' THE EFFECT OF THYROTROPIN RELEASING HORMONE AND SMETHYL THYROTROPIN RELEASING HORMONE ON GROWTH AND HORMONAL RELEASE IN THE GROWING .CALF fiissertation fer the Degree of Ph. D. MICHIGAN STATE UNIVERSITY RODNEY KENNETH MCGUFFEY 197.5 I?“‘$ 1L, l t " P ' (3(1L.2 ' 1’ . ‘ {‘fi'P {vat This is to certify that the thesis entitled The Effect of Thyrotropin Releasing Hormone and 3Methyl Thyrotropin Releasing Hormone On Growth And Hormonal Release In The Growing Calf presented by Rodney Kenneth McGuffey has been accepted towards fulfillment of the requirements for Ph.D. Dairy Science and Jegree in . . . Institute of Nutrition I 7/? / I ,1///://%g;/7LM/ I 1v 5‘ Major professor Date 47/5;/7 5—, 0-7639 -B _ HUAG S annex BINDERI In: I ' LIBRARY amoans I NSPRIIIBPORI IMOIIGAN 7’“ . Wk.“ effec+ thyro- fema 500 ' , \I ‘7 m... THE EFFECT OF THYROTROPIN RELEASING HORMONE AND 3METHYL THYROTROPIN RELEASING HORMONE ON GROWTH AND HORMONAL RELEASE IN THE GROWING CALF BY Rodney Kenneth McGuffey An experiment was designed: (l) to determine the effects of thyrotropin releasing hormone (TRH) and 3 methyl thyrotropin releasing hormone (3MET) on growth in calves; (2) to determine serum concentrations of growth hormone (GH), thyroxine, prolactin, and insulin during chronic injection of TRH or 3MET; and (3) to determine the effects of age and time after feeding on serum hormone concen- trations. Thirty-nine Holstein calves, 30 males and 9 females, received daily intramuscular injections of either 500 ug of TRH, 500 ug of 3MET or 0.85% saline beginning at 45 days of age and continuing to 135 days of age. Weight gain and feed intake were measured at 15-day intervals. Jugular blood was sampled at 4 ages (45, 60, 105, and 135 days) at frequent intervals after injection for the deter- mination of serum concentrations of GH, thyroxine, pro- lactin, and insulin. Nine calves, 3 from each treatment, were 5] muscle (P<0.0 calves were n cant b calves from d greate WET g IP<0.0 PEriod ine, a feedir of in: while Serum facto; Rodney Kenneth McGuffey were slaughtered and tissue weights and gastrocnemius muscle and liver nucleic acids determined. Calves receiving TRH gained 7.8 kg more weight (P<0.05) and consumed 28.8 kg feed (P<0.05) than control calves while differences between 3MET and control calves were not significant. Feed to gain ratio was not signifi- cant between treatments. Weight gain and feed intake of calves receiving TRH were greater (P<0.01) than controls from day 76 to 90 and continued to be nonsignificantly greater than controls through day 135. Calves receiving 3MET gained less weight (P<0.0l) and consumed less feed (P<0.0l) than control calves from day 45 to 60 but other period differences were not significant. Serum concentration of insulin but not GH, thyrox- ine, and prolactin in control calves was increased by feeding (P<0.0l). In control calves, serum concentration of insulin, thyroxine, and prolactin increased (P<0.01) while GH decreased (P<0.0l) with age. The increase in serum prolactin concentration was related to environmental factors moreso than age of calf. Overall, serum GH concentration in calves given TRH was greater than that of calves given 3MET or saline at 5, 10, 15, and 30 minutes after injection. Calves given 3MET had greater (P<0.05) serum GH concentration than calves given saline at 10 and 15 minutes post-injection. From 45 to 480 minutes post-injection serum GH concentra— tion was not different between treatments. injecti was 47. and 62. calves 105 tha and 8.1 the in! 105, a 60% (p 72% (p 9iVEn 30, 45 than t greate inlem relea: and t; Rodney Kenneth McGuffey The difference between maximum GH (ng/ml) after injection and at injection at days 45, 60, 105, and 135 was 47.5, 35.8, 38.0, and 35.1 for TRH and 20.9, 16.8, 4.3, and 62.4 for 3MET, respectively. The difference for calves given TRH was greater (P<0.05) at days 45, 60, and 105 than that of 3MET. Overall serum thyroxine concentration was increased 3.60, 4.64, and 0.81 ng/ml-hr for calves receiving TRH, 3MET, and saline injections, respectively. The increase was greater (P<0.01) for TRH and 3MET than that for saline. At day 45 serum thyroxine concentration was increased 6.25 and 8.65 ng/ml-hr for TRH and 3MET. Relative to day 45, the increase in serum thyroxine concentration on days 60, 105, and 135 was decreased 50 (P<0.08), 55 (P<0.05), and 60% (P<0.05) for TRH and 50 (P<0.05), 77 (P<0.01) and 72% (P<0.05) for 3MET, respectively. Overall serum prolactin concentration in calves given TRH was greater (P<0.01) than that of saline at 15, 30, 45, and 60 minutes post-injection and greater (P<0.05) than that of 3MET at 60 minutes. Calves given 3MET had greater (P<0.05) serum prolactin concentration than saline— injected calves at 15, 30, and 45 minutes. Prolactin release was not depressed by repeated injections of TRH and 3MET. Overall serum insulin (ng/ml) was 1.51, 1.19, and 1.14 for calves given TRH, 3MET, and saline, respectively, and the difference between TRH and the other two treatments was signi concentre treatment serum in: were HOt' from cal when exp tion of O<0.05) receivir in live} between in calv Saline. for cal lover I differs balanCE Rodney Kenneth McGuffey was significant (P<0.05). At days 45 and 60 serum insulin concentrations were relatively stable and similar between treatments. At days 105 and 135 variable responses in serum insulin concentration to treatments especially TRH were noted. Tissue weights were greater on an absolute basis from calves receiving TRH but these differences disappeared when expressed on a percent of body weight with the excep- tion of the thyroid gland. Thyroid weight was greater (P<0.05) in calves receiving TRH than that from calves receiving 3MET or saline. Concentration of RNA and DNA in liver and gastrocnemius muscle were not different between treatments but muscle RNA/DNA ratio was 50% greater in calves receiving TRH than that of calves receiving saline. Total liver and muscle nucleic acids were greater for calves receiving TRH. Overall plasma non-esterified fatty acids were lower (P<0.05) in calves receiving TRH with the major difference at day 135. Plasma glucose and nitrogen balance were unaffected by treatment. THE EFFECT OF THYROTROPIN RELEASING HORMONE AND 3METHYL THYROTROPIN RELEASING HORMONE ON GROWTH AND HORMONAL RELEASE IN THE GROWING CALF BY Rodney Kenneth McGuffey A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Dairy Science 1975 IESCE O executi I: m ‘luscr encoura AC KNOWLED GMENTS I would like to acknowledge the invaluable assis- tance of all persons involved during the planning, execution, analysis, and preparation of data for this manuscript. Dr. J. W. Thomas for his guidance, advice, and encouragement throughout my graduate program. Dr. E. M. Convey for his ever positive attitude shown throughout this research and his highly appreciated compliments and critiques and most of all his profes- sionalism. Dr. L. L. Bieber for his advice for the non- esterified fatty acid assay and his enthusiasm. Dr. W. G. Bergen for amino acid analysis and his stimulating conversation. Dr. Ron Gendrich, Abbott Laboratories, for supplying TRH* and 3MET*. Ms. Eileen Bostwick for her cooperation in helping with hormone analysis. Dr. Roger Neitzel for computer programming and statistical advice. ii W ‘I L )1 *r l. U ) sample 1. AI ' ,1 I‘LVL lKj Mr. Tim Middleton, Richard Greening, John Johns, and Ms. Jane Carstairs for their help in collection of samples. Dr. C. A. Lassiter for making the facilities at Michigan State University available for this research. My wife Patty whose patience, encouragement, and love made my graduate experience easier. iii LIST OF LIST OE II. III. TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . . LIST OF FIGURES. . . . . . . . . . I. INTRODUCTION . . . . . . . . II. REVIEW OF LITERATURE. . . . . . Description of Growth. . . . Changes in Chemical Constituents during Growth. . . . . III. Nutritional Requirements of Growth Endocrine Requirements of Growth . Growth Hormone . . . . . . . Thyroid Hormones . . . . . . Growth Promotants . . . . . . The Hypothalamus and its Hormones. ThyrotrOpin Releasing Hormone . . Thyroid Hormones and Growth. . . Control of Growth Hormone Release. Metabolic Actions of Growth Hormone Control of Insulin Release . . . Metabolic Actions of Insulin . . Protein Metabolism. . . . . . MATERIALS AND METHODS . . . . . Design of Experiment . . . . . Animals. . . . . . . . . Treatments. . . . . . . . iv Page vi ix 13 14 15 17 22 23 29 31 33 37 41 42 43 43 43 45 VI_ VII. h Page Collection of Samples . . . . . . . 46 Blood. . . . . . . . . . . . 46 Nitrogen Balance . . . . . . . . 47 Tissues . . . . . . . . . . . 48 Laboratory Analysis. . . . . . . . 49 Plasma Glucose. . . . . . . . . 49 Non-esterified Fatty Acids. . . . . 49 Hormone Determinations. . . . . . . 51 Growth Hormone. . . . . . . . . 51 Prolactin . . . . . . . . . . 52 Insulin . . . . . . . . . . . 53 Calculation of Standard Curve and Results . . . . . . . . . . 54 Thyroxine . . . . . . . . . . 54 Determination of Tissue Nucleic Acids. . 56 IV. RESULTS . . . . . . . . . . . . 59 Weight Gain, Feed Intake, and Their Ratio. . . . . . . . . . . . 59 Serum Hormones . . . . . . . . . 62 Growth Hormone. . . . . . . . . 65 Thyroxine . . . . . . . . . . 83 Prolactin . . . . . . . . . . 97 Insulin . . . . . . . . . . . 110 Remaining Parameters . . . . . . . 117 Glucose . . . . . . . . . . . 117 Non—esterified Fatty Acids. . . . . 122 Nitrogen Balance . . . . . . . . 124 Tissue Weights. . . . . . . . . 124 Tissue Nucleic Acids. . . . . . . 125 V. DISCUSSION . . . . . . . . . . . 129 VI. CONCLUSIONS . . . . . . . . . . . 141 VII. APPENDIX . . . . . . . . . . . . 143 VIII. BIBLIOGRAPHY. . . . . . . . . . . 147 Table LIST OF TABLES Page Hypothalamic hormones controlling release of pituitary hormones . . . . . . . 24 Composition of ration fed to calves from 30 to 135 days of age . . . . . . ,. 44 Bleeding sequence and samples used for hormone analysis. . . . . . . . . 47 Weight gain (WG), feed intake (FI) and feed to gain ratio (F/G) by 15 day periods for calves receiving daily injections of thyrotrOpin releasing hormone (TRH), 3-methy1 thyrotrOpin releasing hormone (3MET), or saline (Sal) . . . . . . . . . . . . 60 Total weight gain (WG), feed intake (FI) and feed to gain ratio (F/G) in calves receiving daily injections of thyrotrOpin releasing hormone (TRH), 3-methy1 thyrotrOpin releasing hormone (3MET) or saline (Sal) from day 45 to day 135 . . . . . . . . 63 Double split-plot analysis of variance table for testing significance of main effects and their interactions . . 64 Serum growth hormone (GH), prolactin (prl), and insulin (Ins) (ng/ml) at day 45, 60, 105, and 135 in calves receiving saline. . . . . . . . . 68 vi table I‘H Lu 8A. 11. 12. Table 8A. 8B. 10. 11. 12. Ratios to and difference between serum growth hormone at maximum concentra- tion after injection of thyrotrOpin releasing hormone (TRH) or 3 methyl thyrotropin releasing hormone (3MET) concentration at injection for calves at day 45, 60, 105, and 135 . Number of calves at each of 4 ages categorized as to magnitude of increase in serum growth hormone (ng/ml) after injection of thyro- tropin releasing hormone (TRH) or 3 methyl thyrotropin releasing hormone (3MET). . . . . . . . Baseline serum growth hormone (GH) (ng/ml) (average of -10 and 0 min) at day 45, 60, 105, and 135 for calves receiving daily injection of thyrotrOpin releasing hormone (TRH), 3-methy1 thyrotropin releasing hormone (3MET) or saline (Sal) . . . . . . . . Linear regression equations of serum thyroxine concentration (Y) with time (X) (hrs) after injection of thyrotropin releasing hormone (TRH), 3—methy1 thyrotropin releasing hormone (3MET) or saline (Sal) into calves at day 45, 60, 105, and 135 . . . . . . . . . . Average serum insulin concentration in calves after injection of thyro- tropin releasing hormone (TRH), 3- methyl thyrotrOpin releasing hormone (3MET) and saline (Sal) . . Average plasma glucose concentration at 0, 2, 4, and 8 hr after injection of thyrotrOpin releasing hormone, 3-methyl thyrotropin releasing hormone (3MET) or saline . . . . vii Page 79 81 82 86 114 122 13 None c 14. Nit d I t c 15. Org < 16 1111 A‘1- Cc 5-2, Cc A B C E E I I Table 13. 14. 15. 16. Page Non-esterified fatty acids (NEFA) in calves: Age and treatment effects . . . . 123 Nitrogen balance in calves receiving daily injections of thyrotrOpin releasing hormone (TRH), 3 methyl thyrotropin releasing hormone (3MET) or saline (Sal) . . . . . . . . . . 124 Organ weights of calves receiving daily injections of thyrotropin releasing hormone (TRH), 3 methyl thyrotropin releasing hormone (3MET) and saline (Sal). . . . . . . . . . 126 Muscle and liver nucleic acids in calves receiving daily injections of thyrotrOpin releasing hormone (TRH), 3 methyl thyrotropin releasing hormone (3MET) or saline (Sal) . . . . . . . . . . . . . 127 Composition of scintillation fluid . . . . 143 Composition of reagents for radio- immunoassay . . . . . . . . . . . 144 A. 0.05 M PBS-1% BSA pH 7. 4 . . . . . . 144 B. Buffer A1 . . . . . . . . . . 144 C. Guinea Pig Anti- bovine Insulin and Guinea Pig Anti-bovine Growth Hormone . . . . . . . . 144 D. 0.05 M PBS- EDTA pH 7.0 . . . . . 144 E. 0.01 M phosphate buffered saline, pH 7.0 (PBS). . . . . . . 144 F. Monobasic phosphate (0.5m). . . . . . 144 G. Dibasic phosphate (0.5m) . . . . . . 145 H. 1:400 Normal Guinea Pig Serum. . . . . 145 I. Sheep Anti-Guinea Pig Gamma Globulin Antibody (SAFPFF) . . . . . 145 J. Guinea Pig Anti-Bovine Growth . Hormone . . . . . . . . . . . 145 A. Orcinol Reagent . . . . . . . . . 146 B. Diphencylamine Reagent . . . . . . . 146 C. Acetaldehyde Solution . . . . . . . 146 viii L!" o LIST OF FIGURES Figure Page 1. Thyrotropin releasing hormone. . . . . . 26 2. Average serum growth hormone concentration in calves after intramuscular injections of thyrotropin-releasing hormone (TRH), 3 methyl thyrotropin releasing hormone (3MET), or saline (Sal) . . . . . . . 67 3. Serum growth hormone concentration at 45 days of age following intramuscular injection of thyrotrOpin releasing hormone (TRH), 3 methyl thyrotrOpin releasing hormone (3METL.or saline (Sal) . . . . . . . . . . . . . 70 4. Serum growth hormone concentration at 60 days of age following intramuscular injection of thyrotrOpin releasing hormone (TRH), 3 methyl thyrotrOpin releasing hormone (3MET), or saline (Sal) . . . . . . . . . . . . . 72 5. Serum growth hormone concentration at 105 days of age following intramuscular injection of thyrotropin-releasing hormone (TRH), 3 methyl thyrotrOpin releasing hormone (3MET), or saline (Sal) . . . . . . . . . . . . . 74 6. Serum growth hormone concentration at 135 days of age following intramuscular injection of thyrotropin releasing hormone (TRH), 3 methyl thyrotrOpin releasing hormone (3MET) or saline (Sal) . . . . . . . . . . . . . 76 ix Figure 13. Ser 6 j 1 14 Se 15. s. 1 16. s 17. 18. 19, Figure Page 7. Plots of linear regression equations for serum thyroxine concentration combined for all days following intramuscular injection of thyrotropin releasing hormone (TRH), 3 methyl thyrotropin releasing hormone (3MET), or saline (Sal) . . . . . . . . . . . . . 85 8. Plots of linear regression equations for serum thyroxine concentration in calves 45 days of age following intra- muscular injection of thyrotropin releasing hormone (TRH), 3 methyl thyrotropin releasing hormone (3MET), or saline (Sal). . . . . . . . . . 88 9. Plots of linear regression equations for serum thyroxine concentration in calves 60 days of age following intra- muscular injection of thyrotrOpin releasing hormone (TRH), 3 methyl thyrotropin releasing hormone (3MET), or saline (Sal). . . . . . . . . . 90 10. Plots of linear regression equations for serum thyroxine concentration in calves 105 days of age following intra- muscular injection of thyrotropin releasing hormone (TRH), 3 methyl thyrotrOpin releasing hormone (3MET), or saline (Sal). . . . . . . . . . 92 11. Plots of linear regression equations for serum thyroxine concentration in calves 135 days of age following intra- muscular injection of thyrotropin releasing hormone (TRH), 3 methyl thyrotropin releasing hormone (3MET), or saline (Sal). . . . . . . . . . 94 12. Average serum prolactin concentration in calves following intramuscular injection of thyrotrOpin releasing hormone (TRH), 3 methyl thyrotrOpin releasing hormone (3MET), or saline (Sal) . . . . . . . . . . . . . 100 ii, Figure 20. Se Figure Page 20. Serum insulin concentration in calves 135 days of age following intramuscular injection of thyrotropin releasing hormone (TRH), 3 methyl thyrotrOpin releasing hormone (3MET), or saline (Sal) . . . . . . . 121 xii fed a 0f 1e m6nt, affec StIOr for j incr gnal I . INTRODUCTION Growth is a phenomenon of all species. Like many other scientific phenomena, description of growth cannot be embodied in simple terms and present a complete definition. Yet, the definition of growth is more simple than a com— plete description of factors causing growth (or lack of). Long ago animal producers recognized that animals fed a "superior" ration grew faster than animals fed one of lesser quality. Other factors such as genetic improve- ment, special breeds, and environment have been shown to affect growth. More recently growth has been shown to be strongly influenced by hormones both synthetic and natural. Today, the human pOpulation has created competition for both land and feedstuffs for animal production. An increase in animal production on less land and from lower quality rations has become a necessity to meet the pressure of this expanding population. To meet pOpulation demands for increased production the previous factors affecting growth, i.e. nutrition, superior genetic strains or capabilities, environment, and specific growth stimulants will have to be integrated in some mar first it growth. answere superic of inc1 parame that m some manner. For this to occur animal scientists must first formulate basic, simple questions about animal growth. Only after these questions are satisfactorily answered can the above factors be integrated to produce superior and faster growing animals. This study was designed to investigate a new source of increasing growth in young calves and determine hormonal parameters resulting from this treatment and explore those that may alter growth. " I sell my 3111 per C01: II. REVIEW OF LITERATURE Description of Growth Brody (1945) analyzed growth curves of pOpulations of yeasts, flies, individual pumpkins, and rats and found growth curves from each species similar and that they coincided in his mathematical expression of growth. He divided the growth curve into two phases: (1) a self- accelerating phase and (2) a self-inhibiting phase. The self-accelerating phase is characterized by reproducing units to reproduce at a constant percentage rate when allowed to do so. In the absence of inhibiting factors the percentage growth rate remains constant. Finally, there comes a time when inhibiting factors override the accel- erating factors until growth processes cease. This idealized description of growth was mathe- matically described by Laird (1966a, 1966b). Growth before and after birth was described by an equation having an exponential and linear component. The exponential component described hyperplasia or an increase in cell numbers while the linear component described hypertrophy or an increase in cell size. da ti si tr LID OL 82 m V: Enesco and Leblond (1962) studied growth of many tissues in rats from birth to 160 days of age. From their data they divided tissue growth into three phases. First, tissues grew by hyperplasia with minute changes in cell size; secondly, growth was characterized by both hyper- trophy and hyperplasia and finally, a period that continued until mature body weight was attained where growth was characterized by hypertrophy with hyperplasia playing only a small part in the increase in size. Changes in Chemical Constituents During Growth Chemical composition of an animal changes through- out the growth process. At birth, water constitutes about 82% of fat-free body weight, protein about 14%, and minerals about 4% (Forbes, 1968). During growth water decreases, protein increases, and they plateau at 70-75% and 21-24%, respectively. Mineral contribution to body weight remains relatively constant on a percentage basis during growth. Palsson and Verges (1952) reported the order of development is from inside to outside, i.e. central ner- vous system, bone tendon, muscle, intermuscular fat, and finally subcutaneous fat. At birth the central nervous system is essentially complete. Changes in percentage of bone, fat, and muscle then constitute body components undergoing postnatal changes. ment matt oft bo< an: Berg and Butterfield (1968) described the develop- ment of bone, muscle, and fat in cattle from birth to maturity. At birth, bone represented a greater prOportion of the carcass than at any other time. Fat, conversely, represented a greater prOportion of the carcass at maturity than at any other time. Muscle represented a greater proportion of the carcass intermediate in development. Factors such as nutrition, breed, and sex affected the rate of growth of tissues but not order of development. Moulton (1923) introduced the concept of chemical maturity and defined it as the age which the concentration of water, protein, and ash become constant in the fat free cell. Armsby and Moulton (1925) reported the age mammals reach chemical maturity as follows: rat, 50 days; guinea pig, 50 days; swine, 150 to 300 days; cattle, 150 to 300 days; and humans, 500 to 1000 days or at approximately 3.9 to 4.6% of the total expected life span of each species. Reid et a1. (1955) summarized published data on body composition of the bovine as influenced by sex, age, and breed. A total of 256 cattle representing 139 beef (133 males) and 117 dairy (2 males) animals ranging in age from 1 to 4860 days were used to establish relationships of body fat, water, protein, and ash. Fat and water were the body constituents exhibiting the most variation. Expression of water, protein, and ash on a fat-free empty body weight basis tended to make these components relatively constant. (1) V stant fat c varic conte basis of 3 betwe ash < ever. corr: Issp. Thus basi Reid with the fed Wit} Iqu mail Of 1 Body constituents were divided into two groups: (1) variable, which included fat and water; and (2) con- stant, which included protein and ash. Correlation between fat content (%) and water content (%) for 256 cattle of various ages was -0.987. Expression of protein and ash content on the fat-free dry basis rather than fat-free basis reduced variation of these components by a factor of 3 and 2, respectively. There were no differences between breed or sex of cattle in amount of protein and ash content when expressed on the fat-free dry basis. How- ever, age of animal was found to be highly significantly correlated with protein and ash content (-0.42 and +0.42, respectively) when expressed on the fat-free dry basis. Thus, expression of protein and ash on the fat-free dry basis gave the most accurate prediction of these components. Reid et a1. (1955) presented a series of equations so that with an accurate estimate of body water content and age of the animal, body composition and body energy from cattle fed under different nutritional regimes can be estimated with a relatively high degree of precision. Nutritional Requirements of Growth Quantitatively and probably qualitatively, nutrient requirements for growth are different than those for maintenance. Relative to maintenance, a larger prOportion of protein, energy, minerals, and vitamins is required for a young growing animal than a mature animal on a body weight growinc nutrie: nutrit reali: normal intake in ga. these weigh these weight basis. A decrease in growth occurs in a young growing animal when any single or combination of these nutrients is sub-optimal. Osborne and Mendel (1915) demonstrated that under- nutrition in the growing period delayed growth. Upon realimentation growth resumed at a more rapid rate than normally observed. McCay et a1. (1935) restricted caloric intake in rats at weaning to allow approximately 10 grams in gain every 2 to 3 months. At 766 or 911 days of age these rats were fed ad libitum and an increase in body weight was noted. Final body weights were however less in these animals than those fed at a normal rate from weaning and these restricted animals lived longer. Meyer and Clawson (1964) reported 5 month-old sheep and 1 to 2 month-old rats were able to maintain body weight, body fat, and body protein percentage when fed 52% of the amount consumed by ad libitum fed animals. When refed an amount of feed to give the same total intake as consumed by ad libitum controls, restricted rats and sheep did not attain the same body weight as controls, and final body energy in restricted rats but not sheep was less than controls. When fed to the same body weight as controls, restricted animals had a higher body fat thus greater final body energy than controls. The time required to reach control weight was prOportional to severity of restriction. suckling Rat pups litters suming lactati dams (S grew 15 proteir and its decreag 0f mUS< decrea in aCC' deerea tiOn 0 muSCIQ resalt McCance and Widdowson (1962) correlated growth of suckling rats with the protein content of maternal milk. Rat pups in litters of 16 grew less than did rat pups in litters of 4 (Sinha et al., 1973). Pups from dams con- suming 50% of ad libitum intake during gestation and lactation grew slower than did pups from ad libitum fed dams (Stephan et al., 1971). Weanling rats fed 6, 12, or 18% crude protein diets grew less in 14 days than did similar rats fed 24% crude protein (Howarth, 1972). Weight of gastrocnemious muscle and its DNA, RNA, and protein content decreased with decreasing dietary protein level. Relative to accumulation of muscle constituents on the 24% crude protein diet, decreasing dietary protein resulted in a greater decrease in accumulation of DNA than protein. RNA accumulation decreased intermediate to DNA and protein with the excep- tion of the 6% diet where there appeared to be a loss of muscle RNA. Feeding a protein free diet for 14 days resulted in a marked loss of muscle RNA but insignificant changes in DNA and protein content from that initially present. Howarth and Baldwin (1971) fed weanling rats ad libitum and restricted some to 50 to 60% of ad libitum intake. As a result gastrocnemius muscle weight and RNA, DNA, and protein content, were less in the restricted group. This dietary restriction resulted in lowered 32P-orthophosphate incorporation into RNA and DNA and \_ reduce: Refeed of pro eratic DNA 53 Dieta intak by h} aCCun muScJ net ( adeql reduced protein synthetic capacity of muscle microsomes. Refeeding the restricted group resulted in delayed recovery of protein synthetic capacity with no compensatory accel- eration due to dietary restriction, yet rates of RNA and DNA synthesis during refeeding were greater than normal. Trenkle (1974) observed a reduced growth rate in rats when fed either a calorie or protein deficient diet. Limiting protein or energy resulted in reduced muscle growth, reduced DNA, RNA, and protein content. Limiting either nutrient singly or together did not change protein/ DNA and muscle weight/DNA ratios. During dietary restric- tion muscle continued to grow but the rate of growth was retarded. Dietary restriction limited accumulation of muscle constituents but had no effects on rate of accumu- lation relative to muscle growth. Muscle is of primary interest in meat production. Dietary restrictions such as reduced protein or energy intake influences muscle growth. During tissue growth by hyperplasia, dietary restriction limits total DNA accumulation therefore limiting future growth capacity of muscle although concentrations of muscle constituents may not decrease. To attain maximum growth during hypertrOphy adequate nutrition is imperative early in life. For reasons such as time, money, and animal size the amount and detail of investigation into large animal growth is limited. There is considerable literature where growth rate under various nutritional regimes has ‘\ been qL nechan: cellul; and 1a? influe tions growth nent c Morris growt} Energ effiC Cons: incrs Fret. 10 been quantitated. Since many basic anabolic and catabolic mechanisms are common to all animal species, patterns of cellular growth would be expected to be similar for farm and laboratory animals. This section will review the influence of nutrition on ruminant growth and make assump- tions concerning nutritional alterations of ruminant growth based on data from laboratory animals. Jones and Hogue (1960) in a 2 x 3 factorial experi— ment compared energy and protein fed at 90 and 120% of Morrison standards with and without oral stilbestrol on growth in lambs. Lambs fed the high protein ration (regardless of energY) had higher average daily gain (0.34 lb) than those fed the low protein ration (0.28 lb) and ate more feed than lambs fed the low protein level. No significant effect on weight gain occurred with different energy levels. Lambs fed high protein-high energy gained faster than other combinations with low energy-high protein the most efficient combination for growth. Lambs fed high energy—low protein gained slower and were the least efficient of all treatment groups with a much lower expected consumption of estimated net energy (78.6%). Stilbestrol increased average daily gain and feed efficiency in all protein and energy combinations. These authors concluded that low protein intake relative to energy influences food intake through the adverse ratio of the two nutrients. Baile and Forbes (1974) concluded from a review of the literature that growing cattle can compensate for diet dilut. feed ; birth ll dilution by eating more as long as dilution does not limit feed intake through its effects on rumen fill. Willey et a1. (1952) fed fattening steers low and high energy rations (58.5 and 64.5 therms respectively) with low and high levels of fat (3.0% and 7.5%) in the diet with a 12.5% crude protein content in all diets. Steers did not differ in average daily gain but animals fed high levels of fat required about 45.4 kg less feed per 45.4 kg gain during the fattening period. Struedemann et al. (1968) raised beef calves from birth to slaughter on five nutritional levels as follows: Treatment Group_ Very Very Restricted Restricted Normal High High Weaning age, days 170 240 240 240 240 Milk produc- tion of dam Average Low Average Average High Supplemental feed --- --- --- --- Creep Between 170 and 240 days of age calves in the very restricted group were raised on limited hay and pasture with calves receiving creep feeding between 90 and 240 days of age. Calves in restricted, normal, high, and very high gained .04, .16, .25, and .23 kg per day more than very restricted calves, respectively. When placed in the feedlot there was no significant difference in average daily gain for calves from the dif- ferent nutritional regimes. The time required to reach \. growi WhEn requ; C0031 heav the inCr gain 12 a final weight of 430 kg was less for calves in the higher nutritional levels (high or very high) but, the amount of feed per unit of gain was less for the lower nutritional levels (normal, restricted, very restricted). As the level of nutrition increased during early life, carcasses from these animals tended to contain a greater percentage of lean and bone and a lower percentage of fat. Waldman et a1. (1971) fed steers ad libitum or 70% of ad libitum intake. Steers fed ad libitum had higher average daily gain but less carcass protein at comparable weights than steers fed 70% ad libitum. Gardner et a1. (1960) compared the efficiency of growth of suckling single or twin lambs to 90 days of age when ewes were fed 95 or 115% National Research Council requirements for digestible energy. Lambs whose dams consumed the higher level of energy were significantly heavier at 90 days of age than lambs whose dams consumed the lower level of energy. The heavier lambs had a 12% increase in body energy gain and a 10% increase in protein gain. Body composition in lambs was not affected by energy intake of the dam. Johns (1974) fed weanling lambs for 60 days either a 7% or 15% crude protein (C.P.) ration having equal digestible energy density. Lambs fed the 15% C.P. ration gained more, consumed more, and had a lower feed/gain ratio than did lambs fed the 7% C.P. ration. Lambs fed the 5 m1. .Otc his) by I inc' me& (1 . 13 15% C.P. ration had heavier liver and gastrocnemius muscle. Total liver RNA, DNA, and protein were increased with the higher protein diet while liver RNA/DNA ratio was unaffected by protein level. Similar results were obtained for muscle except that muscle RNA/DNA ratio was increased by the high protein diet. Total essential amino acids (TEAA) per gram of wet tissue were increased in muscle but not liver in the 15% C.P. ration. Total non-essential amino acids (TNEAA) were increased in muscle but not in liver for the 7% C.P. ration. Plasma TEAA increased and TNEAA decreased with feeding the 15% C.P. diet. These results indicate that muscle which is of economic interest for meat production is more adversely affected by low protein diets than is liver. Endocrine Requirements of Growth Cushing (1912) observed a marked loss in body weight following removal of the anterior pituitary from growing animals. Evans and Long (1922) restored growth of hypophysectomized rats to normal with the injection of anterior pituitary extracts. Since that early research, growth hormone (GH) has been isolated (Li and Evans, 1944) and identified as the single most important anterior pituitary hormone in improving growth in hypophysectomized animals. The following discussion will review the effects of hormones on altering growth in animals. on bon (b) in 1963). ject c IEpor Pitui and 1 slang Slau< Year tOta tOta 14 Growth Hormone Growth hormone affects growth in two ways: (a) acts on bone to cause bone growth especially long bones and (b) increases the retention of ingested nitrogen (Nalbandov, 1963). The possible cellular mechanism of GH is the sub- ject of a latter part of this review. Baird et a1. (1952) using 2 lines of swine bred to gain at different rates found an increased pituitary GH activity of the line that gained faster. At any age or body weight the GH content of the pituitary was highest in the faster gaining line. Armstrong and Hansel (1956) reported a positive correlation between GH/gram of pituitary tissue or total pituitary GH/lOO lb body weight and rate of growth for the 16 week period previous to slaughter in dairy heifers. Purchas et a1. (1970) slaughtered bulls at monthly intervals from birth to one year of age and found neither plasma GH concentration, total GH in circulatory fluid, pituitary GH concentration, total pituitary GH nor pituitary GH per unit body weight to be closely correlated with observed rate of growth. Measures of serum GH in cattle (Grigsby, 1973) and lambs (Johns, 1974) were negatively correlated with growth fed feedlot type rations. The concentration of serum GH is greater in young growing animals than older growing animals (Armstrong and Hansel, 1956; Purchas et al., 1970; and Trenkle, 1971b). Nalbandov (1963) and Curl et a1. (1968) have suggested the: and GH he: ra‘ ob pr ra SC 15 there is less GH per unit of body weight in heavier animals and this results in growth statis. In agreement with this hypothesis, Trenkle (1971b) observed a 5-fold decrease in GB secretion rate in cattle from 3 to 17 months of age. Machlin (1972) injected 1.1 mg/kg body weight purified porcine GH into pigs and noted death in 8 of 12 pigs due to liver damage, kidney degeneration, and stomach hemorrhage. When this GH preparation was injected into rats at 10 times the dosage given pigs no lethality was observed. A dose of 0.13 mg GH/kg body weight to pigs produced no fatal symptoms. This dose increased average daily gain and percentage lean cuts and decreased feed/gain ratio and dressing percentage. The hypothalamic peptide somatostatin, which inhibits release of GH from the pituitary, decreased body weight gains in male rats during 8 days of injection (Brazeau et al., 1974). Thyroid Hormones Brody (1945) thyroidectomized a calf at 50 days of age. At maturity this animal's mature body weight was half normal and energy metabolism per unit body area was 40% of normal. Scow (1959) thyroidectomized (T) and hypophysec- tomized (H) rats at 21 and 26 days of age, respectively. At 56 days of age either GH, thyroxine, or both were given daily for 36 days. Rats without pituitaries and thyroids gained 8.0 grams but when given 0.5 mg GH or 2.5 mg v ‘1‘ o 51' 68, £01 rat 16 thyroxine alone or in combination each day the gain was 68, 27, and 81 grams, respectively. For the 70 day period following removal of these glands gains for HT, H and T rats were 32, 35, and 67 grams respectively. Quantitation of muscle myosin and collagen showed different sites of action for thyroxine and GH in HT rats. Myosin to collagen ratio was 12 in HT rats given 2.5 mg thyroxine alone and this ratio decreased to 9.3 when 0.5 mg GH was also pro- vided. Scow (1959) suggested thyroxine to be myotropic and GH to be collagenotropic. Trenkle (1974) reported that GH and thyroxine given in combination completely restored muscle DNA synthesis in hypophysectomized rats to levels comparable to that of normal rats of the same age. Pituitary thyroid-stimulating hormone (TSH) con- centration like GH is highest in young cattle and decreases with age (Armstrong and Hansel, 1956). Heifers fed a high plane of nutrition tended to have higher pituitary TSH content than those fed a low plane of nutrition. Armstrong and Hansel (1956) found a positive correlation between pituitary TSH content and rate of growth during the 16 week period previous to slaughter in heifers. Hatch et al. (1972a, 1972b) classified steers and lambs by thyroid status, i.e. hypo-, eu- or hyperthyroid from estimates of thyroid activity of the animals. Steers classified hyperthyroid gained significantly more weight during the 28 day period following classification than either hypo- or euthyroid steers while lambs showed no ‘5 () 17 difference in weight gain following thyroid status clas- sification. During a 147 day feedlot period steers classified hyperthyroid gained 5.6 kg and 3.7 kg more than hypo- or euthyroid classed steers, respectively. Growth Promotants The suggestion that hormones have a profound influence of growth led researchers to begin looking for agents that would stimulate growth by alteration of endogenous hormone production. Although not included in this review, the reader is reminded of growth stimulation by antibiotic additions to feed especially in monogastric animals. In 1954, two reports appeared describing increased weight gain following addition of the synthetic estrogen diethylstilbestrol (DES) to feed of ruminants (Burroughs et al., 1954; Clegg and Cole, 1954). Surprisingly, DES had little or no positive effects on milk production in ruminants (Wrenn and Sykes, 1957) or increasing weight gain in monogastric animals. Clegg and Cole (1954) postulated that the action of stilbestrol in promoting growth may be mediated by pituitary hormones. These workers noted larger pituitaries from animals implanted with stilbestrol. With the introduction of the radioimmunoassay to endocrinology, scientists were better able to determine the role DES played in altering hormone levels. CT) C1 18 Davis et a1. (l970a,b,c) in a series of papers noted a high degree of similarity of action between purified ovine GH and DES. With fasting sheep, the authors noted an initial decline in plasma non-esterified fatty acids (NEFA) and glucose concentrations followed by an elevation at one hour following injection of 15 mg GH. Eight hours following GH injection, plasma NEFA and glucose concentrations reached maximum values while plasma urea nitrogen and free amino acid nitrogen concentrations were decreased from the level at injection. There was no change in plasma insulin concentration as the result of GH injection (Davis et al., 1970a). Davis et al. (1970b) proceeded to do a longer trial in which 10 mg of GH was injected daily for 9 days. A 9 and 7 day control period preceded and followed GH treatment, respectively. Plasma NEFA were elevated by daily injections of GH. Plasma insulin and glucose con- centrations showed an initial increase after GH treatment began but both were depressed midway through the treatment period. A high positive correlation (0.95) was found between insulin and glucose concentrations. Again plasma urea nitrogen concentration decreased and nitrogen reten- tion increased more during GH treatment. The third paper (Davis et al., 1970c) reported on the metabolic effects of DES. Two 3 week periods during which 4 mg DES per day was administered were spaced 19 between 3 one-week control periods giving a sequence as follows: control, DES, control, DES, and control. Glucose and insulin concentrations in plasma tended to increase weekly over each 3 week DES treatment period and decline during control periods. Weekly means of plasma urea nitrogen concentration and nitrogen retention were inversely related (r = -0.95). Plasma urea nitrogen : concentration decreased and nitrogen retention increased with DES treatment. Trenkle (l970b) fed finishing rations to fattening steers and observed no effect of ration on plasma GH con- centration and only a slight increase in plasma insulin concentration. Addition of DES (10 mg/day) to the ration increased plasma GH and insulin concentrations with no effect on protein-bound iodine. DES feeding to lambs for 2 weeks resulted in a 32% increase in serum GH (Hutcheson and Preston, 1971). Steers fed DES gained significantly more weight and had 15.7% larger pituitaries than control animals. Davis and Garrigus (1971) fed DES either continually (7 weeks) or intermittently (3 weeks fed, 1 week without, 3 weeks fed) to lambs. Nitrogen retention was signifi- cantly increased in both DES treatment groups with inter- mittent DES being significantly greater than continual DES. During the week without DES, nitrogen balance remained equal to continual DES and when DES feeding was resumed a further stimulation in nitrogen retention occurred. As prev101 nitrog 3 week ment, gradua and P1 plasma after to E is I eSt] 12g wit] int. inc 20 previously reported (Davis et al., 1970c), plasma urea nitrogen concentration was decreased in sheep fed DES for 3 weeks. Between 3 and 7 weeks of continuous DES treat- ment, plasma urea nitrogen concentration increased gradually to a similar concentration of controls. Hutcheson and Preston (1971) also found DES to initially depress plasma urea nitrogen followed by a return to control values after 6 weeks of DES feeding. In 1973, legal action banned the use of DES for ruminants because of its possible carcinogenic activity. Later that year an injunction by the courts allowed use of DES in ruminants until the matter could be settled legally. As a result the search for other growth promoting agents (natural and synthetic) has intensified with the identifi- cation of some active compounds. To date the mechanism of each has not been elucidated but certain agents show sufficient promise to warrant further research. A few will be discussed briefly. Zearalonol or Zeranol (same) is similar in structure to DES and is the lactone of resorcylic acid. In nature it is produced by a mold which grows on corn and it has known estrogenic activity. Sharp and Dyer (1971) observed a 12% increase in feedlot gain of steers and lambs implanted with zearalonol with a decrease in feed/gain ratio and no intake differences. Borger et a1. (1973) noted a 7.8% increase in gain of steers implanted with 36 mg Zeranol at l and 84 days of a 169 day feedlot trial. Implanted steers bad 51 trols steers implar estrac faster terone 100 It positi and be as not 9rOWth QIEVat produe not in Stand 21 had significantly higher serum GH concentration than con- trols with no difference in insulin. Goodrich and Meiske (1973) implanted Holstein steers with a variety of growth promotants. Steers implanted with DES, 200 mg progesterone plus 20 mg estradiol benzoate, or Zeranol gained 17.0, 15.0, and 5.9% faster than controls. An implant of 120 mg of testos- terone plus 24 mg DES did not improve gain. Feed per 100 lb of gain was less with those treatments showing a positive response in weight gain. Raun et a1. (1974) added monensin a compound produced by Streptomyces cinnamonensis to feedlot rations and observed a 14 to 15% improvement in feed/gain ratio. The exact mechanism producing the positive response is not completely understood but a 45% increase in ruminal propionic acid production has been observed (Richardson et al., 1974). The composition of carcass gain was not affected by monensin treatment (Potter et al., 1974). The previous section has shown that growth rate and body composition can be altered by many factors such as nutrition, breed, sex, hormones, and compounds termed growth promotants. There is general consensus that an elevation of serum hormones especially insulin and GH produce an increase in rate of gain and more often than not improved feed utilization. To more completely under- stand mechanisms regulating growth, improved methods for Chan assc brai of t Optic The l conte stalk The h fiber Porta neuroe latiOr Provid the "n stated SYche fibers hUmOrs Carriec‘ inhibit Was lat cortiCo' 22 changing growth rate and patterns and evaluating changes associated with improved growth are needed. The Hypothalamus and Its Hormones Located in the diencephalon at the base of the brain, the hypothalamus forms the floor and lateral walls of the third ventricle and is bordered anteriorly by the optic chiasma and posteriorly by the mammillary bodies. The hypothalamus is connected to the pituitary by a stalk containing the portal vessels. Located dorsal to the stalk is an expanded area known as the median eminence. The hypothalamus consists of a series of nuclei with nerve fibers running toward the pituitary stalk and ending at the portal vessels. Green and Harris (1947) made a major discovery in neuroendocrinology when they described the portal circu— lation of the hypothalamus and pituitary. This discovery provided the final connection for the anatomical basis of the "neurohumoral" concept for pituitary secretion. Simply stated this hypothesis suggested that neurohumors were synthesized in hypothalamic nuclei and stored in nerve fibers of the median eminence. At this point the neuro- humors were released into the portal circulation and carried to the anterior pituitary causing release or inhibition of release of pituitary hormones. This theory was later given impetus with the discovery of a corticotrophin-releasing factor (Saffran and Schally, 1955). To dat descri of pit strate These tase i extrac the ir rats a thyroj (1966) amino IUtere TRH t< 23 To date nine other hypothalamic hormones have been described that control release or inhibition of release of pituitary hormones (Table 1). Thyrotropin Releasing Hormone ThyrotrOpin-releasing hormone (TRH) was demon- strated by Schrieber et a1. (1961) in Czechoslovakia. These workers noted an increase in pituitary acid phospha- tase in vitro when rat pituitaries were treated with acid extracts of bovine hypothalamic tissue. When media from the incubation mixture was injected into hypophysectomized rats an increase in the uptake of radio-iodine by the thyroid was observed (Schrieber et al., 1962). Not until five years later did Schally et a1. (1966) report TRH to be made of equimolar amounts of 3 amino acids: glutamic acid, histidine, and proline. Interestingly enough, Schrieber et a1. (1962) had reported TRH to be composed of 9 amino acids. Boler et a1. (1969) and Burgus et a1. (1970) reported the structure of TRH isolated from porCine and ovine hypothalamic tissue, respectively. The structure is shown in Figure l and undoubtedly the cyclized glutamic acid and the prolineamide group contributed to the long delayed structural determination. Today, TRH is made in a relatively large and synthetic TRH has all the functions of natural TRH. Vale et a1. (1971) reported an analog of TRH having a methyl group in the 3-N position on the imidazole M: H 24 TABLE l.-—Hypotha1amic hormones controlling release of pituitary hormones. Hypothalamic Hormone Corticotropin (ACTH)-releasing hormone Thyrotropin (TSH)-releasing hormone Luteinizing hormone (LH)-releasing hormone Follicle stimulating hormone (FSH)-releasing hormone Growth hormone (GH)-releasing hormone Growth hormone (GH)-releasing-inhibiting hormone Prolactin release-inhibiting hormone Prolactin—releasing hormone Melanocyte-stimulating hormone (MSH) release-inhibiting hormone Melanocyte-stimulating hormone (MSH) releasing hormone 25 .oQOEHos mcflmmoaou Camoupouwne .H onsmflm $353-8“. s: 228.1% I o \ / N _._z z «:0 :w NIW p / _ \ / \zlllflwlxolllelwlxo oflo «Ioluzo o n_v _ _ «zo.|u~zo It... ring relea syntl syste The ; mole: inhi have cell pora incr Pitt caus tiss una: incl reL tha and SYn is 0f TRH inc 27 ring of histidine to be 8-fold more potent than TRH in releasing TSH from mice pituitaries in_vivo. Mitnick and Reichlin (1972) reported in_yiyg synthesis of TRH to require an ATP dependent soluble enzyme system. The authors named the enzyme "TRH synthetase." The incorporation of amino acids into the tripeptide molecule was not inhibited by the protein synthetic inhibitors cycloheximide and puromycin. Labrie et a1. (1972) and Wilber and Shaw (1973) have reported TRH to be bound to membranes of pituitary cells. Wilber (1971) observed a TRH stimulated incor— poration of 14C-glucosamine into TSH in yitrg when the incubation medium was assayed for TSH but not when pituitary tissue was assayed. Protein synthetic inhibitors caused a decrease in the appearance of label into TSH in tissue and medium, but total TSH secreted into medium was unaffected by inhibitors. When thyroxine was added to the incubation medium TRH stimulated synthesis of TSH but release was inhibited. Bowers et a1. (1968) demonstrated that triiodothyronine inhibited TRH-induced TSH release and the effect was abolished by incubation with protein synthetic inhibitors. This result suggests that a protein is involved in triiodothyronine inhibition. The thyroid status (hypo-, eu-, or hyperthyroid)’ of an animal affects the response of the pituitary to TRH-induced TSH release. Wilber and Shaw (1973) reported increased TRH binding to pituitary membranes in the hyp‘ stai and TRH thy] (197 fron trii and lati Reic Plas: hYPO‘ Peak stra1 in mi et a1 injec ttho day l thyro. 9t a1. aftEr Unrela a biPh 28 hypothyroid state and decreased binding in hyperthyroid state relative to the euthyroid state. Vale et a1. (1971) and Wilber (1971) reported the amount of TSH released from TRH stimulation to be less when either triiodothyronine or thyroxine was incubated with pituitary explants. Smith (1974) observed a decreased TRH-induced prolactin release from bovine pituitary cell cultures incubated with triiodothyronine or thyroxine. Martin and Reichlin (1970), Adams et a1. (1973), and Debeljuk et a1. (1973) measured an increase in circu— lating TSH after administration of TRH. Martin and Reichlin (1970) were also able to measure an increase in plasma TSH by electrical stimulation of the medial basal hypothalamus. In each case the rise in TSH was rapid and peak levels reached within 30 minutes after TRH admini- stration. The response of thyroxine to TRH has been reported in mice (ChOpra and Solomon, 1973) and cattle (Convey et al., 1973a and Vanjonack et al., 1974). Twice daily injections of 1 ug of TRH for 7 days increased serum thyroxine concentrations at 2 hours after TRH only on day l with no change in thyroid size or 1311 uptake by the thyroid noted in mice (ChOpra and Solomon, 1973). Convey et al. (1973a) reported maximal thyroxine response 5 hours after TRH injection and that magnitude of response was unrelated to dose given. Vanjonack et a1. (1974) noted a biphasic response of thyroxine to TRH. An increase of inc11 With: from time trol. was 3 Pitu: Inje( Preve PTU ( The t weigh 29 107% in plasma thyroxine was observed 30 minutes after TRH administration followed by a decline to only 10.8% increase above controls at 2 hours after administration. At 4 and 6 hours after TRH administration, plasma thyroxine concentration was 34 and 41% greater than controls. Thyroid Hormones and Growth Peake et a1. (1973) made rats hypothyroid by including propylthrouracil (PTU) in the drinking water. Within 12 days protein bound iodine (PBI) had decreased from 4.0 to less than 1.6 ug/100 ml. During this same time period pituitary GH content decreased to 75% of con- trol. After 40 days of PTU treatment pituitary GH content was less than 10% of control. When PTU therapy ceased pituitary GH concentration returned to pretreatment levels. Injection of thyroxine thrice weekly into PTU treated rats prevented the decline in GH. Rats rendered hypothyroid by PTU grew lewer than normal controls. Armstrong and Hansel (1956) slaughtered heifers at l, 16, 32, 48, 64, and 80 weeks of age and observed greater pituitary TSH activity in younger animals than in older animals. Mixner et a1. (1966) using cattle noted a decline with age in PBI, thyroxine turnover, and thyroid secretion rate (TSR) when expressed per 100 kg body weight. The total TSR increased with age which coincided with an increase in body weight. For each 1% increase in body weight, there was a 0.60% increase in TSR. omge Youse et a1 Thoma reduc l g t resul becom in se Press 30 Administration of exogenous thyroxine increases oxygen consumption (Mukherjee and Mitchell, 1951; and Yousef and Johnson, 1966) and milk production (Thomas et al., 1954; Moore, 1958; and Yousef and Johnson, 1966). Thomas (unpublished observations) noted a 0.33 kg per day reduction in gain in 6 to 10 months old calves when fed 1 g thyroprotein per day. The exact mechanism of increased milk production resultant from therprotein feeding in unknown. Animals become hyperthyroid and may exhibit a 2-3 fold increase in serum thyroxine concentration (Shaw et al., 1975 in press). No change in the concentration of serum prolactin, GH, or total corticoids were noted during 35 days of therprotein feeding. There was an increase in excretion of endogenous nitrogen and creatinine nitrogen during thyroprotein feeding. Maracek and Feldman (1973) have observed an increase in glucose utilization and decreased insulin half-life in hyperthyroid rabbits. Blaxter et a1. (1949) and later Tucker and Reece (1961) suggested thyroid secretion to be the limiting factor in milk production but no concrete evidence presently exists for this idea. Stimulation of milk production by supplemental thyroxine administration ceases after 3-8 months of continued thyroxine administration (Thomas et al., 1974). mild the l Mart: (Will hypo Dail weig et a COUC CODS COWS et a (Vin imp} GH I an j intc trat et a BOWE 31 Control of Growth Hormone Release The release of growth hormone can be illicited by mild electrical stimulation of the ventromedial nucleus of the hypothalamus (Kokka et al., 1972; Martin, 1972; and Martin, 1974). A GH-releasing hormone has been prOposed (Wilber and Porter, 1970). Brazeau et a1. (1973) isolated a peptide from ovine hypothalami that inhibited GH release, named somatostatin. Daily injections of the synthetic peptide reduced body weight gain in 180 gm rats over an 8 day period (Brazeau, et al., 1974). Conflicting evidence appears in the literature concerning TRH-induced GH release. TRH has been shown to consistently increase serum GH concentration in lactating cows (Convey et al., 1973b) ovariectomized heifers (Smith et al., 1974; Smith, 1974) and 4 to 5 month old heifers (Vines et al., 1974). Smith (1974) reported estradiol implants to sensitize overiectomized heifers to TRH induced GH release. Davis and Borger (1972) failed to observe an increase in serum GH concentration after TRH injections into lambs. Administration of TRH increased serum GH concen- trations in acromegalics (Saito et al., 1971; and Schalch et al., 1972) and normal humans (Fleischer et al., 1970; Bowers et al., 1971; and Torjesen et al., 1973). However, Andei obsei dent] 22-24 mente measr 35° t highe in ci tempe grain serum day, in sh as gr twei Or 14 Ship 32 Anderson et a1. (1971) and Saito et a1. (1971) failed to observe an increase in GB after TRH in normal humans. Yousef et al. (1969) and Trenkle (1971b) indepen- dently estimated the plasma half-life of GH in cattle to be 22-24 minutes. Mitra et a1. (1972) found elevated environ— mental temperature prolonged GH half-life in cattle. Their measured half-life was 26 minutes at 18° and 39 minutes at 35° but GH secretion rate was decreased by 43% at the higher temperature. Their data suggest a slight decrease in circulating GH concentration at higher environmental temperatures. Purchas et a1. (1971) reported heifers fed 0.9 kg grain per day between 4 and 10 months of age had higher serum GH concentration than heifers fed 4.5 kg grain per day. Trenkle (l970a) found plasma GH concentration higher in sheep fed hay than in sheep fed 30 or 70% of their diet as grain. Trenkle (1970b) reported plasma GH concentration remained unchanged in cattle fed finishing rations for 80 or 142 days. These results indicate a negative relation- ship between circulating GH and dietary energy content. Sinha et a1. (1973) adjusted litter size to either 4 or 16 rat pups at birth. At 21 days of age pups from the smaller litter were larger, had 50% larger pituitaries, and 3 times higher pituitary GH concentration than pups from the larger litter. Pups from the smaller litter had an increased rate of incorporation of 3H-leucine into GH. When pups from the larger litter were weaned and allow feren GH co intak GH co weigh purif GH fc contr amoun restr GH Cc maras exper from ShEeP Serum rumin 9I0wt 33 allowed 2 weeks of free access to food, then the dif- ferences in body weight, pituitary size, and pituitary GH concentration disappeared. Maternal restriction of feed intake (to 50% of control) resulted in decreased pituitary GH content in newborn rat pups (Stephan et al., 1971). Machlin (1972) restricted feed intake in 8 pigs weighing 90 kg and injected 4 pigs with porcine GH purified to have no lipolytic activity. Pigs receiving GH for 21 days gained 10 kg more weight than restricted controls receiving saline with both groups consuming equal amounts of dry matter. The author suggested caloric restriction inhibited endogenous GH production. Plasma GH concentration is elevated in kwashiorkor but not in marasmus (Raghuramulu and Jayo Rao, 1974). These results with pigs plus the results from rat experiments previously mentioned are at odds with results from cattle (Trenkle, 1970a; and Purchas et al., 1971) and sheep (Trenkle, 1971a) with respect to caloric intake and serum GH level. This difference between monogastric and ruminant species may be responsible for the favorable growth response seen in ruminants to diethylstilbestrol. Metabolic Actions of Growth Hormone Houssay and Biasotti (1931) observed a lessening of the diabetic symptoms in depancreatized dogs when these dogs were hypophysectomized. They postulated a substance 34 was released from the pituitary which antagonized insulin action. Since that time numerous workers have shown GH to be both glycogenolytic and lipolytic. Davis et a1. (l970a) observed an increase in plasma glucose after daily injec— tions of GH. Radloff and Schultz (1966) administered GH to goats and observed an increase in blood glucose. Rathgeb et a1. (1970) noted a hyperglycemic state in chronic GH-treated dogs. Head et a1. (1970) and Manns and Boda (1965) have administered GH to calves and sheep and failed to observe an increase in plasma glucose concen- tration. The reason for the difference in response is not apparent. Goodman (1965) administered GH to hypophysectomized rats and observed a two-fold increase in glucose uptake, CO2 production from glucose and conversion of glucose carbon into fatty acids by adipose tissue 30 minutes after GH administration. At 60 minutes after GH administration glucose conversion to fatty acids was not different from controls while uptake and transport were still increased. However, glucose uptake, CO production from glucose, and 2 glucose conversion to fatty acids were all decreased 3.5 hours after GH administration. Goodman (1968) classified the early and late actions of GH on adipose tissue as being insulin-like and anti- insulin—like, respectively. The early effects include increased glucose transport, CO production, and glucose 2 conversion to glycogen and occur within one hour after GH util and chai celi Kos1 inci hyp< this the musc The: of,, hch sugg neeE and anin 35 administration. The late effects include reduced glucose utilization, decreased glucose conversion to fatty acids, and fatty acid release from adipose tissue and these changes occur more than two hours after GH administration. Many early workers observed a growth increase in hypophysectomized animals when given an injection of pituitary extract. One of the major changes that OCCurred when the extract was given was an improved nitrogen balance. This led to the hypothesis that a material in the extract improved nitrogen or protein utilization. In hyp0physectomized animals there is a decrease in cellular protein and RNA content and amino acid transport. Kostyo (1964) and Rillema and Kostyo (1971) have observed increased amino acid accumulation in diaphragm muscle from hypophysectomized rats when treated with GH. However, this effect on amino acid transport is not necessary for the stimulation of protein synthesis by GH. Beach and Kostyo (1968) found an increase in total muscle DNA in hypophysectomized rats given GH injections. There was no differences in concentration of DNA per mg of wet muscle between hypophysectomized controls and hypOphysectomized rats given GH. Cheek and Hill (1970) suggested GH administration to hypophysectomized rats was necessary for normal muscle nuclear proliferation. Jasper and Brasel (1973) found GH treatment of hyp0physectomized animals increased liver DNA polymerase activity. 36 Kostyo (1966) failed to observe an increase in the incorporation of uridine into RNA from diaphragms of GH treated hypophysectomized rats. Jefferson and Korner (1967) found an increased incorporation of orotic acid into liver nucleic acids during perfusion with GH. Cheek and Hill (1970) found an increase in the amount of RNA in muscle after GH treatment of hypophysectomized rats. Kostyo and Rillema (1971) suggested the simulatory effect of GH on protein synthesis was related to the ability of ribosomes to promote peptide bond formation. This sug- gestion is in agreement with the earlier finding of Florini and Breuer (1966) in which a single injection of GH into hypophysectomized rats increased ribosomal activity before a stimulation in RNA polymerase activity was observed. Regardless of the mechanisms of GH (and certainly more than one is involved) the stimulation of amino acid incorporation into protein by GH has been confirmed by many studies (Kostyo, 1964; Jefferson and Korner, 1967; Kostyo, 1968; Reeds et al., 1971; Rillema and Kostyo, 1971; and Jaspar and Brasel, 1973). Kostyo (1968) suggested the early action of GH on protein synthesis involved the synthesis of a few specific proteins which caused the later stimulation of protein synthesis observed after GH treatment in hypophysectomized animals. 37 Control of Insulin Release The release of insulin by pancreatic beta cells is influenced by neural inputs, plasma metabolites, and hormones. This section will discuss these factors as they related to insulin release. The islets of Langerhans are innervated by sympa- thetic and parasympathetic nerve fibers (Honjin, 1956; and Esterhuizen et al., 1968). Insulin secretion is increased by cholinergic agents (Malaisse et al., 1967) and blocked by atrOpine (Porte et al., 1973) a drug which blocks cholinergic receptors. Adrenergic agents have mixed effects due to differential receptor types. Drugs that stimulate alpha adrenergic receptors inhibit insulin release while insulin release is increased by beta- adrenergic drugs (Porte et al., 1966; Burr et al., 1971; and Frohman et al., 1967). The destruction of the ventromedial nucleus (VMN) of the hypothalamus leads to hyperphagia and obesity in rats (Cox et al., 1968; Goldman et al., 1970; and York and Bray, 1972) and ruminants (Baile and Mayer, 1969). This condition is associated with hyperinsulinemia (Frohman and Bernardis, 1968; Frohman et al., 1969). Several lines of evidence indicate that the hyperinsulinemia is a cause of the hyperphagia and obesity. Animals made obese and hyperphagic by insulin treatment fail to gain further weight with VMN lesion (Hoebel and Teitelbaum, 1966). Pancreatic beta-cell destruction prevents VMN lesioned animals from 38 becoming obese unless excess insulin is administered (York and Bray, 1972). Finally, after obesity developed in VMN lesioned animals a subdiaphragmatic vagotomy reversed the obesity and hyperphagia and produced normal sized rats once again (Powley and Opsahl, 1974). That the central nervous system (CNS) is involved in insulin secretion was further substantiated recently (Curry and Joy, 1974). These workers were able to measure an increase in insulin secretion by electrical stimulation of the VMN during glucose infusion into the pancreas. When VMN stimulation ceased, insulin secretion returned to a rate similar to that in animals receiving only glucose. Manns and Boda (1965) injected either prolactin or GH into male castrate sheep and failed to observe an increase in plasma insulin levels. Similarly, Davis et al. (1970a) failed to observe an increase in plasma insulin con— centration with the injection of ovine growth hormone. It would appear that acute injections of either GH or prolactin fail to elevate plasma insulin in ruminants. This lack of response to exogenous hormones does not however rule out the possibility that prolonged endogenous changes in cir— culating hormones have a direct effect on insulin release or synthesis. Curry and Bennett (1973) reported the diphasic pattern of insulin release following glucose perfusion of the pancreas was reduced 70% and 35%, for the first and second phase, respectively by hypOphysectomy. SEC SOB": inh Pam Stin Stin HOri 39 Administration of ACTH and hydrocortisone for 7 days before perfusion restored both phases to normal while GH admini- stration restored only the second phase. Rathgeb et a1. (1970) administered GH to dogs and observed no change in plasma insulin concentration. Continued daily GH administration produced an increase in plasma insulin concentration with dogs exhibiting many diabetic symptoms. Young (1963) in similar experiments reported increased insulin requirement of dogs made diabetic by GH treatment. Koerker et a1. (1974) have reported insulin secretion to be decreased during intravenous infusion of somatostatin to baboons. These and other workers (Curry et al., 1974; Devane et al., 1974) have proposed a direct inhibition by somatostatin on insulin release from the pancreas. The best known effector of insulin release is undoubtedly glucose. Ingestion of a high carbohydrate meal gives rise to insulin postprandial (Manns and Boda, 1967; and Trenkle, 1970a). Intravenous infusion of glucose or perfusion of the pancreas with glucose results in a rise of circulating insulin (Trenkle, 1970a, Stern et al., 1971; and Feldman and Jackson, 1974). The volatile fatty acids propionate and butyrate stimulate insulin release while acetate has little stimulatory effect in ruminants (Manns and Boda, 1967; Horino et al., 1968; and Trenkle, 1970a). Horino et a1. shee Plas with inte Corr plas accc Wit} and but} phys inc: 197: eff, Phe] TYr< his] et 90a- 40 (1968) found VFAs to have no stimulatory effect on insulin. release in non-ruminant species. Trenkle (1970a) con- cluded VFAs especially propionate and butyrate when infused intravenously stimulate insulin release and that diets affect the concentration of these fatty acids and in this manner diet affects insulin release by altering the pattern of VFAs produced in the rumen. Bassett et a1. (1971) fed sheep diets differing both quantitatively and qualitatively. Plasma insulin concentration was most highly correlated with digestible organic matter intake (r = 0.74) and intestinal non-ammonia nitrogen digested (r = 0.74). Correlation between individual or total VFA production to plasma insulin concentration was less (0.40-0.51). Acetate accounts for 95% of peripheral plasma VFA concentration with propionate and butyrate removed by the liver (Bergmann and Wolff, 1971). Thus, the significance of propionate and butyrate as stimulators of insulin release under normal physiological conditions remains speculative. Intravenous infusion of amino acids to ruminants increase serum insulin (Stern et al., 1971; and Davis, 1972), or with other species arginine is the most effective amino acid in causing release while leucine and phenylalanine also stimulate release (Davis, 1972). Tyrosine, valine, isoleucine, and phenylalanine were most highly correlated to plasma insulin concentration. Stern et a1. (1971) reported a higher serum insulin for mature goats than weanling or suckling goats. Young et a1. (1970) 41 failed to observe a statistical increase in insulin in calves from birth to 105 days of age but reported highly variable plasma insulin concentrations for the calves. Metabolic Actions of Insulin Even more so than with GH, insulin exerts a powerful influence on the intermediary metabolism of carbohydrates, fats, protein, and amino acids. The action of insulin on carbohydrate metabolism is probably foremost when one discusses insulin action. In diabetic animals insulin increases metabolism of carbohydrates, lipids, protein, and amino acids. Volumes have been written concerning insulin action on all aspects of metabolism. For a more detailed analysis of the specific metabolic action of insulin reviews covering that aspect will be cited for the reader. The administration of exogenous insulin has been shown to lower blood glucose in a variety of species. Resultant to this decrease in blood glucose is an increase in glucose transport into a variety of tissues (muscle: Morgan et al., 1965; adipose: Rodbell et al., 1968; and liver: Fritz, 1972), glycogen synthesis from glucose (Whelan and Cameron, 1964; and Larner et al., 1968) and inhibition of gluconeogenesis (Jefferson et al., 1968; and Hanson et al., 1975). has acid trar late and pres bee] in 1 and (Je lat pat DNA stu to Of the an let lat dat COI‘ SYr 42 Protein Metabolism Insulin lowers circulating plasma amino acids and has more of an effect on essential than nonessential amino acids (Luck et al., 1928; and Cahill et al., 1972). The transport of many amino acids has been shown to be stimu- lated by insulin (Kipnis and Noall, 1958; and Manchester and Young, 1958). Urea production is reduced in the presence of insulin (Exton et al., 1971) and insulin has been demonstrated to increase the rate of protein synthesis in muscle (Manchester, 1970; and Morgan et al., 1971) and also to decrease protein degradation in muscle (Jefferson et al., 1974). v The mechanism whereby protein synthesis is stimu- lated is not clear but a number of steps in the synthetic pathway are stimulated by insulin. RNA synthesis but not DNA synthesis is increased by insulin. In perfusion studies with muscle, Morgan et a1. (1971) found insulin to increase the level of polysomes and decrease the level of monosomes suggesting insulin involvement in peptide chain initiation. Davey and Manchester (1969) observed an increased activation of the amino acyl t-RNA for leucine and tyrosine suggesting a role for insulin regu- lation of protein synthesis by peptide elongation. These data provide evidence that the action of insulin in controlling protein synthesis is not confined to one particular step but acts at each step along the protein synthetic pathway to make protein synthesis more efficient. Anim stud; orig Thes desi gene Pure Thes aCCQ Shav dail COnt Tab] tent 0n 6 III. MATERIALS AND METHODS Design of Experiment Animals A total of 39 Holstein calves were used in this study. Nine intact male calves and nine female calves originated from the Michigan State University dairy herd. These calves were assigned to a randomized complete block design (Sokal and Rohlf, 1969) according to sex and genetic history. The remaining 21 intact male calves were purchased at one week of age from a nearby dairy herd. These calves were assigned to treatment at 45 days of age according to their body weight at that time. Calves were housed individually in pens with wood shavings for bedding. All calves were fed whole milk 2X daily at 0800 and 1600 hours to 30 days of age with free access to alfalfa hay, calf starter, and water. At 30 days of age the evening feeding was dis- continued and the calves offered the ration shown in Table 2. This ration had a calculated crude protein con- tent of 15.4%, 73% TDN, and 3250 Kcal/kg digestible energy on a dry matter basis. The ration was fed as a complete 43 TA 1 Cor Oat SO} Cor Biol 44 TABLE 2.--Composition of ration fed to calves from 30 to 135 days of age. Ingredients Corn Oats Soybean meal Corn cobs Molasses Alfalfa meal T.M. salt Dicalcium phosphate CaCO 3 K2504 MgO Vit. ADEa 30 to 135 39 10 15 25 +++ aVit. ADE to provide: A: D: E: 11,000 I.U./kg of ration 1,100 I.U./kg of ration 137 I.U./kg of ration mixe The give IRON ints weis eve] giv: 135 Fla: 9ra. vat day tio Cal fee to 45 mixed ration with corn cobs as the roughage source to give a more uniform ration throughout the experimental period. The ration was fed once daily at 1300 hours at a rate to give about 10% feed refusal. After 3 to 4 days the morning milk feeding was discontinued. All calves were weighed at 30 days and at 15 day intervals thereafter to 135 days of age. Feed given was weighed daily and that not eaten was weighed and discarded every third day. Consumption was summed by 15 day periods giving weight gain and feed intake data for 7 periods. At 135 days of age, calves were removed from individual pens, placed in a common pen, and fed hay and any available grain. Thirty days later, calves were weighed for obser- vation of residual effects of treatments. Treatments Beginning at 45 days of age and continuing to 135 days of age, calves received a daily intramuscular injec- tion of either of the following solutions (5 ml): (a) thyrotropin-releasing hormone (TRH) 500 ug (b) 3—N-methy1 histidyl-TRH (3MET) 500 ug (c) Saline (0.9 g/100 ml) Calves were injected within 30 minutes of their once daily feeding using a 5 m1 syringe and a 20 gauge needle. Sufficient TRH, 3 MET, and saline were prepared to give enough volume to inject for one week. TRH and CO re ju da an tC Tu le wa to Wi sa 30 an Ca 0b 6p] im 46 3 MET were dissolved in isotonic saline to give a final concentration of 100 ug/ml and stored at 4°C. Collection of Samples 11.929. To determine the pattern of hormone release as a result of injections, blood was obtained by indwelling jugular cannula implanted the evening before the following day's collection. The jugular area of the neck was shaved and washed with 70% ethanol. A 10 gauge needle was used to puncture the vein and a sterile 35 cm cannula (Vinyl IV Tubing, Clay Adams, Inc., New York) inserted into the vein leaving about 10 cm to the exterior. The exposed cannula was flushed with 3.5% sodium citrate, sealed, and affixed to the neck area with tag cement (Nasco, Fort Atkinson, Wisconsin) on 6.6 x 13.2 cm adhesive tape. 0n the day of collection of sequential blood samples, the following regime was used: (1) approximately 30 minutes prior to injection about 5 m1 of fluid (citrate and blood) were removed and discarded to insure proper cannula function. (2) The cannula was resealed with 3.5% sodium citrate until the following sample was obtained. (3) At time intervals indicated in Table 3 approximately 10 ml of blood were withdrawn and dispensed into 15 x 85 mm glass test tubes. (4) Step 2 was repeated. Blood was stored overnight at 4°C and sera obtained by centrifugation at 2500 x g for 30 minutes. Plasma for TABL Horn Groe ho: Thy] Insx age tlms the was sod Wit 47 TABLE 3.--Bleeding schedule and samples used for hormone analysisl. Time relative to injection2 Hormone -10 0 5 10 15 30 45 60 90 120 240 480 Growth hormone X X X X X X X X X X X X Prolactin X X X X X X Thyroxine X X X X Insulin X X X X X X lCalves were bled at 45, 60, 105, and 135 days of age and hormone concentration determined at the designated time (X) for all ages. 2Calves received an IM injection at 0. Remaining times are minutes relative to injection. the determination of glucose and nonesterified fatty acids was obtained by mixing blood with potassium oxalate and sodium fluoride, chilling immediately, and centrifuging within one hour at 2000 x g for 30 minutes. Male calves were bled at 45, 60, 105, and 135 days of age. Female calves were bled at 60 days of age only. Plasma was obtained at 60, 105, and 135 days of age. Serum and plasma were stored at -20°C in 7 dram vials until analyzed. Nitrogen Balance To determine the effects of treatments on nitrogen retention, 5 calves per treatment were placed in metabolism 48 cages at 90 days of age. After a 3 day adjustment period urine and feces were collected for 7 days. Urine was collected in bottles containing 25 m1 of 50% H2804. Daily 10% aliquots of urine were pooled and stored at 4°C until analyzed. All feces voided were collected daily and stored at 4°C. At the end of the 7 day collection period feces were weighed and a sample obtained for the deter- mination of dry matter, nitrogen, and ash. Calves were weighed at the end of the 7 day collection period and returned to their pens. Each calf was offered an amount of feed that approximated 90% of their ad libitum intake during the previous week. After 3 days feed intake was adjusted and maintained at that level for the remaining 7 day collec- tion. Feed refusal was weighed and a sample obtained for dry matter, nitrogen, and ash determination. A11 feed was weighed prior to the collection period and stored at 4°C. Samples were obtained for dry matter, nitrogen, and ash. Tissues A total of 9 calves (3/treatment) was designated for slaughter at the end of the trial. Because of a prior commitment, 3 calves (one/treatment) were slaughtered at approximately 105 days of age and the remaining animals were slaughtered at 135 days of age. Animals were trans- ported by truck to the abattoir on the morning of slaughter. 49 Calves were stunned with a blow to the head, raised and exsanguinated. The abdomen was opened and the internal organs removed. The liver was removed immediately, weighed, samples taken from different lobes and frozen in liquid nitrogen. Other tissues removed included the pituitary, adrenals, testis, thyroid, pancreas, and gastrocnemius muscle. All tissues were weighed, frozen in liquid nitrogen, and stored at -20°C until analyzed. Laboratory Analysis Plasma Glucose Glucose in plasma was assayed using glucose oxidase (Worthington). A plasma volume of 0.5 ml was deproteinized by addition of 1.0 ml of 1.8% Ba(OH)2 (w/v) and 1.0 of 2.0% ZnSO4 (w/v). After centrifugation at 2000 x g for 30 minutes duplicate 0.5 m1 aliquots of the supernatant were incubated at 37°C for 45 minutes with 3.0 ml of glucose oxidase. At the end of the incubation period, 1 ml of 0.1 N HCl was added to stOp the reaction. Optical density was obtained for all at 400 nm using a Gilford spectrophotometer fitted with a flow through cell. Water was used as a blank. Nonesterified Fatty Acids (NEFA) NEFA were determined using the method of Ho (1970) as modified by Bieber (1974). NEFA were extracted from 50 0.2 m1 plasma with 1.0 m1 of Dole extraction mixture (isopropanol:heptane:lNHZSO4, 40:10:l) (Dole, 1956). Samples were vortexed and placed in ice. After 10 minutes of standing, 0.2 m1 of heptane and 0.2 m1 H20 were added, vortexed, and placed in ice to allow the separation of organic and aqueous phases. An aliquot of the heptane layer (0.2 ml) was removed and placed in a 1.5 ml poly- propylene centrifuge tube with an attached cap (Brinkman Instruments, Inc., Westbury, N.Y.). Chloroform (0.8 ml) was added to each tube, the tube capped and placed on ice. Nickel reagent was made by dissolving 0.4050 g of NiC12‘6H2) in 100 m1 of 1 M triethanolamine giving a final Ni concentration of 1 mg/ml of reagent. Radioactive 63Ni (1.17mCi/100ugNi; Amersham Searle Corp., Arlington Heights, IL) was added to give approximately 106 cpm per 0.1 m1 of Ni reagent. Nickel reagent (0.1 ml) was added to each tube, vortexed vigorously for 45 seconds, and placed in an ice bath. The organic and aqueous phases were separated by centrifugation at 500 x g for 5 minutes. The aqueous phase was removed by aspiration and a 015 m1 aliquot of the organic phase transferred to a scintillation vial and evaporated to dryness. Ten ml of scintillation fluid 63Ni counted in a Nuclear (Appendix Table l) were added and Chicago liquid scintillation counter. Palmitic acid was used as standard for calculation of unknown NEFA concentration. The concentration of NEFA 51 in plasma was obtained from an equation derived by the regression of palmitic acid (Y) in concentrations from 0 to 100 nmoles on counts corrected for blank activity (x). Standards were carried through all the manipulations as for serum. Hormone Determinations Growth Hormone Serum samples described in Table 3 were thawed overnight at 4°C. Dilution of the sample in phosphate- buffered saline, 1% bovine serum albumin (PBS-BSA), pH 7.4 was accomplished using an automatic pipette (Micromedic Systems Inc., Philadelphia, PA). Because of different concentrations of hormone in serum samples, a range of dilutions (10 ul-250 ul) was used with 500 ul final volume of the serumrPBS-BSA mixture. Composition of all reagents used in radioimmunoassays is shown in Appendix Table 2. Growth hormone (GH) was quantitated using the double antibody radioimmunoassay of Purchas et a1. (1970). After the appropriate dilution (lo-250 ml) was obtained, 200 ul of guinea pig antibovine growth hormone serum (GPABGH) was added to each tube (except background and total count tubes). After a 24 hour incubation at 4°C, 100—200 ul (depending on specific activity) of 125I—GH was added to all tubes. Following a second 24 hour incubation at 4°C, 200 ul of sheep antiguinea pig gamma globulin 52 (SAGPGG) was added to all tubes (except total counts). A11 tubes were incubated for an additional 72 hours. At the end of the 72 hour incubation, 2 ml of 0.01 M PBS, pH 7.0 was added to each tube (except total counts) followed by centrifugation at 2500 x g for 30 minutes. The liquid was decanted, tubes inverted to drain for 30 minutes or longer, dried and the precipitate counted in a Nuclear-Chicago Model 4230 autogamma scintil- lation counter. A standard curve was constructed for each assay with NIH-GH-B 2 ranging from 0.1 to 5.0 ng/tube as a standard. Regression coefficients for the standard curve were calculated on the C.D.C. 3600 or 6500 and entered into an Olivetti calculator (Programma 101, Olivetti Underwood New York, NY) which corrected for dilution and each serum GH concentration was then calculated. Prolactin Prolactin was determined in serum samples described in Table 3 (KOprowski and Tucker, 1971). Sera (10 to 100 ul) was added to a tube, diluted to 500 ul with PBS-BSA, then 100 ul of guinea pig antibovine prolactin serum (1:30,000) added to all tubes (except background and total count tubes) followed by mixing and incubating 24 hours at 4°C. Following the initial incubation, 100 ul 125 of I-prolactin (20,000 cpm) was added to all tubes, mixed and incubated for an additional 24 hour period. Fol mi) prc sta NI} Ins Ta} M1 20( (G) grs 24 of Sea of f0: SA< mi: ha} Cu: 53 Following this incubation, 100 ul of SAGPGG was added, mixed and incubated for 72 hours. The remainder of the procedure was similar to that for GH determination. A standard curve was constructed for each assay with NIH-P-l as a standard. Insulin Insulin was obtained in serum samples described in Table 3 using the method described by Grigsby (1973). After dilution of serum samples (150-250 ul) to 500 ul, 200 ul with buffer of guinea pig antibovine insulin (GPABI, l:105,000) was added to each tube (except back- ground and total count tubes), mixed and incubated for 24 hours at 4°C. Following the initial incubation 100 ul of 125 I-insulin (approximately 16,000 cpm, Amersham Searle Co., Arlington Heights, IL) with a specific activity of 50 uCi/ug were added to all tubes, mixed, and incubated for 24 hours at 4°C. After this incubation 200 ul of SAGPGG were added to all tubes (except total counts), mixed and incubated for 96 hours at 4°C. Subsequent handling was similar to that for GH assay. A standard curve was constructed using highly purified bovine insulin (Eli Lilly and Co., Indianapolis, IN, lot 795372, 24.2 units per mg) at a concentration ranging from 0.04 to 5.0 ng of insulin per tube. Calcx Curve toa tube para whic coef ente time wit} line eXp; 011 New ho: USE ta; Ins 54 Calculation of Standard Curve and Results The autogamma counter was electronically connected to a teletype (Teletype Corp., Skokie, IL) which printed tube number and counting time. In addition these two parameters were simultaneously punched onto a paper tape which was used later to calculate results. Regression coefficients were generated by computer (C.D.C. 6500) by entering hormone concentration and corresponding counting time. Coefficients generated described a sigmoid curve with a negative slope and included cubic, quadratic, and linear terms. The proportion of within assay variation explained by the coefficients exceeded 0.98 in all assays. Regression coefficients were entered into an Olivetti calculator (Programma 101, Olivetti Underwood, New York, NY) which corrected for dilution and calculated hormone concentration of unknown sera. The paper tape was used to enter tube number and counting time via a punch tape editor (Beckman Model 6912 Tape Editor, Beckman Instruments, Inc., Fullerton, CA). Thyroxine Thyroxine was quantitated using Tetra-Sorb kits donated by Abbott Laboratories, North Chicago, Illinois. A mixture of 1 m1 of plasma and 2 ml of absolute ethanol were vortexed vigorously for 20 seconds, allowed to stand in ice for 10 minutes, and centrifuged at 2000 x g for 15 minutes at 0°C. A 0.3 ml aliquot of the ethanolic 55 supernatant was added to polyprOpylene tubes supplied in the kit and dried at 45-50°C with a continuous flow of air. After the tubes were dried, 1 ml of thyroid binding 1251 was added to each tube, mixed globulin-thyroxine- gently avoiding foaming, and incubated for 12 minutes at room temperature. After this incubation period, tubes were placed in a water bath maintained at 0 to 1°C with ice. After 5 minutes a sponge impregnated with a resin which 125I-thyroxine was added to each tube and binds displaced the liquid absorbed into the sponge. The tubes and its contents were incubated for 60 minutes in the water bath. At the end of the 60 minute incubation period liquid was removed from the tubes and each tube washed three times with cold distilled water. To determine thyroxine concentration random tubes were counted for 1 minute during the 60 minute incubation period to determine initial counts added to tubes. After washing, all tubes were counted for one minute. Remaining 125I-thyroxine from TBG-lzsl- counts represented displaced thyroxine. The ratio of final counts to initial counts gave a percent uptake from the TBG-125 I-thyroxine. Percent uptake was converted into uncorrected thyroxine concentra- tion from the graph of percent uptake vs. thyroxine concentration supplied with each kit. Since ethanol does not extract thyroxine from serum completely efficiency of extraCtion was determined as 56 follows. To 0.2 m1 of Tea-125 I-thyroxine, 1 m1 of serum was added and incubated for 10 minutes at 45°C. Tubes were counted for one minute, 2 ml of absolute ethanol added, mixed for 30 seconds, and allowed to stand for 5 minutes. Following centrifugation, 1 ml of supernatant was added to polypropylene tubes and counted for one minute. Extrac- tion efficiency was calculated as follows: final counts x 3.2 initial counts x 100 Extraction efficiency was 79, 77 and 76% determined on 3 different days. An extraction efficiency of 77% was used. The product of the reciprocal of extraction efficiency and uncorrected thyroxine concentration yielded corrected serum thyroxine concentration. Determination of Tissue Nucleic Acids Liver and muscle were used for the determination of RNA and DNA by methods outlined by Munro and Fleck, (1966). Each tissue was homogenized in cold distilled water in the ratio of 20:1 and 10:1 for liver and muscle, respectively. Four samples representing different sec- tions of liver and two samples from different areas of the gastrocnemious muscle were assayed in duplicate. Two ml of homogenate were dispensed into glass centrifuge tubes and 5 ml of cold 2.5% (w/v) perchloric acid (PCA) added. Tubes were vortexed and placed in an ice at car bro was pel wer. Tube the] The the and The orig PCA. 5.0 aCCc minu incu iCe. minu deCa: WaSh. waSh: brOUc 57 ice bath for 10 minutes, vortexed again, and centrifuged at 35,000 x g for 15 minutes. The supernatant was dis- carded and 5 ml of cold 1% PCA added. The pellet was broken apart, vortexed, and centrifuged. After the second centrifugation, the supernatant was discarded, 4 ml of 0.3 N potassium hydroxide added, pellet broken apart, and tubes incubated at 37°. Tubes were agitated frequently until all tissue was digested. Tubes were removed from a water bath and placed in ice; then 5 ml of 5% cold PCA added, vortexed, and centrifuged. The supernatant was added to 25 m1 graduated tubes and the pellet washed twice with 5 ml of 5% cold PCA, vortexed, and centrifuged at 35,000 x g for 10 minutes each time. The washings were added to the tubes containing the original supernatant and volume brought to 20 ml with 5.0% PCA. This fraction represented RNA. To the pellet remaining after washing, approximately 5.0 ml of 10% PCA was added. The extraction of DNA was accomplished by incubating the pellet at 70°C for 25 minutes shaking at the beginning, middle and end of the incubation period. After incubation, tubes were placed in ice. When cold, centrifugation at 35,000 x g for 15 minutes produced the DNA-containing supernatant. This was decanted into 10 ml calibrated tubes. The pellet was washed with 4.8 ml of cold 10% PCA and centrifuged. The washings were added to the original supernatant and volume brought to 10 ml by addition of 10% PCA. col ali and rea 12. and coc era Spe usi the of Tat Tat PCA ml 0n 30° ten Gil Wit 58 The concentration of RNA was determined by a colorimetric procedure utilizing orcinol. A two m1 aliquot of the 20 m1 RNA solution was added to test tubes and 2 ml of orcinol reagent (Appendix Table 3A) added. A reagent blank of 2.0 ml of 5% PCA and RNA standards of 12.5, 25.0, 37.5, and 50.0 mg per ml were used for deriving a standard curve. Marbles were placed on top of each tube and tubes incubated in a boiling water bath for 30 minutes, cooled in running cold water, allowed to reach room temp- erature, and optical density determined using a Gilford Spectrophotometer at 680 nM. DNA concentrations were determined colorimetrically using diphenylamine and acetaldehyde. A 2 ml aliquot of the 10 m1 DNA solution was added to test tubes and 2.0 m1 of 4.0% diphenylamine in glacial acetic acid (Appendix Table 3B) and 0.1 m1 of acetaldehyde solution (Appendix Table 3C) were added and mixed. A reagent blank of 10% PCA and DNA standards of 12.5, 25.0, 37.5, and 50.0 mg per ml were treated in the same manner. Marbles were placed on top of the tubes and samples incubated for 16 hours at 30° in a water bath. Tubes were removed, cooled to room temperature, and optical density determined at 595 nM in a Gilford Spectrophotometer. Duplicate determinations agreed within 10%. all to of are dii cal low per grc day les tha: dif 9&1: Sal.- IV. RESULTS Weight Gain, Feed Intake and Their Ratio A 15 day pre-treatment period (31-45 days of age) allowed adequate time for calves to be weaned and adjusted to the experimental ration. Weaning was usually accom- plished by 35 days of age and treatment began at 45 days of age. Weight gain, feed intake and feed to gain ratio are shown in Table 4 for each 15 day period. Although differences between groups were not significant (P>0.10) calves assigned to receive saline gained more and had a lower feed to gain ratio during the 15 day adjustment period than calves assigned to TRH or 3 MET treatment groups. During the first 15 day treatment period (46-60 days of age) weight gain of calves injected with 3 MET was less (P<0.01) and feed to gain ratio was higher (P<0.01) than calves receiving saline. Total feed intake was not different (P>0.10) between groups. Differences in weight gain, feed intake, and feed to gain ratio between TRH and saline treated calves were not significant (P>0.10). 59 ~:£.:..F.|F . ’28.»; CCCFZLPOZ 0:.—.r..fl..~_-c~ey.4 Cflinvhbnvifixifmu “who M..ChU.~.~.J..«.fiCN \AerJO thN>HM~UM~N Titu>fi~ohv MOM TCGTNCL «some n; >2 A3\n: Carri Caz? Cu Talon“ mote. .aen: Oxfiu2w Used .333» 2.4!: u23_URII.? ”15¢an 6O .HHmum>o paw mpofluom mchHmEmn ozu Mom mHmEHcm 0 com mxmc moH smsoucu mamsflcm ma co comma Bmzum mom mcmoz N .cmos mo uouuo cumpcmum H .Ho.vm .Houucoo Eoum acouowwfip >HUCMUAMHcmHm mumfluomuomsm ucmquMHp nufl3 cEsHoo oEMm ca mEmuH mfl .mo.vm .Houucoo Eoum ucouowmap waucmoflmflcoflm mumfinomuomsm ucouommwp nuflz cadaoo osmm cw mEouH om Ho.m ov.m ms.v H~.m vo.m aom.~ mm.a o\m o.num.oo o.me.om m.~Hm.Hm s.¢flo.mq m.auo.sm o.dflo.mm H.~Hm.ma foxy Hm m.auo.mfl m.¢flm.oa v.auo.aa mm.oHo.vH o.oflo.ma am.oHH.oa H.4Hs.m foxy oz Ham oa.m vm.m HH.¢ so.m mm.m msa.o om.m o\n m.mum.ao m.nHm.mm m.mua.om o.muo.oq m.~Ha.om m.~Hm.om m.mua.ofi loss Hm o.mum.oa m.&HH.mH m.aflo.mfl ma.oflm.ma H.aHm.mH mo.oum.o o.wuo.s loss oz emzum Hm.m om.m Hm.v mo.m os.m £u Hanuoslm .Ammev ecosuon mafimmoflmn camouuouxgu mo mcofluooncw wafimp mcw>aooou mo>Hmo now mpofluom woo ma >9 A0\mv oflumu cfimm ou comm can .AHmV oxmucfl comm .Aozv camm unmflo3nl.v mamas to di tw ti th 0t 42 th gr tC tr pe m f. 61 Weight gain, feed intake, and feed to gain ratio for the second 15 day period (61-75 days of age) were not different between treatment groups. During this period two calves receiving TRH were sick with respiratory infec— tions and required veterinary attention. On the average these two calves gained no weight during the period while other calves in the TRH group averaged 14.2 kg gain and 42.7 kg feed intake. Deletion of these two calves from the period totals resulted in an average gain which was greater than that of calves receiving saline. This trend toward greater gain in calves given TRH relative to con- trols continued without exception throughout successive periods. During the third 15 day period calves injected with TRH gained more weight (P<0.01) than controls and differences in gain for calves given 3 MET and saline approached significance (P<0.10). However, differences in feed intake and feed to gain ratio between treatments were not significant. During the remaining periods calves given TRH gained more weight, consumed more feed and had a lower feed to gain ratio than calves given saline but in no instance were treatment differences within a period significant. During the last 3 periods (91-135 days of age) period weight gains, feed intake and feed to gain ratios for calves given 3 MET or saline were virtually identical. From 91 to 105 days of age 5 calves from each treatment pei cor to ca] sal peI gi\ flies in the eit Was 3 M the 62 were used for a nitrogen balance trial. Calves used in this balance study gained less weight than calves kept in individual pens for each treatment and average period gain was less than the preceding or subsequent period. Gain was similar between treatment groups during each of the remaining two periods. Overall, performance for the 90 day experimental period is shown in Table 5. Calves receiving TRH gained 7.8 kg more weight (P<0.05) than saline-injected calves, consumed 28.8 kg more feed (P<0.05), but had a higher feed to gain ratio (P>0.10). On a daily basis TRH-injected calves gained 87 grams (0.10 lb.) per day more than did saline-injected calves during the 90 day experimental period. Differences in growth parameters between calves given saline and those given 3 MET were not significant. Analysis of weight gain during the 90 day experi- mental period adjusted by 15 day pretreatment gain resulted in negligible adjustments (less than 1.0 kg) of actual treatment means. The level of significance was similar (f = .05) for treatment effects on weight gain using either 2-way ANOVA or covariance analysis. Serum Hormones Double split-plot analysis of variance (Table 6) was calculated 3 ways as a result of failure to receive 3 MET late in the trial. In the first analysis (Analysis I) the hormonal response of 6 animals from each of 3 treatments 63 TABLE 5.--Total weight gain (WG), feed intake (FI), and feed to gain ratio (F/G) in calves receiving daily injections of thyrotropin releasing hormone (TRH), 3—Methy1 thyrotropin releasing hormone (3MET), or saline (Sal) from day 45 to day 135. wo FI F/G Treatment n kg a l a TRH 13 89.1 13.6 313.1 :12.6 3.51:0.29 3-MET 9 79.3b:5.2 276.8bil4.9 3.49:0.35 Sal 13 81.3bi3.6 284.3b:11.4 3.41:0.40 ab Means not sharing like superscripts significantly different P<.05. 1 Standard error of mean. on days 45, 60, and 105 were used. In the second analysis (Analysis II), 6 animals given TRH or saline at days 45, 60, 105, and 135 were used. Finally, a third analysis (Analysis III) was based on 3 animals from each treatment on all days. Generally the results of each analysis relating significance of main effects (treatment, day and time) and the interaction of main effects were similar for each analysis. Exceptions to this generality were related to a decrease in degrees of freedom for those effects tested. The following discussion will concern itself with Analysis I and II because of the greater number of animals involved. 64 TABLE 6.-—Doub1e split-plot analysis of variance table for testing significance of main effects and their interactions. 1 Treatment (T) T-l l 2 Animals/ Treatment (A/T) A(T-l) 2-1 3 Day (D) D-l 3 4 T X D (T-l) (D-l) 4-3—1 5 A/T X D A(T-l) (D-l) 5-2-4+1 6 Time (M) M-1 6 7 T X M (T-l) (M-l) 7-1-6 8 A/T X M A(T-l) (M-l) 8-2-7+1 9 D X M (D-l) (M-l) 9-3-6 10 T X D X M (T-l) (D—l) (M-l) 10-2-4-9+1+3+6 11 A/T X D X M A(T-l) (D-l) (M-l) Total-Z Adj SS 12 Total ATDM-l Total 65 Growth Hormone (GH) The effect of age and time after feeding on serum GH concentration was determined by split-plot analysis of variance using 6 calves given saline and sampled on each day tested. There appeared to be an increase in serum GH concentration occurring between 60 and 90 minutes after saline injection which persisted through 240 minutes but the increase was not significant at any time. Injection and feeding occurred within 30 minutes of each other. Serum GH at four ages is shown in Table 7. Means are the average of 12 samples per calf for 6 calves each day. At 45 days of age serum GH averaged 17.5 ng/ml during the 8 hour period following injection. Serum GH decreased to an average of 13.2 ng/ml at 60 days of age and this decrease approached significance (P<0.10). The decrease in serum GH occurring at 105 and 135 days of age from 45 days of age was significant (P<0.01). Serum GH concentration of calves given TRH, 3 MET, or saline injections are shown in Figure 2. Serum GH con- centration after TRH was greater (P<0.01) than 3 MET or Sal at 5 minutes after injection and persisted through 30 minutes. Injections of 3 MET resulted in a greater (P<0.05) serum GH concentration than did saline injection at 10 and 15 minutes post-injection. Maximal GH concen- trations were attained between 10 and 15 minutes after TRH and 3 MET injection. But at 30 and 45 minutes after 3 MET and TRH respectively differences in GH concentrations 66 qum .Aammv ocflamm no .Aemzmv ocosuon mcflmmoaou cflmouuouhcu chuoE m .Ammev ocoauoc mcflmmoaouucflmouuoumcu mo mcofluoomcw smasomse Imuucfl Houmm mo>amo ca soflumuucmocoo mGOEuon Luzoum Eamon ommuo>¢ > I .m ousmflm 67 5:83. Lots .55. cow 9%. ON. cm 00 no on m. o. m o d . (.1 q q S‘— _ _ _ . _ _ u \ 4% I Ems. n all. 1m... I 444mm>0 0. ON on O¢ Om (um/bu) H9 wmas 68 TABLE 7.--Serum growth hormones (GH), Prolactin (prl) and insulin (Ins) (ng/ml) at day 45, 60, 105, and 135 in calves receiving saline. Days Hormone n MSE 45 60 105 135 GB 72 17.5a 13.2ab 11.6b 11.3b 113.4 Prl 36 6.6b 7.5b 27.4a --—- 284.1 Ins 36 1.04B 1.03B 1.32B 2.13A 0.69 abP<0.05; ABP<0.01. Means within a row not sharing same superscript significantly different. MSEmeans square error. n = number of samples used for determination of hormone concentration at each day. were not significantly different than those for saline injected calves and this relationship existed to 480 minutes post-injection. Serum GH after TRH, 3 MET, and saline for each sampling date is shown in Figures 3, 4, 5, and 6. These data illustrate the effectiveness of TRH in causing :release of pituitary GH throughout the 90 day experi- mental period. Detectable increases in serum GH concen— tration from pre-injection levels occurred on each sampling date after TRH and 3 MET injections. On all days observed, serum GH concentration after TRH was greater (P<0.05) than pre-injection levels at 10 and 15 minutes post-injection but serum GH concentration after 3 MET was 69 DevxaRu .Aammv ocflamm Ho .Aemzmv oCOEuoc msflmmoaou camouuouhsu stuoa m .Ammfiv mCOEuo: mcflmmoaou camouuouwnu mo coHpoomcH Hmasomse IMHHCH mcflonHom mom mo whom we um coflumnucoocoo oCOEuo: nuzoum Esnom .m ousmflm 4“ IIL 70 Omv c2898. Etc .52 ovm 0N. om cm 0? on m. 0 0.- — - q - q q _ - oucsqm I oucbzm I @uCImF I mv >40 .V . 0. ON On 0? On 00 (um/bu) H9 wmas 71 .Aaomv ocflHMm no .Aemzmv ocoEnoz mcflmmoaou camouuoumcu Hmcuoe m .Ammev ocoauoc mcammoaou Camouuouwnu mo coflpoomcfi “Madonna nmuucw mcfl3oHH0m mom mo wasp om um coflumnucoocoo oCOEuon :u3oum Esmmm .v ousmflm 72 8s. 528.5. Coco .55. O¢N ON. 0m 00 a? On n. O 0... q . _ _ q . a _ _ d L mu: 44m I m2. 52... I .. "C IKE. I OO >40 O. ON Om O¢ (um/bu) H9 wmas 73 .Aammv ocHHMm no .Asmzmv o:o&uoc msflmmoaou Camouuouxnu Hmsuoe m .Ammev oCOEuon mcflmmmawulcfimouuoumnu mo cofluoomcfl smasomsfi Imuucw mcHBOHHow omm mo mmmc moa um coflumnusoocoo oGOEHoz suzouo Esuom .m onsmflm 74 8.8%.. Loco 6.5. Omv OvN ON. 00 00 mg on n. O O... - q - u — - — - if ‘ i||..lli ii I. 0?: 44m I 0»: PM: n 0'6 O_uc IE... I I 00. >40 O. ON On 0? On (um/fin) H9 wmas 75 .Aamm. ocflamm no .Aemzmv occhon mcflmmoaou camouuou>£u Hmcuofi m .Ammev oCOEuoc mcflmmoaou Camoupoumcu mo coHuoonCH Hmasomss Imuucfl msfl3oaaow mom mo wasp mma um coflumuucoocoo OGOEHOS nuBoum Esuom .o ousmflm 76 5.80.... Loco 6.5. 09¢ O¢N ON. GO om m¢ 0n n. O 0.- 1 q d d u q u A q 1 A’v i l bu c 44m I nu: PM: n I was It... I l 09 >43 J L 0. On O? On 00 ON (rm/bu) H9 mules 77 greater (P<0.05) than pre-injection levels at 5 minutes post-injection for only days 60 and 135. Serum GH concentration of calves given TRH was greater (P<0.05) than for those given saline at 15 minutes post-injection at day 45 (Figure 3). A difference in serum GH concentration of at least 20 ng/ml between TRH and saline occurred at 5, 10, 15, and 30 minutes post- injections, but only differences at 15 minutes were significant. The greatest difference in serum GH concen- tration after giving 3 MET or saline was 16.9 ng/ml at 15 minutes post—injection but the difference was not significant. At day 60 (Figure 4), serum GH concentration in calves after TRH was greater (P<0.05) than those given saline at 5, 10, 15, 30, and 45 minutes post-injection. Calves injected with 3 MET had lower (P<0.05) serum GH concentration than calves injected with saline at 10 minutes pre-injection. At no times post-injection was serum GH concentration in calves given 3 MET significantly greater than comparable values for calves given saline. Serum GH concentration is shown in Figure 5 for calves at day 105. At 5, 10, and 15 minutes post-injection serum GH concentration of TRH-injected calves exceeded that for saline calves (P<0.05). However, at 60, 90, and 120 minutes post-injection serum GH concentration in saline treated calves exceeded that of TRH and 3 MET-injected calves. Serum GH concentration after 3 MET injection 78 increased only 4.9 ng/ml at 10 minutes post-injection from the level at injection. At day 135 (Figure 6), serum GH concentration was greater (P<0.05) in calves given TRH than those given saline at 10 and 15 minutes post-injection. Serum GH concentration in calves given 3 MET was greater (P<0.05) than those given saline at 5 and 10 minutes post-injection. The difference in magnitude and consistency of increases in serum GH concentration resulting from TRH and 3 MET injections is illustrated further by either the ratio or difference in maximum GH concentration occurring by 30 minutes post-injection when compared to GH concentration at injection (Table 8A). At 45, 60, 105, and 135 days the ratio was 6.3, 6.1, 6.9, and 5.7 respectively for TRH and 2.1, 3.7, 2.6, and 7.3 for 3 MET. At 45, 60, 105, and 135 days of age the difference for TRH was 47.5, 35.8, 38.0, and 35.1 ng/ml but only 20.9, 16.8, 4.3, and 62.4 ng/ml for 3 MET, respectively. The ratio and difference were not significant for TRH treatment between days. The difference was greater (P<0.05) at day 135 than at day 105 while the ratio at day 135 was greater (P<0.05) than day 45 and 105 in calves receiving 3 MET. Ratios and differences for TRH were greater (P<0.05) than corresponding values for calves given 3 MET at day 45, 60, and 105 but not at day 135. The magnitude of increase in GH concentration during the 30 minute period post-injection of TRH and 3 MET 79 TABLE 8A.--Ratios to and differences between serum growth hormone at maximum concentration after injection of thyrotropin releasing hormone (TRH) or 3 methyl thyrotropin releasing hormone (3MET) concentration at injection for calves at day 45, 60, 105, and 135 Days 45 60 105 135 TRH 6.3a 6.1a 6.9a 5.7 Ratio 3MET 2.1Bb 3.7ABb 2.6Bb 7.3A TRH 47.5a 35.8a 38.0a 35.1 Difference (“g/m1) 3MET 20.9ABb 16.8ABb 4.3Ab 62.4A abP<.05. Means for ratio or difference within a day not sharing same superscript significantly different among treatments. ABP<.05. Means within a row significantly dif- ferent between days. 80 for each calf are categorized in Table 8B. At day 45, serum GH in each calf after TRH injections increased by at least 15 ng/ml with 3 of 6 exhibiting an increase serum GH greater than 50 ng/ml. In contrast, serum GH after 3 MET injections increased more than 15 ng/ml in'only 2 of 6 calves. At day 60 TRH caused an increase in serum GH which exceeded 15 ng/ml in 7 of 9 calves while only 4 of 9 calves injected with 3 MET had a comparable increase in serum GH. At day 105, a decrease in number of calves responding with an increase in serum GH concentration to TRH or 3 MET was observed. Thus serum GH increased more than 10 ng/ml in only 5 of 10 calves given TRH while 1 of 6 calves given 3 MET responded with an increase of 10 ng/ml or greater. Of the 5 calves exhibiting an increase greater in serum GH than 10 ng/ml, 4 responded with an increase of 40 ng/ml or greater. At day 135 all 6 calves given TRH responded with an increase in serum GH of 15 ng/ml or greater while the 3 calves given 3 MET responded with an increase of 30 ng/ml or greater. Serum GH at —10 and 0 minutes were averaged to estimate basal concentrations for each treatment at each day and overall (Table 9). Calves injected with TRH had lower nonsignificant GH concentrations than that of saline at each day sampled and the overall mean for TRH was less (P<0.05) than saline. Calves injected with 3 MET had lower (P<0.05) basal GH concentration than saline at 105 days of 81 TABLE 88.--Number of calves at each of 4 ages categorized as to magnitude of increase in serum growth hormone (ng/ml) after injection of thyrotrOpin releasing hormone (TRH) or 3 methyl thyrotrOpin releasing hormone (3MET). Days Increased (ng/ml) above 45 60 105 135 Baseline TRH 3MET TRH 3MET TRH 3MET TRH 3MET <5.0 - 2 2 l 3 4 - - 5.0-10.0 - - - l 2 l - - 10.1-15.0 - 2 - 3 - 1 - - 15.1-20.0 1 — l l - - 2 - 20.1-30.0 - 1 2 2 1 - 1 - 30.1-40.0 2 - - l - - 1 l 40.1-50.0 - - l - 2 - l 1 >50.0 3 1 3 - 2 - l l n 6 6 9 9 10 6 6 3 82 TABLE 9.--Baseline serum growth hormone (GH) (ng/ml) (average of -10 and 0 min) at day 45, 60, 105, and 135 for calves receiving daily injection of thyrotropin releasing hormone (TRH), 3-methy1 thyrotrOpin releasing hormone (3MET), or Saline (Sal). Days 45 60 105 135 Average ----------- GH (ng/ml)----------- TRH 11.5 8.5 8.0AB 7.1 8.6B 3MET 13.9a 7.1ab 4.3Ab 11.6ab 8.7AB Sal 18.9a 14.2ab 9.2Bb 11.3b 12.9A Average 14.8a 9.9ab 7.6b 9.8a ab P<.05. Means within a row not sharing same super— script significantly different. ABP<.05. Means within a column not sharing same superscript significantly different. 83 age. Overall, the magnitude of difference in serum GH concentration between 3 MET and saline treatments was similar to the magnitude of difference in serum GH concen- tration between TRH and saline and approached significance (P<0.10). Thyroxine Linear regression equations combining data from all test days and their graphic presentation for changes in serum thyroxine concentration with time after treatment are shown in Figure 7. Daily injections of TRH or 3 MET but not saline resulted in significant regressions (P<0.0005) indicating serum thyroxine concentration increased after injection of TRH or 3 MET. The y-intercepts for TRH and 3 MET regressions were lower than that of saline with the difference between 3 MET and saline significant (P<.05). The lepe of each regression equation was used as an estimate of potency of treatments. Slopes ranked in order of magnitude (largest to smallest) were 3 MET, TRH, and saline with slopes for calves given 3 MET and TRH each greater (P<.01) than saline injected calves but not dif- ferent from each other (P>.10). Table 10 lists linear regression equations for each treatment at each day. Lines described by these equations and actual treatment means are plotted in Figures 8, 9, 10, and 11. At day 45, injection of 3 MET and TRH resulted in significant regressions for serum Figure 7. 84 Plots of linear regression equations for serum thyroxine concentration combined for all days following intramuscular injection of thyrotrOpin releasing hormone (TRH), 3 methyl thyrotropin releasing hormone (3MET) or saline (Sal). Serum Thyroxine (ng/ml) 85 Overall R2 TRH f 3 MET I SAL Y 77.3 4' 3.60X .22 69.3 + 4.64X .27 = 8|.6 + 0.8l X .0! HO IOO 90 Hrs. after Injection Sign of Regress n <.0005 3| <.OOO5 24 O.I8I 32 - .— 3 MET .— TRH ‘— SAL 86 TABLE 10.--Linear regression equations of serum thyroxine concentration (Y) with time (X) (hrs) after injection of thyrotropin releasing hormone (TRH): 3 methyl thyrotrOpin releasing hormone (3MET), or Saline (Sal) into calves at day 45, 60, and 135. 2 Sign. of Day 45 BL. Regress. TRH Y 72.1 6.25 (X) 37 .002 3-MET Y 47.7 8.65 (X) .66 <.0005 Sal § 73.9 1.29 (X) .03 .423 Day 60 TRH Y 81.5 3.15 (X) .21 .004 3-MET § 84.1 4.41 (x) .26 .002 Sal Y 75.5 0.71 (X) .02 .410 Day 105 TRH Y 64.7 2.81 (X) .33 <.0005 3-MET Y 61.3 1.96 (X) .14 .069 Sal Y 69.8 0.64 (X) .03 .290 Day 135 TRH Y 97.6 2.50 (X) .25 .014 3-MET Y 86.0 2.47 (X) .31 .097 Sal Y 112.4 0.81 (X) .06 .212 Figure 8. 87 Plots of linear regression equations for serum thyroxine concentration in calves 45 days of age following intramuscular injection of thyro- tropin releasing hormone (TRH), 3 methyl thyrotropin releasing hormone (3MET), or saline (Sal). T, M, and 8 symbols on graph represent actual thyroxine means for the three respective treatments. Serum Thyroxine (ng/mI) 88 Sign of DAY 45 F!2 Regress n TRH f: 72.: +6.25 x .37 .002 6 3 MET X= 47.7 + 8.65 X .66 <.OOO5 6 SAL Y = 73.9 + l.29 X .03 .423 6 ISO - M I30 - o—TRH T . .—3 MET IIO - T 90 — " s. S - Ao—SAL s 70 T l 50M L . I . O 2 4 8 Hrs. after Injection 89 Figure 9. Plots of linear regression equations for serum thyroxine concentration in calves 60 days of age following intramuscular injection of thyrotropin releasing hormone (TRH), 3 methyl thyrotropin releasing hormone (3MET), or saline (Sal). T, M, and 8 symbols on graph represent actual thyroxine means for the three respective treatments. Serum Thyroxine (ng/ml) 90 Sign of DAY 60 R2 Regress n TRH 2: 8|.5 + 3.15 x .2: .004 9 3 MET I: 84.I +4.4: x .26 .002 9 SAL Y = 75.5 + 0.7: x .02 .410 9 l20 llO I00 90 Hrs. after Injection Figure 10. 91 Plots of linear regression equations for serum thyroxine concentration in calves 105 days of age following intramuscular injection of thyrotropin releasing hormone (TRH), 3 methyl thyrotropin releasing hormone (3MET), or saline (Sal). T, M, and S symbols represent actual thyroxine means for the three respective treatments. 92 . Sign of DAY l05 R Regress n TRH 9: 64.7 +2.8I x .33 wow IO 3 MET 9: 6|.3+I.96 x .14 .069 6 SAL 9 = 69.8 + 0.64 x .03 .290 I0 Serum Thyroxine (rig/ml) Hrs. after Injection Figure 11. 93 Plots of linear regression equations for serum thyroxine concentration in calves 135 days of age following intramuscular injection of thyro- trOpin releasing hormone (TRH), 3 methyl thyro-l tropin releasing hormone (3MET), or saline (Sal). T, M, and S symbols represent actual thyroxine means for the three respective treatments. Serum Thyroxine (ng/ml) 94 DAY I35 TRH i: 97.6 + 2.50 x 5 MET I: 86.0 +2.47 x SAL Y = ::2.0 +0.8: x IOO 90 sol; _ l l 2 4 Hrs. after Injection Sign of R2 Regress .25 .OI4 .3l .097 .06 .2l2 quail: 95 thyroxine. The y-intercept for 3 MET was significantly less than either saline or TRH initially. This is purely random since calves had received no prior injections and had been assigned to treatments by weight at 45 days of age or by genetic background at birth. Using slopes of regression equations as an estimate of potency, 3 MET treatment was the most potent followed by TRH and saline. Slopes for 3 MET and TRH injections were greater (P<0.05) than saline but not different from each other. Examina- tions of the actual means shown in Figure 8 indicate that the increase in serum thyroxine induced by TRH was faster than that induced by 3 MET but the effect of 3 MET per- sisted longer. In fact, serum thyroxine concentration appears to be increasing even at 8 hours after 3 MET injection. At day 60 (Figure 9), injection of TRH and 3 MET resulted in significant regressions. Comparisons of y-intercepts between treatments at day 60 were not signifi- cant. The y-intercepts were greater for TRH and 3 MET at day 60 than at day 45 but when compared to day 45, only 3 MET was greater (P<.01). The slope of the regression equation describing serum thyroxine response to injection of 3 MET was greater (P<.05) than that for saline but not different from that for TRH. Differences in the lepe for TRH and control regression equations approached significance (P<.08). The patterns of actual means for 3 MET and TRH were similar at each time but always greater for 3 MET. 96 Injection of TRH at day 105 produced a highly sig- nificant regression (P<0.01) while that for 3 MET injection only approached significance (P<0.10) (Figure 10). Again, regression for saline was not significant. The y-intercepts for regression equations for 3 MET and TRH but not saline were lower (P<0.05) than comparable values at 60 days of age. At day 135 injection of TRH resulted in a signifi- cant regression (P<0.01) (Figure 11). Due to small numbers involved, the regression for 3 MET treatment only approached significance (P<0.10) while that for saline was not significant (P<0.10). Serum thyroxine concentration at time zero (y-intercepts) for saline was greater (P<0.01) than that of TRH and 3 MET treatments and the difference between y-intercepts for TRH and 3 MET approached significance (P<0.10). Differences in slopes between the two treatments were not significant (P>0.10). Serum thyroxine concentration for calves given saline at day 135 was greater (P<0.01) than comparable values for all previous days with similar y-intercepts at 45, 60, and 105 days. Differences in y-intercepts on different days followed a similar pattern for both TRH and 3 MET treatments. Intercepts at day 60 were greater than at day 45, but decreased at day 105 and increased at day 135. Saline treated calves had similar slopes of regression equations for each test day which indicates a 97 relatively constant thyroxine output from day 45 to 135. Injection of 3 MET or TRH on day 45 produced a marked stimulation of thyroxine output (indicated by slopes of equations) but this response diminished with repeated injections. Difference in magnitude of slopes between day 45 and 60 was significant for 3 MET (P<0.05).and approached significance for TRH (P = 0.10). By day 105 differences in slopes from day 45 were significant for TRH and 3 MET treatments and remained significantly different from day 45 at day 135. Slopes at days 60, 105, and 135 were not different between days for TRH and 3 MET treatments. The decrease in thyroxine response to TRH and 3 MET occurred early in the repeated injection scheme followed by smaller depressions at later dates. Prolactin Results of split-plot analysis of variance were similar for analysis I and analysis II. Therefore only analysis I which includes all treatments will be discussed. Average serum prolactin concentration increased with age (Table 7). This was determined by using all prolactin values from 6 calves injected with saline at days 45, 60, and 105. Calves at day 105 had greater (P<0.01) serum prolactin concentration than those at 45 or 60 days of age. Time relative to saline injection and day X time interaction did not affect (P>0.10) serum prolactin concentration. 98 Overall changes of serum prolactin concentration smitflh time after injection of TRH, 3 MET, or saline are shown in Figure 12. Injection of TRH resulted in an iaaczrease (P<0.01) in serum prolactin concentration above cxbrrtrols at 15, 30, 45, and 60 minutes post-injection. Prolactin (ng/ml) in calves injected with TRH declined to ‘4IL.3 at 30 minutes and remained within 5 ng/ml of this at 45 and 60 minutes post-injections. Injection of 3 MET lSesulted in an increase (P<0.05) in serum prolactin con- <2entration relative to saline at 15, 30, and 45 minutes Igost-injection while at 60 minutes post-injection the (difference in serum prolactin concentration between calves {given 3 MET and saline approached significance (P<0.07). .At.60 minutes post-injection serum prolactin concentration in TRH-injected calves was greater (P<0.05) than that of <3alves injected with 3 MET. The differences in serum pro- lactin concentration between calves receiving TRH and 3 MET at earlier times were not significant. The response of serum prolactin to each treatment (at.each time on the four test days is shown in Figures 13, 14, 15, and 16. At day 45 (Figure 13) calves receiving YTRH had significantly greater serum prolactin at 15 1minutes (P<0.01) and 30 minutes (P<0.05) than at the time :of injection. Compared to calves given saline, serum prolactin concentration in TRH-treated calves was greater (P<0.05) at each post-injection time. Calves receiving 3 MET had greater (P<0.05) serum prolactin concentration 99 ..Hmm. osflamm Ho .Aamzm. oCOEuo: mcflmmoaou Camouuouhcu Hmsuos m .Amme. oCOEuo: mcflmmoaou camonuouwcu mo cofiuooflcfl Hmasomsfi :muucH OGHSOHHOM mo>Hmo CH mc0wumuucoocoo cfiuomaoum Eduom omnuo>4 .NH mucosa 100 OO 04 c0209.... Etc .55. On 0. O O... 44m .lll hm: n I Imh ell. ......4¢w>O O. ON On O¢ Om (jut/5U) ugioolmd wmas 101 .Aamm. ocflamm no .Aemzm. ocosuon mcflmnoaou camouuouanu Hmsuoa m .Amma. ocOEHoc mcfimmoaou Camouuouxzu mo coauooncw unasomsamuucfl mcwonHow omm mo mzmc mv mo>amo ca cofiuouusoocoo cwuomHoum Eduom .ma musoem 102 c0509.... Etc .55. / 0»... 44m III. on: .55.» I on: I”: I 04 >40 0. , . . . II 5 O Q I O. ON On 04 On (nu/fin) ugioojmd wmas 103 .Aamm. ocflamm no .Aemzm. wooeuoc mcflmmoaou cflmonuonmcu Hmnuofi m .Amme. oCOEuoc mcflmmoaou camouuouacp mo cowuooflca Hoasomsamuucfl mcfl3oHHom mom mo wasp om mo>amo ca coaumuucoocoo cwuomaoum Esuom .ea messes 104 5.8%... Etc .55. 00 no on m. o o... 7 _ _ . _ , /\Ii ‘1‘ O— i ON 1 on one 44m I 1 mac .55. m I O? mu: ImE. I om >amo CH coflumuucoocoo caponaoum Esuom .mH ouooflm 106 8.85.5 Lots .55. OO 05 On 0. O O... .i — — - — ON O¢ 0.23% 1|. one he: I :oo. 0....sz I no. >3 (gm/Em) ugioojmd UJI‘IJGS 107 Serum prolactin concentration in calves 135 days of age following intramuscular injection of thyrotropin releasing hormone (TRH), 3 methyl thyrotrOpin releasing hormone (3MET), or Saline (Sal). Figure 16. Serum Prolactin (ng/ml) 108 DAY :35 :40 - I20 - 0—5 TRH n86 9—0 3METn=3 H SAL n87 IOO 80 60 L l I l J ‘IO 0 IS 30 45 60 Min. after Injection 109 than controls at 30, 45, and 60 minutes post-injection. .Although serum prolactin concentration was increased at each time after 3 MET injection, the concentration at 15 minutes after injection was the only value which differed (P<0.05) from the level at injection. At day 60 TRH injection resulted in an increase (P<0.05) in serum prolactin concentration at 15 and 45 but not at 30 and 60 minutes post-injection when compared to the level at injection (Figure 14). Compared to saline injected calves, serum prolactin concentration in TRH- treated calves was greater (P<0.05) at 15 minutes post- injection only. In contrast, there were no significant differences in serum prolactin concentration between 3 MET and control calves at any post-injection time and although serum prolactin concentration was increased over the pre- injection level, post-injection differences were not significant. At day 105, calves receiving TRH had greater (P<0.05) serum prolactin concentration at all times after :injection than did control calves. Serum prolactin con- <:entration was increased (P<0.05) relative to pre-injection level at all times post-injection in calves receiving TRH. The increase in serum prolactin concentration in calves injected with 3 MET was not significant relative to that of control calves at all times post-injection and serum prolactin concentrations after 3 MET injection were not greater than that at injection. 110 At day 135, serum prolactin concentration at 15 Ininutes after TRH injection was greater (P<0.05) than that (of control calves. At no other times post-injection were there any significant differences between TRH and saline treatments although calves receiving TRH averaged 57.1, 31.0, and 28.0 ng/ml more prolactin in serum collected at 30, 45, and 60 minutes post-injection respectively than did controls (Figure 16). Serum prolactin concentration increased after 3 MET injection but this increase was not significant at any time post-injection. Four of 7 calves receiving saline at day 135 were sampled in mid-July while all calves receiving 3 MET were sampled in late March and early April. Therefore comparison of serum prolactin Jbetween the two treatments is not justifiable due to amo cfl coflumuucmocoo CHHSmGH thmm mmwum>¢ .na musmflm 113 9.6mm... 8:0 .55. 8v 0% om. pm on o — q q ,4 ‘ \ 1 ON._ 1 0i. \\ 1 09. 1 00.. L oo.~ 1 CNN 1 O¢.N (lw/bu) uunsul wmas 114 TABLE 11.--Average serum insulin concentration in calves after injection of thyrotropin releasing hormone (TRH), 3 methyl thyrotropin releasing hormone (3MET), and saline (Sal). 1 Treatment Analysis TRH 3-MET Sal -------------- ng/m12--------------- I 1.51a : 0.12 1.1910 i 0.11 1.14b : 0.06 II 1.82A : 0.12 ----- 1.36B : 0.08 ab P<.05. Means not sharing same superscript within a.row significantly different. ABP<.Ol. Means not sharing same superscript within (a.row significantly different. 1Analysis I. 6 Animals per treatment at day 45, 60, and 105. Analysis II. 6 Animals per treatment at day 45, 60, 105, and 135. 21 ng equals 24.4 uU/ml crystalline bovine insulin. 115 Figure 18. Serum insulin concentration in calves 45 (A) and 60 (B) days of age following intramuscular injection of thyrotropin releasing hormone (TRH), 3 methyl thyrotropin releasing hormone (3MET), or Saline (Sal). Serum Insulin (ng/ml) |.60 l.50 L40 |.30 I20 I. I 0 l.00 0.90 0.80 0.70 116 DAY 45 III w l I l J I 0 30 60 120 240 480 2.20 2.00 l.80 |.60 4 L40 l.20 P - - l.00 0.80 7 DAY 60 0— TRHn=9 o—o 3METn=9 0—0 SALn=9 l l l ' J 0 30 60 l20 240 480 Min. after Injection 117 or treatment were found, but at 105 and 135 days of age responses to treatments were different than those noted at days 45 and 60 and these are shown in Figures 19 and 20, respectively. On day 105 at 60 minutes post-injection, calves injected with 3 MET had higher (P<0.05) serum insulin concentrations than saline-injected calves and the difference in serum insulin concentration between TRH and saline approached significance (P<0.lO). Serum insulin concentration was markedly higher with either TRH or 3 MET injections than with saline during the 4 hour interval following injection. Serum insulin concentration in calves receiving saline was higher (P<0.05) at 480 minutes post-injection than at all previously measured times for calves receiving saline. At day 135 calves injected with TRH had greater (P<0.05) serum insulin concentration at 60 minutes post- injection than calves given 3 MET or saline. Although there was a greater difference in serum insulin concen— tration between calves injected with TRH and those given 3 MET or saline at 120 minutes than at 60 minutes statis- tical evaluation showed these differences non-significant. Remaining Parameters Glucose Average plasma glucose is shown in Table 12 for each of 4 intervals according to treatments and are averaged for days 60, 105, and 135. No significant Figure 19. 118 Serum insulin concentration in calves 105 days of age following intramuscular injection of thyrotrOpin releasing hormone (TRH), 3 methyl thyrotropin releasing hormone (3MET), or saline (Sal). Serum Insulin (ng/ml) 119 DAY l05 3.40 - a 3.20 - 3.00 I- .._. TRHn=IO 2'80 '" o-—o 3MET n=6 2.60 _ H SAL "3'0 2A0. 2.20 _- 2.00 _. \ I.80 . I.60 L40 l.20 - |.00 " Ll—J‘ L 4 1 0 30 60 l20 240 480 Min. after Injection 120 Figure 20. Serum insulin concentration in calves 135 days of age following intramuscular injection of thyrotropin releasing hormone (TRH), 3 methyl thyrotrOpin releasing hormone (3MET), or saline (Sal). Serum Insulin (ng/ml) 121 DAY I35 3.80 I- 340 - 3.00 — 2.60 r- 2.20 l.80 l.40 |.00 I I 1 l I 0 30 E50 120 240 480 Min. ofter Injection 122 TABLE 12.--Average plasma glucose concentration at 0, 2, 4, and 8 hr after injection of thyrotropin releasing hormone (TRH): 3 methyl thyrotropin releasing hormone (3MET), or saline (Sal). Time (Hrs) Treatment 0 2 4 8 1 TRH 52.2 54.5 58.0 60.6 3-MET 50.4 57.1 55.7 58.8 Sal 52.3 58.5 55.8 57.4 1 mg/100 ml differences in plasma glucose due to time, treatment, or day were noted. Plasma glucose concentration was higher at all times after injection than at injection. This increase with time was probably related to calves consuming a large portion of their daily feed soon after injection more than any other factor. Non-esterified Fatty Acids (NEFA) In analysis I, main effects and their interactions were not significant. Analysis II resulted in significant differences for treatment and day. Calves injected with TRH when compared to saline-injected calves had lower (P<0.05) plasma NEFA overall (Table 13). This lower plasma NEFA in TRH-injected calves was due mainly to values obtained on day 135. Plasma NEFA increased with age as 123 TABLE l3.--Non-esterified fatty acids (NEFA) in calves: Age and treatment effects. Age in Days 60 105 135 MSE --------------- ueq per liter----------------- NEFA 168.9b 182.0ab 191.1a 219.6 n = 48 1 Treatment TRH Sal MSE ------------- ueq per liter---------------- b a NEFA 170.6 190.7 226.9 n = 72 abP<.05. Means not sharing same superscript within a row significantly different. lAnalysis II data: 3 methyl thyrotropin releasing hormone not included. MS E mean square error. 124 calves at day 60 had lower (P<0.05) plasma NEFA than comparable values at days 105 and 135 (Table 13). Nitrogen Balance During the 3 day adjustment period feed intake decreased as much as 30% in some calves. The range in dry matter intake for the collection period was 2.43 to 3.71 kg per day with TRH, 3 MET, and Sal averaging 3.40, 2.86, and 2.92 kg intake per day, respectively. No significant differences in any aspect of N-balance were found (Table 14). TABLE l4.--Nitrogen balance in calves receiving daily injections of thyrotropin releasing hormone (TRH), 3 methyl thyrotropin releasing hormone (3MET), or saline (Sal). Treatment TRH 3 MET Sal N-intakel 81.0 66.1 67.5 N-balancel 30.4 20.4 25.6 - N igggigzgfix 100 54.2 45.7 53.4 1 grams per day. Tissue Weights Due to a commitment to provide carcasses for a teaching class on a predetermined date a complete block of calves was slaughtered at 110 days of age. The remaining 125 blocks designated for slaughter were sacrificed at 135 days of age. Table 15 gives the weights of several endocrine glands, liver, and gastrocnemius muscle expressed as total weight and as percent of body weight. With the exception of the adrenal glands all organs in calves injected with TRH were heavier than other treat- ments but these differences were minimized when expressed as a percent of body weight. The exception was the thyroid gland. There was a marked enlargement of the thyroid from calves receiving TRH and differences due to treatments were significant when expressed as total weight (P<0.05) or as a percent of body weight (P = 0.05) (Table 15). Tissue Nucleic Acids Muscle and liver nucleic acids are shown in Table 16. Statistical analysis resulted in no significant differences but trends will be discussed. The concentra- tion of DNA in liver and muscle was greatest for calves receiving Sal and least for TRH-injected calves. The con- centration of RNA in liver was greatest for calves receiving TRH and least in muscle for calves receiving 3 MET. Calves receiving TRH had the highest RNA to DNA ratio in both tissues while calves receiving saline had the lowest ratio. When liver and muscle weights are used to give total nucleic acid content differences became more marked especially the RNA fraction. 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