. H ‘I'VJ - l q'.;'_\}.-) 2-5} 1‘I‘o.-I-._a I II. ._._c._.) . I. , ,I, r, p“ _I‘r_ H .... . "g'fii‘\$fi‘}’.‘.‘ ','.' 3p - ' . . V I“ ‘I ‘A . ’ . ~ ..~ ....JO: my. a: ,. " ‘ - ... ,. . . . ‘ I v . I.. ‘ SERUM PROLACTIN, GROWTH HORMONE AND f GLUCOCORTICOIDS IN HUI-STEIN cows AND THEIR , RELATIONSHIP TO MILK PRODUCTION Thesis for the Degree of Ph. D; ,1 MICHIGAN STATE UNIVERSITY JAMES ANDREW KOPROWSKI 1972 LIBRARY Michigan State ‘tlfliVCC§WY' _!p‘ This is to certify that the thesis entitled Serum Prolactin, Growth Hormone and Glucocorticoids in Holstein Cows and Their Relationships to Milk Production presented by James Andrew Koprowski has been accepted towards fulfillment of the requirements for PhD degreein Dairy Solence A; Mad Mqim professox Date May I9, I972 0-7639 ABSTRACT SERUM PROLACTIN, GROWTH HORMONE AND GLUCOCORTICOIDS IN HOLSTEIN COWS AND THEIR RELATIONSHIP TO MILK PRODUCTION BY James Andrew Koprowski Endocrine changes in heifers and lactating Holstein cows were measured during different lactational and/or reproductive states and were related to milk production. Beginning 4 weeks postpartum, each of 55 cows were bled at 4-week intervals for the duration of lactation or until week 44 which ever occurred first. On a given bleeding date, blood samples were collected via tail vein puncture 2 to 4 hours before, immediately after and 1 hour after milking. To study the effect of the estrous cycle on serum prolactin and growth hormone (GH), 12 cows were bled, three times around milking, on the day of first postpartum estrus, days 2, 4, 7, 9, ll, 15, 18, 20 and again on the day of subsequent estrus. Beginning 30 days after conception 28 heifers were bled once every 30 days until 270 days of gestation to monitor serum hormone content throughout pregnancy without concurrent lactation. Serum prolactin James Andrew Koprowski and GH (ng/ml) were quantified by double antibody radioim- munoassays and glucocorticoids (ng/ml) by a competitive protein binding assay. Serum prolactin increased in response to milking. This response to milking (serum prolactin immediately after milking minus serum prolactin 2-4 hours before milking) was largest at 8 weeks of lactation (77 ng/ml) then gradually decreased as lactation advanced until at 32 weeks prolactin was no longer released in response to milking. Similarly, glucocorticoids were released following milking. Gluco- corticoids before milking averaged 5.7 ng/ml whereas immedi- ately after milking they averaged 8 to 12 ng/ml during the first 32 weeks of lactation then declined to 7 to 9 ng/ml during the remainder of lactation. In contrast, milking did not alter serum GH. However, serum GH content decreased (P<0.0l) as lactation advanced. These data imply that serum prolactin, GH and glucocorticoids may become limiting to milk production during advanced lactation. Overall simple correlations of serum prolactin and milk yield for pre-milking samples were very low (r = -0.03), although similar correlations for samples collected immedi- ately after and 1 hour after milking (r = 0.36 and 0.18, reSpectively) were highly significant (P<0.01). Within stage of lactation correlations between serum prolactin and milk yield were not meaningful during early lactation but after 12 to 24 weeks the correlations between the two variables / James Andrew Koprowski were positive and ranged from 0,08 to 0.48. These data also may be interpreted to indicate that prolactin becomes lim- iting to milk production during the later stages of lactation. Overall correlations of GH and milk production were low and non-significant (P>0.05) i.e. r = -0.01, 0.10 and 0.07 for the three samples collected around milking. Within stage of lactation correlations between GH and milk yield during the first 20 weeks of lactation were not significant. However, between 24 and 44 weeks of lactation a negative relationship between serum GH and milk production was evident. As with prolactin, glucocorticoids measured immedi- ately after milking were significantly (P<0.05) correlated with milk production (r = 0.19). However, no meaningful relationships between serum corticoids and milk production were observed within any stage of lactation. No consistent relationships were observed between serum prolactin and GH throughout pregnancy in non-lactating heifers with milk production during the subsequent lactation. In lactating cows the highest correlations of serum prolac- tin and glucocorticoids with milk yield were observed for the blood samples collected immediately after milking. This may suggest that the correlation between hormones measured in pre—lactating animals and subsequent milk yield would be greater if the animals' ability to release these hormones had been challenged. James Andrew Koprowski Season of the year significantly effected prolactin levels in both heifers and lactating cows. Serum prolactin was highest during the warm summer months (averaging 74 ng/ml) and lowest (averaging 35 ng/ml) during the cold winter months. In contrast GH and corticoid levels were not effected by season of the year. Non-pregnant, lactating cows had elevated (P<0.0l) serum prolactin whereas there were no differences (P>0.05) in prolactin values of lactating cows during the various stages of pregnancy. Likewise, serum glucocorticoid content was not affected by stage of pregnancy although GH increased (P<0.0l) as pregnancy advanced. This increase in GB may reflect metabolic changes in the dam with advancing pregnancy and may not be directly related to lactation. This may explain why negative correlations were observed between GH and milk yield as lactation advanced. Neither cow's age nor sex of the fetus had any significant effects on serum prolac— tin, GH nor corticoid levels. Neither milk production nor serum prolactin content were affected by stage of the estrous cycle. However serum GH was higher (P<0.05) during the estrogenic phase (4.3 ng/ml) of the cycle than during the luteal phase (3.3 ng/ml). Ele- vated levels of estrogens at this time may be responsible for this transient rise in serum GH content. SERUM PROLACTIN, GROWTH HORMONE AND GLUCOCORTICOIDS IN HOLSTEIN COWS AND THEIR RELATIONSHIP TO MILK PRODUCTION BY James Andrew Koprowski A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Dairy Science 1972 L1?" B IOGRAPHICAL SKETCH of James A. Koprowski I was born on July 5, 1944 in Dunkirk, New York. Having completed my elementary and secondary education in the Dunkirk public school system, I entered the New York State College of Agriculture at Cornell University and received a Bachelor of Science degree in June 1965. I accepted a graduate research assistantship in the Department of Food Science at Michigan State University and received the Masters of Science degree in March 1968. My thesis was entitled "Studies of the Detection of Dimethylnitrosoamine in Lake Michigan Chub." Desiring to gain a better background in the physi- ology of animal protein production I shifted my emphasis to the physiology of milk secretion. In September 1968 I was granted a National Institute of Health Predoctoral Fellowship to continue my studies. I am completing the requirements for the Ph.D. degree in May 1972. ii ACKNOWLEDGMENTS I would like to acknowledge and extend deep appreci- ation to Dr. H. Allen Tucker for his constant encouragement and unending enthusiasm which enabled me to complete my graduate studies. I would like to thank the Dairy Depart- ment and Dr. C. A. Lassiter for providing funds and facili- ties for my graduate training. A debt of gratitude is extended to Drs. E. M. Convey, H. D. Hafs and W. D. Oxender for their interest and advice throughout my graduate program. I am also grateful for the advice and consent of my committee members Drs. S. D. Aust, R. D. Dukelow, W. L. Frantz, J. L. Gill and L. D. McGillard. I am grateful for the advice and computer programing afforded by Dr. R. Neitzel. For their assistance in the laboratory and in collecting blood samples I would like to thank my colleagues Dr. L. A. Edgerton, W. G. Ingalls, M. C. Pratt, V. G. Smith and J. Zolman. The financial support rendered by the National Institutes of Health predoctoral fellowship is appreciated. Finally, I would like to thank my wife Jeannene and children Peter and Kathy Jo for the sacrafices they endured during the completion of this project. iii TABLE OF CONTENTS Page BIOGRAPHICAL SKETCH 0 0 O O O O O O I O O 0 ii ACKNOWLEDGMENTS . . . . . . . . . . . . . iii LIST OF TABLES . . . . . . . . . . . . . . vii LIST OF FIGURES o o o o o o o o o o o o o X LIST OF APPENDICES . . . . . . . . . . . . xi INTRODUCTI ON 0 O O O O O O O O O O O O O 1 REVIEW OF LITERATURE . . . . . . . . . . . . 5 Terminology . . . . . . . . . . . . . . 5 Hormonal Requirements for Lactational Performance . 5 Mammary Gland DevelOpment . . . . . . . . . 5 Lactogenesis . . . . . . . . . . . . . 7 Maintenance of Milk Secretion . . . . . . . 8 Galactopoietic Factors . . . . . . . . . . 9 Endocrine Changes During Lactation . . . . . . 10 Endocrine Changes During Gestation . . . . . . l7 Endocrine Changes During the Estrous Cycle . . . . 22 MATERIALS AND METHODS . . . . . . . . . . . 25 Experimental Animals and Design . . . . . . . 25 Experiment l.--Endocrine Changes During Lactation . . . . . . . . . . . . . 25 Experiment 2. --Endocrine Changes in Heifers During Gestation . . . . . . . . . . . . 28 Experiment 3.--Endocrine Changes During the Estrous Cycle in Lactating Cows . . . Blood Sampling and Processing Procedures Radioimmunoassays (RIA) . . . . . . Prolactin . . . . . . . . . . Antibodies . . . . . . . . . O O O O O O u ...; Radioiodination . . . . . . . . . . 32 Radioimmunoassay . . . . . . . . . 36 Selection of Assay Conditions . . . . . . . 38 Validation of Assay . . . . . . . . . . 40 iv Growth Hormone (GH) . . . . . . . Antibodies . . . . . . . . Radioiodination . . . . . . Radioimmunoassay . . . Selection of Assay Conditions . . Validation of Assay . . . . Protein Binding Assay for Glucocorticoids Preparation of Binding Protein . . . Serum Corticoid Extraction Procedure . Assay . . . . . . . . Statistical Methods . . . . . . . RESULTS AND DISCUSSION . . . . . . . . . . . Experiment 1. --Endocrine Changes During Lactation . Serum Prolactin . . . . . Relationship of Serum Prolactin to Milk Yields . . Effects of Stage of Lactation on Serum Prolactin . Seasonal Effects on Serum Prolactin . . . . . Effects of Stage of Gestation on Serum Prolactin . Effect of Fetal Sex on Serum Prolactin . . . Effect of Lactation Number on Serum Prolactin . . Effect of Genetic Herd Classification on Serum Prolactin . . . . . . . . . . . . Serum Growth Hormone (GH) . . . . . Relationship of Serum Growth Hormone (GH) to Milk Yields . . . . . . . . . . . . . Seasonal Effects on Serum Growth Hormone (GH) . . Effects of Stage of Gestation on Serum Growth Hormone (GH) . . . . . . . Effect of Fetal Sex on Serum Growth Hormone (GH) . Effect of Lactation Number on Serum Growth Hormone (GH) . . . . . . Effect of Genetic Herd Classification on Serum Growth Hormone (GH) . . . . . . . . . Serum Glucocorticoids . . . . . . Relationship of Serum Glucocorticoids to Milk Yields . . . . . . . . . . Seasonal Effects on Serum Glucocorticoids . . . Effects of Stage of Gestation on Serum Glucocorticoids . . . . . . . . . Effect of Fetal Sex on Serum Glucocorticoids . . Effect of Lactation Number on Serum Glucocorticoids Effect of Genetic Herd Classification on Serum Glucocorticoids . . . . . . . . . . . . Experiment 2.--Endocrine Changes in Heifers During Gestation . . . . . . . . . Serum Prolactn . . . . . . . . . . . Serum Growth ormone (GH) . . . . . . . . . Page 42 42 43 43 45 45 45 45 46 47 48 53 53 53 S9 65 66 69 71 71 72 73 77 79 79 83 83 83 83 86 88 88 92 92 93 93 93 99 Page Experiment 3.--Endocrine Changes During the Estrous Cycle in Lactating Cows . . . . . . . 102 Serum Prolactin . . . . . . . . . . . . 103 Serum Growth Hormone (GH) . . . . . . . . . 105 SUMMARY AND CONCLUSIONS . . . . . . . . . . . 106 BIBLIOGRAPHY O O O O O O O O O O O O O O 110 APPENDIX 0 O O O O O O O O O O O O O O O 123 vi Table l. 10. 11. LIST OF TABLES Page Average serum prolactin in cows, reared under loose housing conditions, throughout ' lactation . . . . . . . . . . . . 27 Titration of guinea pig anti-bovine prolactin (GPABP) and two sources of sheep anti-guinea pig gamma globulin (SAGPGG) for the prolactin radioimmunoassay . . . . . . . . . . 41 Effect of loglo and reciprocal transformations on the homogeneity of prolactin variance through lactation . . . . . . . . . . 51 Average serum prolactin and DHI milk yields in cows through lactation . . . . . . . . 55 Average prolactin response to milking and subsequent return to basal values 1 hour after milking throughout lactation . . . . 57 Overall correlations of serum prolactin and loglo prolactin with several estimates of milk production . . . . . . . . . . 60 Within stage of lactation correlations between serum prolactin or loglo prolactin with total milk of the five individual milkings . . . 63 Adjusted prolactin means for stage of lactation using seasonal effects, seasonal effects squared and stage of pregnancy as covariates . 67 Adjusted prolactin means for seasonal effects using stage of lactation and stage of pregnancy as covariates . . . . . . . . 68 Adjusted prolactin means for stage of pregnancy using seasonal effects, seasonal effects squared and stage of lactation as covariates . 70 Average serum growth hormone (GH) in cows throughout lactation . . . . . . . . . 75 vii Table Page 12. Adjusted growth hormone (GH) means for stage of lactation using seasonal effects, seasonal effects squared and stage of pregnancy as covariates . . . . . . . . 76 13. Within stage of lactation correlations between serum growth hormone (GH) or log 0 GH with total milk of the five individuaI milkings . . 78 14. Adjusted growth hormone (GH) means for seasonal effects using stage of lactation and stage of pregnancy as covariates . . . . . . . 80 15. Adjusted growth hormone (GH) means for stage of pregnancy using seasonal effects, seasonal effects squared and stage of lactation as covariates . . . . . . . . . . . . 81 16. Average serum glucocorticoids in cows throughout lactation . . . . . . . . . . . . . 85 17. Adjusted glucocorticoid means for stage of lactation using seasonal effects, seasonal effects squared and stage of pregnancy as covariates . . . . . . . . . . . . 87 18. Within stage of lactation correlations between serum glucocorticoids or loglo glucocorticoids with total milk of the five individual ' milkings . . . . . . . . . . . . . 89 19. Adjusted glucocorticoid means for seasonal effects using stage of lactation and stage of pregnancy as covariates . . . . . . . 90 20. Adjusted glucocorticoid means for stage of pregnancy using seasonal effects, seasonal effects squared and stage of lactation as covariates . . . . . . . . . . . . 91 21. Average serum prolactin and growth hormone (GH) throughout pregnancy in heifers . . . . . 94 22. Correlation coefficients of serum prolactin and loglo prolactin through pregnancy with milk production . . . . . . . . . . . . 98 viii Table Page 23. Correlation coefficients of serum growth hormone (GH) and loglo GH through pregnancy with milk production . . . . . . . . . . . . 101 24. Average serum prolactin, growth hormone (GH) and milk yield during the estrous cycle in lactating cows . . . . . . . . . . . 104 ix LIST OF FIGURES Figure Page 1. Elution profile of iodinated prolactin after passage through Bio-Gel P-60. The first peak represents iodinated prolactin and the second peak represents free iodine . . . . . . . . 35 2. Dose-response curves for NIH ovine and bovine prolactin and for ovine, bovine and caprine sera . . . . . . . . . . 39 3. Recovery of exogenous NIH-Bl-prolactin added to lOOuliters bovine serum diluted 1:16 . . 44 4. Pre- (L——A), immediately after (o——o) and 1 hour after milking (o-—o) serum prolactin and milk yield (kg/day) (D-—-U) throughout lactation . . . . . . . . . . . . 54 LIST OF APPENDICES Appendix Page I. Composition of reagents used in radio- immunoassay . . . . . . . . . . . 124 II. Composition of liquid scintillation fluids 0 O O O O O C I O O O O O 127 xi INTRODUCTION In many overcrowded, underdeveloped countries the primary source of protein is cereal grains. Malnutrition in these areas is due mainly to the poor quality of these proteins which usually lack adequate amounts of one or more of the essential amino acids. In contrast, in developed countries a large part of the dietary protein is furnished by milk, dairy products and meat which contain adequate amounts of the essential amino acids. In addition to its high quality protein, milk also contains an abundant supply of carbohydrate, fat, minerals and vitamins and has been appropriately called nature's most nearly perfect food. Whether measured in terms of pounds consumed, in dollar value, or in nutritional value dairy products are one of our most important foods. Even so, the question has been raised whether animals will continue to provide future food needs since animals are relatively inefficient in converting feed to edible products for human consumption. Milk production, based on both energy and protein produc- tion, is one of the most efficient processes for converting plant materials into animal food products.- Theoretically at least, the dairy cow appears to be the domestic animal best adapted to live in the world of the future. Major advances in genetics, management, nutrition and physiology during the last few decades have greatly increased the efficiency of milk production and future research should continue this trend. But for dairying to survive, dairy scientists of all disciplines must work together to develop even more efficient production programs. Drastic changes in management practices will also be needed. From a physiological standpoint there are several avenues which can be explored in an attempt to increase the efficiency of milk production. Probably the most obvious example is hormonal supplementation during lacta- tion. Classical examples of hormonal treatments to stimu- late production are diethyl-stilbesterol implants in poultry and beef cattle to stimulate weight gain and feed efficiency and the feeding of thyroprotein (iodinated casein) to stimu— late milk production in dairy cows. In addition, growth hormone (GH) stimulates milk yields. However, additional research is necessary to develop programs whereby hormone supplementation will be an economically feasible means of stimulating milk production. A brief summary of hormonal relationships and their practical application in the pro- duction of meat, milk and eggs has been published (Casida, 1959). Another way of increasing the efficiency of dairying would be to develop better means of selecting replacement heifers and bulls. A major advance in dairying would be forthcoming if a simple but accurate screening test could be developed to detect genetically inferior or superior milk producing animals at a relatively early age. Such a test when used in combination with common culling practices might reduce the costs of raising heifers as well as sire evaluation programs. Classically, measurements of hormones were restricted to bioassays of the endocrine glands. These assays had several limitations, mainly lack of specificity, sensitivity, and the limited number of samples that could be analyzed in a reasonable period of time. Within the past 5 years a major breakthrough in endocrinological research has been attained with the development of the radioimmunoassay (RIA). RIAs combine the sensitivity of radioisotope techniques with the specificity of immunochemical procedures. The sensitivity of the GH and prolactin RIA's, used in our laboratory, are approximately 1000x as sensitive as their respective bioassays. Thus, RIA's are applicable for rapidly assaying large numbers of blood or tissue samples, and they are relatively inexpensive. The study reported herein involved Holstein- Friesian heifers and cows in various reproductive and/or lactational states. The objectives of this study were to measure normal serum hormone content during these physi- ological states and to investigate possible relationships between these endocrine parameters and milk production. Hopefully, this pilot study will provide new insight into some of the endocrine factors affecting lactation. The long term goals behind such research are to develop economically feasible methods for identifying superior or inferior animals to increase the efficiency of milk production. REVIEW OF LITERATURE Terminology Lactation is defined by Webster (1959) as the se- cretion and yielding of milk by the mammary gland. Milk secretion refers to the intracellular synthesis of milk in the alveolar epithelium of the mammary gland and the subse- quent passage of milk into the alveolar lumen from the cytoplasm. Milk removal corresponds to the second part of the definition and involves both active and passive withdrawal of milk from the mammary gland. The term lactogenic refers to those factors responsible for the initiation of lactation whereas galactopoietic indicates the ability of factors to enhance an already established lactation (Cowie, 1961). In this review I will generally restrict the discussion to the hormonal factors affecting milk secretion including lactogenic and galactopoietic hormones with special reference to ruminants and more specifically to dairy cattle. Hormonal Requirements for Lactational Performance Mammary Gland Development Since mammary cell numbers greatly influence subse- quent milk production (Tucker, 1969), a brief review of the hormones regulating mammary growth is warranted. 5 Hormones of the ovary, pituitary and placenta are the most important regulators of mammary growth during pregnancy. In a series of classical experiments using hypophysectomized, adrenalectomized, ovariectomized rats Lyons et a1. (1958) determined the specific hormonal requirements for mammary development. Estrogen, proges- terone, growth hormone (GH) and prolactin were found to be the basic hormones necessary for complete lobular-alveolar development. However, there are considerable species dif- ferences in the roles played by the pituitary and ovarian hormones and the effects of their interaction on mammary growth (Cowie, 1957; Benson et al., 1959; Cowie and Folley, 1961; Jacobsohn, 1961; Meites, 1966; Baldwin, 1969). In intact ruminants, estrogen alone stimulated lobular—alveolar growth but estrogen and progesterone were necessary for optimal mammary develOpment (Meites et al., 1951). Progesterone also lessened the undesirable features of uninhibited estrogen administration. For a review of the effects of estrogen administration to ruminants see Sykes, 1953. Sinha and Tucker (1969) showed that the mammary glands of heifers grew at accelerated rates even before puberty and that during recurring estrous cycles mammary development was greatest during the estrogenic phase of the cycle. Sud et al. (1968) injected various quanti- ties of estrogen and progesterone to determine the require- ments for maximal udder development in ovariectomized heifers. They obtained mammary development qualitatively similar but quantitatively less than that of normal preg- nancy. They concluded that the absolute doses of proges- terone and estrogen were of more importance for mammary growth than the ratio between the two hormones. Their fail- ure to completely mimic pregnancy may have represented an imbalance of pituitary hormones. Lactogenesis The initiation of lactation is a very complex phenomenon probably involving the interactions of several hormones. The classical work of Stricker and Grueter (1928) first suggested that the anterior pituitary played a major role in lactogenesis. Subsequently, Riddle et al. (1933) isolated and purified a lactogenic factor from pituitary extracts that caused proliferation of the pigeon crop mucosa and called it prolactin. Several reviews on the hormonal requirements for the initiation of milk secretion indicated that the primary hormones involved were prolactin and cortisol or corticosterone and that the requirements were species dependent (Benson et al., 1959; Cowie, 1961; Meites, 1961 and 1966; Cowie, 1969; Baldwin, 1969). Recently, excellent reviews concerning all aspects of lacto- genesis (Reynolds and Folley, 1969) and the hormonal con- trol of lactogenesis (Denamur, 1971) have been published. In the hyppphysectomized goat, Cowie (1969) found that combinations 5f sheep prolactin, bovine GH, glucocorticoids, and tri-iodothyronine were highly lactogenic and restored milk yield to prehypOphysectomy levels. This work suggested that GH played an important role in lacto- genesis, a concept which is not too surprising since bovine GH has such a marked galactopoietic potency in cows in declining lactation (Hutton, 1957). Tucker and Meites (1965) consistently initiated lactation in pregnant heifers with well developed udders by injections of an adrenal cortical steriod (9-f1uoroprednisolone acetate). Maintenance of Milk Secretion Numerous studies utilizing hypophysectomized animals demonstrated that prolactin, GH and adrenal corticoids were the basic hormones necessary for the maintenance of lacta- tion; however, hormones of the thyroid, parathyroid and pancreas were required for maximal milk secretion (Benson et al., 1959; Cowie, 1961; Cowie and Folley, 1961; Meites, 1966; Baldwin, 1969). Hypophysectomy caused a marked depression in milk secretion in the goat (Cowie and Tindal, 1960; Cowie et al., 1964). Cowie et a1. (1964) reported that full milk secretion could be maintained after hypophy- sectomy by hormone therapy consisting of prolactin, GH, thyroxine, insulin and an adrenal cortical steroid. Although oxytocin is not essential for milk synthesis it is necessary for milk removal from the mammary gland (Cowie, 1961). However, the exact extent or the mechanism whereby each of the above hormones contributed to the preservation of the functional integrity of the alveolar cells of the lactating gland is unknown. Galactopoietic Factors Grueter and Stricker (1929) first demonstrated that ox pituitary extracts increased milk production in cows. Several workers (Cotes et al., 1949; Flux et al., 1954; Brush, 1960; Campbell et al., 1964) reported that adrenalcorticotrOphic hormone (ACTH) or glucocorticoids caused temporary declines in milk production in cows. However, Roy (1947) reported that injections of ACTH increased milk production. Lind (1969) observed that feeding flumethasone (a synthetic glucocorticoid) at rela- tively low dosages may also increase milk production in cows. Many workers have found that GH is galactopoietic when administered to cows (Cotes et al., 1949; Hutton, 1957; Donker and Petersen, 1951 and 1952; Chung et al., 1953; Wrenn and Sykes, 1953). Presently, thyroprotein is the only hormone approved by the Federal Food and Drug Administration to be used commercially in dairy cows to stimulate lactation. The galactopoietic effects of thyroprotein were reviewed by Thomas (1953) and Moore (1958). Thyroprotein feeding during the declining phase of lactation usually resulted in a 10-20% increase in milk production, if feed intake was also increased. Maximal increases in milk yield usually 10 occurred during the first 60 days of thyroprotein supple- mentation after which milk production declined. The beneficial effects of thyroprotein often disappeared after 2-4 months of treatment and subsequent milk production was generally below that normally expected. Provided feed intake was increased there was usually little dif- ference in total milk production over an entire lactation between cows fed thyroprotein and those fed at a higher plane of nutrition. Thus from the standpoint of total milk production thyroprotein feeding does not appear to be economically feasible at the present time. Physiologically, thyroprotein usually resulted in some weight loss, increased heart and respiration rates but no serious effects on health, longevity or reproduction were noticed. Although prolactin was essential for the initiation and maintenance of milk secretion it had little or no effect on milk yields of lactating cows (Folley and Young, 1940; Cotes et al., 1949; Wrenn and Sykes, 1953) or ewes (Morag et al., 1971). Endocrine Changes During Lactation Although prolactin is classically known as the lactogenic hormone and generally thought of as the "milk. producing" hormone, very little data are available which- attempt to relate milk production with prolactin secretion. Reece and Turner (1937) generally found more prolactin in ll pituitaries from lactating cows than in pituitaries from . non-lactating cows. They also observed higher levels of pituitary prolactin in dairy cows when compared with beef cows of similar lactational and/or reproductive states. However, it is difficult to assess the true meaning of these results, especially those for lactating cows, because the pituitaries were obtained from cattle at a slaughter-house and exact information on stage of lactation or time from last milking were not known. As previously stated with the recent development of the radioimmunoassay (RIA) it is possible to measure serum hormone concentrations as well as pituitary hormone fcontent. Several laboratories have developed RIA's for measuring prolactin in ruminants. Some of the RIA's specifically developed to measure bovine or ovine prolactin were found to cross-react with prolactin of the other species and with caprine prolactin. Thus, most of these assays can be used interchangeably to quantify prolactin in cattle, sheep and goats (Arai and Lee, 1967; Bryant and Greenwood, 1968; Johke, 1969a; Schams and Karg, 1970; Koprowski and Tucker, 1971). Serum prolactin is relatively high during the latter stages of pregnancy and at parturition (see Endocrine Changes During Gestation). In the ewe, Arai and Lee (1967) observed a sharp postpartum rise in serum prolactin which was maintained during lactation whereas McNeilly (1971) 12 reported a gradual decline in serum prolactin reaching control values by 4 weeks postpartum. Ingalls et a1. (1971) observed a decline to prepartum values in serum prolactin of heifers by 60 hours after calving and prolac— tin remained relatively low to the first postpartum estrus and were similar to values observed in non-lactating animals (Johke, 1970 and 1971; Schams and Karg, 1970). In Women, Berle and Apostolakis (1971) observed an in- crease in plasma prolactin during the 4 days following parturition before it began to decline to basal levels, whereas Pronaszko (1963) reported a steady decrease in urinary mammotrOphic activity of lactating women during the first few weeks after birth. Prolactin secretion in mammals is regulated by the hypothalamic prolactin inhibiting factor (PIF) which acts _directly on the anterior pituitary to inhibit prolactin synthesis and release. In addition, other hormones may affect prolactin secretion by their direct actions on the hypothalamus and/or pituitary (Meites and Nicoll, 1966). A variety of specific as well as non-specific stimuli have been reported to cause a rapid but short-lived prolactin release in ruminants, regardless of lactational state. Stressful stimuli i.e. pain, forced restraint and emotional disturbance have been associated with prolactin release (Johke, l969a,b and 1970; Bryant et al., 1970; Butler et al., 1971; Raud et al., 1971; Tucker, 1971). 13 Sexual excitement in both males and females also elevated serum prolactin values (Bryant et al., 1970; Convey et al., 1971). An acute release of prolactin was observed in lactating ewes and cows after feeding (Bryant et al., 1970; Johke, 1970); however, whether this response was due to feeding or to a conditioned reflex associating feeding with subsequent milking, remains to be tested. Plasma prolactin increased 2-fold within 6 hours after feeding, then declined to barely detectable levels (1-2ng/m1) after 46 to 60 hours of fasting (McAtee and Trenkle, 1971). These authors also observed that arginine infusions resulted in an immediate increase in plasma prolactin whereas glucose or 2-deoxyglucose (inhibits glucose utilization) infusions had no effect on plasma prolactin. Milking appears to be one of the vstrongest stimuli for prolactin release in lactating cows although sham milking and/or udder washing without milking also stimulates serum prolactin levels (Johke, 1969a,b and 1970; Bryant et al., 1970; Fell et al., 1971; Koprowski and Tucker, 1971; Tucker, 1971). Udder or brisket washing also evoked the release of prolactin in unbred heifers and non-lactating (dry) cows (Koprowski et al., 1971). Koprowski et a1. (1972) observed a diurnal rhythm of circulating prolactin values in lactating cows. This exemplifies the impoitance of collecting blood samples at 14 approximately the same time each day to minimize changes in prolactin values which may be associated with circadian variations. However, circadian changes accounted for only 8% of the total variation in serum prolactin whereas dif- ferences within cows accounted for 45% and differences among cows accounted for 47%. Following the cestation of an infusion of exogenous prolactin into lactating cows Tucker et a1. (1972) observed a 25 minute half-life for serum prolactin. Schams and Karg (1970) and Johke (1969b) estimated the half-life of pro- lactin to be 25 to 29 minutes in lactating cows and 32 minutes in a pregnant, non-lactating animal (Johke, 1969b). Schams and Karg (1970) also found, in daily samples taken for extended periods of time, a higher relationship between prolactin concentration and season of year than between prolactin and stage of lactation or milk yield. In fact, these authors were unable to demon- strate any meaningful relationships between blood pro- lactin values and milk production; thus, they concluded that blood prolactin was a poor estimator of lactational performance in dairy cattle. Grosvenor (1971) observed that the rate of mammary gland refilling in the rat was dependent upon the amount of circulating prolactin. The amount of prolactin released at suckling influenced the rate of mammary refilling during the immediate post-suckling period. 15 In the cow, plasma prolactin was highest 5 to 15 minutes after the start of milking, returning to control values within 30 to 60 minutes after milking (Tucker, 1971). The magnitude of the prolactin response to milking was greatest during early lactation and declined as lactation advanced (Johke, 1970; Fell et al., 1971). This suggests that prolactin may be directly involved in the maintenance of milk secretion during advancing lactation but whether or not prolactin directly stimulates mammary refilling in the bovine, as Grosvenor (1971) reported in the rat, remains to be tested. Recently, Pershin et a1. (1969) observed increased prolactin and GH in the blood of lactating cows following thyroxine administration. They claimed the resulting increase in milk production was closely associated with the increase in blood prolactin and not associated with changes in GH. Furthermore, Gotsulenko (1968) observed an increase in milk secretion immediately after injecting prolactin into the arterial system supplying the mammary gland of goats. The relationship between circulating GH and lacta- tional performance is just beginning to be explored. Yousef et a1. (1969) attempted to gain a better understanding of the role of GH in bovine endocrinology by measuring GH secretion rates (GHSR) in non—lactating (dry) cows. These authors estimated GHSR by two methods; (1) the disappearance 16 rate of injected unlabeled GH or (2) the disappearance rate of injected 125 I labeled GH. Using these two methods these authors calculated GHSR's of 19.1 and 20.3 mg/day/animal respectively. The half—times were 22.5 and 24.4 minutes, respectively, as determined by the 2 methods. Fitko et a1. (1969) measured the t% of 131 CH during the distributive and metabolic phases in high and low producing cows as well as in dry cows. During the distri- butive phase t% was 25.5 minutes in high producing cows, 19.1 minutes in low producers and 18.5 in dry cows whereas during the metabolic phase t% was 329.5 minutes for high producers, 256.1 minutes for low producers and 250.2 minutes for dry cows. The authors suggested that the delayed degradation and disappearance of GH from the blood of high producing dairy animals enhances the lactogenic effect of GH. These studies support the thesis that GH plays a role in lactation and indicates the need for further research in this area. Although it is established that glucocorticoids are important for milk secretion (see Maintenance of Milk Secretion) very little data are available on endogenous corticoid levels during lactation in the bovine. However, milking and exteroceptive stimuli associated with milking increased circulating corticoid values (Wagner, 1969; Wagner and Oxenreider, 1971; Paape et al., 1971; Smith et al., 1972). l7 Adrenal secretions are probably rate limiting to lactational intensity in rats because Thatcher and Tucker (1968) observed that pituitary ACTH and adrenal corticoster- oid content (Thatcher and Tucker, 1970c) decrease markedly as lactation advances. Furthermore, the normal decreases in lactational intensity, as lactation advances, can be prevented with exogenous administration of glucocorticoids (Thatcher and Tucker, l970a,b). In contrast circulating corticosterone was not altered during lactation, although there was a marked increase between the virginal and the lactating state (Thatcher and Tucker, l970c). Endocrine Changes During Gestation During pregnancy the prolactin and GH secreting cells (acidophils) of the bovine anterior pituitary become granulated but at a slower rate than animals during the estrous cycle (Jubb and McEntee, 1955). These same authors also witnessed hypertrophy and other signs of acidophil hyperactivity between the 4th and 8th months of pregnancy, after which the acidophils generally become smaller and tend to concentrate granules. From these data it is impossible to determine which hormone(s) (prolactin, GH or both) these cells are actively secreting at this time. However, Goluboff and Ezrin (1969) using differential staining techniques studied the effect of pregnancy on the prolactin and GH secreting cells of the human pituitary. 18 They found that the prolactin secreting cells were present in significant numbers only during pregnancy and the post- partum period whereas there was a concomitant decrease in the number of GH secreting cells during these times. The prolactin content of the pituitary increased during late pregnancy (Bates et al., 1935; Reece and Turner, 1937), suggesting an increase in the rate of production of the hormone. However, as with all pituitary content data this interpretation is subject to controversy since an increase in pituitary content of a hormone may result from an increase in production or decreased release or a combi— nation of the two processes. Studies of prolactin content in the pituitaries of several species suggested that pituitary prolactin content remains low during pregnancy, increases slightly just prior to parturition and increases sharply after parturition (Meites and Turner, 1948). The literature concerning circulating pituitary hormones in pregnant ruminants is limited. Serum prolactin in dairy heifers was low and quite variable during the first 75 days of pregnancy (Wettemann and Hafs, 1971). Oxender and Hafs (1971) reported that serum prolactin increased 59% during the third trimester of pregnancy in heifers. Likewise, in the ewe, Saji (1966) and McNeilly (1971) observed increasing concentrations of circulating prolactin during the last half of pregnancy although Arai and Lee (1967) reported aigradual decrease in serum prolactin 19 during this time. Blood levels of prolactin rise sharply during the 2 days before and at the time of parturition in cows and heifers (Schams and Karg, 1970; Arije et al., 1971; Edgerton and Hafs, 1971b; Ingalls et al., 1971); in goats (Johke, 1971); and in ewes (Arai and Lee, 1967; Davis et al., 1971; McNeilly, 1971). The placenta is generally classified as an endocrine organ and plays an important role in the physiology of pregnancy. The human placenta produces a polypeptide hormone that is chemically and immunologically related to human GH. The placental factor has been identified and named human placental lactogen (HPL) (Josimovich and Mintz, 1968). HPL has also been called human chorionic somatomammotrophin (HCS) and chorionic growth hormone- prolactin (CG—P). This hormone has luteotrophic, GH and prolactin-like effects which may contribute to mammary development and to the altered carbohydrate metabolism associated with advancing pregnancy. Grumbach et a1. (1968) called HPL the growth hormone of pregnancy because of its effect on carbohydrate metabolism, and in addition Denamur (1971) suggested that HPL may play a vital role in lactogenesis. Excellent reviews on HPL secretion and its biological and immunochemical properties have been published (Grumbach et al., 1968; Josimovich and Mintz, 1968). Presently, it is not known whether a similar placental hormone exists in the bovine. 20 HPL can be detected in blood between the 5th and 8th weeks of pregnancy, it then gradually increases in concentration as gestation advances reaching maximum values during the third trimester (Samaan et al., 1966; Spellacy et al., 1966; Saxena et al., 1968; Singer et al., 1970; Varma et al., 1971). Abnormally low HPL values found in women with threatened abortion correlated closely with the clinical outcome of their pregnancies and measurement of serum HPL promises to be a useful prognostic index of placental function (Saxena et al., 1968; Singer et al., 1970). HPL values rise during labor with a sharp increase at the end of the third stage of labor (Singer et al., 1970). These same authors observed a 14.5 minute half- time disappearance of HPL from the maternal serum during the first 30 minutes after delivery. Using bioassays, the mammotrophic activity in urine (Pronaszko, 1963) and prolactin in blood of humans (Berle and Apostolakis, 1971) were found to increase gradually as pregnancy advanced. Contrary to the work of Singer et a1. (1970), Berle and Apostolakis (1971) reported that prolactin in the blood decreased during labor. In addition, the latter authors observed increasing concentrations of prolactin up to 4 days following birth, then prolactin values decreased as lactation advanced. Berle and Apostolakis (1971) suggested that prolactin may not play an important role in the maintenance of lactation but may 21 be important for the initiation of lactation in women. Likewise, Pronaszko (1963) found decreasing mammotrophic activity in the urine of women during the first few weeks of lactation. Bovine GH remained unchanged throughout gestation (Oxender and Hafs, 1971), increased sharply at parturition and exhibited elevated values for 36 hours postpartum before decreasing to prepartum levels (Ingalls et al., 1971). These same authors observed a marked rise in serum prolac- tin 2 days prior to this GH rise. Plasma GH was relatively low in the ewe throughout pregnancy (Bassett et al., 1970), and in the rat, Schalch and Reichlein (1966) and Dickerman (1971) found that circulating GH did not change during pregnancy. In humans normal or reduced levels of GH were observed in the blood during pregnancy (Yen et al., 1967; Spellacy and Buhi, 1969; Varma et al., 1971). Tyson et a1. (1969) suggested that these low GH values may be a conse- quence of the high HPL levels which may suppress GH release. The capacity of the human pituitary to secrete GH in response to various challenges was low during the last trimester of pregnancy (Yen et al., 1967; Tyson et al., 1969; Spellacy et al., 1970) but normal GH release was attained by 6 weeks postpartum. Studies in ruminants suggest reduced adrenal cortical activity for the greater part of gestation 22 (Paterson and Hills, 1967; Bassett and Thorburn, 1969). However, increased serum cortiosteroid concentrations were observed during late pregnancy in cows (Heitzman et al., 1970; Adams and Wagner, 1970). Glucocorticoid adminis- tration during advanced gestation induced parturition in ruminants (Adams and Wagner, 1969; Fylling, 1970); thus reiterating the importance of glucocorticoids in the initiation of parturition and lactogenesis. Endocrine Changes During the Estrous Cycle Cyclic changes in the granulation of the acidophilic cells of the pituitary were observed during the bovine estrous cycle (Jubb and McEntee, 1955). Acidophils started discharging their granules about 3 days after estrus with peak degranulation occurring 10 days after estrus. If pregnancy did not ensue, regranulation commenced slowly until proestrus when it was slightly accelerated. However, definitive conclusions can not be drawn from these data regarding the secretion of prolactin or GH because it was not determined whether the prolactin and/or GH secreting acidophils were undergoing these changes. In gilts, Day et a1. (1959) observed a linear increase in pituitary prolactin content from day 2 to day 19 of the estrous cycle. Pituitary prolactin in heifers decreased significantly between estrus and day 2 and then gradually rose until the subsequent estrus (Sinha and 23 Tucker, 1969). That the pituitary changes are reflected in the blood concentrations was observed by Swanson and Hafs (1971) who found increasing serum prolactin concen- trations within 4 days preceding estrus, and peak values on the day of estrus, which subsequently decreased during diestrus. Similarly, serum prolactin in ewes was signifi- cantly higher during proestrus and the day of estrus than during metestrus or diestrus (Reeves et al., 1970). In contrast, Schams and Karg (1970) did not detect differences in serum prolactin during the estrous cycle in lactating cows. Raud et a1. (1971) also failed to find a relation- ship between circulating prolactin and stage of the estrous cycle in cattle when blood samples were collected via jugular puncture. However, to minimize the stress associ- ated with venipuncture, the same authors collected samples via indwelling jugular canulas. The prolactin values from the canulated animals were less erratic and a typical proestrus estrus prolactin peak was observed. Several workers have reported that stressful stimuli will cause prolactin release in ruminants (Johke, 1969a,b and 1970; Bryant et al., 1970; Butler et al., 1971; Raud et al., 1971; Tucker, 1971). The function of prolactin in the cow during estrus, ovulation and luteal growth is not known. To my knowledge there has been no data published on circulating growth hormone (GH) levels during the estrous cycle in ruminants. 24 In the rat, circulating prolactin (Niswender et al., 1969a; Amenomori et al., 1970; Neill, 1970) and GH (Dickerman, 1971) is elevated during estrus. Neill (1970) found that stress associated with different blood collection techniques may stimulate serum prolactin in rats thus masking the cyclic changes in prolactin during the estrous cycle. Prolactin activity was found in the blood during the first 5 and the last 10 days of the human menstrual cycle but no activity was found during days 6 to 18 (Simkin and Arce, 1963). Furthermore, the mammotrophic activity of human urine was at least twice as high during the second half of the menstrual cycle as during the first half (Pronaszko, 1963). Increased human GH levels have been observed during the ovulatory and premenstrual phases of the cycle (Frantz and Rabin, 1965; Spellacy et al., 1969). The physiological significance of these changes in human prolactin or GH during the menstrual cycle remains to be determined. As with GH, the literature on adrenal hormones during various physiological states in the bovine is very limited. Swanson et a1. (1972) observed elevated serum cortisol (the principal glucocorticoid) during estrus with low values at midcycle. MATERIALS AND METHODS Experimental Animals and Design Experiment l.--Endocrine Changes During LactatiBn This study was conducted to monitor serum hormone content in lactating Holstein cows through a complete lactation and to investigate possible relationships between serum hormones and milk production. At the onset of this study I did not know what blood sample would be the best estimator of serum prolactin to correlate with milk pro- duction. Because preliminary work revealed that prolactin is released in response to milking I decided to take a pre- milking sample to estimate resting or unstimulated hormone content, an immediate post milking sample to measure pro- lactin released by milking and a third sample 1 hour after milking to gain an indication of prolactin disappearance rate back to basal levels following the milking response. A total of 75 Holstein cows from the Michigan State University herd were used in this study. Starting 4 weeks postpartum, each cow was bled at 4-week intervals for the duration of lactation or until week 44, whichever occurred first. On a given bleeding date, blood samples were collected 2 to 4 hours before, immediately after and 1 hour 25 26 after the PM milking. Daily milk weights and monthly Dairy Herd Improvement Association (DHI) records were available for each cow. When this experiment was initiated all cows were housed and milked in a stanchion barn. However, due to other research commitments, 20 cows were subsequently allocated to loose housing facilities. Under these condi- tions the animals had to be caught and moderately restrained before blood samples could be withdrawn. During the summer months the cows were brought in from pasture before the pre-milking samples were taken thus adding another variable into the experiment. Because serum prolactin is so variable and appears to be released from the pituitary by superfluous external stimuli, I questioned the merits of using these cows. However, I continued to sample these cows to see if my convictions were justified. Pre-milking serum prolactin for the 20 cows at loose housing averaged 57 ng/ml, 66 ng/ml immediately after milking and 37 ng/ml 1 hour after milking (Table 1). Cor- responding prolactin values for the 55 cows in the stanchion barn were 55, 82 and 41 ng/ml respectively. Overall, cor— relation coefficients for pre-milking, post-milking and 1 hour post-milking prolactin and DHI milk production were -0.19, -0.10 and -0.09 (P>0.05) for cows in loose housing and -0.07, 0.31 (P<0.01) and 0.14 (P<0.01) respectively for the other cows. The within stage of lactation prolactin .m:0npm>ummbo mo MODES: u zQ .mm H Gmmzm 27 Anne omnem Anne mmnem Anne emnnen en Aenc annex lone mmnenn xenc emnemn on Amnc e nom lens a nnm Anne nnnme . em Aenv nnnmm Leno e nme Aenc ennee mm Acme m new lone ennne Acme mnnem em Aemv m nmm Acme nnnee Acme onnmm em Acme mnnne Lone ennee Acme emnme em 10ml m new Acne mmnme lane mnnem en Acme m nan Lone m nme Acme nnnmm an Anne nnnmm lane a nee lane ennmm e nxnnc N non QAONC e nee n1mnv m nen e menxnns menxnns menxnne eonnmnomn umumm Hmpmm Imnm mo H90: OGO .cmEEH xmmz mcfluomaonm Esuom .GOHumuoma usocmsounu .mCOHuHccoo mcwwso: mmooa Hmpcs common .m3oo CH chomHoum Esnwm mmmum>4ii.a mamme 28 averages (all three samples) for cows in loose housing, were very erratic between 4 and 36 weeks of lactation then increased to over 100 ng/ml and remained around this level to the end of lactation. Not only was the overall prolac- tin variance of cows in loose housing about twice as large as the corresponding variance of cows in stanchions but the variances within stages of lactation were also larger for cows in loose housing than for those of the other group. Likewise, within stage of lactation correlations with milk production were also highly variable. In contrast meaning- ful trends in the data of cows at the stanchion barn were observed. Because of these discrepancies data from the cows at loose housing were omitted from the study. Growth hormone (GH) assays were performed on 10 of the cows at loose housing and no differences were noted between the two groups were seen. These data support the observation by Tucker (1971) that GH was not affected by stressful stimuli. Experiment 2.-—Endocrine Changes in Heifers During Gestatian The cows in the previous experiment were bred beginning 60 days postpartum, therefore the effects of lactation on hormone content were confounded with pregnancy. The purpose of this study was to monitor and characterize serum prolactin and GH concentrations throughout pregnancy in non-lactating Holstein heifers. A second objective was 29 to correlate serum prolactin and GH during pregnancy with subsequent lactational performance. The 28 heifers used were born during July and August 1968 and were hemimastectomized at 5 months of age as part of another experiment. They were artifically inseminated between October 15 and December 17, 1969 and subsequently freshened between July 20 and September 16, 1970. Blood samples were collected between 12 and 2 PM, at 30-day intervals beginning 30 days after breeding and continuing through 270 days of gestation. Because samples were collected once a week the range at any sampling interval during pregnancy was i 4 days. Following parturi- tion, the heifers were milked and daily milk weights were recorded through the first 60 days of lactation. Experiment 3.--Endocrine Changes During the Estrous Cycle in Lactating Cows Estrogen has been implicated as a stimulant of prolactin and GH secretion but in pharmacological doses estrogen is detrimental to milk secretion. This experi- ment was conducted to study the in zizg_effects of the estrous cycle on serum prolactin and GH under resting and milking conditions. Blood was collected from 12 lactating Holstein cows on the day of first postpartum estrus (Day 0), days 2, 4, 7, 9, ll, 15, 18, 20 and again on the day of subsequent estrus (Day 00). On each of these days blood 30 samples were obtained 2 to 4 hours before, immediately after, and 1 hour after the PM milking. The concentration of prolactin and GH were correlated with stage of estrous, cycle and milk production. Blood Sampling and Processing Procedures In the lactating cow and heifer studies (Experiments 1, 2 and 3) tail vein (coccygeal vein) blood was obtained by veni-puncture, using a 20 m1 draw-BD Vacutainer (165 x 16 mm, Becton Dickinson and Co., Rutherford, N.J.) and a 1.5 inch 20 ga BD Vacutainer needle. Immediately after collection blood was transfered from the vacutainer to a polypropylene centrifuge tube and centrifuged within 15 minutes of collection at 6,500 x g for 15 minutes. The supernatant fluid was poured into 4 dram plastic vials and stored at -20 C until assayed for hormone content. Upon thawing for hormone assay, most samples contained large fibrin aggregates, therefore they were recentrifuged at 6,500 x g for an additional 15 minutes to remove this material. This procedure of immediately centrifuging the blood samples, rather than letting them clot for a day, was incorporated because I wanted to minimize metabolism of glucocorticoids by enzyme systems in plasma and red blood cells. 31 Radioimmunoassays (RIA) Prolactin A double RIA procedure for bovine prolactin was developed, with modifications, according to the procedures reported by Niswender et al. (1969b) for bovine LH. Descrip- tions of this assay system have been published (Tucker, 1971 and Koprowski and Tucker, 1971). Antibodies.--Antibodies to bovine prolactin (NIH-Bl)* were induced in guinea pigs by repeated subcutaneous injec- tions of 2 mg of prolactin in the scapular region. Each injection consisted of 2 mg prolactin emulsified in 2 ml of 0.85% NaCl and 2 m1 of Freund's Complete Adjuvant (Difco Lab., Detroit, Mich.) and each guinea pig received this mixture once every 2 to 3 weeks except that Freund's Incomplete Adjuvant was used after the initial injection. Blood was collected via heart puncture under ether anes— thesia using a 10 ml syringe and a 1.5 inch 18 ga needle at 2 to 3 week intervals after the 9th week. Antisera, recovered by centrifugation at 6,500 x g for 15 minutes, were pooled and stored at -20 C. This guinea pig anti- bovine prolactin serum (GPABP) is commonly refered to as the first antibody. * Supplied by the National Institutes of Health, Endocrinology Study Section, Bethesda, Maryland. 32 The second antibody, sheep anti-guinea pig gamma globulin (SAGPGG) was developed as follows. Fifty mg guinea pig gamma globulin (Fraction II, Pentex Inc., Kankakee, Ill.) dissolved in 2.5 ml distilled water was emulsified in an equal volume of Freund's Complete Adjuvant and injected subcutaneously in multiple sites over the scapular region of a sheep. Similar injections of gamma globulin emulsified in Freund's Incomplete Adjuvant were continued at 3-week intervals for 9 weeks after which serum was collected at monthly intervals. At each bleeding 400 to 600 ml of blood were obtained via jugular puncture with a 13 ga California bleeding needle. Serum was processed as previously described (see Blood Sampling and Processing Procedures). Radioiodination.--RIA's are based upon the competi- tion between a labeled and unlabeled protein for a limited number of antibody binding sites. If a homogeneous antibody is not used the two competing hormones must be identical in every respect save for the label to compete in a dose response fashion. Because it is almost impossible to obtain a homogeneous antibody it becomes important that homogeneous antigens are used for radioiodination. A highly purified bovine prolactin preparation would be the ideal hormone to iodinate for this assay. Swanson (1970) reported that NIH-Bl-prolactin is not a homogeneous protein because after disc gel electrophoresis he found 4 bands. Subsequently he attempted to purify the NIH-prolactin by passage through 33 Sephadex G-100 and by electrofocusing but was unsuccessful. The bands seen after electrophoresis could represent co- polymers of bovine prolactin subunits, contaminating proteins or a combination of these. However, the sensitivity and repeatability of the assay would indicate that NIH-Bl- prolactin is sufficiently pure for RIA usage. NIH-Bl-prolactin was iodinated by a modification of the method of Greenwood et a1. (1963). Radioiodination was accomplished in a 1 m1 glass vial and consisted of adding 5 ug of prolactin in 5 ul distilled water to 25 ul of 0.5M sodium phosphate buffer pH 7.5 (Appendix I.A.l). One mCi of 125 I (50 mCi/ml, Iso-Serv Division of Cambridge Nuclear Corp., Cambridge, Mass.) was added and the contents gently mixed. More efficient iodinations were obtained when 125I was used within 1 week of shipment. Twenty ug chloramine-T (Eastman Organic Chemicals, Rochester, N.Y.), (Appendix I.A.3) were added and the reaction mixture gently agitated for exactly 2 minutes by finger tapping before 125 ug of sodium metabisulfite (Appendix I.A.4) were added to stop the reaction. To promote quantitative recovery of iodinated hormone, 25 ul of a 0.01 M phosphate buffer in 0.14 M NaCl (PBS), (Appendix I.B.l) containing 2% bovine serum albumin (BSA), (Nutritional Biochemicals Inc., Cleveland, Ohio), (PBS-2% BSA) were added followed by 100 pl of transfer solution (Appendix I.A.S). The mixture was layered beneath the 0.05 M phosphate buffer (Appendix 34 I.A.2) on a l x 12 Om glass column packed with Bio-Gel P-60 (Bio Rad Labs, Richmond, Calif.). The column had been equilibrated with 0.05 M phosphate buffer pH 7.5 and then 2 m1 PBS-2% BSA were added and eluted with 0.05 M phosphate buffer. Seventy ul of rinse solution (Appendix I.A.6) were added to the vial, recovered and layered on the column. The column was eluted under gravity with 0.05 M sodium phosphate buffer and 15 1 ml aliquots were collected in disposable glass culture tubes (12 x 75 mm) containing 1 m1 PBS-2% BSA. The elution profile was determined by quantifying the radioactivity of 10 ul from each of the 15 tubes in an automatic gamma counter (Nuclear Chicago Corp., Des Plaines, 111.). I A typical elution profile consisted of 2 peaks (Figure l). The first peak (usually tubes 4-6) contained the iodinated hormone whereas the second peak (usually tubes 10-14) contained the free 125I. The peak 125I— prolactin tube was used in the prolactin RIA. Iodinated prolactin was quite stable and could be satisfactorily stored at 4 C up to 1 month after preparation. Common practice was to discard the iodinated preparation after 1 month although it can be used after this time if re- 125 purified by separating the free I and the radiation 125 125 damaged I-prolactin from the I-prolactin on a 1 x 12 cm column of Sephadex G-100 (Pharmacia Fine 35 500- 400- ‘i’soo- 9 X E \ E .‘i‘zooL Ioob o 2 4 e 8 IO I2 l4 ml of effluent Figure l.--Elution profile of iodinated prolactin after passage through Bio-Gel P-60. The first peak represents iodinated prolactin and the second peak represents free iodine. 36 Chemicals Inc., Piscataway, N.J.). The elution procedures and profiles are similar to those in the original iodination except that a third peak, representing the radiation damaged hormone, is evident. Radioimmunoassay.--Bovine serum must be diluted 1:2 to 1:10 with PBS-1% BSA (Appendix I.B.3) to be within the workable range of the standard curve. One hundred and 200 pl of each diluted serum sample were added with a Hamilton microliter syringe (Hamilton Co., Whittier, Calif.) to disposable glass culture tubes (12 x 75 mm) which contained an appropriate amount of PBS-1% BSA to yield a volume of 500 pl. Close agreement of dilution (volume) duplicates provides evidence of specificity of the assay. Standards were also dispensed with Hamilton microliter syringes into the assay tubes. Four sets of standards were evenly distributed within each assay. A set of standards was composed of 10 tubes containing 0.1, 0.2, 0.5, 0.8, 1.0, 1.5, 2.0, 2.5, 3.0, or 4.0 ng of NIH-Bl-prolactin (Appendix I.B.4). Each assay tube received 100 pl of a l:30,000 dilution of the'prolactin antibody (GPABP), (Appendix I.B.6) was agitated and then incubated for 24 hours at 4 C. Although the GPABP serum.was diluted 1:30,000 it was diluted 1:400 with respect to normal guinea pig control serum (GPCS) which contains gamma globulin, (Appendix 1.8.5). This dilution of GPCS provided adequate mass 37 with which the second antibody could react. Bovine 125I- prolactin was diluted with PBS-1% BSA so that 100 pl contained approximately 25,000 cpm. One hundred ul of the diluted iodinated prolactin solution were added to each assay tube which was then agitated and incubated at 4 C for an additional 24 hours. Since bovine prolactin-GPABP complexes are soluble at the concentrations employed, an immunoprecipitation step utilizing SAGPGG was used. One hundred ul of SAGPGG (Appendix I.B.7) were added to each assay tube at an ap- propriate dilution to optimally precipitate the guinea pig gamma globulin. After a 72 hour incubation at 4 C, 3 ml of PBS at 4 C were added to each assay tube. The precipitated hormone was separated from the free hormone by centrifuging each assay tube at 2,500 x g for 30 minutes in a refrigerated centrifuge with a swinging bucket rotor (Sorvall Model RC-3, Ivan Sorvall, Inc., Norwalk, Conn.). The supernatant fluid was decanted and the tubes allowed to drain for 30 minutes before the remaining fluid was 125I-prolactin removed with absorbent tissue. The amount of precipitated was quantified in an automatic gamma counter (Nuclear Chicago Model 1185). Samples were counted for 10,000 counts or for 10 minutes, whichever accumulated first. This information was automatically typed on paper and punched on tape by a teletypewriter (Teletype Corp., Skokie, I11.). The standard curve was calculated by multiple 38 regression analysis on a Control Data Corporation (CDC) 3600 computer. Standard prolactin data fit linear, quadratic and cubic components of the regression equation; correla- tions of fit (R2) were consistently between 0.98 and 0.99. These regression coefficients were manually entered into an Olivetti computer (Programme 101, Olivetti Underwood, New York, N.Y.). The sample number and counting time for each unknown were automatically entered into the Olivetti computer from the punched tape via a Beckman Model 6912 Tape Editor (Beckman Instruments, Inc., Fullerton, Calif.) and the prolactin concentrations (ng/ml) were computed. Control tubes were included in each assay to deter- mine background radioactivity (tube containing l:400 GPCS in lieu of first antibody, GPABP), total counts added (tube receiving only 125I-prolactin) and total precipitate (tube containing no unknown or standard). These tubes served as a type of "quality control" on the assays for determining non-specific binding (background) and per cent binding (obtained by dividing total precipitate by total counts). A typical standard curve is illustrated in Figure 2 where the quadruplicate standards were averaged and plotted as 125I—prolactin precipitated at each standard 125 the per cent of dose compared with the I-prolactin precipitated in the total precipitate tubes. Selectiongof Assay Conditions.--Although Yalow and Berson (1968) reported that maximum sensitivity of the 39 .mnmm mcwnmwo cam mcn>on .mcn>o How can anuomaonm mcfi>on can mcfi>o mHz now mm>uso oncommmnlmmoali.m onsmflm 3:3 635 .t 3 £829.“. 52 o: _.0 0.0? 0.0. 0.. - \II' 5.8.2.. - .m- 52 6?... 2:33\‘ . \‘ E38 2.38 \‘ . 63: 3:6 d c 1822.. -312 L 0 ON o¢ ecsom 5.8.2.152 om .6 g. 00 00. 4O assay can be obtained when one-third of the labeled hormone is bound, the prolactin assay is routinely used with anti- body concentrations that bind 40 to 50% of the available iodinated hormone. Because the binding changed with dif- ferent iodinated preparations, several dilutions of both first (GPABP) and second antibody (SAGPGG) were used to titrate the proper dilutions to insure approximately 50% binding. The first antibody was routinely used at a l:30,000 dilution whereas the second antibody was always diluted (1:3 to 1:6) to yield maximum precipitation since each bleeding contained a different titer. An example of a double titration of both first antibody and two bleedings of second antibody is illustrated in Table 2. On the basis of these data, the first antibody would be used at a l:25,600 dilution and either source of the second anti- body at a 1:3 dilution. The 1:25,600 dilution of SAGPGG would be used even though the 1:12,800 dilution resulted in a higher per cent binding, because the added sensitivity and specificity of the more dilute antisera would compensate for the slight difference in binding. It is also evident that antisera B had a higher titer than antisera A. Validation of Assay.--A typical standard curve is shown in Figure 2. The useful range of assay sensitivity for measuring bovine prolactin was between 0.2 and 4.0 ng (80 to 20% binding). Increasing levels of bovine serum gave a dose reSponse curve parallel to the standard curve 41 .uummm mnucoa Hmum>mm cmxmp mmmcm cmNHcsEEH mEmm wry Eonm mmcflpmoHe pcmnmwMHp ozu ucmmmummn m can «a .pcson capomHonmIH mo ucmo Hmmm mmH mm em mm nm mm mm oom.nmun me am me me me on eoe.mmun em en mm es nm me . oom.~nun m an. m 41w m 34 mmfimw M 0 sun mun Nun eonnsnno moomoam no connsnno .mmmmmocsEEHOHpmn canomaoum new mom Awwmo¢mv CHHDQon mafimm mam omensmiflucm mmmcm mo mmousom 03¢ cam Ammmmwv snuomHonm mcn>0bIflpcm mam mmcflsm mo COHDMHDHBII.N mqmma 42 (Figure 2). When varying amounts of NIH-Bl-prolactin were added to bovine serum they were quantitatively recovered (Figure 3). Precision of the RIA.was obtained by including a common serum sample in each assay. The prolactin content of this common serum was determined 45 times in 16 assays from October 1970 to February 1971 and averaged 35.9 i 1.0 ng/ml. Specificity of the assay was also satisfactory. NIH—bovine LH, TSH or ovine FSH at levels up to 1000 ng/ tube did not interfere with this system although NIH-B -GH 12 in excess of 100 ng/tube did interfere. This quantity of GE is at least 200 x greater than the GH content in the amount of bovine serum normally used in this assay. Thus, the assay is highly sensitive and specific for bovine prolactin. Dose response curves for NIH-SB-prolactin, ovine serum and caprine serum were parallel to similar curves of NIH—B -prolactin and bovine serum (Figure 2). The 1 crossreactivity of the bovine prolactin RIA with ovine and caprine hormones suggests the possibility of using this system to measure prolactin in sheep and goats. Growth Hormone (GH) The radioimmunoassay for bovine GH was developed by Purchas (1970). Antibodies.--Anti-bovine GH (GPABGH) was developed in guinea pigs by repeated subcutaneous injections of 43 NIH-Blz-GH.* The immunization and bleeding procedures were similar to those used in the prolactin assay for GPABP. The second antibody (SAGPGG) used in the GH assay was the same as that described for the prolactin assay. Radioiodination.--The procedure to iodinate bovine GH was similar to that previously described for prolactin, therefore only the variations in procedures will be noted. Five ug of NIH-Blz-GH in 5 ul distilled water was 125 reacted with one mCi I for 2 minutes in the presence of 75 ug chloramine-T (Appendix I.A.3). Iodinated GH 125 appeared to be more labile than I-prolactin and had to be repurified, 2 to 3 weeks after iodination on Sephadex G-100 as previously described for 125 I-prolactin. Radioimmunoassay.--Normally, bovine serum contains such low quantities of GH that serum could be assayed without dilution. Four sets of standards were included in each assay. A set of GH standards was composed of 10 tubes containing 0.1, 0.3, 0.5, 0.8, 1.0, 1.5, 2.0, 3.0, 4.0 or 5.0 ng of NIH-BlZ-GH (Appendix I.B.4). Each assay tube received 200 ul of first antibody (GPABGH), (Appendix I.B.6), 100 pl 125I-GH solution and 200 pl of second anti— body (SAGPGG), (Appendix I.B.7) according to the same incubation schedule as the prolactin assay. * Supplied by the National Institutes of Health, Endocrinology Study Section, Bethesda, Maryland. 44 .mHuH pennant Esnmm mcn>on mnmuwanooa on terms cwuomaonmIHmImHz msocmmoxm mo mumboommII.m mnsmnm 384 5.8.95 9. e n N _ o _ N 005352 5829:. a: neu>ooue 800. . 39603 L n L c 45 Selection of Assay Conditions.--The first antibody was used at a 1:3,200 dilution and the second antibody at a 1:3 to 1:6 dilution to optimally precipitate the labeled GH. Incubation intervals and conditions were identical to those in the prolactin assay. Validation of Assay.--Purchas (1970) described the sensitivity, accuracy and crossreactivity of the GH assay. In addition, Purchas et a1. (1970) compared estimates of pituitary GH content measured by bioassay with those measured by RIA. Protein Binding Assay for Glucocorticoids Total serum corticoids were quantified by a com- petitive protein binding assay (Murphy, 1967). The assay procedures were developed in our laboratory and recently reported by Smith et al., 1972. Preparation of Binding Protein Dog plasma obtained from terminal class operations at the Michigan State Veterinary Clinic was the source of binding globulin. In preparation for assay usage, 2.5 ml dog plasma were diluted to 100 ml with double glass distilled water. Florisil (8 gm, 60-100 mesh; Fisher Scientific Co., Fair Lawn, N.J.) was added and the contents stirred on a magnetic mixer for 15 minutes to absorb any endogenous steroids that were present in the dog plasma. After precipitating the Florisil by centrifugation (10 46 minutes at 5000 x g) the supernatant was decanted and diluted to a suitable concentration for assay purposes. For all assays reported herein a 1% concentration of dog 3H at 20,000 cpm/ml in 1% plasma was used. Cortisol-1,2- dog plasma was used as the competitor. Once prepared and mixed gently the competitor was stored at 4 C and used within a week after preparation. Serum Corticoid Extraction Procedure To estimate extraction efficiency approximately 1500 cpm of cortisol-1,2-3H in ethanol was placed into 20 m1 extraction vials fitted with polyethylene screw caps (Rochester Scientific Co., Inc., Rochester, N.Y.). The extraction vials and caps were previously silated with 5% chlorotrimethylsilane (Eastman Kodak Co., Rochester, N.Y.) in toluene. Total counts added were determined by placing equal volumes of cortisol-1,2-3H into duplicate scintillation vials; 5 ml of steroid scientillation fluid (Appendix II.A) were added; and radioactivity counted for 10 minutes in a liquid scintillation spectrophotometer (Nuclear Chicago Model, Mark I). One ml of serum was added to the extraction vials and extracted with 6 m1 redistilled 2,2,4- trimethylpentane (iso-octane) (Mallinckrodt Chemical Works, St. Louis, Mo.) by stirring on a vortex mixer (Deluxe Mixer, Scientific Prod., Evanston, Ill.) at high speed for 2 minutes. 47 The iso-octane—progestin fraction was removed by disposable pipettes and the extraction procedure was repeated a second time. Under these conditions iso- octane extracts less than 3% of added 3H-cortisol. There- after, samples were extracted twice with 6 ml of redis- tilled dichloromethane by vortexing at moderate speed for 2 minutes. The dichloromethane-corticoid fraction was transfered by disposable pipettes to silated 12 ml conical tubes and dried under nitrogen to approximately 3 ml. m To determine extraction efficiency, 500 pl of the dichloromethane extract was evaporated in scintillation vials, and radioactivity was quantitated. Using Hamilton syringes, dilution duplicates (500 and 1000 ul) of the dichloromethane extract were pipetted into silated culture tubes (12 x 75 mm). Duplicate standard curves; 0.0, 0.1, 0.5, 1.0, 1.5, 2.0 and 5.0 ng of cortisol per tube were included in each assay. Culture tubes containing unknowns or standards were dried under nitrogen and 1 ml of 1% dog plasma-cortisol-l,2-3H was added. The contents of each tube were mixed for 15 seconds and incubated for 18 to 24 hours at 4 C. Unbound 3H-cortisol was removed from the reaction mixture by adding 1 ml dextran coated charcoal [1.25% Charcoal-Carbon Decolorizing Neutral Norit (Fisher 48 Scientific Co., Fair Lawn, N.J.), 0.125% dextran 150 (Pharmacia Fine Chemicals, Uppsala, Sweden)] in water which had been double distilled in glass. The contents of each tube were mixed and incubated for 5 minutes from the onset of adding the dextran-coated charcoal; each tube was then centrifuged at 2500 x g for 10 minutes. Supernatant fluid (0.5 ml) was removed with an automatic diluter (Model D- 1000, York Instrument Corp., New York, N.Y.), then flushed from the diluter tip with 10 m1 Bray's scintillation fluid (Appendix II.B) (Bray, 1960), and radioactivity counted for 10 minutes in the liquid scintillation spectrOphotometer. The amount of corticoid in each sample was calculated (after correcting for extraction efficiency) by interpolation between standards. A common serum sample and water blank were included in each assay as an internal check on repeatability and non-specific interference. For 28 assays this common serum averaged 2.2 i 0.1 ng/ml and the water blank was consistently less than 0.05 ng/ml. Statistical Methods Basic statistics (means, standard errors of means and correlations) were calculated on a CDC 3600 computer, utilizing the Basic Statistics (BASTAT) and Least Squares Multiple Regression Routines. These programs were developed by the Michigan State University Agricultural Experiment Station. One of the basic assumptions in the analysis of 49 variance, that errors be uncorrelated, may be violated in these analyses since repeated measurements are made on individual animals. To the extent that this occurred, the type I error was underestimated (Gill and Hafs, 1971). Because I did not know the best manner to handle my data I decided to test the effects of data transforma- tions before attempting any correlations or other statisti- cal analyses of serum hormone content with milk production. Precedence for data transformations originate from the fact that many hormones act in a log dose relationship. However, an even more important reason was that the means for the 4-weekly prolactin samples taken throughout lacta- tion were positively correlated (r=0.64) with their variances (greater means are accompanied by greater variances) (Gill, 1971). Usually under conditions of heterogeneous variance a logarithmic transformation will make the variance inde- pendent of the mean, which results in homogeneous variances (Steel and Torrie, 1960). Tucker (1971) used a reciprocal transformation to reduce the heterogeneity of the variance and normalize the distribution of serum hormone data in experiments centered around the release of prolactin at milking. Thus I decided to compare the variances of un- transformed data with variances obtained from the same data using log10 and reciprocal transformations. To make these comparisons, for each of the three samplings around milking, I calculated the prolactin 50 variance (82) for each of the 11 sampling periods throughout lactation and then computed an overall variance for each of the three prolactin estimates. The same procedure was followed for the log10 and reciprocal transformations. Simple F-ratios (S2 larger/S2 smaller) were calculated for the three average prolactin samples at each period of lactation. Individual F-ratios for the untransformed as well as the transformed data are summarized in Table 3. Overall, F-ratios for the untransformed data for tfluapre-,immediately'after and 1 hour after milking samples were 1.6, 1.4 and 1.4 respectively. Comparable values for the loglo transformed data were 1.2, 1.2 and 1.1 respec- tively. Thus, on the average the loglo transformation reduced the heterogeneity of the variance. Likewise, the reciprocal transformation yielded F-ratios of 1.4, 2.2 and 1.3 for the samples taken before, immediately after and 1 hour after milking, respectively. Since the recipro- cal transformation did not reduce the heterogeneity of the variances I decided to conduct future analyses, including correlations, using untransformed and log10 transformed data. In Experiment 1 an analysis of covariance with unequal numbers program was used to determine the effects of season of year, stage of lactation and stage of pregnancy on serum hormone levels. Seasonal effects were analyzed including stage of lactation and stage of pregnancy as 51 no mam .mocmflnm> some m>fluommmmu mufi sufl3 mOCMflHm> sumo mo % HH NMP owumulm msu mucmmmnmmn Hones: sommm A ummuma mmv m.H m.m v.a H.H N.H N.H v.H v.H m.H ommum>¢ m.H m.H o.H H.H N.H m.H N.H ¢.H m.a we H.H ~.m m.H H.H H.H H.H m.H m.H m.a ov H.H H.m n.H H.H o.H ~.H N.H H.N m.H mm H.H m.H m.m H.H H.H ~.H N.H H.H H.H mm n.H m.H m.H N.H o.H m.H H.H H.H o.~ mm H.H m.H m.a o.H H.H o.H H.H H.H N.H vm N.H w.H H.H o.H H.H N.H H.H H.H H.H om m.H m.H H.H v.H N.H m.H N.m ¢.H m.H ma o.H N.m H.H H.H n.H o.H o.H o.H N.H NH m.H m.m m.H v.H m.H o.H v.N H.H m.H m m.H v.H H.H o.H m.H m.a m.H h.H . h.m v 3322 33:5 93an 33:? 33:? 3335 333.2 33:2 95:2 838.03 Hmumm umumm Imnm monum umumm Imum umumm Hmumm Imam mo Moon wco .UmEEH noon mco .omEEH moo: moo .UmEEH Mmmz coflmeH0mmcmuu coflum8“0mmcmuu mumo Hmooumfiomm oamoq omEH0mmcmuucD . m.coflumuomH nmsounu mUCMflum> cfluomaoum mo wwflmcmwoaon map so mQOHquHommcmuu Hmooumflomu cam camoa mo pomMMMII.m mqmde 52 covariates. Likewise, the effects of stage of lactation and stage of pregnancy were analyzed separately holding the other 2 parameters as covariates. However, because the seasonal effects on serum prolactin exhibited a signifi- cant quadratic response, the square of the seasonal com- ponent was incorporated as a third covariate in assessing the effects of stage of lactation and stage of pregnancy on serum prolactin content. To test for significant differences in serum hormones and milk yields due to stage of estrous cycle in Experiment 3, I partitioned, by orthogonal contrasts, the 9 degrees of freedom for stage of cycle as follows: 1. days 0, 20 and 00 versus all others (estrogenic phase versus luteal phase of cycle). 2. day 20 versus days 0 and 00 (proestrus versus days of estrus). 3. day 0 versus day 00 (first estrus versus subse- quent estrus). 4. days 2, 4 versus days 7, 9, ll, 15 and 18 (metestrus versus diestrus). 5. day 2 versus day 4 (early metestrus versus late metestrus). 6. days 7, 9 and 11 versus days 15 and 18 (early diestrus versus late diestrus). 7. day 7 versus days 9 and 11. 8. day 9 versus 11. 9. day 15 versus day 18. RESULTS AND DISCUSSION Experiment l.--Endocrine Changes During Lactation Endogenous hormone changes during different physi- ological states and their relationship to lactation are beginning to be researched. While this study was in pro- gress several papers were published concerning prolactin secretion and its relationship to lactational physiology (Schams and Karg, 1970; Johke, 1969a,b, 1970 and 1971). However, conclusions drawn from these data are restricted due to the limited number of animals studied. Studies of endogenous growth hormone and glucocorticoids and their relationship to lactation are also very scarce. Therefore, the purpose of this experiment was to study these hormones during various physiological states and to relate these values to milk production in the bovine. Serum Prolactin Average serum prolactin for the three samples col- lected before, immediately after and 1 hour after milking for each 4-week period throughout lactation are shown in Figure 4 and listed in Table 4. At 4 weeks of lactation pre-milking serum prolactin averaged 33 ng/ml; gradually increased to 68 ng/ml at 16 weeks of lactation, then 53 54 .coflumwomH usosmsouzu ADIIDY Axm©\oxv camw% xHflE cam CapomHonm Enumm AOIIOV mcHxHHE Hmpmm H50: H van AOIIOV umumm hampmflomEEH .A4ll0.05). In contrast, serum prolactin content measured immediately after milking was positively and significantly (P<0.01) related to milk production (r = 0.36). A significant (P<0.01) relationship between serum prolactin 1 hour after milking and milk production .Ho.ovm n aH.OAH .mo.ovm n mo.oxum 60 m .pcmoflmflcmwm mum mucmwoflmmmoo coaumawnuoo Hamo .cfiuomaoum How mafiamfimm Hmuwm mafixaflzo .cwpomHonm How mcHHmEmm mHOMmQ Amvmcwxawza .mCOHum>Hmmno mo umnEdzm ha.o vH.o mm.o Hm.o mHH.OI no.0: Auz mun ummg wanucoav Hma Hm.o ha.o mm.o mm.o v0.0: No.0: Am+vv mcflamfimm . Hmumm ham .5 om.o mH.o mm.o wm.o v0.0: mo.ou Amlav Hmuoe .w ma.o oa.o nm.o vm.o mo.ou no.0: 0mm: chase .m om.o mH.o wm.o mm.o mo.o| mo.on omdz wcoomm .v m~.o ma.o mm.o mm.o v0.0: No.0: 0mm: pmuflm .m mm.o ma.o mm.o nm.o vo.on Ho.o| 9mm: #muwm .m om.o na.o om.o mm.o mo.ou vo.o| 3mm: 039 .H mmcflxafla stofl>flch .Houm camoq .Honm .Houm camoq .Houm .Honm camoq .Houm Gama» xaflz mlammv mcflxafle 6.816vamcflxafla umnum 6.6x4mmvmcflxafie “whom .UmEEH Imum Mao: mco .cowuosooum xHHE mo mmumaflumm Hmum>mm npfl3 :Hbomaong camoH paw cHuomHoum Eamon mo mQOHumamuHoo Hamnm>o|n.m mamma 61 (r = 0.19) was also evident. These data may suggest that the prolactin response to milking and the subsequent return to low values are important in regulating lactational performance. Within a given sampling time (i.e., pre-milking, immediately after or 1 hour after milking) correlation coefficients for each of the seven individual milkings with serum prolactin were essentially the same. However, correlations with DHI milk weights tended to be lower than similar correlations using individual milking data. This slight discrepancy was probably due to the fact that the individual milking data represented actual milk production at each sampling whereas the DHI values were taken once a month and did not correspond as closely to actual produc- tion at the time each blood sample was obtained. Correlation coefficients of untransformed and loglO transformed prolactin data were remarkably similar, but the log10 transformation generally resulted in correlation coef- ficients of slightly larger magnitude. For example, cor- relation coefficients for pre-milking samples of prolactin and log10 prolactin with the two milkings before sampling were -0.04 and -0.06, respectively; for samples taken immedi- ately after milking r = 0.33 and 0.36 for prolactin and loglo prolactin, respectively; and for samples taken 1 hour after milking r = 0.17 and 0.20 for prolactin and loglo prolactin, respectively. Based on these observations I used the sum of the five individual milkings to estimate milk production 62 in future calculations involving the relationship of serum prolactin and milk production. Within stage of lactation correlations between serum prolactin and log10 prolactin with total milk yield for the five individual milkings are contained in Table 7. Pre- milking serum prolactin was not significantly (P>0.05) related to milk production for the first 20 weeks of lac— tation. However, during the later stages of lactation (24-44 weeks) the correlation coefficients were positive and tended to be larger than during early lactation. Sig— nificant (P<0.01) correlations were present at 24 weeks (r = 0.35) and 40 weeks (r = 0.40). A similar pattern was observed in the blood samples collected immediately after and 1 hour after milking. The time of transition toward an increased degree of relation— ship between milk yield and serum prolactin was between 16 and 20 weeks of lactation for blood samples taken immediately after milking and between 8 and 12 weeks for blood samples obtained 1 hour after milking. Correlations involving the loglo transformed data were essentially the same as those for the untransformed data. These data may indicate that prolactin becomes rate limiting for milk production as lactation advances; at least high serum pro- lactin immediately after milking and 1 hour after milking is associated with greater milk production especially during the declining phases of lactation. 63 .Ho.0vm Q .mo.0vmm 00.0 00.0 hH.0 HH.0 mm.o hm.o mm 00 whm.o wom.0 0H.0 H~.o va.o 900.0 00 00 900.0 Dov.o 0N.o wom.o v~.o ma.o ovlmv 0m mom.0 0N.o mm.o 0N.0 0H.0 0H.o omImv mm mm.o mH.o hH.0 00.0 mH.0 0H.o omlmv mm mam.o omm.o mH.o mH.0 Q0m.o Qmm.0 mm:vm 0N mH.0 ha.o vm.o whm.o H0.0: m0.0 0m om 0H.o ma.o 0H.o 00.0 00.0: 0H.o: vmlmm 0H 0N.0 mam.0 No.0 no.0 Ho.0 H0.0 mm:Hm NH HH.0 00.0 no.0: m0.o: No.0 0H.0 mm:am m 0H.0 No.0 m0.0: Ho.o 00.0: 00.0: mm:am v .Honm .Honm .Houm .Houm .HOHQ .Houm .>Hmmno GOAumuoma oamoq camoq onOA m0 mo :1: umnadz xmmz mcHxHHE Hmumm mafixHHE kumm maHxHHE H90: mco .omEEH :mum .mmcflxfifle Hmswfl>flcafi m>Hu map 00 xHHE Hmnou rung afluomfioum oaooa Ho capomaonm Eoumm cmwBqu mQOHumHonmoo coflumwomH mo oompm cflnpfl3::.n mamae 64 Grosvenor (1971) showed in the rat that the rate of mammary refilling immediately after nursing was dependent upon the amount of prolactin in the peripheral circulation at this time. Applying this concept to my data, it is possible that the prolactin released at milking stimulated milk synthesis during the immediate post-milking period in proportion to the amount of prolactin released. Thus, as the prolactin response to milking decreases a concomitant decrease in milk production follows. This concept is purely speculation on my part. The cause and effect relationship of prolactin on milk production must be tested by specifically designed experiments. Fitko et a1. (1969) reported a longer half-life for OH in high milk producing cows than in low producers. They theorized that the difference in milk production may result from the longer time GH had to exert its galactopoietic effects in the higher producers. Similarly, in my study it appears that high serum prolactin 1 hour after milking is associated with greater milk production. Presently, I do not know if this difference is due to the magnitude of the prolactin response to milking, or due to different degradation rates among animals or both. However, the rather high within stage of lactation correlations of prolactin with milk pro- duction suggest that differences in degradation rates may be important since prolactin released by milking diminishes as lactation advances. Although there is evidence that acute 65 prolactin administration does not stimulate lactational performance (see Review of Literature Galactopoietic Factors) it would be interesting to speculate, in view of this evi- dence, what would happen to milk production if prolactin were administered for extended periods of time during the latter stages of lactation. Effects of Stage of Lactation on Serum Prolactin Significant correlations between prolactin and milk production with advancing lactation suggested the possi- bility that prolactin is related to stage of lactation and may have little to do with milk production per ES“ Overall pre-milking serum prolactin was not related to milk produc- tion (P>0.05) although it was significantly correlated with stage of lactation although it was significantly correlated with stage of lactation (r=0.12, P<0.0l). Serum prolactin measured immediately after milking was highly correlated with milk production (P<0.01) but negatively related to stage of lactation (r = -0.37, P<0.0l). Stage of lactation was not related (r = -0.06, P>0.05) to serum prolactin measured 1 hour after milking. Since the effects of stage of pregnancy and season of year on prolactin concentration were unknown, each of these variables were analyzed separately by covariance analysis. Adjusted prolactin means for stage of lactation using seasonal effects, seasonal effects squared and stage of 66 pregnancy as covariates are listed in Table 8. Serum pro- lactin increased linearly (P<0.01) with advancing stages of lactation for pre-milking and 1 hour post milking samples. Prolactin values measured immediately after milking decreas— ed linearly (P<0.01) from the 8th to the 44th week of lac- tation. As previously shown in Figure 4 this response to milking appears real and related to stage of lactation and milk production. However, the underlying mechanisms responsi- ble for these changes remain unknown. Seasonal Effects on Serum Prolactin Seasonal effects were assessed including stage of lactation and stage of pregnancy as covariates. Adjusted monthly prolactin means from the covariance analysis are contained in Table 9. Seasonal effects were highly significant and fit a quadratic regression equation for the three sampling times (P<0.01) with maximal concentrations of prolactin occurring in the summer months and minimal estimates being recorded in the cold months. Bryant et a1. (1970) and Schams and Karg (1970) reported preliminary evidence which supports my data of high prolactin during the warm months and low values during the cold months. Milk production is known to decrease during the summer. Although serum prolactin content is increased at this time, the inLrease in serum prolactin following milking (19 ng/ml) is less than that released after milking during 67 TABLE 8.--Adjusted prolactin means for stage of lactation using seasonal effects, seasonal effects squared and stage of pregnancy as covariates. Serum prolactin Week Number Immed. One hour of of Pre- after after lactation observ. milking milking milking -------------- ng/ml------------- 4 42 ' 41 101 37 8 43 46 121 34 12 42 45 105 38 16 43 58 87 44 20 43 59 82 40 24 44 54 72 44 28 42 44 70 42 32 42 60 62 42 36 39 65 56 44 40 36 71 58 46 44 26 78 56 46 68 TABLE 9.-—Adjusted prolactin means for seasonal effects using stage of lactation and stage of pregnancy as covariates. Serum prolactin Number Immed. One hour of Pre- after after Month observ. milking milking milking -------------- ng/ml-----------—— January 33 25 68 27 February 30 24 67 26 March 33 34 80 29 April 41 52 82 29 May 44 62 90 33 June 43 62 94 49 July 42 104 123 78 August 39 99 102 70 September 39 72 73 50 October 35 36 66 34 November 30 29 41 25 December 33 35 54 30 69 the winter (33 ng/ml) when milk production is higher. These data fit my concept that the prolactin released following milking has pronounced effects on milk production whereas basal values are of minor importance. Because seasonal effects on serum prolactin fit a quadratic response, the square of the seasonal component was incorporated as a third covariate in assessing the effects of stage of pregnancy and stage of lactation on serum prolactin content. Effects of Stage of Gestation on Serum Prolactin Mean serum prolactin concentrations in samples col— lected 1 hour before (P<0.03) and 1 hour after milking (P<0.01) were larger in non-pregnant cows than in pregnant cows (Table 10). Prolactin samples measured immediately after milking did not differ (P>0.05) between pregnant and nonOpregnant cows. Dividing pregnancy into trimesters revealed that stage of pregnancy had little if any effect on serum prolactin (P>0.05). Oxender (1971) observed increasing concentrations of serum prolactin with advancing stages of gestation in Holstein heifers. However, he reports that physical stress of handling and surgery may have inflated his prolactin estimates and masked possible effects due to stage of gestation. Thus I conclude that serum prolactin is relatively unaffected by stage of preg- nancy although prolactin was greater in non-pregnant cows. 70 TABLE 10.--Adjusted prolactin means for stage of pregnancy using seasonal effects, seasonal effects squared and stage of lactation as covariates. Serum prolactin Month Number Immed. One hour of of Pre- after after pregnancy observ. milking milking milking -------------- ng/ml—----------- 0a 177 70 88 52 l 43 58 66 42 2 41 46 73 38 3 41 44 80 38 4 39 42 79 36 5 33 42 77 28 6 29 27 83 31 7 24 44 73 24 8 12 65 84 20 9 3 96 68 18 aNon—pregnant. 71 Effect of Fetal Sex on Serum Prolactin Cows chosen for this analysis were bred early enough in their lactation so that information for at least 7 months of concurrent lactation and pregnancy was available. All blood samples collected after conception were used in this analysis. Of the 33 cows studied 14 had male fetuses and 19 had female fetuses. Milk production did not differ between the two groups (P>0.05) averaging 22 kg/milking. Likewise, serum prolactin between the two groups was not significantly different (P>0.05) and averaged 54 and 56 ng/ml, respectively. Wettemann (1972) was also unable to find any differences in prolactin during early pregnancy in cows associated with sex of the fetus. However, Oxender and Hafs (1971) reported higher serum prolactin in heifers with male fetuses than in heifers with female fetuses. The reason for this discrepancy is not readily apparent. Effect of Lactation Number on Serum Prolactin Another variable known to effect milk production is age or the number of lactations a cow has completed. For example, total milk production per lactation generally increases until the cow is about 8 years old, but the increase after the 5th year is relatively minor. After this peak, production generally decreases at an increasing rate with advancing age. I used lactation number as an index of a cow'slage; usually a cow was 2-3 years older than the number of lactations she had completed. Older 72 cows in this study produced more milk (P<0.01) than younger cows through the 28th week of lactation; however, no sig- nificant differences (P>0.05) were observed between 32 and 44 weeks of lactation. This relationship is consistent with previous work since older cows reach a much higher peak of lactation but their persistency is relatively poor whereas younger animals generally do not attain as high a production level as older cows but they are more persistent. Serum prolactin va;ies for pre-, immediately after and 1 hour after milking did not differ (P>0.05) among cows with different lactation numbers. Thus, I conclude that lactation number (age) had no effect on serum prolactin content in lactating cows. Effect of Genetic Herd Classification on Serum Prolactin As part of another study, cows in the Michigan State University herd were randomly assigned to a best or worst breeding group of sires. The long term goals of this genetic study were to measure the stimulatory or detrimental effects of this breeding program on milk production. Most of the cows utilized in my study were parental stock, thus the effects of this breeding program should be minimal. Of the 55 cows, 29 were in the best breeding group and 26 were in the worst group. Serum prolactin for all three blood samples collected around milking was significantly higher (P<0.01) in the worst 73 breeding group (64 ng/ml) than in the best group (56 ng/ml). However, the prolactin response to milking (prolactin immediately after milking minus pre-milking prolactin) and the post-milking return to basal values (prolactin immediately after milking minus prolactin 1 hour after milking) were not different (P>0.05) between the two genetic groups. These data suggest that the prolactin response to milking did not differ between the two groups but the difference in prolactin levels can be attributed to higher basal levels in the worst breeding group of cows. Milk production was also greater (P<0.01) in the worst breeding group (28 kg/ milking) than in the best group (25 kg/milking). The difference in milk production may, at least in part, be attributed to differences in age between the two groups; the worst group were significantly (P<0.01) older than the best breeding group of cows. At the time of this writing the best group of cows are averaging about 100 kg more milk per lactation than the worst group. It would be interesting to remeasure serum prolactin in these animals when there is such a pronounced difference between the two groups in milk production. Serum Growth Hormone (GH) Growth hormone analyses were conducted on 26 of the 55 cows in this experiment. Average GH values for samples collected before, immediately after and 1 hour after milking : for each 4—week period throughout lactation are summarized 74 in Table 11. Overall, pre-milking GH averaged 4.2 ng/ml, 4.4 ng/ml for samples collected immediately after milking and 4.2 ng/ml for samples taken 1 hour after milking. Unlike prolactin, serum GH content was not affected by milking (P>0.05); an observation previously reported by Tucker (1971). Visual inspection of the data revealed that GH tended to decrease as lactation advanced. Adjusting the data for stage of lactation by covariance analysis using seasonal effects, seasonal effects squared and stage of pregnancy as covariates resulted in a similar but non- significant decrease in serum GH with advancing lactation (Table 12). For a physiological interpretation of these data it is important to know if this is a meaningful trend. Therefore I conducted a linear regression analysis of the GH concentrations throughout lactation for the three sampling times. On the basis of these analyses the lepe of each regression was significantly different from zero (P<0.01). Thus, I conclude that serum GH content decreases with advancing lactation. The decreasing serum GH content with advancing lactation in combination with the decreasing pro— lactin response to milking during advancing lactation may be contributing to the lower milk production during the latter stages of lactation. This agrees, at least in part, with previous observations that exogenous administration of GH to lactating cows will stimulate milk production although 75 TABLE ll.—-Average serum growth hormone (GH) in cows throughout lactation. Serum GHa'b Week Immed. One hour of Pre- after after lactation milking milking milking ---------------- ng/ml----------------—- 4 4.0:0.4 5.8:0.9 5.5:0.7 8 4.8:0.6 5.3:l.0 4.6:0.6 12 4.1:0.5 4.5:0.8 4.6:l.l l6 4.4:0.5 4.4:0.6 4.3:0.5 20 4.1:0.5 4.2:0.5 3.8:0.5 24 3.8:0.5 4.0:0.5 3.8:0.5 28 4.1:0.5 4.0:0.6 3.9:0.5 32 4.1:0.5 4.0:0.5 4.0:0.5 36 4.2:0.5 4.0:0.5 4.2:0.5 40 4.4:0.5 4.1:0.5 4.1:0.5 44 3.8:0.6 3.7:0.6 3.7:0.6 Average 4.2:0.2 4.4:0.2 4.2:0.2 aMean : SE. bN = 26 observations/mean; except week 44 = 20 observations. 76 TABLE 12.--Adjusted growth hormone (GH) means for stage of lactation using seasonal effects, seasonal effects squared and stage of pregnancy as covariates. Serum GH Week Number Immed. One hour of of Pre- after after lactation observ. milking milking milking -------------- ng/ml------—------ 4 22 4.9 6.4 6.0 8 22 5.9 5.9 5.1 12 22 5.0 5.3 5.4 16 22 5.3 5.1 5.0 20 22 4.8 4.8 4.5 24 22 4.1 4.6 4.3 28 22 4.2 4.4 4.3 32 22 3.8 4.1 4.1 36 22 3.7 3.8 3.9 40 22 3.6 3.5 3.6 44 18 3.1 3.3 3.3 77 exogenous prolactin had no effect on milk production (see Review of Literature GalactOpoietic Factors). Relationship of Serum Growth Hormone (GH) to Milk Yields Correlations of serum GH content and total milk pro- duction of the five individual milkings will be used since correlations using other estimators of milk production were essentially identical. Overall correlations of serum GH and loglOGH with total milk production of the five individual milkings were very low i.e., r = -0.01, 0.10 and 0.07 for pre—, immediately after and 1 hour post milking GH samples respec- tively; r=0.03, 0.07 and 0.06 for respective samples using loglOGH. Within stage of lactation correlations between GH and loglOGH with milk production are summarized in Table 13. During the first 20 weeks of lactation, GH and milk yield were generally positively related but there were no sig- nificant correlation coefficients. However, between 24 and 44 weeks of lactation the correlation coefficients, for the three blood samples, were consistently negative. Significant relationships (P<0.05) between GH and milk yield were evident from the 32nd to the 40th week of lactation for all three samples collected around milking (r = -0.39 to -0.46). Cor- relations of log10 GH and milk yield were essentially the same as those for the untransformed data. Strict interpre- tation of these data suggest that GH and/or the factors 78 .00.0v0 u 00.0A00 .mo.0v0 u 00.0A00 .mcowpm>00mbo om 00 x003 pmwoxm xmcowum>00mno on u 20 00.0: 00.0: 00.0: 00.0- 00.0: 00.0: 00 00.0: 000 0: 000 0: 00 0: 00 0: 000 0: 00 00.0: 00.0: 00.0: 00.0: 00.0: 00.0: om 00.0: 00.0: 00.0: 00.0: 00.0: 00.0: mm 00.0: 00.0: 00.0: 00.0: 00.0: 00.0: om 00.0 00.0: 00.0 00.0: 00.0 00.0: 00 00.0 00.0 00.0 00.0 00.0 00.0 om 00.0 00.0 00.0 00.0: 00.0 00.0 00 00.0 00.0: 00.0 00.0 00.0 00.0 00 00.0 00.0 00.0 00.0 00.0 00.0 0 00.0 00.0 00.0 00.0 00.0 00.0 0 COH#M#UMH 00000 00 00000 mo 00000 mo 00 0003 000x000 00000 000x005 00000 00000E H503 MCO .mvwEEH IQHAH 0.00000005 0mso0>0000 0>00 000 00 0000 00000 0003 00 00000 00 A300 macho: £03000 8500m cmmsumn mCOHuMHmHHOO COHpMpoma wo mmmum cflnuflz:l.m0 mqmde 79 responsible for GB release during advanced lactation may be detrimental to milk production, at least high serum GH within a given stage of lactation was associated with low milk production. In contrast prolactin appears to be rate limiting to milk production during the latter stages of lactation. But as proposed below (Effects of Stage of Gestation on Serum Growth Hormone) these negative correla- tions may reflect a change in metabolism of the animal as a result of pregnancy rather than lactation per 52' Addi- tional eXperiments will be necessary to determine if this is a cause and effect relationship because previous work (see Review of Literature Galactopoietic Factors) illustrated that exogenous GH administration was beneficial to milk production. Seasonal Effects on Serum Growth Hormone (GH) Table 14 contains the adjusted GH means for seasonal effects using stage of lactation and stage of pregnancy as covariates. Season of the year did not consistently effect any of the GH samples (P>0.05). Effects of Stage of Gestation on Serum Growth Hormone (GH) adjusted GH means for stage of pregnancy using seasonal effects, seasonal effects squared and stage of lactation as covariates are found in Table 15. Stage of pregnancy as analyzed by covariance analysis did not effect serum GH content in either of the three samples (P>0.05). 80 TABLE l4.--Adjusted growth hormone (GH) means for seasonal effects using stage of lactation and stage of pregnancy as covariates. Serum GH Number Immed. One hour of Pre- after after Month observ. milking milking milking —————————————— ng/ml————-—----—-— January 20 4.0 4.4 4.2 February 19 4.1 4.5 5.1 March 21 4.5 4.1 3.8 April 22 4.6 4.0 3.6 May 22 4.3 4.3 4.2 June 22 4.3 4.5 4.5 July 22 4.3 4.4 4.3 August 20 4.6 4.9 4.7 September 21 4.6 5.2 5.0 October 19 5.1 5.3 5.0 November 14 4.3 4.7 4.6 December 16 4.8 6.5 5.6 81 TABLE 15.--Adjusted growth hormone (GH) means for stage of pregnancy using seasonal effects, seasonal effects squared and stage of lactation as covariates. Serum GH Month Number Immed. One hour of of Pre- after after pregnancy observ. milking milking milking -------------- ng/m1--—--------- 0a 81 4.0 4.5 4.3 l 22 3.9 3.9 3.9 2 22 4.3 4.6 4.3 3 22 4.3 4.5 4.1 4 22 4.5 4.8 4.6 5 19 4.6 t 4.7 4.6 6 18 5.0 4.9 5.2 7 18 5.4 5.4 5.2 8 18 6.1 6.0 5.7 9 11 5.1 5.1 4.4 aNon-pregnant. 82 However, visual inspection of the adjusted means suggested that serum GH increased as pregnancy advanced. Linear regression analysis of GH concentrations throughout preg- nancy revealed that the lepe of each regression was sig- nificantly different from zero (P<0.01). Thus, I conclude that serum GH content increases with advancing stages of gestation. Similarly, Oxender (1971) reported a non- significant increase in GR with advancing stages of preg- nancy in heifers. Perhaps the increase in serum GH with advancing pregnancy is associated with the increasing demands of the fetus for nutrients from the mother. To summarize, I have evidence which suggests that during the latter stages of lactation, prolactin response to milking decreases and average GH values also decline. These changes are associated with declining milk production. However, during the latter stages of lactation the within stage of lactation correlations between serum GH and milk production were consistently negative suggesting an inverse relationship between these two variables. But, the data used in the correlation analysis were uncorrected for stages of concurrent pregnancy. Possibly higher serum GH and lower milk production in pregnant cows may contribute to this nega- tive relationship of GH and milk yield within the latter stages of lactation. 83 Effect of Fetal Sex on Serum Growth Hormone (GH) Nineteen cows were analyzed in this study, 10 had male fetuses and 9 had female fetuses. Serum GH from cows with male fetuses averaged 4.9 ng/ml after conception and were not different (P>0.05) than GH in cows with female fetuses (4.6 ng/ml). In contrast, Oxender and Hafs (1971) reported significantly more GH in serum from cows with male fetuses than from cows with female fetuses. Effect of Lactation Number on Serum Grow H Hormone (GH) Lactation number or the age of a cow did not sig- nificantly effect (P>0.05) serum GH content. Likewise age did not effect serum prolactin content. Effect of Genetic Herd Classification on Serum Growth Hormone (GH) Of the cows analyzed for GH, 15 were in the best breeding group and 11 in the worst breeding group. Serum GH averaged 4.3 ng/ml for cows in the best breeding group and was not significantly different (P>0.05) than that in the worst breeding group of cows (4.1 ng/ml). Serum Glucocorticoids Twelve cows that had lactated for 44 weeks were chosen for total glucocorticoid analysis. Pre-milking corticoids averaged 3.4 ng/ml at 4 weeks of lactation, peaked at 20 weeks (9.3 ng/ml) then fluctuated between 5 84 and 7 ng/ml before decreasing to 4.3 ng/ml at 44 weeks of lactation (Table 16). Corticoid samples collected immedi- ately after milking averaged 10.8 ng/ml at 4 weeks of lactation increased to 16.2 ng/ml at 12 weeks then gradu- ally decreased to 8.7 ng/ml at the end of this experiment. Samples taken 1 hour after milking were lower and more stable averaging 2.5 ng/ml at 4 weeks of lactation and 2.4 ng/ml at 44 weeks. Overall, serum glucocorticoids for the 12 cows averaged 5.7 ng/ml for pre-milking samples, 11.1 ng/ml for samples taken immediately after milking and 2.6 ng/ml 1 hour after milking. Wagner (1969), Wagner and Oxenreider (1971), Paape et a1. (1971) and Smith et a1. (1972) also observed that milking increased serum corticoid levels in cows, but these authors did not present data comparing the response to milking during different stages of lac- tation, which are presented below. Corticoids measured immediately after milking were relatively constant between the 4th and 32nd week of lac- tation averaging 10 to 13 ng/ml of serum glucocorticoids. However, corresponding values following milking averaged 6.6, 9.0 and 8.7 ng/ml for the 36th, 40th and 44th week of lactation respectively. A similar decline in serum prolactin measured immediately after milking was observed in these same cows but the response began to decrease earlier, some- time between the 8th and 12th week of lactation. After the 85 TABLE l6.--Average serum glucocorticoids in cows throughout lactation. Serum glucocorticoidsa’b Week Immed. One hour of Pre- after after lactation milking milking milking ---------------- ng/ml----------------- 4 3.4:0.5 10.8:l.4 2.5:0.3 8 4.7:l.2 10.7:l.5 2.6:0.3 12 5.6:l.2 16.2:2.3 3.3:0.7 l6 6.2:l.5 13.1:3.2 3.0:0.8 20 9.3:2.4 11.6:l.7 2.3:0.2 24 5.9:0.7 10.3:2.0 2.3:0.2 28 5.4:l.0 12.4:2.4 2.9:0.3 32 4.8:0.6 12.9:1.4 2.7:0.3 36 6 9:1.4 6.6:0.9 2.4:0.3 40 5 5:0.8 9.0:l.l 2.2:0.3 44 4 3:0.5 8.7:l.6 2.4:0 2 Average 5.7:0.4 ll.l:0.6 2.6:0.1 aMean : SE. bN = 12 observations/mean. 86 32nd week of lactation and prolactin response to milking was essentially zero a time when the corticoid response de- creased. Although serum GH was not released in response to milking, GH values did decline with advancing stages of lactation. Whether these data mean that prolactin, GH and glucocorticoids are rate limiting to milk production as lactation advances must await further investigations. Corticoid means adjusted for stage of lactation using seasonal effects, seasonal effects squared and stage of pregnancy as covariates are summarized in Table 17. Stage of lactation as analyzed by covariance analysis did not effect serum corticoids (P>0.05). Linear regression analysis of the adjusted average corticoid concentrations throughout lactation revealed that the slope of the regression for pre-milking samples throughout lactation was significantly different from zero (P<0.01) but the slopes of the regressions for samples collected immedi- ately after and 1 hour after milking were not different from zero (P>0.05). Relationship of Serum Glucocorticoids to Milk Yields Correlation coefficients between serum corticoids and total milk yield of the five individual milkings were -0.14 and 0.01 (P>0.05) for samples collected before and 1 hour after milking respectively, the coefficient for samples collected immediately after milking was 0.19 (P<0.05). 87 TABLE l7.--Adjusted glucocorticoid means for stage of lactation using seasonal effects, seasonal effects squared and stage of pregnancy as covariates. Serum glucocorticoids Week Number Immed. One hour of of Pre- after after lactation observ. milking milking milking --------------- ng/m1------------ 4 11 2.5 11.1 2.5 8 11 3.7 10.7 2.7 12 11 4.8 15.6 3.4 16 11 5.2 13.2 3.2 20 11 9.1 10.8 2.3 24 11 5.9 10.6 2.3 28 11 5.6 10.6 2.9 32 11 5.4 13.3 2.9 36 11 8.4 7.3 2.2 40 11 7.1 9.3 2.2 44 10 6.5 9.5 2.5 88 Similarly the highest correlations between serum prolactin and milk production were observed with the samples taken immediately after milking. Within stage of lactation correlations between serum corticoids or loglo corticoids and milk production are summarized in Table 18. No consistent relationships between serum corticoids or log10 corticoids and milk production are evident. This lack of relationship between serum corticoids and milk yield in cattle agrees with similar findings in lactating rats (Thatcher and Tucker, l970c). Seasonal Effects on Serum Glucocorticoids Adjusted glucocorticoid means for seasonal effects using stage of lactation and stage of pregnancy as covari- ates are listed in Table 19. Season of the year did not alter serum glucocorticoid content (P>0.05). Similarly, season of the year did not effect serum GH but serum pro- lactin was higher during the warm summer months than during the cold winter months. Effects of Stage of Gestation on Serum Glucocorticoids Covariance analysis revealed that stage of pregnancy did not have a significant (P>0.05) effect on either of the three corticoid samples collected around milking (Table 20). As previously described these means were adjusted using season, season squared and stage of lactation as covariates. 89 .0c.ovm .mo.ovm 00.0A00 . 0 mm 0A 3 .mcoflum>umm30 NH u 20 0503 000 .UQEEH mm.0 0m.0 00.0 00.0 300.0 300.0 00 00.0: 00.0: 00.0 00.0 00.0 00.0 00 00.0 00.0 00.0 00.0 00.0 00.0 00 mm.o: mm.o: 00.0: 00.0: 00.0: 00.0: mm 00.0 00.0 300.0 00.0 00.0: m~.0: mm 00.0 00.0 3N0.o: 000.0: 00.0 00.0 00 00.0 00.0 00.0 0N.o 00.0: mo.o: on 00.0: 0N.o: 00.0: 00.0: 00.0: 00.0: 00 00.0: 00.0: 00.0: 00.0: 0N.o: 00.0: NH 00.0 00.0: 00.0: 00.0: 00.0 00.0 0 00.0 mo.o: 00.0 00.0: 300.0: 300.0: 0 m000000000 mofloofluuoo m©00000000 0000000000 m000003000 m000000000 000000000 :00000 :00000 :00000 :00500 :00000 :00000 00 00300 00000 00003 3003 000x002 00000 0000000 00000 J: 0000005 I000 0.00000005 00000>0000 0>00 000 00 x00E 00000 0003 000000000000000 00000 HO mUHOUH#HOUOOSHO ESHmm C003#wfl mCOHumflmuhoo COH#M#OMH m0 mm0#m CHEHHBII.mH mqm<8 90 TABLE l9.--Adjusted glucocorticoid means for seasonal effects using stage of lactation and stage of pregnancy as covariates. Serum glucocorticoids Number Immed. One hour of Pre- after after Month observ. milking milking milking -------------- ng/ml------------- January 10 4.9 8.7 2.5 February 9 6.8 14.0 2.3 March 10 7.0 13.0 3.3 April 11 7.5 11.7 2.4 May 11 6.1 9.6 2.8 June 11 4.6 10.5 2.3 July 11 5.8 11.9 2.6 August 9 5.1 13.1 2.7 September 11 6.9 8.4 2.6 October 11 4.4 13.2 2.9 November 7 5.9 10.2 2.5 December 9 5.1 8.8 2.8 91 TABLE 20.--Adjusted glucocorticoid means for stage of pregnancy using seasonal effects, seasonal effects squared and stage of lactation as covariates. Serum glucocorticoids Month Number Immed. One hour of of Pre- after after pregnancy observ. milking milking milking -------------- ng/ml------------- 0a 46 7.5 11.5 2.8 l 11 8.5 10.8 3.3 2 11 5.0 9.8 2.2 3 11 4.9 8.9 2.2 4 11 3.0 15.4 2.8 5 8 4.9 11.5 2.7 6 8 4.1 12.9 2.5 7 8 3.8 7.9 2.3 8 4 3.0 8.3 2.8 9 2 2.4 7.4 2.0 aNon-pregnant. 92 Regression analysis of the pre-milking corticoid samples revealed that serum corticoids significantly decreased as gestation advanced, but the slope of the regressions for samples collected immediately after and 1 hour after milking were not different from zero (P>0.05). The significance of the decrease in pre-milking samples and not the other samples is not readily apparent. These corticoid data are in contrast to serum GH which increased with advancing pregnancy whereas prolactin was unaffected by stage of pregnancy although non-pregnant cows had significantly higher concentrations of prolactin in their blood than pregnant cows. Effect of Fetal Sex on Serum Glucocorticoids Ten cows were analyzed in this study, five had male fetuses and five had female fetuses. As with serum pro- lactin and GH sex of the fetus did not effect serum corticoid content (P>0.05) after conception. Glucocorticoids averaged 5.9 ng/ml for cows with male fetuses and 6.1 ng/ml for cows with female fetuses. Effect of Lactation Number on Serum Glucocorticoids Lactation number or age did not have a significant effect (P>0.05) on serum corticoids in these 12 cows. However, as with the other variables measured, a more reliable estimate of these effects on corticoid values could be obtained ;_; 1f the number of cows sampled were increased. 93 Effect of Genetic Herd Classification on Serum Glucocorticoids In this analysis five cows were in the best breeding group and seven cows in the worst breeding group. Pre- milking serum corticoids were significantly larger in cows from the best breeding group (6.9 ng/ml) than comparable samples from the worst breeding group (4.4 ng/ml). However, serum corticoids from samples collected immediately after and 1 hour after milking did not differ (P>0.05) between the two breeding groups. Thus it appears that there are no real differences in serum glucocorticoids between the two grOUps. Experiment 2.--Endocrine Changes in Heifers DuringfiGestation In the previous experiment the effects of pregnancy on serum hormone content were confounded with lactational effects. Therefore, this study was conducted to characterize serum prolactin and GH throughout pregnancy in 28 non- lactating Holstein heifers and to correlate hormone values through pregnancy with each heifer's subsequent lactational performance. Serum Prolactin Prolactin concentrations (ng/ml) throughout preg- nancy are shown in Table 21. Serum prolactin was fairly constant during the first 90 days of gestation (9 ng/ml), I then increased (P30.05) almost linearly to 44 ng/ml at 94 TABLE 21.--Average serum prolactin and growth hormone (GH) throughout pregnancy in heifers. Days pregnant Prolactina'b GHa’b --------------- ng/m1------------- 30 9: l 2.8:0.1 60 9: l 2.7:0.1 90 9: 1 3.1:0.2 120 18: 4 2.6:0.1 150 25: 5 2.6:0.1 180 37: 5 2.6:0.1 210 44: 4 2.7:0.1 240 111:14 2.6:0.2 270 76: 9 2.8:0.2 Average 38: 2 2.7:0.1 aMean : SE. bN = 28 observations/mean. 95 210 days, peaked at 240 days (111 ng/ml) before decreasing (P<0.05) to 76 ng/ml at 270 days of gestation. Singer et a1. (1970) reported a similar profile for human placental lactogen (HPL) during hormal pregnancies in women. The decline (P<0.05) in serum prolactin (35 ng/ml) between 240 and 270 days of gestation in my heifers corresponds to a similar but non-significant decrease in serum HPL observed by Singer et a1. (1970) between 38 and 40 weeks of preg- nancy in women. The biological significance of these observations is unknown, although the decreasing content of HPL may result from a loss in the ability of the human placenta to synthesize HPL with impending parturition. It has not been determined if the bovine placenta secretes any of the prolactin we measured in the serum. Serum prolactin increased (P<0.01) during each successive trimester of pregnancy; averaging 9, 27 and 77 ng/ml prolactin during the lst, 2nd and 3rd trimesters, respectively. Oxender (1971) observed elevated serum pro- lactin values in heifers during the last trimester of pregnancy but his values (even those in early pregnancy) are several times greater than corresponding values reported here. More than likely the higher values reflect the stressful conditions of handling and surgery used during collection of his samples. Results of Experiment 1 showed that season of the year affected serum prolactin. Prolactin values were 96 highest during the summer months and lowest during the winter months. Because all of the above heifers were bred and subsequently calved within a 2-month period, stage of pregnancy was confounded with seasonal effects. Serum prolactin was lowest during the first trimester which occurred during the months of December through March when serum prolactin would be expected to be low. Likewise, highest concentrations of prolactin occurred during the third trimester of pregnancy which occurred during the summer months when serum prolactin would be expected to be highest. To further clarify this question, on December 8, 1970, I bled 6 heifers in the first trimester of pregnancy and 6 in the third trimester; serum prolactin averaged 10 ng/ml for both groups. Thus, seasonal effects may be the dominant factor responsible for prolactin differences seen during pregnancy in the 28 non-lactating heifers. After freshening, daily milk weights were obtained for each heifer through the first 60 days of lactation. Each heifer's total milk production for the first 30 and second 30 days of lactation were correlated with their respective prolactin and log10 prolactin values for the individual 30 day sampling intervals during pregnancy. There does not appear to be a consistent relationship between serum prolactin or log10 prolactin throughout Pregnancy With milk production during the subsequent lac- tation. Correlation coefficients were generally negative 97 and low although a significant relationship was seen at 150 days of gestation (Table 22). The biological sig- nificance of these observations do not appear to be meaningful at this time. Thus, I conclude that serum prolactin as measured during pregnancy in this study is not a practical means of predicting lactational performance in first calf heifers. In Experiment 1 overall correlations of pre-milking prolactin values with milk production were relatively low (r = 0.02); however, prolactin values taken immediately after milking (r = 0.36) and to a lesser degree those taken 1 hour after milking (r = 0.18) were significantly related to milk yield. This would suggest that future experiments should measure serum prolactin values after the animal's ability to release prolactin has been challenged. If a uniform prolactin-releasing stimulus could be repeatedly administered to a series of animals then it may be possible to obtain more meaningful correlations between prolactin and future milk production. Injections of several different hormones or possibly electrical or mechanical stimulation of the udder may be tried as stimuli to evoke prolactin responses in dairy heifers. Rank correlations were utilized to estimate the repeatability or to gain an indication of how consistent the prolactin values of a given heifer were throughout pregnancy in relation to the other animals. Based upon 98 TABLE 22.-~Correlation coefficients of serum prolactin and log 0 prolactin through pregnancy with milk pro uction. Prolactin Logl0 prolactin Days of Milk 1st Milk 2nd Milk 1st Milk 2nd pregnancy 30 days 30 days 30 days 30 days 30 —o.13 -0.l9 -o.02 -o.12 60 ' -o.12 -o.29 -o.15 -o.31 90 0.12 0.07 0.10 0.11 120 -o.01 -o.02 -o.02 -o.04 150 -o.41b -o.42b -0.26 -o.38b 180 ~o.17 -0.08 -o.07 0.03 210 -0.17 -o.23 -o.14 -o.22 240 0.11 —0.08 0.10 -o.05 270 0.07 -o.09 0.01 0.07 ar>o.32 = po.37 = P<0.05. 99 1 her serum prolactin content, each heifer was ranked from 1 to 28 for each of the 9 sampling periods. Correlations between the rankings within a sampling period and among each of the subsequent periods did not reveal any meaning- ful relationships, suggesting that even though average serum prolactin increased during advancing pregnancy this increase was not consistent among heifers. Sex of the fetus does not appear to influence maternal prolactin levels. Serum prolactin for heifers with male fetuses averaged 34 ng/ml, which was not dif- ferent (P>0.05) than the 40 ng/ml average for heifers with female fetuses. This observation supports the data observed in Experiment 1 which was conducted on lactating cows. In contrast, Oxender and Hafs (1971) found significantly higher concentrations of serum prolactin in heifers with male fetuses than in heifers with female fetuses. Serum Growth Hormone (GH) Average GH values throughout gestation are shown in Table 21; GH averaged 2.9 ng/ml during the first trimester of pregnancy, then decreased (P<0.05) and remained rela- tively low during the last 2 trimesters (2.6 and 2.7 ng/ml respectively). Circulating levels of GH have been ob- served to remain unchanged throughout pregnancy in the rat (Schalch and Reichlein, 1966; Dickerman, 1971), in the ewe (Bassett et al., 1970) and in the bovine (Oxender, 1971). 100 Low levels of GH observed in women during pregnancy have been attributed to negative feedback suppression of GH secretion by high circulating concentrations of HPL and to the effects of elevated serum levels of steroids, especially the glucocorticoids, which may be inhibitory to GH secretion (Spellacy and Buhi, 1969; Tyson et al., 1969). Correlations between serum GH and logloGH for each of the nine sampling periods during pregnancy with the first and second 30-day total milk production records were low and mostly negative with significant relationships occurring at 60 days of gestation (Table 23). Physiologi- cally, these data indicate'that serum GH values throughout pregnancy are not acceptable indicators of subsequent lactational performance. Rank correlations, based on serum GH content, of the heifers between successive stages of gestation revealed that there were no meaningful relationships between suc- cessive GH values within heifers. Serum GH averaged 2.7 ng/ml for all samples in this experiment. On the average GH concentrations in heifers with female fetuses were not different (P>0.05) than GH levels in heifers with male fetuses. Likewise, no difference in serum GH was seen due to sex of fetus in lactating cows during pregnancy in Experiment 1. In 101 TABLE 23.--Correlation coefficients of serum growth hormone (GH) and loglo GH through pregnancy with milk production. GH LoglO GH Days of Milk lst Milk 2nd Milk lst Milk 2nd pregnancy 30 days 30 days 30 days 30 days 30 0.07 0.17 0.05 0.15 60 -0.33 -0.40 —0.34 —0.41b 90 0.27 0.22 0.24 0.20 120 0.09 -0.04 0.06 -0.10 150 0.12 0.05 0.17 0.05 180 -0.03 —0.20 -0.02 -0.20 210 -0.04 -0.19 0.04 -0.l7 240 —0.13 -0.16 -0.08 -0.15 270 -0.00 —0.10 -0.03 -0.14 ar>0.32 = P0.37 = p<0.05. 102 contrast, Oxender and Hafs (1971) reported higher serum GH in cows with male fetuses than cows with female fetuses. Experiment 3.--Endocrine Changes During the Estrous Cycle in LactatinggCows This study was undertaken to investigate possible relationships between serum prolactin, GH and milk produc- tion with stage of the estrous cycle. Some dairymen associate psychic estrus with a temporary decline in milk production although most experimental evidence does not support this concept (Copeland, 1929). Injections of large doses of estrogens are known to inhibit lactation. Thus, the decrease in milk production during estrus has been attributed to relatively large amounts of circulating estrogens at this time. Proposed mechanisms of estrogen action include alteration of the release of prolactin and/ or CH from the anterior pituitary or impariment of the functionality of the milk ejection reflex. In addition, the estrogen surge has been implicated in decreasing feed consumption and increasing physical activity, all resulting in the disruption of normal mammary function (for reviews see Cowie, 1961 and Smith, 1964). The purpose of this experiment was to study the effect of changing endogenous estrogen levels on serum prolactin and GH under basal and milking conditions. 103 Serum Prolactin Average serum prolactin and milk yields during the estrous cycle are summarized in Table 24. Although milk production was lower during the estrogenic phase (32.9 kg/ day) than during the luteal phase (33.2 kg/day) of the cycle this difference was not statistically significant (P>0.05). Serum prolactin values taken before, immediately after and 1 hour after milking did not change significantly (P>0.05) during the estrous cycle. This is rather surprising since estrogen has such a stimulating effect on prolactin secretion (Meites and Nicoll, 1966) and the fact that serum prolactin is elevated in ewes and heifers during the estro- genic phase of the estrous cycle (Reeves et al., 1970; Raud et al., 1971; Swanson and Hafs, 1970). However, like the results of this study, Edgerton and Hafs (1971a) also failed to detect a rise in prolactin during the estrogenic phase of the cycle in lactating cows thus suggesting that prolactin secretion during the estrous cycle in lactating cows may be controlled by a different mechanism(s) than in non-lactating animals. Serum prolactin averaged 59 ng/ml for the pre— milking samples, 82 for the samples taken immediately after milking and 50 ng/ml 1 hour after milking. These values are similar to comparable samples obtained in Experiment . . .i . . . . 1, thus indicating that the animals in this experiment were 104 .msuumm ucosvmmnsm mo mono .msuumm mo woo 9 .mm H cmem m.OH~.mm m.oao.v ~.oas.m ~.owo.m q Mom o “mm a “mm mmmum>4 o.mko.mm m.ows.¢ a.ow¢.m m.owo.m omkmo mmkms makes ooo o.mwm.mm m.oam.m m.oam.m m.owfl.q sakmm makma makes om H.mkm.mm m.owm.m h.¢km.m a.owa.m naksm magma Nfiflmm ma m.~ao.~m H.Hkfl.v v.0Ho.m w.owo.m makmm omflmm sawam ma ~.~Hm.~m m.owm.m m.ows.~ a.owm.~ makmm mmkmm «Hues Ha m.~w~.mm o.owm.m m.owm.m m.oam.m Nflkmm nmumoa sakes a m.mam.sm a.owm.v a.owm.m m.oam.~ NHkmv omkom m “we a m.mflm.vm w.owm.m a.owm.m H.0Mm.m m “we makes makms v m.mflm.mm m.onm.m m.oam.m «.0Hm.~ a “ma makam oawsv N a.ma~.mm q.HHm.m m.HHm.m H.Hks.m m “mm makes HHHHS no MIAMS\mkV mcflxflfls mcflxafla mcaxafis mcfixaas maflxaas mcaxafls Macao tamwm Hmpwm Hmpmm Imnm kumm Hmuwm Imum msouumm xaflz noon mco .meEH H50: mco .UmEEH mo woo MAHE\msv mm MAHE\msV sfluomaoum .msoo mafiumuoma CH maomo msouumm map moauso tamflm xHHE pom Amwv msoEHo: cpsoum .cfluomaonm Ednmm mmmuo>¢nl.vm mqmme 105 normal and exhibited a normal prolactin response to milking. Furthermore, changing estrogen levels during the estrous cycle (estrogenic vs. luteal phase) had no effect (P>0.05) on the quantity of prolactin released in response to milking. Serum Growth Hormone (GH) Serum GH was significantly higher (P<0.05) during the estrogenic phase (day of estrus, day 20 and day of subsequent estrus) of the cycle than during the luteal phase (days 2 through 18). It appears that the relatively high levels of estrogen may be stimulating GH secretion at this time. Estrogenic compounds, especially diethylstil- besterol, have been observed to stimulate GH secretion in humans (Frantz and Rabkin, 1965) and in ruminants (for a review see Hafs et al., 1971). In addition, GH did not appear to be released in response to milking thus confirming our earlier observations in Experiment 1 and also those of Tucker (1971). SUMMARY AND CONCLUSIONS Changes in serum hormone levels in heifers and lac- tating cows were monitored during various lactational and/ or reproductive states. In addition serum hormone content and loglo hormone content were correlated with milk pro- duction. Overall, serum prolactin averaged 52 ng/ml for pre-milking samples, 82 ng/ml immediately after milking and 41 ng/ml 1 hour after milking. Thus serum prolactin increased in response to milking but the magnitude of this response resembled a typical lactation curve. That is, the largest responses were observed during early lactation (77 ng/ml at 8 weeks) then gradually decreased as lactation advanced (essentially no responses were observed after 32 weeks). A 0.96 correlation was observed for the average serum pro- lactin content measured immediately after milking at each stage of lactation with its corresponding average milk yield. Serum glucocorticoids also increased in response to milking. Corticoid samples taken before and 1 hour after milking averaged 5.7 and 2.6 ng/ml, respectively, whereas samples taken immediately after milking averaged 11.1 ng/ml. Corticoid values immediately after milking ranged between 10 and 13 ng/ml during the first 32 weeks of lactation then declined averaging 7 to 9 ng/ml for the remainder of lac- tation. 106 107 In contrast to both prolactin and glucocorticoid data, serum GH was not increased in response to milking; GH values averaged 4.2, 4.4 and 4.2 ng/ml for pre-, immedi- ately after and 1 hour after milking samples. Overall correlations of milk production with pre- milking serum prolactin were low (r = -0.03); however pro- lactin and milk yields were significantly (P<0.01) related for samples collected immediately after and 1 hour after milking (r = 0.32 and 0.18 respectively). During the early stages of lactation, within stage of lactation correlations of serum prolactin with milk production were low and failed to reveal any meaningful trends. In contrast, serum pro- lactin was positively related to milk production during the latter stages of lactation. Overall correlations of serum GH and milk production were low (P>0.05) for each of the three samples collected around milking (r = -0.01, 0.10 and 0.07 respectively). As with prolactin, GH and milk production were not related (P>0.05) during early lactation but serum GH and milk pro- duction were negatively related after the 32nd week of lactation. This inverse relationship between GH and milk yield may reflect changes in maternal metabolism associated with pregnancy rather than with stage of lactation. Overall correlations between serum corticoids and milk production were non-significant (P>0.05) for pre- and 1 hour post milking samples (r = -0.14 and 0.01 respectively) 108 but a highly significant relationship (r = 0.19) for samples collected immediately after milking was observed. No meaning- ful relationships were observed between serum glucocorticoids and milk production within any stage of lactation. No consistent relationships were observed between serum prolactin and GH values measured throughout pregnancy r. i with milk production during the subsequent lactation. I would suggest that future experiments designed to study the relationship between serum hormones and milk pro- duction should use samples collected after hormone-releasing “W J stimuli have been applied to the animals because milking— stimulated levels of prolactin and corticoids were more closely related to milk production than basal samples. Season of the year had a significant (P<0.01) effect on serum prolactin levels in both heifers and lactating cows. Serum prolactin concentrations were highest during the warm summer months and lowest during the cold winter months. However, season of the year did not affect serum GH nor corticoid levels. Serum prolactin content was not affected by stage of pregnancy (P>0.05) although prolactin levels in non- pregnant cows were higher than those in pregnant cows (P<0.01). Glucocorticoid concentrations were also unaffected by stage of pregnancy whereas serum GH content increased (P<0.01) as pregnancy advanced. This increase in serum GH may not be related to lactation but may merely reflect 109 metabolic changes in the dam associated with advancing con- current pregnancy. Neither age of cow nor sex of the fetus had any effect (P>0.05) on serum prolactin, GH nor glucocorticoid content. Both serum prolactin content and milk production were unaffected (P>0.05) by stage of the estrous cycle. 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APPENDICES 123 APPENDIX Appendix I. Composition of reagents used in radioimmunoassay. A. Reagents for radioiodination l. 0.5 M sodium phosphate buffer, pH 7.5 Monobasic (0.5M) Add 69.005 g NaH2P04'H 0 to distilled water. Dissolve, dilute to 1 iter. Dibasic (0.5 M) Add 70.98 g NaZHP04 to distilled water. Heat to dissolve, then dilute to 1 liter. Mix monobasic and dibasic to give pH 7.5. Dispense in 1 ml portions, store at -20 C. Store the monobasic and dibasic buffers at 4 C. 2. 0.05 M sodium phosphate buffer, pH 7.5 Solution A NaH2P04.H20 . . . . . . . . . . . 2.78 g Merthiolate . . . . . . . . . . . 0.01 g Dilute to 100 ml with distilled water. Solution B NaHPO °7H 0 . . . . . . . . . . 26.825 g Merthiola e . . . . . . . . . . 0.05 g Dilute to 500 ml with distilled water. Use 16 ml Solution A, 84 ml Solution B, dilute to 400 ml with distilled water. Adjust pH to 7.5 with NaOH, if necessary. Store at 4 C. 3. Chloramine-T, l ug/ul for prolactin and 3 ug/ul for CH Upon receiving chloramine-T, dispense into small, tightly sealed vials, cover with foil, and store at -20 C. Dilute 10 mg* chloramine-T to 10 ml with 0.05 M NaP04, pH 7.5 buffer. Use within 30 minutes of preparation. Discard chloramine-T remaining in vial. * 30 mg for GH 4. Sodium metabisulfite, 2.5 ug/ul Dilute 25 mg Na28205 to 10 ml with 0.05 M NaP04, pH 7.5 buffer. Use within 30 minutes of preparation. 5. Transfer solution Sucrose . . . . . . . . . . . . . . . 1. KI ._2 . . . . . . . . . . . . . . . 0. Dilute to 1 ml with distilled water. Dispense in 1 m1 portions, store at -20 C. 69 19 124 125 Rinse solution Sucrose . . . . . . . . . . . . . . 0 KI . . . . . . . . . . . . . . . 0 Bromphenol blue . . . . . . 0 Dilute to 10 ml with distilled water. Dispense in 1 m1 portions, store at -20 C. B. Reagents for radioimmunoassay l. 0.01 M phosphate buffered saline, pH 7.0 (PBS) NaCl . . . . . . . . . . . . . . . 143 g Dibasic phosphate . . . . . . . 260 ml (See Appendix A.l) Merthiolate . . . . . . . . . . 1.75 g Dissolve in distilled water and transfer to a large container. Dilute to 17.5 liters with distilled water. Adjust pH to 7.0, if necessary, store at 4 C. 0.05 M EDTA-PBS, pH 7.0 Disodium ethylenediamine-tetraacetate (EDTA) . . . 18.612 9 Add approximately 950 ml PBS. Adjust pH to 7.0 with 5 NaOH while stirring. Dilute to 1 liter, store at 4 C. ‘PBS-l% bovine serum albumin (PBS-1% BSA) Add 990 ml PBS to beaker. Add 10 g BSA (Fraction V 35% sterile Solution, Nutritional Biochemicals Corp., Cleveland, Ohio). Mix over magnetic mixer. Store at 4 C. Hormone standards (prolactin and GH) PBS-1% BSA is used to dilute prolactin and GH; hereafter it will be referred to as buffer. Rinse a small screw-cap vial with buffer, dry. Weigh 200-400 g NIH-Bl-P or NIH-BlZ-GH on Cahn Electrobalance and transfer hormone to the screw-cap vial. Add 0.85% saline to 1 mg/ml. Add buffer to 9 volumetric flasks. Using Hamilton microliter syringes, add appro- priate volumes of the 1 mg/ml stock hormone to volumetric flasks to obtain the following concentrations: Prolactin- 0.2, 0.4, 1.0, 1.6, 2.0, 3.0, 4.0, 5.0, 6.0 and 8.0 ng/ml. GH- 0.2, 0.6, 1.0, 1.6, 2.0, 3.0, 4.0, 6.0, 8.0 and 10.0 ng/ml. Add buffer to final volume in each volumetric flask. 126 Dispense each standard in quantities suitable for one assay. Freeze in Dry Ice-ethanol, store at -20 C. For use, thaw rapidly with 38 C water. 1:400 guinea pig control serum (GPCS) Obtain blood from guinea pig that has not been used to develop antibodies. Allow blood to clot, recover serum and store the serum in convenient quantities at -20 C. Add 2.5 m1 of appropriate serum to a 1 liter volumetric flask, dilute to 1 liter with 0.05 M PBS-EDTA, pH 7.0. Divide into 100 ml portions and store at -20 C. Guinea pig anti-bovine prolactin (GPABP), identified in our laboratory as antibody I or guinea pig anti-bovine GH (GPABGH). Dilute time antisera to 1:400 with 0.05 M PBS- EDTA, pH 7.0. Dispense in small quantities, store at -20 C. On day of use, dilute the 1:400 antisera to the required concentration using 1:400 (GPCS) as diluent. Anti—gamma globulin Use sheep anti-guinea pig gamma globulin (SAGPGG) in prolactin and GH assays. Dilute antisera to required concentration with 0.05 M PBS—EDTA, pH 7.0. Store at 4 C or at -20 C. Appendix II. 127 Composition of liquid A. Steroid scintillation fluid 1968). Naphthalene PPO . . . . POPOP . . . Xylene . . p-dioxane . . Mix until dissolved. scintillation fluids. (Hafs and Armstrong, B. Bray's scintillation fluid (Bray, Naphthalene PPO . . . . Dimethyl POPOP Ethylene glycol Methanol . . p-dioxane . . Mix until dissolved. 1960) 480 g 30 g 0.3 g 2000 ml 2000 ml 240 g 16 g 0.8 g 80 ml 400 m1 3264 ml "11111111131111.1111