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Carruthers has been accepted towards fulfillment of the requirements for Ph. D. degree in Dairy Science (/Anudwa Major professor Date /¢1fla:/78 O7 639 0mm: FINES ARE 25¢ PER DAY . PER 1m Return to book drop to remove . this checkout from your record. EFFECTS OF SUCKLING ON POSTPARTUM OVULATION, LUTEINIZING HORMONE, FOLLICLE-STIMULATING HORMONE AND PROLACTIN IN HOLSTEIN CONS By Terry David Carruthers A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Dairy Science 1979 ABSTRACT EFFECTS OF SUCKLING ON POSTPARTUM OVULATION. LUTEINIZING HORMONE, FOLLICLE—STIMULATING HORMONE AND PROLACTIN IN HOLSTEIN COWS By Terry David Carruthers Suckling-induced postpartum (PP) anovulation in beef cows pre- vents many cattlemen from attaining an economically desirable 12-month calving interval. How suckling inhibits PP ovulation is not known. Elucidation of this mechanism may lead to successful strategies for inducing early PP ovulation and estrous cycles in suckled cows. The objective of this thesis was to explore possible endocrine mechanisms by which suckling could delay PP ovulation in cows. This research was conducted using dairy rather than beef cows because of availability and amenability to handling and sampling procedures. Experiment I was designed to confirm effects of suckling on the PP intervals to ovulation and observed estrus in Holstein cows. Treatments consisted of nonsuckled controls milked twice daily (NS-2X, n = 5), suckled cows milked twice daily (S-2X, n = 6) and nonsuckled cows milked four times daily (NS-4X, n = 5). Calves were free to nurse except while the milking machine was attached and during periods of estrus detection. Mean intervals to first PP ovulation did not differ between NS-ZX and NS-4X treatments (l9.4 t 3.3 vs 23.0 i 5.3, P > .25), whereas S-2X cows ovulated later (38.7 1 4.9 days, P < .05) than nonsuckled cows. The PP interval to first observed estrus was Terry David Carruthers prolonged in S-2X cows compared to NS—2X cows (50.2 i 4.3 vs 39.0 i 2.4 days, P < .06) but not compared to NS-4X cows (44.8 i 6.1 days, P > .25). On days 7 and l4 PP, blood was collected at frequent intervals for 4.5 h centered on the l600 h milking. Serum was assayed for lute- inizing hormone (LH) and prolactin, with selected samples assayed for total glucocorticoids, follicle-stimulating hormone (FSH) and estradiol-l7B. Mean serum LH was lower in S-2X cows than in NS-2X or NS-4X cows (.7 vs l.4 or 1.3 ng/ml, P < .05), but LH in NS-2X and NS-4X cows did not differ (P > .25). During the anovulatory period, LH was released in an episodic pattern. The reduced mean LH in suckled cows resulted from decreased frequency and amplitude of epi- sodic LH peaks (P < .05). Mean serum LH concentrations increased from day 7 to l4 PP across all treatments (.9 vs l.4 ng/ml, P < .05), due to increased frequency and amplitude of LH peaks. Basal and milking-induced serum prolactin was greater on day l4 than on day 7 PP, but was not affected by suckling. No effects of suckling were apparent on serum FSH, glucocorticoids, estradiol-l78 or progesterone. In Experiment II, I examined how suckling decreased episodic LH secretion in early PP cows. Treatments consisted of nonsuckled milked controls (NS-2X, n = 8) and milked cows suckled gg_libitum by two calves (S-2X, n = 9). Blood was collected at l5-min intervals for 8 h on day 13 PP, then cows were killed on day l4 postpartum. Hypo- thalami and pituitaries were collected for in vitro studies. Experiment 11 confirmed that suckling reduced the frequency and amplitude of episodic LH peaks. Suckling did not significantly Terry David Carruthers alter serum FSH, prolactin, progesterone, estradiol-l78 or gluco- corticoids. Hypothalamic content of gonadotropin-releasing hormone (GnRH) did not differ between S-2X and NS-ZX cows on day l4 PP (ll8.2 i l6.3 vs lll.4 i l9.5 ng/hypothalmus, P > .25). Suckling did not affect the LH, FSH or prolactin content of pituitaries from cows killed l4 days postpartum. Pituitary explants were challenged jg_yjtrg_with GnRH (25 ng/ml) or K+ (59 mM) in continuous-flow superfusion. Suckling reduced significantly LH but not FSH released in response to either GnRH or potassium. Because similar amounts of tissue were superfused and tissue LH contents were not different in suckled and nonsuckled cows, it appears that LH in pituitaries of suckled cows is less releasable than that of nonsuckled cows. Reduced LH secretion in suckled cows did not appear to result from a specific reduction in response to gonadotropin-releasing hormone. In overview, delayed PP ovulation in suckled cows is associa- ted with reduced episodic secretion of luteinizing hormone. The reduced LH secretion does not reflect depleted hypothalamic GnRH or pituitary LH content, but may result from reduced hypothalamic GnRH secretion leading to reduced LH mobilization and secretion. To my wife Carolyn. ii ACKNOWLEDGMENTS I wish to express my appreciation to my major professor, Dr. H. D. Hafs, for his encouragement and advice during the course of my studies at Michigan State University. I am also grateful to the other members of my graduate committee, Drs. R. H. Douglas, J. L. Gill, G. L. Waxler and E. M. Convey, for their efforts on my behalf during my course of studies and the preparation of this thesis. I am especially indebted to Dr. E. M. Convey for his advice and assistance in designing and executing my research as well as for his valued friendship. My fellow graduate students, the faculty and staff of the Dairy Science Department have all contributed immensely to the success of my graduate studies at Michigan State and I am very grateful to them. I wish to thank the Department of Dairy Science, Michigan State University for providing facilities and funding for my research and the Medical Research Council of Canada for the Fellowship which has supported me during my graduate studies. iii TABLE OF CONTENTS ACKNOWLEDGMENTS LIST OF TABLES . LIST OF FIGURES LIST OF APPENDICES INTRODUCTION REVIEW OF LITERATURE . General Factors Influencing the Intervals to First Postpartum Ovulation and Estrus in Cows Suckling . Lactational Intensity Nutrition . . Age and Parity . Other Factors Summary . Postpartum Reproductive Physiology and Endocrinology. Effects of Suckling . . . . . . . Uterus and Cervix . Adrenals . Ovaries . Corpus Luteum of Pregnancy . Follicular Growth . Postpartum Ovarian Steroid Secretion Ovarian Responsiveness to Exogenous Gonadotropins . Effects of Exogenous Steroids on Postpartum Intervals . . . . . Hypothalamo- -Pituitary. Axis . Hypothalamus . Anterior Pituitary Gland Hormone Content Anterior Pituitary Hormones in Serum of. Postpartum Cows . Effects of GnRH on Gonadotropins. and Post- partum Intervals . iv Page vii ix xi Summary MATERIALS AND METHODS General . . Blood Collection and Handling . Assays . . Statistical Analysis . . Experiment I: Effects of Suckling on Postpartum Intervals and Serum Hormone Concentrations in Holstein Cows . . . . . . . . . . . . . Experiment 11: Effects of Suckling on the Hypothalamo- Pituitary Gonadotropic Axis of Early Postpartum Holstein Cows . . . . . . . . In vivo In V1tro . . Tissue Collection . . . ExtractionoflJl, FSH and Prolactin from Pituitary Tissue . Hypothalamic Tissue Trimming and GnRH Extraction . . Pituitary Superfusion Method RESULTS AND DISCUSSION Experiment I: Effects of Suckling on Postpartum Intervals and Serum Hormone Concentrations in Holstein Cows . . Animal Characteristics and PP Intervals to Ovulation and Estrus . Serum Hormone Concentrations Luteinizing Hormone . Follicle- -Stimulating Hormone Prolactin . . Progesterone, Estradiol- 178 and Gluco- corticoids Summary . . . . . . . . . . . . . Experiment II: Effects of Suckling on the Hypothalamo- Pituitary Gonadotropic Axis of Early Postpartrum Holstein Cows . . Animal Characteristics and Slaughter Data . Serum Hormone Concentrations Luteinizing Hormone . Follicle- -Stimulating Hormone Prolactin . . Progesterone, Estradiol- l78 and Glucocorticoids . Summary . Page 39 42 42 42 43 45 49 Page Hypothalamic Gonadotropin-Releasing Hormone (GnRH) Content . . . . . . . . . . . . 78 Anterior Pituitary Gland . . . . . . . . . 8l Height and Hormone Content . . . . . . . 8l In_Vitro Superfusion: Response to Gonadotropin- Releasing Hormone and K . . . . . . . 83 Summary . . . . . . . . . . . . . . . 86 GENERAL DISCUSSION . . . . . . . . . . . . . . 88 SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . 94 APPENDICES . . . . . . . . . . . . . . . . . 96 LITERATURE CITED . . . . . . . . . . . . . . . 134 vi Table TO. F.l. LIST OF TABLES Intervals to first postpartum (PP) ovulation and estrus in cows: Literature review . . Characteristics of suckled (S) and nonsuckled (NS) Holstein cows milked twice (2X) or four times (4X) daily: Experiment I . . . Intervals to first postpartum ovulation and estrus in suckled (S) and nonsuckled (NS) Holstein cows milked twice (2X) or four times (4X) daily Summary characteristics of serum LH profiles in suckled (S) and nonsuckled (NS) cows on days 7 and l4 postpartum (PP) . Serum concentrations (x t SE) of progesterone, estradiol-l78 and glucocorticoids in suckled (S—2X) and nonsuckled (NS-2X) cows on days 7 and l4 postpartum (PP) . . . . . . . . Characteristics of suckled (S-2X) and nonsuckled (NS-2X) postpartum (PP) Holstein cows used in Experiment II . . . . . . . . . Characteristics of LH secretory profiles in suckled (S- 2X) and nonsuckled (NS— 2X) cows on day 13 postpartum . . . . Serum hormone concentrations (x 1 SE) in suckled (S-2X) and nonsuckled (NS-2X) Holstein cows on day l3 postpartum . . . . . Content and gross distribution of hypothalamic GnRH in suckled (S- 2X) and nonsuckled (NS- 2X) cows killed on day l4 postpartum . . Content of LH, FSH and prolactin in anterior pitui- taries of suckled (5- 2X) and nonsuckled (NS- 2X) cows killed on day l4 postpartum . Cross reaction of other bovine pituitary hormones in the follicle-stimulating hormone radioimmunoassay . vii Page 50 52 58 65 68 71 79 80 82 TIB Table Page F.2. Assay recovery of FSH added to bull serum . . . . l20 6.1. Mean (1 SE) concentrations of quality control stan- dards in estradiol-l78 radioimmunoassay . . . . l27 G.2. Cross reactioncrfnmjor steroids in the estradiol-l78 radioimmunoassay . . . . . . . . . . . . l29 H.l. Stock solutions used for Kreb's ringer bicarbonate buffer . . . . . . . . . . . . . . . l32 H.2. Volumes of stock solutions required for l.32 liters of Kreb's ringer bicarbonate buffer containing 5.9 mM K+ (KRB-5.9) or 59 mM KT (KRB-59) . . . . l33 viii Figure LIST OF FIGURES Diagram of the ventral surface of the bovine brain with the dissection boundaries (anterior- posterior and lateral) of the hypothalamic tissue blocks removed for extraction and radioimmunoassay of gonadotropin- releasing hormone . Schematic diagram of the apparatus used for in vitro superfusion of bovine pituitary explants . Luteinizing hormone profiles in serum of nonsuckled twice daily milked (NS-2X) cows on days 7 and 14 postpartum, (* = designated peak) . Luteinizing hormone profiles in serum of suckled twice daily milked (S-2X) cows on days 7 and 14 postpartum, (* = designated peak) Luteinizing hormone profiles in serum of nonsuckled four times daily milked (NS—4X) cows on days 7 and 14 postpartum (* = designated peak) 6. Characteristics of serum luteinizing hormone (LH) \I o 00 profiles in suckled (S) and nonsuckled (NS) cows milked twice (2X) or four times (4X) daily . . . Prolactin in serum of suckled (S- 2X) and nonsuckled (NS- 2X) postpartum (day 7 and 14 pooled) holstein cows for 2 h before and after start of afternoon milking . Prolactin in serum of holstein cows (suckled and non- suckled treatments pooled) for 2 h before and after start of afternoon milking on days 7 and 14 postpartum . . . . . 9. Characteristics of serum luteinizing hormone (LH) profiles in suckled (S- 2X) and nonsuckled (NS- 2X) cows on day 13 postpartum ix Page 44 46 55 56 57 59 62 63 72 Figure 10. 11. 12. 13. 14. F.1. Concentrations of follicle-stimulating hormone in serum of suckled (S-2X) and nonsuckled (NS-2X) cows on day 13 postpartum Mean concentrations of prolactin in serum of suckled (S- 2X) and nonsuckled (NS- 2X) cows on day 13 postpartum . . Serum concentrations of Estradiol- 178 in suckled (S- 2X) and nonsuckled (NS- 2X) cows from day 4 to day 14 postpartum. . In_vitro secretion of luteinizing hormone (LH) in response to gonadotropin-releasing hormone (GnRH) or K challenge (solid bar) by pituitary pieces from suckled and non- suckled cows on day 14 postpartum Ifl_vitro secretion of follicle- stimulating hormone (FSH) in response to gonadotropin- -releasing hormone (GnRH) or KT challenge (0 to 30 min) by pituitary pieces from suckled (S) and non- suckled (NS) cows on day 14 postpartum Parallelism of serum, superfusion media, pituitary extracts and follicle-stimulating hormone reference preparation (NIH-FSH-Bl) in the bovine follicle-stimulating hormone radioimmunoassay . Page 73 75 77 84 87 117 LIST OF APPENDICES Appendix Page A. RADIOIMMUNOASSAY FOR BOVINE LUTEINIZING HORMONE (LH) . 97 B. RADIOIMMUNOASSAY FOR BOVINE PROLACTIN . . . . . . 100 C. EXTRACTION AND RADIOIMMUNOASSAY OF PROGESTERONE . . 103 D. RADIOIMMUNOASSAY OF GONADOTROPIN-RELEASING HORMONE . 107 E. EXTRACTION AND COMPETITIVE PROTEIN BINDING RADIOASSAY OF TOTAL GLUCOCORTICOIDS . . . . . 110 F. RADIOIMMUNOASSAY 0F BOVINE FOLLICLE- STIMULATING HORMONE (FSH) . . . . . . . . 114 G. EXTRACTION, CHROMATOGRAPHY AND RADIOIMMUNOASSAY OF ESTRADIOL-17B . . . . . . . 121 H. STOCK SOLUTIONS AND MIXING RATIOS FOR MAKING KREB'S RINGER BICARBONATE BUFFERS USED IN PITUITARY SUPERFUSIONS . . . . . . . 13D xi INTRODUCTION Females of many species undergo a period of reproductive acyclicity following parturition. The length of this anovulatory period may vary dramatically and be influenced by many factors among which nutrition, season and lactational or suckling intensity have been demonstrated to be of major importance. In cows, it appears that suckling is the principle stimulus for a prolonged postpartum (PP) anovulatory period although concurrent nutritional and/or environmental factors may exacerbate the suckling effect. A 12-month calving interval is associated with maximum eConomic return for both beef and dairy cows; howeven.current industry averages range from 13 to 14 months. Since a 12-month calving interval includes the 280-day bovine gestation there remains only 85 days during which ovulatory ovarian activity must be reinitiated and conception occur. In most management schemes, breeding does not occur until detection (by bull or man) of the first PP behavioural estrus; however, ovulation not estrus is the critical prerequisite to conception. This is particularily apparent in view of recent advances in ovulation synchronization and timed artificial insemination which have reduced or eliminated the need for estrus detection. Unfortunately, these techniques are of limited practical value in acyclic cows. The aim of this thesis research was to examine the influence of suckling on bovine PP reproduction and endocrinology in order to elucidate the mechanism by which suckling delays the first PP ovulation. Understanding the basic mechanisms controlling the interval to first PP ovulation in cows and the manner by which suckling delays this ovulation is important to development of management schemes to optimize PP reproduction in cattle. REVIEW OF LITERATURE General The PP intervals to first ovulation and first observed estrus in cattle have been summarized by Casida (1971) and more recently by Stevenson (1977). The reported PP intervals for milked, suckled and nonlactating beef and dairy cows are summarized in Table 1. Mean intervals to first PP ovulation ranged from 16 to 71 days whereas first observed PP estrus was reported to have occured after mean intervals of 20 to 84 days. The percentage of cows which did not exhibit detectable estrous behavior at the first PP ovulation ranged from 25 to 100% (Hackett et_gl,, 1973; Hartigan et_gl,, 1974; King et_gl,, 1976), reasons for these "silent" ovulations are unclear. However;the absence of appreciable concen- trations of serum progesterone prior to the first PP ovulation may have been involved in the reduced estrual behaviour since pre- exposure to progesterone has been shown to increase the behavioural response to exogenous estrogens (Melampy et_gl,, 1957). Estrus without subsequent ovulation was infrequent in cattle (Labhsetwar et_gl:, 1963; Casida, 1971) and frequency of "silent" ovulations decreased with both increasing PP interval and number of preceeding ovulations (Casida and Nisnicky, 1950; Kidder et_gl,, 1952; Stevenson and Britt, 1977a). Delays in PP rebreeding that result Table 1. Intervals to first postpartum (PP) ovulation and estrus in cows: Literature review. Cow Lactation Interva1c(days) to PP typea statusb Reference Ovulation Estrus Saidduddin et a1., 1968 D NL ---- 30.0 Casida & Venzke, 1936 D M-2X 41.0 ---- Clapp, 1937 D M-2X ---- 46.0 Olds et a1., 1949 D M-2x ---- 30.0 Herman & Edmondson, 1950 D M-2X ---- 57.0 Buch et a1., 1955 D M-2X ---- 33.0 Trimberger & Fincher, 1956 D M-ZX 44.0 50.0 Fosgate et a1., 1962 D M-2X 30.0 48.0 Menge et a1., 1962 D M-2X 20.0 34.0 Morrow et a1., 1968 D M-2X 16.4 ---- Saiduddin et a1., 1968 D M-2X ---— 23.5 Morrow et a1., 1969 D M-2X 22.9 ---- Moller, 1970b 0 M-ZX 35.9 ---- Edgerton & Hafs, 1973 D M-2X ---- 36.0 Williams et a1., 1973 D M-2X 18.0 55.0 Britt et a1., 1974 D M-2X 19.5 39.3 Manns & Richardson, 1976 D M-2X 19.0 ---- Stevenson, 1977 D M-2X 17.6 26.8 Goodale et a1., 1978 D M-2X 19.1 38.2 ClapP, 1937 D M-4X ---- 69.0 Clapp, 1937 0 S-1 ---— 72.0 Saiduddin et a1., 1968 0 S-1 ----- 45.0 Moller, 1970b D S-2X 60.6 ---- " " 0 S-3 61.8 ~--- " " 0 S-4 71.0 ---- Graves et a1., 1968 B NL ---- 30.7 McCartor, 1972 B NL ---- 36.7 England et a1., 1973 B NL 21.3 38.6 Bellows et a1., 1974 B NL ---- 20.2 LaVoie et a1., 1974 B NL ---- 30.0 Hiltbank & Cook, 1958 B M-2X 36.0 54.0 Lasley & Bogart, 1943 8 S-1 ---- 80.0 Niltbank 8 Cook, 1958 8 S-1 53.0 84.0 Harnick, 1955 8 S-1 ---- 61.0 Table 1 (cont.) Cow Lactation Intervalc(days) to PP typea statusb Reference Ovulation Estrus Foote et a1., 1960 8 S-1 61.5 81.0 Foote & Hunter, 1964 8 S-1 44.0 49.0 Foote & Saiduddin, 1964 8 S-1 38.0 46.0 Graves et a1., 1968 8 S-1 ---- 73.0 Oxenreider, 1968 8 S-1 36.0 45.0 Brown et a1., 1973 8 S-1 55.0 48.0 McCartor, 1972 8 S-1 ---- 67.0 England et a1., 1973 8 S-1 43.3 65.0 Bellows et a1., 1974 8 S-1 ---- 40.4 LaVoie & Moody, 1974 8 S-1 ---- 62.0 Corah et a1., 1975 8 S-1 ---- 49.0 Humphrey, 1977 8 S-1 ---- 76.0 Clemente et a1., 1978 B S-l ---- 41.0 Holness et a1., 1978 8 S-1 ---- 70.5 Oxenreider, 1968 8 S-2 55.0 55.0 a0 = dairy breed, 8 = beef breed. bNL = nonlactating (weaned < 14 days PP), M = milked, S = suckled; 2X or 4X refers to number of milkings or sucklings per day and the single number after S refers to the number of calves per cow. c . . . Mean w1th1n experiment. from undetected estrus are amenable to management procedures which improve the level of estrus detection (Esslemont, 1974; Britt, 1975; Stevenson and Britt, 1977b) or eliminate it entirely by use of timed insemination following ovulation synchronization (Inskeep, 1973; Lauderdale, 1974; Roche, 1976). Ovulation is required for conception and there is little evidence (excluding cystic ovarian disease and pyometra) that ovarian ovulatory cyclicity is interrupted prior to conception once the first spontaneous PP ovulation has occurred. For these reasons, understanding the mechanisms controlling the first PP ovulation and the factors which influence the timing of this event are crucial to intelligent manipulation of bovine PP reproduction. Various factors that have been suggested to influence PP intervals in cows will be reviewed in the next section. Factors Influencing the Intervals to First Postpartum Ovulation and Estrus in Cows Suckling The effect of suckling on reproduction has been known for many years. Seyle and McKeown (1934) recognized that suckling affected the occurrence of estrus in rats. Clapp (1937) reported that cows which suckled their calves exhibited a longer period between calving and first estrus than milked cows. Based on the literature tabulated in Table 1, it can be seen that beef cows had mean PP intervals to ovulation ranging from 21.3 to 61.5 days compared to 16.4 to 71.0 days for dairy cows. Also, suckled cows had mean PP anovulatory periods of 36.0 to 71.0 days whereas non- suckled (milked or nonlactating) cows ovulated 16.4 to 44.0 days postpartum. Nonsuckled beef cows may ovulate by day 7 PP (Han and Moody, 1974), an interval even shorter than that of milked dairy cows. Also, the suckled dairy cow may have a PP anovulatory period lasting 60 to 71 days (Holler, 1970b). In both beef and dairy cows suckling by two or more calves was more effective in delaying PP ovulation than suckling by a single calf (Moller, 1970b; Bellows et_gl,, 1974). This may have resulted from an increased frequency of suckling. However, even once daily suckling by a single calf had more effect than twice daily milking (Moller, 1970b), suggesting that the influencecn’the calf may be more complex than frequency of milk removal alone. In conclusion, in both beef and dairy cows, suckling by one or more calves will cause a major increase in the interval to the first PP ovulation. Lactational Intensity In milked cows, an increase in frequency of milking from twice to four times daily has been shown to increase the PP inter- val to estrus (Clapp, 1937; Niltbank and Cook, 1958); effects on the PP interval to first ovulation have not been reported. Non- lactating PP cows exhibited first ovulation and estrus as early as day 7 PP (Han and Moody, 1974). Heifers which were mastectomized prior to calving exhibited first PP estrus earlier and presumably ovulated earlier than nonsuckled intact controls (LaVoie et a1., 1974; Short et;gl,, 1976; Samuelson et_gl,, 1977), suggesting a possible effect of the mammary gland itself on PP reproduction. In summary, although milking will prolong the interval to first PP ovulation and estrus, its effects are much less marked than those of suckling, probably because milking mimics only a portion of the complex of stimuli associated with a suckling calf. Nutrition A number of studies have demonstrated that reduced energy intake in the face of recommended levels of other nutrients resulted in prolonged PP intervals to ovulation and estrus (Wiltbank et_al,, 1962, 1964; Wagner and Oxenreider, 1971; McCartor, 1972). Prepartum energy balance appears more critical than PP energy intake since cows subject to high prepartum energy levels followed by restricted PP intake (H:L) had shorter intervals to estrus than L:H, L:L or H:H (Wiltbank et_gl,, 1962, 1964). This may have reflected the importance of body condition at calving in determining the body weight change PP as reported by Oxenreider and Wagner (1971) and the reported shorter PP anovulatory period in cows losing the least body weight during the early PP period (Menge et_gl,, 1962). Wiltbank and coworkers (1962) suggested that a reduction in dietary energy may reduce pituitary gonadotropin production and/or release. McClure (1968) speculated that low energy intake reduces circulating glucose concentrations and that lack of glucose may reduce gonadotropin production by the pituitary. A recent report by Radford et a1. (1978) does not support this concept since serum glucose concentrations did not differ between suckled and nonsuckled beef cows during the first 100 days postpar- tum. Suckled cows in this same experiment had lower serum concen- tration of LH and markedly prolonged PP intervals to ovulation and estrus when compared to nonsuckled controls. It should also be noted that reduced energy intake did not delay onset of PP ovarian activity in milked cows (Gardner, 1969; Whitmore et_gl,, 1974; Corah et_§1,, 1974; Ray et_gl,, 1974) or in nonlactating cows (Oxenreider and Wagner, 1971). The effects of excess energy on PP ovulation and estrus are less definite. Wiltbank et_gl, (1964) reported that a diet supplying 150% of National Research Council energy requirements resulted in prolonged intervals to first PP estrus in suckled beef cows. Morrow et_gl, (1969b) increased the PP interval by about nine days in dairy cows fed “liberal" amounts of concentrate. However, Holness and coworkers (1978) were unable to influence PP reproduction in lactating beef cows fed 130% National Research Council energy requirements. In practical terms, overfeeding of cows to an extent that impairs PP reproduction is an unlikely occurrence. Adequate nutrition, primarily pre- and postpartum energy intake, would appear to be important in achieving optimum PP reproductive performance in cattle. However, the literature does not suggest that inadequate energy intake is the major mechanism by which suckling causes prolonged PP anestrus. 10 Age and Parity Parity has been reported to influence the interval to first estrus by some (Hammond and Sanders, 1923; Casida and Wisnicky, 1950) but not all investigators (Warnick, 1955; Foote et_gl,, 1960). Older cows with more than four calvings tended to have slightly longer PP intervals than cows with two to four calvings. However, a more marked effect of parity was evident with first-calf heifers. The prolonged PP intervals to ovulation and estrus in young primi- parous heifers may have reflected insufficient energy intake to meet the demands of growth and concurrent lactation (Menge et_gl,, 1962). It appears that age and parity have variable but relatively minor effects on PP reproduction which may in fact derive from confounding nutritional inadequacies. Other Factors Genetic variation in interval to first PP ovulation has been measured with a reported hereditability estimate of approxi- mately .2 (Menge et_§l,, 1962; Saiduddin et_gl,, 1968). The interval to first estrus was prolonged in cows with genetically high milk production potential as compared to cows with genetically low production potential (Whitmore et;gl,, 1974). In contrast to other species such as the sheep, season appears to have minimal if any affect of PP anestrus in cattle. A few researchers have reported that cows calving in winter had longer intervals to first estrus than those calving during other seasons 11 (Chapman and Casida, 1937; Buch et_gl,, 1955; Carman, 1955). How- ever,other workers have been unable to support this finding (Herman and Edmondson, 1950; Wiltbank and Cook, 1958). Periparturient diseases (dystocia, retained placenta, metritis, ketosis, severe mastitis) have been associated with delayed first PP ovulation (Callahan et_gl,, 1971; Morrow, 1971). However, the delays were relatively minor and the results of Morrow may have been confounded with effects of milk yield, age and parity. Summary The intervals from parturition to first ovulation and estrus may vary greatly among cows, and a number of factors have been shown to influence them including suckling, lactational in- tensity, nutrition, age, parity, genetics and periparturient disease. Suckling appears to be the most effective inhibitor of PP ovulation in both beef and dairy cows, and although the suckling effect may be compounded by other concurrent factors as in underfed and growing heifers, it does not appear that suckling exerts its effect indirectly via the other factors discussed above. The next section of this review will expand upon the repro- ductive physiology and endocinology of PP cows and the influence of suckling on this system. 12 Postpartum Reproductive Physiology and Endocrinology: Effects of Suckling Normal PP physiology and endocrinology will be described for the major components of the reproductive system and the effects of suckling will be stressed. Emphasis will be on cows, with relevant information from other species cited where gaps exist in the literature on cattle. Uterus and Cervix Information regarding uterine involution in cows has been reviewed (Morrow et_gl., 1969a; Moller, 1970a). The majority of reports have been based upon rectal palpation of uterine size, tone and fluid content. These researchers have found that gross involution of the uterus and cervix was completed by 20 to 25 days PP unless complicated by pathological conditions. Based on histological criteria, other researchers concluded that uterine involution was essentially complete by 20 to 30 days postpartum (Gier and Marion, 1968; Wagner and Hansel, 1969). Most authors have also concluded that involution takes about 5 days longer in multiparous than in primiparous cows (Rasbeck, 1950; Morrow et_el,, 1966). Wiltbank and Cook (1958) reported that suckling delayed uterine involution whereas Wagner and Hansel (1969) concluded that suckling had no effect on involution. In studies based on uterine histology as well as gross characteristics, Riesen et_gl,, (1968) and Lauderdale et a1. (1968) reported that suckling significantly 13 increased the rate of uterine involution in dairy and beef cows, respectively. Although suckling has been shown to hasten uterine involu- tion in some studies (possibly thru the release of oxytocin), there is no evidence that the uterus limits the onset of PP ovarian ovulatory cyclicity in suckled cows. Also, based on histological examination of the uterus on day 20 or 70 after calving in ovari- ectomized (24 h PP) and intact animals, Sawhney et_gl, (1966) concluded that the ovaries had little influence on the uterus during the early PP period. Adrenals Glucocorticoids measured in serum of PP dairy cows milked twice daily were found to average 3.2 i .3 ng/ml from 1 to 8 weeks postpartum (Edgerton and Hafs, 1973). Milking resulted in a significant increase in total serum glucocorticoids (Smith et_§l,, 1972; Koprowski and Tucker, 1973). However, the glucocorticoid response to milk removal did not differ between cows that were suckled and those that were milked (Wagner and Ozenreider, 1971). Wagner and Hansel (1969) reported that adrenals of suckled cows had lower contents of progesterone and cortisol than did adrenals of milked cows and suggested reduced synthesis or increased secretion of these steroids in suckled cows. Adrenal hypertrophy was not seen in the suckled cows as might be expected if chronic hyperstimula- tion had occurred, and in a subsequent study (Wagner and Oxenreider, 1971) suckling did not affect adrenal size or steroid content. 14 In serum collected from cows over a 24-h period, glucocorti- coids increased to >10 ng/ml more frequently in milked and suckled cows than in nonlactating cows (Wagner and Oxenreider, 1971), suggesting that possibly the frequency of suckling-induced adrenal stimulation could be involved in suppression of PP ovarian activity. The adrenal glands have been shown to secrete both glucocorticoids and progesterone (Short, 1960; Resko, 1968); how- ever, there is no evidence that adrenal progesterone secretion is increased by the presence of a suckling calf in the PP cow. I conclude that although adrenal secretion is stimulated by suckling, the adrenals do not appear to be principle mediators of suckling's effects on PP reproduction in cattle. Ovaries Corpus Luteum of Pregnancy The corpus luteum of pregnancy regresses rapidly during the few days immediately prior to and following parturition in cows. Little if any functional capability exists following parturition as measured by gross size on rectal palpation (Morrow et_al., 1966), progesterone content (Labhsetwar et_gl,, 1964; Oxenreider, 1968), histological structure (Wagner and Hansel, 1969) or serum proges- terone concentrations (Erb et_gl,, 1968: Stabenfeldt, 1969; Edgerton and Hafs, 1973; Corah et_gl,, 1974). Suckling has no influence on the corpus luteum of pregnancy and PP anestrus is not a result of prolonged maintenance of the pregnancy corpus luteum. 15 Follicular Growth Follicular development reached a nadir at the time of parturition with only scattered small follicles present on the ovaries (Labhsetwar et_gl,, 1964). Following calving there was rapid resumption of flflliculargrowth, and developing follicles weme present on the ovaries within a few days postpartum (Cole and Hughes, 1946; Marion and Gier, 1968; Saiduddin et_gl,, 1968). Labhsetwar et_§1, (1964) reported that follicles of mature size had developed by day 21 PP, and follicular fluid weight increased as early as day 10 PP then remained constant thru day 30 postpartum (Saiduddin et_gl,, 1968). These authors found no effects of suckling on ovarian follicular fluid weight on days 10, 20, or 30 postpartum. Wagner and Oxenreider (1971) reported that suckled cows averaged 16.0 i 3.3 days until follicles grew to >10mm in diameter, significantly longer than nonlactating cows which averaged 9.0 i 1.6 days. Milked cows were intermediate at 13.0 i 1.4 days. In conclusion, there appears to be morphologically adequate follicular development in cows by as early as one week PP, and suckling delays follicular growth only about one week. Apparently follicular growth up to at least the preovulatory stage is not markedly slowed by suckling, and retarded follicular growth does not appear responsible for the prolonged PP anovulatory period in suckled cows. This suggests that follicle-stimulating hormone (FSH) secretion is not imparied in suckled cows but rather that 16 luteinizing hormone (LH) secretion is affected since ovulation, and not follicular growth, is delayed. Postpartum Ovarian Steroid Secretion One to two days prior to parturition serum progesterone decreased precipitously from >5 ng/ml to <1 ng/ml at the time of calving (Stabenfeldt et_el,, 1970; Henricks et_gl,, 1972; Edgerton and Hafs, 1973; Smith et_gl,, 1973). In some studies, progesterone remained low or undetectable from calving until corpus luteum formation following the first PP ovulation (Arije et_gl,, 1974; Echternkamp and Hansel, 1973; Stevenson, 1977). Several other investigators have reported transitory (3 to 5 day) increases (>1 ng/ml) in serum progesterone concentration which return to baseline immediately before the first apparent PP estrus (Pope et_ gl,, 1969; Donaldson et_gl,, 1970; Tribble et_gl,, 1973; Corah et_gl., 1974; LaVoie et_gl,, 1976; Humphrey et_gl,, 1976; Castenson et_gl,, 1976). Based on visual inspection of the ovaries, Castenson et_gl, (1976) concluded that in seven of eight beef heifers the pre-estrus progesterone increase they observed had been due to a corpus luteum formed following a prior "silent" ovulation. To a less marked degree, the corpus luteum lifespan has been reported to be shorter during the first PP estrous cycle than in subsequent cycles (Morrow et_gl,, 1966; Marion and Gier, 1968; Pope et_gl,, 1969; Corah et_gl,, 1974; Stevenson, 1977). The source of the transitory progesterone increase wasrkn:definitively determined in the other studies. However, Humphrey et a1. (1976) 17 collected blood at 6-h intervals and were unable to show an increase in estradiol or a preovulatory-like LH surge before the pre-estrus progesterone increase. They speculated that the source of the pro- gesterone may have been partially luteinized anovulatory follicles. In support of this view, Wagner and Hansel (1969) described a thickening and infolding of granulosa cells in large follicles which approached but did not reach maturity in PP cows. They sug- gested that this phenomenon may have represented unsuccessful or partial luteinization. Although the majority of investigators who observed a tran- sient progesterone increase prior to first estrus used suckled beef cows, this phenomenon has been described in nonlactating beef and milked diary cows; therefore it does not appear to be a direct results of suckling. Other progestogens in serum have not been extensively char- acterized during the bovine PP period although in one report by Tribble et_gl, (1973) serum concentration of ZOB-hydroxypregn-4-ene- 3-one was found to be increased prior to the first PP LH surge and l7-hydroxypregn-4-ene-3,20-dione concentration peaked at or near standing estrus. The study was limited to three primiparous nonlactating heifers. Estradiol-17B, estrone and total estrogen concentrations increased linearly in serum during the last 25 to 30 days of gestation. These estrogens were derived from the placenta and not the ovary (Henricks et_gl,, 1972; Stellflug, 1972; Edqvist et;gl,. 1973; Smith et a1., 1973). Following a peak immediately prior to 18 parturition, estrogen concentrations in serum declined to low values by four to eight days postpartum (Robinson et_gl,, 1970; Arije et_gl., 1974; Echternkamp and Hansel, 1973; Smith et_gl., 1973; Corah et_gl,, 1974; Stevenson, 1977). Serum estrogen levels remained relatively low during the PP interval until immediately prior to the first PP ovulation when they increased to typical proestrus concentrations of 12 to 25 pg/ml (Henricks et_al,, 1972; Echternkamp and Hansel, 1973; Humphrey et_gl., 1976; Stevenson, 1977). The low amplitude fluctuations in estrogen secretion during the PP period may have reflected follicular growth and atresia occurring at this time. Humphrey (personal communication, 1978) measured serum estrone concentrations in PP suckled beef cows and found discrete peaks which lasted one to three days and occurred at approximately 10- to 14-day intervals from calving until first ovulation. These peaks of estrone may represent waves of follicular growth occurring in the absence of adequate concentrations of luteinizing hormone. In support of this view, Nadaraja and Hansel (1976) reported that the major estrogen secreted in heifers passively immunized against LH was estrone. No reports in the literature have demonstrated a direct influence of suckling on serum estrogen concentrations during the PP anovulatory period. Profiles of ovarian steroids in serum of suckled cows have not been demonstrated to differ from those in nonsuckled cows, except that the interval from parturition to the acute preovulatory 19 follicular growth and estradiol-178 secretion is prolonged in suckled cows. Ovarian Responsiveness to Exogenous Gonadotropins Casida et_gl, (1943) demonstrated that the ovaries responded with follicular growth and ovulation to exogenous gonadotrOpins injected early in the PP period of suckled beef cows. Oxenreider (1968) was also able to induce ovulation in lactating beef cows within one week of calving using a combination of pregnant mares serum gonadotropin (PMSG) and human chorionic gonadotropin (HCG). A single intravenous injection of HCG on days 5, 15 or 25 PP in suckled Hereford cows caused a reduction in the interval to first ovulation from 47 days in saline controls to 29 days in HCG-treated cows (Foote engl,, 1966). However, it appeared that the HCG- induced corpus luteum had a shorter than normal lifespan in some cows and there was a failure to reovulate in some animals following regression of the induced corpus luteum. Porcine-FSH and HCG injections induced corpora lutea formation in 90% of anestrus PP beef cows when treatment was commenced 30 to 40 days PP (Rovira et_gl,, 1978). Wettemann et_gl, (1978a) found that the ovaries of anestrous beef cows on day 45 PP secreted both estradiol and progesterone in response to PMSG injection. Echternkamp (1978) was unable to demonstrate an influence of suckling on ovarian steroid secretion following PMSG treatment on day 42 PP in beef COWS . 20 Fertility was increased in suckled cows treated with PMSG following synchronization with short-term progesterone treatment (Mulvehill and Sreenan, 1977); however,ear1ier work by Brown et_gl, (1972) found no effects of exogenous gonadotropins on fertility following treatments with progesterone and estradiol valerate in PP cows. In summary, only limited direct comparisons of ovarian response to exogenous gonadotropins have been made between suckled, milked and nonlactating PP cows. However, since the ovaries of suckled cows will respond to exogenous gonadotropic stimulation with follicular growth and ovulation very shortly after parturition, it would appear that suckling's effect on PP reproduction does not result from inability of the ovaries to respond to an appropriate gonadotrOpic stimulus. Effects of Exogenous Steroids on Postpartum Intervals Attempts to reduce the interval to first ovulation, estrus and conception with progestogens and/or estrogens have been made with varying degrees of success (Ulberg and Lindley, 1960; Foote and Hunter, 1964; Saiduddin et;gl,, 1968; Hill et;gl,, 1972; Brown et_gl,, 1972; Britt et;§l,, 1974; Lavoie and Moody, 1974; Cupin et_gl,, 1975). From these and other studies it would appear that short-term (7 to 14 day) progestogen administration with or without estrogen was the most consistently effective treatment for reducing the PP intervals to estrus and conception in beef and dairy cows. 21 Searle Agriculture Inc. (Elburn, 11.) has widely promoted its Synchro-Mate-B (SMB) treatment for synchronization of ovulation and estrus in PP beef cows. The SMB treatment consists of an implant (6 mg of the synthetic progestogen Norgestomet) placed subcutaneously in an ear for 9 days plus an intramuscular injection of 3 mg Norgestomet and 6 mg estradiol valerate on day one of the implant. This treatment is frequently used in combination with 24- or 48-h temporary weaning at the time of implant removal and timed insemination 48 to 60 h thereafter. This series of procedures has been found to increase the percentage of treated cows conceiving early in the breeding season (Mares et_gl,, 1977; Smith et_gl,, 1977; Kaltenbach et_gl,, 1978). Walters and coworkers (1977) reported that SMB was relatively ineffective in thin anestrus beef cows and that complete weaning in conjunction with SMB was more effective than only 48-h weaning after SMB. Also, in the study of Mares et_gl, (1977), the cows which were classified as noncyclic or anestrus had pregnancy rates 15 to 20% lower than those of cyclic cows. I conclude that although short-term progesterone treatment will synchronize cycling beef and diary cows, it is only partially effective at inducing cyclic activity in anestrus beef cows and its efficacy is markedly impaired by high suckling intensity and/or low energy intake. 22 Hypothalamo-Pituitary Axis Hypothalamus The major factors controlling secretion of the pituitary gonadotropins LH and FSH as well as prolactin are the hypothalamic- releasing factors which are secreted by modified neurons within the medial-basal hypothalamus and delivered to the anterior pitui- tary via the hypothalamo-hypophyseal portal circulation. The historical develOpment and current concepts of the neuroendocrine theory are beyond the scope of this thesis; however, they have been extensively reviewed (Convey, 1973; Schally et_el,, 1975, 1976; Arimura, 1976). To date, the evidence suggests that secretion of LH and FSH is controlled by a single stimulatory hypothalamic-releasing factor, the decapeptide gonadotropin-releasing hormone (GnRH; Schally et_gl,, 1975). First isolated from hypothalami of swine (Schally et_gl,, 1971) and sheep (Amoss et_gl,, 1971), the amino acid sequence of GnRH has been identified and synthesized (Matsuo et_gl,, 1971). Species differences in its structure have not been reported. The ability of GnRH and/or its synthetic analogues to release both LH and FSH has been reviewed (Convey, 1973; Schally et_gl,, 1975, 1976; Arumura, 1976). Zolman and Convey (1972) demonstrated that GnRH caused LH release from bovine pituitary tissue jfl_vjtrg using a continuous- flow superfusion system. I vivo, both heifers and bulls secreted LH in a dose-dependent manner in response to increasing amounts of intravenously injected GnRH (Zolman et a1., 1973, 1974). FSH 23 release in response to synthetic GnRH injection has been demon- strated in cows (Akbar et_gl,, 1973). Gonadotropin secretion in response to exogenous GnRH administered to PP cows and the effects of GnRH on PP intervals to ovulation and estrus will be reviewed later. GonadotrOpin-releasing hormone has been localized and quantified in the bovine hypothalamus (Estes et_gl,, 1977) and median eminence (Kizer et;gl,, 1976). Estes and coworkers (1977) found GnRH content to be greatest in hypothalamic sections corres- ponding to the median eminence and pituitary stalk with lesser concentrations located bilaterally in the anterior hypothalamus. Comparisons of hypothalamic GnRH content and localization in suckled and nonsuckled cows have not been reported; however, hypothalami of lactating PP rats have less GnRH bioactivity than those of nonlactating PP rats (Minaguchi and Meites, 1967). The possibility that suckling affects hypothalamic control of LH secretion has been suggested by two types of experiments. First, suckling has been demonstrated to reduce the post-ovariectomy increases in serum LH and FSH in PP rats (Smith and Neill, 1977) and rhesus monkeys (Weiss et;§1,, 1976). Echternkamp (1978) recently reported that the frequency of episodic LH secretion and the minimum LH concentration observed on day 30 PP was greater in nonsuckled than in suckled beef heifers which had been ovariecto- mized on day three postpartum. If the post-ovariectomy gonadotropin increase represents predominantly an increased hypothalamic secre- tion of GnRH then these studies suggest that suckling may act to 24 inhibit GnRH release. Secondly, Short 23221: (1974) reported that intramuscular injection of estradiol benzoate (10 mg) tended to be less effective in stimulating LH surgesirlsuckled beef heifers at 2 weeks PP than in nonsuckled controls; however, this trend was not evident at 4, 6 or 8 weeks postpartum. A recent study by Radford et_gl, (1978) demonstrated a more consistent failure of suckled anestrus PP cows to respond to estrogen injections (500 pg estra- diol benzoate in oil) with a LH surge at 6 weeks PP as compared to 14 weeks postpartum. Comparisons between suckled and nonsuckl ed cows in this experiment are questionable since nonsuckled cows had ovulated and therefore been exposed to corpus luteum progester- one secretion prior to the estrogen challenge whereas suckled cows had not ovulated nor been exposed to elevated progesterone concentrations. Although results in the cow are somewhat incon- sistent, there is a suggestion that suckling may act to prevent estrogen-induced LH surges, an effect that probably involves inhibition of hypothalmic GnRH secretion. Anterior Pituitary Gland Hormone Content Early reports on gonadotropins in the PP cow were concerned primarily with anterior pituitary content as measured by bioassay. Pituitary LH content has been reported to be low at parturition and to increase PP (Labhsetwar et_gl,, 1964; Saiduddin and Foote, 1964; Quevedo et_gl,, 1967; Saiduddin et_gl,, 1968; Wagner, Saatman and Hansel, 1969). Pituitary FSH content peaked at parturition and 25 then decreased following calving (Labhsetwar et_gl,, 1964; ‘Saiduddin et_gl,, 1968). Pituitary FSH content increased again to concentrations equivalent to those in late pregnancy by day 15 of the first and subsequent PP estrous cycles (Saiduddin et_el., 1968). Thus, the decline in pituitary FSH during PP anestrus may have resulted from decreased steroid feedback at this time. Secretion of FSH appears to be adequate at this time as reflected by the observed PP resumption of follicular growth. Suckling did not affect pituitary LH or FSH content in beef or dairy cows (Labhsetwar et;gl,, 1964; Saiduddin et;gl,, 1968). Pituitary prolactin bio-activity as measured by the pigeon crop assay did not differ when measured on day 1, 10, 20 or 30 PP in dairy cows (Riesen et_gl,, 1968), nor on day 3 or 15 following the first PP estrus in beef cows (Lauderdale et;gl,, 1968). In the above studies suckled dairy but not beef cows had a slight but significant decrease in pituitary prolactin activity when compared to nonsuckled controls. Thyroid-stimulating hormone content varied from 3.1 to 4.2 USP mU/mg anterior pituitary tissue in PP cows and differences due to suckling or PP interval were not significant (Wagner et_gl,, 1969). In conclusion, pituitaries of suckled cows do not appear markedly different in pituitary hormone content from those of non- suckled cows and would not appear to be the limiting factor in the onset of PP reproductive cyclicity. 26 Anterior Pituitary Hormones in Serum of Postpartum Cows With the development of specific and sensitive radioimmuno- assays for anterior pituitary hormones, recent studies have concen- trated on the characterization of their serum profiles during the PP period. Serum LH concentrations were low during late pregnancy and immediately PP in samples collected at daily or weekly intervals (Erb et_gl,, 1971; Arije et_gl,, 1974; Edgerton and Hafs, 1973; Ingalls et_gl,, 1973). Concentrations of LH increased within one to two weeks PP in serum of nonsuckled cows (Echternkamp and Hansel, 1973; Edgerton and Hafs, 1973; Ingalls et_gl,, 1973; Randel et;gl,, 1976). When blood was collected at 15- to 60-min intervals, distinct but brief increases in serum LH were reported to occur prior to the first PP preovulatory LH surge (Schams et_el,, 1972; Humphrey et_gl,, 1976; Stevenson, 1977; Goodale et_gl,, 1978). These brief episodic releases of LH resulted in average peak serum concentrations of 1.5 to 4 ng/ml which declined to baseline within 15 to 30 min (Stevenson, 1977; Goodale et_gl,, 1978). Controlled studies on the effects of suckling on episodic release of LH have not been reported; however, their onset appears to be delayed in the suckled beef cow (Humphrey et_gl,, 1976). Only limited information is available on serum FSH concen- trations in cattle during any reproductive stage due to a lack of satisfactory radioimmunoassays. Recently, Dobson (1978a, b) des- cribed changes in serum FSH concentrations during the bovine 27 estrous cycle and PP period in milked dairy cows. Postpartum concentrations of FSH were similar to those occurring during the luteal phase of the estrous cycle. Schallenberger (1977) has reported on serum FSH during the PP period and described wavelike peaks of FSH occuring at 12- to 14-day intervals. Neither con- centration nor pattern of FSH secretion was affected by suckling in this study. Characteristics of these two FSH radioimmunoassays and the physiological results reported await confirmation in other laboratories. Control of prolactin secretion in cattle has been reviewed by Karg and Schams (1974). A distinct peak of prolactin occured around parturition and then concentrations decreased to prepartum baseline values by 2 to 3 days postpartum (Schams and Karg, 1969; Johke, 1971; Ingalls eILQlJ, 1973; Karg and Schams, 1974). Milk removal, either by suckling or milking, was one of many extrinsic stimuli shown to cause acute prolactin release in cows (Johke, 1969, 1970; Karg and Schams, 1970; Fell et_gl,, 1971; Tucker, 1971; Koprowski and Tucker, 1971). The abrupt PP decrease in serum prolactin seen in cows is distinct from the sustained PP hyperprolactinemia seen in suckled rats (Amenomori et_gl,, 1970), goats (Johke et_gl,, 1971) and sheep (Kann et_gl,, 1977). The difference probably arises from differences in suckling intensity and frequency since restricted suckling caused a rapid fall in serum prolactin in these species (Amenomori et_gl,, 1970; Johke et a1., 1971). 28 There is no conclusive evidence of a role for prolactin in controlling bovine reproductive processes with the exception of mammogenesis and lactogenesis. 0n the contrary, Hoffman et_gl. (1974) were unable to demonstrate a luteotrOphic action of prolac- tin in the cow, and inhibition of prolactin secretion with the ergot alkaloid 08-154 (2-bromo-a-ergocryptine-methansulphonate, Sandoz, Switzerland) did not affect the estrous cycle of cows (Karg and Schams, 1974). In addition, recent studies on PP cows have used CB-154 to suppress endogenous prolactin without effects on the response to estrus synchronization at 50 days PP (Kaltenbach et_gl,, 1977; Williams and Ray, 1978) or the PP interval to estrus (Clemente et_gl,, 1978; Gimenez et_gl., 1977; Williams and Ray, 1978). Also, Clemente et_gl, (1978) were unable to influence occurrence of the first PP estrus by passive immunization of suckled cows with prolactin antiserum. Suckling may selectively delay the episodic release of LH in PP cows without altering circulating concentrations of follicle- stimulating hormone. However, controlled contrasts between suckled and nonsuckled cows are lacking and the mechanism by which suckling affects LH and FSH secretion is not clear. Serum prolactin may be increased marginally in suckled cows but does not appear to be involved in suckling-induced PP anestrus in cattle. Effects of GnRH on Gonadotropins and Postpartum Intervals Pelletier (1976) has reviewed the influences of GnRH on LH and FSH secretion in domestic animals including cows. Treatment 29 of dairy cows with GnRH caused release of LH (Kaltenbach et_gl,, 1974; Zolman et_gl,, 1974; Kesler et_gl,, 1977). Ovulation followed by recurrent estrous cycles occurred when GnRH was given 14 to 20 days after calving in dairy cows (Schams et_gl,, 1973; Britt et_gl,, 1974; Manns and Richardson, 1976; Garverick et_gl., 1978). Dairy cows responded to GnRH injection with reduced LH release during the first 1 to 2 weeks PP possibly reflecting low pituitary LH stores (Kesler et;gl,, 1977). The LH response was linearly related to the log of the GnRH dose injected into cycling beef cows suckling calves (Schams et_gl,, 1974; Webb et_al,, 1977). In anestrus suckled beef cows, maximal LH release in response to GnRH injection increased until day 20 PP; a pattern similar to that observed for early PP dairy cows (Irvin et_gl., 1977; Webb et_el,, 1977). A single GnRH injection caused anestrus beef cows to undergo follicular growth and luteinization or ovula- tion as indicated by progesterone secretion. However, these cows frequently failed to establish normal ovulatory cycles following this brief increase in serum progesterone (Fonseca et_gl,, 1977; Irvin et_§l,, 1977; Wettemann gt;313, 1978a). A second GnRH treat- ment 10 to 14 days after the first resulted in ovulation and initiation of cyclic activity in a high proportion of these cows (Fonseca et_gl,, 1977; Webb et_gl., 1977). Lishman et_gl, (1977) were unable to increase the LH released in response to 300 ug of GnRH in 30-day PP suckled beef heifers by priming injections of FSH nor was the interval to first estrus reduced. In a study using 24-day PP beef cows, 32-h but not 30 24-h weaning prior to 300 pg of GnRH caused an increase in LH re- leased as compared to nonweaned controls. The PP interval to ovulation was not affected and exceeded 70 days (Inskeep et_al,, 1977). Fertility in suckled beef cows synchronized with SMB treatment and weaned between implant removal and insemination was not improved by injection of 125 pg GnRH at 30 h post implant removal (Kaltenbach et_gl,, 1978). Estrogen enhanced the LH release in response to GnRH infusion (Ellicott et_§l,, 1978) or injection (Irvin et_gl,, 1978) in early PP beef cows. Hyperpro- lactinemia induced by multiple injections of thyrotropin-releasing hormone did not effect the GnRH-induced LH release in ewes (Kann et_el,, 1976) or beef cows (Nancarrow and Radford, 1976). Follicle-stimulating hormone release in response to GnRH has not been described for either suckled or nonsuckled PP cows. Direct comparisons of LH release in response to GnRH have not been reported for suckled versus nonsuckled cows during the early PP period. Echternkamp (1978) was unable to demonstrate an influence of suckling on LH secretion in response to 7, 150 or 300 pg of GnRH injected on day 42 PP in beef heifers. Since the pituitary of the suckled cow responds to GnRH with LH secretion as early as l to 2 weeks PP, well before the first spontaneous PP ovulation occurs, the depressed serum LH and delayed ovulation does not appear to reflect pituitary insensitivity to GnRH but rather may be a result of decreased GnRH release from the hypothalamus. 31 Summary Suckling has an inhibitory effect on PP reproduction in beef and dairy cows that appears to be discrete from that of other factors such as nutrition. The end result of this inhibition is a prolonged PP annovulatory and anestrus interval in suckled cows which leads to calving intervals that exceed the optimum 12 months. The mechanism(s) by which the suckling stimulus or complex of stimuli delays ovulation is unknown. Neither the uterus nor adrenals appears to play a major role in inhibition of ovulation by suckling although adrenal secretion is stimulated by suckling. Ovaries of suckled anestrus cows will ovulate in response to exogenous or endogenous gonadotropins, suggesting that their failure to ovulate spontaneously is secondary to inadequate gonado- tr0pin secretion. Serum prolactin in suckled cows may be elevated over that of controls; however, this hyperprolactinemia is not great nor has it been demonstrated to have antigonadotropic actions in the cow. It must be remembered that results obtained in PP sheep, primates and rats may not extrapolate well to the PP cow since prolactin has been demonstrated to play a significant role in PP anestrus in the former species but not in the cow. Changes in FSH secretion have not been well characterized in the PP cow but do not appear to be involved in suckling-induced anestrus. This view is supported by the presence of follicular growth in early PP nonsuckled and suckled cows and by similar 32 pituitary FSH content in nonsuckled and suckled cows. In addition, limited available data suggests that concentrations of FSH in serum of suckled cows are apparently similar to values observed during the bovine estrous cycle. In contrast, serum LH concentrations are low in early PP cows and although definitive studies have not been reported, suckling appears to delay onset of episodic LH secretion. Since pituitaries of suckled cows secrete reduced amounts of LH in response to exogenous GnRH only during the first 1 to 2 weeks PP, suckling-induced depression of serum LH after this time may reflect a decreased pituitary stimulation as a result of suckling. The major trOphic factor involved in acute release of LH is believed to be hypothalamic gonadotropin-releasing hormone. Although the hypothalamic distribution of GnRH has been reported for the bovine, no studies in cattle have reported the effects of suckling on the content or distribution of GnRH in the hypothalamus. Hypo- thalamic GnRH bioactivity was found to be reduced by suckling in PP rats. The possibility of a suckling-induced hypothalamic dysfunction, failure to release GnRH in response to stimuli such as estrogen injection or ovariectomy, has been suggested in studies on suckled ewes, monkeys and rats as well as in cattle. Since dairy cattle were readily available and the liter- ature suggested that suckling effectively increased the PP anovulatory period in dairy as well as beef cows, Experiment I was designed with the following objectives: 33 l. to determine the suitability of the PP dairy cow as a model system for studying PP anovulation and anestrus caused by a suckling calf and 2. to characterize the effects of suckling on PP inter- vals and serum concentrations of reproductively signi- ficant hormones. Based on the results of Experiment I and the literature reviewed, a second experiment was designed to investigate the hypothesis that suckling-induced depression of serum LH was a result of altered hypothalamic GnRH content or release. More slaecificly the objectives of Experiment 11 were to determine the effects of suckling by two calves on: 1. hypothalamic GnRH content and gross distribution, 2. pituitary LH, FSH and prolactin content, 3. jn_vitro pituitary secretion of LH and FSH in response to GnRH or elevated K+ concentrations, 4. serum profiles of LH, FSH and prolactin, as well as 5. estimates of ovarian and adrenal activity by measure- ment of serum estradiol-178, progesterone and glucocorticoid concentrations. It was felt that these experiments would lead to an increased understanding of both the level at which and the mechan- ism by which the suckling stimulus acts to delay the first PP ovulation in cow. This would be important in designing logical and effective management regimes for controlling PP reproduction in COWS . MATERIALS AND METHODS General Blood Collection and Handling Blood samples were collected as indicated in each experiment by either percutaneous puncture of the coccygeal artery or vein using a 20 G needle or via jugular vein cannulae (Medical polyvinyl tubing, sive v-lO; Bolab Inc., Derry, N.H.) which were inserted at least 12 h prior to sample collection. Blood samples were allowed to clot at room temperature for 4 to 8 h then stored at 4 C for 24 to 48 h before harvesting serum. After centrifuging at 2000 x g for 30 min, serum was decanted into 12 x 75 mm culture tubes, capped and stored at -20 C until assayed. Assays Detailed protocols for the competitive binding assays used in this research as well as the validations of the follicle- stimulating hormone (FSH) and estradiol-178 radioimmunoassays are contained in Appendices A thru G. In overview, luteinizing hormone (LH), prolactin, proges- terone and gonadotropin-releasing hormone (GnRH) were measured using previously described double antibody radioimmunoassays (Convey et_gl,, 1976; Tucker, 1971; Convey et_gl,, 1977; Estes et_gl,, 1977). Total serum glucocorticoids were determined by competitive protein binding assay as described by Smith e331. (1973). 34 35 Bovine FSH was quantified by a homologous radioimmunoassay using antiserum (rabbit anti-bovine FSH-82) and highly purified bovine FSH (FSH-HS-2) generously provided by Dr. K. W. Cheng.1 The assay was performed as previously described (Cheng, 1978) with three modifications. First, the purified bovine FSH was iodinated using chloramine-T rather than the lactoperoxidase method used by Cheng. The iodination method has been described (McCarthy, 1978) and consisted of reacting 5 pg FSH, l mCi Na‘zsl and 5 pg chloramine-T for 120 sec at 25 C in a total volume of 30 pl of phosphate (.5M) buffered (pH 7.4) saline. The reaction was quenched with 12.5 pg sodium metabisulfate (2.5 pg/pl in water) and 1001M transfer solution (16% sucrose and 1% KI in water). Products 'were separated immediatley on a .8 x 15 mm column of Biogel P-60 (BioRad Laboratores, Richmond, CA) using phosphate (.OlM) buffered (pH 7.0) saline (.14M) containing .1% bovine serum albumin (BSA) as eluant. The second modification was in using a total sample volume of 500 p1 for all standards and unknowns. Finally, normal rabbit serum (excess gamma globulin required for double antibody precipitation) was included with the first antibody rather than being added separately at the time of second antibody addition. Estradiol-l78 was assayed using antiserum (rabbit anti- 125 estradiol-l78-11B-hemisuccinate: BSA, #930) and I labelled 1University of Manitoba, Winnipeg, Manitoba, Canada. 36 estradiol-178-lla-tyrosine methyl ester as tracer.2 Radioiodina- tion and assay procedures were similar to those described by England et_§l, (1974) and are detailed in Appendix G. Notable modifications included use of diethyl ether for serum extraction, rather than benzene, and chromatography using commercially pre- pared columns (Ouik-Sep Extended LH-20 Columns, Isolab Inc., Akron, OH). Protein content of tissue extracts was determined by dye- binding assay (BioRad Protein Assay; RioRad Laboratores, Chemical Division, Richmond, CA) as described by Bradford (1976). Statistical Analysis Radioimmunoassay results were calculated using a Fortran language interactive program on the Michigan State Univeristy time-share computer system. Raw data were first corrected for background and expressed as percent of total binding or zero standards. Then standard curve means were fitted to a third order polynomial equation using least squares methods. Unknown concentrations were estimated by solving this equation for each unknown. Appropriate corrections were made for volume assayed, dilutions and extraction efficiency. Statistical analyses of serum hormone concentrations were performed as described by Gill (1978a, b). Double split-plot 2Antiserum and unlabelled estradiol-tyrosine methyl ester conjugate were provided by Dr. K.ll Kirton of The Upjohn Co., Kalamazoo, MI. 37 analysis of variance with repeated measures over time was the basic model used. Treatment group, sampling period and sampling time (within period) were the main effects. Homogeneity of variance was tested and data transformed to logarithms where apprOpriate. Treatment means were compared using appropriate tests: f-tests of orthogonal contrasts, Scheffé's interval, Student's-t, etc. Experiment I: Effects of Suckling on Postpartum Intervals and Serum Hormone Concentrations in Holstein Cows For reasons of availability and manageability, the dairy cow was more amenable than the beef cow for studies on postpartum (PP) reproductive endocrinology which involve frequent blood sampling and animal handling. Based on this premise, the rationale behind this experiment was to determine the suitability of the suckled Holstein cow as a model for studying the mechanisms of suckling-induced PP anovulation, a problem of economic importance in the less easily studied beef cow. The experiment had these specific objectives: 1. to characterize the PP intervals to ovulation and estrus, 2. to measure the chronic and acute ‘fluctuations in serum gonadotropins, prolactin and steroids during the early PP period in our experimental population of milked Holstein cows, and 3. to determine the effects of a single suckling calf on these functional and endocrine parameters. Multiparous Holstein cows from the Michigan State University dairy herd were assigned at calving to one of three treatment 38 groups. Cows in treatment I were nonsuckled controls milked at 12-h intervals (NS-2X) and those in treatment 11 were suckled gd_ libitum by one calf and milked at 12-h intervals (S-2X). Treat- ment III cows were not suckled and were milked at 6-h intervals (NS-4X). All animals calved in individual box stalls and at approximately 24 h PP nonsuckled cows (I and III) were moved to tie stalls located in the same barn. Suckled cows (II) remained in their box stalls with their calves and were tied in the same manner as the nonsuckled cows. Beginning on day two PP, all cows were milked in their stalls using a portable milking machine and vacuum pump. Calves were free to nurse at all times except while the milking machine was attached and during periods of estrus detection. A11 cows were fed individually a ration of corn silage (30% dry matter), concentrate mix (11 to 15% crude protein), alfalfa hay (88% dry matter) and haylage (60% dry matter) calcu- lated to meet 100% NRC (1971) requirements for lactating cows. Animals were not involved in concurrent nutrition experiments. Estrous detection consisted of visual observation from 0600 to 0800 h and from 1700 to 1800 h during which periods the cows were penned outdoors with a minimum of five other cows and a testosterone-treated teaser heifer (Kiser et_gl,, 1977; Stevenson and Britt, 1977b). Each animal was also fitted with a Mate Master rump-mounted heat detector (Stevenson and Britt, 1977). Estrus was defined as standing to be mounted by other cows or the teaser heifer. 39 Ovaries were palpated pe§_5egtgm_twice weekly to record follicular growth, ovulation and corpus luteum development. Prior to each palpation a 10-ml tail vein blood sample was collected; later this serum was assayed for progesterone content and results were used in conjunction with palpation data to determine time of first ovulation. For statistical analysis, time of first ovulation was set at 48 h prior to the first increase in progesterone to greater than 1 ng/ml for more than two consecutive samples. At weekly intervals from day 7 PP until day 28 PP or first ovulation whichever was earliest, blood samples were collected from indwelling jugular cannulae at frequent intervals for 4.5 h centered on the 1600 h milking. Serum from these samples was assayed for luteinizing hormone and prolactin, with selected samples from each period also assayed for total glucocorticoids, follicle-stimulating hormone and estradiol-178. Experiment II: Effects of Suckling on the HypothaTamo-Pituitary Gonadotropic Axis of Early Postpartum Holstein Cows Based on Experiment I, this experiment was designed to determine the mechanisms by which suckling affected the hypothalamo-pituitary luteinizing hormone (LH) axis. The ig_vjyg_portion was essentially a modification of Experiment I with suckling intensity increased by use of two calves and frequent blood sampling only on day 13 postpartum. Its objec- tive was to confirm the relationships between suckling and serum hormone concentrations observed in Experiment I. 40 The rationale for the jfl_vitro studies was to define the 1evel(s) at which the suckling stimuli alter LH secretion by measuring serum, pituitary and hypothalamic components of the LH control system within each animal of the suckled and nonsuckled treatments. A direct functional assessment of the anterior pituitary was obtained by measuring the LH release in response to maximal doses of gonadotropin-releasing hormone (GnRH) and elevated potassium ion (K+). Receptor-mediated LH release as demonstrated by response to GnRH was compared to LH released by 59 mM K+ and used to evaluate the functional adequacy of the pituitary gonado- trOphs' GnRH receptors. In Vivo Healthy, pregnant Holstein cows were purchased in April, 1977 and maintained on pasture until they calved during May. Supplemental feed consisting of alfalfa hay and haylage: corn silage (1:2) was provided prepartum. At parturition, cows were assigned alternately to be suckled gg_1ibitum by two calves (n=9, S-2X) or nonsuckled as controls (n=8, NS-2X) with each suckled and subsequent nonsuckled cow being considered as one treatment pair for future sampling and slaughter assignment. From calving until slaughter on approximately day 14 PP all cows were handled as follows. Within 2 to 4 h of calving, cows were moved from pasture to tie stalls and calves were removed from nonsuckled dams and fostered onto the respective suckled cow of that treatment pair. Calves were restricted to the area of 41 their dams by fences enclosing a 2 x 5 m area with the cow tied at one end. Animals were maintained under natural photOperiod and fed a ration of alfalfa hay, corn silage and 11 to 14% crude protein concentrate mix to meet or exceed 100% NRC requirements. Water was supplied §g_libitum. In an attempt to minimize differ- ences in nutritional drain caused by lactation (i.e. suckled cows possibly producing less milk than nonsuckled cows), cows in both groups were machine-milked at 0600 and 1800 h daily; milk weights were recorded at each milking. Body weights were recorded during the early afternoon on days 4, 8 and 12 PP, as well as on the morning of slaughter. Neither feed nor water were restricted prior to weighing or slaughter and cows were milked at the scheduled time on the morning of slaughter. Tail vein blood was collected prior to each morning milking. On day 12 PP cows were fitted with jugular cannulae. Jugular blood was sampled at 15-mi n intervals from 0815 to 1200 and from 2015 to 2400 h on day 13 PP for characterization of serum LH, prolactin, and follicle-stimulating hormone (FSH) profiles. Treatment pairs, consisting of one suckled and one non- suckled cow, were sampled together and then killed between 0700 and 0800 h on day 14 PP in the Michigan State Univeristy Meat Science abbatoire. Animals were stunned using a captive bolt pistol and then rapidly exsanguinated. 42 In Vitro Tissue Collection Hypothalami with pituitary stalks attached were collected within 22 i 1.5 min of stunning, frozen on Dry Ice then stored at -60 C until trimmed, extracted and extracts assayed by radioimmuno- assy for gonadotropin-releasing hormone content. Whole pituitaries were removed from the sella turcica and sectioned midsagittally, then the anterior pituitary tissue was dissected free and weighed. One half of the anterior pituitary was frozen on Dry Ice and stored at -60 C until extracted and extracts assayed for LH, FSH and prolactin content. The remaining half was sliced 1 mm thick in the saggittal plane using a Staddie- Riggs microtome and diced with a scalpel blade into 1 mm3 pieces. Diced tissue was placed in tissue culture medium (TC 199, Appendix H) until used in the superfusion studies. All pituitary tissue preparation was carried out at room temperature (20 to 24 C). Ovaries and uteri were collected at slaughter and evalu- ated for gross ovarian activity and degree of uterine involution. Extraction of LH, FSH and Prolactin fFom Pituitary Tissue Pituitary tissue (150-200 mg) was weighed and placed in 15-ml plastic tubes with .5 m1 of carbonate (.05 M) buffer con- taining 18.6 g/l iter (ethylenedinitrilo)-tetracetic acid disodium salt (NaZEDTA,leO.O), and homogenized with a polytron tissue homogenizer (three 5-sec intervals at highest speed). Homogenate was transferred to a 50-ml plastic screw cap tube and the small 43 tube and homogenizer were washed with four 5-ml washes of buffer. Each wash was transferred to the 50 ml tube. Homogenates were shaken overnite at 4 C, the pH was reduced to 8.5 with l N HCl and the extracts centrifuged at 2000 x g for 30 minutes. Super- nates were decanted and frozen. No measure of extraction effi- ciency was made, and protein content of the extracts was determined (Bradford, 1976). Supernates were diluted 10 to lOOOX with the appropriate assay buffer and then dilution duplicates (10,20,40, 80,160pl) assayed for LH, FSH and prolactin. Results were expressed as total content (mg hormone/pituitary) and as concen- tration (pg hormone/mg tissue). Hypothalamic Tissue Trimming and GnRH Extraction Frozen hypothalami were trimmed and divided into three pieces designated preoptic (P0), anterior hypothalamic (AH) and mediobasal hypothalamic (MBH) which also included the pituitary stalk. Anterior-posterior and lateral boundaries are shown in Figure 1, a ventral veiw of the bovine brain; dorso-ventrally the trimmed tissue was 5 to'6 mm deep. Tissue blocks were kept frozen during and after trimming. GnRH was extracted as follows. Tissue blocks were weighed, placed in lS-ml plastic centrifuge tubes with 5 ml (PO and AH) or 10 m1 (MBH) of cold 2N acetic acid and homogenized by 2 x 30 sec bursts of a polytron tissue homogenizer (Type PT 10 OD; Brinkmann Instruments, Westbury, N.Y.). The homogenizer blade was rinsed 44 POA = Preoptic Area AHA = Anterior Hypothalamic Area MBH = Medial Basal Hypothalamus M8 = Mammillary Bodies +15 POA +10 AHA + 6 MBH Figure 1. Diagram of the ventral surface of the bovine brain with the dissection boundaries (anterior-posterior and lateral) of the hypothalamic tissue blocks removed for extraction and radioimmunoassay of gonadotropin-releasing hormone. 45 with 5 ml of 2N acetic acid which was pooled with the homogenate. Prior to homogenization, 10,000 cpm of 3H-GnRH (19.6 Ci/mM, Abbott Laboratories) was added to each tube for determining recovery. Individual homogenates were frozen until all tissue was processed. Then homogenates were thawed and centrifuged for 10 min at 10,000 x g in a refrigerated centrifuge (Sorvall RC-ZB). Supernates were decanted into 20-ml glass scintillation vials, frozen, lyophilized and stored at -20 C. Prior to assay, the extracts were reconstituted to original volume with phosphate (.OlM) buffered (pH 7.0) saline containing .1% (w/v) pigskin gelatin (#5247; Eastman Kodak G., Rochester,N.Y.). An aliquot was counted in a liquid scintillation spectrophotometer (Model 6860 Mark 1., Nuclear, Chicago) for calculation of procedural losses. Pituitary Superfusion Method A multichannel ig_yitrg_superfusion system was used to ccnnpare the LH and FSH responses of anterior pituitary tissue from suckled and nonsuckled cows to GnRH or elevated K+ concen- tration. A schematic diagram of the system used is shown in Figure 2; it is similar to that used by Zolman and Convey (1972). Media were maintained at 37 C in a thermoregulated waterbath and con- tinuously gassed with 02:C02 (95:5). Medium was drawn from the reservoir (A) to the tissue chambers (C) thru efferent tubings (B; Tygon tubing, 1/32 inch I.D. x 3/32 inch 0.0.; Arthur H. Thomas Co., Philadelphia, PA) which were also submerged in the waterbath. The chambers consisted of 4-cm lengths of larger bore 46 11.11- .mu:m_nxw Xemuwsuwa mcw>on mo cowmzmcmaam.muw_> :_ com vow: mapmceaqm as» we Emcmmwu ovumewnom .N mesde copuwppou cowpumcd mcompoacm mwuoz assa ueu_apm_cma mm=_n=u acetac+< meaneocu mammwh mmcwnzu ucmgmmem cwo>cmmmg meow: .25), whereas S-2X cows had a mean interval to first ovulation + of 38.7 _ 4.9 days which was significantly greater than nonsuckled cows (P < .05). The overall average interval to PP ovulation for nonsuckled cows (21.2 days) agrees with that reported for M-2X dairy cows (mean - 25.3 days; range = 16.4 to 44.0 days) as summarized in Table l and is similar to the 21.3 days reported by England et_gl, (1973) for nonlactating beef cows. The interval to first PP ovulation in the suckled cows (38.7 days) is shorter than that reported by Moller (1970b) for dairy cows nursing multiple calves (64.5 days). However, if the intervals for suckled cows are expressed as percentages of the intervals for nonsuckled controls within each experiment, the results are remarkably similar (181% vs 180%). The interval to first ovulation in S-2X cows also lies Table 3. 52 Intervals to first postpartum ovulation and estrus in suckled (S) and nonsuckled (NS) Holstein cows milked twice (2X) or four times (4X) daily. Intervala to first postpartum Treatment Ovulation Observed Estrus NS-2X 19.4 i 3.3 39.0 i 2.4 (n=5) . b c S-2X 38.7 t 4.9 50.2 i 4.3 (n=6) NS-4X 23.0 i 5.3 44.8 i 6.1 (n=5) aDays, x t SE. b S-2X > NS-ZX and NS-4X, P < .05. Cs-2x > NS-ZX but not NS-4X, P < .06. 53 within the range of mean intervals reported for suckled beef cows in Table 1 (36.0 to 61.5 days). The PP interval to first observed estrus was increased in S-2X cows compared to NS-2X cows (50.2 i 4.3 vs 39.0 1 2.4 days, P < .06), but not significantly compared to NS-4X cows (44.8 i 6.1 days, P > .25). Nonsuckled cows (NS-2X vs NS-4X) did not differ significantly in the mean interval to first observed estrus. Across all treatments, interval to first estrus was longer than interval to first ovulation (P < .01 by paried t-test) as would be expected since 81% of the cows ovulated one or more times before showing estrous behaviour. The high incidence of silent ovulations during the early PP period is in agreement with the literature (Hackett et_gl,, 1973; Hartigan et_gl,, 1974; King et_gl,, 1976). The occurrence of estrus associated with the first PP ovulation in three of six S-2X cows versus none of ten NS cows may reflect the greater PP interval to ovulation in this treatment group since increasing PP interval has been associated with reduced frequency of silent ovulation (Casida and Wisnicky, 1950; Stevenson, 1977). I conclude that the presence of a calf allowed to suckle §g_libitum delays the first PP ovulation in Holstein cows milked twice daily. Suckling also causes an increase in the interval to the first PP estrus. Increasing the milking frequency from twice to four times daily did not significantly alter the PP intervals to ovulation or estrus in this experiment. 54 Serum Hormone Concentrations Luteinizing Hormone Serum from jugular vein blood collected at 15-min intervals from 1400 to 1800 h on days 7 and 14 PP was assayed for LH content. Analysis of variance was performed using a double split-plot model with repeat measure over time; main effects were treatment group, period (day PP sampled) and time of sample. There was no signifi- cant treatment by period interaction. Mean serum LH concentration was lower in S-ZX cows than in NS-2X or NS-4X (.7 vs 1.4 ng/ml, P < .05) but NS-2X and NS-4X did not differ significantly (1.4 vs 1.3 ng/ml, P > .25). Mean serum LH concentration increased significantly between day 7 and day 14 PP across all three treat- ments (.9 vs 1.4 ng/ml, P < .05). Figures 3, 4 and 5 present the individual serum LH pro- files on days 7 and 14 PP for five cows from the NS-2X, S-2X and NS-4X treatments. Episodic releases (peaks) of LH were apparent upon inspection of plotted data and were identified by visual appraisal since no appropriate and valid statistical method of selection was available. Increases which were considered as peaks are marked with asterisks. Data for one randomly selected cow from the six S-2X animals were not presented for expediency in drawing the graphs. Characteristics of the serum LH profiles are summarized in Table 4 and Figure 6. The following criteria were calculated for each 4.5-h sampling period; mean LH concentration (ng/ml), coeffi- cient of variation of the LH concentration (%), frequency of 55 Day? PP Doyl4PP 5‘ .11 1318 5’1: 1318* I- * b 3- 3. * 1- )- '1r 1 l “l l l l 1 L l * f 5* 1334 5) 1334 :2 b b * E 2"I 1 * 3:* .1 1: v .FL 1 1 a L 'rr 1 L j 1_. c C) *' 1* '12; Sr 1142 5’ 1142 g l- l- 5 3~ 3- o . . g .. 1. W L l 1 L I J: .J 5- 1331 5’ 1331 E F F a 31— 31- ‘3 r”: * pm le”\‘Scnvnfl/A‘xm~ |- l i _L #L I 1 J_ * i l J_ l b 5 1358 b 'I- r- * 3 ‘I' . 1.]__.L__.l.—1l—-I—— “HOC115CKJIGIID FHDODMNJO Time of Day (h) Figure 3. Luteinizing hormone profiles in serum of nonsuckled twice daily milked (NS-2X) cows on days 7 and 14 postpartum, (* = designated peak). 56 Day7PP Doyl4 PP 5" I- _ £308 _ race 3" r- - L *, |I- pM— l l l l 1 l l 1 l J 5" 1- , 1314 _ 13p; E 3" v- \ "' 1- O 1|: i- 5 |bM’ .- 8 1 l 1 l L I 1 1 l l E 5’ " 5 3 0 |P W ZN I 1 1 VI 1 I L L l l —J 5 g - 1347 1. 1347 h 0 -- h m 3% 'M .- * L 1 1 1 1 M 5p _ . 1P961 . 1196 3" r- . _ * 11 '.W—’ ~_A/\ WA I l L l 1 l l l l 1400 1500 1600 1f00 1800 1400 1500 1600 1100 1800 Time of Day (M Figure 4. Luteinizing hormone profiles in serum of suckled twice daily milked (S-2X) cows on days 7 and 14 postpartum (* = designated peak). Serum LH Concentration (no/ml) Figure 5. 57 s- * L 1358 * 111 3- * Ir- 1 _1 L l 5 1195 ‘l' | W A__ J l L j l l 5- __ 1341 31- i- * * I'M 1 1 1 l l 51- _ 1355 15* 1* 1- * Ir J 4 l L 1400 1500 600 1700 1800 tiny 1‘1 FWD «* : 1353 . * * .1. r '- l l I l : l l 1 j l 1- _ 1341 __ t W p- 1 J 1 1 1 _ 1355 * -1* W l 1 l l 1 1400 1500 1600 1700 1800 Time of Day (111 Luteinizing hormone profiles in serum of nonsuckled four times daily milked (NS-4X) cows on days 7 and 14 postpartum, (* = designated peak). 58 .mcppmmmn mcwuzpocw Aps\mcv mesoPFQEm xoma :4 mmmcw>< v .cowcon mcwpasmm s1m.¢\zoo\xmma :4 nonchmumu appozmw> Lo consazu .mmPQEmm m_ mg» cw cowumcucmocou :4 mguhtocopumwgm> yo pcwwuwmemou “coupon n .uowcma s1m.¢ m cm>o mpm>cwucv :w21mp um umuomppou mmpnswm mp ea A_E\mcv mmmgo> .2). The peak milking- induced glucocorticoid release was greater on day 14 than on day 7 PP (12.9 i 2.8 vs 5.9 i 1.5 ng/ml, P < .05), although nonsuckled and suckled cows did not differ significantly either on day 7 PP (6.9 1 1.6 vs 5.2 i 2.5 ng/ml) or day 14 PP (10.5 t 2.6 vs l4.l8 i 4.7 ng/ ml). These results do not support a direct role for the ovarian (progesterone and estradiol-T78) or adrenal (total glucocorticoids) steroids as mediators of suckling's effect on LH secretion or on delay of PP ovulation and estrus. Summary This experiment demonstrates that stimuli provided by a single calf allowed to suckle gg_libitum approximately doubles the interval to first PP ovulation in milked dairy cows. Suckling significantly increased the interval to first PP observed estrus; however, the effect was less dramatic than on ovulation because there tended to be a lower incidence of ovulation without observed estrus in the suckled cows. Increased frequency of milking did ruat alter the PP intervals to ovulation or estrus. Suckling resulted in delayed PP resumption of episodic LH secretion; both LH peak amplitude and frequency were reduced resulting in lower mean and coefficient of variation for serum LH Concentration. Serum concentrations of FSH, prolactin, progester- OHE, estradiol-T78 and total glucocorticoids did not differ 67 significantly between suckled and nonsuckled cows. Howeven,basal prolactin and milking-induced prolactin and glucocorticoid con- centrations were greater on day 14 than on day 7 PP in both treat- ments. It would appear that suckling suppresses episodic LH secretion during the PP period and this in turn may be responsible for the increased interval to first PP ovulation in these cows. Experiment 11: Effects of Suckling‘on the Hypothalamo-Pituitary,Gonadotropic Axis in Early Postpartum Holstein Cows Animal Characteristics and Slaughter Data Charateristics of Holstein cows used in Experiment II are sumnarized in Table 6. No information was available on past milk production or on reproductive histories of these animals. Treat- ment means for day PP killed, body weight at slaughter and percent body weight change between day 4 PP and slaughter did not differ significantly between nonsuckled (NS-2X, n=8) and suckled (S-2X, n=9) cows. Milk removed by twice daily machine milking of all cows during the two weeks between calving and slaughter averaged 20.8 : l.l kg/day for nonsuckled cows and 5.9 i 1.7 kg/day for cows suckled .ag libitum by two calves. Suckling frequency was estimated by observation during the periods of intensive blood sampling; each cow was observed for an average of 26 i 4 h and the calculated suckling frequency was 68 Table 6. Characteristics of suckled (S-2X) and nonsuckled (NS-2X) postpartum (PP) Holstein cows used in Experiment 11. Calving Paritya Body Day PP Slaughter % wt Treatment Cow Date condition killed wt (kg) changec NS-2X 818d 5-1 0 1 15 389 4.6 (n = 8) 821 5-7 1 2 16 482 -5.4 607e 5-28 l 3 16 515 -0.4 609 5-14 1 3 12 441 --- 611 5-21 0 l 17 514 -0.6 825 5-7 1 4 12 527 .0 615 5-13 0 l 12 405 -1.0 822 5-16 0 2 16 427 2.1 x .5 2.1 14.5 462 -0.1 SE .2 .4 .8 19 1.2 S-2X 816 5-7 0 2 16 494 -0.8 (n = 9) 820 5-13 1 2 13 516 5.1 819 5-27 0 2 15 450 .8 608 5-24 1 1 14 595 -1.2 616 5-29 0 1 15 444 -0.9 612 5-6 0 2 13 432 2.6 824 5-1 1 2 15 509 1.3 817 5-10 1 2 15 518 -1.3 612 6-16 0 3 16 461 -2.0 x .4 1.9 14.7 491 .4 SE .2 .2 .4 17 .8 aHeifer = o, cow = 1. bSubjective evaluation: thin = 1, fair = 2, good = 3, fat = 4. CPercent body weight change = (slaughter wt - day 4 PP wt) X lOD/day 4 PP wt. d818 was the smallest heifer and had the largest calf, required assistance andretainedpflacenta for 36 hours. e609 had breech presentation which required assistance. 69 9.9 i 1.0 suckling episodes/24 h. This result assumes that the suckling activity observed from 0800 to 1200 and 2000 to 2400 h is representative of the remainder of the day. Based upon uterine measurements and subjective appraisal of uterine content and endometrium on day 14 PP, there was a tendency for suckled cows to have smaller more involuted uteri. Uteri of nonsuckled cows appeared to contain a greater volume of fluid contents and more inflammatory-like changes such as severe endometrial hyperemia and purulent material than those of suckled cows. Ovaries of both suckled and nonsuckled cows showed evi- dence of follicular growth. The majority of follices were small to medium in size (<10 mm diameter) although in five of seven suckled cows and four of six non-suckled cows at least one ovary contained a follicle >10 mm in diameter. No luteal tissue was detected in any cow except for the regressed corpus luteum of pregnancy on the ovary ipsilateral to the previously gravid uterine horn. Differences in ovarian status due to suckling were not apparent although detailed evaluation of ovarian weight, follicular fluid weight and ovarian histology or steroid contents was not performed. Serum Hormone Concentrations Luteinizing Hormone Luteinizing hormone was measured in serum collected at 15-min intervals between 0815 to 1200 and 2015 to 2400 h on the 70 day before slaughter, approximately day 13 postpartum. Secretory profiles of LH were characterized as described in Experiment I and results are summarized in Table 7 and Figure 9. No change in LH secretion was associated with the time of day at which samples were collected for characterization of the secretory profile. However, suckled cows had lower (P < .05) mean serum LH concen- trations (.9 i .1 ng/ml) than nonsuckled controls (1.6 i .3 ng/ml). The lower mean LH concentration resulted from both peak frequency and amplitude being reduced (P < .05) in suckled vs nonsuckled cows (.9 i .2 peaks/4 h, 1.8 i .1 ng/ml vs 2.1 i .2 peaks/4 h, 3.1 i .9 ng/ml). Follicle-Stimulating Hormone Follicle-stimulating hormone was assayed in serum from blood collected at 0815, 0915, 1015, 1115, 2015, 2115, 2215, and 2315 h on day 13 PP, results are shown in Figure 10. There was no significant difference between morning and evening samples nor did suckled cows differ much from nonsuckled cows (67.3 1 6.9 vs 59.1 1 4.6 ng/ml, P > .3). Serum from tail vein blood samples collected on day 7 and 13 PP had average FSH concentrations of 65.2 1 7.6 and 66.6 i 12.6 ng/ml for suckled cows and 56.2 i 6.3 and 66.1 i 7.8 ng/ml for nonsuckled cows. There were no signi- ficiant differences due to day PP or suckling. These results are in agreement with those of Dobson (1978a, b) for milked PP Holstein cows and support the contention that FSH secretion is not 71 Table 7. Characteristics of LH secretory profiles in suckled (S-2X) and nonsuckled (NS-2X) cows on day 13 postpartum. NS-2X (n = 8) s-2x (n = 9) LH AM PM AM PM Meana 1.6 e .3 1.6 e 3 9 e .1 9 e 1 % cvb 39 i 11 37 i 6 36 i 7 34 e 5 Peak frequencyc 2.1 t .4 2.1 e 3 .8 x 2 1.1 e .3 Peak amplituded 3.4 i 1.1 3.8 t 7 1.9 e 2 1.7 x .1 aAverage (ng/ml) of 16 samples/cow collected over a 4-h period. bPercent coefficient of variation of the LH concentration. cMean number of visually determined 1J1 peaks/cow/4-h sampling period. dAverage LH peak amplitude (ng/ml) including baseline. 72 Overall Moon 2" 'OOL Coefficient of Variation E \ 'l s 3 2 " I " I 350*- :t 5’ (3 ~' 4 '1; F2 0 AM PM o my Pink Annpfiiude 4;. .\ \ i 1o 52 e 2- * II III 3 == 3: ’35 " \l :.. 7 ”7 o .. I PIA Figure 9. Characteristics of serum luteinizing hormone (LH) profiles in suckled (S-2X) and nonsuckled (NS-2X) cows on day 13 postpartum. 73 120- NS-Zx \ - '\ E \ 80". . ‘ .~~ E1 .\_\~—_. /:7":- Ix)j>‘<>§t e r -// =>-‘<é\/7 . \.__2-<,<. : _.-\‘/ g 40_ //\ 'S<\E:>- .2) in suckled and nonsuckled cows (6.6 1 .8 vs 5.1 1 1.2 ng/ml). Serum concentrations of glucocorticoids were only slightly higher in this experiment than those reported for the first 8 weeks PP in milked Holstein cows (Edgerton and Hafs, 1973). These results also agree with the work of Wagner and Oxenreider (1971) who reported no difference between mean serum concentrations of glucocorticoids in milked and suckled cows. These results do not support the view that suckling-induced changes in adrenal 77 Q 5" 5 ‘Ch S 4- a). 1\ NS-Zx 1 J. E S-Zx/ 13 14.1 2- 0%; ‘ ' ‘ ' 4 8 12 I3 Day Postpartum Figure 12. Serum concentrations of estradiol-178 in suckled (S-2X) and nonsuckled (NS-2X) cows from day 4 to 14 postpartum. 78 glucocorticoid secretion are responsible for the depressed LH secretion and delayed PP ovulation in suckled cows. Summary Mean serum hormone concentrations in suckled and non- suckled cows on day 13 PP are summarized in Table 8. The only significant change in serum hormones associated with suckling was a reduction in frequency and amplitude of episodic LH secretion which was reflected in decreased mean serum LH concentration. These results do not support a role for increased serum prolactin or glucocorticoids in suckling-induced suppression of episodic LH secretion, nor does ovarian progesterone or estradiol-17B secre- tion appear to be directly involved. Hypothalamic GonadotrOpin-Releasing Hormone (GnRH) Content Hypothalamic content of GnRH is summarized in Table 9. There was no significant difference in the total hypothalamic GnRH content of suckled and nonsuckled cows (118.2 1 16.3 vs 111.4 1 19.5 ng/hypothalamus, P > .2), nor did suckling affect the gross hypothalamic distribution of gonadotropin-releasing hormone. The distribution of GnRH was similar to that reported by Estes gt_gl, (1977) with >95% of the total content confined to the medial-basal hypothalamus and pituitary stalk. The bilateral anterior or preop- tic concentration of GnRH reported by Estes gt_al. (1977) was not apparent in this study. The large size of the preoptic tissue block may have resulted in a dilution effect that masked the Table 8. 79 Serum hormone concentrations (x 1 SE) in suckled (S-2X) and nonsuckled NS_2X) Holstein cows on day 13 postpartum. Treatment Significance Serum hormone NS-2X (n 8) S-2X (n 9) level LH 1.6 1 .3 .9 1 .l P < .01 (ng/ml) FSH 59.1 1 .6 67.3 1 6.9 N.S. ("g/ml) Prolactin 46.2 1 .9 59.6 1 13.4 N.S. ("g/m1) Progesterone .09 1 .02 .13 1 .03 N.S. (nglml) Estradiol-l7 3.0 1 4 2.9 1 .3 N.S. (Pg/m1) Glucocorticoids 5.1 1 2 6.6 1 .8 N.S. ("g/m1) 80 Table 9. Content and gross distribution of hypothalamic GnRH in suckled (S-2X) and nonsuckled (NS-2X) cows killed on day 14 postpartum. Treatment group Hypothalamic area NS-2X (n = 8) S-2X (n = 9) Preopticb’e 3.9 t .8 nga 4.2 i .4 ng Anteriorc’e 2.9 1 .4 ng 3.8 i .5 ng Medial 16as111d’e 104.6 1 18.6 ng 110.1 1 15.5 ng Total 111.4 1 19.5 ng 118.2 1 16.3 ng aNg/tissue block, x 1 SE. b+10 to +15 mm anterior to the center of the pituitary stalk. c+6 to +10 mm anterior to the center of the pituitary stalk. dMamillary bodies to +6 mm anterior to the center of the pituitary stalk. eAll blocks extended laterally on each side to the lateral sulci or 6 mm whichever was least and to a depth of 5 to 6 mm. 81 relatively discrete regions defined previously by assay of indi- vidual 500 micron serial sections. To my knowledge, this thesis is the first report of hypo- thalamic GnRH content in early PP cows or of direct comparison of hypothalamic GnRH in suckled and nonsuckled ruminants of any species. Although tissue content is difficult to interpret without knowledge of secretion rates, these findings suggest that reduced episodic LH secretion in suckled cows does not result from inade- quate stores of hypothalamic gonadotropin-releasing hormone. Anterior Pituitary Gland Weight and Hormone Content Mean anterior pituitary weights and total contents and tissue concentrations of LH, FSH and prolactin in suckled and non- suckled cows are summarized in Table 10. As demonstrated previously using bioassay procedures (Labhsetwar gt_al,, 1964; Saiduddin gt_al,, 1968), suckling did not affect pituitary content of LH or follicle-stimulating hormone. The trend towards greater FSH content in suckled cows would not appear to result from de- creased secretion since serum FSH concentrations were at least as great in suckled cows as in nonsuckled cows 1 day before slaughter. Unlike the report of Riesen gt_gl, (1968), there was no decrease in pituitary prolactin content or concentration as a result of suckling. The possibility of a change in the bioactivityzradio- immunoactivity ratio as a result of suckling cannot be excluded, but to my knowledge this has not been examined in suckled animals. 82 Table 10. Content of LH, FSH and prolactin in anterior pituitaries of suckled (S-2X) and nonsuckled (NS-2X) cows killed on day 14 postpartum. Treatment group Significance Criteria NS-2X (n = 8) S-2X (n = 9) level LH a mg b 2.3 1 .3 2.9 i 3 N.S. ug/mg 1.2 i .1 1.3 i l N.S. FSH mg 17.9 i 3.2 24.3 1 1.5 P < .1 ug/mg 9.8 i 1.9 10.8 i .8 N.S Prolactin mg 45.8 i 11.5 53.1 i 10.1 N.S ug/mg 23.9 i 5.0 25.5 i 4.4 N.S Pituitary weight g 1.9 i 1 2 3 + .2 P < 12 amg of hormone/anterior pituitary bug of hormone/mg of anterior pituitary tissue (wet weight) 83 It does not appear that suckling causes decreased pulsatile LH secretion by reducing the total pituitary stores of LH as measured by radioimmunoassay. In Vitro Superfusion: Response to Gonadotropin-Releasing Hormone and K+ Luteinizing hormone secretion from superfused pituitary pieces challenged with 25 ng/ml GnRH or 59 mM K+ is shown is Figure 13. Analysis of variance showed that the main effects of treatment (NS-2X vs S-2X) and time were highly significant (P < .01) as were the time by treatment and challenge (GnRH vs K+) by time inter- actions. There was no significant main effect of challenge nor were treatment by challenge or treatment by challenge by time interactions significant (P > .5). These results may be summarized by the following points. 1. less LH was secreted from pituitaries of suckled cows than from those of nonsuckled+cows in response to jg_vitro GnRH (25 ng/ml) or K (59 mM) challenge, 2. GnRH challenge resulted in secretion of amounts of LH similar to those secreted in response to K challenge in both suckled and nonsuckled cow pituitaries, and 3. the time-course of LH release was more prolonged fol- lowing 30-min exposure to GnRH than after similar exposure to elevated K in both suckled and nonsuckled cow pituitary tissue. Since pituitary content of LH in suckled cows did not differ from that in nonsuckled controls, the reduced in vitro secretion of LH in response to both specific and non-specific stimuli may reflect a reduction in the "releasable portion" of the 84 Sunkied-SS MM K+ wormed-59 mM K+ 888 JJAAAA Ill I j J I l l l 'an, mean 1 519/11} (3 I [Suckled-25 no/ml GnRH Nonsuckled-25 11971111 GnRH 120 LH (rig/from 8 500506090120-300 506090120 Minutes From Start of Challenge Figure 13. In vitro secretion of luteinizing hormone (LH) in response to gonadotropin- -releasing hormone (GnRH) or K+ challenge (solid bar) by pituitary pieces from suckled and nonsuckled cows on day 14 postpartum. 85 LH found in pituitaries of suckled cows. Based on work by Zolman (1973) and Kesner (personal communication), the duration of expo- sure and concentrations of both GnRH and K+ used in this experiment were assumed to be greater than those which would elicit maximal LH secretion. This assumption was not tested. However, the lack of a significant challenge effect supports the concept that both stimuli caused maximal LH secretion of a common releasable pool. Zolman (1973) reported that the effects of GnRH and 1<+ on LH secretion jn_vitro were not additive, a finding also suggestive of a single releasable LH pool. The lack of a treatment by challenge interaction would suggest that suckling's reduction of the releas- able LH pool is not a specific effect on GnRH receptors unless the amount of releasable LH is a direct reflection of the number of GnRH receptors present. The more prolonged release of LH following GnRH challenge is consistent with a receptor mediated mechanism in comparison with the electrochemical membrane depolarization caused by elevated K+ concentration. Follicle-stimulating hormone content was assayed in the fractions collected beginning -5, 25, 55, 85 and 115 min relative to the start of the ig_vitrg_challenge. The only significant main effect was time (P < .001) indicating that FSH was secreted in response to jg_vjtrg_challenge but that the amount released was not affected by the type of challenge (GnRH vs K+) or the j_.vjvg_ treatment (NS-2X vs S-ZX). The only significant interaction was time by challenge (P < .001) resulting from less abrupt and more . prolonged FSH release following GnRH than K+ stimulation, a 86 finding consistent with the receptor vs electrochemical mediation of the two stimuli. Time-course of FSH release or response to jn_vitro challenge was not affected by suckling treatment jg_vivo. Results of in_vitro FSH response to GnRH and K+ in suckled and non- suckled cows on day 14 PP are summarized in Figure 14. Summary Results in this experiment confirm the finding in Experi- ment I that suckling causes a suppressioncfl’episodic LH secretion in early PP Holstein cows as reflected by decreased overall mean and reduced frequency and amplitude of episodic LH release. Although the sampling procedures differed slightly from those in Experiment I (i.e. no milking challenge during sampling; morning and evening sampling periods) and suckling intensity was doubled (two calves), I found no effect of suckling on serum concentra- tions of FSH, prolactin, progesterone, estradiol-17B and gluco- corticoids. These results agree with those of Experiment I. The reduced i vivo secretionon 0;: c: AFmrzmuiszv :owumsmamca mucmcmmmg mcosco; mcwumpzswum10FUwP—om we: muumcaxm Acmuwzuma .mwums cowmamcqum .Ezgmm $0 EmwFoF—mcme ._.u mcamwu 3:... 231.3... 12.5 a: 93 em .3 2 m m.~ — CNN b — F D u n n i“ o... .. a .0... s O... a 2 Do. a .II coco fl 0000 r 8 m o“ 000 M .0... u .l :3: 3323...; 25...»... o 1 8 M 33:53 53.3... a .. M 81:9. .538... 3...: o .4 m 32.3 .35.. a... .. 9 :33 :8 32.35.... 0 1 8 3...: 5.... a 21:31:... . .3 can a: on mu .1 P b L- . b ..~ :lv alas, 118 Table F-I- CCOSS reaction of other bovine pituitary hormones in the follicle-stimulating hormone radioimmunoassay. Preparationa Relative Immunoreactivityb bFSH-LER-lO72c 100.0% FSH-NIHd~B1 9.0% TSH-NIH-B6 5. % LH-NIH-BB .4% GH-NIH—Bl7 N.D.e Prolactin-NIH-B4 N.D. aFSH follicle-stimulating hormone; LH = luteinizing hormone; TSH thyroid-stimulating hormone; GH =growth hormone. bCalculated at 50% specific binding. CPurified bovine FSH provided by Dr. L.EL Reichert. dNIH hormone preparations provided by Endocrine Study Section, Hormone Distribution Program,National Institutes of Health. eLess than 10% inhibition of specific binding by 1000 ng. 119 have been characterized for this antibody by Cheng (1978). Table F.2 shows the recovery of 1000 ng/ml of NIH-FSH-Bl added to bull serum containing 68 ng/ml of endogenous activity. Recovery averaged 96.1% for volumes of serum from 10 to 160 microliters. High (133.5 ng/ml) and low (57.1 ng/ml) standard sera had inter- assay C.V. of 15.8 and 15.1% respectively, with intraassay C.V. of 11.2 and 13.2%. 120 Table F.2. Assay recovery of FSHa added to bull serum. Serum volume Immunoassayable FSH (ng/ml) Recovery assayed (ul) b (%) Total Corrected 10 9.2 8.5 85.1 20 20.8 19.5 97.3 40 42.5 39.8 99.5 80 81.4 76.0 95.0 160 176.5 165.5 103.3 Average 96.1 aFSH = follicle-stimulating hormone, NIH-Bl, 1 pg added per ml of bull serum. bCorrected for endogenous FSH activity of 68.0 ng/ml in bull serum. APPENDIX G EXTRACTION, CHROMATOGRAPHY AND RADIO- IMMUNOASSAY OF ESTRADIOL-17B 121 APPENDIX G EXTRACTION, CHROMATOGRAPHY AND RADIO- IMMUNOASSAY 0F ESTRADIOL-178 (modified from England et a1., 1974. J. Clin. Endocr. Metab. 38:42) Extraction 1. Individual recoveries were monitored for each quality control and unknown sample using 3H-(2,4)~1701-estradiol (Amersham Corp., 42 Ci/mM) as an internal standard. Tritiated estradiol- 170 (5,000 cpm in 20 pl of phosphate (.01 M) buffered (pH 7.0) saline (.14 M) containing .1% Knox gelatin (PBS-K)) was added to each extraction tube (15-ml conical bottom glass tubes with teflon stoppers) plus four scintillation vials. An additional 80 pl of PBS-K was added to the scintillation vials. 2. Samples were pipetted into extraction tubes, vortexed and allowed to equilibrate with the internal standard at room temperature for at least 1 hour. 3. Diethyl ether (10 ml; Mallinckrodt Inc., anesthesia grade) from a freshly opened can was added to each extraction tube and after stoppering, extraction was performed by shaking in a horizontal position for 45 min at 60 oscillations/min on a platform shaker. 4. Following extraction, phases were separated by centrifugation (10 min at 1000 x g) and the aqueous phase was frozen by 122 123 immersing in a Dry Ice: methanol bath; then, the ether phase was decanted into 12-ml conical bottom glass tubes. Extracts were evaporated to dryness in a vacuum oven (28 inches vacuum, 45 C) and sides of tubes rinsed once with 1 m1 of fresh ether. Quality control standards included a water blank, serum blank (ovariectomized cow), added mass standard (serum plus 10 pg/ml of unlabelled estradiol-17B) and high standard serum (estrus cow). One such set of standards was inclued at the beginning and end of each assay. Up to 32 unknown samples were run in each assay, serum volumes were between .5 and 3.0 ml depending upon the expected estra- diol concentrations. Chromatography, 1. Extracts were chromatographed thru Sephadex LH-20 columns (OS-44 Quik-Sep Extended LH-ZO Columns, Isolab Inc., Akron, OH.). These commercially prepared columns are 8 mm ID x 253 mm long and contain 1000 mg of Sephadex LH-20 (Pharmacia Fine Chemicals, Uppsala, Sweden). Freshly mixed, glass distilled benzenezmethanol (90:10 v/v, Burdich and Jackson Lab. Inc., Muskegon, MI.) was used as column solvent. Columns were prepared for use by equilibrating with 25 ml of solvent then samples were transferred from the conical drying tubes to the columns using a 9 inch Pasteur 124 pipette (one/sample) and three aliquots of column solvent (300, 200 and 200 pl). Unwanted materials were eluted from the columns by an additional 5.5 ml of solvent following which a 4.2-ml fraction containing the estradiol peak was collected into a 12 x 75 glass culture tube. The column solvent was evaporated in a drying oven and the residue redissolved in 600 p1 of PBS-K. Columns were washed with an additional 40 ml of column solvent perfore reuse or storage in a glass cylinder filled with column solvent. Radioimmunoassay 1. The column fractions in PBS-K were aliquoted using a Hamilton syringe, 2 x 200 01 into 12 x 75 mm culture tubes for assay and l x 100 p1 into a scintillation vial for determination of recovery of internal standard. Scintillation fluid (5 ml; 3a7OB Preblend, RPI Corp., Elk Grove Village, 11.) was added to scintillation vials and radioactivity quantified in an auto- matic liquid scintillation spectrophotometer. Assay buffer was PBS-K and total sample volume was 200 pl; each assay consisted of four total count tubes, eight total binding tubes (zero standards), four nonspecific binding tubes (500 P9/tube of estradiol-178) and three standard curves of estradiol- 17 (stock solution = .32 ng/ml in PBS-K, curve range = .32 to 64.0 pg/tube) in addition to duplicate assay tubes from each of up to 40 samples (quality control and unknowns). 125 3. Two hundred pl of first antibody1 (1:200,000 in PBS-K) were added to each tube, mixed by gentle vortexing and assay incu- bated at room temperature (=22 C) for 90 min then placed in an icebath for 30 minutes. 4. One hundred pl of ice cold tracer2 (125-I-estradiol~17B-lla- tyrosine methyl ester; 20,000 cpm/100 pl) were added to each tube, mixed and incubation continued on ice for 45 minutes. Estradiol conjugate was radioiodinated using the chloramine-T method as described for this preparation by England et_al. (1974). 5. One ml of dextran T70 (.025%, Pharmacia Fine Chemicals, Uppsala, Sweden)-coated charcoal (.25%) in ice cold PBS was added to all but the total count tubes, vortexed briefly and allowed to incubate on ice for 30 min then transferred to cold carriers and centrifuged (10 min, 2,000 x g, 4 C) to precipitate the charcoal. Supernates containing antibody-bound 125I-tracer were immediately decanted into 12 x 75 mm tubes and capped. 6. Radioactivity in total count tubes and supernates was quanti- fied in an automatic gamma radiation spectrophotometer. Each tube was counted for 10 min or 10,000 counts, whichever occurred first. lRabbit anti-estradiol-l7B-llB-hemisuccinate-bovine serum albumin, #930 supplied by Dr. K. T. Kirton, The Upjohn Co., Kalamazoo, MI. 2Uniodinated estradiol conjugate supplied by Dr. K. T. Kirton, The Upjohn Co., Kalamazoo, MI. 1. 126 7. Estradiol-178 immunoreactivity/assay tube was corrected for serum volume extracted and recovery efficiency off the column to give estradiol concentration in p9/ml of serum. General All glassware except pipettes and columns was oven baked at 600 F for 4 h before use, a process that significantly decreased the occurrence elevated water and serum blanks. Assay Validation 1. In 10 assays, total binding (zero standards) averaged 34.1 1 1.9% of the total counts added and nonspecific binding (excess cold standard) averaged 3.1 1 .3%. Standard curve intercepts (80%, 50% and 20%) averaged 2.4 1 .2, 9.7 1 .7 and 42.6 1 3.3 pg/ml respectively in these 10 assays. Table G.1 summarizes the values obtained for the water blanks, serum blanks (ovariectomized cow) and high serum standard (estrus cow). Unknowns were not corrected for either water or serum blanks. Insufficient assays were available for calculation of the interassay coefficient of variation (CV). However, the intraassay CV for the ovariectomized and estrus standard sera in assays containing samples from the experiments in this thesis were 23.2% and 11.3% respectively. Procedural losses ranged from 5 to 25% as calculated from the 3H-estradiol-l7o1 recovery and sera were corrected individually. 127 Table G.1 Mean (1 SE) concentrations of quality control standards in estradiol-178 radioimmunoassay. Quality control standard n Assayed estradiol-178 Distilled water 30 .4 1 .l pg/tube Ovariectomized cow serum 28 1.1 1 .2 pg/ml Estrus cow serum 20 20.7 1 2.3 pg/ml 128 These losses included those due to adsorption to glass, ex- traction inefficiencies, and chromatography and transfer losses. No correction was made for the mass of 3H-tracer in the tubes since 60,000 cpm of the estradiol-170 was found to cause less than 5% inhibition of the total binding tubes in the assay. Cross reactivity of some important steroids in the estradiol~ 178 assay are summarized in Table 6.2. Recovery of estradiol-178 added to ovariectomized cow serum (10 pg/ml) was 91.5 1 1.5% (n = 10) when corrected for 1.1 pg/ml of endogenous immunoreactivity in theowariectomized COW SEY‘UlTl. 129 Table G.2. Cross reaction of major steroids in the estradiol-17B radioimmunoassay. Steroid Relative immunoreactivitya Estradiol-178 100.0% Estrone 4.04% Estriol .12% Progesterone .OO4% Testosterone .OO4% Dihydrotestosterone .OO4% Corticosterone N.D.b Cortisol N.D. Androstenediol N.D. Cholesterol N.D. aCalculated at the 50% inhibition level on the standard curve. bLess than .001% cross reaction. APPENDIX H STOCK SOLUTIONS AND MIXING RATIOS FOR MAKING KREB'S RINGER BICARBONATE BUFFERS USED IN PITUITARY SUPERFUSIONS 130 APPENDIX H STOCK SOLUTIONS AND MIXING RATIOS FOR MAKING KREB'S RINGER BICARBONATE BUFFERS USED IN PITUITARY SUPERFUSIONS (from: Zolman, 1973) Table H.1 lists the stock solutions and their compositions. All solutions were made using double—distilled water and were stored at 4 C until used to make the appropriate buffers. The glucose solution was sterilized by filtration thru a Nalgene filter unit (.2 m pore size, No. 120-0020, Nalge Sybron Corp., Rochester, N.Y.), and all stock solutions were remade at weekly intervals. The volumes of individual stock solutions required to make 1.32 liters of either 5.9 mM or 59 mM K+ Kreb's Ringer bicarbonate buffer are listed in Table H.2. The NaHCO stock solution must be 3 gassed with CD for at least 1 h before use and the final buffer 2 solutions must be gassed with 02:CO2 (95:5) for at least 30 min and equilibrated to 37 C befbre and during use. Gonadotropin- releasing hormone challenge medium was obtained by adding 1 m1 of PBS-BSA containing 6.25 pg gonadotropin-releasing hormone (GnRH) to 249 ml of KRB-5.9; 1 ml of PBS-BSA without GnRH was added to the KRB-59. 131 Table H.l. Stock solutions used for Kreb's Ringer bicarbonate buffer. Solution Reagenta Percent (v/b) I NaCl .90% II KC1 1.15% III CaC12 1.22% IV KH2P04 2.11% v MgSO4°7H20 3.82% VI NaHCO3 1.30% VII Glucose 5.00% aReagent grade chemicals purchased from Mallinckrodt Chemical Works, St. Louis, MO. 133 Table H.2. 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