l we" 1.235:me «3.3:.- :...m 9“»? {g era. .2 unwraty l. ,i v—wfi— 7—1 This is to certify that the dissertation entitled INFLUENCE OF SUCKLING AND DIET ON ENDOCRINE PHYSIOLOGY OF POSTPARTUM BEEF COWS presented by Gary L. Faltys has been accepted towards fulfillment of the requirements for Ph.D. degree in Animal Science Date July 19, 1985 MS U i: an Affirmative Action/Equal Opportunity Institution 0-12771 MSU LIBRARIES .—:—. RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. INFLUENCE OF SUCKLING AND DIET ON ENDOCRINE PHYSIOLOGY OF POSTPARTUM BEEF COWS by Gary L. Faltys A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Animal Science 1985 ABSTRACT INFLUENCE OF SUCKLING AND DIET ON ENDOCRINE PHYSIOLOGY OF POSTPARTUM BEEF COWS by Gary L. Faltys Two experiments were conducted to determine the influence of suckling and dietary energy on concentration of gonadotropins and steroids in the serum and estradiol-17B (E2) receptors in the anterior pituitary and hypothalamus of postpartum beef cows. Experiment I_ Twenty-six cows in postpartum anestrus were either allowed to nurse their calves ad libitum or were separated from their calves at d 35 postpartum (PP). Serial samples of blood were collected twice daily between d 35 and 41 PP. Baseline and pulse frequency of L3 in serum increased (P<.01) following weaning while the amplitude of the pulses remained unchanged. The secretion of cortisol did not differ between treatment groups within 72 h postweaning. Experiment.££ Sixty-nine multiparous crossbred cows were fed either a low (602 NRC) or high (1202 NRC) energy ration 85 d before calving and during the PP period. One-half of the cows in each group were allowed to nurse a calf during the PP period while the remaining cows were separated from their calves at 21 d PP. Cows were slaughtered on d 14, 23 and 25 PP and the pituitaries and hypothala- mi removed. Neither calf removal nor diet increased B receptors in the pituitaries or hypothalami. Concentrations of LH in the anterior pituitaries increased (P<.Ol) from d 14 to 25 PP independent of diet or suckling. ACKNOWLEDGEMENTS I wish to express my thanks and appreciation to Drs. E. M. Convey, R. E. Short and R. B. Staigmiller for their advice and guidance during the course of my graduate program. Appreciation is also expressed to Drs. R. L. Fogwell, D. R. Romsos and H. D. Ritchie for serving on my graduate committee. Special thanks to Larry Chapin and Brad Knapp for their assistance in completing the sta- tistical analysis of the data. I would like to extend a special thanks to the laboratory per- sonnel of the Large Animal Reproduction Laboratory and Fort Keogh Livestock and Range Research Laboratory for their technical assistance. Gratitude is expressed to Naomi Orestad for typing this dissertation and for her never-ending patience. I appreciate the excellent work that you have done. Special gratitude is extended to my parents for their love, understanding and encouragement. Most of all, I would like to express my deep appreciation and love to my helpmate, Lynne. You have made it all worthwhile. Gary L. Faltys ii TABLE OF CONTENTS LIST OF TABLES O O O O I O O O O O O O O O O O O O O O O O O O 0 LIST OF FIGURES O O O O O O O 0 O O O O O O O O I O O O O O O 0 Chapter I. INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . II. LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . Influence of Suckling on Postpartum Reproduction . . . . Influence of Nutrition on Postpartum Reproduction . . . . Influence of Body Condition and Weight on Postpartum ReprOdUCtion O O O O O I O I C O O O O O O O O O O O O 0 Factors Influencing Postpartum Reproduction . . . . . . . Effect of Suckling on Endocrine Systems During POBtpartumPerIOdo00000000000000so... Effect of Nutrition on Endocrine Systems . . . . . . . . suerOOOO0.0000000000000000000 III. RELATIONSHIP BETWEEN SUCKLING AND THE SECRETION OF LH, CORTISOL AND CORTISOL-BINDING GLOBULIN IN BEEF COWS . . . IV. INFLUENCE OF SUCKLING AND DIET ON ESTRADIOL-17B RECEPTORS AND PITUITARY LH IN BEEF COWS O O O O O O O O O I C O O O V. GENERAL DISCUSSION . . . . . . . . . . . . . . . . . . . LIST OF REFERENCES 0 O O O O O O O O O O O O O O O O O O O O O 0 iii iv 10 23 26 28 48 67 74 LIST OF TABLES TABLE PAGE III. RELATIONSHIP BETWEEN SUCKLING AND THE SECRETION OF LH, CORTISOL AND CORTISOL-BINDING GLOBULIN IN BEEF COWS 1. Influence of weaning on occurrence of LH surges8 and postpartum interval to ovulation in lactating beef cows . . . 35 2. Influence of weaning on secretory characteristics of LH8 . . 37 3. Influence of weaning on secretory characteristics of COttiBOlaoooooooo00.00000000000000042 4. Influence of calf removal on binding affinity and capacity OfCBGfOtCOttBOlaooo0.000000000000000046 IV. INFLUENCING OF SUCKLING AND DIET ON ESTRADIOL-17B RECEPTORS AND PITUITARY LH IN BEEF COWS 1. Number of cows assigned to experiment and slaughtereda . . . S3 2. Least-square analysis of estradiol receptors (ER) in posterior hypOthalamua o o o o o o o o o o o o o o o o o o o 57 3. Least-square analysis of estradiol receptors (ER) in anteriorhYPOthalam‘lsoooooooooooooooocoo.6O 4. Least-square analysis of estradiol receptors (ER) in anteriorpituitary.....................61 5. Least-square analysis of LH in anterior pituitary . . . . . . 62 iv LIST OF FIGURES FIGURE PAGE III. RELATIONSHIP BETWEEN SUCKLING AND THE SECRETION OF LH, CORTISOL AND CORTISOL-BINDING GLOBULIN IN BEEF COWS l. Secretion of luteinizing hormone in nonsuckled cows (calves weaned d 35 pOStpartum)o o o o o o o o o o o o o o o o 38 2. Concentrations of cortisol in serum of suckled cows before (60 min), during (60 min) and after (60 min) a suckling event. Pooled standard error for each period was .48, .58 and .44, respeCtively o o o o o o o o o o o o o o o o o o o o o o o o 40 3. Secretion of cortisol in nonsuckled cows (calves weaned d 35 postpartum) O O O I I O O O O O O O O O O O O O O O O O O 43 4. Secretion of cortisol in suckled cows from d 35 to 41 postpartum o o o o o o o o o o o o o o o o o o o o o o o o o o 44 IV. INFLUENCE OF SUCKLING AND DIET ON ESTRADIOL-17B RECEPTORS AND PITUITARY LH IN BEEF COWS 1. Weight change in cows fed low energy (601 NRC) or high energy (1201 NRC) diets o o o o o o o o o o o o o o o o o o o o o o o 55 2. Concentration of estradiol receptors in the anterior hypothalamus of beef cows on d 23 and 25 postpartum . . . . . 58 3. Concentration of estradiol receptors in the anterior hypothalamus and pituitary of beef cows on d 23 and 25 postpartum o o o o o o o o o o o o o o o o o o o o o o o o o o 59 4. Concentration of luteinizing hormone in the anterior pituitary of postpartum beef cows . . . . . . . . . . . . . . 63 CHAPTER I INTRODUCTION INTRODUCTION A goal for the cattle industry is to produce a healthy calf every year from each cow inseminated. However, actual calf crops are approximately 702 of expectations and the major cause of the lowered calf crop is the failure of the cow to become pregnant (Bellows et al., 1979). A prolonged period of time from parturition to first postpartum ovulation (postpartum interval) may cause a large percentage of cows to be anovulatory during the breeding season and, therefore, may reduce the number of cows that are pregnant (Symington, 1969; Wiltbank, 1970). The length of the postpartum interval varies in dairy and beef cows. Postpartum intervals less than 20 d and longer than 100 d have been reported in cattle (Wiltbank, 1970; Short et al., 1972; Dunn and Kaltenbach, 1980). A variety of factors contribute to the length of the postpartum interval and include suckling stimuli, nutrition, age of dam, dystocia, breed and season. Identifying all of the factors affecting postpartum intervals and determining asso- ciated costs are formidable tasks for scientists, economists and livestock producers. The intent of the following review is to identify and discuss factors which affect duration of postpartum anovulation in cattle with emphasis on regarding effects of suckling and nutrition on postpartum endocrine and ovarian events. CHAPTER II LITERATURE REVIEW LITERATURE REVIEW Influence gf_ Suckling _o_n_ Postpartum Reproduction The inhibitory effect of suckling and(or) presence of a calf on reproductive function was first reported by Clapp (1937). Similar observations have been reported in numerous studies since that time (Graves et al., 1968; Oxenreider, 1968; Wagner and Oxenreider, 1971; Short et al., 1972; Bellows et al., 1974). Wettemann et a1. (1976; 1978) reported that cows nursing two calves had a longer interval from calving to first estrus than cows with only one calf (94 vs 67 d). Milking delayed postpartum ovula- tion but delays were less than observed with suckling. Oxenreider and Wagner (1971) showed that Holstein cows milked twice daily had an extended postpartum interval as compared to nonlactating cows (46 vs 24 d) but a shorter postpartum interval than suckled cows (46 vs 52 d). These studies demonstrate that removal of milk from the udder or stimuli associated with this event inhibit reproductive function. Furthermore, the intensity of the stimulus influences the length of the postpartum interval. Short et a1. (1972) reported that postpartum intervals were longer for suckled and nonsuckled cows than for mastectomized cows (65 and 25 vs 12 d, respectively) suggesting that the presence of a lactating gland is sufficient stimulus to extend postpartum intervals. However, mammary denervation did not reduce 5 the interval from calving to estrus indicating that the lactating gland is extending postpartum intervals by means other than sensory stimuli from the udder (Short et al., 1976). The possibility exists that inhibition of reproductive function by suckling, lactation and(or) presence of a calf is mediated through endocrine organs. This will be discussed in subsequent sections. Attempts have been made to shorten the postpartum interval by permanently weaning calves, controlling the frequency of suckling or temporarily separating calves from cows. Weaning calves ((60 d of age) shortens postpartum anovulation of cows (Smith. and. Vincent, 1972; Laster et al., 1973; Bellows et al., 1974; Lusby and Parra, 1981). Removing calves from dams at parturition reduced the interval from calving to first estrus by 40 d (Short et al., 1972) while calf removal on d 10 postpartum reduced postpartum intervals by 33 d (Clemente et al., 1978) when compared to suckled cows. Suckling once daily (30 min) beginning at 21 d postpartum induced cows to exhibit estrus earlier (20 d) than suckled cows (Reeves and Gaskins, 1981). Similar results were reported when once-daily suckling was initiated at 30 d postpartum (Randel and Welker, 1976; Randel, 1981; Bluntzer, 1982). Cows temporarily separated from their calves for 48 h (d 21 to 80 postpartum) exhibited estrus earlier than suckled cows (Beck et al., 1977; Beck et al., 1979; Odde et al., 1982; Tervit et al., 1982). Calves that nursed once daily or were temporarily separated from their dams had daily rates of gain that were comparable to calves that suckled continuously. These management schemes offer the benefits of 6 reducing the postpartum interval in the cow as well as a continued supply of milk for the calf. .An increased incidence of short first estrous cycles ((11 d) among cows nursed once daily was observed by Reeves and Gaskins (1981). Odde et a1. (1980) reported that 802 of early-weaned cows showed short estrous cycles (7 to 10 d) compared with 12 normally nursed. These observations indicate that, even though postpartum interval can be shortened by once-daily suckling or temporary calf removal, the subsequent estrous cycle is abnormal. Influence _o_f_ Nutrition gt; Postpartum Reproduction Optimum reproductive performance of cows depends on adequate nutrition (Parr and Klemmedson, 1924; Hafez, 1959; Bellows, 1966). Cows receiving prepartum diets that are low in energy have longer intervals from calving to first estrus than cows fed high prepartum levels of energy (Wiltbank et al., 1962; Dunn et al., 1969; Falk et al., 1975). The percentage of heifers exhibiting estrus 80 d after calving was greater (92 vs 732) if the cattle were fed 3.6 kg of total digestible nutrients (TDN) before calving (120 d) rather than 2 kg TDN (Dunn et al., 1969; Wiltbank, 1970). Bellows and Short (1978) reported that cows fed a high (6.4 kg TDN) daily feed level for 90 d before calving exhibited estrus earlier postpartum than cows fed a low (3.3 kg TDN) daily feed level (50 vs 60 d). Contrary to these studies, Corah et a1. (1974) observed that prepartum nutrition did not 7 signficantly influence the interval to first estrus in heifers or cows. These authors suggested that the results may be explained by the fact that the cattle were in excellent body condition. Level of energy in the diet after calving also affects the expression of postpartum estrus. Wiltbank (1970) reported that the percentage of cows exhibiting estrus at 80 d postpartum was greater if the cows received 10 or 6 kg TDN following calving rather than 3.2 kg TDN (93 and 92 vs 812, respectively). High energy diets fed following calving, however, do not overcome effects of low prepartum dietary energy (Wiltbank et al., 1962; 1964; Bellows and Short, 1978). Supplemental protein has been shown to decrease postpartum inter- vals. Williams et a1. (1980) reported that heifers fed isocaloric diets that were high in crude protein (.96 kg, CP) had a shorter interval to first estrus than animals receiving a low crude protein diet (75 vs 86 d). Dairy cows fed diets that were high in protein (19.32 CP) had shorter postpartum intervals than cows receiving medium (16.32 GP) or low (12.72 CP) protein diets (Jordan and Swanson, 1979). Treacher et al. (1976) reported that dairy cows fed 752 of the recom- mended protein requirement for 8 wk before and 8 wk following par- turition 'had longer intervals to first estrus than cows fed the recommended level of protein (46 vs 35 d). Influence 2£_Body Condition and Weight 22_Postpartum.Reproduction Body condition and weight before parturition influence the reproductive performance of postpartum cattle (Langley and McCarthy, 8 1981; Graham et al., 1982; Post et al., 1982). Wettemann et a1. (1981) reported that postpartum intervals were correlated with the percentage decrease in body condition (r-.6l) and body weight (r-.58). These findings suggested to the authors that body weight or condition score could be used to estimate the length of the postpartum interval. Sows that lose large quantities of weight and backfat during lactation have a higher incidence of anestrus through 70 d after weaning than those that experience minimal losses (Reese et al., 1982a; b). Contrary to these results, Lishman et al. (1974) found that weight loss in Merino and Merino-cross ewes during an 84-d lac- tation period had little effect on the length of the postpartum inter- val (range 39 to 53 d). Whether body condition and weight change affect the length of the postpartum interval independent of nutrition or whether these effects interact remain to be determined. Furthermore, research is needed to determine how a loss in body condition and weight affect the endocrine system of postpartum cows. Factors Influencing_Postpartum Reproduction Young cows have a longer interval from calving to first estrus than older cows. The average postpartum interval in a breeding season was 53.4 d in cows which were 5 yr or older, 69.2 d in 4-yr-old cows, 66.8 d in 3-yr-old cows and 91.6 d in cows calving at 2 yr of age (Wiltbank, 1970). Inskeep and Lishman (1979) reported that a greater 9 percentage of mature cows ()4 yr) had a palpable CL between d 30 and 99 postpartum than 2- and 3-yr-old cows. Other researchers were unabLe to demonstrate a difference in postpartum interval among age groups (Warnick, 1955; Smith and Vincent, 1972). Body' condition and(or) nutrition of cows used in the above studies were not described and, therefore, may account for differences within and among reports. Dystocia not only increases calf mortality but also increases the postpartum interval and lowers subsequent fertility of the dam (Wiltbank et al., 1961; Anderson and Bellows, 1967; Brinks et al., 1973; Laster et al., 1973). Doornbos (1978) reported that increasing the duration of labor by 10 min increased postpartum interval by 2 d and decreased pregnancy by 0.62. The interval from parturition to estrus may be affected by breed. Angus cows exhibited estrus earlier postpartum than Hereford and Shorthorn cows (Wiltbank et al., 1961) and Brahman-cross cows (Reynolds, 1967). Contrary to these reports, Casida et a1. (1968) found no difference in the interval from parturition to first estrus between Angus and Hereford cows. Similarly, Laster et al. (1973) found no effect of breed on interval to the first postpartum estrus in Hereford-, Angus- and Charolais-cross cows. Body condition, nutrition and(or) age of cows used in the above studies were not described and, therefore, may account for differences within and among reports. Cows calving in the spring had a shorter interval to first estrus than cows calving in the fall (Loyacano et al., 1972). Hot temr perature and high humidity were considered to be major factors 10 contributing to the seasonal effects on postpartum reproduction in this study. Monty and Wolff (1974) reported that Holsteins calving during the hot summer had longer intervals from parturition to pregnancy than cows calving during cool ambient temperatures. Duration of estrus was reduced in postpartum dairy cows exposed to hot environmental temperatures and may be a contributing factor to lowered fertility in cows calving during the summer months (Monty and Wolff, 1974; Wolff and Monty, 1974). Effect 9_f_ Suckling 93 Endocrine Systems During Postpartum Period I. Gonadotropins and gonadotropin-releasing hormone (GnRH). Reproductive endocrine function in the cow becomes operative as the postpartum period progresses. Pituitary content and plasma con- centrations of luteinizing hormone (LH) are low at parturition and increase concomitant with the postpartum period (Labhsetwar et al., 1964; Saiduddin et al., 1968; Wagner et al., 1969; Erb et al., 1971; Edgerton. and 'Hafs, 1973). .Anterior pituitary content of LHL‘was reported by Cermak et a1. (1983) to be low through d 15 postpartum (<.4 ‘mg/pituitary) but increased. by’ d 30 postpartum. (1.2 to 1.6 mg/pituitary). Serum LH concentration increases within the first 2 wk postpartum. Kesler et a1. (1977) reported that serum concentrations of LH increased from 1.1 ng/ml on d 2 postpartum to 3.5 ng/ml by d 18 postpartum in anovulatory dairy cows. Similarly, Goodale et a1. (1978) reported an increase in plasma LH concentrations from d 2 to 13 ll postpartum. These observations suggest that pituitary content and serum concentrations of LH during the early postpartum period ((15 d) may be a limiting factor in the return to estrous cycles in postpartum cows. Available information on pituitary content and serum concentr- ations of follicle-stimulating hormone (FSH) is limited and contradic- tory. Cermak et al. (1983) reported that anterior pituitary content of FSH did not change significantly during the postpartum period and was comparable to the content in cycling beef cows (1.2 to 2.5 mg/pituitary). In contrast, others have reported that pituitary FSH content was high at parturition and decreased with time in postpartum dairy cows (Labhsetwar et al., 1964; Saiduddin et al., 1968). Cermak et al. (1983) utilized beef cows as an animal model while the latter authors used dairy cows. lufferences in body condition, weight and nutrition between breeds may explain differences between the studies. Plasma concentrations of FSH were higher on d 50 postpartum (1393522 ng/ml) than on d 5 postpartum (128i22 ng/ml) in anovulatory beef cows (Gauthier et al., 1982). Webb et al. (1980) found that concentrations of FSH remained low during the first 2 wk postpartum. and then increased in two of four dairy cows. Dobson et a1. (1978) have shown that serum concentrations of FSH declined from early postpartum (0 to 20 d) to late postpartum (21 to 48 d) in 502 of the dairy cows (110i37 to 84 334 ng/ml). Concentrations of FSH in the remaining cows did not change over time. Large variation in FSH concentrations reported in these studies make interpretation of the results difficult if not 12 impossible. An experiment designed to examine the preovulatory secre- tion of FSH during the postpartum period has not been conducted. Suckling may not influence the content of gonadotropins in the anterior pituitary of cows. Carruthers et a1. (1980) reported that the pituitary content of LH and FSH on d 14 postpartum of suckled dairy cows were comparable to levels observed in milked dairy cows. Pituitary content of LH and FSH in beef cows were not changed within 96 h following weaning (Walters, 1981). Contrary to these results, Cox and Britt (1982) have reported that anterior pituitary content of LH in sows increased 2.5-fold during the first 60 h postweaning. These authors reported that anterior pituitary content of FSH remained unchanged following weaning (60 h). Weaning calves from cows increased serum concentrations of LH and FSH during the 96-h postweaning period (Smith et al., 1977; Forrest et al., 1979; Walters et al., 1981). Increased serum LH following wean- ing is primarily a result of increased frequency of LH pulses (Walters et al., 1981). Similarly, an increase in number of LH pulses occurs before the first ovulation in suckled beef cows (Humphrey et al., 1976; Rawlings et al., 1980) but occurrence is delayed relative to nonsuckled cows. Amplitude of episodic peaks of LH increased follow- ing separation of calves from cows (Carruthers and Hafs, 1980; Carruthers et al., 1980). These studies indicate that suckling reduces serum concentration of LH by reducing the frequency and ampli- tude of LH pulses. 13 Information regarding change in the pulsatile release of FSH during the postpartum period is limited. Schallenberger (1977) observed peaks of FSH every 12 to 14 d during the postpartum period of dairy cows. Others have suggested that pulsatile release of FSH during the postpartum period is absent in beef cows (Garcia-Windsor and Inskeep, personal communication). Release of gonadotropins from the anterior pituitary is controlled by GnRH, a releasing hormone isolated in the hypothalamus (Schally et al., 1971; Zolman and Convey, 1972; Kaltenbach et al., 1974; Estes et al., 1977; Carruthers et al., 1980). Recent experiments have demon- strated GnRH pulsatile release in rats during the estrous cycle and in ovariectomized sheep utilizing a push-pull perfusion cannula (Levine and Ramirez, 1982; Levine et al., 1982). Information regarding the pulsatile release of GnRH from the hypothalamus of cows during the postpartum period is unavailable. The push-pull perfusion cannula will offer scientists a way of measuring GnRH release in postpartum cows. Cermak et a1. (1983) found that the content of GnRH in the total hypothalamus proper and preoptic area of the hypothalamus in suckled beef cows did not change from 1 to 45 d postpartum. Moss et a1. (1980) and Carruthers et a1. (1980) reported that hypothalamic GnRH content did not differ between nonsuckled and suckled ewes and cows (one offspring per dam). Contrary to these studies, Minaguchi and Meites (1967) demonstrated that the hypothalamus of suckled rats had 14 less GnRH content than nonsuckled rats. Difference between observa- tions of Minaguchi and Meites (1967) and those of Moss et a1. (1980) and Carruthers et al. (1980) may be due to species differences or a difference in suckling stimuli caused by nursing of a litter vs single offspring. II. Pituitary responsiveness The ability of the anterior pituitary of cows to respond to exo- genous GnRH with a release of LH increases with time postpartum. A single injection of GnRH (100 to 500 ug) induced an LH release in cows during the first 10 d postpartum but maximal response was not obtained until d 20 postpartum (Irvin et al., 1977; Kesler et al., 1977; Webb et al., 1977; Fernandes et al., 1978; Schallenberger et al., 1978). Suckling stimuli reduce ability of the pituitary to release LH in response to GnRH. Administration of GnRH (250 ug) following calf removal induced an LH release that was greater than the LH release observed in suckled cows (Troxel et al., 1980; Carter et al., 1980). Smith (1977) reported that milked postpartum dairy cows released more LH in response to GnRH than dairy cows that were suckling four calves. Carruthers et a1. (1980) observed that pituitaries from nonsuckled cows released more LH in vitro in response to GnRH than pituitaries from suckled cows. The ability of the anterior pitutiary to respond to exogenous estradiol-17B with a release of LH increases with time postpartum. A 15 single injection of estradiol benzoate (1 mg) did not cause a release of LE on d 2 postpartum in dairy cows but induced a release of LH (>20 ng/ml) in 66.6 and 40.02 of the cows on d 9 and 16 postpartum (Forrest et al., 1981). An increase in the number of estradiol receptors in the anterior pituitary from d l to 15 postpartum (Cermak et al., 1983) may account for the acquired responsiveness of the anterior pituitary to exogenous estradiol. The observations of Forrest et al., (1981) may also be due to an increase in the amount of LH that can be released over time from the pituitary in response to exogenous hormone treatment. Suckling stimuli reduce the ability of the pituitary to release LH in response to estradiol-178. Short et a1. (1979) reported that estradiol-induced LH release was greater in nonsuckled than suckled cows during the first 2 wk postpartum. However, peak LH concentra- tions following estrogen administration was not affected by suckling stimuli from wk 3 through 6 postpartum. Therefore, the inhibitory effect of suckling on estrogen-induced release of L8 is not a limiting factor in initiation and maintenance of estrous cycles beyond 3 wk postpartum. III. Ovarian activity and steroids Ovarian follicular development is positively correlated with time postpartum in cows. Spicer et a1. (1983) reported that the number of follicles 4 to 7.9 mm in diameter increased between 7 and 42 d post- partum. Saiduddin et a1. (1968) observed that the number of follicles 16 (>5 mm) on d 1 was less than the mean number on d 10, 20 and 30 post- partum (1.1 vs 4.4). Percent of nonatretic follicles in dairy cows belonging to the class of small diameter follicles ((.22 mm) decreased between 15 and 35 d postpartum; whereas, percent of medium (.29 to .67 mm) and large follicles increased from 37.4 to 47.22 and from 11.5 to 17.32 (Dufour and Roy, 1983). Increase in size and number of follicles between parturition and first ovulation reflect variable follicular growth as well as atresia during the preovulatory postpar- tum period (Morrow, 1969). Weaning increases follicular growth and development during the early postpartum period. Bellin et a1. (1982) reported that the number and average diameter of follicles on d 5 postpartum‘were greater in nonsuckled than suckled beef cows. Similarly, Carter et a1. (1980) reported that the number of medium and large follicles and follicular volume on d 5 postpartum were greater in nonsuckled than suckled beef cows. In contrast, weaning did not affect follicle numbers, volume of largest follicle or total follicular fluid weight on d 10 and 20 post- partum (Saiduddin et al., 1968). Weaning may stimulate an acute increase in follicular growth during the early postpartum period. However, increased follicular growth in response to weaning does not continue beyond d 10 postpartum. Serum concentrations of estrogen in cows decrease following par- turition and remain low until the preovulatory increase in estrogen (Henricks et al., 1972; Arije et al., 1974; Stevensen and Britt, 1979). Variation in serum estradiol between parturition and first l7 ovulation reported by Echternkamp and Hansel (1973) may reflect variable follicular growth and atresia during the preovulatory post- partum period. Weaning calves within 24 h of birth did not increase serum con- centrations of estrogen between parturition and d 14 postpartum (Carruthers and Hafs, 1980; Carruthers et al., 1980). Estrogen con- centrations of follicular fluid on d 5 postpartum in suckled cows were not significantly different from levels in cows that had their calves weaned following parturition (Carter et al., 1980). In contrast, Bellin et a1. (1982) reported that concentrations of follicular estro- gens on d 5 postpartum were greater in nonsuckled cows than suckled cows. An explanation for the differences in concentrations in folli- cular estrogens between the studies is unavailable. Serum concentrations of progesterone in cows decline following parturition to levels that are less than 1 ng/ml (Edqvist et al., 1974; 1978; Smith et al., 1973; Wise et al., 1975). This reduction in serum progesterone is a result of no luteal function by 7 d postpar- tum. Serum concentrations of progesterone have been observed to increase in beef cows 2 to 4 d before the onset of estrous cycles (Donaldson et al., 1970; Arije et al., 1974; Humphrey et al., 1976; Lavoie and Moody, 1976; Dobson, 1978). This increase in progesterone is believed to be secreted by ovarian luteal tissue (Donaldson et al., 1970; Castenson et al., 1976) and has been shown to be preceded by a release of LH (Webb et al., 1980). A similar increase in LH followed by an increase in progesterone have been observed in prepubertal 18 heifers before the onset of estrous cycles (Gonzalez-Padilla et al., 1975). These authors suggested that the increase in LH and(or) pro- gesterone before puberty serves as a ”primer for further maturation” of the hypothalamo-pituitary-ovarian axis. Increases in LH and pro- gesterone before normal estrous cycles (21 d) in postpartum cows may act to prime an endocrine system that has been noncyclic during gesta- tion and the postpartum period. IV. Glucocorticoids and cortisol-binding globulin (CBG). Concentrations of glucocorticoids decrease after parturition and remain low (<.10 ng/ml) through 85 d postpartum (Edgerton and Hafs, 1973; Ellicott et al., 1981). Synthesis of cortisol does not appear to change during the postpartum interval because adrenal weight and content of cortisol remain unchanged through 30 d postpartum (Wagner et al., 1969). Variation in serum cortisol concentrations during the postpartum period may be influenced by milk removal and(or) suckling. Milking stimulated a twofold or greater transient increase in serum glucocorticoids (Koprowski and Tucker, 1973; Smith. et al., 1973; Carruthers and Hafs, 1980). Wagner and Oxenreider (1972) showed that glucocorticoids increased in serum collected frequently (S-min inter- vals for 45 min after initiation of suckling) from dairy cows that had been separated from their calves. Dunlap et a1. (1981) reported that cortisol in serum collected (15-min intervals for 45 min) from beef cows that had been separated from their calves only tended to increase l9 (P>.06) after the initiation of suckling. Concentrations of glucocor- ticoids in serum collected (l-h intervals for 8 h on d 13 postpartum) from dairy cows suckled ad libitum and milked (2X daily) did not differ from cows that were milked (Carruthers et al., 1980). Differences among the studies may be explained by the following: (1) Blood collection frequency. GlucocorticoLd concentrations fluctuate episodically (two to three pulses/6 h, personal observation) in suckled beef cows and sampling may have to be frequent and prolonged to resolve differences in concentration or patterns of secretion; (2) How the suckling stimulus was administered. Serum glucocorticoids have been measured in blood collected following a period during which the calves were separated from their dams (Wagner and Oxenreider, 1971; 1972; Ellicott et al., 1981). Therefore, it is likely that the observed increase in glucocorticoids is due to stimuli associated with reuniting calf and dam; (3) Animal model. The use of beef or dairy cows and whether the dairy cows were milked and frequency of milking may change concentrations of cortisol. Glucocorticoids inhibit reproductive function by inhibiting the secretion and(or) action of gonadotropins (Selye, 1939). Administra- tion of glucocorticoids inhibit the preovulatory gonadotropin surges in cycling rats (Baldwin and Sawyer, 1974), the estrogen-induced release of LH in ovariectomized rats (Baldwin, 1979) and the GnRH- induced release of LH from cultured pituitary cells (Padmanabhan et al., 1982). Smith et a1. (1971) observed that cortisol implanted into the medial preoptic area of rats inhibited ovulation induced by 20 electrical stimulation of that site. This study leads to the hypothe- sis that the inhibitory effect of glucocorticoids on gonadotropin release may be mediated via effects on the brain. A specific binding protein with an affinity for cortisol (CBG) is found in serum of cattle (Seal and Doe, 1966). Cortisol, which is bound to CBG, is biologically inactive and is protected from catabo- lism (Sandberg et al., 1966). Keech et a1. (1983) found that CBG- binding capacity (ng cortisol/m1) was higher in serum of nonsuckled than suckled cows (34.9 vs 25.8). Gala and Westphal (1965) reported that concentrations of CBG in rats were greater after weaning than during lactation. These data support the hypothesis that suckling inhibits secretion of CBG. Increase in concentrations of C36 following weaning would decrease the amount of cortisol capable of interferring with gonadotropin secretion. V. Prolactin. Serum concentrations of prolactin decrease in suckled cows from peak concentrations during parturition (245 ng/ml) to levels that remain low ((30 ng/ml) during an 85-d postpartum period (Edgerton and Hafs, 1973; Peters et al., 1979; Kodagli et al., 1980; Humphrey et al., 1983). Prolactin secretion between parturition and first ovula- tion is pulsatile with a few pulses of prolactin attaining concentra- tions in excess of 200 ng/ml (Chang et al., 1981; Humphrey et al., 1983). Prolactin is released in a daily biphasic pattern in cattle with low concentrations of prolactin observed between 0900 and 1300 h 21 and significantly higher concentrations between 1900 and 2300 h (Webb and Lemming, 1981). Chang et a1. (1981) reported that the number of pulses of prolactin observed during a 24-h period did not change from d 45 to 73 postpartum (range .75 to .91 pulses/h) in suckled cows but amplitude of pulses increased during the postpartum interval (239 vs 333 ng/ml). Suckling has been reported to increase serum prolactin con- centrations in beef cows (Johke, 1969; Falk et al., 1971; Karg and Schams, 1974) and sows (Mulloy and Malven, 1979). Convey et a1. (1983) reported that prolactin increase in beef cows accompanied suckling 67, 96 and 952 of the time on d 14, 28 and 42 postpartum, respectively; 'The increase in the percentage of suckling-associated release of prolactin with time postpartum may represent specific development of the mechanism by which prolactin is released by suckling. Convey et a1. (1983) suggested that prolactin release is frequently coincidental with suckling events but can also be released spontaneously at other times. Baseline and suckling-induced release of prolactin were the same for cows suckling one or two calves indi- cating that the amount of prolactin released in response to suckling is not associated with the amount of stimulation. Stimuli associated with milking initiate a release of prolactin in dairy cows. Carruthers and Hafs (1980) found that prolactin con- centrations increased from low baseline levels ((12 ng/ml) to peak concentrations on d 7 (20.5 ng/ml) and 14 (43.9 ng/ml) prepartum within 13.6 min after premilking udder wash. Release of prolactin in 22 response to milking was largest at 8 wk of lactation and then gradu- ally decreased as lactation advanced (Koprowski and Tucker, 1973). Goodman et a1. (1979) reported that the presence of a calf (suckling not permitted) decreased basal milking-induced and TRH-induced prolactin release (68, 69 and 292, respectively) in dairy cows (2 to 3 d postpartum) as compared to cows separated from calves. Similarly, Akers and Lefourt (1984) observed that the release of pro- lactin in response to milking during the first week postpartum was greater in dairy cows separated from their calves than when calves were present. Thus, tactile stimulation of the teats increases secretion of prolactin but presence of a calf immediately adjacent to their dam but without suckling is inhibitory to secretion of prolac- tin. Suppression of prolactin secretion by administration of CB-154, an ergot alkaloid, had no effect on the length of the postpartum interval (Cummins et a1. 1977; Montgomery, 1982). Contrary to these studies, Short et a1. (1978) reported that administration of CB-154 shortened the postpartum interval in beef cows. An explanation for the difference in results among studies is unavailable. Williams and Ray (1980) showed that injections of CB-154 decreased serum prolactin concentrations in postpartum beef cows but did not affect serum con- centrations of LH, estradiol-178 or progesterone. 23 Effect‘QEDNutrition'22_Endocrine_§ystems I. Gonadotropins and GnRH Terqui et al. (1982) reported that underfeeding cows 2 mo before calving reduced body weight at calving and lowered mean plasma con- centrations of LH and FSH on d 30 postpartum compared to cows fed ad libitum. Cows fed a high energy diet (1502 National Research Council [NRC]) had greater mean concentrations of LH on d 14 and 50 postpartum than cows receiving a diet containing 1002 of the recommended energy requirements (Echternkamp et al., 1982). Pulse frequency of LH in cows fed excess energy (1502 NRC) was greater than in cows fed low energy (1002 NRC) between (I 24 and 84 postpartum. Furthermore, magnitude of LH pulses differed between treatment groups on d 60 and 84 postpartum. These results show that energy intake acts to shorten the postpartum period by increasing LH secretion. Concentrations of LH and FSH in the pituitary were less in cows fed high energy diets than cows receiving medium or low energy diets (Moss et al., 1982). Pituitary LH and FSH was similar for cows re- ceiving medium or low energy diets. A possible explanation for lower LH in pituitaries of high energy group is that release of gonadotro- pins from the pituitary is enhanced. Moss et a1. (1980) also reported that the content of GnRH in the stalk median eminence, preoptic area and hypothalamus proper did not differ among the three treatment groups. This suggests that GnRH concentrations in the hypothalamus is 24 not a limiting factor to reproductive functions. However, effects of diet on secretion of GnRH is unknown. II. Steroids Corah et a1. (1974) reported that heifers fed high energy (17.6 mCal digestible energy, DE/d) or low energy (11.4 mCal DE/d) diets had similar levels of progesterone during the first 21 <1 postpartum. Progesterone levels among dairy cows fed high energy or maintenance rations were not different during any day of the estrous cycle following parturition (Folman et al., 1973). Contrary' to these reports, Gombe and Hansel (1973) reported that concentrations of pro- gesterone in serum and CL and response of CL to LH was reduced during the second and third estrous cycle of heifers fed 702 of Morrison's requirements compared to heifers fed an adequate diet. Imakawa et a1. (1983) fed heifers either low (6.8 mCal metabolizable energy [ME/d1), moderate (12.7 mCal ME/d) or high (19.2 mCal ME/d) energy for six estrous cycles. Serum progesterone concentrations increased linearly as energy levels increased when analyzed for the six estrous cycles. These authors reported that in vitro progesterone production by cor- pora lutea cells, when incubated with human chorionic gonadotropin, was less in heifers fed low energy than moderate or high energy rations. Hill et a1. (1970) showed an immediate decline in pro- gesterone concentrations occurring within 5 d after a reduction in feed intake by heifers. Others have reported that serum progesterone 25 concentrations in cows increase (Dunn et al., 1974) or remain the same (Apgar et al., 1975) when diets are restricted for successive estrous cycles. An explanation for differences among studies is not immedi- ately apparent but could possibly be due to diets that were not iso- nitrogenous, duration that cattle were fed diets or condition of animals. Energy restricted the last trimester of pregnancy and during lac- tation of cattle did not affect size of ovarian follicles (Lishman et al., 1979) or serum concentrations of estradiol-178 (Corah et al., 1974; Dunn et al., 1974; Lishman et al., 1979). Therefore, it is unlikely that energy restriction delays estrous cycles during the post- partum period by decreasing serum concentrations of estrogens. During periods of energy restriction, corticosteroids are impor- tant in catabolism of muscle protein into amino acids which are used as a substrate for gluconogenesis (Trenkle, 1978). One might hypothe- size that animals in'negative energy balance or animals fed diets restricted in energy would have greater serum concentrations of gluco- corticoids than animals adequately fed. Hall et a1. (1984) reported that cortisol concentrations in serum tended to be greater in postpar- tum cows fed a low energy than a high energy diet. A highly signifi- cant negative correlation between weight change of lactating ewes and free cortisol in serum was observed by Barnett and Star (1981). Whether an increase in serum concentrations of cortisol during nega- tive energy balance is due to an increase in secretion of cortisol by 26 adrenals, decrease in the secretion of CBG by liver, or a combination of the two events is unknown. Summary Weaning calves markedly reduces the interval from parturition to first ovulation. Weaning calves at 21 d postpartum results in an increase in basal concentration and pulse frequency of LH within 96 h postweaning followed by a preovulatory LH surge. Thus, the endocrine changes that precede, and presumably result in first ovulation post- partum occur during a short period of time (4 d). An understanding of how weaning acutely alters LH secretion is not well understood. A possible explanation is that inhibitory effects of suckling are medi- ated by a hormone that inhibits the secretion of gonadotropins (e.g., cortisol) and that weaning causes a decrease in concentration or effects of the hormone. However, it is uncertain if cortisol is released in response to sucking in beef cows or if concentrations of cortisol decrease following weaning. An alternative hypothesis is that suckling prevents an increase in LH secretion by inhibiting estrogen receptors in the hypothalamus and pituitary. Weaning likely increases the number of estradiol receptors in the hypothalamo- hypophyseal axis enabling low concentrations of estradiol to increase secretion of LH. Effects of weaning on pattern of secretion of FSH in postpartum cows have not been elucidated. Experiments were conducted to examine the following remaining questions: (1) Does weaning alter the release of gonadotropins and 27 steroids in postpartum cows? (2) Do concentrations of cortisol decrease postweaning and, if so, does this decrease precede changes in secretion of gonadotropins? (3) Do the number of hypothalamic and hypophyseal estrogen receptors increase in response to weaning and high energy diets? CHAPTER III RELATIONSHIP BETWEEN SUCKLING AND THE SECRETION 0F LH, CORTISOL AND CORTISOL-BINDING GLOBULIN IN BEEF COWS 28 29 Summary The relationship between suckling and the secretion of luteinizing hormone (LH), cortisol and CBG was investigated in multi- parous, crossbred (Hereford x Angus x Simmental) cows. Twenty-six postpartum anovulatory cows were suckled ad libitum by their calves or were separated from their calves at d 35 postpartum (PP). Serial samples of blood (lS-min intervals for 6 h) were collected twice daily between d 35 and 41 PP. Cortisol, LH, progesterone and CBG were assayed in harvested serum. Baseline and pulse frequency of LH increased (P<.01) following weaning while the amplitude of the pulses remained unchanged. Cortisol concentrations did not change following the initiation of the suckling event. The secretion of cortisol did not differ between treatment groups within 72 h postweaning. Cortisol baseline concentrations decreased in both the nonsuckled and suckled cows between d 35 and 38 PP. CBC-binding affinity and capac- ity were the same for both treatment groups and d 35 and 38 PP. Data are interpreted to suggest that secretion of cortisol is not changed by suckling or weaning and it is unlikely that cortisol is an inhibi- tor of LH secretion during the postpartum period in beef cows. Introduction The interval of postpartum anovulation is extended by suckling in dairy (Mollar, 1970) and beef (Wiltbank and Cook, 1958; 30 Oxenreider, 1968; Short et al., 1972) cows; however, the mechanism involved is unclear. Weaning calves increases LH (Forrest et al., 1979; Smith, 1977) and follicle stimulating hormone (Walters et al., 1981) in serum of beef cows during the 96-h postweaning period. Elevated mean levels of serum LH may be a consequence of increased numbers of pulses of LH following weaning of calves (Walters et al., 1981). Wagner and Oxenreider (1972) reported higher concentrations of glucocorticoids in serum of suckled cows than nonsuckled or milked cows. In contrast, others (Carruthers and Hafs, 1980; Dunlap et al., 1981) could not demonstrate differences in glucocorticoid con- centrations between suckled and nonsuckled cows. Whether suckling alters glucocorticoid and LH concentrations independently or whether observed changes in glucocorticoids and LH are associated remains to be determined. A specific binding protein with a high affinity for cortisol (CBG) has been observed (Seal and Doe, 1966). Cortisol, which is bound to CBG, is biologically inactive. Therefore, only unbound cor- tisol (free) is able to interact with its receptor in target cells. In rats, total corticosteroids are increased during lactation while CBG concentrations are decreased (Gala and Westphal, 1965). Following weaning, there is an acute increase in CBG while total cor- ticosteroids decrease. These shifts in concentrations of cor- ticosteroids and CBG after weaning precede and may account for resumption of estrous cycles. 31 The following study was conducted in postpartum beef cows: (1) to determine the secretory pattern of LH and cortisol and binding of CBG for cortisol from weaning to the preovulatory LH surge; and (2) to determine whether concentrations of cortisol increase following suckling. Materials and Methods Twenty-six multiparous crossbred (Hereford x Angus x Simmental) anovulatory cows were assigned randomly on d 34 PP to be suckled (S) or nonsuckled (NS). Calves from S cows (n88) were allowed to nurse ad libitum while calves from NS cows (n-12) were removed from their dams at 35 d PP. Jugular blood was collected daily via cannula at 15-min intervals between 0600 to 1200 h and 1800 to 2400 h on d 35 to 41 PP. In addition, a single blood sample was collected at 0200, 0400, 1400 and 1600 h on d 35 to 41 PP. Serum was stored at -4°C. Concentrations of LH, cortisol and CBG were determined by validated radioimmunoassay techniques. Ovaries of all cows were examined rectally for presence of a corpus luteum (CL) on d 34 PP. Six cows with a palpable CL were removed from this experiment. Samples of blood were collected and ovaries were examined every third day from d 42 PP until a palpable CL was present. Concentrations of progesterone in serum greater than 1 ng/ml were interpreted to indicate presence of a CL. Interval to ovulation was reported as the number of days from calving to presence of a CL. 32 Cows were fed 15.5 kg of a balanced ration consisting of corn silage and high moisture corn from calving to the completion of the experiment. The ration was balanced to maintain the weight of lac- tating cows (475 kg) and provided 11.5 MCal of net energy (1002 NRC). Body condition was independently scored from 1 (thin) to 10 (obese) on d 41 PP by two technicians using techniques described by Bellows et al., 1971. The cows were acclimated to surroundings and restrained 2 wk before the intensive bleeding period. Animals were maintained as a group until d 34 PP when each cow and her calf were individually housed in adjacent pens. Suckling events (time of init- iation and completion) were recorded for each suckled cow between 0600 to 1200 and 1800 to 2400 on d 35 through 41 PP. Assay Procedures. Serum concentrations of LH were quantified by double antibody radioimmunoassay (RIA) previously described by Convey et a1. (1976). All samples collected were analyzed in duplicate using bovine LH (NIH-LH—BB) as a standard. Intraassay and interassay coefficients of variation were 10.8 and 14.32. Concentrations of serum cortisol were determined by RIA using procedures validated by Purchas (1985). Diurnal variation in cortisol release ‘was not observed on d 35 and 38 PP. Therefore, only samples collected between 0600 and 1200 h of remaining days were assayed. Intraassay and interassay coefficients of variation were 4.5 and 12.22. Cortisol-binding capacity of serum samples collected at 1000 h on d 35 and 38 was determined using a modification of a procedure described by Pegg and Keane (1969), which is based on the principle 33 of the competitive protein-binding assay of Murphy (1967). These samples represent CBG-binding and affinity characteristics following calf removal and preceding the preovulatory LH surge and are assumed to reflect any acute change in CBG in response to weaning. A stan- dard curve was established utilizing cortisol concentrations ranging from .25 to 200 ng/ml. Serum to be analyzed for CBG was diluted with H20 and tritiated cortisol added such that the resulting plasma- isotope solution contained .04 m1 plasma plus .4 ng tritiated cor- tisol. Serum isotope solution was added to tubes containing stan- dards, contents of these tubes were mixed then allowed to stand for 5 min at 45°C. The tubes were then placed in an ice bath, allowed to stand for 10 min and then .5 m1 dextran-coated charcoal was added to each tube. Tubes were vortexed, centrifuged (400xg) and 1 m1 of the supernatant counted in a liquid scintillation counter. Binding af- finity and capacity were determined from ‘plots and calculations described by Scatchard (1949). Coefficients of variation among and within assays were less than 102. Serum concentrations of progesterone were quantified by RIA as described by Convey et a1. (1977). All blood samples collected fol- lowing palpation were analyzed in the same assay. Progesterone con- centrations (>1 ng/ml) were used to determine the occurrence of ovulation. Statistical Analyses. Cortisol and LH data were plotted and baseline (ng/ml), pulse frequency (#/6 h) and amplitude (ng/ml) were 34 determined for each 6-h sampling interval. A peak is defined as a value that is greater than the preceding value by an amount equal to the 952 confidence interval for the assay. Decay of a hormone pulse (peak to baseline) must be consistent with the known half-life of the hormone in question. Baseline is the lowest point between peaks and amplitude is the peak height minus the baseline. Daily mean baseline, pulse frequency, amplitude, binding affin- ity and capacity of measured hormones were calculated for both suckled and nonsuckled cows on d 35 and 38 PP. Differences in the secretory characteristics between treatment groups and between d 35 and 38 PP were determined by student t test (Steel and Torrie, 1960). To test the hypothesis that weaning contributes to variation in pre- surge LH and cortisol release over time, the slope of linear regression lines were compared to lines with zero slope (McClave and Dietrich, 1982). Results and Discussion Weaning calves at 35 d PP shortened the interval from calving to ovulation in beef cows an average of 12.7 d (table 1). This supports other studies that have shown that suckling prolongs the postpartum anestrous period (Graves et al., 1968; Oxenreider, 1968; Wagner and Oxenreider, 1971; Short et al., 1972). Body condition did not differ (P>.l) between NS (i - 4.5) and S (I - 4.0) cows and likely did not influence the interval from calving to ovulation. The mechanism by 35 TABLE 1. INFLUENCE OF WEANING ON OCCURRENCE OF LH SURGES(8) AND POSTPARTUM INTERVAL TO OVULATION IN LACTATING BEEF COWS Interval to Proportion ovulation (days) Treatment exhibiting LH surge 'fi’i S.E. Nonsuckled 11/12 45 i .6 Suckled 1/8 58 i 1.2 a Day 35 to 42 d postpartum. 36 which suckling affects duration of the postpartum interval is not completely known. The proportion of nonsuckled cows exhibiting an LH surge within 7 d after weaning was greater (P<.Ol) than contemporary suckled cows (table 1). The nonsuckled cow that did not exhibit an LH surge and the suckled cow that did were not considered in further analysis of preovulatory secretory patterns of hormones. Baseline LH concentrations and the number of LH pulses observed (within a 6-h interval) increased (P<.05) within 3 d of weaning while amplitude of LH pulses remained unchanged (table 2). These data agree with reports that LH concentrations and pulse frequency increase 96-h after weaning (Forrest et al., 1979; Smith et al., 1977; Walters et al., 1981). Changes in the secretory pattern of LH before the LH surge in nonsuckled cows were best described by a linear regression line (R2>.85). A regression of either presurge baseline or pulse fre- quency of LH plotted against time exhibited a positive slope (P<.01, figure 1). Less than 632 of the variation of pulse amplitude measured over time is accounted for by either a first, second or third order polynomial (figure 1). The linear regression line formed by LH amplitude tended to increase (P<.1) over time. The pattern of secretion of LH in suckled cows (table 2) did not change over time and, therefore, the slope of the regression lines (data not shown) did not differ (P>.l) from zero slope. These data are interpreted to show that following weaning baseline concentrations increase in response to an increase in pulse frequency but are not influenced by .(I~ 37 TABLE 2. INFLUENCE OF WEANING ON SECRETORY CHARACTERISTICS OF LHa 1— Day Baseline Amplitude Treatment postpartum (ng/ml) Pulses/6 h (ng/ml) 35 .7b 2.5d 2.0f Nonsuckled 38 1.0c 3.6e 1.7f 35 .7b 1.9d 2.5f Suckled 38 .6b 1.9d 2.1f (.09) (.28) (.19) a Data from cows exhibiting an LH surge by d 38 PP were not used in these comparisons. Value within parentheses is a pooled standard error of secretory characteristics. b,c,d,e,f Means within column with different superscripts differ (P<.05). Ru QJ .Aasuummumoa mm m poemoB mm>Hmov m3ou ponusmco: GH escape: wcfiuficfimuna mo COHumuuom .H ouswam megawats umOuutml m>.l) with levels mea- sured before initiation of suckling (60 min) or levels observed after 4O .hao>fiuuommou .qc. pom mm. .wqy was moaned some now House wumwumuw wmaoom .u:o>o wawaxUSm m Ange oov nouns mam AcfiE oov mufiusv .Aofia oov ouomon m3oo mmeoam mo annom GH Homfiuuou mo coaumuucouaou ._.zu>m OZDXODm . Er: . . ozaa mmobm .N munwflh ‘f’ F3 00 U) I‘m/9N) 'Iosusoo op 41 the suckling event (60 min after the suckling period; figure 2). These data support the findings of Carruthers et a1. (1980) and Dunlap et a1. (1981) but contradict studies of Wagner and Oxenreider (1971; 1972). Serum concentrations of cortisol reported here were collected from beef cows nursing a calf ad libitum while Wagner and Oxenreider (1971; 1972) measured glucocorticoids in blood collected from dairy cows following a period during which the calves were separated from their dams. Reuniting the calf with her dam and the use of dairy cows as an animal model may account for elevated glucocorticoid concentrations observed in the latter study and might explain differences between the two studies. Baseline concentrations of cortisol decreased (P<.05) between d 35 and 38 PP in both nonsuckled and suckled cows, while pulse fre- quency and amplitude of cortisol remained unchanged (table 3). A regression of cortisol baseline concentrations of nonsuckled cows plotted against time was best described by a first order polynomial (R2-.67) and exhibited (P<.01) a negative slope (figure 3). Regression lines formed by cortisol pulse amplitude and pulse fre- quency of nonsuckled cows measured over time were described by second R2-.93) and third (R2-.70) order polynomials, respectively (figure 3). The slope of the linear regression lines did not differ signifi- cantly (P>.l) from zero slope. The secretion of cortisol in suckled cows plotted over time was described by third order polynomials (R2>.70, figure 4). The slope of the linear regression lines formed by cortisol baseline and pulse 42 TABLE 3. INFLUENCE OF WEANING ON SECRETORY CHARACTERISTICS OF CORTISOLa g Day Baseline Amplitude Treatment postpartum (ng/ml) Pulses/6 h (mg/m1) 35 6.72b 3.14d 11.54e Nonsuckled 3s 2.04c 2.57d 8.68e 35 5.19b 2.28d 13.38e Suckled 3s 2.84c 3.14d 9.898 (1.10) (.27) (.39) a Data from cows exhibiting an LH surge by d 38 PP were not used in these comparisons. Value within parentheses is a pooled standard error of secretory characteristics. b,c,d,e Means within column with different superscripts differ (P<.05). 43 Aasuumaumom mm m enamoz mo>Hmov msou uoaxunmcoc ca Homaunoo no coaummomm .m ouswfim UOKDW I.— umOuum m>.l). c,d Means in the same column with different superscripts differ (P<.1). 47 cortisol concentrations between treatments were not observed. CBG- binding affinity did not differ (P>.l) between treatment groups on d 35 and 38 PP (table 4). CBG-binding capacity (ng/ml) tended to be lower (P<.l) in S cows on d 35 PP than S cows on d 38 PP or NS cows. Others (Keech et al., 1983) have reported that CBG-binding affinity did not differ between cows nursing calves and cows that had their calves removed at 21 d. However, Keech et a1. (1983) also reported that binding capacity was greater in nonsuckled cows than suckled cows (34.9 vs 25.8 ng/ml). In the present study, baseline and pulse frequency of LH increased following calf removal at 35 d PP. Amplitude of pulses did not change after weaning. Cortisol baseline concentrations decreased in both the nonsuckled and suckled cows. Secretion of cortisol did not differ between treatment groups for 72 h after weaning and was not influenced by suckling events. CBG—binding affinity and capacity did not differ between treatment groups or d 35 and 38 PP suggesting that the amount of free cortisol does not change within 72 h of weaning. These data do not support the hypothesis that suckling increases cortisol concentrations or that cortisol is an inhibitor of LH secretion. CHAPTER IV INFLUENCE OF SUCKLING AND DIET ON ESTRADIOL-17B RECEPTORS AND PITUITARY LH IN BEEF COWS 48 49 Summary Influence of suckling and dietary energy on estradiol-178 (E) receptors and concentrations of LH in the pituitary of beef cows was examined in a 2 x 2 factorial experiment. Main effects were: (1) dietary energy (60 and 1202 NRC); and (2) suckling (suckled and nonsuckled). Sixty-nine multiparous crossbred cows were fed either a low (602 NRC) or high (1202 NRC) energy ration 85 d before calving and during the postpartum (PP) period. One-half of the cows in each dietary group were allowed to nurse a calf during the PP period while the remaining cows had their calves weaned at 21 d PP. Jugular blood was collected every 6 h on d 12, 13, 21, 22, 23 and 24 PP. Cows were slaughtered on d 14, 23 and 25 PP and pituitaries and hypothalami were collected. Ovaries were inspected at slaughter for evidence of an ovulation. An interaction (P-.02) of suckling and time after weaning affected the number of E receptors in the posterior hypothal- amus (PH). Concentration of estradiol-17B receptors was not dif- ferent (P>.1) between d 23 and 25 in the anterior hypothalamus (AH) and anterior pituitary (AP) due to either suckling or diet. However, concentration of E receptors in the AR was influenced by day post- partum showing an increase (PI-.01) from d 23 to 25 irrespective of treatment. Concentration of estradiol-178 receptors in the AH, PH and AP on d 14 did not differ (P>.1) from E receptors on d 23 and 25 PP. Concentration of LH in the AP increased (P-.01) from d 14 to 25 PP. Serum concentrations of E were below the sensitivity of the assay ((2 pg/ml) for anovulatory cows in all treatment groups on all 50 days. In summary, concentration of E receptors in the PH is affected by an interaction of suckling and time after weaning and con- centration of E receptors in the AH and AP are unaffected by suckling or dietary energy. Introduction The interval of postpartum anestrus and anovulation is extended by suckling (Clapp, 1937; Oxenreider, 1968; Short et al., 1972) and diets low in energy (Wiltbank et al., 1962; Dunn et al., 1969; Bellows and Short, 1978). Recent reports have demonstrated that con- centrations and release of luteinizing hormone (LH) were increased by calf removal (Carruthers et al., 1980) and by feeding high energy rations (Echternkamp et al., 1982). Increases in concentration and release of LH culminate in an LH surge and ovulation (Walters et al., 1982; Faltys et al., 1983). The mechanisms associated with the inhibitory influence of suckling and nutrition on the release of LH have yet to be elucidated. Ability of the anterior pituitary to release LH in response to estradiol increases between d 2 and 10 postpartum (Forrest et al., 1981) and is greater in nonsuckled than suckled cows early postpartum (Short et al., 1979). An increase in responsiveness of the anterior pituitary to estradiol may be due to an increase in the number of estradiol receptors. Cermak et a1. (1983) reported that the con- centration of pituitary receptors for estradiol increased from d l to 51 15 PP and appeared to precede release of substantial amounts of LH in beef cows. The increase in release of LH in response to weaning and high energy is possible due to an increase in the ability of the anterior pituitary to release LH and(or) the ability of the hypothalamus to release GnRH. in response to low' endogenous levels of lestradiol. Weaning and high energy may increase E receptors in the hypothalamo- hypophyseal axis enabling low levels of estradiol to release LH from the AP of the postpartum cows. Therefore, the objectives of this study are to determine the effects of suckling and dietary energy on concentration of E receptors in the AH, PH and AP of beef cows and to determine changes in numbers of receptors for estradiol during the postpartum period. Materials and Methods Sixty-nine crossbred beef cows were randomly assigned to either a high energy (HE; 1202 NRC) or low energy (LE; 602 NRC) treatment group. .A ration containing corn silage (IFN 3-28-250) and barley (IFN 4-00-549) was fed to cows in the HE treatment group from 85 d before calving through the PP period. Grass hay (IFN 2-00-262) was fed to cows in the LE treatment group for a comparable period of time. One-half of the cows in each feed group were suckled and their calves were allowed to nurse ad libitum throughout the experiment (S) while the remaining cows had their calves weaned 21 d PP (NS). 52 Animals were weighed every 14 d from the beginning of the experiment until calving. Body condition was independently scored from 1 (thin) to 10 (obese) by two technicians using techniques described by Bellows et a1. (1971). Jugular blood (10 m1/sample) was collected from six HE and five LE cows at 0600, 1200, 1800 and 2400 h on d 12 and 13 PP. Blood samples were collected from the remaining cows at 0600, 1200, 1800 and 2400 h from d 21 PP to slaughter. Serum was stored at -4°C until concentrations of LH, progesterone and estradiol-17B were determined via validated radioimmunoassay techniques. Cows were slaughtered on d 14, 23 or 25 PP (table 1). Pituitaries and hypothalami were collected and placed on ice within 20 min of slaughter. Tissues were dissected, homogenized and centri- fuged as described by Class et a1. (1984). The resulting super- natants were frozen in 52 dimethyl sulfoxide (DMSO) until E receptor numbers were determined by radioreceptorassay. Ovaries were inspected for corpora lutea (CL) at the time of slaughter. Animals were eliminated from statistical analysis if a CL was detected or if elevated serum concentrations of LH (>25 ng/ml) or progesterone (>1 ng/ml) were detected. Assay Procedures Concentrations of LH in serum and anterior pituitaries were quantified by double antibody radioimmunoassay (RIA) previously TABLE 1. NUMBER OF COWS ASSIGNED TO EXPERIMENT AND SLAUGHTEREDa 53 Diet Days, PP Nonsuckled Suckled 14 - 5 (5) Low 23 8 (6) 6 (5) 25 8 (7) 8 (7) l4 - 6 (6) High 23 5 (3) 6 (5) 25 10 (7) 7 (4) 3 Number of cows slaughtered and used in statistical analysis appear in parentheses. 54 described by Niswender et al. (1969) and modified by Staigmiller et a1. (1979). All serum samples collected were analyzed in duplicate. Intraassay and interassay coefficients of variation of a serum pool from cyclic cows were 5.12 and 9.22, respectively. Supernatant from the anterior pituitaries was diluted with PBS-Gel and assayed for concentration of LH in duplicate in the same assay. Serum concentra- tions of estradiol-178 were quantified by procedures described by England et a1. (1974) and modified by Anthony (1983). Intraassay and interassay coefficients of variation were 4.42 and 10.82, respec- tively. Cytosolic B receptors were determined by radioreceptorassay pre- viously described by Class et a1. (1984). Interassay coefficient of variation of a cytosol pool from anterior pituitaries was 12.52. Statistical Analysis Analysis of variance and Harvey's least-squares analysis (Harvey, 1960) were used to identify differences among treatments in: (l) the number of receptors for E per gram tissue; and (2) differ- ences in concentrations of LH in the anterior pituitary. Results Cows fed a diet low in energy for 85 d before calving and throughout the postpartum period lost 6.82 of their initial body weight (figure 1). Cows fed a ration high in energy for a comparable 55 .muoww Aomz NQNHV kmmmCm ewe: no Aomz Noov hwuoco 30H wow mzou ca mwcmsu uswfioz .H ouswfim mvfimzs NF 0— m m .V N . q . _ _ J - O x\\ 1 00¢ m E ......... mxxx m IIIE: H 1 x3 ......... Ex; 1 09V 11111.5 ) M 9 1m ( .................. E ........ D 1 com. ....... m---------m- mini. - >ommzw IQI ID: >cmmzm 30.. -i..- 1 0mm 56 interval gained 4.2% over their initial body weight. Condition score of cows at slaughter fed the low energy ration averaged 2.7 in com- parison with a score of 7.0 for cows fed the high energy ration. A suckling by d 23 vs 25 interaction (P=-.02) was observed for concentration of E receptors in the PH (table 2). Concentration of estradiol receptors in the PH of NS cows decreased from d 23 to 25 following weaning but increased from d 23 to 25 in S cows (figure 2). Concentration of B receptors in either AH or AP did not differ (P>.1) between nonsuckled and suckled cows on d 23 or 25 PP (figure 3). Concentration of receptors for estradiol on d 14 PP did not differ (P>.1) from the number of receptors for estradiol on d 23 and 25 PP in any tissue examined (tables 2, 3 and 4) but concentration of B receptors in the AH increased (P-.01) from d 23 to 25 PP (figure 3). Dietary energy did not change (P>.1) concentration of receptors for E in any tissues examined (tables 2, 3 and 4). A suckling by d 23 vs 25 interaction (PI-.04) was observed for the concentration of LH in the AP (table 5). Concentration of LH in NS cows decreased from d 23 to 25 following weaning but increased from d 23 to 25 in S cows (figure 4). Concentration of LB in the AP of cows did not differ between d 23 and 25 PP but concentrations of LE on both d 23 and 25 were greater (P-.Ol) than measured on d 14 PP (figure 4). Serum concentrations of estradiol-178 in anovulatory cows used in this study were below the sensitivity of the assay ((2 pg/ml) and are not published in this report. TABLE 2. LEAST-SQUARE ANALYSIS OF ESTRADIOL RECEPTORS (ER) IN POSTERIOR HYPOTHALAMUS ER/gfitissue Source df ms F ratio Feed (F) 1 3048.71 2.31 Suckling (S)/day (D) 4 2300.83 1.74 F x S/D 4 368.69 .28 Error 45 1323.55 Contrast df P value Low vs high energy 1 .13 D 14 vs d 23 + d 25 l .31 D 23 vs d 25 1 .44 F x d 14 vs d 23 + d 25 l P>.50 P x d 23 vs d 25 l .47 S 1 P>.50 S x d 23 vs d 25 1 .02 Low vs high energy x S 1 P>.50 F x S x d 23 vs d 25 1 P>.50 58 .Eauumnumoa mm ocm mm o :o m3oo moon mo mSEmHmauoa%£ uofiumumoa wsu ca muouamomu Howwmuumm mo cowumuucmoaoo .N ouawwm mm EDEEBE m>.5 F x S x d 23 vs d 25 l .16 60 61 TABLE 4. LEAST-SQUARE ANALYSIS OF ESTRADIOL RECEPTORS (ER) IN ANTERIOR PITUITARY ER[gfitissue Source df ms F ratio Feed (F) 1 234415.88 1.68 Suckling (S) x day (D) 4 104326.67 .75 F x S x D 4 133868.83 .96 Error 45 139437.46 Contrast df P value Low vs high energy 1 .31 Day 14 vs d 23 + d 25 1 P>.5 Day 23 vs d 25 1 .29 F x d 14 vs d 23 + d 25 1 .12 F x d 23 vs d 25 l P>.5 S 1 P>.5 S x d 23 vs d 25 1 .35 Low vs high energy x S l P>.5 F x S x d 23 vs d 25 l .39 62 TABLE 5. LEAST-SQUARE ANALYSIS OF LH IN ANTERIOR PITUITARY W Source df ms F ratio Feed (F) 1 .16 .16 Suckling (S) x day (D) l 3.13 3.15 F x S x D 4 1.01 1.01 Error 45 .99 - Contrast df P value Low vs high energy 1 .42 Day 14 vs d 23 + d 25 1 5.01 Day 23 vs d 25 l .32 F x d 14 vs d 23 + d 25 1 .20 F x d 23 vs d 25 l .49 S 1 .06 S x d 23 vs d 25 1 .04 Low vs high energy x S 1 .18 F x S x d 23 vs d 25 1 P>.5 CZ! NONSUCKLED m SUCKLED . l, ‘t 23 DAYS POSTPARTUM teinizi \\\\\\\\\\ —K\\\\\\ LL 14 O M N v- ( anssu 89/9") H‘I ng hormone in the anterior pituitary of postpartum beef cows. 0‘ w 64 Discussion Understanding the distribution of E receptors in the brain is essential to assessing the roles of estradiol in regulating reproduc- tive function. In this study, concentration of estradiol receptors in the AP were 5 to 10 times greater than levels observed in the AH and PH. Similarly, Class et a1. (1984) reported that the concentra- tion of E receptors in the AP of castrate ewes in May were greater than concentrations observed in the AH or PH. These data are inter- preted to suggest that estrogen has a greater role in controlling the secretion of LH from the AP than controlling the secretion of GnRH from the anterior or posterior hypothalamus. However, the median eminance region of the hypothalamus which has a prominant role in release of LHRH has concentrations of E receptors that are comparable to concentrations reported in the AP (Glass et al., 1984). Suckling increases the length of the postpartum anovulatory period (Graves et al., 1968; Short et al., 1972; Bellows et al., 1974) and inhibits gonadotropin secretion in postpartum cows (Smith et al., 1977; Forrest et al., 1979; Walters et al., 1982). Number of LH pulses and concentrations of LH in the serum. of beef cows increases within 4 d after weaning (Walters et al., 1982; Faltys et al., 1983). Data from this study show that concentration of estra- diol receptors in the AH and AP of NS cows did not change within 4 d after weaning and were comparable to levels observed in S cows. These results are interpreted to show that previously observed increases in release of LB in response to weaning are not related to 65 and are likely not a result of changes in estradiol receptors in the AH or AP. An explanation for the suckling by d 23 vs 25 interaction on the number of estradiol receptors in the PE is unavailable. Cows receiving prepartum diets that are low in energy have longer intervals from calving to first estrus than cows fed high pre- partum levels of energy (Wiltbank et al., 1962; Dunn et al., 1969). The mechanism by which nutrition affects the reproductive endocrine system is not well understood. Echternkamp et a1. (1982) reported that cows fed a high energy diet (1501 NRC) had greater concentra- tions of LB and numbers of LH pulses during the postpartum period than cows receiving diets containing 1001 of the recommended energy requirements. ‘Data from the current study show that concentrations of estradiol receptors did not differ between the two feed groups in any of the tissues examined on d 14, 23 and 25 PP. An interpretation of these data is that effects of dietary energy on postpartum release of gonadotropin is not mediated by changes in concentrations of estradiol receptors. Concentration of E receptors in the AP on d 14 did not differ from E receptors on d 23 and 25 PP. Cermak et al. (1983) have shown that concentration of E receptors in the AP decreased between (1 15 and 45 PP after increasing from d l to 15 PP. Serum concentrations of estradiol were not reported by Cermak et a1. (1983) and, there- fore, changes in estradiol due to follicular growth and atresia could have affected the number of E receptors during the postpartum period. Serum concentrations of estradiol reported in this study remained low 66 (<2 ng/ml) and, therefore, it is unlikely that numbers of E receptors were changed by varying levels of estradiol. Concentration of LH in the AP of NS cows decreased from d 23 to 25 in S cows. These results may reflect an increase in release of LH from the AP into the blood of NS cows and a continued increase in concentration of LH in AP of S cows. Concentration of L8 in the AP increased between d 14 and 25 PP. Anterior pituitary content of LH was reported by Cermak et al. (1983) to be low through d 15 PP but increased by d 30 PP. Decreased pituitary content of LH during the early postpartum period may be a.]Jndting factor in initiation and maintenance of ovarian activity. In summary, an interaction of suckling and time after weaning determined the concentration of E receptors in the PH. Suckling did not change E receptors in the AH and AP and dietary energy did not change E receptors in any tissue examined. Concentration of L8 in the AP increased from d 14 to 25 PP. CHAPTER V GENERAL DISCUSSION 67 68 General Discussion The influence of suckling and dietary energy on endocrine events in postpartum beef cows was examined in two experiments. This sec- tion will discuss the results of these experiments in relationship to previous research and will offer possible explanations for the results. The interval of postpartum anovulation was extended by suckling. Weaning at 35 d PP caused cows to have a shorter postpartum anovula- tory period than cows that were suckled ad libitum. Similar obser- vations have been reported in earlier studies (Clapp, 1937; Graves et al., 1968; Oxenreider, 1968; Wagner and Oxenreider, 1971; Short et al., 1972; Bellows et al., 1974). The means by which suckling inhib- its reproductive function during the postpartum period remain obscure. It is logical to assume that the influence of suckling on the length of the postpartum interval is mediated by neural stimula- tion resulting in inhibition of the hypothalamo‘hypophyseal-ovarian axis. However, mammary denervation did not reduce the interval from calving to estrus, indicating that postpartum intervals are prolonged by means other than sensory stimuli of the udder (Short et al., 1976). This does not exclude the possibility that neural stimuli associated with presence of a calf (i.e., smell, sight, tactile, etc.) may prolong postpartum intervals. The possibility exists that inhibition of reproductive function by suckling, lactation and(or) presence of a calf is mediated through endocrine organs. Weaning increased the frequency of LH pulses and 69 nadir concentrations of LH within 7 d after weaning. The increase in nadir concentrations of L3 is likely a result of the increase in the number of pulses. The change in the pattern of release and con- centration of LH culminate in a preovulatory LH surge within 7 d after calf removal. An increase in pulse frequency of L1! in cows following weaning of their calves is likely in response to an increase in the frequency of GnRH released from the hypothalamus (Walters, 1981). If first ovulation postpartum is dependent upon resumption of episodic secretion of frequent pulses of GnRH then ovu- lation could be induced by giving injections of GnRH at frequent intervals. Short et al. (1981a) were successful in inducing an ovu- lation (within 8 d of treatment) in suckled beef cows by injections of GnRH every 2 h for 4 (1 starting on d 20 postpartum. When the study was repeated in cows averaging 90 kg less body weight, exoge- nous GnRH did not induce ovulation in suckled cows (Short et al., 1981b). The results are interpreted to suggest that suckling pro- longs the postpartum interval of beef cows by reducing the frequency of pulsatile GnRH release from the hypothalamus and that cows in poor body condition are unable to respond to pulsatile release of GnRH with an ovulation. Determining the influence of different body con- ditions on the ability of suckled cows to ovulate in response to intermittent injections of GnRH is an objective for future research. Further discussion concerning increases in release of LH following weaning is speculative but may' offer explanations for endocrine changes that occur in response to weaning. Walters (1981) 70 proposed that for ovulation to occur, pulses of LHRH must be released frequently from the hypothalamus. An increase in the pulsatile release of GnRH following calf removal may ”prime" the pituitary and increase the amount of gonadotropin released into the plasma pool. Pituitary "priming” is defined as the increased capacity' of the pituitary to release LH in response to GnRH after previous frequent exposures to small quantitites of exogenous GnRH (Padmanabhan et al., 1980). Repeated exposure of the pituitary to GnRH may increase the ”releasable pool” of gonadotropins in the pituitary or increase the number of pituitary GnRH receptors or both and may bring about the preovulatory LH surge following weaning. Neurotransmitters of the central nervous system may also be involved in regulating release of LH. Administration of dopamine and seratonin inhibits the pulsatile release of LH in rats. (Kamberi et al., 1970; Schneider and McCann, 1970; Arendash and Gallo, 1978; Gallo, 1978) while administration of norepinephrine has been reported to increase (Porter et al., 1976) or decrease (Gallo and Drouva, 1979; Negro-Vilar and Ojeda, 1978) the release of LB in rats. Future research should test the influence of suckling on concentration of neurotransmitters in the hypothalamus of beef cows. Concentration of cortisol in cows before initiation of suckling did not differ from levels observed after suckling nor were pattern of release or concentration of cortisol in suckled cows different from nonsuckled cows. These data do not support the hypothesis that suckling induced a release of cortisol from the adrenal glands and 71 that cortisol was inhibiting the release of gonadotropins in suckled cows. Levels of cortisol in suckled and nonsuckled cows were greater on d 35 PP than levels observed on d 38 PP. Although cows were acclimated to their surroundings, stress associated with initiation of blood collection and related handling procedures contributed to high levels of cortisol on d 35 PP. The idea that cortisol is released in response to stress and unfamiliar experimental procedures is not unprecedented. Echternkamp (1984) reported that beef cows that had been stanchioned for the first time had levels of cortisol that were 10 times greater than cows that had been accustomed to the procedure. The cortisol-CBG complex renders the hormone biologically inac- tive and incapable of interacting with cortisol receptors (Sandberg et al., 1966). 'It is possible that changes in binding affinity or capacity of CBG for cortisol could affect the amount of unbound cor- tisol able to inhibit LH release without any changes in total con- centrations of cortisol. Keech et al. (1983) reported that binding capacity of CBG was greater in cows that had their calves weaned than suckled cows suggesting that there was a greater concentration of unbound cortisol available to interact with cortisol receptors in suckled cows than nonsuckled cows. Data reported in the present study showed that CBG-binding affinity and capability did not change following weaning. An explanation for differences in CBG-binding capacity between the two studies is unavailable. 72 Weaning and diet did not change the concentration of estradiol-178 receptors in the hypothalamus or anterior pituitary. Because cows utilized in the experiment did not exhibit a preovula- tory LH surge, it is uncertain if any change in the number of estradol-l7B receptors occurred after d 25 PP and before the preovu- latory LH surge. It is unknown if an increase in estradiol-178 receptors is a prerequisite for the occurrence of a preovulatory LH surge and is a subject for future research. Serum concentrations of estradiol-17B reported in experiment 2 remained low (<2 Pg/ml) on d 23 and 25 PP. Others have reported that estrogen levels in postpartum cows remain low and increase immed- iately before ovulation (Henricks et al., 1972; Arije et al., 1974; Stevensen and Britt, 1979). It is likely that cows were slaughtered before any rise in estradiol-178 occurred. It is possible that increases in estrogens before ovulation may increase the con- centration of its own receptor. Therefore, an increase in estradiol-178 receptors would be secondary to and dependent on any increase in levels of estradiol that occurred following calf removal. In summary, reestablishment of estrous cycles in postpartum cows is dependent on an increase in the release of LH. Release of cor- tisol is not increased by suckling and is not associated with gona- dotropin secretion in nonsuckled cows. Concentration of estradiol- 178 receptors in the hypothalamus and pituitary of postpartum cows does not appear to be influenced by calf removal or energy in the diet 0 LIST OF REFERENCES 74 Literature Cited Akers, R. M. and A. M. Lefcourt. 1984. Effect of presence of calf on milking induced release of prolactin and oxytocin during early lactation of dairy cows. J. Dairy Sci. 67:115. Anderson, D. C. and RJ A. Bellows. 1967. Some causes of neonatal and postnatal calf losses. J. Anim. Sci. 26(Suppl. I):94l. Anthony, R. V. 1983. Fetal growth of beef calves: I. Effect of prepartum crude protein on birth weight, blood metabolites and steroid concentrations. 11. Effect of sire on prenatal deve- 1opment of the calf and related placental characteristics. Ph.D. Dissertation. University of Wyoming, Laramie. Apgar, J., D. Aspros, J. E. Hixon, R. R. Saatman and W. Hansel. 1975. Effect of restricted feed intake on the sensitivity of the bovine corpus luteum to LH in vitro. J. Anim. Sci. 41:1120. Arendash, G. W. and R. V. Gallow. 1978. Serotonin involvement in the inhibition of episodic luteinizing hormone released during electrical stimulation of the midbrain dorsal raphe nucleus in ovariectomized rats. Endocrinology 102:1199. Arije, G. R., J. N. Wiltbank and M. L. Hopwood. 1974. Hormone levels in pre- and post-parturient beef cows. J. Anim. Sci. 39:338. Baldwin, D. M. 1979. The effect of glucocorticoids on estrogen- dependent luteinizing hormone release in the ovariectomized rat and on gonadotropin secretion in the intact female rat. Endocrinology 105:120. Baldwin, D. M. and C. H. Sawyer. 1974. Effects of dexamethasone on LH release and ovulation in the cyclic rat. Endocrinology 94:1397. Barnett, J. L. and M. L. Star. 1981. Relationship between plasma corticosteroids and weight change in recently parous lactating and dry sheep. Australian J. Agr. Res. 32:487. Beck, T. W., R. P. Wettemann, E. J. Turman. 1977. The influence of 48-hour calf separation on calf growth rate and milk production in postpartum range cows. Oklahoma Agr. Exp. Sta. 101:39. 75 Beck, T. W., R. P. Wettemann, E. J. Turman, R. Totusek. 1979. Influence of 48 hour calf separation on milk production and calf growth in range cows. Theriogenology 11:367. Bellin, M. E., M. M. Hinshelwood, G. M. Robinson, R. L. Ax and E. R. Hauser. 1982. Distribution of follicles and intrafollicular estrogen in suckled and non-suckled postpartum beef cows. J. Anim. Sci. 55(Supp1. I):338. Bellows, R. A. 1966. Improving reproductive efficiency in beef cattle. Vet. Scope 11:2. Bellows, R. A. and R. E. Short. 1978. Effects of precalving feed level on birth weight, calving difficulty and subsequent fer- tility. J. Anim. Sci. 46:1522. Bellows, R. A., R. E. Short, D. C. Anderson, B. W. Knapp and 0. F. Pahnish. 1971. Cause and effect relationships associated with calving difficulty and birth weight. J. Anim. Sci. 33:407. Bellows, R. A., R. E. Short and R. B. Staigmiller. 1979. Research areas in beef cattle reproduction. In: H. W. Hawk (Ed.) Beltsville Symposia in .Agricultural Research: Animal Reproduction. pp 3-18. Allanhald, Osmnn and Co., New Jersey. Bellows, R. A., R. E. Short, J. J. Urick and 0. F. Pahnish. 1974. Effects of early weaning on postpartum reproduction of the dam and growth of calves born as multiples or singles. J. Anim. Sci. 39:589. Bluntzer, J. 8., J. R. Beverly, P. G. Harms, J. L. Fleeger, D. W. Forrest. 1982. Effects of once daily suckling on return to estrus and pregnancy in postpartum primiparous Brahman percen- tage cows. J. Dairy Sci. 65(Supp1. I):236. Brinks, J. S., M. E. Olson and E. J. Carroll. 1973. Calving dif- ficulty and its association with subsequent productivity in Herefords. J. Anim. Sci. 36:11. Carruthers, T. D. and H. D. Hafs. 1980. Suckling and four-time daily’ milking: Influence of ovulation, estrus, and. serum luteinizing hormone, glucocorticoids and prolactin in postpartum Holsteins. J. Anim. Sci. 50:919. Carruthers, T. D., E. M. Convey, J. S. Kesner, H. D. Hafs and K. W. Cheng. 1980. The hypothalamo-pituitary gonadotrophic axis of suckled and nonsuckled dairy cows postpartum. J. Anim. Sci. 51:949. "".".""""!’.'=:&é)l 76 Carstairs, J. A., D. A. Morrow and R. S. Emery. 1975. Energy and phosphorus influence on postpartum estrus and metabolites. J. Dairy Sci. 58:750. Carter, L., D. J. Dierschke, J. J. Rutledge and. E. R. Hauser. 1980. Effect of gonadotropin-releasing hormone and calf remo- val on pituitary-ovarian function and reproductive performance in postpartum beef cows. J. Anim. Sci. 51:903. Casida, L. E., W. E. Graves, J. W. Lauderdale, J. W. Riesen, S. Saiduddin, E. R. Hauser and W. J. Tyler. 1968. Studies on the postpartum cow. Univ. of Wisconsin Res. Bull. 270:54. Castenson, P. E., A. M. Sorensen, Jr., C. R. Cobos and J. L. Fleeger. 1976. Source of postpartum P and 208-0HP preceding estrus in heifers. J. Anim. Sci. 43(Supp1. I):227. Cermak, D. L., T. Braden, J. Manna, G. D. Niswender and T. M. Nett. 1983. Contents of hypohalamic GnRH, pituitary FSH and LH, and pituitary receptors for GnRH and estradiol in postpartum suckled beef cows. Proc. West. Sec. Amer. Soc. Anim. Sci. 34:215. Chang, C. H., T. Gimenez and D. M. Henricks. 1981. Modulation of reproductive hormones by suckling and exogenous gonadal hormones in young beef cows postpartum. J. Reprod. Fertil. 63:31. Clapp, H. 1937. A factor in breeding efficiency of dairy cattle. Proc. Amer. Soc. Anim. Prod. 30:259. Clemente, P. F., R. E. Short, R. B. Staigmiller and R. A. Bellows. 1978. Effect of precalving nutrition, early weaning, CB-154 and antiprolactin treatment on postpartum interval length in beef cows. J. Anim. Sci. 47(Suppl. I):351. Convey, E. M., W. E. Beal, B. E. Seguin, K. J. Tannen and V. C. Lin. 1976. Gonadotropin releasing hormone induced hormone release after prostaglandin F20 in heifers. Proc. Soc. Exp. Biol. Med. 151:84. Convey, E. E., T. W. Beck, R. R. Weitzel, E. F. Bostwick and H. D. Hafs. 1977. Negative feedback control of bovine serum luteinizing hormone (LH) concentration from competion of the preovulatory LH surge until resumption of luteal function. J. Anim. 8C1. 45:792. Convey, E. M., H. A. Tucker and R. E. Short. 1983. Acute effect of suckling on gonadotropin, prolactin and glucocorticoid con- centrations in serum of intact and ovariectomized beef cows. Theriogenology 20:661. ,‘L‘N... ‘- ‘ufllfis-‘BWA” ' ' 77 Corah, L. R., T. G. Dunn and C. C. Kaltenbach. 1974. Prepartum and postpartum levels of progesterone and estradiol in beef heifers fed two levels of energy. J. Anim. Sci. 39:380. Cox, N. M. and J. H. Britt. 1982. Relationships between endogenous and gonadotropin-releasing hormone, gonadotropins, and follicu- lar development after weaning in sows. Biol. Reprod. 27:70. Cummins, L. J., J. K. Findlay and R. A. S. Lawson. 1977. An attempt at the use of CB-154 (ergocryptine) to reduce postpartum anoestrus in Hereford heifers. Theriogenology 8:193. Dobson, H. 1978. Radioimmunoassay of FSH in the plasma of postpar- tum dairy cows. J. Reprod. Fertil. 52:45. Donaldson, L. E., J. M. Bassett and G. D. Thorburn. 1970. Peripheral plasma progesterone concentration of cows during puberty, oestrous cycles, pregnancy and lactation and the effects of undernutrition or exogneous oxytocin on progesterone concentrations. J. Endocrinol. 48:599. Doornbos, D. Eh 1978. Bovine postpartum reproductive performance after obstetrical assistance. M.S. Thesis.. iMontana State Univ., Bozeman. Dufour, J. J. and G. L. Roy. 1983. Distribution of ovarian follicu- lar populations during early postpartum in the dairy cow. J. Anim. 8C1. 57(Supp1. I):3310 Dunlap, S. E., T. E. Kiser, G. B. Rampacek, R. R. Kraeling and F. N. Thompson. 1981. Effect of suckling on cortisol, progesterone and luteinizing hormone in postpartum beef cows. Theriogenology 16:185. Dunn, T. G., J. E. Ingalls, D. R. Zimmerman and J. N. Wiltbank. 1969. Reproductive performance of 2-year-old Hereford and Angus heifers as influenced by pre- and post-calving energy intake. J. Anim. 8C1. 29:719. Dunn, T. G. and C. C. Kaltenbach. 1980. Nutrition and the postpar- tum interval of the ewe, sow and cow. J. Anim. Sci. 51(Supp1. II):29. Dunn, T. G., J. Rone, C. C. Kaltenbach, L. S. van der Walt, M. L. Riley, A. M. Akbar. 1974. Hormone changes during underfeeding of beef cows. J. Anim. Sci. 39:206. Echternkamp, S. E. 1984. Relationship between LH and cortisol in acutely stressed beef cows. Theriogenology 22:305. 78 Echternkamp, S. E., C. L. Ferrell and J. D. Rone. 1982. Influence of prepartum and postpartum nutrition on luteinizing hormone secretion in suckled postpartum beef heifers. Theriogenology 18:283. Echternkamp, S. E. and W. Hansel. 1973. Concurrent changes in bovine plasma hormone levels prior to and during the first post- partum estrous cycle. J. Anim. Sci. 37:1362. Edgerton, L. A. and H. D. Hafs. 1973. Serum luteinizing hormone, prolactin, glucocorticoid and progestin in dairy cows from calving to gestation. J. Dairy Sci. 56:451. Edqvist, L. E., L. Ekman, B. Gustafsson and E. D. B. Johansson. 1973. Peripheral plasma levels of oestrogens and progesterone during late bovine pregnancy. Acta Endocrinol. 72:81. Edqvist, L. E., H. Kindahl and G. Stabenfeldt. 1978. Release of prostaglandin F20. during the bovine peripartal period. Prostaglandins 96:111. Ellicott, A. R., D. M. Henridks, T. Gimenz and T. E. Kiser. 1981. Suckling induced cortisol secretion in young beef cows. Theriogenology 16:469. England, B. G., G. D. Niswender and A. R. Midgley, Jr. 1974. Radio- immunoassay of estradiol-178 without chromatography. J. Clin. Endocrinol. Metab. 38:42. Erb, R. E., A. H. Surve, C. J. Callahan, R. D. Randel and H. A. Garverick. 1971. Reproductive steroids in the bovine. VII. Changes postpartum. J. Anim. Sci. 33:1060. Estes, K. S., V. Padmanabhan and E. M. Convey. 1977. Localization of gonadotropin-releasing hormone (GnRH) within the bovine hypothalamus. Biol. Reprod. 17:706. Falk, D. G., R. E. Christian, R. C. Bull and R. G. Sasser. 1975. Prepartum energy effects on cattle reproduction. J. Anim. 8C1. 41:267. Faltys, G. L., R. L. Fogwell, R. E. Short and E. M. Convey. 1983. Influence of suckling on secretory patterns of ‘LH.Iand con- centrations of cortisol in lactating beef cows. Proc. Amer. Soc. Anim. Sci. 57(Supp1. l):335. Fell, L. R., C. Beck, M. A. DeBlockey, J. M. Brown, K. J. Catt, I. A. Cumming and J. R. Goding. 1971. Prolactin in the dairy cow during suckling and machine milking. J. Reprod. Fertil. 24:144. 79 Fernandez, L. C., W. W. Thatcher, C. J. Wilcox and E. P. Call. 1978. LH release in response to GnRH during the postpartum period of dairy cow. J. Anim. Sci. 46:443. Folman, Y., M. Rosenberg, Z. Herz, M. Davidson. 1973. The rela- tionship between plasma progesterone concentration and concep- tion in postpartum. dairy cows maintained on two levels of nutrition. J. Reprod. Fertil. 34:267. Forrest, P. K., H. J. Irvin, H. A. Garverick. 1981. Effect of time postpartum on estrogen and gonadotropin releasing hormone induced luteinizing hormone release in dairy cows. J. Anim. Sci. 53(Suppl. l):3l8. Forrest, D. W., C. C. Kaltenbach and T. G. Dunn. 1981. Estriol and estradiol-178 induced LH release in ovariectomized cows and ewes. J. Anim. Sci. 52:1106. Forrest, D. W., W. M. Moseley, C. C. Kaltenbach, T. G. Dunn, R. E. Short and R. B. Staigmiller. 1979. Serum LH response to estrone or short-term calf removal in postpartum beef cows. Proc. West. Sec. Amer. Soc. Anim. Sci. 30:227. Gala, R. R. and U. Westphal. 1965. Corticosteroid-binding globulin in the rat: Possible role in the initiation of lactation. Endocrinology 76:1079. Gallo, R. V. 1978. The effect of blockade of dopamine receptors on the inhibition of episodic luteinizing hormone release during electrical stimulation of the arcuate nucleus in ovariectomized rats. Endocrinology 102:1026. Gallo, R. V. and S. V. Drouva. 1979. Effect of intraventricular infusion of catecholamines, steroid-primed rats. Neuroendocrinology 29:149. Gauthier, D., M. R. Blane, M. Petit and J. Pelletier. 1982. FSH and LH variation in beef cows during the postpartum period. Reprod. Nutr. Development 22:283. Glass, J. D., R. P. Amann and T. M. Nett. 1984. Effects of season and sex on the distribution of cytosolic estrogen receptors within the brain and the anterior pituitary gland of sheep. Biol. Reprod. 30:984. Gombe, S. and W. Hansel. 1973. Plasma luteinizing hormone (LH) and progesterone levels in heifers on restricted energy intakes. J. Anim. Sci. 37:728. .1 80 Gonzalez-Padilla, E., J. N. Wiltbank and G. D. Niswender. 1975. Puberty in beef heifers. I. The interrelationship between pituitary, hypothalamic and ovarian hormones. J. Anim. Sci. 40:1091. Goodale, W. S., H. A. Garverick, D. J. Kesler, C. J. Bierschwal, R. G. Elmore and R. S. Youngquist. 1978. Transitory changes of hormones in plasma of postpartum diary cows. J. Dairy Sci. 61:740. Goodman, G. T., H. A. Tucker and E. M. Convey. 1979. Presence of the calf affects secretion of prolactin in cows. Proc. Soc. Exp. Biol. Med. 169:421. Graham, J. F., 1982. The effect of body condition of beef cows at calving and post calving nutrition on calf growth rate and cow fertility. Proc. Australian Soc. Anim. Prod. 14:309. Graves, E. W., J. W. Lauderdale, E. R. Hauser and L. E. Casida. 1968. Relation of postpartum intervals to pituitary, gonadotro- pins, ovarian follicular development, and fertility in ‘beef cows. Wisconsin Agr. Exp. Sta. Bull. 270:23. Hafez, E. S. A. 1959. Reproductive capacity of farm animals in relation to climate and nutrition. J. Amer. Vet. Med. Assoc. 135:606. Hall, J. B., T. D. Kiser, W. L. Jenkins, C. S. Whisnant, R. S. Lowrey and T. G. Althen. 1984. Effect of energy level on calechola- mine, insulin, cortisol and luteinizing hormone concentrations in postpartum beef cows. J. Anim. Sci. 59(Suppl. I):324. Harvey, W. R. 1960. Least-squares analysis of data with unequal subclass numbers. USDA, ARS BUll. 20-8. Henricks, D. M., J. F. Dickey, J. R. Hill and W. E. Johnson. 1972. Plasma estrogen and progesterone levels after mating and during late pregnancy and postpartum in cows. Endocrinology 90:1336. Hill, J. R., D. R. Lamond, D. M. Henricks, J. F. Dickey and J. D. Niswender. 1970. The effects of undernutrition on ovarian function and fertility in beef heifers. Biol. Reprod. 2:78. Humphrey, W. D., Co Co KaltenbaCh, To Go Dunn, D. R. Koritnik and G. D. Niswender. 1983. Characterization of hormonal patterns in the beef cow during postpartum anestrus. J. Anim. Sci. 56:445. Humphrey, W. D., D. R. Koritnik, C. C. Kaltenbach, T. G. Dunn and G. D. Niswender. 1976. Progesterone and LH in postpartum suckled beef cows. J. Anim. Sci. 43:290. 81 Imakawa, K., R. J. Kittok and J. E. Kinder. 1983. The influence of dietary energy intake on progesterone concentrations in beef heifers. J. Anim. Sci. 56:454. Inskeep, E. K. and A. W. Lishman. 1979. Factors affecting postpar- tum anestrus in beef cattle. In: H. W. Hawk (Ed.) Beltsville Symposia in Agricultural Research: Animal Reproduction. pp 3-18. Allenhald, Osmun and Co., New Jersey. Irvin, H. J., H. A. Garverick, A. A. Zaid, D. J. Kesler, B. N. Day and R. S. Youngquist. 1977. LE response to GnRH in postpartum dairy cows. J. Anim. Sci. 45(Suppl. 1):172. Johke, T. 1969. Prolactin release in response to mdlking stimulus in the cow and goat estimated by radioimmunoassay. Endocrinol. Japan 16:179. Jordon, E. R. and L. V. Swanson. 1979. Effect of crude protein on reproductive efficiency, serum total protein and the albumin in the higheproducing dairy cow. J. Dairy Sci. 62:58. Kaltenbach, C. C., T. G. Dunn, T. E. Kiser, L. R. Corah, A. M. Akbar and G. D. Niswender. 1974. Release of FSH and LH in beef heifers by synthetic gonadotrophin releasing hormone. J. Anim. Sci. 38:357. Kamberi, I. A., R. S. Mical and J. C. Porter. 1970. Effect of anterior pituitary perfusion and intraventricular injection of catecholamines and. indolamines on I)! release. Endocrinology 87:1. Kanchev, L. N., H. Dobson, W. R. Ward and R. J. Fitzpatrick. 1976. Concentrations of steroids in bovine peripheral plasma during the oestrous cycle and the effect of betamethasone treatment. J. Reprod. Fertil. 48:341. Kaprowski, J. A. and H. A. Tucker. 1973. Bovine serum growth hor- mone, corticoids and insulin during lactation. Endocrinology 93:645. Karg, H. and D. Schams. 1974. Prolactin release in cattle. J. Reprod. Fertil. 39:463. Keech, C. A., R. E. Short and E. M. Convey. 1983. Corticoid-binding globulin (CBG) concentrations increase in beef cows postweaning. J. Anim. 8C1. 57(Supp1. I):3470 82 Kesler, D. J., H. A. Garverick, R. S. Youngquist, R. G. Elmore and C. J. Bierschwal. 1977. Effects of days postpartum and endogenous reproductive hormones on GnRH-induced LH release in dairy cows. J. Anim. Sci. 45:797. Kodagali, S. B., B. R. Deshpande, C. R. Sane, A. R. Sheth, GG. V. Shah and B. A. Gadgil. 1982. Serum prolactin levels of post- partum Gir cows. Indian J. Dairy Sci. 33:185. Kaprowski, J. A. and H. A. Tucker. 1973. Bovine serum growth hor- mone, corticoids and insulin during lactation. Endocrinology 93:645. Labhsetwar, A. T., W. E. Collins, W. J. Tyler and L. E. Casida. 1964. Some pituitary-ovarian relationships in the postpar- turient cow. J. Reprod. Fertil. 8:85. Langley, 0. H. and D. MacCarthy. 1981. Relationships between body condition score of dairy cows and the intervals from calving to oestrus and conception. 11th Int. Congress on Diseases of Cattle, Tel Aviv. Laster, D. B., H. A. Glimp and K. E. Gregory. 1973. Effects of early weaning on postpartum reproduction of cows. J. Anim. Sci. 36:794. LaVoie, V. A. and E. L. Moody. 1976. Suckling effect on steroids in postpartum cows. J. Anim. Sci. 43(Suppl. I):292. Levine, J. E. and V. D. Ramirez. 1982. ‘Luteinizing ‘hormone- releasing hormone release during the rat estrous cycle and after ovariectomy, as estimated with push-pull cannulae. Endocrinology 111:1439. Levine, J. E., F. P. Kwok-Yuen, V. D. Ramirez and G. L. Jackson. 1982. Simultaneous measurement of luteinizing hormone releasing hormone and luteinizing hormone release in unanesthetized, ovariectomized sheep. Endocrinology 111:1449. Lishman, A. W., S. M. J. Allison, R. L. Fogwell, R. L. Butcher and E. K. Inskeep. 1979. Follicular development and function of induced corpora lutea in underfed postpartum anestrous beef cows. J. Anim. Sci. 48:867. Lishman, A. W., W. J. Stielaw and W. A. Botha. 1974. Reproduction in the ewe in relation to plane of nutrition, body mass. 1. Incidence of oestrus between lambing Iand reconception. Agroanimalica 6:25. 83 Loyacano, A. L., C. K. Vincent, W. A. Nipper and J. Pontif. 1972. Effects of supplemental energy during breeding and season of breeding on reproductive performance in beef cattle. Louisiana Livestock Producers Day Rep. 12:127. Lusby, K. S. and A. A. Parra. 1981. Effects of early weaning on calf performance and on reproduction in mature cows. Oklahoma Agr. Exp. Sta. 107:550 McClave, J. T. and F. H. Dietrich. 1982. Statistics. 2nd Ed. Dellen Publishing Co., Santa Clara, CA. Minaguchi, H. and J. Meites. 1967. Effects of suckling on hypotha- lamic LH-releasing factor and prolactin inhibiting factor, and on pituitary LH and prolactin. Endocrinology 80:603. Mollar, K. 1970. Uterine involution and ovarian activity after calving. New Zealand Vet. J. 18:140.. Montgomery, G. W. 1982. Influence of suckling frequency and bromo- criptine treatment on the resumption of ovarian cycles in post- partum beef cattle. Theriogenology 17:551. Monty, D. E., Jr. and L. K. Wolff. 1974. Summer heat stress and reduced fertility in Holstein-Friesian cows in Arizona. Amer. J. Vet. Res. 35:1495. Morrow, D. A. 1969. Postpartum ovarian activity and involution of the uterus and cervis in dairy cattle. Vet. Scope 14:2. Moss, G. E., T. E. Adams, G. D. Niswender and T. M. Nett. 1980. Effects of parturition and suckling on concentrations of pituitary gonadotropins, hypothalamus GnRH and pituitary respon- siveness to GnRH in ewes. J. Anim. Sci. 50:496. Moss, G. E., J. R. Parfet, M. A. Kiekman, R. P. Lemenager and K. S. Hendrix. 1982. Pituitary luteinizing hormone (LH), follicle- stimulating hormone (FSH), serum progesterone (P) and hypothala- mic gonadotropin-releasing hormone (GnRH) in beef cows in varied body condition. J. Anim. Sci. 55(Supp1. I):374. Mulloy, A. L. and P. V. Malven. 1979. Relationships between con- centrations of porcine prolactin in blood serum and milk of lac- tating sows. J. Anim. Sci. 48:876. Murphy, B. E. P. 1967. Some studies on the protein binding of steroids and their application to the routine micro and ultra- micro measurements of various steroids in body fluid by com- petitive protein-binding radioassay. J. Clin. Endocrinol. Metab. 24:919. '7.- ~-. _ 84 Niswender, G. D., L. E. Reichert, Jr., A. R. Midgley, Jr. and A. V. Nalbandov. 1969. Radioimmunoassay for bovine and ovine luteinizing hormone. Endocrinology 84:1166. NRC. 1976. Nutrient Requirements of Domestic Animals, No. 4. Nutrient Requirements of Beef Cattle. Fifth Revised Ed. National Academy of Sciences-National Research Council, Washington, DC. Negro-Vilar, A. and S. R. Ojeda. 1978. Catecholaminergic and steroidal modification of LHRH and somatostatin (SRIF) release by median eminance (ME) in vitro. Endocrinology 102:459A. Odde, K. G., G. H. Kiracofe, R. S. Schalles. 1982. Effect of limited suckling on reproductive performance and milk production and weight gains and suckling behavior of calves. J. Anim. Sci. 53(Supp1. I):353. Odde, K. G., H. S. Ward, C. H. Kiracofe, R. M. McKee and R. J. Kittok. 1980. Short estrous cycles and associated serum pro- gesterone levels in beef cows. Theriogenology 14:105. Oxenreider, S. L. 1968. Effects of suckling and ovarian functions on postpartum reproductive activity in beef cows. Amer. J. Vet. Res. 29:2099. Padmanabhan, V., C. Keech and E. M. Convey. 1982. Cortisol and adrenocorticotropin (ACTH) as potential inhibitors of bovine LH secretion. J. Anim. Sci. 55(Supp1. l):376. Padmanabhan, V., J. S. Kesner and E. M. Convey. 1980. LH secretion by bovine pituitary cells after consecutive LHRH challenges in vitro. J. Anim. Sci. 51(Supp1. l):313. Parr, V. V. and G. S. Klemmedson. 1924. An economic study of the cost and methods of range cattle production in northeastern range area of Texas. Economics and Bureau of Animal Industry. Pegg, P. J. and P. M. Keane. 1969. The simultaneous estimation of plasma cortisol and transcortin binding characteristics by a competitive protein binding technique. Steroids 14:705. Peters, A. R., S. Vyvoda and G. E. Lamming. 1979. Comparison of plasma prolactin levels in the milked and suckling cow. J. Endocrinol. 83:27. 85 Porter, J. C., N. Ben-Jonathon, C. Liver, R. L. Eskay and A. J. Winters. 1976. Interrelationship of CSF hypophyseal portal vessels and hypothalamus and their role in the regulation of anterior pituitary function. In: T. C. Anad Kuman (Ed.) Neuroendocrine Regulation of Fertility. p 71. Karger, Basel. Post, T. B. and R. B. Morrish. 1982. Relationships between inter- vals to first oestrus and postpartum weight gains in Brahman- cross cows and their calves. Proc. Australian Soc. Anim. Prod. 14:613. Purchas, R. W., S. A. Zinn and H. A. Tucker. 1985. A simple method for separating unbound and bound cortisol in radioimmunoassay. Anal. Biochem. (in press). Randel, R. D. 1981. Effect of once-daily suckling on postpartum interval and cowbcalf performance of first-calf Brahman x Hereford heifers. J. Anim. Sci. 53:755. Randel, R. D. and G. A. Welker. 1976. Once daily suckling effect on cow-calf performance. J. Anim. Sci. 43(Suppl. I):301. Rawlings, N. C., L. Weis, B. Todd, J. Manns and J. H. Hyland. 1980. Some endocrine changes associated with the postpartum period of the suckling beef cow. J. Reprod. Fertil. 60:301. Reese, D. E., B. D. Moser, E. R. Peo, Jr., A. J. Lewis, D. R. Zimmerman, J. E. Kinder and W. W. Stroup. 1982a. Influence of energy intake during lactation on the interval from weaning to first estrus in sows. J. Anim. Sci. 55:590. Reese, D. E., B. D. Moer, E. R. Peo, Jr., A. J. Lewis, D. R. Zimmerman, J. E. Kinder and W. W. Stroup. 1982b. Influence of energy intake during lactation on subsequent gestation, lac- tation and postweaning performance of sows. J. Anim. Sci. 55:867. Reeves, J. J. and C. T. Gaskins. 1981. Effect of once-a-day nursing on rebreeding efficiency of beef cows. J. Anim. Sci. 53:889. Reynolds, W. L. 1967. In: T. J. Cunha, A. C. Warnick and M. Koger (Ed.) Factors Affecting Calf Crop. Breeds and Reproduction. Saiduddin, S. J. W. Riesen, W. J. Tyler and L. E. Casida. 1968. Relation of postpartum. interval to pituitary gonadotropins, ovarian follicular development and fertility in, dairy cows. Wisconsin Res. Bull. 270:15. 86 Sandberg, A. A., H. Rosenthal, S. L. Schneider and W. R. Saunwhite, Jr. 1966. Protein-steroid interaction and their role in the transport and metabolism of steroids. In: G. Pincos, T. Nakao and J. F. Tait (Eds.) Steroid Dynamics. Academic Press, New York 0 Scatchard, G. 1949. The attraction of protein for small molecules and ions. Ann. NY Acad. Sci. 51:660. Schallenberger, E. 1977. Radioimmunological determination of FSH in cattle - methodological development work and physiological results. Ph.D. Dissertation, Ludwig-Maximilian Univ., Munchen, German Federal Republic. Schallenberger, E., D. Schams and K. Zottneier. 1978. Response of lutropin (LH) and follitropin (FSH) to the administration of gonadoliberin (GnRH) in pregnant and post-partum cattle including experiments with prolactin suppression. Theriogenology 10:35. Schally, A. V., R. M. G. Nair, T. W. Redding and A. Arimura. 1971. Isolation of the LH and FSH-releasing hormone from porcine hypothalami. J. Biol. Chem. 246:7230. Schneider, H. P. G. and S. M. McCann. 1970. Mono- and indolamines and control of LH secretion. Endocrinology 86:1127. Seal, U. S. and R. P. Doe. 1966. Corticosteroid-binding globulin: biochemistry, physiology and phylogeny. In: G. Puncos, T. Nakao and J. F. Tait (Eds.) Steroid Dynamics. pp 63-90. Academic Press, New York. Selye, H. 1939. The effect of adaption to various damaging agents on the female sex organs in the rat. Endocrinology 25:615. Short, R. E., R. A. Bellows E. L. Moody and B. E. Howand. 1972. Effects of suckling and mastectomy on bovine postpartum repro- duction. J. Anim. Sci. 34:70. Short, R. E., E. M. Convey, R. B. Staigmiller and R. A. Bellows. 1981b. Effects of intermittent small-dose injections of GnRH in anestrous beef cows. J. Anim. Sci. 53(Suppl. l):366. Short, R. E., R. D. Randel, R. B. Staigmiller and R. A. Bellows. 1979. Factors affecting estrogen-induced LH release in the cow. Biol. Reprod. 21:683. Short, R. E., R. B. Staigmiller, J. K. Baber, J. B. Carr and R. A. Bellows. 1976. Effects of mammary denervation in postpartum cows. J. Anim. Sci. 43(Suppl. I):304. 87 Short, R. E., R. B. Staigmiller, R. A. Bellows and J. B. Carr. 1978. Effect of suckling and a prolactin inhibitor (CB-154) on post- partum anestrus in the cow. Proc. West. Sec. Amer. Soc. Anim. 8C1. 29:308. Short, R. E., D. L. Walters, E. M. Convey, R. B. Staigmiller and C. C. Kaltenbach. 1981a. Role of pulsatile LH release in resump- tion of ovarian cycles in postpartum beef cows. In: EEF Seminar, Factors Influencing Fertility in the Postpartum Cow, Freising, Germany. Smith, E. R., J. Johnson, R. F. Weick, S. Levine and J. M. Davidson. 1971. Inhibition of the reproductive system in immature rats by intracerebral implantation of cortisol. Neuroendocrinology 8:94. Smith, J. F. 1977. Effect of suckling on the onset of oestrus, ovu- lation and LH response to GnRH in the post-partum cow. New Zealand Med. J. 86:401. Smith, L. E. and C. K. Vincent. 1972. Effects of early weaning and exogenous hormone treatment on bovine postpartum reproduction. J. Anim. 8C1. 35312280 Smith, M. F., D. L. Walters, P. Go name and J. No Wiltbank. 1977. LH levels after steroids and/or 48 hr calf removal in anestrous cows. J. Anim. Sci. 45(Suppl. I):209. Smith, V. G., L. A. Edgerton, H. D. Hafs and E. M. Convey. 1973. Bovine serum estrogens, progestins and glucocorticoids during late pregnancy, parturition and early lactation. J. Anim. Sci. 36:391. Spicer, L. J., K. Leung, E. M. Convey, J. Gunther, H. A. Tucker and R. E. Short. 1983. Secretion of luteinizing hormone (LH), follicle hormone (FSH) and prolactin (PRL) and changes in ovarian folliculogenesis and follicular fluid (FF) hormones during postpartum. anestrus in beef cattle. J. .Anim. Sci. 57(Supp1. I):375. Staigmiller, R. B., R. E. Short, R. A. Bellows and J. B. Carr. 1979. Effect of nutrition on response to exogenous FSH in beef cattle. J. Anim. Sci. 48:1182. Steel, R. G. D. and J. H. Torrie. 1960. Principles and procedures of Statistics. McGraw-Hill Book Co., New York. 88 Stevenson, J. A. and J. H. Britt. 1979. Relationships among luteinizing hormone, estradiol, progesterone, glucocorticoids, milk yield, body weight and postpartum ovarian activity in Holstein cows. J. Anim. Sci. 48:570. Symington, R. B. 1969. Factors affecting postpartum fertility in cattle with special emphasis on the hormonal aspects of the problem in ranch cows in Southern Africa. Proc., South African Soc. Anim. Prod. 8:29. Terqui, M., D. Chupin, D. Gauthier, N. ‘Perez, J. Pelot and P. Mauleon. 1982. Influence of management and nutrition on post- partum endocrine function and ovarian activity in cows. Martinus Nijhoff 304. Terv1t, H. R., J. F. Smith, P. Go GOOld, K. R. Jones and Jo J. D. Vandien. 1982. Reproductive performance of beef cows following temporary removal of calves. New' Zealand Soc. .Anim. iProd. 42:83. Treacher, R. J., W. Little, K. S Collis and A. J. Stark. 1976. The influence of dietary protein intake on milk production and blood composition of high-yielding dairy cows. J. Dairy Res. 43:357. Trenkle, A. 1978. Relation of hormonal variations of nutritional studies and metabolism of ruminants. J. Dairy Sci. 61:281. Troxel, T. R., D. J. Kesler, R. C. Noble and S. C. Carlin. 1980. Ovulation and reproductive hormones following steroid pretreat- ment, calf removal and GnRH in postpartum suckled beef cows. J. Anim. 8C1. 51:652. Wagner, W. C. and S. L. Oxenreider. 1971. Endocrine physiology following parturition. J. Anim. Sci. 32(Suppl. I):1. Wagner, W. C. and S. L. Oxenreider. 1972. Adrenal function in the cow. Diurnal changes and the effects of lactation and neurohy- pophyseal hormones. J. Anim. Sci. 34:630. Wagner, W. G., R. Saatman and W. Hansel. 1969. Reproductive phy- siology of the postpartum cow. II. Pituitary, adrenal and thyroid function. J. Reprod. Fertil. 18:501. Walters, D. In» 1981. Effects of suckling on pituitary and ovarian function in postpartum beef cows. Ph.D. Dissertation. University of Wyoming, Laramie. 89 Walters, D. L., R. E. Short, E. M. Convey, R. B. Staigmiller, T. G. Dunn and C. C. Kaltenbach. 1982. Pituitary and ovarian func- tion in postpartum beef cows. 11. Endocrine changes prior to ovulation in suckled and nonsuckled postpartum cows compared to cycling cows. Biol. Reprod. 26:647. Warnick, A. C. 1955. Factors associated with the interval from par- turition to first estrus in beef cattle. J. Anim. Sci. 14:100. Webb, R. and G. E. Lamming. 1981. Patterns of plasma prolactin in postpartum suckled cows. J. Endocrinol. 90:391. Webb, R., G. E. Lamming, N. B. Hanes and G. R. Foxcroft. 1980. Plasma progesterone and gonadotropin concentrations and ovarian activity in postpartum dairy cows. J. Reprod. Fertil. 59:133. Webb, R., G. E. Lamming, N. B. Haynes, H. D. Hafs and J. G. Manna. 1977. Response to cyclic and postpartum suckled cows to injec- tions of synthetic LH-RH. J. Reprod. Fertil. 50::203. Wettemann, R. P., T. W. Beck and E. J. Turman. 1978. Endocrine response of postpartum anestrus beef cows to GnRH and PMSG. J. Anim. Sci. 47(Supp1. I):399. Wettemann, R. P., K. S. Lusby and E. J. Turman. 1981. Relationship between changes in prepartum body' weight and condition and reproductive performance of range cows. Oklahoma Agr. Exp. Sta. 96:121. Wettemann, R. P., E. J. Turman, R. D. Wyatt, L. Knori and R. Totusek. 1976. Reproductive performance of range cows ‘with 'various suckling intensities. Oklahoma Agr. Exp. Sta. Misc. Publ. 96:121. Williams, G. L. and D. E. Ray. 1980. Hormonal and reproductive pro- files of early postpartum beef heifers after prolactin suppression or steroid-induced luteal function. J. Anim. Sci. 50:906. Williams, Rm al., C. AL. RUder, IL. G. Falk, Rm ‘3. Ball and IL. G. Sasser. 1980. Effect of prepartum and postpartum reproductive performance in heifers. J. Anim. Sci. 51(Supp1. I):337. Wiltbank, J. N. 1970. Research needs in beef cattle reproduction. J. Anim. 8C1. 31:755. Wiltbank, J. N. and A. C. Cook. 1958. The comparative reproductive performance of nursed and milked cows. J. Anim. Sci. 17:640. 9O Wiltbank, J. N., W. W. Rowden, J. E. Ingalls, K. E. Gregory and R. M. Koch. 1962. Effect of energy level on reproductive phenomena of mature Hereford cows. J. Anim. Sci. 21:219. Wiltbank, J. N., W. W. Rowden, J. E. Ingalls and D. R. Zimmerman. 1964. Influence of post-partum energy level on reproductive performance of Hereford cows restricted in energy intake prior to calving. J. Anim. Sci. 23:1049. Wiltbank, J. N., E. J. Warwick, E. W. Vernon and B. M. Priode. 1961. Factors affecting net calf crop in beef cattle. J. Anim. Sci. 20:409. Wise, T. H., A. M. Sorensen, Jr. and J. L. Fleeger. 1975. Quantitation of deoxycorticosterone and its relationship to pro- gesterone in the prepartum bovine. Steroids 26:17. Wolff, L. K. and D. E. Monty, Jr. 1974. Physiologic response to intense summer heat and its effect on the estrous cycle of nonlactating and lactating Holstein-Friesian cows in Arizona. Amer. J. Vet. Res. 35:187. Zolman, J. and E. M. Convey. 1972. Bovine pituitary LH and prolac- tin release during superfusion. Proc. Soc. Exp. Biol. Med. 140:194.