eomuomorm AND GONADAL HORMONES . ASSOCIATED WITH FERTILIZATION AND ' . EMBRYOGENESIS IN THE BOVINE Thesis for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY ROBERT PAUL WETIEMANN 1972 This is to certify that the thesis entitled Gonadotropin and Gonad al Hormones Associated with Fertilization and Em bryogenesis in the Bovine presemed by Robert Paul Wettemann has been accepted towards fulfillment of the requirements for Ph D degree in__D_ai ry Sience [Lleaéayfic(Z/ij§x<;€;;oi// Mfimummuun Dateiebruaw 24, I972 0-7639 ABSTRACT GONADOTROPIN AND GONADAL HORMONES ASSOCIATED WITH FERTILIZATION AND EMBRYOGENESIS IN THE BOVINE BY Robert Paul Wettemann Endocrine changes associated with early pregnancy, estrous synchronization and nonfertile inseminations were studied in four experiments with Holstein heifers and cows. The major objectives were to quantify gonadotropin and gonadal hormones during the first 75 days of pregnancy and to determine whether reduced fertility after estrous syn- chronization is related to altered hormones. Serum luteinizing hormone (LH) and prolactin were quantified by double antibody radioimmunoassays. Estradiol and progesterone were extracted from 10 ml of blood serum and isolated by column chromatography using a l x 32 cm Sephedex LH—20 column eluted with freshly distilled chloro- form: 100% ethanol (96:4). Progesterone was quantified by competitive protein binding assay and estradiol was measured by radioimmunoassay. Jugular blood was obtained from 11 nonpregnant heifers at seven intervals during the estrous cycle after Robert Paul Wettemann an infertile insemination and from 26 pregnant heifers at 18 intervals during the first 75 days of pregnancy. Serum LH concentration in heifers was greatest on the day of estrus (avg. 9.7il.4 ng/ml) when 51% of the heifers possessed LH greater than 4 ng/ml. Between—heifer variation in LH was not significant (P>.10) and LH concen- tration did not vary significantly (P>.10) from day 18 to day 75 of pregnancy. Pregnant heifers had lower serum LH compared to nonpregnant heifers (l.0i.l vs l.2i.l ng/ml, respectively, P<.lO) during days 2 through 11 after insemi- nation. Serum prolactin varied significantly (P<.005) between heifers. Average prolactin concentration of samples from individual heifers ranged from 7:3 to 56il4 ng/ml. Prolactin did not change significantly with day of the estrous cycle or pregnancy. Serum estradiol was high for l or 2 days before estrus and decreased from 12.6i2.4 pg/ml at estrus to 8.4i .6pg/ml by day 4 of pregnancy. Estradiol averaged 6 to 8 pg/ml during days 7 to 75 of pregnancy except on day 40, when four of the eight heifers had clearly elevated estra- diol. Serum progesterone increased from 0.4:.1 ng/ml at estrus to 6.8i.4 ng/ml on day ll. Maximum progesterone (about ll ng/ml) during the first 75 days of pregnancy was attained by day 18; then progesterone decreased signifi- cantly (P<.05) about 20% during the next 5 days and returned to 11 ng/ml by day 35. Robert Paul Wettemann Forty-eight cows were treated in a 2 x 2 factorial experiment with and without MGA and with and without HCG given at estrus. Serum LH was not influenced by MGA, HCG or pregnancy. Similar to the heifers, pregnant cows had lower LH than nonpregnant cows during days 2 through 25 after insemination, but difference in cows was not signifi- cant. Serum prolactin was not affected by MGA, HCG or pregnancy. On the last day of MGA treatment in the cows, proges- terone averaged 0.7:.3 ng/ml and only 3 cows had greater than 1 ng/ml. Progesterone increased from 0.3i.l ng/ml at estrus to 6.9i.6 ng/ml by day 11. Pregnant and nonpregnant cows did not differ (P>.lO) in progesterone concentration during days 2 through ll after insemination. Serum estradiol was greater in MGA treated cows than in control cows (P = .15). On the last day of MGA treatment, 47% of the cows had estradiol concentration comparable to the high values during proestrus in control cows. Within 3 days after MGA withdrawal, estradiol increased as much as 10-fold in some cows. Estradiol concentration was similar in pregnant and nonpregnant cows during days 2 through 11 after insemination. Serum estradiol averaged about 6 pg/ml during days 7 through 75 of pregnancy and, similar to the heifers, 50% of the cows had elevated estradiol between days 30 and 42 of pregnancy. Robert Paul Wettemann Serum concentrations of LH, prolactin and progester— one were not altered after estrous synchronization with MGA. But concentrations of estradiol comparable to those at proestrus were present at MGA withdrawal and possibly began during treatment. Prolonged elevations in serum estradiol during and following progestogen treatment may account for the infertility of inseminations at the synchronized estrus. GONADOTROPIN AND GONADAL HORMONES ASSOCIATED WITH FERTILIZATION AND EMBRYOGENESIS IN THE BOVINE BY Robert Paul Wettemann 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 To My Parents ii ‘4 BIOGRAPHICAL SKETCH of Robert Paul Wettemann I was born on November 12, 1944, in New Haven, Connecticut. I attended public schools in Guilford, Connecticut, graduating in June 1962. In September 1962, I enrolled at the University of Connecticut, majoring in Dairy Science, and received a Bachelor of Science degree in June, 1966. I accepted a graduate assistantship at Michigan State University in September, 1966, and I was granted an National Institutes of Health Predoctoral Fellowship in March, 1968. In September 1968, I received the Master of Science degree and my thesis was entitled, "Endocrine Changes Influencing Sperm Capacitation". I completed the requirements for the PhD degree in February, 1972, and I shall be employed by Oklahoma State University as an environmental physiologist. iii ACKNOWLEDGMENTS The thoughtful guidance and willing assistance of my major professor, Dr. H. D. Hafs are invaluable to me. The facilities and assistance provided by Dr. C. A. Lassiter have made graduate school an enjoyable experience. I appreciate the interest and advice of Dr. H. A. Tucker, Dr. E. M. Convey and Dr. L. J. Boyd during my gradu— ate program. I am grateful for advice and consent given by Drs. L. D. McGilliard, F. M. Rottman and E. P. Reineke. I wish to thank my colleagues Dr. Lee Edgerton, Win Ingalls, Jim Koprowski, Dr. Wayne Oxender, Manley Pratt, Norm Rawlings, Bill Smith, Val Smith, Dr. Lloyd Swanson and Joe Zolman for their assistance at the barn and with labo- ratory tasks. I would like to thank Dr. D. A. Morrow for assistance in performing rectal palpations and Dr. Roger Neitzel for computer programing. I thank Patty Kaneshiro for her skillful laboratory assistance. I greatly appreciate the encouragement and under- standing of my wife Grace, during my graduate studies. I wish to thank the National Institutes of Health for my fellowship and the Endocrinology Study Section of the National Institutes of Health, E. R. Squibb and Sons and The Upjohn Company for gifts of hormones. I am obliged to Dr. S. A. Tillson for an antibody to estradiol. iv TABLE OF CONTENTS DEDICATION '. . . . . . . . . . . . . BIOGRAPHICAL SKETCH . . . . . . . . . . ACKNOWLEDGMENTS . . . . . . . . . . . LIST OF TABLES . . . . . . . . . . . LIST OF FIGURES . . . . . . . . . . . LIST OF APPENDICES . . . . . . . . . . INTRODUCTION 0 O O O O O O O O I O 0 REVIEW OF LITERATURE . . . . . . . . . . Pregnancy . . . . . . . . . . . . Endocrine Changes During the Estrous Cycle . Ovulation and Fertilization . . . . . . Regulation of the Corpus Luteum . . . . . Implantation . . . . . . . . . . . Endocrine Activity During Pregnancy . . . Luteinizing Hormone . . . . . . . . Prolactin . . . . . . . . . . . Estrogens . . . . . . . . . . . Progesterone . . . . . . . . . . Estrous Synchronization with Progestogens . . Administration and Effectivensss . . . . Induced Ovulation . . . . . . . . . Fertility . . . . . . . . . . . . Mode of Action . . . . . . . . . . Rats and Guinea Pigs . . . . . . . . Primates . . . . . . . ." . . . . Sheep . . . . . . . . . . . . cattle O O O O O O O O O O O 0 MATERIALS AND METHODS . . . . . . . . Experimental Animals and Designs . . . . . Experiment I: Changes in Gonadotropin and Gonadal Hormones Associated with Early Pregnancy and after Nonfertile Inseminations V Page ii iii iv viii xi Page Experiment II: Serum Luteinizing Hormone (LH) and Prolactin after MGA Withdrawal . . . . 24 Experiment III: Induced Ovulation and Ova Recovery . . . . . . . . . . . . . 24 Experiment IV: Gonadotropin and Gonadal Hormones after Estrous Synchronization and after Fertile and Nonfertile Inseminations . . . . . 25 Steroid Hormone Assays . . . . . . . . . 27 Isolation of Steroid Hormones . . . . . . 27 Thin Layer Chromatography . . . . . . . 27 Column Chromatography . . . . . . . . 28 Extraction Procedure . . . . . . . . . 32 Estradiol . . . . . . . . . . . . . 32 Protein Binding Assay . . . . . . . . 32 Radioimmunoassay (RIA) . . . . . . . . 34 Comparison of Methods . . . . . . . . 41 Progesterone Assay . . . . . . . . . . 44 Protein Hormone Assays . . . . . . . . . 46 Luteinizing Hormone (LH) . . . . . . . . 46 Prolactin~ . . . . . . . . . . . . . 48 Statistical Analysis . . . . . . . . . . 48 RESULTS AND DISCUSSION . . . . . . . . . . 52 Experiment I: Changes in Gonadotropin and Gonadal Hormones Associates with Early Pregnancy and after Nonfertile Inseminations . . . . . 52 Size and Behavior at Breeding . . . . . . 52 Fertility . . . . . . . . . . . . . 52 Endocrine Changes . . . . . . . . . . 53 LH . . . . . . . . . . . . . . 53 Prolactin . . . . . . . . . . . . 56 Estradiol . . . . . . . . . . . . 57 Progesterone . . . . . . . . . . . 58 Relationships of Hormones . . . . . . . 61 Experiment II: Gonadotropin Levels after MGA Withdrawal . . . . . . . . . . . . . 63 Behavior and Fertilit . . . . . . . . . 63 Endocrine Changes . . . . . . . . . . 63 LH . . . . . . . . . . . . . . 63 Prolactin . . . . . . . . . . . . ’ 66 Experiment III: Induced Ovulation and Ova Recovery . . . . . . . . . . . . . 66 Experiment IV: Gonadotropin and Gonadal Hormones after Estrous Synchronization and Insemination . 73 Reproductive Performance . . . . . . . . 73 Endocrine Changes . . . . . . . . . . 75 LH . . . . . . . . . . . . . . 75 vi Prolactin Estradiol Progesterone GENERAL DISCUSSION SUMMARY AND CONCLUSIONS BIBLIOGRAPHY APPENDICES vii Page 82 87 95 103 116 120 133 10. ll. 12. 13. LIST OF TABLES Page Relative activity of selected steroids in the estrogen protein binding assay . . . . . . 37 Levels of estradiol in peripheral blood serum from Holstein heifers . . . . . . . . . 43 Precision of the determination of LH in analysis of different serum pools in different assays . 47 Precision of the determination of prolactin in analysis of different serum pools in different assays . . . . . . . . . . 49 Split plot analysis of hormones during early pregnancy . . . . . . . . . . . . . 50 Orthogonal contrasts used to partition the 17 degrees of freedom for stage of pregnancy . . 51 Some estrual criteria of nine Holstein heifers after MGA treatment . . . . . . . . . 64 , Serum LH and prolactin in Holstein heifers after MGA treatment . . . . . . . . . 65 Ovulation rate and ovarian characteristics of Holstein cows at induced ovulation . . . . 69 Relation between ova recovery and stage of the estrous cycle cows were started on MGA treatment 0 O O I O O O O O C I O I 7]- Fertility of ova after controlled ovulation of Holstein cows . . . . . . . ,. . . . 72 Reproductive performance of cows after estrous synchronization . . . . . . . . . . . 74 Serum LH in pregnant cows after MGA and HCG treatment . . . . . . . . . . . . . 77 viii Table 14. 15. 16. l7. 18. 19. 20. 21. 22. 23. 24. Page Serum LH in nonpregnant cows after MGA and HCG treatment 0 O O C I O O O O O O I O 79 Serum LH in cows: InfluenCe of genetic classifi- cation, pregnancy and HCG treatment . . . . 81 Serum prolactin in pregnant cows after MGA and HCG treatment . . . . . . . . . . . 83 Serum prolactin in nonpregnant cows after MGA and HCG treatment . . . . . . . . . . 85 Serum estradiol in pregnant cows after MGA and HCG treatment . . . . . . . . . . . 88 Serum estradiol in nonpregnant cows after MGA and HCG treatment . . . . . . . . . . 90 Cows with high serum estradiol . . . . . . 92 Serum estradiol in cows after MGA withdrawal . . 94 Serum progesterone in pregnant cows after MGA and HCG treatment . . . . . . . . . . 97 Serum progesterone in nonpregnant cows after MGA and HCG treatment . . . . . . . . . . 100 Serum progesterone in cows: Influence of genetic classification, pregnancy and HCG treatment . 102 ix Figure 10. 11. 12. 13. LIST OF FIGURES- Elution profile of steroids in an ether extract of bovine serum from a Sephadex LH-20 column using chloroform:ethanol (24:1) as the solvent . . . . . . Standard curve for l7B-estradiol and cross- reactions with estrone and estriol using a rabbit uterine cytosol binding protein Standard curve for 17B-estradiol and cross- reaction with estrone and estriol using a radioimmunoassay (antibody against 1,3,5 (10)- estratriene-3, 178-diol, 178- succinyl-bovine serum albumin) . . . Serum LH in heifers after insemination . Serum prolactin in heifers after insemination Serum estradiol and progesterone during pregnancy in heifers . . . . . . . Serum LH, prolactin, estradiol and progesterone during pregnancy in a typical heifer . Serum LH and prolactin in heifers after MGA Withdrawal . . . C C C . C C . Serum LH in cows before and after insemination Serum estradiol in cows before and after insemination . . . . . . . . . Serum estradiol in MGA treated cows before and after insemination . . . . . . . Serum progesterone in cows before and after insemination O O O O O O O O 0 Serum prolactin in cows before and after insemination . . . . . . . . . Page 29 35 39 54 54 59 59 67 105 108 108 110 110 Appendix I. II. III. IV. VI. VII. VIII. IX. LIST OF APPENDICES Preparation of liquid scintillation fluids Composition of buffers used in estradiol assays O O O O O O O O O O O O Fertility of cattle after estrous Synchronization with progestogens . . . Serum LH and prolactin in pregnant and nonpregnant heifers . . . . . . . Serum estradiol and progesterone in heifers during early pregnancy . . . . . . Some within day correlations between pituitary and ovarian hormones in blood serum during early pregnancy in heifers . . . . . Serum LH and prolactin in heifers carrying female or male fetuses . . . . . . Split plot analysis of luteinizing hormone for Experiment IV . . . . . . . . Split plot analysis of luteinizing hormone for cows in treatments I and II of Experiment IV . . . . . . . . . xi Page 134 135 136 137 138 139 140 141 142 INTRODUCTION Our knowledge of the hormonal changes associated with fertilization and embryogenesis is limited in the bovine. Advances in techniques used in endocrinology within the last 3 years made it possible to measure blood serum luteinizing hormone (LH) and prolactin by radioim- munoassay (RIA). Similarly, progesterone in peripheral serum can be quantified by gas—liquid chromatography, protein binding assay or by RIA, but there were no reports of determination of serum estradiol in cattle. Therefore, I set out to develop techniques to quantify estradiol in blood serum of cattle, and to relate serum estradiol with other steroid and gonadotropin hormones and with fertility in cattle. More specifically, the major objectives of this thesis were to determine ovarian and gonadotropin hormones during the first 75 days of pregnancy in heifers and cows, and to determine whether reduced fertility after estrous synchronization is related to altered hormones. In the future, general acceptance of artificial insemination of cattle probably will depend upon high fertility following estrous cycle synchronization because of increased labor costs, larger herds, and fewer farms. Ovulation can be controlled with progestogens, but pre-I sently fertility following ovulation synchronization is usually reduced 10 to 30 percentage points. This reduced fertility makes estrous synchronization economically un- feasible in most commercial herds. Some possible causes of the reduced fertility after estrous synchronization are (l) altered sperm or ova transport (2) unfavorable uterine environment for sperm or ova (3) altered sperm capacitation (4) aged ova or (5) inhibition of implantation. Possibly, large ovarian follicles known to persist during progestogen treatment may secrete abnormal amounts of estrogen. Or progesterone may be secreted by abnormal luteal or ovarian interstitial tissue. Alteration of sperm or ova transport could be associated with abnormal contractility of the uterus or oviducts caused by altered estrogen secretion or by residual progestogens. An un- favorable uterine environment could be associated with increased estrogen secretion prior to proestrus, higher than normal quantities of progesterone or progestogen present during proestrus. The stimulation of uterine glands by these steroids could alter uterine secretions and also alter sperm capacitation. In other words, most of the hypothetical causes of reduced fertility at the synchronized estrus probably are associated with or caused by altered hormone levels. Information from this study will characterize hormones during early pregnancy in normal cattle, then altered hormone secretion after estrous synchronization can be determined. These data may suggest hormones which are altered in infertile cattle and possibly help to in- crease fertility at synchronized ovulation. REVIEW OF LITERATURE Pregnancy Pregnancy begins at fertilization and terminates at parturition or abortion. During this reproductive stage the secretion of endocrine glands is altered compared with the estrous cycle. Endocrine Changes During the Estrous Cycle Pituitary luteinizing hormone (LH) decreases during estrus (Rakha and Robertson, 1965; Hackett and Hafs, 1969) and plasma LH increases (Anderson and McShan, 1966). The ovulatory surge of LH, which begins shortly before estrus and is maintained about 6 hours (Swanson and Hafs, 1971), may be initiated by increased serum estradiol since estra- diol injection will cause release of LH in cattle (Howland et al., 1971). Recently we demonstrated that the proestrus increase in serum estradiol usually precedes LH release by l or 2 days (Wettemann et al., 1972). Hansel and Snook (1970) and Schams and Karg (1969) reported small midcycle peaks of serum LH occurring on days 8 to 10. Presently, techniques are not available to quantitate FSH in bovine plasma. Pituitary FSH decreases during estrus (Rakha and Robertson, 1965; Desjardins and Hafs, 1968) and Hackett and Hafs (1969) found that this decrease started between days 18 and 20 of the cycle. Therefore, FSH as well as LH probably participate in estrus and ovulation. Pituitary prolactin declines from estrus to day 2 of the Cycle (Sinha and Tucker, 1969) and Swanson et a1. (1972) observed higher serum prolactin at estrus compared with metestrus, diestrus, and proestrus. Raud et a1. (1971) also observed elevated serum prolactin during proestrus or estrus; however, Schams and Karg (1970) found no significant changes in serum prolactin during the estrous cycle in milking cows. Mature nonpregnant cattle exhibit estrus once about every 21 days, or injection of estrogen will induce estrus (Foote and Walker, 1961: Carrick and Shelton, 1969). Estra- diol in peripheral blood serum of heifers increases to about 10 pg/ml during the 2 or 3 days before estrus, and remains at about 30 to 40% of the proestrus level throughout the luteal phase of the estrous cycle (Wettemann et al., 1972). The major urinary estrogens during the estrous cycle are estrone, l7a-estradiol and l7B-estradiol (Garverick et al., 1971) and excretion is greatest from 3 days prior to estrus until 3 days after estrus. During the luteal phase of the cycle, estrone is excreted in greatest quantity, whereas l7d-estradiol and l7B-estradiol predominate near estrus. Progesterone and 20B-hydroxy-pregn-4-en-3-one (208-01) in the corpus luteum were greatest from days 9 to 15 of the cycle, however the concentration of 208-01 was only 2 to 30% of the concentration of progesterone (Mares et al., 1962). Hafs and Armstrong (1968) confirmed that both progesterone and 208-01 concentrations of the corpus luteum were greatest at mid-cycle and steroidogenesis also was greatest from days 11 through 20 of the cycle. Although Gomes et a1. (1963) could not detect progesterone in ovarian venous plasma at estrus using macro techniques, they found that progesterone concentration increased from low levels on day 2 to a maximum on day 15 and then de- creased before the next estrus. At estrus, Plotka et a1. (1967), found 10.1 ng/ml of progesterone in peripheral plasma and a maximum concentration of 25.8 ng/ml on day 14. With more sensitive and precise techniques (Stabenfeldt et al., 1969; Swanson et al., 1972), progesterone concen- tration in peripheral plasma was less than 0.5 ng/ml at estrus and increased to about 7 ng/ml on days 15 to 18 of the estrous cycle. Ovulation and Fertilization Luteinizing hormone causes rupture of the graffian follicle and discharge of the ovum. In heifers, ovulation occurs about 10 hours after the end of estrus and cows ovulate slightly later (Trimberger, 1948). Swanson and Hafs (l971).observed that ovulation occurred about 32 hours after the ovulatory surge of LH in heifers. Fertility is maximal when inseminations are performed 7 to 24 hours before ovulation; reduced fertility results from earlier or later inseminations (Trimberger, 1948). Regulation of the Corpus Luteum Factors controlling the life span of the corpus luteum have been studied extensively. An intriguing question is, what causes the corpus luteum of pregnancy to be maintained whereas the corpus luteum of the estrus cycle regresses within about 3 weeks? The presence of a foreign body which distends the bovine uterus alters the cycle length. If the foreign body is inserted within 2 or 3 days after ovulation, the cycle is shortened but there is no change in cycle length when it is inserted on day 6 or 8 (Yamauchi and Nakahara, 1958). Ginther et a1. (1966) observed that an intra-uterine plastic coil had a unilateral inhibitory influence on the corpus luteum. Injection (SQ) of pharmacological doses of oxytocin during the first week after estrus also shortens the cycle (Armstrong and Hansel, 1959) because corpora lutea fail to develop. Although oxytocin shortens the cycle in unicornual heifers with the retained horn adjacent to the corpus luteum, it is without effect when the retained horn is opposite to the corpus luteum (Ginther et al., 1967). This suggests that at least part of the oxytocin effect is through a local uterine-ovarian relationship. Hyster- ectomy also alters ovarian activity in cattle. Removal of the uterus 5 to 12 days post estrus maintains the corpus luteum for more than 150 days (Wiltbank and Casida, 1956). The pituitary gland has an active role in maintenance of the corpus luteum. When cows are injected with purified bovine LH, the inhibitory effect of concurrently injected oxytocin on luteal tissue is abolished (Hansel and Seifart, 1967), but prolactin, FSH or growth hormone are ineffective in overcoming the luteostatic influence of oxytocin. Injec- tion of human chorionic gonadotropin (HCG) into heifers from day 15 through 26 of the cycle prolongs the cycle (Wiltbank et al., 1961). Exogenous steroid hormones also alter the life eXpectancy of the corpus luteum. The corpus luteum re- gresses early if estradiol valerate is injeCted during days 3 to 9 of the cycle, but if injections are on days 15 or 16 the corpus luteum doesn't regress and large ovarian cysts develop (Wiltbank, 1966). Estrogen injection also will cause regression of corpora lutea in pregnant or hysterectomized heifers. Since injection of human chorionic gonadotropin (HCG) negates the luteolytic effect of estrogen, (Wiltbank, 1966) the estrogen may act by decreasing plasma LH cOncentration. Injection of progesterone during the first 10 days of the cycle shortens the cycle (Woody et al., 1967). Ginther (1970) confirmed the shortening effect of progester- one given early in the cycle and found that simultaneous administration of HCG maintained normal cycle length. Progesterone injections from day 3 to 8 do not alter the cycle but injections after day 8 lengthen the cycle. Thus progesterone administration may inhibit LH release which is necessary for ovulation and corpus luteum growth. Implantation Based on studies in the rat, LH and estrogen may have a causative effect on implantation in bovine. Macdonald et a1. (1967) demonstrated that LH initiates blastocyst implantation in rats and this effect of LH is probably mediated through estrogen since Yoshinaga and Hosi (1961) found that estrogen caused implantation in lactating rats. Hormonal changes have not been related to embryo develop- ment in bovine. An embryo is considered implanted when it is fixed in one position in the uterus. The bovine embryo remains in the uterine cavity and is only loosely attached before formation of the placenta (Hafez, 1969). During formation of the placenta (syndesmo-chorial type), the uterine epithelium is erroded and the chorionic ectoderm comes in direct contact with the vascular uterine connective tissue (Arey, 1954). In cattle, the ovum reaches the uterus about 4 days after estrus and the blastocoele begins forming at 7 days (Winters et al., 1942). About the eleventh or 10 twelveth day, the embryo attaches loosely to the uterine wall and the chorion starts elongating. The placental attachment is gradual, with the first placental plates forming on the chorionic membrane at 30 days and in the body of the uterus at 35 days (Melton et al., 1951). By 35 days the placental attachment is sufficiently developed so that the embryo can receive some of its nourishment through the cotyledons. Endocrine Activity during Pregnancy Luteinizing Hormone.--Recently Edgerton and Hafs (1971) observed lower serum LH concentration during the first 18 days post—insemination in lactating cows that conceived than in cows which did not conceive. Henricks et a1. (1970) observed that nonpregnant cows had higher LH on day 8 and pregnant cows had higher LH on day 15. Randel and Erb (1971) found that LH concentration in serum was low at day 7 of pregnancy and varied little until day 260. But with more frequent sampling, Schams (1969) observed elevated LH at 52 days of pregnancy in one cow and at 61 days in another. Prolactin.--Reports of serum prolactin during pregnancy in the bovine are limited. Serum prolactin didn't differ between pregnant and non pregnant lactating cows during the first 18 days after insemination, but there was extreme variation between animals and days ll (Edgerton and Hafs, 1971). Serum prolactin decreased with advancing pregnancy in sheep (Arai and Lee, 1967) and Davis et a1. (1971) found that serum prolactin in ewes stabilized at the third to fourth month of pregnancy. Estrogens.-—ln vitro evidence summarized by Mellin and Erb (1965) indicates that estrogens can be formed from acetate, cholesterol, progesterone or neutral steroids in the bovine adrenal, ovary and placenta. Estrogens are transformed to less active biological forms and these metabolites are excreted in feces and urine. The major excretory forms are estrone, l7d-estradiol and 17B—estradiol, especially conjugated as sulfates and glucuronides. The study of estrogen activity during early pregnancy until recently has been limited to changes in urinary estro- gens. But estrone in peripheral blood during late gestation has been quantified since concentrations at this time are about 1000 fold greater than during early pregnancy (Robinson et al., 1970). Since Randel et al. (1971a) observed higher urinary estrogen excretion during the first 7 days postbreeding in infertile cows as compared with pregnant cows, they speculated that altered estrogen metabolism may influence tubal transport of ova and the uterine environment. Pregnant cows excreted more 17d- estradiol than nonpregnant cows 42 days after breeding (Randel et al., 1971b) and the excretion of l7d-estradiol on day 42 of pregnancy also was significantly greater than 12 on days 35 and 65 (Randel and Erb, 1971). With advancing pregnancy from day 65 to day 230, excretion of l7d-estradiol and estrone increased 20-fold and 6-fold, respectively. Excretion of 17B-estradiol was less than that of 17a- estradiol and estrone, and it increased only slightly after mid pregnancy. Ovariectomy on days 111 to 251 of pregnancy does not significantly decrease urinary estrogens (Erb et al., 1968c), so the ovary isn't the major source of estrogen during late pregnancy. Findlay and Cox (1970) found low concentrations of unconjugated estrogens in fetal sheep, but conjugated forms of l7d-estradiol and 17B-estradiol with lesser quantities of estrone were present. Total estrogen concentration in plasma increased in both male and female ovine fetuses from 70 to 125 days of gestation. By perfusion in vivo of the auto—transplanted ovary of the ewe, Rado et a1. (1970) determined that 17B-estradiol was the major phenolic steroid synthesized from either testos- terone or androstenedione. Though smaller amounts of estrone were also produced, l7a-estradiol, estriol or conjugated estrogens were not isolated. Progesterone.--Progesterone concentration in corpora lutea and ovarian veins were used as indicators of ovarian function before techniques were available to quantitify peripheral plasma progesterone. Erb et al. (1968b) deter- mined that the corpus 1uteum contained 94% of the total l3 progestogens in ovaries and found no relationship between progesterone concentration in jugular plasma, ovarian venous plasma and content of the ovary during pregnancy. Although progesterone concentration in ovarian venous plasma decreases during pregnancy, jugular plasma levels increase (Erb et al., 1968a). Zimbelman et a1. (1961) observed a decreased percentage of functional cells in corpora lutea on day 23 of pregnancy compared with days 14 and 18, but Wickersham and Tanabe (1967) concluded that the functional activity of the corpus luteum is constant throughout pregnancy based on luteal weights, progesterone concentration and d9 £939 progesterone synthesis. Although the corpus luteum is necessary for normal pregnancy, abortions usually do not occur when ovariectomy takes place after 200 days of pregnancy although gestation is usually shortened by about 2 weeks (Estergreen et al., 1967). Early parturition could be caused by the decrease in plasma progesterone observed after ovariectomy performed late in pregnancy (Erb et al., 1968c). Tanabe (1970) reported that more progesterone is needed to maintain pregnancy after the corpus luteum is enucleated during early pregnancy than during mid pregnancy. Apparently extraovarian progesterone in the bovine increases as pregnancy progresses. The source of progesterone could be the placenta (Ainsworth and Ryan, 1967) or the adrenal (Stormshak and Erb, 1961). l4 Shemesh et a1. (1968) first observed an influence of the conceptus on plasma progesterone at 19 days after breeding, and Henricks et a1. (1970) reported higher pro- gesterone in pregnant than nonpregnant cows from 10 to 14 days after breeding. In another study, Henricks et al. (1971a) observed higher plasma concentration of progester- one in pregnant heifers by 9 days after estrus than in nonpregnant heifers. There is disagreement as to the progesterone secretion pattern during pregnancy. Early workers found that progesterone concentration in peripheral plasma was relatively constant from day 32 to day 250 of pregnancy (Short, 1958). Randel and Erb (1971) observed a rapid increase in plasma progesterone concentration from 2 ng/ml at estrus to 9 ng/ml by day 7 of pregnancy, followed by a linear increase until day 42. Then plasma progesterone decreased from 20 ng/ml on day 42 to 10 ng/ml on day 125 and then increased to 24 ng/ml by day 200. Stabenfeldt et a1. (1970) found that plasma progesterone concentration was stable from 140 to 200 days of pregnancy but the level of progesterone appeared lower than during 10 to 20 days of pregnancy. Estrous Synchronization with Progestogens Soon after progesterone was isolated from corpus luteum tissue (described by Petrow, 1970), Makespeace et a1. 15 (1937) determined that injection of progesterone would inhibit ovulation after mating in estrogen primed rabbits. Willett (1950) injected heifers with progesterone starting on day 14 or 15 of the estrous cycle and inhibited estrus during the l3-to 17-day treatment. Estrus occurred about 5 days after progesterone withdrawal, but only 50% of the heifers conceived. Although fertility decreased after progesterone injections, the infertility was temporary and limited to the estrus following treatment (Trimberger and Hansel, 1955). In a recent review (Petrow, 1970), the structure and biological activity of contraceptive proges- togens are described. Administration and Effectiveness Progesterone is not used to synchronize estrous cycles because it is only slightly active orally and it is costly to produce relative to alternatives. Many orally active progestogens have been synthesized. Although the effective dose of different progestogens varies, decreased fertility is observed at the first estrus after withdrawal of all progestogens including progesterone. Progestogens have been administered by injection, orally and vaginal pessaries, but use of implants maybe more efficient in most management practices. Since melengestrol acetate (MGA, The Upjohn Co.) has been studied extensively and is the progestogen used 3n Tfiw .l‘w 16. in my experiments, I shall discuss its biological effects in cattle. To my knowledge, except for potency, biological action of MGA resembles that of most other progestogens. Whether injected or ingested, MGA is a potent synthetic progestogen (Zimbelman and Smith, 1966a). Heifers exhibit estrus 3 to 6 days after the last feeding of MGA, depending partly on the dose. The number of animals with detectable corpora lutea decrease during MGA treatment as the number of animals with follicles increase (Zimbelman and Smith, 1966b). Follicular size increases during treatment and based on proestrus behavior, vaginal mucus and fern patterns in cervical mucus, there appears to be increased estrogenic activity.. During MGA treatment, estrus and ovulation can be induced in heifers by injection of estrogens (Smith and Zimbelman, 1968). Although injection of gonadotropins will also cause ovulation during MGA treatment, functional corpora lutea do not always develop. Induced Ovulation Graves and Dziuk (1968) used 6d-methyl-l7a-acetoxy- progesterone (MAP) to synchronize estrus in cattle, then induced ovulation by HCG injection 60 hrs after the last feeding of MAP. Ovulation occurred about 40 hrs after HCG injection and resultant ova were fertilizable. Although induction of ovulation with HCG after estrus synchronization did not influence incidence of estrus or conception, Baker l7 and Coggins (1968) found that it eliminated the problem of estrus detection and reduced the time required for re- straining and inseminating cows. Fertility Progestogens can effectively synchronize estrus and ovulation in cattle but fertility at the first estrus after progestogen feeding usually is reduced significantly. Results from fertility trials using various progestogens are presented in Appendix Table III. A summary of these data indicated reduced fertility at first estrus after synchronization in 16 experiments; 420 control cows averaged 55% conception at first service but 609 treated cows had only 42% cOnception. Zimbelman et a1. (1970) summarized data for 5 years involving 24 studies in which 1853 cows were synchronized with MGA and there were 537 control cows. Conception rate from inseminations at the first synchronized estrus was about 70% of the rate of controls. This reduction of fertility after estrous synchronization greatly limits the usefulness of progestogens for this purpose. Mode of Action Rats and Guinea Pigs.--Labhsetwar (1968) observed that when rats were given chlormadinone, a potent synthetic progestogen, ovaries as well as ovarian interstitial tissue atrOphied and ovarian compensatory hypertrOphy was blocked in unilaterally spayed rats. Both pituitary LH and FSH 18 concentration were increased by progestogen injection, suggesting that release of these hormones is blocked but synthesis is not altered. Although plasma LH was depressed after progestogen treatment (Schally et al., 1968), adminis- tration of luteinizing hormone releasing factor MEG? caused elevation of plasma LH. Since LRF can overcome the blocking effect of progestogens on LH release, progestogens probably affect blood LH at the level of the hypothalamus or higher brain centers.’ Based upon intracranial implants of MAP in female guinea pigs, progestogens can act directly on the anterior pituitary to inhibit ovulation (Malven and Ruiz-Diaz, 1971). While MAP implants into the arcuate nucleus of the hypo- thalamus were partially efflective in inhibiting ovulation, implants in other areas of the hypothalamus had no effect. Primates.--Ovulation was blocked in cycling Rhesus monkeys by injection of progesterone (Spies and Niswender, 1971). The ovulatory surge of LH was inhibited by pro- gesterone injections but basal LH levels were not altered. Similarly, the ovulatory surge of LH was suppressed during administration of progestogens in women (Mishell and Odell, 1971; Saunders et al., 1971), but basal urinary excretion of LH was not depressed (Larsson-Cohn et al., 1970). Increased urinary excretion of estrogen also was observed when women were treated with progestogens (Larsson-Cohn et al., 1970). Llerena et a1. (1969) found a greater 19 concentration of LH but not FSH in peripheral plasma than in ovarian venous plasma of women during the menstrual cycle. This difference in LH concentration was not apparent when women were on progestogen treatment. Therefore, LH may be metabolized by the ovary during a normal menstrual cycle but not when ovulation is blocked by progestogens. §h§_p,--Injection of female sheep with progesterone for 10 days did not alter blood serum LH as determined by ovarian ascorbic acid depletion assay (McDonald and Clegg, 1967), but elevated serum LH occurred 4 to 6 days after withdrawal in both intact and ovariectomized ewes. Exogenous progestogen did not alter cyclic changes in luteal function of ewes when treatment started after formation of corpora lutea (Smith and Robinson, 1969). However when progestogen was given during formation of the corpus luteum, full luteal size was not attained and early luteal regression occurred. Pelletier and Thimonier (1969) observed that the ovulatory surge of LH was smaller than normal after treat- ment of ewes with progestogens and estrogen secretion also was altered. Using fluorimetry, Smith and Robinson (1970) noted decreased estrogen in ovarian venous blood at the synchronized estrus after treatment with a low dose of progestogen but the level of estrogen in plasma of ewes treated with a higher dose was similar to controls. Recently 20 Smith and Allison (1971) reported that the synchronized estrus was shorter than a normal estrus, and daily maximum cervical mucus secretion occurred before rather than after the onset of estrus. Although the daily maximum secretion of mucus was less than normal after treatment, the total volume produced during proestrus and estrus was similar. This alteration of cervical mucus secretion may be related to changes in gonadal hormone secretion. Cattle.--Administration of MGA to cattle did not influence follicular activity, in the presence of a corpus luteum,but follicular fluid weights increased up to 3-fold in the absence of a corpus luteum (Zimbelman, 1966). Although pituitary FSH was not altered significantly, pituitary LH was increased by MGA treatment. Hill et a1. (1971) also observed more large follicles in heifers treated with MGA and basal plasma LH tended to be higher in treated than control heifers. Plasma progesterone concentrations during MGA treatment were similar to comparable days during the estrous cycle. Limited data (Guthrie et al., 1970) suggest that follicles may luteinize and become atretic during MGA treatment. Lamond et al. (1971a) observed higher progesterone after MGA withdrawal than that usually found in the follicular phase of the estrous cycle. When heifers were fed MGA for several months, adrenal weight, plasma cortisol, adrenal cortisol concen- tration and adrenal fasiculata layer widths decreased .m-‘.-mz’ I‘h . ‘7 21 compared with controls (Purchas et al., 1971). Average daily gain in carcass weight was greater but plasma growth hormone concentration was significantly lower in MGA treated animals. Heifers also had increased mammary DNA and RNA compared with control heifers after several months of MGA treatment (Pritchard et al., 1972). MATERIALS AND METHODS Experimental Animals and Designs Holstein heifers or cows were housed either in Michigan State University's stanchion barn or in loose t housing. In the stanchion barn, cows were observed at least once daily, in the morning, for signs of estrus. Heifers and cows in loose housing were observed for signs ; of estrus twice daily; between 7:30 and 8:30 am and between 5 and 6 pm. For estrous synchronization experiments, melengestrol acetate (MGA, The Upjohn Co., Kalamazoo, Michigan) premix was mixed with a concentrate mixture so the daily dose could be administered in 2 pounds of grain mix. Experiment I: Changes in Gonadotropin and Gonadal Hormones Associated with Early Pregnancy and after Nonfertile Inseminations. The purpose of this experiment was to quantify endocrine activity during early pregnancy. Twenty-eight heifers, 14 to 15 months of age and during approximately their seventh estrous cycle, were managed in loose housing for this study. Semen from Baron, a bull with high fer- tility, was used for all artificial inseminations during the breeding period (October 15, 1969 to November 26, 1969). When estrus (standing heat) occurred before noon, heifers 22 23 were inseminated in the late afternoon of the same day. Those heifers first observed in estrus after noon were inseminated the next morning. If heifers did not conceive to the first insemination, the above breeding regime was repeated at the next estrus. Heifers not conceiving to the second insemination were removed from the study. Jugular blood was obtained by venipuncture from all heifers when first observed in standing heat (day 0). Then each heifer was bled in the afternoon on days 2,4,7, ll,l8,20,22,25,30,35,40,42,45,50,60,63 and 75 of pregnancy or until she returned to estrus. Blood was transferred from the syringe to polypropylene centrifuge tubes containing 31.7 mg oxalic acid and centrifuged at 650 xg for 15 minutes to remove the blood cells. Plasma was dacented into another centrifuge tube containing 27.8 mg calcium chloride and stored at 5 C to allow clotting. Within 1 to 2 days the clotted plasma was centrifuged and the serum was transferred to 7—dram plastic vials and stored at -20 C until assayed for hormone concentration. Pregnancy was confirmed by rectal palpations 40 to 60 days postinsemination. Withers height and body weight also were recorded at the beginning of the breeding period. 1 Unless stated otherwise, these experimental details (insemination time, bleeding regime, preparation of serum and pregnancy diagnosis) were applicable in all subsequent experiments. 24 Experiment II: Serum Luteinizing Hormone (LH) and Prolactin after MGA Withdrawal This experiment was conducted to determine when the ovulatory surge of LH occurs after oral administration of MGA and if prolactin is elevated at the synchronized estrus. Beginning February 1, 1970, nine heifers were fed 1.0 mg of MGA daily for 18 days in individual stalls. After the last feeding, the heifers were moved to loose housing, cohabited with 35 heifers of similar age, and observed twice daily 1.l_~_.s—“‘e— .r’ flmrestrous. JUgular blood was obtained each afternoon for 3 8 days, starting on the last day of MGA administration. Heifers were inseminated at the first estrus after treat- ment and at the subsequent estrus. Palpations per rectum at 13 days after MGA withdrawal determined if ovulation had occurred. Experiment III: Induced Ovulation and Ova Recovery This experiment was designed to estimate when estrus synchronized cows ovulate after injection of Human Chorionic Gonadotropin (HCG) and to determine the fertility of the ova. Twenty—five first calf heifers at 60 to 90 days post- partum were group fed 1.0 mg MGA each, daily for 14 days. They were injected with 2500 IU of HCG (Squibb Chorionic GonadotroPin, E. R. Squibb and Sons, Inc., New York) on the morning of day 17 (3 days after the last MGA feeding) and inseminated on the morning of days 18 and 19. The time of ovulation was determined by twice daily (8 am and 5 pm) 25 palpations starting on the afternoon of day 17. If a fol— licle was present at a palpation and absent at the next palpation, ovulation was estimated to occur midway between the two observations. On the morning of day 21, the cows were transported 11 kilometers to a local abattoir and killed; reproductive tracts were recovered and returned to our laboratory. The corpus luteum was measured and _ :M‘j ova were flushed from the oviducts and uteri. A blunt 16 ga needle was inserted into the ovarian end of the ”we“: r ‘ ' I oviduct or the cervical end of the uterine horn. Ova were collected from the tubouterine end of the oviduct or from the uterus in a watch glass as warm medium TC 199 (Difco, Detroit, Michigan) was flushed through. Ova were observed for cleavage with a dissecting microsc0pe (X35) and by phase contrast or dark field illumination (X250). Experiment IV: GonadotrOpin and Gonadal Hormones after Estrous Synchronization and after Fertile and Nonferti 1e Inseminations . The purpose of this experiment was to quantify serum hormones after MGA treatment and to relate hormones with fertility. A second objective was to relate endocrine changes during early pregnancy with fetal development. Between 30 and 60 days post-partum, the uterus and structures on the ovary were palpated to determine if the uterus was involuted and if the cow was having estrous cycles. Sixty-cows were randomly assigned to five groups and a genetic herd classification as to best and worst 26 sires was blocked across treatments. Cows in groups I, II, and III received 1.0 mg of MGA daily for 18 days starting on the first day of the treatment month. Cows in group I were injected with 2500 IU of HCG at 8 am on the third day after the last MGA feeding, and inseminated approximately 12 and 24 hours after HCG injection. Cows in group II were injectedwith HCG when first observed in estrus after MGA withdrawal and inseminated about 12 hours later. Group III cows were treated similarly to those in group II, but HCG was omitted. Cows in groups IV and V were treated similarly to cows in groups II and III, respectively, but estrous cycles were not synchronized with MGA. So that average insemination time post-partum would be the same in synchronized and control cows, cows in groups IV and V were not inseminated until the eleventh of each month (41 to 71 days post- partum). Jugular blood was obtained on the last day of MGA administration and 2 days later, as well as at the same intervals as heifers in Experiment I. Since cows in group I were not inseminated with regard to estrus, the day-0 blood sample was taken immediately prior to HCG injection. Anestrus controls and cows which failed to exhibit estrus within 7 days after MGA withdrawal were removed from the experiment. 27 Steroid Hormone Assays At the initiation of this study, estradiol had never been quantified in bovine peripheral blood. The possibility of using thin layer chromatography and column * chromatography for purification of estradiol was investi- gated. I validated a protein binding assay for estradiol, similar to that described by Korenman (1968) and Korenman et a1. (1969) for human plasma, and an estradiol radio- immunoassay using an antibody described by Tillson et a1. (1970) to quantify bovine serum estradiol. Progesterone was measured using a protein binding assay described by Murphy (1967) and modified by Swanson et a1. (1972). Isolation of Steroid Hormones Thin Layer Chromatography.—-Satisfactory isolation of standard estrone, estradiol and estriol was achieved by two dimentional chromatography using silica gel thin layer plates or Eastman chromagram sheet 6060 (Distillation Products Industries, Rochester, N.Y.) with cyclohexane: ethyl acetate in the first dimension (1:1) and cyclohexane: ethyl acetate:ethanol (45:45:10) in the second dimension. High solvent blank values were obtained in the protein binding assay for estradiol when both types of chromatograms were eluted with nanograde ethyl acetate, ethanol or methanol. After trying numerous modifications of the techniques, I discOntinued using thin layer chromatography gluta 1 “T _. __.-—-.—~T 28 for purification of micro quantities of estradiol because I encountered high solvent blanks by protein binding assay. Evidently, substances from the thin layer chromatogram interfere with the estradiol assay. This observation was confirmed by others at the Second Karolinska Symposium (Diczfalusy,.l970). Column Chromatography.--Column Chromatography on Sephadex LH-20 (Pharmacia Fine Chemicals, Inc.) in a l X 32 cm column gave satisfactory separation of progesterone, estrone, estradiol and estriol and low column solvent blank values using freshly distilled benzene:methanol (85:15). The elution pattern was similar to that described by Mikhail et a1. (1970). But corticosterone eluted with estrone and cortisol eluted with estradiol. Since there was slight cross-reaction with cortisol in the protein binding assay for estradiol this solvent system was un— satisfactory. After testing several ratios of chloroform:ethanol, we determined that 24:1 gave adequate separation of the six steroids of major interest (Figure 1). They were eluted in the following order: progesterone, corticosterone, estrone, cortisol, estradiol and estriol. Although 20a-01 (20a-hydroxypregn-4-ene-3-one) and l7a-hydroxyprogesterone (l7a—hydroxypregn-4-ene-3, 20-dione) are eluted slightly later than progesterone, they were included in the 2.5 m1 29 .ucm>H0m mnu mm Aauvmv Hocmcum "EHOwOHoHno mcflms cESHoo omlmq xwpmcmmm m Eonm Esuwm ocfl>on wo uomuuxm Hmcum cm as mpfloumum mo maflmoum coeusamll.a ousmflm 30 2323» “—o .2 09 Om. ON On On 0. + 3.53 353:8 48.586: ( mzoEmm\ mzommhmoorrmool': uzomwhmmeoma IV 00_ com com :uanlua nu 13d [Howls-Hg WdO 31 progesterone fractions routinely collected. MGA eluted with progesterone in this separational system. Glass columns (1.0 X 40 cm) were packed with 32 cm of LH—20 which had equilibrated in the solvent system for at least 3 hours.‘ Cotton plugs were placed in the topsand bottoms of the columns to prevent floating of the I. LH-20. The bottoms of the columns were fitted with teflon stOpcocks and the tops had 14/35 ground glass joints so 250 m1 separatory funnels with teflon stopcocks could be 15.-1...- —....__‘-. mu“ 4 Ar J ’ — I attached as solvent reservoirs. Using a fraction collector with an 11.5 inch drum (ISCO model 567, Instrumentation Specialities Co., Lincoln, Nebraska) and 12 X 75 mm disposable test tubes, fractions were collected from six columns simultaneously. Although only one column was mounted over a volumeter (ISCO model 400), 2.5 ml fractions were collected from all six columns because flow rate among the columns differed no more than about 5%. Between samples, each column was flushed with about 250 m1 of solvent. Reagent grade chloroform and 100% ethanol were distilled within 2 days of use. During the course of my research, flow rates of the columns varied from 0.4 to 0.8 ml per min, with freshly poured columns eluting faster. Columns were repacked after 8 to 10 samples, re-using the LH-20, and repacked with new LH-20 after about 20 samples. 32 Extraction Procedure.-—Because of the sensitivity of these assays, glassware was washed with detergent and rinsed in tap water, distilled water, glass distilled water and distilled methanol before use. Ten ml of serum was added to a 30-ml culture tube with a screw cap and teflon liner. About 3,000 dpm estradiol -2,4,6,7-3H (New England Nuclear, 95 c/mM) and 2,000 dpm of progesterone -1,2-3H (New England Nuclear, 50 c/mM, and purified by column chromatography) were added to the serum and mixed gently. The serum was mixed vigorously with about 18 ml of freshly opened anhydrous diethyl ether for 2 min and stored at -20C for 3 hours before the ether extract was decanted. The extraction procedure was repeated and the combined ether extract was dried under nitrogen. Steroids from the ether extracts were dissolved in 0.5 m1 of chloroform:ethanol (24:1) and layered on columns. After fractions (2.5 ml) were collected from‘the column, radioactivity was determined (Scintillation Fluid, Appendix I) in 0.5 ml of each fraction to locate steroids and to calculate procedural losses of steroids. .Approximately 85% of the progesterone and 90% of the estradiol tracers were recovered from the column. .Estradiol Protein Binding Assay.--The estradiol protein 13inding procedure was developed by Korenman (1968) and Pfiorenman et a1. (1969). Uteri were obtained from estrous 33 rabbits and homogenized for 1 min in three volumes of buffer A (Appendix II) at 4C in a Waring blender. The homogenate was centrifuged at 3,000 xg for 20 min and the supernatant fluid was recentrifuged at 95,000 xg for 90 minutes. The supernatant cytosol was frozen at -20C in aliquots useful for single assays. 1 Standards were diluted in redistilled 200 proof ethanol so that 100 pl contained 0, 10, 20, 40, 60, 80, I L : 100, 120, 160, or 320 pg of estradiol. Two standard curves fig" Il’ . were used in each 48 tube assay and the first fraction collected from the LH-20 column was assayed as a column blank to check purity of solvent and cleanliness of reagents and glassware. Standard 17B-estradiol (Sigma Chemical Co.), column blanks and 4 ml (the two fractions containing the maximum 3H-estradiol) of the eluate con- taining estradiol from the LH-20 column for each unknown were dried under nitrogen in disposable glass test tubes (12 X 75 mm). During drying, the walls of the tubes were rinsed twice were chloroform:ethanol (24:1). After addition of 0.8 ml of buffer A and 0.2 ml of ethylene glycol to each tube, 60,000 dpm of 3H-estradiol (95 c/mM) in 0.01 ml of buffer A was added and the tubes were mixed vigorously for 5 seconds.- Then 0.025 to 0.040 ml (depending upon the titer of estrogen binding protein) of cytosol from rabbit uteri was added, the tubes were mixed gently for 5 sec and incubated at 4C for about 15 hours. 34 To separate free and bound estradiol, 1 ml of .25% Dextran 150 (Pharmacia, Uppsula Sweden) and 2.5% carbon decolorizing neutral Norit (Fisher Scientific Co.) was added in buffer A to each tube. The tubes were shaken gently for 5 sec and incubated for 10 minutes at 4C, then centrifuged at 2,500 xg for 5 minutes. A 0.5 ml sample of the supernatant fluid was mixed with 10 ml of Bray's solution (Bray, 1960; Appendix I) and radioactivity was quantified in a liquid scintillation spectrometer (Nuclear Chicago Corp. Mark I). The mass of estradiol in unknowns was calculated by interpolation between standards and corrected for procedural losses. Figure 2 illustrates a typical standard curve for 17B-estradiol and cross-reactions with estrone and estriol. Cortisol and corticosterone also competed with estradiol in the protein binding assay (Table 1), but little cross reaction occurred with progesterone and MGA. When 500 or 1,000 pg of l7B-estradiol was added to 10 ml serum samples, 88:13 (n=9) and 60:6 (n=12)% were recovered by the protein binding assay. Radioimmunoassay (RIA).--For RIA of estradiol, we used an antibody (Antibody SLC-6X generously supplied by Dr. S. A. Tillson and associates, Worcester Foundation for Experimental Biology, Shrewsbury, Mass.) which was obtained by immunization of sheep with the conjugate l,3,5(10) 35 .ANHQV camuonm mchCHQ Homou>o mafiumus uwnnmu m gnaw: Hoeuwmw cam maouumm LDHB mcofluomoulmmouo paw Hoflcmupmmlmha Mow m>mso pumpcmumlu.m musmflm 36 33 32 ton canoe—mm (°/o) punoq Iowansa "He 000. ON» om oc om o. I . 4 _ _ _ _ 1 ON .. ow .0623 .. om :23 low _o_.:mm, LOO. 37 TABLE l.--Re1ative activity of selected steroids in the estrogen protein binding assay. Steroid Relative Activitya Estradiol 1.00 Corticosterone 0.004 Cortisol 0.006 Melengestrol Acetate <0.0001 Progesterone 0.0004 pg of estradiol bound a , . . Relative Act1v1ty — pg of steroid X bound estratriene-3,l78-diol,17B-succinyl—bovine serum albumin. Some of the prOperties of this antibody and its reactions with various steroids have been reported (Tillson et al., 1970). Standard and unknown estradiol were dried in assay tubes as described for protein binding assay. Then 0.1 m1 antibody diluted 1:15,000 in buffer AA (Appendix II) was added to each tube. After gentle vortex mixing for 5 seconds, the tubes were incubated for 30 min at room temperature. About 60,000 dpm of 3H-estradiol (95 c/mM) was added to each tube in 0.1 ml buffer AA, the tubes were shaken for 5 sec and incubated at 4 C for 3 to 4 hours. Then, 0.1 m1 of buffer AA containing 0.5% gelatin was added to each tube. Free and bound estradiol were "1.5.. 38 separated, radioactivity was quantified and mass of estradiol was calculated as described for the protein binding assay. Because of the small quantity of estradiol in 10 ml of serum, only one estimate on each unknown could be performed. For each unknown, the first fraction collected from the LH-20 column was assayed as,a column blank to check purity of solvents and cleanliness of reagents and glassware. Column solvent blanks never exceeded 10 pg estradiol, and they were not subtracted from estradiol determined in the lO-ml serum unknowns. When 40, 80, or 160 pg 17B-estradiol were added to lO-ml serum samples, 72:18 (n=4), 98:9 (n=8) and 110:8 (n=10)%, respectively, were recovered. A serum sample from a steer was assayed with each group of extractions. Twenty-three determinations on the pool of steer serum averaged 5.1 : 0.4 pg/ml. Ten pg of estradiol was easily distinguished from no hormone with 95% confidence (n=4). Therefore, allowing for procedural losses and for an aliquot to determine recovery, the sensitivity of the assay was about 2 pg/ml serum. Within assay coefficient of variation was 2.3% and the between assay coefficient of variatipn was 3.8% as determined from ten standards in duplicate in each of eight assays. Figure 3 illustrates a typical standard curve for l7B-estradiol and cross- reactions with estrone and estriol determined by RIA. 0'1. .hrq‘! 1’... $11 u?! I ell-I'll. 39 .vnc How muouum Unaccmpm u mammE mum mwcflom pmuuoam .ACHESQHM Enumm mcfl>onlamcfloosm Inna .HonumsH .mumcmflupmupmmuxoavm.m.a umafimmm wponflwcmv >mmmmocsfififloflcmu m mcflms Hofluumm paw mcoupmw cuHB wooeuommnlmmono paw Howpmuummlmha How m>uso cumpcmumul.m ousmflm 4O 33 on... .2. scoot—mm Om O¢ ON O. 0mm. Own _ d OCOLumw V/ ,/./.v/ 3...?me _ _ _ — ION HC— .... m... -3 m. m. a. ,m 00 u D. “In Om oo. . 41 Since the antiserum was not specific for estradiol, but crossreacted with estrone and estradiol, isolation of the steroids in blood sera was required. To determine which column fractions contained radioimmunoactive estrogen, a serum extract was fractionated on an LH-20 column and the first 24 fractions (about 60 ml) were assayed by estradiol RIA. Competition for immunological activity in the RIA occurred in four peaks in the elute from the column. The first was fraction 2, soon after the void volume; there was a small amount of activity in the two fractions (numbers 7 and 8) after progesterone and the other two peaks were associated with estrone and estradiol, I assayed only four fractions eluted from the column after estradiol, so there may be other immunoreactive materials ‘which are eluted after estradiol in Serum extracts. Ether extracts of steer serum were assayed for estradiol by RIA and compared with steer serum extracts purified by column chromatography. Although the values for the ether extracts were about 3.5 times the values obtained after purification of the sample on LH-20, the overestimation (17.7 : 2.51 pg/ml, n=3) was consistent. Comparison of Methods.--The RIA resulted in more precise recovery of estradiol added to sera (described in assay method); it also had lower values than protein binding for solvent blanks from LH-20 columns (1.2 : 0.1, n=70 vs 2.6 : 0.6, n=27, pg/ml, respectively). 42 Estradiol was quantified by RIA and by protein binding assay in two different sets of blood sera col- lected during different estrous cycles from eleven heifers (Table 2). Based upon RIA, estradiol was lowest (3.0 pg/ml) on day 2 and relatively constant during the luteal phase of the estrous cycle, averaging about 3.6 pg/ml from days 2 through 11. Then estradiol increased (P<.05) to 4.8 pg/ml at 3 days before estrus, continued to increase (P<.05) to 9.7 pg/ml 0.5 days before estrus and remained high on the day of estrus (8.4 pg/ml). Changes in estradiol determined by protein binding assay resembled those deter- mined by RIA but quantities were greater at all times during the estrous cycle. But I do not know if higher estradiol in samples quantified by protein binding assay reflects assay or sample differences. The within day correlation of average estradiol values during the estrous cycle as determined by RIA with averages obtained by protein binding determination was 0.85 (P<.01). Similar to my data, Echternkamp and Hansel (1971) found 8.1 pg/ml of estradiol at estrus when estradiol was purified on Sephadex LH-20 and quantified by RIA. Swanson (1970) in our laboratory, quantified estrone, estradiol and estriol during the estrous cycle but lack of precision for his assays contributed to large variance and very high values. .m.m H ammzo .msuumo mo map mm3 o amen .mposumfi O3e may Hem poms mhmz mumm mo mumm HGOHOMMHQM. 43 m.HHm.s v v.oww.m OH Ha m.HHv.s a o.owm.m OH R n.mfim.m a q.owm.m OH 4 m.awn.m m m.owo.m OH N m.mwm.~a e m.aw¢.m a o N.~Hm.oa m m.mwn.m m m.- o.~HG.HH N H.st.m AA A. m.mwo.a « n.awp.> . m m- a.mwm.m m o~.aflm.v v m- 1H2\mm Ace Aaexmmv .Acc mafichm cflmuonm wmmmmocsafiwoficmm nwaowo moosumz >mmm< Hoapmuumm >wo .mHOMMmc cflmumaoc Eoum Esumm cooHQ Hmumcmflumm CH Hoflpmnwmo mo mHm>qul.m mqmH ucmfiflummxm Ge cumocmvm 0:99 .mmlmq mHz mmz HH cam H unmEflummxm cw pumpcmum maem no.0 H mm.m mm.~ .om.m .vh.m .mo.m .mm.~ m m vm.m em.m a m HH.o H om.o HH.H .No.H .Nm.o .mm.o .mm.o am a mo.o « mm.o mm.o .mo.H .mo.a .mm.o ma H .m.m A new: mmmmmm Hmnpfl>flch mo mwaommm UHMUCMDm doom Edumm .mhmmmm ucmHmMMHU CH maoom Esumm ucmHOMMHU mo mammamcm CH ma mo GOHDMGHEHmumo mcp mo coflmflomumll.m mqmde 48 Prolactin The procedure used to quantify prolactin was develOped by Tucker (1971) and validation of the assay has been reported (Koprowski and Tucker, 1971). Standards were diluted in 1% bovine serum albumin so 0.5 ml would contain 0, 0.1, 0.2, 0.5, 0.8, 1.0, 1.5, 2.0, 2.5, 3.0, or 4.0 ng of prolactin. Four sets of standards were included in each set of 300 to 500 assay tubes. One group of standards prepared from NIH Prolactin-Bl (Supplied by . u.._- .9..- vw—ir Ti" l- .0 0 the National Institute of Health, Endocrinology Section, Bethesda, Maryland) was used for analysis of Experiments I and II. And another group of standards from NIH Prolactin-B2 was used for analysis of samples in Experi- ment IV. Because of the high concentration of prolactin in some samples relative to the sensitivity of the assay, sera were diluted 1:2 to 1:20 in 1% bovine serum albumin. Each serum sample was assayed in dilution duplicate and a standard cow serum sample was included in each assay. Examples of precision of the prolactin assay are listed in Table 4. Statistical Analysis When repeated measurements are made on animals in each treatment group, a split plot analysis is desirable (Gill and Hafs, 1971). The analysis used for Experiment I 49 ‘ 1 _ LI}! . -32 at. . '1'-lll11'|5 .) I:- I .mm cHHomHoum mHz mmB >H HcmEHHmmxm CH pHMUGMHm oxen .Hm :HHomHoum mHz mm3 HH cam H chmEHHmmxm CH pumpcmum onem m.mH H m.woa ¢.mm .m.mm .m.mna .m.oo .o.moa m m N.mN H h.mNH m.om .m.moa .m.mmm .m.vm .m.ama QN N m.mma m.mmH H m m.mm H m.hmv c.5vm .o.oov .H.Ohv .m.mbv 6H H .m.m H cmmz m>Mmm¢ HMDUH>HUGH mo mHHDmmm pHMUCMHm Hoom Eduwm .mmmmmm Hcmummch CH mHoom Enumm HcmHmHMHU mo mHmMHmcm :H :Huomaoum mo coHHmcHEHmuwp may mo conHomHmll.w mqmfie 50 to determine the influence of pregnancy on serum LH and prolactin is shown in Table 5. Predetermined orthogonal contrasts were used to partition the 5 degrees of freedom for periods. Another set of orthogonal contrasts (Table 6) was used to partition the 17 degrees of freedom for the days of pregnancy. Within-day correlations between LH and estradiol, LH and progesterone, LH and prolactin, estradiol and progesterone, estradiol and prolactin, and prolactin and progesterone after insemination were determined in pregnant and nonpregnant heifers. A split plot analysis similar to that in Table 5 was used for Experiment IV except there were four between block treatments (genetic classification, MGA, HCG, and pregnancy) and one within block treatment (stage of preg- nancy). Orthogonal contrasts listed in Table 6 were used to partition the 17 degrees of freedom for stage of pregnancy. TABLE 5.—-Split plot analysis of hormones during early pregnancy. Source of Variation df Between Heifers (36) Treatments 1 Cows Within ' 35 Within Heifers (176) Periods 5 T X P 5 Heifers X P (error) 166 51 TABLE 6.--Orthogona1 contrasts used to partition the 17 degrees of freedom for stage of pregnancy. Source of Variation df Days of pregnancy 17 Days 0,2,4,7,ll, XE 18,20,22,25,30,35,40,42,45,50, 60,63,75 . ‘1 Days 0,2,4 XE 7,11 ‘ 1 Day 7 X5 11 1 Day 0 XE 2,4 1 Day 2 XE 4 1 Days 18,20,22,25,30,35,40,42 XE 45,50,60,63,75 1 Days 18,20,22 gs 25,30,35,40,42 1 Among days 18,20,22 2 Among days 25,30,35,40,42 4 Days 45,50 XE 60,63,75 1 Days 45 25 50 1 Among days 60,63,75 2 {J .. .u gain—q ' \ l”. Aun'.‘ -‘ RESULTS AND DISCUSSION Experiment I Changes in Gonadotropin and Gonadal Hormones Associated with Early Pregnancy and after Nonfertile Inseminations Size and Behavior at Breeding The body weight (367 : 5 kg) of the heifers in this experiment compared closely to Morrison's (1957) standards but their height at the withers (119 : 1 cm) was slightly less. Standing heat (estrus) was first observed in the morning in 76% of the heifers (n=37). Similarly, Swanson et a1. (1972) first observed 62% of their heifers in standing heat in the morning and Trimberger (1948) reported 70% first showing estrus in the morning. Fertility Since heifers first observed in estrus in the morning were inseminated in the afternoon by design, 76% of the heifers were inseminated in the afternoon and the remainder in the forenoon. (Sixty-eight per cent of the 28 heifers conceived at the first insemination and fertilities of forenoon and afternoon inseminations were similar (71 and 67%, respectively). The interval from onset of standing 52 53 heat to time of insemination was similar in heifers that became pregnant and those that did not; 10 : 2 hours. An average of 1.33 inseminations was performed per conception. Endocrine Changes .L§.-4Serum LH concentration was greatest on the day of estrus, averaging 8.7 : 1.6 ng/ml in heifers that con- ceived and 12.0 : 3.2 ng/ml in heifers that failed to conceive (Figure 4, Appendix Table IV). Since the ovulatory surge of LH in cattle occurs between 3 hr before and 6 hr after the onset of standing heat and persists 6 to 10 hours (Schams and Karg, 1969; Henricks et al., 1970; Swanson and Hafs, 1971), LH concentration on the day of estrus is dependent on the intervals between blood sampling. Nineteen of 37 blood samples taken on the day of estrus contained more than 4.0 ng/ml LH, indicating that part of the ovulatory surge of LH was detected in 51% of the heifers sampled when first observed in estrus. Swanson et a1. (1972) detected 46% of the ovulatory discharges of LH when heifers were bled when first observed in estrus. Although serum LH (Figure 4 and Appendix Table IV) appeared to decrease with advancing pregnancy, LH concen- tration did not change significantly (P>.lO) from day 18 to 75. Similarly, Randel and Erb (1971) did not observe any significant changes in plasma LH from day 7 to day 260 of pregnancy. There was little among-heifer variation of 54 Figure 4.--Serum LH in heifers after insemination. Figure 5.--Serum prolactin in heifers after insemination. 55 ”PT I :04 I E I \ 9'1 g | NON-PREGNANT I J 2“I /'\ I , \ PREGNANT I I .— ll I l Ill 1 l I ll 1 l l l l 0 ll 20 30 40. 50. 60 75 DAYS AFTER ESTRUS 30- : \ PREGNANT E I \ I. / \ I \ I 2’ 20 I. \ V \ I I z ‘ I :- ‘\ Iv.._——N0N-PREGNANT 0 or. 3 IO— 0 c: 0. ill I l Ill 1 I ll ll 1 l l l J 0 ll 20 30 4O 50 6O 75 DAYS AFTER ESTRUS 56 LH (P?.10) during early pregnancy and after the day of estrus only three samples contained more than 4 ng/ml; one each on days 11, 30 and 40 and all in different heifers. Schams (1969) found elevated LH in one sample from each of two cows bled three times daily during the first 2 months of pregnancy. Serum LH concentration was greater in nonpregnant heifers (P<.10), averaging 1.2 : .1 ng/ml during days 2 through 11 after insemination compared with 1.0 : .1 ng/ml in pregnant heifers (Figure 4 and Appendix Table IV). Edgerton and Hafs (1971) also observed higher LH in non- pregnant cows compared with pregnant cows during days 2 through 11 after insemination. In a study with five pairs of cows, Henricks et a1. (1970) found higher LH in non- pregnant cows 8 days after estrus but higher LH in pregnant cows at 16 days after estrus. Greater concentration of serum LH may be caused by less steroid hormone feedback at the pituitary so more LH is released. On day 18, non- pregnant heifers had elevated LH (2.0 : .4 ng/ml), possibly associated with proestrus increases described by Swanson et a1. (1972) and Garverick et a1. (1971). Prolactin.--Serum prolactin concentration was more variable than LH and differed significantly (P<.005) among heifers. Average prolactin concentration of all samples from individual heifers ranged from 7:3 to 56:14 ng/ml. But milk production during days 30 to 60 of the subsequent 57 lactation was not related significantly (r=.07) with average serum prolactin during the first 75 days of pregnancy. Although prolactin tended to decrease with advancing pregnancy (Figure 5 and Appendix Table IV), stages of preg- nancy did not differ significantly (P>.lO). Stress associated with jugular puncture occasionally may release prolactin from the pituitary (Tucker, 1971; Raud et al., 1971), so sampling blood by a non-stressful method or after uniform stress may be necessary to determine precisely prolactin changes associated with early pregnancy. During the first 3 days of pregnancy in the rat, serum prolactin is higher than during the following 18 days (Amenomori et al., 1970). A gradual decline in prolactin levels during pregnancy also has been observed in sheep (Arai and Lee, 1967; Davis et al., 1971). In contrast to serum LH, prolactin was higher (P<.10) in pregnant than in nonpregnant heifers during the first 18 days of pregnancy (Figure 5 and Appendix Table IV). From 2 to 11 days after estrus, prolactin averaged 24:3 ng/ml in pregnant heifers and 16:3 ng/ml in nonpregnant heifers. Estradiol.--Sera from 8 of the 26 pregnant heifers were selected to isolate and quantify estradiol and pro- gesterone. Concentration of estradiol in heifers did not differ significantly during early pregnancy (P>.lO). Estradiol decreased from 12.6:2.4 pg/ml at estrus to 8.4:.6 58 pg/ml by day 4 of pregnancy (Figure 6 and Appendix Table V). Although estradiol concentration on day 2 was similar to that at estrus in these pregnant heifers, estradiol was markedly reduced by day 2 of the estrous cycle (Wettemann et al., 1972). During days 18 to 75, estradiol averaged 6 to 8 pg/ml except for day 40 when estradiol averaged 11.1:2.3 pg/ml (P<.01) because four of the eight heifers had elevated estradiol on day 40. More frequent sampling of blood near day 40 would be necessary to determine if elevated estradiol occurs in all pregnant heifers near this time. Randel et al. (1971b) observed elevated urinary estrogens at day 42 of pregnancy in cows. Increases in serum estradiol at this stage of pregnancy could be related to growth of the placenta. Progesterone.--Serum progesterone increased dramati- cally from 0.4 ng/ml on the day of estrus to 6.8 ng/ml on day 11 (Figure 6 and Appendix Table V). This rapid increase is similar to changes observed during the estrous cycle (Stabenfeldt et al., 1969; Swanson et al., 1972) and during early pregnancy (Henricks et al., 1971a). Progesterone concentration differed significantly among days 18, 20 and 22 of pregnancy (10.8:1.3, 9.2:.9 and 8.1:.9, respectively, P<.05). During the estrous cycle, serum progesterone is usually greatest after day 15 but before day 18, (Stabenfeldt et al., 1969; Swanson et al., 1972). Therefore the decrease in serum progesterone from day 18 to day 22 of pregnancy may 59 Figure 6.--Serum estradiol and progesterone during pregnancy in heifers. FIgure 7.--Serum LH, prolactin, estradiol and progesterone during pregnancy in a typical heifer. 6O 2.59.. 2535 2.505 mzomwhmmoomm 2:04.51.“ mzommpmwooma mmes42 wwmz news 7 _ _ _ _ 14 H _ _ q _ 15 J L _ 1n + T ’ m _ ’ m 5 _ , m - m .A . 1 fl 3 10 A 10 E 6 , R 6 _ "n m a , E . O a m M r10 N \- 15 H A n“. _ 5 O . L R 1 O N L W ”0 m E \HuO s 4 / G _ 4 E O. G l m . l M 0 I“\\ h 10 P 13 I, — 3 l m I’ l 10 I- “0 H2 KH\ m II 12 I T _ l S _ ll H E I l” l L l l / /‘.IIII" In] E _ E . _ _ 0 ml. _ r H E O 2 0 8 6 4. 2 O 8 6 4 2 O O 0 .l l . I. 3 2 .l :52: 49925.”. 252:: 1.. :52: Jo_o.05), this trend suggests a shift in the regulation of steroid hormones at about day 18 to 20 of pregnancy. Serum LH and prolactin during the first 75 days of pregnancy were not influenced by the sex of the fetus (Appendix Table VII). Also, sex of fetus (5 female and .t 63 3 male fetuses) did not influence estradiol and progesterone in the eight animals studied. Experiment II Gonadotropin Levels after MGA Withdrawal Behavior and Fertility 2“ —v—-"—‘3‘r Only three of the nine heifers were observed in standing heat within 6 days after MGA withdrawal (Table 7). aha: {'3' This low incidence of estrus could be related to moving “no-1h"- the heifers from the stanchion barn to the loose housing barn on the day of MGA withdrawal. Of the three heifers inseminated at the synchronized estrus, only one conceived. Seven of the eight nonpregnant heifers returned to estrus an average of 24.7 : .8 days after MGA withdrawal. Thus the length of estrous cycle after synchronization appeared to be normal in length. Fertility was normal at the second estrus; of seven heifers inseminated, six conceived. Endocrine Changes . LH.--The ovulatory surge of LH was detected in six of the nine heifers; it occurred 2 to 7 days after the last MGA feeding (Table 7). The per cent LH peaks detected was similar to 51% of the LH peaks observed by bleeding heifers on the day of estrus (Experiment I). BeCause of the variation in interval from the last MGA feeding to the ovulatory surge of LH, average LH values by days after MGA withdrawal is not meaningful (Table 8). The ovulatory surge .UQHMCHEmeH uoz .comouAIV .HcmcmmHQHA+vQ 64 o .omuomumo uozo .Homemenu «02 Ho zoo ummom m.He.em >.H~.m m.Hm.m mm H ommz + «N u m m a + mm u m _ m m Homommuo + m m e + mm Hz oz oz m Hz oz Hz oz oz m + mm Hz 5 . oz 4 + mm , Hz m . oz m + mm Hz b oz N I em on oz ooz H Amzmov xmzmoc Amzmoc Aozc now: How: QmsHHmm msHpcmum Qmsuumm xmmm mchamum com um com. umH no mo umH HHHHHpnmo on moo: zuHaHnumo 0» m mqmde TABLE 8.—-Serum LH and 65 prolactin in Holstein MGA treatment.a heifers after Days After Last LH Prolactin MGA Feeding (ng/ml) 0b 0.30 : 0.11C 52.3 : 12.8 1 1.02 : 0.07 40.8 : 13.2 2 2.09 : 1.03 30.2 : 21.1 3 1.06 : 0.11 11.9 : 1.8 4 1.22 : 0.17 48.4 : 20.7 5 2.57 : 1.10 25.4 : 6.2 6 1.19 : 0.32 32.0 : 12.3 7 2.75 : 1.66 6.0 : 2.2 aNine observations (heifers) at each day. bLast day of treatment. CMean : S.E. 66 of LH was analyzed further by adjusting LH values to the day of peak serum LH (Figure 8). Maximum LH was 8.2 : 2.3 ng/ml, which is similar to 8.7 : 1.6 ng/ml observed in fertile heifers (Experiment I). Basal values were about 1 ng/ml. Thus the ovulatory surge of LH as determined by once daily bleeding after MGA treatment appeared normal (Swanson et al., 1972) in magnitude and duration, lasting less than a day. . i Prolactin.--All six heifers that had detectable LH 1:1", av peaks after MGA withdrawal also had elevated prolactin on the day before or on the day of the LH peak. Elevated prolactin near the time of estrus has been reported (Swanson et al., 1972; Raud et al., 1971). Serum prolactin increased significantly (P<.05) from 29 : 13 ng/ml 2 days before the LH peak to 83 : 30 ng/ml the day before the LH peak, then decreased to 18 : 3 ng/ml by the day after the LH peak (Figure 8). This increase in prolactin at the synchronized estrus appears less variable and of greater magnitude than serum prolactin during estrus (Swanson and Hafs, 1971). Experiment III Induced Ovulation and Ova Recovery Ovulation occurred in 22 of 25 cows when HCG was injected 3 days after the last MGA feeding, but ova were recovered from only 7 cows (Table 9). I feel that ova were recovered from only 31% of the cows because ova 67 .Hm3MHpLHH3 «02 umpmm mummHmc CH GHHUMHOHQ pom mg Enummln.m mHDmHm 68. (um/bu) N|10V108d OO._ xfio 5 out? mo mmoumm 225 I. N... O __u. 2:04.40"; . NI _ O_ (um/60) H'l 69 .:OHHmmHmm maHMU mUHBH an cmHmEHHmmm H.oHH.H H.0HH.H OHo.H Aeovnmpomsmam um go no muHm H.0Hm.H H.0Ho.~ H.OHo.H . raucomumas>o ems» maoHHHoH Ho muHm mHoo MHHG onm Ammuomsmamnmuo one mHom mHmm ofiom loom umoo use mmer ooHumHs>o em ma m . . mooHumHs>o Ho Honesz mm ma 4 n . mzoo Ho Hmnasz HMHOB pmum>oomm pmum>oomm Hoz m>o m>o 038 .coHHmHs>o twosocw , _ Hm mzoo chpmHom mo moHHwHHmHomnmco cmwmm>o paw mumu COHHMHD>OII.m mamme 70 recovery was performed too 1ate--at 4 days after HCG injection. Wagner et a1. (1968) found 79% of the ova in the oviducts of CAP treated cows 3 to 3.5 days after the onset of estrus. In my hands, ova recovery from the uterus is considerably less efficient than that from the oviduct, and some ova were in the uterus when the cows were slaughtered. However, I cannot exclude rapid ova transport as a possible cause of low ova recovery after MGA treatment. Ova recovery, but not ovulation rate, may be related to the stage of the estrous cycle at the start of MGA treatment. The data in Table 10 suggest that ova were not recovered when the cows were proestrus or estrus (days 19 through 0) or mid diestrus (days 6 through 14) at the outset of MGA treatment. But ova were recovered from cows when MGA was started during metestrus and late diestrus (60 and 50%, respectively). Ovulation was synchronized quite well, occurring 36:3 hr after HCG injection or about 60 hr before slaughter (Table 9). Graves and Dzuik (1968) estimate that ovulation occurred in synchronized cows about 40 hr after HCG given 1.5 to 2.5 days after the last MAP feeding. During the ‘estrous cycle, ovulation occurred 20 to 32 hr after the LH peak (Schams and Karg, 1969; Swanson and Hafs, 1971). Although the ovulatory follicles were slightly larger in cows from.which ova were not recovered (2.0:.1 cm, 71 TABLE 10.--Re1ation between ova recovery and stage of the estrous cycle cows were started on MGA treatment. Day of Cycle MGA Started Cows 'Cows Ovulated Ova Recovereda (no) (me) (%) (no) (%) 19 through 0 ‘3 3 100 0 0 1 through 5 5 , 5 100 3 60 6 through 14‘ 6 5 83 0 0 15 through 18 9 8 89 4 50 Total 23b 21 91 7 33 aPer cent recovered based on the number of cows that ovulated. bStage of the cycle was not available on two cows. Table 9), they were not significantly different (P>.lO) from follicles in cows from whiCh ova were recovered (1.6:.1 cm). Corpora lutea averaging 1.1:.1 cm in diameter were observed at slaughter on all ovaries that had ovulated. Four of the seven cows from which ova were recovered ovulated between the first and second breeding and had 2- to 8-cell fertilized ova in their oviducts (Table 11). In contrast to rabbits (Pritchard, Wettemann and Hafs, 1970) sperm transport was not inhibited in these MGA treated cows. A fragmented ovum was observed in the oviduct of one cow and in the uterus of another. These fragmented ova may or may not have been fertilized since Chang (1967) demon— strated that fragmented rabbit ova may or may not develop ‘1‘”.- A '- ND! ““33." I. " 72 .xH>H00 0H HoHumumom pmuHmommp Enomm n .300 chH EOHH ©0H0>000H 0H03 m>o 039m omnomEomHm moumuo ma no one maamo o HosoH>o ma no one maamo m Hoan>O ma no mav omuHHHuummcD m0H0HD ma no omav maamo m HODUH>O 5 mm pmucofimmnm H000H>o ma om omov pouHHHuuowca HoscH>o Hmumm mm ma mow maamo N H05©H>o n mm now Amnsosv Amusonv . coHumcHfimmcH Hmuzmsmam mHHHHHuom coHHmooq 0cm 0H0m0m muonm Honasz 300 0>O COHfiMHSx/O .0300 chumaom m0 coHumH5>o pwaaouucoo HmHmm m>o m0 >HHHHHH0EII.HH mqmge 73 into viable young. The unfertilized ovum recovered from cow 408 could be expected since ovulation occurred about 29 hr after the second insemination, probably well after the end of the fertile life of the sperm. Cow 413 also had an unfertilized ova which may be due to the fact that sperm were deposited posterior to the cervix at the insemi- nations, because the breeding catheter could not be passed through the cervix. Therefore, fertility appears normal in cows that ova were recovered from. Experiment IV Gonadotropin and Gonadal Hormones after Estrous Synchronization and Insemination Reproductive Performance This experiment was designed to study endocrine changes after MGA and HCG treatment and it was recognized at the onset that fertility data would be limited by the number of cows available. Cows in this study were started on MGA treatment an average of 55 days postpartum. Anestrus controls and cows which failed to exhibit estrus within 7 days after MGA withdrawal were removed from the experiment. Of the 65 cows assigned at random to the five treatments, 11 to 13 cows per treatment completed the study (Table 12). Ninety per cent of the cows exhibited estrus within 7 days after MGA withdrawal, but few cows conceived at the first insemination; an average of 20% of the treated cows and .w-ptt- 5' r'.‘ . . ' J 74 .pouumum m03 <02 ESHHmmHmom mwmoc .coHHmwocoo H0O AmcoHHmcH60mch m00H>H0m0 .mDHHmm EH p0>H0mQ0 HmHHm cocB Q0>Hm worn .mcHoomm €02 Hmma Hmemm mmmp m c0>Hm 00mm Hm mm mm am so woo mzmo omH NMH Hma HHH Hma ammo when m.m o.m m.m e.m m.m oo\m m w m H N A.ocv ucmcmmnm mzoo. NH MH HH HH HH .E.oov mzoo Hounooox>v oomA>Hv «oszHHV ooz+¢oszHc nooz+oolec o o QOHH0HHHO Homeummne .coHHmNHcounocmm msonumm Hmpwm m300 mo 00cmEH0mumm 0>Huospoum0mll.ma mqmfie 75 25% of the control cows. Days from parturition to con— ception (days open) and the number of inseminations per conception were not altered significantly by the treatments (Table 12). Of the 12 cows which conceived to the first insemination, six were in the genetic group inseminated with semen from best sires and the other six were insemi- nated with semen from worst sires. Endocrine Changes By plan, cows in treatment I were not observed for estrus; they were inseminated at 12 and at 24 hr after HCG given 3 days after the last MGA feeding. Therefore they were omitted from the split plot analysis (Appendix Table VIII). LH.--Serum LH averaged 1.0 ng/ml (n=33) on the last day of MGA treatment, similar to values observed during days 2 through 18 in nonpregnant cows. LH did not differ (P>.lO) between the last day of MGA treatment and 2 days later. The ovulatory surge of LH (:_4.0 ng/ml) was detected in only 23% (n=70) of the samples taken on the day of estrus, whereas I detected 51% of the surges in heifers (Experiment I). Ovulatory surges occur between 3 hr before and 6 hr after the onset of estrus (Schams and Karg, 1969; Henricks et al., 1970; Swanson and Hafs, 1971), so the peak of LH is less likely to be detected if cows are not bled early in estrus. It was more difficult to 76 determine the onset of estrus in cows at the stanchion barn than in heifers at loose housing. Failure to deter- mine the onset of estrus in the cows may be related to low fertility and to the low proportion of cows in which an LH peak was observed compared to the heifers (Experiment I). The ovulatory surge of LH averaged 13.2 : 9.0 ng/ml (n=10) in cows that conceived and 2.5 : .6 ng/ml (n=36) in nonpregnant cows (Tables 13 and 14). Serum LH was not influenced directly by genetic classification, pregnancy or MGA (Appendix Table VIII). 'Cows treated with HCG had less serum LH (P<.05) than non-treated cows. But this difference was caused by less LH in HCG—treated than non-treated cows prior to HCG injection. Therefore, the HCG could not have caused the difference in LH. A genetic by pregnancy interaction (P<.05) resulted; among best cows the nonpregnant had more LH while among worst cows the pregnant had more LH (Table 15). The physiological meaning of this interaction as well as the interaction between genetic classification, pregnancy and HCG (P<.005) are not apparent. The day by pregnancy interaction was significant (P<.005); LH was similar in pregnant and nonpregnant cows during proestrus, greater in pregnant than nonpregnant cows at estrus but less in pregnant than in nonpregnant $1 77 m o Hm.o m.0Hm.H H.HN.H v.0 QHH.o mm H.o Hn.o N.OHm.o H.Hm.o m.o H.Hm.o mm H.o Hm.o mm.OHm.o m.Ho.o m.o H.Hv.o om mo Hm.o m.0Hm.o OHh.o m.o H.Hm.o ma N.o Hm.o H.0Hh.o OHm.o m.o H.Hm.o HH m.o Hm.o H.0Hm.o H.Hm.o v.0 H.H~.o o m.o Hv.o om.0Hm.o H.Hm.o o.H OHv.o v N.o Ho.o mN.OHm.o H.Hm.o v.0 N.Hm.o m H.mmHH.mm N.HHH.m w.Ho.H H.m N.MHH.m monumm mOHH.H m.o H.Hm.o HI 00.H 0v.m ml 0m.o 0N.H v.0 H.Hn.o ml 0m.o 0m.o «1 0w.o 0m.H m: 0m.o on N.Ho.H m.o H.Hm.o m+H0mno 039m .C0HH0>H0mno 0C00 . . u C00 m m + 20 .mCHHmm CH ©0>H0mao HmHHw 0H03 m300 C0C3 C0>Hm oumo .mCHpmww COS Hmma 0CD Hmumm whee m C0>Hm womb .Amuov Houuoou .Aqnov 000m .Ammuov «oz .Aanov 00oz + <02 .Amnov boom + «026 NNr-lr-lr-JCNr—l O O OOOOOOOOr—l r—l Ho.H H.0Hv.o v.Hm.o «.0 OHN.o H¢.H H.0Ho.o H.Hn.o m.o H.He.o He.o SOHm.o OHo.H v.0 OHm.o Hq.o OHm.o QHm.o m.o N.Hq.o Hm.o. OHo.o mh.o v.0 N.Hv.o Hm.o om.OHo.o N.Hm.o v.0 OH~.o Hm.o H.0He.o mm.o m.o o.Hm.o “v.0 m.OHo.H OHn.o 0.0 H.H~.o Hv.o ®H.0Hm.o N.Hm.o h.o OHN.o mm mm .oo om mv Nw 0v mm om 79 AVON.OHm.o Amvm.Hn.o 1H1 m.o Amvm.OHm.o omm Avvm.on.o Amcm.H¢.o lav H.n Amvm.on.o 0mm Cecq.OHe.o Amcq.Ho.H Amcm.OHo.H oom Aevm.OHm.H Amvm.Ho.o Cay m.H Amvm.0Ho.H oma AmvH.OHm.o Amc¢.Hm.o Amvm.OHe.o COACH.OHA.O AmVN.OHm.o HH AmVH.on.o AmVH.Ho.o luv OHm.o AmVN.OHm.o room.OHH.H e Amvm.OHH.H Amvm.Hm.o AmVH.OHo.o AOHVN.OHO.H AmvH.OHo.H v AmcH.OHo.H EoVH.Ho.H AmVH.OHe.o AQHVN.OHO.H Cove.OHe.H m Amvm.on.H Amvv.Hm.H Amvo.mHm.e Aoavm.OHH.m Amvm.OHv.H mCHHmm AHV m.H AHV m.m Avvm.OHm.H Amvv.OHo.H AmVH.OHN.H HI nae v.H Amcm.OHH.H AmVH.OHo.H Nu ANVH.OHO.H 1H1 m.o Amvo.OHm.H A¢VN.OHm. AmVH.OHm.o mu 1H1 m.m ANVN.OHN.H AeVV.OHH.H v: AHV m.o Amvv.OHH.H mu lav m.o Amvm.HH.H Amvm.HHm.H .ou AmVB.HHo.N AmVN.OHH.H CovH.OHm.H m+Hmmno HOC 0H03 HMCH m300 HCMCmmumCOC HOW mmsHm> .msuumm CH ©0>H0meo HmHHm 0H03 m300 C0C$ C0>Hm 00m U .Am300v mCoHHm>H0mno m0 HmeECC 0CH 0H Hmmmu mmmoCHCmumm CH muonECC 0CH «.m.m H C0020 Q .mCHpmmw «02 HmmH 0CH kumm mwmo m C0>Hm 00mm AHV n.m Amvv.mHm.h ANVH.OHo.o AHV N.v AS owmo 3; M; 35.34 8.5.34 3; 03$ :3 034 A: To Amvo.mHm.m Amvv.HHv.m AmVH.0Ho.H AmVN.OHm.o AHV v.0 Amvm.OHm.H AHV m.o $5.384 $3.3m; $5.534 E 03.0 Avvm.OHm.H AHV v.0 AHV m.o Amvm.HHo.N Amvh.0Hm.N Amvn.0Hm.H “my on.o Amvw.HHh.H Amvm.0Hv.o mCHHmm 81 r. 1:1“. N.Hm.H o.HHo.m m.Hv.H H.Hm.o 0mmu0>< v.Ho.H m.Hw.H n.mHm.m m.Hm.H H.Hm.o HOHHCOO H.HH.H H.HN.H H. Hm.o m.Hm.H mH.Hm.o mom 1He\mov 000H0>C HCMCmonmCoz HCMCmmum HCowmumCoz HCMCmmum Hmuoz . Hmmm CoHHMOHHHmmMHO 0HH0C00 .HC08H00HH 00m 0C0 WOCMCmmHQ .CoHHmoHHHmmmHo UHHmam m0 monusCH “m300 CH ma Edummll.mH mquB 82 cows from days 2 through 25 after insemination. This agrees with my finding that nonpregnant heifers also had greater serum LH than pregnant heifers. Edgerton and Hafs (1971) also reported a greater concentration of LH in nonpregnant cows compared with pregnant cows. Values for serum LH in nonpregnant cows on days 18,20,22 and 25 after estrus are listed in Table 14. These samples were from cows not observed in estrus between days 18 and 25. When cows were sampled daily before estrus, increased LH was detected before estrus (Table 14), probably because we did not detect estrus among the cows as carefully as among the heifers where the LH surge usually occurred after the onset of estrus. Prolactin.--Serum prolactin was not influenced (P>.lO) by genetic classification, pregnancy, MGA or HCG. Although days after insemination differed significantly (P<.01) no meaningful pattern of prolactin changes could be established (Tables 16 and 17). Prolactin ranged from 20:4 ng/ml on the day of estrus to 37:8 ng/ml on day 22 of pregnancy. Other values were intermediate between these extremes until day 75. Prolactin values were similar in pregnant and nonpregnant cows (Tables 16 and 17). Increases in prolactin near estrus, which have been reported for heifers (Raud et al., 1971; Swanson et al., 1972) were not observed in these cows. [pl-15.5; r- _ 83 m HON O HON O HOO NN O HOH ON HHHNN NHHOO O HOO NN HHHOH NN OHHHN ON HNH O HOO ON m HOH ON OHNHNN Om HON N HNN Om OHHOH OH H HOH OHHON H HNN OH O HON HH O HNN N HON OHHNN OH O HON N H HNH Om HOH NNHOO NO m HNN O O Hmm Om HOH H HON Om N HNH N O HON O HNH HHHHN NH N HOO OOHHmo HN HON ON O HNN H- 0OH N- 0NN mNH Om ONHON m- 0NO 0ON O- 0ON mHH O- mON O- O HON ON O HNN N+00 OHCmepmmue .HC08H00HH our pCm <02 Houwm m300 HCmCmmum CH AHE\va CHpomHOHm Ednomll.mH mqmfle 84 .m.m + .0C0HH0>H0000 039 C002 . mCOHnwmkrhmeO meSBm H .C0HH0>H0000 0CO0 U .05HH00 CH ©0>H0000 H0HHH 0H03 0300 C0C3 C0>Hm Oomo .mCHp00H CO: H00H 05H H0HH0 0900 m C0>Hm mum Q .Hmuov HouHooo .AOnov U0oz .ANnov <02 .AHnov 0000 + 002 .ANuov 0000 + 4020 ONHOO OHHNO O HON OH H HH ON ONHOO OHHNO O HON NO NHHNH OO O HOH OO HON OHHOO NH O HOH OO O HO OHHOO O HNN Om 0OO OO H HNH O HNN HHHOO NO OHHOO OH O HOH ON HOH OHHOO Om N HOH NO HHHOH O HNN 0ON NO OHHOO OH O HNN N HHO NHHHO OO O HNN OO O HOH ONHNO O Hem ON N HO OO 85 AOONHHOO AOOO HON 1H0 H COOO HON 0ON AOVONHOO AOOO HON AHV OO AOVOHHOO ONN COCO HOO COCO HON COCO HHO OON AOOOHHOO COCO HNN AHO ON AOVOHHOO OOH AOOOHHOO AOOOHHON COCO HON AOHVOHHOO HOOO HON HH COCO HON COCO HON COCO HON AOHOO HOO AOOHHHHO O AOVOHHOO COCO HOH AOOO HON AOHOHHHOO COCO HOO O AOVOHHNO COCO HON COCO HOO AOHVNHHNO AOOOHHOO N AOOOHHNO AOVO HON AOOO HON AOVO HNN AOVO HON OOHHmm AHV ONH AHV NH AOOOHHOO AHO ON AOOO HON H- AHO NN ANVOHHON AOVOHHON N- ANVNOHOO AHO NH COCONHNO AOOOHHON HOOO HON O- AHO NN ANON HHH AOOOHHOO O- CHO OH ANON HNH O- AHO NO ANOO HON ANONOHNOH O- AOVO HNN AOVOHHON AOOO HON N+<0z HOOH HOVOHHOO AOHVOHHNO HOOO HON <02 HOOH HOHHoo0. 000m <0: 0000 + <0: 0000 + 00: >00 HCOEHO0H9 .HC0EH00HH our 0C0 COS H0HH0 0300 HC0C00HQC0C CH HHE\mCV CHH00H0HQ ECH0mII.OH mqm¢9 .05HH00 CH 00>H0000 HOC 0H03 H0CH 0300 HC0Cm0HmC0C How 00CH0>0 Ho H00ECC 0CH 0H H0H0H 0000CHC0H0Q CH 0H0QECC 0CH 86 .203002 0C0HH0>H0000 .m.m H C0020 .05HH00 CH 00>H0000 H0HHH 0H03 0300 C0C3 C0>Hm 00: Q .mCH000H 002 000H 0CH H0Hm0 0200 m C0>H0 0000 Amvm HMH HOVO HON AmvaHHm 2H0 HH Aux-v Our #3?- OVVNNNTO AVVO flmm HthHHmm HOOOHHOO HMVOHHmH Hmvm Hm Hmvm HmH HNVH HO AmVOHHmH AHV Hm HOVONHOO AHV ON A: v.1 N..N-. AMOS HON Amvv HON COCO HNN COCO HON COCO HOH EOVNHHNO COCONHOO COOHHHHN COCO HOH AOOO HNO AOOOOHOO 1H0 OO. 1H0 NH ANOO HOO COVNNHHO ANOO HON 1H0 OH 2H0 OO “H” CH0 HmOAO HOV mfiw MN «34/944n1 COCO HOH OOHHOO CNOO HOH H- AOONHHOO N- AOOO HOH O- AOVHHHOO O- ANOO HOH O- 0 l O- nmvw HH¢ CON Cm O m H H O m How-m \‘u/‘l ll" 87 The lactating pregnant cows in this experiment had greater prolactin (28:1 ng/ml, n=184) than the nonlactating pregnant heifers in Experiment I (21:1 ng/ml, n=423), and the trend for decreasing serum prolactin with increasing duration of pregnancy observed in heifers was not apparent in cows. Differences in serum prolactin between cows and heifers could be related to lactation, since milking causes a transitory increase in serum prolactin (Tucker, 1971). Similar to heifers, the among cow variation in serum pro— lactin was large; cow averages ranged from 6:1 to 89:16 ng/ml. Significant two and three way interactions of days with the four main effects (pregnancy, genetics, HCG, MGA) are difficult to interpret because of much day variation in prolactin concentration. Estradiol.--Serum estradiol was greater in MGA- treated cows than in control cows (P = .15, Tables 18 and 19). This difference was apparent from proestrus until 2 days after estrus, but at no other time. None of the other main effects, two way or three way interactions approached significance. Only one of the non-MGA treated cows had greater than 22 pg/ml of estradiol and this elevation occurred on day 42 of pregnancy. But 13 samples from 13 different cows after MGA treatment had greater than 35 pg/ml estradiol (Table 20). During the estrous cycle in heifers, 88 m.0Hm.m O.HHo.m H.HHH.m m.mHm.O mm O.NHH.O O.NHH.O O.HH0.0 0.0 0O.O NN O.HH0.0 0.0H0.0 O.HH0.0 0.0 0.0HO.N ON 0O.HHO.O 0.0HN.O O.HHN.O 0.0 0.0H0.0 . OH 0.0HH.O 0.0H0.0 N.HH0.0 N.OH 0O.O HH 0.0HH.O O.HHO.O O.HH0.0 0.0 O.NH0.0 O H.HH0.0 OO.HHO.O OHO.O 0.0H O.NH0.0 O O.OHN.O OO.HHO.O 0.0H0.0 H.OH 0.0HO.N N 0O.OHO.OH OO.OHO.O O.OHO.O 0.00 O.NOOHN.OOO 000000 0O.NHN.OH 0.0H O.HHO.OH H- 0O.HH N- 0O.O 0O.OH O.NH N.HH0.0H O- 0O.O 0O.O O- 0O.O 0O.O O- 0O.O O- 0.0HO.NH 0.0H O.HHO.OH N+<0z 0000 0.0HN.O O.NH ON.HHO.OH 000 0000 HOHHooo 0000 002 0000 + 002 0000 + 002 >0Q M#C®E#MGHB .HC0EH00HH 002 0C0 «02 H0HH0 0300 HC0Cm0HQ CH AHE\mmv H0H00H000 ECH0m-I.0H mam<9 89 .0C0HH0>H00Q0 00HC90 . mGOHHMNVHGmQO 033% .C0HH0>H00Q0 0CO0 .m.m H C002 U .0CH000 CH 00>H00Qo HmuHm 0H03 0300 C033 C0>Hm 0020 .O0H0000 000 HmOH 00H 00000 0000 O 00>HO 000 Q .AOuCO HOHHooo .AOucv 0000 .ANnov 000 .AHuoO 0000 + 002 .HNro 0000 + 0000 O.HH0.0 OO.HHO.O O.OHN.O N.O N.OHO.H ON O.HH0.0 0.0H0.0 N.OH0.0H 0.0 N.HH0.0 OO O.HH0.0 OO.HHH.O O.NHH.O 0.0 0H.O OO O.HHO.O OO.HHO.O O.NHO.O 0.0H OHO.N OO O.HHN.O 0.0H0.0 H.HH0.0 0.0H 0.0HO.H OO O.OHH.O OO.HHHN.OH O.NH0.0 0.0 N.OHO.H NO 0.0H0.0 O.HH0.0 0O.O N.NH 0.0H0.0 OO O.HHO.O O.HHO.O O.NHN.O N.OH OHO.N OO O.OHO.O O.HH0.0 0.0HH.OH O.HH N.OHH.O OO 90 AOO0.0HO.H AOO0.0HH.O 0H0 0.0H AOOO.O HO.O OON “OOH.NH0.0 0N0 OHN.N 0H0 N.OH AOVN.O HO.HH ONN AOVO.NHO.O AOOO.NHO.O COCO.N HO.O OON COCO.HHN.N AOOO.OHN.O 0H0 O.O AOOO.H HO.O OOH AOOO.OHN.O COCO.HHO.O COCO.H HO.O COON.H HH.O COCO.O HH.O HH COCO.HHO.O AOOO.OHO.O COCO.H HN.O AOHOO.H HH.O COON.O HO.HH O AOVO.OHO.O AOOO.OHO.O AOO0.0 HO.O AOHOO.H H0.0 AOOH.N HH.O O AOOO.HHO.O AOOO.HHO.O AOOH.H HO.O AOHOO.OOHO.OO COCO.H HH.O N ANOO.NHO.OH AOOO. HO.O COCO.NHHH.ON COCO.ONHH.OO AOOO.OOHO.NO OOHHmm 0H0 O.OH 0H0 0.0 AOOH.O HO.OH ANON.N HO.OH AOON.H HO.OH H- AHO O.O CNOO.O HO.OH COCO.N HO.O N- CNOO.OHO.N 0H0 0.0 AOON.O HO.OH AOOO.H HO.HH AOON.OOHO.OO O- CHO N.O ANOO.O HO.O AOVO.O HO.O O- CHO O.O 0H0 O.NH O- AHO 0.0 ANOO.HHO.O . ANVO.O HN.HH O- AOVO.H HO.HH COCO.N HN.HH COCO.H HO.OH N+00z 0000 AOOO.N HH.HH COCO.N HO.O AOVN.OOHO.OO 000 0000 HOHHco0 0000 000 0000 + 000 0000 + 000 >00 HC0EH00H9 .HC0EH00HH 000 0C0 <02 H0H00 0300 0C0C0000C0C CH AHE\0QV H0H00H000 8000mlu.mH 00049 91 .05H000 CH 00>H00Q0 HOC 0H03 00:0 0300 HC0C00H0C0C HOH 005H0>0 mo H0QECC 000 OH H000H 0000£HC0H00 CH 0H0QE5C 000 .05H000 CH 00>H00Qo HmuHH 0H03 0300 C003 C0>Hm 000 .OoH0000 000 HOOH .203000 0C0HH0>H00Q0 0.0.0 H 00020 Q 000 H0000 0>00 m C0>H0 0000 AHV Amvm.OHw.0. ANV0.0H0.0H HHV m.h 0.0 Aqvm.NHv.0 CNOO.HH0.0 Amvm.HHm.m ANON.OH0.0 AOON.HHO.O 0.m 2000.0Hm.m 2H0 m.v AOOO.H HN.O COOO.N H0.0 AOON.O H0.0 COC0.0NH0.0N 2H0 m.NH HNVO.o H0.0 HOV m.v Ahvo.H Hmvm.o HOVO.N HOVO.H HOVO.o Amvo.m 0H0 Ho.h H0.0 Hm.0H H0.0 HOVH.H HN.O 2H0 m.m Amvm.HHHo.HN Hmvm.m H0.NH Amvm.H Hm.v ANVO.H HN.O CHH v.m msnumm TABLE 20.--Cows with high 92 (>35 pg/ml) serum estradiol. Cow Treatment Day Estradiol (no) ' (pg/ml) 765 MGA + HCGa 3 days post MGA 44 851 " " " " " " 100 957 " " " " " " 610 958 " " Last day of MGA 598 977b " " 3 days post MGA 690 1027 " " Day 7 43 1037 " " Day 20 44 824p MGA + HCGC Estrus 60 946 " " Estrus 458 998 " " Day 2 356 897 MGA Estrus 38 897 MGA Day 22 88 1005 MGA Estrus 104 771b HCGb Day 42 40 'aHCG 3 days after last MGA feeding. bPregnant, other cows were nonpregnant. CHCG when first observed in estrus. 93 estradiol concentration exceeds 10 pg/ml only at proestrus and estrus (Wettemann et al., 1972). On the last day of MGA treatment 47% of the cows had greater estradiol (30.1: 19.6 pg/ml) than control cows at proestrus. When this elevation in estradiol began during MGA treatment is unknown. Most cows had clearly elevated estradiol during the 2 to 3 days after the last feeding of MGA (Table 21), but only 10% of the cows had estradiol greater than 10 pg/ml by 8 days after MGA. This decrease in estradiol with time after MGA treatment may be attributed to LH release from the pituitary after MGA withdrawal or to injections of HCG; both cause ovulation, removing the source of estradiol. Both pregnant and nonpregnant MGA treated cows had high estradiol during proestrus and estrus compared with controls. The duration of elevated estradiol during MGA treatment could be related to infertility. High con- centrations of estradiol for a prolonged period before estrus could alter uterine and oviducal secretions and contractility, creating a hostile environment for gametes. The onset of elevated serum estradiol may be related to the day of the estrous cycle when progestogen treatment is started. For example, if a large follicle is present at the outset, it may be maintained and secrete estrogen during treatment. Similarly, fertility after progestogen treatment also may be related to the day of the estrous 94 OH o.H H0.0 0H m 0H 0.0 HO.NH mm O om H.NOHH.OO OH 0 cm H.H H0.0 om m om H.m Hm.m 0 O on m.omHN.mm OH m 00 o.H H0.NH om m 9O 0.0HHH.om om o AHe\O00 He\Oo OHM 00H3 0300 Ho 00: 00000 00HQE00 Ho HC00 H00 00 H .0>¢ H0QECZ 0>00 .HmzmHO0HH3 000 .Hmw-HMM m300 SH HOHUMMflmm ESHQMIloHN memANH. 95 cycle when progestogen treatment is started (Wagner et al., 1968; De Bois and Bierschwal, 1970). During days 2 through 11 after insemination, estradiol concentration was similar in pregnant and non— pregnant cows (Tables 18 and 19). In contrast, on day 2 in pregnant heifers, estradiol resembled that at estrus (Figure 6). Differences in estrus detection between the cows and the heifers, as described previously, may be responsible for the difference in estradiol at day 2. Estradiol during pregnancy ranged from 4.9:.6 pg/ml on day 20 to 8.6:3.9 pg/ml on day 42. Similar to heifers, 50% of the pregnant cows had elevated estradiol between days 30 and 42. More frequent sampling would be required to test adequately if growth of the placenta and embryo influence estradiol near day 40. Progesterone.--Serum progesterone averaged 0.7 ng/ml (n=32) on the last day of MGA treatment and only 3 cows had more than ng/ml. The three cows with 4.5 to 6.5 ng/ml progesterone at MGA withdrawal did not exhibit estrus until 6 to 9 days later. Hill et a1. (1971) and Britt and Ulberg (1972) found about 5 ng/ml progesterone at the end of 14 days of MGA when MGA was started on day 14 of the estrous cycle, although corpora lutea had regressed. My cows were at all days of the estrous cycle "when MGA treatment started. Also, I used column 96 chromatography to isolate progesterone from other endogenous steroids which might erroneously inflate progesterone values. Progesterone averaged O.3i.l ng/ml at estrus and increased to 6.9:.6 ng/ml by day 11 in both pregnant and nonpregnant cows (Tables 22 and 23). This rapid increase in progesterone concentration is similar to the change I observed in pregnant heifers and similar to that described by Stabenfeldt et al. (1969), Henricks et al. (1971a), and Swanson et al. (1972). Progesterone values in pregnant and nonpregnant cows did not differ during days 2 through 11 after insemination. Henricks et al. (1971a) reported higher progesterone by nine days after insemination in pregnant cows compared to nonpregnant cows. In my cows, progesterone decreased beginning about 5 to 6 days before the nonpregnant cows returned to estrus, and progesterone decreased further to 2 ng/ml by 2 days before estrus. Progesterone decreased from day 20 to 22 and to day 25 of pregnancy (10.9fl.5, l0.0i1.2 and 8.8il.0 ng/ml, respectively). This decrease is similar to one detected in heifers (Figure 6) occurring between days 18 and 22 of pregnancy, possibly representing initiation of luteal regression which is terminated by the conceptus in the uterus by about day 25. From day 30 through day 75 of pregnancy, progesterone plateaued and averaged about 9.5 ng/ml. 97 m.mww.m m.OHm.m m.owm.n m.HHv.o mm m.mwm.m m.ofiv.> o.HHm.mH N.MH m.owm.m mm m.mum.0H o.mww.HH m.qwh.mH h.o v.me.n om mm.HHH.m m.onm.m m.oan.m >.HH m.owm.m wH m.mwo.n O.me.w H.OHm.o 5.5 wH.m HH w.o«n.m v.mnm.m H.Ofio.m v.v o.vfio.m n m.onm.H mm.owv.m w.OHv.m v.H m.owm.o v H.OHm.o mH.owm.o m.owm.o H.o mH.o m owo moHH.o H.OHH.o N.O OHH.o msnumm mowH.o N.O owo HI mo ml mo N.O owH.o m: mv.v mm.v v: mv.n 0 mn mv.m Aw: H.NHH.N N.O one ~+uwmbo mmucem .mcoHum>HmeO 039m .coHum>Hmeo mcom .m.m H cam: 98 U .mfihfimw CH ©®>H®wQO #mHHM mhm3 m300 C033 C®>Hm UUEO .mchmmm ¢wz ummH may Hmbwm mwmp m cm>Hm.wom Q .Amuav Houuqoo .Avncv ooom .Amncv mos .Aanv coon + aw: .Amncv boom + «02m m.mfih.m mH.~HH.m m.HHN.HH ~.m w.nfim.OH ma >.~H¢.m O.NH5.HH m.onm.a h.m m.m“H.OH mo m.mfio.m mm.OHn.n m.HH~.OH N.HH mo.m om H.~Hm.m mm.HHo.m N.HHm.OH m.HH m.mfis.n om n.HHm.NH m.HHo.m n.mfin.~H m.qH m.on¢.m ms m.owm.m mw.owm.m ~.mHN.NH m.nH H.Hfim.m ms w.HHN.m m.oam.OH . mo.m H.MH m.ono.m ow e.HHv.n m.o“m.mH m.mfim.m m.mH m.mHo.o mm o.mflm.m mm.onH.NH m.mwo.m N.NH m.vum.o om 99 Asvo.mns.m Amvm.o«m.H AHV o.m Amvo.mfls.m 6mm Amvn.mfim.v AHV o.m AHV m.m Amon.mfim.m 6mm Aqvm.mfim.m Amvv.HHn.m AmVH.mH~.N wow AqVF.HHm.q Amvm.onm.o AHV H.m Amvm.mflv.m cwH Asvm.HHm.m Amvm.HH>.m AmVH.HHH.n Amvm.HHm.w AmVN.Hfim.m HH AmVN.HHH.v Amvm.HHm.v Aavm.ono.¢ AOHVm.HHm.v Amvm.onm. a Amvm.OHm.H AmVF.OHm.H Amvo.mfiv.m AoHVo.HHm.H AmVH.owv.o s AmV«.OHm.o Amvm.owm.o AFVH.OHm.o AOHVN.OHm.o Amy OHH.o m Asv¢.oflm.o AmVN.OHm.o AmvH.OHm.o AOHVH.OHN.O Imam.owm.o msuumm AHV o AHV o Avvo.OHs.o ANVH.OHN.O Ame one H- AHV o Ame onm.o Ame OHH.o m- Amvo.mHH.m AHV o.m AmVH.oH~.o Avvm.OHm.o Amy OHH.o m- AHV o ANVH.OHN.O Ame ONH.o v- AHV o Amvm.o«¢.o m- AHV m.oa Amva.HHo.N Amvm.mflm.m w- Amvo.Hnm.H AmVH.OHm.o Ame one ~+ummbo umHHm mums mzoo CmCB Cm>Hm mum mo HmQECC on“ on mommy mommCquHmm CH mHmQESC pr .msnumm CH pm>ummbo UOC mumB ume mBOo uCMCmmHmCOC new mmCHm>p Q .wCHpmmm «oz umMH me umumm m>MU m Cm>Hm Guam .Amzoov mCngm>nmmbo “.m.m H Gmmzo AHV N.O Amvo.NHH.N Amvn.mwn.m E . We Avv owm.o Amvm.owm.o Amvm.QMh.o Amvv.mwm.m Amvm.HHm.m AHV ~.m Amvo.NHH.m AHV v.5 3:434 3:636 $5.306 “$936.6 A: o Amvh.mmo.OH AHV m.m AmVH.OHN.o KETZMK 8353.0 Amvm.HHm.m Avvh.HHm.n Amvm.mwm.m AHV o.m So @434” So one A2 N.Hnmé $5.034 3:436 $5.3m...“ AHV m.m msmumm 101 The interaction between genetic classification, pregnancy and HCG was significant (P<.05). Nonpregnant cows in all treatment combinations had similar progesterone concentration but best pregnant cows and worst pregnant cows given HCG had greater progesterone than worst preg- nant cows without HCG (Table 24). A similar interaction was observed for serum LH (Table 15). Comparing LH and progesterone in the eight treatment combinations, cows with higher progesterone had lower LH and vice versa except for worst pregnant cows given HCG. These same treatment combinations also caused the genetic by preg— nancy interaction to be significant (P<.05). I do not comprehend the physiological meaning of these interactions. 102 m.Hm.m m.Hm.m m.nm.m m.Ho.m mmmum>a m.wm.w m.Hm.m m.wm.m v.Hm.m >.Hv.m HOHuCoo m.Hm.v m.Hm.m n.wm.n v.wo.m we.Hm.w mom Aflsxmcv mmmum>< “CmCmmHmCoz uCMCmmHm #CMCmmumCoz quCmmHm pmuoz ummm CoHumonHmmMHU OHumcmw .ngEumme 00m pCm moCMCmmum .CoHuMOHMHmmmHo oHmemm mo moCmCHmCH umzoo CH mConmummmoum Enummll.vm mamCB GENERAL DISCUSSION Estrus was first observed in the morning in 76% of. the heifers and serum LH was greatest at this time. Elevated LH (34 ng/ml) was detected in 51% of the heifers bled when first observed in estrus. Only 23% of the LH surges were detected in cows sampled similarly, probably because of less meticulous estrous detection among the cows. This concurs with observations that the ovulatory surge of LH occurs between 3 hr before to 6 hr after the onset of estrus and perSists for 6 to 10 hours (Schams and Karg, 1969; Henricks et al., 1970; Swanson and Hafs, 1971). Because of the short duration of the peak, LH concentration at estrus is dependent on the interval between blood sampling; there is no evidence from my data on heifers or cows that the shape or magnitude of the LH surge is related to fertili- ty. Sera concentrations of LH, prolactin, estradiol and progesterone were not influenced by genetic classification. The majority of the cows in this study were foundation cows in the genetic experiment, so the influence of best or worst sires would not be present until the next gener- ation.' 103 104 As described earlier, cows in treatment I of Experiment IV were omitted from the split plot analysis because they were not observed for estrus and were insemi- nated at 12 and at 24 hr after HCG given 3 days after the last MGA feeding. Cows in treatment II (given HCG when observed in estrus) were compared with cows in treatment I (Appendix Table IX). Serum LH, prolactin, estradiol and progesterone were similar after these two treatments. Therefore the graphs in this section (except for estradiol) depict average hormone concentration for cows in all five treatments. Serum LH varied little among heifers (P>.lO), and it did not decrease (P>.lO) with advancing pregnancy in heifers (Figure 4) or cows (Figure 9). None of the pregnant cows had elevated LH (34 ng/ml) after estrus and only three samples from three different pregnant heifers were elevated after estrus. Randel and Erb (1971) observed no significant changes in serum LH from day 7 to day 260 of pregnancy, but Schams (1969) detected increases in serum LH at day 52 in one cow and day 61 in another with more frequent sampling. Nonpregnant heifers had more LH (P<.10) than pregnant heifers during days 2 through 11 after insemination, similar to a report for cows (Edgerton and Hafs, 1971). Nonpregnant cows in my experiment also had higher serum LH from days 2 through 11 after insemination, but this difference was not 105 .CoHuMCHEmmCH kuwm pCm mHOMmQ mzoo CH mH Enummll.m musmHm 106 mnmhmw aubud mo mmommm m>.lO). Lower LH in pregnant heifers or cows probably is not related to estradiol or progesterone feed- back at the pituitary or hypothalamus because I detected no differences in serum estradiol or progesterone between pregnant and nonpregnant cows during days 2 through 11 after insemination (Figures 10, ll, 12). Prolactin concentration differed among heifers (P<.005). But average prolactin in heifers during the first 75 days of pregnancy was not related significantly to milk production in the subsequent lactation (r=.07). Serum prolactin did not vary significantly with day of the estrous cycle or day of pregnancy in cows or heifers (Figures 5 and 13). However, after heifers were synchronized with MGA, a significant increase in prolactin occurred at the time of the ovulatory surge of LH. Increases in pro- lactin at estrus have been reported in heifers (Raud et al., 1971; Swanson et al., 1972) and prolactin decreases during pregnancy were reported in rats (Amenomori et al., 1970) and sheep (Arai and Lee, 1967; Davis et al., 1971). Serum estradiol was lowest during the luteal phase of the estrous cycle and increased beginning about 3 days before estrus to maximal values on the day before or on the day of estrus (Table 2). Estradiol and progesterone were correlated significantly only on the day before estrus (r=-.57, P .05). The ovulatory surge of LH usually occurred 1 or 2 days after the first clear evidence of rising "Fun—‘- t. M‘Am b 'v s . 108 Figure lO.—-Serum estradiol in cows before and after insemination. Figure 11.--Serum estradiol in MGA treated cows before and after insemination. ESTRADIOL (pg/ml) ESTRADIOL (pg/ml) 109 50~ 4o- 30- 20r- NONPREGNANT PREGNANT ESTRUS Io*- fi/dy’ 0 111111 111111 [111 11 1. -6 0 ll 20 30 4O 50 60 75 DAYS BEFORE OR AFTER ESTRUS U54m4) (531.3) 50- I 4 .. 0 II n H II : 20 :P | NONPREGNANT :9 ‘ R’/4V/ PREGNANT 'l \ 1' 537903 ‘ I\ _ ‘ l‘ . '0 . W 1 01111111 llllllllll II J -6 0 ll 20 3O 4O 50 60 75 DAYS BEFORE OR AFTER ESTRUS 110 Figure 12.—-Serum progesterone in cows before and after insemination. Figure 13.-—Serum prolactin in cows before and after insemination. PROLACTIN (ng/ml) l6- I4- : PREGNANT E I2- \ U 5 IO- uJ <2; 8 - a: “J NONPREGNANT E} 6" rr/¢p// ‘9 | 2 4 - 4 o- ‘ ESTRUS 2 _ VLIX/ O l l l I l l l l l l l l I l 1_ -6 0 ll 20 30 4O 50 60 75 DAYS BEFORE OR AFTER ESTRUS 70- 60-7 l 50 " || NON PREGNANT - PRE NA T 40 || [- G N BOr- || 1 20- ! \ESTRUS IO- 0 :11111 lilnl‘Lilll 11 J -6 0 ll 20 30 4O 50 60 75 111 DAYS BEFORE OR AFTER ESTRUS 112 estradiol. Injection of 10 mg estradiol caused increased serum LH 20 to 24 hours later in cows (Howlands et al., 1971) and similar data were described in sheep (Goding et al., 1969). Therefore my data suggest that estrogen secretion normally regulate the ovulatory LH surge in cattle. Pregnancy did not alter serum estradiol in control or MGA treated cows until after day 11 when proestrus 1W““ ’1-- increases began only in nonpregnant cows (Figures 10 & 11). Randel et al. (1971) reported altered excretion of urinary estrogens compared with pregnant cows during the first 9 days after breeding in cows which did not conceive. At present, the relationship between serum estradiol and urinary estrogens has not been established. The cause of infertility may determine whether estrogen concentration is altered after insemination.) For example, cows with ovarian cysts may have increased serum estradiol and be infertile, but cows that do not conceive because they are inseminated too late may have normal estradiol levels. During days 18 through 75 of pregnancy, estradiol averaged 7 pg/ml in heifers except for day 40 when four of eight heifers had elevated estradiol (11.1:2.3 pg/ml). Similar to heifers, 50% of the pregnant cows had elevated estradiol between days 30 and 42. Luteinizing hormone initiates blastocyst implantation in rats (Macdonald et_al., 1967), probably mediated through estrogen secretion since 113 Yoshinaga and Hosi (1961) found that estrogen causes implantation in lactating rats. Shaikh (1971) reported increased estrone and estradiol in ovarian venous blood from rats on day 4 after breeding; the day of implantation. Therefore, possibly the increased estradiol I found in some cows may facilitate implantation in cattle. More frequent sampling between days 35 and 45 of pregnancy would be necessary to examine precisely the role of estradiol. Serum progesterone was similar (P>.lO) in pregnant and nonpregnant cows through day 11 after insemination (Figure 12). Henricks et al. (1970) reported significantly greater serum progesterone in pregnant cows than nonpreg- nant cows from 10 to 14 days after insemination, but pro- gesterone was similar in the two groups on day 16. No conclusive differences in progesterone between pregnant and nonpregnant cows have been described until after day 16. Progesterone began decreasing about 5 to 6 days before estrus in cows. Lamond et al. (1971b) observed greatest progesterone in beef cattle 3.5 to 5 days before estrus. Maximum progesterone (about 11 ng/ml) during the first 75 days of pregnancy was attained by day 18 in heifers and by day 20 in cows, then progesterone decreased significantly (P<.05) about 20% during the next 4 or 5 days. This decrease in serum progesterone may represent the initial phases of luteal regression, terminated by .the conceptus about day 22 to 25. Heifers had greater 114 progesterone (P<.005) during days 45 through 75 of preg- nancy than during days 20 through 42, but this difference was not observed in cows. Ovulation occurred 36:3 hr after HCG given 3 days after the last MGA feeding. Twenty-two of the 25 cows ovulated. But I recovered ova from only 31% of the cows at slaughter 4 days after HCG injection. Fertility appeared normal in the few cows that ova were recovered from. Wagner et al. (1968) and Hill et al. (1970) recovered a greater percentage of ova 3 to 3.5 days after insemination but fertility was decreased by progestogen treatment. The ovulatory surge of LH which occurred 2 to 7 days after MGA withdrawal, was not altered by MGA treatment. Basal levels of LH on the last day of MGA treatment were similar to levels during proestrus. Hill et a1. (1970) found higher LH during MGA treatment than in controls. In my eXperiments, serum LH after the synchronized estrus did not differ from control cows. Estradiol was greater in pregnant and nonpregnant MGA treated cows than in control cows (Figures 10 & 11). On the last day of MGA treatment, 47% of the cows had greater than 10 pg/ml estradiol, similar to levels normally observed during proestrus. About 40% of the cows had estradiol greater than 35 pg/ml after MGA withdrawal. The major endocrine abnormality I found after estrous cynchronization with MGA isla prolonged proestrus 115 period (possibly beginning during MGA treatment) typified by high serum concentration of estradiol after withdrawal. This elevated estradiol is accompanied by low progesterone, but this should not be a problem while the cow receives exogenous progestogen. To return a synchronized cow to the normal estrous endocrine state, it may be necessary to remove the influence of the excess serum estradiol; possibly with an antiestrogen to compete with estradiol for binding sites at target tissues. wigs-1:1. I.” {Hi a-IF'PI. ‘. 3.". . .. _.. . 1 SUMMARY AND CONCLUSIONS Endocrine changes associated with early pregnancy and after nonfertile inseminations were studied in 28 heifers and 58 cows. Estrus was first observed in the morning in 76% of the heifers and fertility did not differ between forenoon and afternoon inseminations. An average of 1.33 inseminations were performed per conception. Serum LH concentration was greatest on the day of estrus averaging 8.7il.6 ng/ml in heifers that conceived and 12.0i3.2 ng/ml in nonpregnant heifers. There was little among heifer variation in LH and the concentration of LH did not differ (P>.lO) from day 18 through day 75 of pregnancy. Pregnant heifers had lower serum LH (P<.10) during days 2 through 11 after insemination compared to nonpregnant heifers (1.0:.1 vs 1.2:.1 ng/ml, respectively). Serum prolactin differed significantly (P<.005) among heifers. Average prolactin concentration ranged from 7:3 to 56:14 ng/ml for samples from individual heifers. Changes in prolactin could not be related to stage of estrous cycle or pregnancy. In contrast to LH, prolactin was greater (P<.10) in pregnant heifers (24:3 ng/ml) than in nonpregnant heifers (16:3 ng/ml) during the first 18 days of pregnancy. 116 d“£_.-LA.-‘ -' I v 117 Serum estradiol decreased from 12.612.4 pg/ml at estrus to 8.4:.6 pg/ml by day 4 of pregnancy. During days 7 through 75 of pregnancy, estradiol averaged 6 to 8 pg/ml except on day 40 (11.1i2.3 pg/ml) when four of the eight heifers had elevated estradiol. Increased estradiol con- centration at day 40 of pregnancy may be related to growth of the conceptus. Serum progesterone increased (P<.005) from 0.4 ng/ml at estrus to 6.8 ng/ml on day 11, and differed sig- nigicantly (P<.05) among days 18, 20, and 22 of pregnancy (10.811.3, 9.2:.9 and 8.1:.9 ng/ml, respectively). This decrease in progesterone resembles the initial stages of luteal regression about day 18 of the estrous cycle. Possibly the conceptus terminates this luteal regression about day 22 of pregnancy. On day 18 of pregnancy, serum LH and progesterone were highly correlated (r = .87, P<.01). Progesterone was significantly (P<.005) greater during days 45 through 75 of pregnancy than from days 18 through 42, suggesting an extra-ovarian source of progesterone beginning about day 42. The ovulatory surge of LH in synchronized heifers occurred 2 to 7 days after the last MGA feeding and appeared similar in magnitude and duration to LH peaks observed at control estrus. Serum prolactin was elevated (P<.05) on the day before or on the day of the LH peak. 118 Ovulation was synchronized by HCG injected 3 days after the last MGA feeding. Twenty-two of 25 cows ovulated 36:3 hr after HCG. Only 31% of the ova were recovered, but fertility appeared normal in cows from which ova were re- covered. Endocrine changes after estrous synchronization and insemination were studied in 58 cows. Serum LH was not influenced by genetic classification, pregnancy or MGA. The day by pregnancy interaction was significant (P<.005); pregnant and nonpregnant cows had similar LH during proestrus, LH was greater in pregnant cows at estrus, but LH was lower in pregnant than nonpregnant cows during days 2 through 25 after insemination. Serum prolactin in cows was not affected signifi- cantly by genetic classification, pregnancy, MGA or HCG (P>.lO). Prolactin concentration ranged from 20:4 ng/ml at estrus to 34:8 ng/ml on day 22 of pregnancy, but no meaningful physiological pattern of prolactin changes could be established. Progesterone averaged 0.7 ng/ml on the last day of MGA treatment and only 3 of 32 cows had greater than 1 ng/ml. Serum progesterone increased significantly (P<.Ol) from 0.31.1 ng/ml at estrus to 6.9:.6 ng/ml by day 11, both in MGA and in control cows. Progesterone did not differ (P>.lO) between pregnant and nonpregnant cows during days 2 through 11 after insemination. 119 The MGA treated cows had greater serum estradiol than control cows (P = .15). During the estrous cycle of control cows, estradiol exceeded 10 pg/ml only at proestrus and estrus. On the last day of MGA treatment, 47% of the cows had estradiol concentration comparable to or greater than that during proestrus and estrus in controls. When this elevation in estradiol began during MGA treatment is unknown. After MGA withdrawal, serum estradiol increased to greater than 100 pg/ml in some cows. Estradiol concentration was similar in pregnant and nonpregnant cows during days 2 through 11 after insemi- nation. During pregnancy estradiol ranged from an average of 4.9i.6 pg/ml on day 20 to 8.6:3.9 pg/ml on day 42. Similar to heifers, 50% of the pregnant cows had elevated estradiol between days 30 and 42, possibly related to growth of the conceptus. I conclude that a major endocrine abnormality after estrous synchronization with MGA is prolonged proestrus typified by high levels of estradiol, possibly beginning during MGA treatment and persisting for 2 to 7 days after MGA withdrawal. BIBLIOGRAPHY 120 BIBLIOGRAPHY Ainsworth, L. and K. J. Ryan. 1967. Steroid hormone trans— formations by endocrine organs from pregnant mammals. II. Formation and metabolism of pro- gesterone by bovine and sheep placental preparations - in vitro. Endocrinol. 18:1349. E H i i Amenomori, Y., C. L. Chen and J. Meites. 1970. Serum prolactin levels in rats during different repro— ductive states. Endocrinol. 86:506. Anderson, R. R. and W. H. McShan. 1966. Luteinizing hormone levels in pig, cow and rat blood plasma during the estrous cycle. Endocrinol. 78:976. Arai, Y. and T. H. Lee. 1967. A double-antibody radio- immunoassay procedure for ovine pituitary prolactin. Endocrinol. 81:1041. Arey, L. B. 1954. Developmental Anatomy. p. 147. W. B. Saunders Co., Philadelphia, Pa. Armstrong, D. T. and W. Hansel. 1959. Alteration of the bovine estrous cycle with oxytocin. J. Dairy Sci. 42:533. Baker, R. D. and E. G. Coggins. 1968. Synchronization of estrus and artificial insemination in beef cattle. Can. J. Animal Sci. 48:303. Boyd, L. J. 1970. Effects of Feeding Melengestrol Acetate (MGA) on occurrence of estrus, fertility and milk yield in dairy cows. J. Animal Sci. 31:751. Bray, G. 1960. A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter. Anal. Biochem. 1:279. Britt, J. H. and L. C. Ulberg. 1972. Peripheral plasma progesterone levels during and subsequent to melengestrol acetate (MGA) administration to dairy heifers. J. Reprod. Fert. (in press). 121 122 Carrick, M. J. and J. N. Shelton. 1969. Oestrogen- progesterone relationships in the induction of oestrus in spayed heifers. J. Endocrinol. 45:99. Chang, M. C. 1967. Personal communication. Christensen, D. S., J. N. Wiltbank and M. L. Hopwood. 1971. Blood hormone levels during the bovine estrous cycle. J. Animal Sci. 33:251. Davis, 8. L., L. E. Reichert, Jr. and G. D. Niswender. 1971. Serum levels of prolactin in sheep as measured by radioimmunoassay. Biol. Reprod. 4:145. De Bois, C. H. W. and C. J. Bierschwal, Jr. 1970. Estrous cycle synchronization in dairy cattle given a 14- day treatment of melengestrol acetate. Am. J. Vet. Res. 31:1545. Desjardins, C. and H. D. Hafs. 1968. Levels of pituitary .FSH and LH in heifers from birth through puberty. J. Animal Sci. 27:472. Dhindsa, D. 8., A. S. Hoversland and E. P. Smith. 1967. Estrous control and claving performance in beef cattle fed 6-methyl—l7-acetoxy-progesterone under ranch conditions. J. Animal Sci. 26:167. Diczfalusy, E., Ed. 1970. Steroid Assay by Protein Binding. Transactions of the Second Karolinska Symposia on Research Methods in Reproductive Endocrinology. p. 291. Drewry, K. J., K. Hawkins, C. J. Kaiser, L. A. Nelson and R. Peterson, Jr. 1968. Estrus synchronization trials with a commercial beef cow herd. Research Progress Report, Purdue University. 340:1. Echternkamp, S. E. and W. Hansel. 1971. Plasma estrogens, luteinizing hormone, and corticoid in postpartum cows. J. Dairy Sci. 54:800. Edgerton, L. A. and H. D. Hafs. 1971. Serum luteal hormone and prolactin in cows after successful and unsuc- cessful inseminations. J. Dairy Sci. 54:799. Erb, R. E., V. L. Estrogreen, Jr., W. R. Gomes, E. D. Plotka and O. L. Frost. 1968a. Progestin levels in corpora lutea and progesterone in ovarian venous and jugular vein blood plasma of the pregnant bovine. J. Dairy Sci. 51:401. 123 Erb, R. E., V. L. Estergreen, Jr., W. R. Gomes, E. D. Plotka and O. L. Frost. 1968b. Progestin content of ovaries and the effect on assessment of luteal activity in the bovine. J. Dairy Sci. 51:411. Erb, R. E., W. R. Gomes, R. D. Randel, V. L. Estergreen, Jr., and O. L. Frost. 1968c. Effect of ovariectomy on concentration of progesterone in blood plasma and urinary estrogen excretion rate in the pregnant bovine. J. Dairy Sci. 51:420. Estergreen, V. L., Jr., 0. L. Frost, W. R. Gomes, R. E. Erb and J. F. Bullard. 1967. Effect of ovariectomy on pregnancy maintenance and parturition in dairy cows. J. Dairy Sci. 50:1293. Fahning, M. L., R. H. Schultz, E. F. Graham, J. D. Donker and H. W. Mohrenweiser. 1966. Synchronization of oestrus in dairy heifers with 6a-methy1-l7a- acetoxy-progesterone and its effect on conception rate. J. Reprod. Fert. 12:569. Findlay, J. K. and R. I. Cox. 1970. Oestrogens in the plasma of the sheep foetus. J. Endocrinol. 46:281. Foote, W. D. and L. Walker. 1961. Influence of estrogen treatment on ovarian activity in post-partum dairy cows. J. Animal Sci. 20:671. Garverick, H. A., R. E. Erb, G. D. Niswender and C. J. Callahan. 1971. Reproductive steroids in the bovine. III. Changes during the estrous cycle. J. Animal Sci. 32:946. Gill, J. L. and H. D. Hafs. 1971. Analysis of repeated measurements of animals. J. Animal Sci. 33:331. Ginther, O. J. 1970. Effect of progesterone on length of estrous cycle in cattle. Am. J. Vet. Res. 31:493. Ginther, O. J., C. O. Woody, K. Janakiraman and L. E. Casida. 1966. Effect of an intra-uterine plastic coil on the oestrous cycle of the heifer. J. Reprod. Fert. 12:193. Ginther, O. J., C. Woody, S. Mahajan, K. Janakiraman and L. E. Casida. 1967. Effect of oxytocin adminis- tration on the oestrous cycle of unilaterally hysterectomized heifers. J. Reprod. Fert. 14:225. -gn Wuudmfim l d.-_ l 124 Goding, J. R., K. J. Catt, J. M. Brown, C. C. Kaltenbach, I. A. Cumming and B. J. Mole. 1969. Radioimmunoassay for ovine luteinizing hormone. Secretion of lute— inizing hormone during estrus and following estrogen administration in the sheep. Endocrinol. 85:133. Gomes, W. R., V. L. Estergreen, Jr., 0. L. Frost and R. E. Erb. 1963. Progestin levels in jugular and ovarian venous blood, corpora lutea, and ovaries of the nonpregnant bovine. J. Dairy Sci. 46:553. Graves, C. N. and P. J. Dziuk. 1968. Control of ovulation in dairy cattle with human chorionic gonadotrophin after treatment with 6a—methyl-l7d-acetoxy-progesterone. J. Reprod. Fert. 17:169. Guthrie, H. D., D. R. Lamond, D. M. Henricks and J. F. Dickey. 1970. Ovarian follicular changes in heifers treated with melengestrol acetate. J. Reprod. Fert. 22:363. Hackett, A. J. and H. D. Hafs. 1969. Pituitary and hypothalamic endocrine changes during the bovine estrous cycle. J. Animal Sci. 28:531. Hafez, E. S. E., Ed. 1969. Reproduction in Farm animals. Lea and Febiger, Philadelphia, Pa. p. 132. Hafs, H. D. and D. T. Armstrong. 1968. Corpus luteum growth and progesterone synthesis during the bovine estrous cycle. J. Animal Sci. 27:134. Hansel, W. and K. H. Seifart. 1967. Maintenance of luteal function in the cow. J. Dairy Sci. 50:1948. Hansel, W. and R. B. Snook. 1970. Pituitary ovarian relationships in the cow. J. Dairy Sci. 53:945. Henricks, D. M., J. F. Dickey and G. D. Niswender. 1970. Serum luteinizing hormone and plasma progesterone levels during the estrous cycle and early pregnancy in cows. Biol. Reprod. 2:346. Henricks, D. M., D. R. Lamond, J. R. Hill and J. F. Dickey. 1971a. Plasma progesterone concentration before mating and in early pregnancy in the beef heifer. J. Animal Sci. 33:450. Henricks, D. M., D. R. Lamond, J. R. Hill and J. F. Dickey. 1971b. Plasma total estrogens and progesterone concentrations during proestrus and after mating in beef heifers. (Abstr.) Fourth Ann. Meeting Soc. Study Reprod., p. 13. 125 Hill, J. R., Jr., D. R. Lamond, D. M. Henricks, J. F. Dickey and G. D. Niswender. 1971. The effect of melengestrol acetate (MGA) on ovarian function and fertilization in beef heifers. Biol. Reprod. 4:16. Howland, B. E., R. E. Short, R. A. Bellows and E. A. Ibrahim. 1971. Effect of ovariectomy and estrogen on serum LH in cows. J. Animal Sci. 33:257. Koprowski, J. A. and H. A. Tucker. 1971. Failure of oxytocin to initiate prolactin or luteinizing hormone release in lactating dairy cows. J. Dairy Sci. 54:1675. Korenman, S. G. 1968. Radio-ligand binding assay of specific estrogens using a soluble uterine macromolecule. J. Clin. Endocrinol. 28:127. Korenman, S. G., L. E. Perrin and T. P. McCallum. 1969. A radio-ligand binding assay system for estradiol measurement in human plasma. J. Clin. Endocrinol. 29:879. Labhsetwar, A. P. 1968. Studies on the mode of action of oral contraceptives: Effect of chlormadinone on pituitary FSH and LH contents of the female rat. J. Reprod. Fert. 17:101. Lamond, D. R., J. F. Dickey, D. M. Henricks, J. R. Hill, Jr. and T. M. Leland. 1971a. Effect of a progestin on the bovine ovary. J. Animal Sci. 33:77. Lamond, D. R., D. M. Henricks, J. R. Hill, Jr. and J. F. Dickey. 1971b. Breed differences in plasma pro— gesterone concentration in the bovine during proestrus. Biol. Reprod. 5:258. Larsson—Cohn, U., E. D. B. Johansson, L. Wide and C. Gemzell. 1970. Effects of continuous daily administration of 0.5 mg of chlormadinone acetate on the plasma levels of progesterone and on the urinary excretion of luteinizing hormone and total oestrogens. Acta. Endocrinol. 63:705. Llerena, L. A., A. Guevara, J. Lobotsky, C. W. Lloyd and J. Weisz. 1969. Concentration of luteinizing and follicle-stimulating hormones in peripheral and ovarian venous plasma. J. Clin. Endocrinol. 29:1083. 126 Macdonald, G. J., D. T. Armstrong and R. O. Greep. 1967. Initiation of blastocyst implantation by luteinizing hormone. Endocrinol. 80:172. Makepeace A. W., G. L. Weinstein and M. H. Friedman. 1937. The effect of progestin and progesterone on ovulation in the rabbit. Amer. J. Physiol. 119:512. Malven, P. V. and R. Ruiz-Diaz. 1971. Inhibition of ovulation by intracranial implants of medroxy- progesterone acetate. J. Animal Sci. 32:919. Mares, S. E., R. G. Zimbelman and L. E. Casida. 1962. f Variation in progesterone content of the bovine ' corpus luteum of the estrual cycle. J. Animal Sci. 21:266. McDonald, P. G. and M. T. Clegg. 1967. The effect of progesterone on serum luteinizing hormone concen- trations in the ewe. J. Reprod. Fert. 13:75. Mellin, T. N. and R. E. Erb. 1965. Estrogens in the bovine- a review. J. Dairy Sci. 48:687. Melton, A. A., R. 0. Berry and O. D. Butler. 1951. The interval between the time of ovulation and attachment of the bovine embryo. J. Animal Sci. 10:993. Mikhail, G., C. H. Wu, M. Ferin and R. L. Vande Wiele. 1970. Radioimmunoassay of plasma estrone and estradiol. Steroids. 15:333. Mishell, D. R., Jr. and W. D. Odell. 1971. Effect of varying dosages of ethynodiol diacetate upon serum luteinizing hormone. Amer. J. Obstet. Gynec. 109:140. Morrison, F. B. 1957. Feeds and Feeding, 22nd Ed., p. 680. Morrison Publishing Co., Ithaca, New York. Murphy, B. E. P. 1967. Some studies of the protein-binding of steroids and their application to the routine micro and ultramicro measurement of various steroids in body fluids by competitive protein-binding radioassay. J. Clin. Endocrinol. 27:973. Niswender, G. D., L. E. Reichert, Jr., A. R. Midgley, Jr. and A. V. Nalbandov. 1969. Radioimmunoassay for bovine and ovine luteinizing hormone. Endocrinol. 84:1166. 127 Oxender, W. D., H. D. Hafs and L. A. Edgerton. 1972. Serum growth hormone, LH and prolactin in the pregnant cow. J. Animal Sci. (in press). Pelletier, J. and J. Thimonier. 1969. Etude de la décharge ovulante par dosage radioimmunologique de la LH plasmatique, chez 1a Brebis normale ou traiteé par un progestagene. C. R. Acad. Sc. Paris. 268D:573. Petrow, V. 1970. The contraceptive progestagens. Chem. Rev. 70:713. Plotka, E. D., R. E. Erb, C. J. Callahan and W. R. Gomes. 1967. Levels of progesterone in peripheral blood plasma during the estrous cycle of the bovine. J. Dairy Sci. 50:1158. Pritchard, D. E., R. P. Wettemann and H. D. Hafs. 1970. Fertility of rabbits after melengestrol acetate administration. J. Animal Sci. 31:729. Pritchard, D. E., H. D. Hafs, H. A. Tucker, L. J. Boyd, R. W. Purchas and J. T. Huber. 1972. Growth, mammary, reproductive and pituitary hormone characteristics of Holstein heifers fed extra grain and melengestrol acetate. J. Dairy Sci. (in press). Purchas, R. W., A. M. Pearson, D. E. Pritchard, H. D. Hafs and H. A. Tucker. 1971. Some carcass quality and endocrine criteria of Holstein heifers fed melengestrol acetate. J. Animal Sci. 32:628. Rado, A., J. A. McCracken and D. T. Baird. 1970. The formation of oestrogens by the autotransplanted ovary of the ewe perfused in vivo with C19 steroids. Acta. Endocrinol. 65:244. Rakha, A. M. and H. A. Robertson. 1965. Changes in levels of follicle stimulating hormone and luteinizing hormone in the bovine pituitary gland at ovulation. J. Endocrinol. 31:245. Randel, R. D. and R. E. Erb. 1971. Reproductive steroids in the bovine. VI. Changes and interrelationships from 0 to 260 days of pregnancy. J. Animal Sci. 33:115. Randel, R. D., H. A. Garverick, R. E. Erb and C. J. Callahan. 1971a. Reproductive steroids in the bovine IV. Urinary estrogen excretion rates from O to 9 days after breeding in fertile and nonfertile cows. J. Animal Sci. 32:1183. 128 Randel, R. D., H. A. Garverick, A. H. Surve, R. E. Erb and C. J. Callahan. 1971b. Reproductive steroids in the bovine. V. Comparisons of fertile and nonfertile cows 0 to 42 days after breeding. J. Animal Sci. 33:104. Raud, H. R., C. A. Kiddy and W. D. Odell. 1971. The effect of stress upon the determination of serum prolactin by radioimmunoassay. Proc. Soc. Exp. Biol. and Med.’ 136:689. Robinson, R., R. D. Baker, P. A. Anastassiadis and R. H. Common. 1970. Estrone concentrations in the peripheral blood of pregnant cows. J. Dairy Sci. 53:1592. Saunders, D. M., S. L. Marcus, B. B. Saxena, C. Beling and E. B. Connell. 1971. Effect of daily adminis- tration of 0.5 mg of chlormadinone acetate on plasma levels of follicle-stimulating hormone, luteinizing hormone, and progesterone during the menstrual cycle. Fertil. Steril. 22:332. Schally, A. V., W. H. Carter, M. Saito, A. Arimura and C. Y. Bowers. 1968. Studies on the site of action of oral contraceptive steroids. I. Effect of antifertility steroids on plasma LH levels and on the response to luteinizing hormone-releasing factor in rats. J. Clin. Endocrinol. 28:1747. Schams, D. 1969. Radioimmunobiologische bestimmung des luteinisierung-shormons (LH) in blutserum von kuher in den ersten zwei monaten der trachtigkeit. Dtsch. Tierarztl. Wschr. 76:561. Schams, D. and H. Karg. 1969. Radioimmunologische LH—- bestimmung im blutserum von rind unter besonderer berucksichtigung des brfinstzyklus. Acta Endocrinol. 61:96. Schams, D. and H. Karg. 1970. Untersuchungen uber prolaktin im rinderblut mit einer radioimmunologischen bestimmungsmethode. Zbl. Vet. Med. 17:193. Shaihh, A. A. 1971. Estrone and estradiol levels in the ovarian venous blood from rats during the estrous cycle and pregnancy. Biol. of Reprod. 5:297. Shemesh, M., N. Ayalon and H. R. Linder. 1968. Early effect of conceptus on plasma progesterone level in the cow. J. Reprod. Fert. 15:161. 129 Short, R. V. 1958. Progesterone in blood. II. Progesterone in the peripheral blood of pregnant cows. J. Endocrinol. 16:426. Sinha, Y. N. and H. A. Tucker. 1969. Mammary development and pituitary prolactin level of heifers from birth through puberty and during the estrous cycle. J. Dairy Sci. 52:507. Smith, J. F. and A. J. Allison. 1971. The effect of exogenous progestagen on the production of cervical mucus in the ewe. J. Reprod. Fert. 24:279. Smith, J. F. and T. J. Robinson. 1969. Luteal function in the Merino ewe and the effect of exogenous pro- gestagen. J. Endocrinol. 44:79. ‘11.!51 hurl. ' Smith, J. F. and T. J. Robinson. 1970. The effect of exogenous progestagen on the levels of free oestrogen in the ovarian vein plasma of the ewe. J. Endocrinol. 48:485. Smith, L. W. and R. G. Zimbelman. 1968. Control of ovulation in cattle with melengestrol acetate. III. Inducing ovulation during MGA treatment. J. Reprod. Fert. 16:73. Spies, H. G. and G. D. Niswender. 1971. Blockade of the ' surge of preovulatory serum luteinizing hormone and ovulation with exogenous progesterone in cycling rhesus (Macaca mulatta) monkeys. J. Clin. Endocrinol. 32:309. Stabenfeldt, G. H., L. L. Ewing and L. E. McDonald. 1969. Peripheral plasma progesterone levels during the bovine oestrous cycle. J. Reprod. Fert. 19:433. Stabenfeldt, G. H., B. I. Osburn and L. L. Ewing. 1970. Peripheral plasma progesterone levels in the cow during pregnancy and parturition. Amer. J. Physiol. 218:571. Stormshak, F. and R. E. Erb. 1961. Progestins in bovine corpora lutea, ovaries, and adrenals during preg— nancy. J. Dairy Sci. 44:310. Swanson, L. V. 1970. Endocrine, behavioral, and ovarian changes in holstein heifers from puberty to breeding size. PHD. Thesis Michigan State University. 130 Swanson, L. V. and H. D. Hafs. 1971. LH and prolactin in blood serum from estrus to ovulation in Holstein heifers. J. Animal Sci. 33:1038. Swanson, L. V., H. D. Hafs and D. A. Morrow. 1972. Ovarian characteristics and serum LH, prolactin, progesterone, and glucocorticoids from first estrus to breeding size in holstein heifers. J. Animal Sci. (in press). Tanabe, T. Y; 1970. The role of progesterone during preg- nancy in dairy cows. Penn. State University Agr. Expt. Sta. Research Bull. 774. Tillson, S. A., I. H. Thorneycroft, G. E. Abraham, R. J. ‘Scaramuzzi and B. V. Caldwell. 1970. Solid phase radioimmunoassay of steroids. £n_"Immunologica1 methods in steroid determination" F. G. Peron and B. V. Caldwell (Ed.) Appleton-Century-Crofts, New York, p. 127. Trimberger, G. W. 1948. Breeding efficiency in dairy cattle from artificial insemination at various intervals before and after ovulation. Univ. of Nebraska Agr. Expt. Sta. Research Bull. 153. Trimberger, G. W. and W. Hansel. 1955. Conception rate and ovarian function following estrus control by progesterone injections in dairy cattle. J. Animal Sci. 14:224. Tripath, V. N. and W. E. Howell. 1969. Effects of group- fed dihydroxyprogesterone acetOphenide in combination with an injection of estradiol valerate, and 'melengestrol acetate on estrus synchronization and conception in beef heifers. Can. J. Animal Sci. 49:113. Tucker, H.A. 1971. Hormonal response to milking. J. Animal Sci. 32: Suppl. I, 137. Wagner, J. F., E. L. Veenhuizen, R. P. Gregory and L. V. Tonkinson. 1968. Fertility in the beef heifer following treatment with 6-chloro-A6-l7 acetoxyprogesterone. J. Animal Sci. 27:1627. Wettemann, R. P., H. D. Hafs, L. A. Edgerton and L. V. Swanson. 1972. Estradiol and progesterone in blood serum during the bovine estrous cycle. J. Animal Sci. (in press). 131 Wickersham, E. W. and T. Y. Tanabe. 1967. Functional status of bovine corpora lutea of pregnancy. J. Dairy Sci. 50:1001. Willett, E. L. 1950. The fertility of heifers following administration of progesterone to alter the estrual cycle. J. Dairy Sci. 33:381. Wiltbank, J. N. 1966. Modification of ovarian activity in the bovine following injection of oestrogen and gonadotrophin. J. Reprod. Fert. Suppl. 1:1. Wiltbank, J. N. and L. E. Casida. 1956. Alteration of ovarian activity by hysterectomy. J. Animal Sci. 15:134. Wiltbank, J. N. and C. W. Kasson. 1968. Synchronization of estrus in cattle with an oral progestational agent and an injection of an estrogen. J. Animal Sci. 27:113. Wiltbank, J. N., J. A. Rothlisberger and D. R. Zimmerman. 1961. Effect of human chorionic gonadotrOpin on maintenance of the corpus luteum and embryonic survival in the cow. J. Animal Sci. 20:827. Wiltbank, J. N., J. C. Sturges, D. Wideman, D. G. LeFever and L. C. Faulkner. 1971. Control of estrus and ovulation using subcutaneous implants and estrogens in beef cattle. J. Animal Sci. 33:600. Winters, L. M., W. W. Green and R. E. Comstock. 1942. Prenatal development of the bovine. Minn. Expt. Sta. Tech. Bull. 151. Woody, C. O., N. L. First and A. L. Pope. 1967. Effect of exogenous progesterone on estrous cycle length. J. Animal Sci. 26:139. Yamauchi, M. and T. Nakahara. 1958. Effects of uterine distention on the estrous cycle of cattle. Japan J. Animal Reproduction. 3:121. Yoshinaga, K. and T. Hosi. 1961. On the delayed implanta- tion in the lactating pregnant rat. Part I. The effect of estrogen. Jap. J. Animal Reprod. 3:93. (cited by Dairy Sci. Abstr. 23:222). Zimbelman, R. G. 1966. Effects of progestagens on ovarian and pituitary avtivities in the bovine. J. Reprod. Fert. Suppl. 1:9. 132 Zimbelman, R. G., J. W. Lauderdale, J. H. Sokolowski and T. G. Schalk. 1970. Safety and pharmacologic evaluations of melengestrol acetate in cattle and other animals: a review. J. Amer. Vet. Med. Assoc. 157:1528. Zimbelman, R. G., R. G. Loy and L. E. Casida. 1961. Variations in some biochemical and histological characteristics of bovine corpora lutea during pregnancy. J. Animal Sci. 20:99. Zimbelman, R. G. and L. W. Smith. 1966a. Control of ovulation in cattle with melengestrol acetate. I. Effect of dosage and route of administration. J. Reprod. Fert. 11:185. Zimbelman, R. G. and L. W. Smith. 1966b. Control of ovulation in cattle with melengestrol acetate. II. Effects of follicular size and activity. J. Reprod. Fert. 11:193. fini- APPEND ICES 133 134 TABLE I.--Preparation of liquid scintillation fluids. A. Steroid scintillation fluid (Hafs and Armstrong, 1968). Naphthalene --------------------------------- 480 g PPO ------------------------------------------ 30 g POPOP --------------------------------------- 0.3 g Xylene -------------------------------------- 2000 ml p-dioxane---é ------------------------------- 2000 ml Mix until dissolved. B. Bray's solution (Bray, 1960) Naphthalene --------------------------------- 240 g PPO ----------------------------------------- 16 g Dimethyl POPOP ------------------------------ 0.8 g Ethylene Glycol ----------------------------- 80 m1 Methanol ------------------------------------ 400 ml p-dioxane ----------------------------------- 3264 ml Mix until dissolved. 135 TABLE II-“'Compositioniof buffers used in estradiol assays. A. Buffer A. (0.01M Tris—HCl, 0.25M sucrose, 0.001M EDTA, pH 8.0). Tris ----------------------------------- 1.211 g disodium EDTA -------------------------- 0.372 g Sucrose------------¥ ------------------- 85.85 g Dissolve the reagents in 800 ml distilled water and adjust the pH to 8.0 with 6 N HCl. Then dilute to 1 liter. Store buffer at -20 C in quantities suffi- cient for single assays. B. Buffer AA. (0.1M phosphate, 0.15M sodium chloride, 0.015M sodium azide, 0.1% gelatin, pH 7.0). Sodium phosphate,_monobasic ----------------- 5.38 g Sodium phosphate, dibasic, heptahydrate ----- 16.35 g Sodium chloride ----------------------------- 9.0 g Sodium azide -------------------------------- 1.0 g 'Gelatin (Knox Gelatin, Inc., Johnston, N. Y.) ------------------- 1.0 g Dissolve the reagents in distilled water and dilute to 1 liter. 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