o o: o- oood ood ond mp Pd oN..o ood wood omod oood .ood oo.d oood Nood o: o- oood Nood omod ow..o oo pd oPNd o ..o oN_..o oood mood oood omod omod o. F o: m. wmod owod omod oood mwod oo ..o oo..o ow.d ..ood wood omod .ood oood N.. or. m. Food owod wmod oood oood oo ..o ow.d mwod owod omod mwod Nmod oood o.o o: m. oo_. mN. 8. 8 m w o . m m.N o m- o 7 m F- oo- . coco .o: xoo>> moclofizo outcoocfio o... 2 9.3.8 $55.2 ..om 39o .888. ....-8 288 Nwod oood Nmod Nmod oood oo...o ow..d or _..o mood owod mwod Nwod mwod o.o oo.. o- oood owod oood oood omod mo Pd owod oood oood owod oNod o Pod mood N. .. om.. o- oNod oood wood oood Nood oood oood omod ..ood wNod oood owod OZ o.o omo o- owod ..ood wwod owod omod oood .. .. ...o mood ..ood oNod oood wNod oNod wd oo.. o- oood oNod oood owod Nmod owod OZ oood omod oood mood oood mood Nd oo.. o- Nood oood mwod oood oood mwod omod wmod oNod ..Nod wNod ..Nod ..ood Pd omF o- mwod mood wood mood owod omod owod owod owod ..mod oNod oood oood om.. o- owod mood oood mood om ...o oo Pd ooNd o .. rd wood wood wwod oood oood o. F oo.. m P oood wo ...o oN o.o oood oo o.o FoNd oNNd mde wmod owod owod owod mmod N... oo.. m .. NN...o o.....o oN_..o oood ..o..d oFNd wo..d oo...o mood Nood Nood oNod mood o.o oo.. mo ..w ...o No ed oood oood oo ...o No o.o ooNd ooNd Nood owod wmod omod owod wd oo.. m P ..w...o om ...o oo o.o owod ..ood m.. Pd _.o ...o w Pd mood owod wwod oood oood Nd oo.. m P om ...o mm ...o ..o Pd wwod mmod mood oN_..o NNFd oood oood 02 02 02 ...o ooo m.. owod omod owod mmod oood wwod oood owod wood mood ..wod wood Nood oo.. m.. ..o rd oood oo ...o wood ooNd NoNd woNd oNNd mmod oood oood oood ..ood o. F oo.. o oN Pd rN..d oood mood ..de woNd ....Nd mo Pd mood oo ...o oood o.. ...o NP rd N. F ooF o oo Pd mo Pd oo o.o ow ...o ooNd ooNd ooNd NoNd oood oood oood oood oood o.o ooo o ..o ...o oo ..o oo Pd oood mo Pd oo...o o oNd m de oood oood oood Nood oood wd oo.. o oood oood oood omod mood o .. o.o oo ...o omod owod owod ..mod omod mmod Nd oo.. o o w ...o mo ...o o w ...o mood wood oN ...o om ...o oo o.o oood oood wood omod wood Pd oo.. o oo...o oo...o oo...o wN...o ..ood oowd oo...o oood oo...o Nowd No...o ..ood oowd ooo o ..mod oood owod omod oood oo «d Nw F .o Nood owod _.wod owod oood oood o. .. oo.. o- omod owod omod Nood ..o..d Nmod oood oowd owod wwod oood oood ..mod Né oo.. o- mood oood ..wod owod Nood oood mo ...o oo ...o oood oood omod owod omod o.o oo.. o.. omod omod oood owod ..ood oood oo o.o mo o.o oood wwod owod ..mod oood wd oo.. o- oood owod mood Nood wood Nood oood oood wwod omod owod owod omod Nd oo.. o- oov mNP oN.. oN m w o w m m.N o m- o ..- m 7 oo- a moon .0: xoo>> Guam—3:0 OCtzflwcfim o5 Ow 050—0. mos—=2 .aw ..OOuw .888. .38 83.8 234 oo F.o wFNd ooFd ooFd oo Fd ode Fde NoNd oNFd oood oo Fd mo F.o oood o.o woF o oNod oNod Nood owod oood mo F.o oo Fd ode Food Nood o F F.o Nood owod wd woF o mo F.o oood oNFd Nood oNFd oo F.o mo F.o oo F.o Nood Nw F.o ooNd mo F.o ooFd Nd woF o oood oood oood Food oood mood oo F.o mo Fd owod owod owod wmod omod F.o wo F o Food oood Food omod Nmod wmod owod omod owod wood owod oood oood woF o oood oood mwod owod Nmod omod Food oood oNod oood wmod oood oNod o. F woF o- oNod oNod oood omod owod Food oNFd mood oNod mood oNod oNod oood N. F woF o- woFd mN F.o oo F.o mood wNFd wm F.o oo Fd ode mood mood oood oood mN F.o o.o woF o- omFd FoFd oFFd ooFd ooFd ooFd med NNFd mood ooFd NNFd mde oood wd woF o- oNod wood Fwod oood Nood Nood Nmod oood oNod oNod Food owod owod Nd woF o- F Fod oNod oNod omod oood Fwod mwod oood Nood Food Nood owod oood F.o woF o- woNd oo F.o FoNd oo Fd oood oood Food Food mood oood oood oood m F F.o wo F o- mood oood oood oN F.o oo F.o ode o FNd oood oood omod omod oood omod o. F om F o F mood oood oo F.o oood oo Fd ode o FNd oood Fwod omod wmod Nmod Fwod N. F om F m F oNFd oF ...o oo F.o oo F.o Fde oNNd woNd ooFd Nmod Nood wood oood omod o.o omF mF oood wood Nood mo F.o oN F.o o FNd med ode Fde owod Fwod mwod omod wd om F m F NF F.o oN F.o wF F.o FoNd ode oNod Fwwd oowd owod owod owod Fwod oood Nd omF mF oNFd oo F.o oood Fo F.o ooNd oo F.o med ooFd oNFd mood Nood oood mood F.o omF mF oo F.o oo F.o om F.o oF F.o Nood Food wood 3 F.o oood mood wood mood omod omF mF mm F.o mNNd oo Fd mo F.o moNd Fde oo F.o mood omod Food wood wood Food o. F omF o omod omod omod Food wad oo F.o NoFd Food omod mmod omod owod Fwod N. F omF o NoFd NF F.o wF Fd Food oood oNFd wo F.o ode oood oood Nood oood Nmod o.o omF o oood omod oood wood mN F.o om F.o ooNd w F F.o Food oood wood mood Nood wd om F o oood oood Fwod omod oood oo F.o Fo F.o oo F.o mo F.o oood Fwod owod omod Nd omF o woFd oo F.o oo Fd oood oood oood oN F.o oF F.o omod omod oood mmod Food F.o omF o owod owod omod oood oood oood oood omod oood oood oood owod mmod o omF o oo F mN F oN F oN m F o F m m.N o m- o F- m F- oo- . oooF. .oc xoo>> ooco..o..o octcoocfio 8.. o. 8.328 882...). ..ow 88o .888. ...-8 83.8 235 Food No F.o oF F.o oood wo F.o oF Fd mde Food Nmod Nood oood Food ooFd Nd ooF m F wmod omod owod omod oF F.o oo F.o oF Fd Food wood oood Fwod owod oood F.o ooF m F omod oood omod omod omod omod Food oood mood oood mwod omod owod ooF mF oood oood mood wad ooNd o FNd oo F.o wood owod Fwod owod mwod Food o. F ooF o wood Nood oood ooFd o FNd o FNd oN F.o oood oood No F.o oood oo F.o ode N. F ooF o ode oN Fd ooFd NoNd N Fmd Fmod ooNd Fo F.o oo F.o oo F.o ode 3 F.o oF F.o o.o ooF o mN F.o ooFd ooFd oo F.o oo F.o ooNd ooNd oo F.o oood oood oood mood oood wd ooF o oood oood Food wood oo F.o 02 oo F.o om F.o oood owod Nood Fmod omod Nd ooF o wood omod oood mmod wood oN F.o ww F.o Nm F.o oood mmod Food mood oood F.o ooF o om F.o wo F.o oo F.o oood Nood mood mood Food mmod oood omod omod owod ooF o oood owod mood oood Nde oo F.o oo F.o Nood mood wmod owod oood oood o. F ooF o- omod omod Fmod NF Fd om F.o ooFd oo F.o omod Nwod owod Fmod Food Fmod N. F ooF o- mood owod oood oood Food oo F.o mo Fd mood oood wwod Fmod Food omod o.o oo F o- owod omod Fmod omod wood oN F.o oF F.o wmod oood Nwod owod owod oood wd ooF o- owod wmod wwod owod mood oo F.o oo F.o wF F.o owod oood owod oood Nood Nd ooF o- wwod mood Nwod omod omod oood wood omod omod Nwod owod wmod omod F.o ooF o- owod omod omod oood owod Fmod mmod omod wwod oood Nwod oood oood ooF o- oood wmod oood oood oood oo F.o oo F.o oood oood oNod oNod oood oood o. F woF m F owod oood Fwod Nood F F F.o mF F.o ode oo F.o NNod oood oNod oNod wood N. F woF mF oood Nmod omod omod mood oF F.o Fo F.o oNFd NNod oNod NNod oood oNod o.o woF mF oood oood oF F.o oood oo F.o No F.o ooFd wm F.o omod omod omod oood oood wd woF m F oood Food owod omod mood oo F.o mF F.o mde Fwod oood oood owod Fwod Nd woF mF mood omod mood oood omod mood oood Food oood wood oNod owod Food F.o woF mF oood omod wmod owod omod omod omod owod oNod Fwod mwod omod mwod woF mF oood oood oo Fd No F.o oood woNd oo Fd oF F.o Food oood oood oood mood o. F woF o oood oood oood wNFd Nde wood oood omod oz owod owod mwod mwod N. F woF o ooF mNF oNF oN mF 9 m m.N o m- o F- m F- oo- a moon .o: .325 oodo=ozo 8.33ch o... 2 9.88 855.2 .3 .86 .388. ...-m 83.8-F 236 mmod wmod oNod Food owod owod owod owod oNod owod mwod mood mood o o FN o- Fwod Nood mwod oood oNFd Nm F.o wo F.o No F.o Fwod mmod omod omod omod o. F NoN m F Food mmod owod oood No F.o ow F.o ooFd wad omod Food oood oood Nood N. F NoN m F omod owod Nmod wood wood Nm F.o No ...o oN F.o Nmod omod mmod oood wood o.o NoN m F mwod oood Fwod owod oood oF F.o ow F.o oo F.o oood oood oood omod Food wd NoN mF mmod omo-o Fwod omod oood No F.o FF F.o FN F.o oood owod Nmod omod omod Nd NoN m F Nood omod Food mo F.o Food oo F.o wF F.o oF F.o owod Food oood oood owod F.o NoN m F oood Nood Nmod omod mmod omod omod oood oood omod Nood oood Food o NoN mF Fwod omod Nmod oo F.o ow F.o oo F.o omod om F.o oood ooNd mwod mwod mood o. F NoN o omod omod wmod wN ...o No Fd oo F.o wo F.o oood Fwod omod oood oood wmod N. F NoN o Food Nood mood ooo-o ow F.o FoNd mo F.o wN ...o omod omod omod oood F F F.o o.o NoN o owod owod owod NN F.o Fm F.o woNd o FNd wo F.o oood oood omod wmod oood wd NoN o mood Food oood ow..d moFd oo F.o o FNd oFNd mo Fd wo F.o o F F.o ooFd omFd Nd NoN o 02 02 02 02 02 02 02 02 02 02 02 02 OZ F.o NoN o owod omod omod mwod omod omod owod omod owod omod Food oood oNFd o NoN o Food oood oood FNFd oo F.o ooN-o ode oo ...o oood wood oood oNFd oN F.o o. F NoN o- mood mood owod oood oood wo F.o ooNd oo F.o oood omod owod Nmod Food N. F NoN o- oood oood omod oood mood o F Fd oo F.o No F.o mood wood No F.o oood No Fd o.o NoN o- oood mood omod oood m F F.o oo F.o oo F.o Nw F.o oood oood owod omod omod wd NoN o- oood oood mmod wo F.o moFd ..de NoNd oo F.o oF F.o NN F.o mF F.o wood oood Nd NoN o- omod Food wmod oood wood oo F.o No F.o oo F.o oood wood mo F.o 02 OZ F.o NoN o- wmod oood Food F F F.o oNFd mN F.o oo F.o No F.o oood mood oood oo F.o FNFd o NoN o.- wood wood mood F F F.o mo F.o ooNd FoNd Fo F.o oood omod oood Nmod oood o. F ooF m F Food oood wood Nood oFFd ooFd omFd omFd Nood oood oood Food NoFd N.F ooF mF F _. F.o o F F.o No F.o oood Fo F.o oo F.o oo F.o om F.o oood omod mood omod omod o.o ooF mF omod wmod omod wood o F F.o ow F.o wo F.o m F F.o oood wmod owod omod wmod wd .. oo F m F oo F mNF oN F oN m F o F m m.N o m- o F- m F- oo- . moon .o: .09.. £62.85 233ch o... 2 9.6.8 3.35.2 ..om .oofi .288. ...-m 83.8 237 88.8 88.8 8888 888.8 8.8 8.8 .88 .8.8 .888 888.8 88.8 888.8 8.8 ~.. -~ 8- 888.8 888.8 888.8 88.8 88.8 8...8 8...8 88.8 888.8 8.88 88.8 88.8 .88 88 -~ 8- 8888 888.8 888.8 888.8 888.8 8...8 3.8.8 8888 8.88 88.8 88.8 8.88 .88 88 -~ 8- 888.8 88.8 88.8 888.8 ....8 ~...8 88.8 8...8 ~88 ~88 888.8 888 888.8 ~.8 -~ 8- 8.88 888.8 ..88 888.8 ..88 88.8 888.8 ..888 888.8 888.8 888.8 8.88 ..888 ..8 -~ 8- 8.88 8888 ~88 ..888 8.8.8 8.8.8 :88 ~88 888.8 8.88 8.88 888.8 .88 -~ 8- .888 $8.8 3.8.8 888 8.8 8...8 .~..8 888 8.8.8 83 8888 .888 888.8 8.. o.~ 8. 88.8 888.8 .888 8.8 88.8 ..~..8 88.8 ~88 .888 8888 .888 888.8 888.8 ~.. 8.~ 8. 888.8 888.8 888.8 88.8 .888 8...8 88.8 888.8 ...88 8.8.8 .888 888.8 8.88 8.8 8.~ 8. 88.8 888.8 .88 8...8 88.8 ....8 88.8 88.8 8888 888.8 888.8 8.88 8.8.8 ..8 8.~ 8. 888.8 8.88 .888 88.8 888.8 8...8 8...8 .888 ...88 888.8 8.88 888.8 888.8 ~.8 8.~ 8. 83 .88 8888 .888 88.8 88.8 88.8 .88 .888 888.8 8.88 888.8 888.8 ..8 8.~ 8. ..888 83.8 8888 888.8 8888 888.8 888.8 888.8 8.88 8888 .88 3.88 888.8 8.~ 8. .88 888 88.8 88.8 88.8 oz .38 8888 888.8 88.8 .88 .88 888 8.. o.~ 8 88.8 8.8.8 888 ..888 888.8 -..8 8.8 888 88.8 .888 888 888.8 888.8 ~.. 8.~ 8 .88 ~88 888.8 8...8 8.8.8 ~...8 ~...8 8888 .888 888.8 8888 ..888 .888 8.8 8.~ 8 888.8 .888 3.88 88.8 .888 88.8 8.8 888.8 88.8 888.8 888.8 888.8 888.8 8.8 8.~ 8 8.88 888.8 3.88 88.8 888.8 888.8 ~88 88.8 88.8 38.8 888 888.8 ~88 ~8 o.~ 8 888 8.8.8 ...88 3.88 .888 88.8 88.8 888 88.8 888.8 888.8 8888 888.8 ..8 8.~ 8 888.8 8.88 8.8.8 ~88 .88 ~88 2.88 .888 888 888 .88 ~88 8~88 o.~ 8 .888 8.88 888.8 8.88 .888 8.8 8.8 888.8 ...88 888.8 888 88.8 888.8 8.. 8.~ 8- 8.88 888.8 888.8 88.8 ..88 888.8 8.8 88.8 .888 8.88 8.88 888.8 888.8 ~.. 8.~ 8- .888 88.8 «88 ~88 ~88 88.8 88.8 88.8 .888 888.8 .88 .88 888.8 8.8 8.~ 8- .888 .888 888.8 888.8 ~88 888.8 .88 88.8 83 ~88 .88 83 888.8 ..8 8.~ 8- 88.8 .888 83 ~88 88.8 88.8 888.8 888 .888 888 888.8 888.8 888.8 ~.8 o.~ 8- 8.88 888.8 ...88 888 ~88 888.8 888.8 888.8 3.88 ...88 $88 888 8.8.8 ..8 8.~ 8- 88. 8. 8. 8 8. 8. 8 8.~ 8 8- 8.- 8.- 88- .8888 .88 .882. mggfico Ottznmcfim 05 Cu GEN—o.. wows—:2 ..Qm ..QQ—w 6.88. ....-8 283 238 68858 .0: o_oEmm8 .>>o ooo: .omoo oatcoocawa mood FFFd wNFd oFFd ode ooFd ooFd ooFd oood Food oood oood oood o.F ooo mF oood oood omod oood F F F.o Nw F.o mo F.o Fo F.o owod owod Nood wood Food N. F ooo mF Nood oF F.o oF F.o wN F.o ooNd wde oo F.o oF F.o mood wood Nood oood oo F.o o.o ooo mF Nmod omod mwod mood No F.o oN F.o oo F.o oood owod omod Nood oood oood wd ooo m F NoFd ooFd ooFd omFd med ooFd ooFd ooFd mFFd oood NoFd ooFd NNFd Nd ooo mF ooFd oNFd Nde oNFd ode ooFd ooFd ooFd oNFd ooFd woFd med ooFd F.o ooo mF oo F.o 02 oo F.o oF F.o oo F.o oo F.o mood NoFd oood 02 oz 02 oF F.o ooo mF oood wood Food ow F.o om F.o oo F.o om F.o oF F.o mood oood oood oz wood o. F NNN mF omod Nood wmod oF F.o ow F.o ooFd oo F.o oood oood oood Food omod oood N. F NNN mF omod owod wmod oood oN F.o ode wF F.o oood oood mwod omod oood omod o.o NNN mF omod Food Fmod oood oo F.o Fwwd Nw F.o oood wmod omod oood wmod wmod wd NNN mF wmod omod Nood Nood oood m F F.o FN F.o Nood omod omod oood oood oood Nd NNN m F oood oood Nmod Food m F F.o mo F.o NF F.o oood omod oood oood mood wood F.o NNN m F 3 F.o Nood Nood Food oood mood wood Food Nood mood Food oood wN F.o NNN m F Nood omod Nmod oood oo F.o mNFd wo F.o oood mwod mmod Food omod oood o. F NNN o wood omod oood oood No F.o Fde NoFd oNFd omod owod mmod mwod omod N. F NNN o Nood wood oood oo F.o oN F.o ode ode oood wNod oood wood oood Nood o.o NNN o 02 oz 02 02 oz 02 oz 02 oz 02 oz 02 oz wd NNN o wood owod mmod Nood oood mo F.o F F F.o mo Fd Nmod omod omod omod Nmod Nd NNN o mmod Food owod oood oood wood oo F.o wood owod omod oood oood oood F.o NNN o wood oood oood Food omod omod Food oood mmod omod omod Nmod omod NNN o wood oood Nood wood No F.o oo F.o Fo F.o wood Food omod owod omod oood o. F NNN o- oo F mN F oN F oN m F o F m m.N o m- o F- m F- oo- 8 88°F. d: .882. 35:83 octnoocfio 8.. 2 9.5.8 882...). ..om 38o .3500. .wF-o flow..- 239 .oo.oo__oo .0: 8.9889. 88.8~ 88.88 8.88 8~.88 8.8.. 8.88.. 3.8 88.8.. 8.88 ~8.- «~8~ ooo. w~...~ o88 wm.Fo mN.oo Fo.wo oo.om oNdm wm.mo oo.om oNdm oo.No ow.oF om.wF .02 ow.oF NNN ~88. 88.o~ .oo. 8.... 8.... 8.8 8.8 88.88 oo... 8...... .o.o. 8...... 88.8~ o.~ 2.8 88 88.~. 88.88 88.8.. 2.8 8.... ~8.88 8~.8 8.8.8. .88. 88... .88. ~8~ ~8.~8 ~8.88 ~8.8~ .88.. 3.8 8.88 .888 8.8... 88.88 88.8.. ~8~ ~8.8~ ~8.8~ o8. .8.8~ .8.8~ 8~.8~ 3.8.. 8...... 8.8.88 .888 88.8.. 8~.8~ 8~.8~ 8~.8~ 8o... 8...... ..8. 8.88 8.88 88.o~ 88.88. 88.88 ......8. 8.8 8.88 88.8w 8F.8~ 88.88 8~.w~ 8..~w 8. R8 o8.- 8888 8..... 88.8.. 88.88 88.8.. 88.~8 8..w~ ~8.8~ 88.8~ .w.o~ 8..w~ 88. 88.88 8.8 88.88. 88.88. 88.8~ 88.88 8..... 888 88.8.. 8.8 8...8. 8.8 88.88 8.. ~8.8~ wo.~8 ~8.8~ .88. .88. 88. .88. ~..- ~w8~ .~.8. .88. .88. So. 8.. 888. 88.8. 88.8. 88.~8 .888 o~.ow .o.o~ .o.o~ w8.8. ...... 3.8. 8...8 8.8. w.. o8.o~ ~w.w~ 8...~ 88.8.. 88.8.. :88 88.88 8.3 8.8 8F..~ o8.o~ 8...~ 88.8~ ~8 88..~ ~8.~8 88..~ w~..o 3.8 8.88 8.8. 88.88 88.8 .88. 8~.8~ 38. 88.88 8 88.8~ 8.8. 8~.8~ 88.8 .88.. 88.88 8.8 8.8.. 8.8. .88. 8.8. 8.8. 88..~ 88 ....8 88.8 .w.~.. 888 88.88. 88.8.. 88.-. 8...8... 88.88 3.8 8~.~8 88.8.. 88.88 88 Fo.o~ ~8.- 88.8~ 8.88 .88.. 8.88 .888 8.88 88.8~ 8.5 8.... ...... ~8.- o8 8...-88 8~.8w 8...88 8:8 8~.88 8.88 8....8. .888 oo.8~ 88.88 88.o~ 8..w~ 8.8.3 88 w~.w~ 88.8~ 88.88 88.8 88.88 :88 88.88 8.8.. oo..... 88.8. 88. 88.8. 88.8~ 88 8.8. .~.ow 8~.88 8...88 8.8.88 888 88.88 88.88 88.~8 88.~8 8.8 88.88 ~8.~.. 8. 88. 8.~. 8. 8 8. 8. 8 8.~ 8 8- 8.- 8.- 88- .88 ooco=mco oatcoocao o. 9:98 882...). 888.8 ~8V8o8 E8588 .8. E8888: .8 ..8; £8. 8.8 o.....ao omoo 3m ox.oc_...ooc_oo on o.F 8 0. 32081. 88:08.88 .8896 88885 .m F-m 858... 240 SAS program to calculate area under the curve of epinephrine challenge and to calculate a segmented curve to a plateau (Rmax response) Data area; title1 'Animal 114 weeks -3, 6, 15, area under curve'; input week anno dose 12a5 t5 t10 t15 120 baseline; baseline=baseline'1 000; TH = baseline; t2a5=t2a5‘1000; =t5*1000; t10=t10'1000; t1 5=t1 5'1 000; t20=120‘1000; cards; -3 1 14 0 0.077 0.045 0.070 0.057 0.054 0.050 -3 114 0.1 0.066 0.063 0.063 0.056 0.065 0.037 -3 114 0.2 0.093 0.077 0.047 0.041 0.047 0.039 -3 1 14 0.4 0.057 0.082 0.063 0.057 0.063 0.036 -3 114 0.8 0.077 0.124 0.072 0.061 0.057 0.044 -3 114 1.2 0.065 0.113 0.088 0.081 0.054 0.042 -3 114 1 .6 0.030 0.089 0.091 0.072 0.030 0.031 6 114 0 0.062 0.037 0.048 0.046 0.046 0.048 6 114 0.1 0.073 0.082 0.062 0.080 0.062 0.045 6 1 14 0.2 0.099 0.105 0.083 0.066 0.062 0.057 6 1 14 0.4 0.039 0.101 0.094 0.087 0.041 0.033 6 114 0.8 0.106 0.128 0.112 0.085 0.059 0.040 6 114 1.2 0.071 0.094 0.126 0.087 0.066 0.040 6 114 1.6 0.046 0.071 0.115 0.105 0.076 0.035 15 1 14 0 0.059 0.092 0.067 0.050 0.052 0.062 15 114 0.1 0.085 0.088 0.067 0.056 0.041 0.041 15 114 0.2 0.076 0.085 0.083 0.059 0.047 0.040 15 114 0.4 0.121 0.142 0.097 0.068 0.043 0.041 15 114 0.8 0.117 0.139 0.144 0.106 0.065 0.040 15 114 1.2 0.077 0.150 0.135 0.081 0.043 0.035 15 114 1.6 0.094 0.126 0.216 0.213 0.168 0.063 Proc print data = area; Data auc; set area; /" Calculation of Area 1 */ If t2a5 <=th then a1=0; /' Calculation of Area 2 */ else a1=(2.5*(.5*(t2a5-th))); lf t5 <=th and t2a5<=th then a2=0; lf t5 <=th and 12a5<=th then a2c=1; If t5>th and t2a5th and t2a5th and th=th and th=<12a5<=t5then a2c=3; If t5>th and t2a5>t5 then aZ=(2.5‘(t5+(.5*(12a5-t5)))-(2.5‘th)) ; If t5>th and t2a5>t5 then a2c=4; If t5<=th and t285>th then aZ=((((t2a5-th)/1285)'2.5)‘(.5*(t285-th))); 241 lf t5<=th and 12a5>th then a20=5; l” Calculation of Area 3 ‘I If t10 <=th and t5<=th then a3=0; If t10 <=th and t5<=th then a3c=1; If t10>th and t5th and t5th and th=th and th=th and t5>t10 then a3=(5*(t10+(.5'(t5-t10)))-(5*th)); If t10>th and t5>t10 then a3c=4; If t10<=th and t5>th then a3=((((t5-th)l‘t5)*5)'((.5*(t5—th)))); lf t10<=th and t5>th then a3c=5; /‘ Calculation of Area 4 *I If t15 <=th and t10<-th then a4=0; If t15 <=th and t10<=th then a4c=1; lf t1 5>th and t1 0th and t10th and th=th and th=th and t10>t15 then a4=(5'(t1 5+(.5‘(t10-t15)))-(5*th)); lf t15>th and t10>t15 then a4c=4; If t15<=th and t10>th then a4=((((t10-th)/t10)‘5)*(.5"(t10—th))); If t15<=th and t10>th then a4c=5; /‘ Calculation of Area 5 */ lf t20 <=th and t15<=th then a5=0; If t20 <=th and t15<=th then a5c=1; lf t20>th and t15th and t15th and th=th and th=th and t15>t20 then a5=(5"(t20+(.5*(t15-t20)))-(5*th)); If t20>th and t15>t20 then a5c=4; If t20<=th and t15>th then a5=((((t15-th)lt15)'5)"'(.5"(t15-th))); If t20<=th and t15>th then a5c=5; /' Summation of all areas ’/ areauc=sum(a1, a2. a3, a4, a5); drop t2a5 t5 t10 t15 120 bl1 bl2 th; Proc print data = auc; Proc plot data = auc; by week; plot areauc‘dose; run; data curve; set auc; /' Fitting a Segmented Model using NLIN ’/ if dose = 0 then areauc = 0; proc nlin method=marquardt; by week; parms a= 437 b= 1603 c=-911; file print; dose0=-.5"b / c; /‘ Estimate Join Point ‘I 242 db=-.5 / c; /" Deriv of dose0 wrt B */ dc=.5*b / c“2; /" Deriv of dose0 wrt C */ if dose