ABSTRACT SERUM PROLACTIN AND GROWTH HORMONE CONCENTRATIONS FOLLOWING THYROTROPIN RELEASING HORMONE IN CATTLE DURING VARIOUS PHYSIOLOGICAL STATES BY Dwight T. Vines Serum prolactin and growth hormone (GH) concentrations were determined following administration of thyrotropin releasing hormone (TRH) during various physiological states. In eight prepubertal heifers prolactin concentrations before TRH averaged 16 ng/ml and increased to a maximum value of 81 ng/ml following TRH administration. Repeatability coefficients for day to day baseline prolactin concentrations and prolactin response curve areas (integrated area of plot of hormone concentrations for 30 min after TRH minus baselines before TRH) following TRH administration were .27 and .61, respectively. These results suggest that there are great differences from day to day in baseline prolactin concentrations and that there is less day to day variation in prolactin response curve areas following TRH administration. However, the correlation coeffi- cient, within days, between baseline and maximum serum prolactin concentrations following TRH administration was .64 (P<.01). Serum GH concentrations in prepubertal heifers prior to TRH averaged 8 ng/ml and increased to a maximum of 21 ng/ml after TRH. Dwight T. Vines Based on averaged GH response curve areas, there was little day to day variation in pituitary responsiveness to TRH. However, because of animal variation, averaged GH response curve areas are misleading. This is demonstrated by the low repeatability coefficient (.35) for GB response curve areas following TRH. Similarly, the day to day repeat- ability coefficient for baseline GH concentration was low (.37). Correlation coefficient between baseline GH and post-TRH maximum GH concentrations was .60 (P<.01). Similar to prolactin, maximum GH con- centrations after TRH on a given day are associated with baseline GH concentrations. Routes of administration (IV, intramuscular (IM) and subcutaneous (sc)).05) following IV or SC administration of TRH. Following IV administration of TRH, serum prolactin and GH concentrations reached peak values more rapidly but declined more precipitously than following IM or SC administration of TRH. Therefore, when a prolonged hormonal response is desired, the SC or IM route of administration of TRH should be used. Dose of TRH affected prolactin and GH response curve areas in six prepubertal heifers. The quantities (post-TRH response curve areas) of prolactin (1515 to 3940 ng ml-lmin) and GH (53 to 667 ng ml—lmin) increased linearly (P<.Ol) with increases in the log of the dose (5 to 100 ug) of TRH. Peak serum prolactin concentrations increased from an averaged pre-TRH concentration of 39 ng/ml to values of 143 to 277 ng/ml Dwight T. Vines after TRH. Maximum quantities of prolactin released were achieved with 50 ug TRH. Pre-TRH GH concentrations averaged 5 ng/ml and increased to values of 11 to 46 ng/ml following TRH administration. Unlike prolactin, the largest GH response curve areas were attained using 100 pg TRH, and it is possible that a larger dose of TRH would result in still greater release of GH. Serum prolactin and GH response curve areas did not differ (P>.05) following administration of TRH on various days of the estrous cycle in six heifers. Peak serum prolactin and GH concentrations increased from averaged pre-TRH concentrations of 6 and 6 ng/ml to 70 and 21 ng/ml after TRH, respectively. Prolactin response curve areas, pre-TRH concentrations and maximum post-TRH concentrations at days 2 and 4 of the estrous cycle "tended" to be less than those at other stages of the estrous cycle. During pregnancy, prolactin response curve areas were not affected (P>.05) by stage of pregnancy following TRH administration in 26 heifers. However, there was a “tendency" for increased pre-TRH pro- lactin concentrations, maximum post-TRH prolactin concentrations and prolactin response curve areas with advancing pregnancy. Pre-TRH pro- lactin and GH concentrations averaged 14 and 4 ng/ml and increased to values of 152 and 11 ng/ml following TRH administration, respectively. Prolactin response curve areas were affected (P<.Ol) by season of year, being 6 to 15 X greater in summer than in fall or spring. Prolactin response curve areas were not affected (P>.05) by stage of lactation (2, 4, 6, 8 and 10 months) or month of pregnancy in 16 lactating cows following TRH administration. However, there "tended" to be greater prolactin release at the 2-month stage of lactation. Peak Dwight T. Vines serum prolactin and GH concentrations increased from averaged pre-TRH concentrations of 16 and 5 ng/ml to values of 234 and 16 ng/ml after TRH, respectively. Month of year affected (P<.Ol) prolactin response curve areas following TRH administration. GH response curve areas were greater (P<.05) at the 2-month stage of lactation when compared with other stages. These data suggest that the capacity of the pituitary to release prolactin and GH after administration of TRH is greater during early lactation and that this increased responsiveness may be associated with increased milk yields during early lactation. However, overall correlations between milk production (average of 5 days preceding TRH injections) and hormone response curve areas at the five stages of lac- tation were -.01 and .15 (P>.05) for prolactin and GH, respectively. Neither month of pregnancy nor month of year affected (P>.05) GH response curve areas following TRH administration to pregnant heifers or lactating cows. In overview, season of the year is more important in determining serum prolactin, but not GH, concentrations than are any of the differ- ent physiological states studied. Furthermore, physiological states which I examined have relatively little effect on basal or TRH-induced releases of serum prolactin or CH. SERUM PROLACTIN AND GROWTH HORMONE CONCENTRATIONS FOLLOWING THYROTROPIN RELEASING HORMONE IN CATTLE DURING VARIOUS PHYSIOLOGICAL STATES BY \, (L. at LC“ h Dwight T. Vines A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Dairy Science 1976 PERSONAL NOTE As man ventures forth, let not his brother be overly critical; yet, let man heed his brother's advice and suggestions. For man is not an island, but a part of the body of mankind who must contribute to mankind's knowledge of life or life will surely die. Let man also honor his father and mother for without them he would not be. I pay tribute to my parents' many sacrifices and their encouragement and hereby dedicate this thesis to them. ii ACKNOWLEDGMENTS The author wishes to express his appreciation to the Department of Dairy Science for providing funds and facilities for his graduate studies. A debt of gratitude is extended to Drs. J. H. Britt, H. D. Hafs, R. Mellenberger and W. D. Oxender for their advice throughout my graduate program. He also would like to express his appreciation for the advice and support of his committee members, Drs. L. Bieber, J. Gill, G. Riegle and ex officio member, E. M. Convey. He expresses a particular debt of gratitude to his major professor, Dr. H. A. Tucker, for his advice, understanding and perseverance in times of difficulty and indecision and for his assistance in preparing this thesis. The author wishes to express a great debt of gratitude to Dr. R. Neitzel for his computer programming assistance and to his fellow graduate students for all-around assistance. iii LIST OF TABLES. LIST OF FIGURES INTRODUCTION REVIEW OF LITERATURE. TABLE OF CONTENTS Prolactin and Growth Hormone Requirements for Lactation Prolactin and Growth Hormone Response to Milking, Suckling and Test Stimulation . . . . . . . Prolactin and Growth Hormone Response Following TRH. Seasonal Effects on Serum Prolactin and Growth Hormone. Prolactin and Growth Prolactin and Growth Prolactin and Growth Hypothalamic Control Hormone Secretion MATERIALS AND METHODS A. Animals . . . Hormone Prior to Puberty. . . Hormone During the Estrous Cycle Hormone During Pregnancy. . . of Prolactin and Growth B. Cannulation, Blood Sampling and Processing Procedures C. Hormone Assays. D. Experimental Procedures and Objectives. . . . Experiment 1. Prolactin and Given Dose of Experiment 2. Repeatability of Changes in Serum GH Concentrations in Response to a TRH . . . . . . . . . . . Effects of Various Doses of TRH on Serum Prolactin and GH. . . . . . . . . Experiment 3 . Effects of Various Doses of TRH Via Different Routes of Administration on Serum Prolactin and GH. . . . . . . . . . . . iv Page vi viii 23 23 23 24 24 24 25 26 Page Experiment 4. To Determine the Effects of an Increased Dose of TRH Via Different Routes of Administration on Serum Prolactin and GH. . . . . . . . . . . . . . 27 Experiment 5. Effect of TRH on Serum Prolactin and GH During the Estrous Cycle . . . . . . . . . . . . 27 Experiment 6. Effect of TRH on Serum Prolactin and GH During Pregnancy. . . . . . . . . . . . . . . 28 Experiment 7. Effect of TRH on Serum Prolactin and GH During Lactation. . . . . . . . . . . . . . . 28 E. Statistical Methods . . . . . . . . . . . . . . 29 RESULTS AND DISCUSSION . . . . . . . . . . . . . . . 31 Experiment 1. Repeatability of Changes in Serum Prolactin and GH Concentrations in Response to a Given Dose of TRH . . . . . . . . . . . . . . . . 31 Experiment 2. Effects of Various Doses of TRH on Serum Prolactin and GH . . . . . . . . . . . . . 33 Experiment 3. Effects of Various Doses of TRH Via Different Routes of Administration on Serum Prolactin and GH . . . . . . . . . . . . . . . . 36 Experiment 4. To Determine the Effects of an Increased Dose of TRH Via Different Routes of Administration on Serum Prolactin and GH . . . . . . . . 44 Experiment 5. Effect of TRH on Serum Prolactin and GH During the Estrous Cycle . . . . . . . . . . . 46 Experiment 6. Effect of TRH on Serum Prolactin and GH During Pregnancy . . . . . . . . . . . . . . 50 Experiment 7. Effect of TRH on Serum Prolactin and GH During Lactation . . . . . . . . . . . . . . 55 GENERAL DISCUSSION . O O O O O O C O O O O C O O O 64 SUMMARY AND CONCLUSIONS. . . . . . . . . . . . . . . 70 BIBLIOGRAPHY O O O O O O O O O O O O O O O O D O 76 Table 10. 11. LIST OF TABLES Time of sampling relative to thyrotropin releasing hormone (TRH) administration. . . . . . . . . . Serum prolactin (PRL) and growth hormone (GH) responses to various doses of thyrotropin releasing hormone (TRH). . . . . . . . . . . . . . . Serum prolactin (PRL) responses to doses of thyrotropin releasing hormone (TRH) and routes of administration. . Serum growth hormone (GH) responses to doses of thyrotropin releasing hormone (TRH) and routes of administration . . . . . . . . . . . . . . Serum prolactin and growth hormone (GH) responses to doses of thyrotropin releasing hormone (TRH) and route Of administration 0 O O O O O O O O O O O 0 Serum prolactin response to thyrotropin releasing hormone (TRH) during the estrous cycle . . . . . . Serum growth hormone (GH) response to thyrotropin releasing hormone (TRH) during the estrous cycle . . . Serum prolactin response to thyrotropin releasing hormone (TRH) during pregnancy . . . . . . . . . Serwm prolactin and growth hormone responses in pregnant heifers to thyrotropin releasing hormone (TRH) during seasons 0 O O O I O O I O O O O O O O O 0 Serum growth hormone (GH) response to thyrotropin releasing hormone (TRH) during pregnancy. . . . . . Serum prolactin and growth hormone (GH) responses to thyrotropin releasing hormone (TRH) on two consecutive days during lactation . . . . . . . . vi Page 25 35 37 41 45 47 49 51 51 54 56 Table 12. 13. 14. 15. 16. Page Serum prolactin response to thyrotropin releasing hormone (TRH) during lactation. . . . . . . . . . 57 Serum prolactin and growth hormone responses to thyrotropin releasing hormone (TRH) during seasons . . . . . . . . . . . . . . . . . 57 Serum prolactin (PRL) response to thyrotropin releasing hormone (TRH) during lactation, pregnancy and month of year. . . . . . . . . . . 59 Serum growth hormone (GH) response to thyrotropin releasing hormone (TRH) during lactation . . . . . . 61 Serum GH response to thyrotropin releasing hormone (TRH) during lactation, pregnancy and month of year . . . . . . . . . . . . . . 62 vii LIST OF FIGURES Figure Page 1. Serum prolactin following IV, IM or SC administration of 25 ug TRH in prepubertal heifers. . . . . . . . . . . . 2. Serum growth hormone following IV, IM or SC administration of 25 ug TRH in prepubertal heifers. . . . . . . . . . . . viii INT RODUCT I ON Milk and dairy products are excellent sources of protein, fats, carbohydrates, minerals and vitamins. With an ever increasing world population that has already surpassed four billion, sources of high quality foodstuffs are of prime importance. But presently many of the world's population consume little or no dairy foodstuffs, due to their high cost or unavailability. One of the factors necessary to make more dairy foodstuffs available is more efficient production of these foodstuffs. In the recent past, the approach to increasing production or the efficiency of production was through either more efficient feeding and management or improvement of the milking potential through genetic selection. Both of these approaches have been worthwhile, since milk production per cow continues to increase each year. However, only very recently has the endocrinological approach to increasing production come to the forefront. Today, we know that growth, reproduction, and lactation are under control of the endocrine system. Prolactin and growth hormone in laboratory species have been implicated as part of a complex of hormones which are essential for mammary growth, lactogenesis and maintenance of lactation. However, their role in lactation in the bovine is still unclear. Therefore, the purpose of these studies was to determine the pituitary's responsive- ness, as measured by serum concentrations of prolactin and growth hormone, to exogenous administration of a naturally occurring hypotha- lamic releasing factor during various physiological conditions. This information should give us insight into the control of prolactin and growth hormone secretion and hopefully produce information that can be applied subsequently to increase milk production. REVIEW OF LITERATURE Prolactin and Growth Hormone Requirements for Lactation Pioneering experiments by Stricker and Grueter (1928) demon- strated that injection of aqueous extracts of anterior pituitaries induced milk secretion in pseudopregnant rabbits. This report suggested that some hormones of the anterior pituitary may be important in the initation of lactation. Following the identification and isolation of prolactin (Riddle et a1., 1933) and growth hormone (Li and Evans, 1944), many investiga- tions were begun to ascertain the function of these two hormones with regard to lactation. Daily injections of sheep prolactin into hypophysectomized rabbits induced milk secretion (Fredrikson, 1939). Lyons (1942) confirmed these results when he demonstrated that a purified ovine anterior pituitary preparation, capable of inducing localized proliferation of the pigeon crop-sac mucosa, initiated milk secretion following injection into a single galactophore of the rabbit mammary gland. Kilpatrick et a1. (1964) also induced lactation with ovine prolactin injection in hypophysectomized, pseudopregnant rabbits. However, prolactin injections alone are not capable of inducing lacta- tion in rats (Talwalker et a1., 1961) or mice (Nandi and Bern, 1961). Cowie (1969) reviewed his work and that of others and concluded that either prolactin or GH in combination with adrenocorticotropic hormone (ACTH) or adrenal corticosteroids would induce lactation in hypophy- sectomized, adrenalectomized and ovariectomized rats and mice. Prolactin has not been shown to be galactopoietic in dairy cows or ewes (Folley and Young, 1940, Sulman and Twersky, 1948, Wrenn and Sykes, 1953, Delouis and Denamur, 1967 and Morag et al., 1971). However, Gotsulenko (1968) observed increased lactational performance in goats following injection of prolactin into the arterial system of the mammary gland. Following injection of ergot alkaloids, which block prolactin release from the pituitary, lactation was depressed in rabbits (Taylor and Peaker, 1976) and rats (Shaar and Clemens, 1972 and Tomogane et al., 1975). However, milk yields were not affected by ergot drugs during established lactation in goats (Hart, 1973) or cows (Karg et al., 1972 and Smith et al., 1974) although serum prolactin concentrations were markedly depressed. Koprowski and Tucker (1973) reported a significant correlation between the magnitude of prolactin released to the milking stimulus and milk production in cows which suggests that prolactin released at milking may influence subsequent milk yields as has been reported for rats (Grosvenor and Mena, 1973). Cowie (1969) reported that prolactin, GH, corticoids and tri— iodothyronine were necessary for complete restoration of lactation in hypophysectomized goats, although prolactin alone was slightly lacto- genic. However, removal of prolactin following restoration of lactation had no deleterious effects on the subsequent lactation, but removal of GH resulted in an immediate decline in milk yield. Furthermore, administration of GH enhanced lactational performance in cows (Cotes et al., 1949, Donker and Peterson, 1951, Chung et al., 1953, Wrenn and Sykes, 1953, Bullis et al., 1965 and Machlin, 1973). Prolactin and Growth Hormone Response to Milking, Sucklingxand Teat Stimulation Milking, suckling and teat or udder manipulation have been associated with increased serum prolactin concentrations. Bryant et a1. (1968) reported a three-fold or more increase in serum prolactin follow— ing milking in goats. Johke (1970) reported a 40- to lOO—fold increase in serum prolactin associated with the milking stimulus in goats. The suckling stimulus released prolactin in ewes (Lamming et al., 1972 and McNeilly et al., 1972) and cows (Fell et al., 1971). Teat stimulation or udder manipulation resulted in prolactin release in cows (Johke, 1970, Fell et al., 1971, Tucker, 1971, Schams, 1972 and Reinhardt and Schams, 1975) and goats (Johke, 1970). Reinhardt and Schams (1975) reported a 3— to 10-fold increase in serum prolactin in four heifers following a 3 minute stimulation of two teats. Johke (1970) reported a rapid increase in serum prolactin associated with the milking stimulus in cows. Peak concentrations were reached 4 to 20 minutes after the start of milking. Prolactin release in response to the milking stimulus decreased with advancing stages of lactation. Tucker (1971) also reported a rapid increase in serum prolactin associated with the milking stimulus. Like Johke (1970), Koprowski and Tucker (1973) found a diminished prolactin response to milking in late lactation. Thyrotropin releasing hormone (TRH) releases prolactin in lactating cows (Convey et al., 1973 and Kelly et al., 1973). Tucker et a1. (1975) reported a rapid increase in serum prolactin with peak concentrations attained 30 minutes after the beginning of a constant infusion of TRH in lactating cows. However, serum prolactin concentra- tions declined from peak values during 6 to 13 hours of continuous TRH infusion. In addition, Reinhardt and Schams (1975) reported eventual declines in serum prolactin concentrations from peak concentrations under continuous TRH infusion in heifers. However, when the milking stimulus (Tucker et al., 1975) or teat stimulation (Reinhardt and Schams, 1975) was superimposed on declining serum prolactin concentrations during continuous TRH infusion, markedly increased serum prolactin con- centrations were re-established. One possible explanation for these data is that milking or teat stimulation probably act through the hypothalamus to release prolactin while TRH probably acts directly on the pituitary to release prolactin. Therefore, the diminished prolactin response to continuous infusion of TRH may be due to refractoriness of the pituitary to TRH while the prolactin response to milking or teat stimulation is unaffected. Tucker (1971) reported lack of a GH surge after application of the milking stimulus in cows. Koprowski and Tucker (1973) confirmed these results when they were unable to detect a GH surge after applica- tion of the milking stimulus for the duration of lactation in 26 cows. However, Hart and Flux (1973) reported increased serum GH in goats in response to the milking stimulus. This difference in response may be due to species variability. Prolactin and Growth Hormone Response Following TRH Isolation of porcine TRH (Schally et al., 1969) was followed quickly with elucidation of its structure (Nair et al., 1970) and with synthesis in the laboratory (Boler et al., 1969). Tashjian et a1. (1971) first reported release of prolactin from cloned rat pituitary tumor cells in culture following TRH. These data suggested to me that TRH might be used as an exogenous and physiological stimulus, since TRH is a naturally occurring hypothalamic releasing factor, to evaluate the capacity of the anterior pituitary to release prolactin. Jacobs et a1. (1971) reported an 11-fold increase in serum prolactin over fasting levels in humans following TRH. Maximal values were reached 15 to 20 minutes post-TRH. Bowers et al. (1971) also reported increased serum prolactin following TRH in humans. These early reports were confirmed by several researchers (Friesen et al., 1972, L'Hermite et al., 1972, Bowers et al., 1972, Gautvik et al., 1973, Hershman et al., 1973 and Noel et al., 1974). Administration of TRH to rats increased serum prolactin (Deis and Alonso, 1973, Mueller et al., 1973, Blake, 1974 and Porteus and Malven, 1974). However, Lu et a1. (1972) reported no increase in serum prolactin following administration of 50 ug TRH for six days although there was a significant increase in pituitary prolactin content. Although these results are contradictory, the increase in pituitary Prolactin content may indicate that TRH enhances prolactin synthesis. Following administration of TRH, serum prolactin increased in sheep (Davis and.Borger, 1972, Debeljuk et al., 1973, Fell et al., 1973 and Moseley et al., 1973) and in cattle (Schams, 1972a, Convey et al., 1973 and Kelley et al., 1973). Takahara et a1. (1974) observed increased serum GH in rats following infusion of TRH into a hypophysial portal vessel. Udeschini et a1. (1976) also reported increased serum GH following TRH in hypophysectomized rats bearing an ectopic pituitary. Convey et a1. (1973) observed that GH increased proportionally to the dose of TRH in lactating cows, whereas, Davis (1975) reported no change in serum GH after TRH in immature ewe lambs. These differences are confusing but may be explained on the basis of species and/or age differences. In humans, GH responses following TRH have been quite variable. In acromegalic humans, serum GH increased following TRH (Saito et al., 1971, Irie and Tsushima, 1972, Cryder et al., 1973 and Faglia et al., 1973). Also, serum GH increased following TRH in patients with depres- sion (Maeda et al., 1975) or anorexia nervosa (Maeda et al., 1976). However, in normal healthy humans the serum GH response to TRH is confusing. Torjesen et a1. (1973) reported increased serum GH following TRH administration to women, but not men. In contrast, Anderson et al. (1971), Saito et al. (1971), Irie and Tsushima (1972) and Maeda et a1. (1975) reported no change in serum GH following TRH in normal healthy humans. In view of the ability of TRH to stimulate prolactin and GH release, this hypothalamic releasing factor has been tested for galacto- poietic activity. For example, Tyson et a1. (1972) reported breast engorgement and bilateral milk letdown in women approximately 2.5 hours after TRH injection. Prior to TRH administration, breast engorgement did not occur in any of the breast-feeding mothers. Convey et a1. (1973) reported an increase of 0.66 kg/cow/day in milk yield in 20 cows receiving TRH. In contrast, Kelley et al. (1973) and Schams et a1. (1974) observed no increase in milk production following TRH in lactat- ing cows although few cows were involved in each experiment and doses of TRH were different. In addition, Adams et a1. (1973) detected no differences in litter weight gain in rats after TRH administration to lactating mothers when compared with litter weight gain in control rats. However, when TRH administration began prior to parturition in cattle, a 10 to 30 percent increase in milk yield occurred during the first 20 days of lactation when compared with the same period for the previous year (Schams et al., 1974). Seasonal Effects on Serum Prolactin and Growth Hormone Several researchers have reported a seasonal effect on serum prolactin concentrations in cattle (Koprowski and Tucker, 1973, Schams and Reinhardt, 1974, Thatcher, 1974, Tucker et al., 1974 and Buttle and Forsyth, 1976) and goats (Buttle, 1974 and Hart, 1975). Serum prolactin is highest in summer and lowest in winter months in these species. The available data suggest that illumination and temperatures are important factors in the manifestation of seasonal cyclicity of serum prolactin. Relkin (1972) observed the effects of diurnal lighting, constant lighting or constant darkness on serum and pituitary prolactin in rats. Constant lighting for 7 days resulted in a 67 percent increase in pitui- tary prolactin when compared with diurnal lighting; on the other hand, serum prolactin was suppressed 25 percent. Constant darkness resulted in increased serum prolactin, but decreased pituitary prolactin. In contrast, Reiter (1975) found in hamsters a 50 percent decrease in serum 10 prolactin after 56 days of near constant darkness (light: dark cycles of 1 hour light and 23 hours darkness) when compared with light: dark cycles of 14 hours light and 10 hours darkness. In cattle, shortening of the photoperiod from 16 to 8 hours over a 12-week interval decreased serum prolactin from a maximum of 57 ng/ml to a minimum of 8 ng/ml. In contrast, lengthening of the photo- period from 8 to 16 hours of light increased serum prolactin from 25 to 100 ng/ml (Bourne and Tucker, 1975). In rams, serum prolactin concen- trations were 16 times greater in rams exposed to 16 hours of daylight as compared with rams exposed to 8 hours of daylight (Pelletier, 1973). Furthermore, Forbes et al. (1975) found an increase in serum prolactin following exposure of castrated male lambs to 16 hours of daylight as compared with 12 hours of daylight in control lambs. Temperature has also been shown to play a role in the regulation of prolactin in several species. Mueller et a1. (1974) reported a 5- to lO-fold increase in serum prolactin following exposure of male rats to 40 C for 30 minutes. Exposure of rats to 4 C for l to 2 hours resulted in a significant decrease in serum prolactin when compared with control rats maintained at 24 C. In contrast, Jobin et a1. (1975) reported an increase in serum prolactin in rats after exposure to 5 C for 32 days. These data are in direct conflict and further research is needed to clarify these results. In heifer calves, a reduction of ambient temperature from 21 to 10 C over a 4-hour period decreased serum prolactin from 13 to 4 ng/ml (Wettemann and Tucker, 1974). When the temperature was increased from 21 to 27 C over a 3-hour period, serum prolactin increased from 8 to 22 ng/ml. Also, chronic exposure to 10 or 27 C for 5 days decreased or 11 increased serum prolactin, respectively, when compared with control values (21 C for 5 days). This early report was confirmed in a second set of experiments by the same authors (Tucker and Wettemann, 1976) using more extreme temperatures. In addition, the expected release of prolactin into serum following TRH administration was suppressed at 4.5 C. Mueller et a1. (1973) reported that plasma GH concentrations were decreased by 70 percent following exposure of rats to 40 C for 1 hour. In contrast, plasma GH concentrations were not significantly altered following exposure of rats to 4 C for l or 2 hours. Tucker and Wettemann (1976) found no significant differences in serum GH in heifer calves exposed to 4.5, 21 or 32 C for nine days although GH tended to increase with increasing temperatures. Serum GH concentrations in lactating cows (Koprowski and Tucker, 1973) or bulls (Tucker et al., 1974) were not affected by the season of the year. Prolactin and Growth Hormone Prior to Puberty, Serum and pituitary prolactin increase markedly with the onset of puberty in rats. For example, in prepubertal female rats Minaguchi et a1. (1968) reported uniformly low pituitary prolactin values at 21, 26 and 31 days of age. However, pituitary prolactin content markedly increased shortly after the onset of puberty. VOogt et a1. (1970) also found low pituitary prolactin values in prepubertal female rats prior to vaginal opening. In addition, serum prolactin concentrations were low prior to vaginal opening, but increased markedly on the day of vaginal opening. Also, serum prolactin concentrations were reduced in 12 prepubertal male rats prior to the beginning of sexual development (Negro-Vilar et al., 1973, Dohler and Wuttke, 1974 and Dohler and Wuttke, 1975). In male lambs, Ravault and Courot (1975) observed relatively stable serum prolactin concentrations from 4 to 9 weeks of age. A rapid increase in serum prolactin was noted at 10 to 12 weeks of age which occurred simultaneously with the beginning of a rapid increase in testicular weight and spermatogenic activity. Sinha and Tucker (1969) reported that pituitary prolactin content increased 333 percent between birth and 3 months of age in heifers with maximum values at 9 months which synchronized with changes in mammary development. Armstrong and Hansel (1956) observed higher pituitary GH con- tent in heifers at birth to one and 16 weeks of age when compared with postpubertal heifers. Similarly, in bull calves, pituitary GH content was greater at 2 and 4 months of age as compared with postpubertal values (Purchas et al., 1970) and these increased pituitary concentra- tions in prepubertal cattle probably reflected increased synthesis and release of GH because plasma GH concentrations were higher at birth but decreased to relatively stable values between 2 and 12 months of age. Prolactin and Growth Hormone During the Estrous Cygle Exogenous estrogen and progesterone enhance pituitary and serum prolactin concentrations. Reece and Turner (1937) reported that exogenous estrogen increased pituitary prolactin content in rats, and Nicoll and Meites (1962) found that estrogen increased prolactin release from rat pituitary explants ig_gi£xg, Chen and Meites (1970) reported that exogenous estrogen increased pituitary and serum prolactin 13 concentrations in ovariectomized rats. Pituitary and serum prolactin concentrations were greater in rats at proestrus and estrus when com- pared with diestrus (Sar and Meites, 1967, Amenomori et al., 1970, VOogt et al., 1970 and Ieiri et al., 1971) which was coincidental with increased plasma estrogen concentrations (Yoshinaga et al., 1969). The mechanism of action involved in increased serum and pituitary prolactin concentrations associated with increased plasma estrogen concentrations is unknown. However, hypothalami from rats killed during proestrus and estrus contained significantly less prolactin-inhibiting factor (PIF) than hypothalami taken from diestrus rats (Ratner and Meites, 1964 and Sar and Meites, 1967). These data suggest that estrogen suppresses PIF synthesis and/or secretion in the hypothalamus. In the ewe, Reeves et a1. (1970) reported significantly higher serum prolactin concentrations at proestrus and first day of estrus when compared with other stages of the estrous cycle. Bryant et a1. (1971) and Davis et a1. (1971) also reported increased serum.prolactin concentrations in ewes on the day of estrus. Koprowski and Tucker (1973) and Wettemann and Hafs (1973) found no difference in serum prolactin concentrations at various stages of the estrous cycle in lactating cows or in heifers. In contrast, increased pituitary prolactin concentrations have been reported for cattle during proestrus and estrus (Sinha and Tucker, 1969). Also, Swanson et a1. (1972) reported increased serum prolactin concentrations around estrus. These data are confusing but the more extensive study by Koprowski and Tucker (1973) would seem to indicate that serum prolactin concentrations are variable during the estrous cycle but there is no particular pattern associated with different stages of the cycle. 14 Although serum prolactin was suppressed slightly during infusion of l7B-estradiol in cows, this hormone increased following cessation of the infusion (Schams and Karg, 1972). In contrast, Beck et a1. (1976) found no differences in serum prolactin in ovarietomized heifers with or without estrogen implants. These authors suggested that estrogen did not influence serum prolactin when present in blood at concentrations comparable to those found during the normal estrous cycle. As reported above, serum prolactin concentrations in many species are diminished during the luteal phase of the cycle when serum progesterone concentrations are increased. Reece and Bivens (1942) measured pituitary prolactin content in ovariectomized rats following estrogen, progesterone, and estrogen-progesterone injections. Progeste- rone increased prolactin content of the pituitary; however, estrogen was more effective in augmenting the prolactin content. Estrogen- progesterone augmentation of pituitary prolactin was intermediate between progesterone and estrogen in effectiveness. Sar and Meites (1968) reported that progesterone increased pituitary prolactin content by 51 percent over controls in ovariectomized rats. Hypothalamic PIF content was suppressed in the progesterone injected rats as compared with controls. Chen and Meites (1970) also reported increased pituitary and serum prolactin concentrations following progesterone injections in rats. These data suggest that progesterone may suppress PIF and thus increase pituitary and serum prolactin concentrations. In bulls, infusion of 5 to 80 mg of progesterone during a l-hour Period resulted in as much as a 20-fold increase in serum prolactin (Schams et al., 1974). However, in ovariectomized heifers with a 15 progesterone pessary, serum prolactin concentrations were not different from concentrations in control heifers (Beck et al., 1976). The physiological role of prolactin during the estrous cycle is still unknown in many species. Although serum prolactin concentrations are increased at proestrus and estrus, blockage of the pre-ovulatory prolactin surge with ergot drugs did not prevent normal ovulation from occurring in ewes (Kann and Denamur, 1974), nor did it affect normal corpora lutea function or estrous cycle length (Niswender, 1974). Changes in estrogen concentrations may also influence GH con- centrations. Serum and pituitary GH concentrations were greater at proestrus and estrus in rats (Dickerman, 1971) and mice (Sinha et al., 1972) when compared with diestrus. In contrast, Ieiri et a1. (1971) reported that GH synthesis and release did not change significantly during the estrous cycle in rats. These data are confusing and further research is needed to clarify the relationship between estrogen and GH during the estrous cycle in rats. In humans, increased GH concentrations were noted during the ovulatory and pre-menstrual phases in women (Spellacy et al., 1969). Koprowski and Tucker (1973) reported increased serum GH during estrus in lactating cows. Administration of diethylstilbesterol increased serum GH in steers (Trenkle, 1970) and rats (Lloyd et at., 1973). However, in ovariectomized heifers, Beck et a1. (1976) found no differences in serum GH between heifers with and without estrogen implants. Davis and Borger (1974) reported that the injection of progeste- rone appeared to enhance the secretion of GH in ovariectomized ewes. 16 But in ovariectomized heifers, Beck et a1. (1976) reported that serum GH concentrations were not significantly changed by progesterone administration. Prolactin and Growth Hormone Durixg Pregnancy The function of prolactin during pregnancy in cattle is unknown. Wettemann and Hafs (1971) found that serum prolactin did not change significantly during the first 75 days of pregnancy. Although serum prolactin concentrations were not significantly different at 90, 180 and 260 days of gestation, serum prolactin tended to increase at the third trimester of pregnancy (Oxender et al., 1972). Furthermore, numerous researchers have observed increases in pituitary and serum prolactin concentrations shortly before parturition which may be associated with initiation of lactation. Bates et a1. (1935) and Reece and Turner (1937a) reported that pituitary prolactin content increased during late pregnancy in cattle. Johke et a1. (1971) found that serum prolactin ranged from 8.3 to 19.6 ng/ml during 4 to 55 days prepartum in cows. However, on the day before parturition, serum prolactin increased to a maximum of 137 ng/ml, but it declined shortly after parturition. Schams and Karg (1970) and Ingalls et a1. (1973) also observed increased serum prolactin just prior to parturition in cows and heifers. In the ewe, McNeilly (1971) observed increased serum prolactin concentrations during the last third trimester of pregnancy. In con- trast, Arai and Lee (1967) reported a gradual decline in prolactin during advancing pregnancy. Davis et a1. (1971) also reported decreasing or low serum prolactin in ewes during advancing pregnancy, but serum 17 prolactin began to increase gradually 3 to 5 weeks prior to parturition with a dramatic increase 3 days prior to parturition. Burd et a1. (1976) also reported a dramatic increase in serum prolactin at parturition. In the goat, serum prolactin increased just prior to parturition (Johke et al., 1971). Although these data are confusing, perhaps because of con- founding seasonal effects, the bulk of the data indicate that serum prolactin concentrations increased during late pregnancy. Serum GH concentrations did not change significantly during pregnancy in cows (Oxender et al., 1972). Ingalls et al. (1973) found no change in serum GH for the interval 26 to 9 days before parturition. However, serum GH increased as parturition neared and was maximal at parturition. In contrast, Koprowski and Tucker (1973) reported a linear increase, although quantatively small, in serum GH with advancing pregnancy. Plasma GH was relatively low in the ewe throughout pregnancy (Bassett et al., 1969). Schalch and Reichlin (1966) and Dickerman (1971) observed no change in serum GH concentrations throughout pregnancy in the rat. Collectively, these data suggest that serum GH concentrations remain relatively stable throughout pregnancy but may increase around parturition. Hypothalamic Control of Prolactin and Growth Hormone Secretion Regulation of prolactin secretion is exerted mainly via the hypothalamus, and this appears to involve the actions of PIF, a possible prolactin-releasing factor (PRF), catecholamines, serotonin or perhaps other biogenic amines- all produced in the hypothalamus (Meites and Clemens, 1972). Early studies by Everett (1954) and Nikitovitch-Winer and Everett (1958) indicated that transplantation of the pituitary 18 underneath the kidney capsule resulted in continuous and increased pro- lactin secretion, as indicated by prolonged maintenance of luteal func- tion in rats. Transplantation of l to 4 heterologous anterior pituitaries underneath the kidney capsule of female hypophysectomized- ovariectomized rats, resulted in continuous prolactin release during 10 weeks (Chen et al., 1970). Rats with no pituitary grafts had barely detectable concentrations of serum prolactin while increasing the number of pituitary transplants per kidney increased serum prolactin concentrations. These observations were the initial bases for postulating the long-term control of prolactin secretion by the hypothalamus was inhibiting, and that the hypothalamus secretes a PIF. Placement of bilateral lesions in the median eminence, or anterior or posterior hypothalamus of ovariectomized or intact rats resulted in significant increase of serum prolactin above control values (Chen et al., 1970 and Welsch et al., 1971). This suggests that all of these areas participate in regulation of prolactin secretion. Halasz et a1. (1962) have defined this region as the "hypophysiotropic area" and it is believed to control basal secretion of anterior pituitary hormones. Transection of the hypophysial stalk interrupts anterior pituitary blood supply from the hypothalamus. Kanematsu and Sawyer (1973) observed increased plasma prolactin after hypophysial stalk section in rats and since prolactin secretion is enhanced when the pituitary is transplanted to the kidney or following hypothalamic lesions, this further substantiated the presence of a hypothalamic inhibiting factor. Talwalker et a1. (1963) reported decreased prolactin release from rat pituitary incubates after addition of crude hypothalamic l9 extracts. Hypothalamic extracts from sheep, cattle, pigs and humans also were demonstrated to inhibit release of prolactin (Schally et al., 1967). Infusion of hypothalamic extracts into pituitary portal vessels decreased plasma prolactin concentrations in rats while increasing plasma concentrations of luteinizing hormone (LH) and follicle stimu- lating hormone (FSH) (Kamberi et al., 1971). In addition, the prolactin responses to hypothalamic extracts were related to the concentration of the extract in a negative-dose response manner. Takahara et a1. (1974) observed suppressed serum prolactin following the infusion of a purified porcine PIP preparation via a hypophysial portal vessel in the rat. This purified PIF preparation contained a high content of catecholamines. The mechanism of action of PIF is still unknown. Ca++ ions have been shown to be essential for prolactin release from the pituitary £2. yiExg_(Parsons, 1969). Nicoll (1971) suggested that PIF acts on the prolactin secreting cell membrane to inhibit Ca++ influx. Parsons (1969) suggested that the cell depolarizes spontaneously when it is freed from hypothalamic influence, with a resultant increase in Ca++ entry into the cell and consequent release of secretory granules. Thus, PIF may act by preventing spontaneous depolarization of the cells which secrete prolac- tin. Kamberi et a1. (1971) reported decreased serum prolactin follow- ing the injection of dopamine into the third ventricle of the rat. Serum prolactin was suppressed by 30, 53 and 58 percent at 10, 20 and 30 minutes, respectively, following dopamine injections when compared with pre-injection prolactin concentrations. However, by 120 minutes post-injection, serum prolactin was suppressed by only 8 percent. Epinephrine and norepinephrine also suppressed serum prolactin following 20 intraventricular injection. In contrast, dopamine, epinephrine and norepinephrine, perfused into a hypophysial portal vein, failed to alter serum prolactin. These findings suggest that neither dopamine, epinephrine nor norepinephrine affect prolactin release by direct action on the anterior pituitary but indirectly through the hypothalamic- hypophysial complex. In agreement with these results were the findings that systemic administration of L-dopa suppressed serum prolactin con- centrations, increased hypothalmic PIF content and resulted in prolactin-inhibiting activity in the serum of rats (Lu and Meites, 1972). Shaar and Clemens (1974) observed that rat hypothalamic extracts significantly inhibited pituitary prolactin release 32.23339, These authors further indicated that the prolactin inhibiting activity of hypothalamic extracts can be totally accounted for by the amount of endogenous catecholamines in the hypothalamus. The evidence for the possible existence of PRF is much less convincing than that for PIF. Several researchers have reported the possible existence of PRF (Meites et al., 1960, Mishkinsky et al., 1968 and Nicoll et al., 1970). However, various other researchers have cast doubts on the existence of PRF. In contrast to the inhibiting role of catecholamines on prolac- tin secretion, the biogenic amine, serotonin, has been shown to increase serum prolactin. Kamberi et a1. (1971) reported that injection of serotonin and melatonin into the third ventricle of the brain of male rats stimulated release of prolactin. In addition, neither melatonin nor serotonin infusion into the anterior pituitary resulted in a significant alteration of plasma prolactin. Lu and Meites (1972) reported increased serum prolactin following the injection of 21 tryptophan, 5-hydroxy-tryptophan or melatonin into rats. Serotonin failed to alter serum prolactin but this may be related to its inability to pass the blood-brain barrier. The pineal gland has been implicated recently in the regulation of prolactin secretion (Reiter, 1974 and Blask and Reiter, 1975). White et a1. (1974) reported that the pineal gland can act as a supplemental source of hypothalamic-releasing hormones. As reported previously, the hypothalamus contains TRH which can increase serum prolactin when given exogenously. Whether TRH plays a role in the regulation of prolactin under normal physiological conditions remains to be seen. The regulation of serum GH by the hypothalamus is less clearly understood than for prolactin. The secretion of GH from the pituitary is under the control of a hypothalamic GH-releasing factor (GRF) and GH-inhibiting factor (GIF) or somatostatin (SRIF). Deuben and Meites (1964) demonstrated for the first time that rat hypothalamic extracts promoted the released of GH from 6-day cultured rat pituitary glands. These results were confirmed by Schally et al. (1965) who reported that the addition of highly purified porcine GRH to rat anterior pituitaries incubated ix zigxg_stimu1ated the synthesis and release of GH. GH-inhibiting factor or somatostatin has been shown by several researchers to inhibit GH release from the pituitary. Krulich et a1. (1968) and Dhariwal et a1. (1969) reported the separation of GIF from other hypothalamic factors. Stachura et a1. (1972) observed that ovine hypothalamic fractions purified on Sephadex G—25 almost completely inhibited synthesis and release of GH in a rat pituitary ig_zi£xg_ system. Similarly, Brazeau et al. (1973) determined that a purified extract from sheep hypothalami inhibited GH release in rats. Brazeau 22 et a1. (1974) also reported that SRIF had an inhibiting effect on GH release induced by isoprenalin or chlorpromazine in rats. In general, catecholamines stimulate GH secretion while serotonin has no effect (Wilson, 1974). Mims et al. (1975) observed increased GH secretion following oral administration of L—dopa in humans. Similarly, serum GH concentrations were increased in Rhesus monkeys following infusion of L-dopa (Chambers and Brown, 1976). MATERIALS AND METHODS A. Animals Prepubertal heifers, 4 to 5 months of age and weighing 125 to 148 kg, were used in the first four experiments. In the fifth experi- ment, heifers exhibiting normal estrous cycles were used. Heifers that were either 3, 6 or 9 months pregnant were used in the sixth experiment. In the seventh experiment, primiparous and multiparous lactating cows were used. Prepubertal, cycling and pregnant heifers were maintained under loose housing conditions prior to start of experimentation with free access to feed and water. Lactating cows were maintained in stanchions with free access to water and were fed according to recom- mended practices for milk production. All cattle were of the Holstein breed. B. Cannulation, Blood Sampling and Processing Procedures All animals were fitted with indwelling polyvinyl jugular cannulae (VilO tubing, Bolab, Inc., Derry, N.H.) on the day prior to experimentation in experiments 1, 2, 3, 4, 6 and 7. In experiment 5, heifers were cannulated on the morning of estrus or one day prior to the first TRH injection on day 15 of the estrous cycle. Cannula were flushed with 3.5 percent sodium citrate containing 50 percent dextrose 23 24 dissolved in 0.85 percent NaCl solution. This solution was used to pre- vent coagulation of blood and thus maintain functionality of the cannula throughout the experimental period. Blood samples were collected at various intervals, depending on experimental protocol, before and after TRH injections. To condition animals to the blood sampling procedure and thereby establish stable baseline values for prolactin (Raud et al., 1971 and Tucker, 1971), blood samples were collected and discarded (not assayed for hormones) at lS-minute intervals for 2 to 3 hours prior to start of experimentation. Thereafter, blood samples were collected for hormone assay. Sera were prepared from the blood samples and stored at -20 C until assayed for prolactin and GH. C. Hormone Assays Serum hormone concentrations were quantified by double antibody radioimmunoassay (RIA) procedures as previously described for prolactin (Tucker, 1971 and Koprowski and Tucker, 1971) and GH (Purchas et al., 1970). D. Experimental Procedures and Objectives Experiment 1. Repeatability of Changes in Serum Prolactin and GH Concentrations in Response to a Given Dose of TRH Experimental Design.--Eight heifers received one intravenous (IV) injection of 10 pg TRH in 10 ml 0.85 percent NaCl on days 1, 2, 4 and 8. Heifers were selected on the basis of uniformity in age and weight. During experimentation, heifers were housed in individual tie stalls. Collections of blood and IV injections were via polyvinyl 25 jugular cannulae. Cannulae were Opened at 1000 hours with blood samples collected for hormones assay (table 1) starting at 1230 hours. Table 1.—-Time of sampling relative to thyrotropin releasing hormone (TRH) administration Experiment Pre-treatmenta Post-treatmenta 1 -30, -15, -10, -5’ O 4' 6' 8' 10' 12' 14' 16, 18, 20, 25, 30, 45, 60 2 arid 3 -30' -25, -20' -15, 4' 6' 8' 10' 12’ 14' -10, -5, O 16' 18' 20' 25' 30 4 -15' -10, -5, 0 4, 6' 8' 10' 15’ 20' 30, 40, 50' 60’ 70' 80, 90, 100, 110, 120 5 and 6 -15, -10, -5, 0 4' 6, 8' 10' 15' 20' 25, 3O 7 -30, -15, -5, O 4' 6' 8' 10' 12' 14’ 16, 18, 20, 25, 30 aMinutes relative to TRH administration Experiment 2. Effects of Various Doses of TRH on Serum Prolactin and GH Experimental Design.--The effects of various doses of TRH on serum prolactin and GH were measured in six heifers. Each heifer received IV injections in a sequence determined at random of either 10 ml 0.85 percent NaCl or 5, 10, 25, 50 or 100 pg TRH in 10 ml 0.85 percent NaCl during six consecutive days. However, only one heifer received a given dose on any day. Heifers were selected, housed, bled and injected as described previously in experiment 1. Cannulae were 26 opened at 0930 or 1030 hours with blood samples collected for hormone assay (table 1) starting at 1130 or 1230 hours. Experiment 3. Effects of Various Doses of TRH Via Different Routes of Administration on Serum Prolactin and GH Experimental Design.--The six heifers used in experiment 2 were employed subsequently to determine the effects of route of administra- tion of TRH on serum prolactin and GH concentrations during 30 minutes after TRH was given. An interval of 24 hours was allowed between the last injections of TRH or saline in experiment 2 and the start of experiment 3. On day one, two heifers were injected IV, two were injected intramuscularly (IM) and two were injected subcutaneously (SC) with one m1 of 0.85 percent NaCl. Between days 2 and 7, each heifer received, in a sequence determined at random, either 10 or 25 pg TRH in 1 ml 0.85 percent NaCl injected IV, Im or SC. Thus, each heifer received each dose of TRH (10 and 25 pg) via each route of administra- tion (IV, IM and SC). Each combination of dose and route of administra- tion was given only once on a given day. Injections of NaCl, via a different route of administration for each heifer, were repeated on day 8. However, since only 2 days were allowed for NaCl injections, each heifer received NaCl via only two of the three possible routes of administration. All other conditions and procedures were as stated in experiment 2. 27 Experiment 4. To Determine the Effects of an Increased Dose of TRH Via Different Routes of Administration on Serum Prolactin and GH Experimental Design.--Results from experiment 3 indicated that serum prolactin may not have reached maximum concentrations during 30 minutes after TRH administration, nor were the doses suffiCiently great to achieve concentrations of prolactin or GH which were as great as those in experiment 2. Thus, four heifers were injected IV or SC in random sequence during four consecutive days with either 1 ml 0.85 percent NaCl or 50 pg TRH in 1 ml 0.85 percent NaCl. However, no combination was administered more than once on a given day. Blood samples were collected for 2 hours after treatment (table 1). All other conditions and procedures were as stated in experiment 2. Experiment 5. Effect of TRH on Serum Prolactin and GH During the Estrous Cycle Experimental Desigp.--Six heifers, exhibiting normal estrous cycles, received IV injections of 33 pg TRH/100 kg body weight in a randomized, complete-block-repeat-measurement-design on days 0 (estrus), 2, 4, 7, 15 and 18 of the estrous cycle. Three heifers received their initial injection of TRH on day 0 and the other three were injected initially on day 15 of the estrous cycle. All heifers were observed for estrus during two estrous cycles prior to the start of experimenta- tion. During these two estrous cycles, all heifers exhibited cycles ranging from 19 to 22 days in length. This was the criterion used for deciding that the estrous cycles were "normal". Heifers were observed for estrus at 0700 and 1700 hours for a 30-minute period after being moved to an exercise lot. During all other times of the experimental 28 period, heifers were housed in individual tie stalls. Collection of blood and IV injections were via polyvinyl jugular cannulae. Cannulae were opened at 0930 hours with blood samples collected for hormone assay (table 1) at 1145 hours. Experiment 6. Effect of TRH on Serum Prolactin and GH During Pregnancy Experimental Design.--Twenty-seven pregnant heifers received IV injections of 33 pg TRH/100 kg body weight according to a 3 x 3 factorial design. TRH was injected at either 3, 6 or 9 months of pregnancy during either November, April or July. Nine heifers received TRH during November, April or July with three heifers each in either the first, second or third trimester of pregnancy. However, only two heifers in the first trimester of pregnancy received TRH during July due to loss of a cannula from one heifer. Thus, each heifer received only one injection of TRH during the experimental period. All heifers were diagnosed pregnant on the basis of rectal palpation by a veteri- narian, with the last breeding date used as the date of conception. Collections of blood and IV injections were via polyvinyl jugular cannulae. Cannulae were opened 2 to 3 hours prior to start of experi- mentation with blood samples collected for hormone assay as shown in table 1. Experiment 7. Effect of TRH on Serum Prolactin and GH During Lactation Experimental Desigp.--Sixteen primiparous or multiparous lactating cows received IV injections of 33 pg TRH/100 kg body weight at 2, 4, 6, 8 and 10 months of lactation. Each cow received TRH on two consecutive days at each stage of lactation to determine if there were 29 any carryover effects on serum prolactin and GH following TRH. Cows were started on the experiment in groups of four. Groups were started in May, September, October and November. Although five cows were dried- off prior to the lO-month stage of lactation, their data are included in the analysis. Collections of blood and IV injections were via polyvinyl jugular cannulae. Cannulae were opened 2 to 3 hours prior to start of experimentation with blood samples collected for hormone assay as shown in table 1. E. Statistical Methods Average baseline concentrations (ng/ml) of prolactin and GH prior to TRH were calculated for each animal on each day. This value was subtracted, within animal, from subsequent corresponding hormone concentrations measured at each post-treatment sampling time. These adjusted values (ng/ml) were plotted against time (minutes) and a third degree least squares polynomial curve was computed. The area under this hormone response curve was integrated and expressed as ng ml-lminutes. In addition, average hormone concentrations at the post-treatment peak and average time (minutes) required to achieve peak hormone concentra- tions were calculated for each experiment. In experiments 1, 2, 3 and 4, data were analyzed by analysis of variance, regression analysis and comparisons of means of areas under individual hormone response curves. Dunnett's procedure was used for unbalanced data in experiment 3 since I was primarily interested in comparing IM or SC route of administration of NaCl or TRH with IV route of administration. Tukey's statistic was used to compare all means (balanced data) in experiments 1, 2 and 4 (Kirk, 1968). In 3O experiment 5, analysis of variance was used. Analysis of variance and Tukey's statistic were used to compare means of the areas under individ- ual hormone response curves in experiment 6. In experiment 7, analysis of variance and analysis of covariance were used. Also used were pre- selected orthogonal contrasts among means of areas under individual hormone response curves. RESULTS AND DISCUSS ION Experiment 1. Repeatability of changes in serum prolactin and GH concentrations in response to a given dose of TRH This experiment was designed to determine repeatability of changes in serum prolactin and GH concentrations in response to a given dose of TRH on days 1, 2, 4 and 8. Serum prolactin concentrations, averaged across the four days of the experiment, increased from a baseline value of 16 ng/ml to a peak of 81 ng/ml at 7 minutes after IV administration of 10 pg TRH. Magni- tude of increase in serum prolactin concentrations, as measured by areas under the response curves (ng ml-lmin), after administration of TRH averaged 1,393, 1,532, 1,381 and 1,585 on days 1, 2, 4 and 8, respec- tively. These means were not significantly different from each other (P>.05). These results extend to prepubertal female calves the finding that administration of TRH releases prolactin in mature bulls (Convey et al., 1973 and Tucker et al., 1974) and cows (Schams, 1972, Convey et al., 1973 and Kelly et al., 1973). Repeatability coefficients of average baseline concentrations for prolactin (within heifer, among four days of sampling) was .27, whereas repeatability of the area under the prolactin response curve after TRH administration was .61. The correlation coefficient (within heifers) between average serum prolactin concentrations before TRH and 31 32 maximum prolactin concentrations after TRH was .64 (P<.01). Thus, the amount of prolactin released from the pituitary following TRH is very closely related to basal or resting serum prolactin concentrations. This implies that the quantity of prolactin released following an exogenous stimulus such as TRH may be related to the rate of tonic release of prolactin from the pituitary. Wettemann and Tucker (1974) and Tucker and Wettemann (1976) also reported that greater concentrations of basal or resting serum prolactin were associated with greater peak heights after administration of TRH. In contrast, Koprowski and Tucker (1973) found a negative relationship between baseline or resting con- centrations of serum prolactin and subsequent milking-induced releases of this hormone. These findings may suggest that milking- and TRH- induced releases of prolactin are acting through different mechanisms. Koprowski and Tucker (1973) reported a correlation coefficient of .36 (P<.01) between serum prolactin concentrations immediately after milking and milk yield. Thus, if serum prolactin concentrations following TRH administration are affected by baseline concentrations, possible use of serum prolactin concentrations following TRH administration as an indicator of potential milk producing ability is not very promising in prepubertal heifers. Baseline serum GH concentrations prior to TRH averaged 8 ng/ml and increased to a maximum of 21 ng/ml at 10 minutes after TRH. These results extend to prepubertal female calves the finding that adminis- tration of TRH releases GH in mature bulls (Tucker et al., 1974) and cows (Convey et al., 1973). The correlation coefficient between serum GH concentrations measured before TRH and maximum GH concentrations after TRH was .60 33 (P<.01). The increase in serum GH concentrations after TRH averaged 148, 102, 102 and 142 ng ml-lmin on days 1, 2, 4 and 8, respectively. There were no significant differences among means (P>.05). Repeatability coefficients for baseline serum GH concentrations and post-TRH response areas (calculated within heifers, among four days of sampling) were .37 and .35, respectively. Tucker et a1. (1974) reported .30 repeatability estimate for basal or resting serum GH con- centrations in mature bulls over a lO-day period. These results agree very closely with the repeatability estimate (.37) that I found in prepubertal heifers. In contrast, the repeatability estimate for serum GH response areas (.35) after TRH was only about one-half that for prolactin (.61). These results demonstrate that the pituitary is capa- ble of releasing more prolactin than GH following TRH administration. This may be related to the different mechanisms controlling the releases of these two hormones since prolactin release is thought to be primarily under tonic inhibition and GH primarily under stimulatory control (Meites and Clemens, 1972 and Wilson, 1974). Experiment 2. Effects of various doses of TRH on serum prolactin and GH This experiment was designed to determine if varying the dose of TRH would affect serum prolactin and GH concentrations and if so to determine the doses where maximum hormone releases were attained. Overall, baseline concentrations of prolactin in serum before TRH averaged 39 ng/ml. Injection of .85 percent NaCl did not affect (P>.05) serum prolactin concentrations. Increasing the dose of TRH from 5 to 100 pg increased peak serum prolactin concentrations from 143 to 277 ng/ml, but had little effect on time (8 to 12 min) required 34 to achieve these peaks (table 2). Doses of TRH and heifers affected (P<.01) the areas under the prolactin response curves. The quantity (response curve areas) of prolactin released increased linearly (P<.Ol) with increases in log of the dose of TRH. Although average prolactin response curve areas appeared to plateau between 50 and 100 pg TRH, the quadratic component of regression for these data was not significant (P>.05). Fell et a1. (1973) and Noel et al. (1974) also reported a dose-response increase in serum prolactin concentrations following TRH administration in ewes and humans. In contrast, Convey et a1. (1973) and Kelly et a1. (1973) reported that serum prolactin concentrations were not clearly related to the dose of TRH administered in lactating cows. These differences may be due to more stable serum prolactin con- centrations in calves than those found in lactating cows; thus, with greater endogenous variation the dose responsiveness to exogenous TRH may have been masked in those studies using lactating cows. Although integrated areas under serum prolactin response curves increased with log of the dose of TRH, eventually a dose (50 pg) was reached where additional TRH (100 pg) did not cause a further increase in prolactin response curve areas. This dose may represent the maximum capacity of the anterior pituitary to release prolactin to a given stimulus. These results are in agreement with those published by Tucker et al. (1975) in which they suggested that a ceiling exists in postpu— bertal heifers and lactating cows for release of prolactin and GH in response to constant infusion or multiple injections of TRH. However, this ceiling does not necessarily represent the maximum capacity of the pituitary to release prolactin since Tucker et al. (1975) showed that stimultaneous application of a second heterologous stimulus such as 35 Table 2.--Serum prolactin (PRL) and growth hormone (GH) responses to various doses of thyrotropin releasing hormone (TRH) Peak TRH Concentration Time to: Are (pg) Hormone (ng/ml) (min) (ng ml. min) 0 PRL --- --- -110 :_128 (.85% NaCl) GH --- --- 7 :_ 15 5 PRL 145 12 1515 :_602 GH 11 5 53 :_ 19 10 PRL 143 8 1729 :_406 CH 15 8 129 :_ 45 25 PRL 211 10 3255 :_833 GH 29 11 355 :_116 50 PRL 265 9 3820 :_816 GB 46 9 555 i_137 100 PRL 277 10 3940 :_448 GH 42 10 667 :_102 aOverall serum hormone concentrations (ng/ml) before TRH averaged 39 for PRL and 5 for GH. bIntegrated area of plot of hormone concentrations for 30 min after NaCl or TRH minus baselines before NaCl or TRH, respectively. Values are means :_standard errors (n=6). milking or injection of prostaglandin F a would induce additional pro- 2 lactin release. These data suggest that the pituitary may become refractory to TRH since the pituitary is still responsive to a second heterologous stimulus. Serum GH concentrations before TRH administration averaged 5 ng/ml. Serum GH concentrations increased to peaks of 11 to 46 ng/ml following injections of 5 toJIKJpg TRH (table 2). These peaks were attained 5 to 11 minutes after TRH was administered. Dosage of TRH and heifers affected (P<.01) the quantity of GH released, as measured by area under the response curve, but NaCl did not (P>.05). Furthermore, area under the GH response curve increased linearly (P<.01) with 36 increasing log of the TRH dose. Convey et a1. (1973) also reported a dose-response increase in serum GH concentrations following TRH admin- istration in lactating cows. Unlike prolactin release from the pituitary, these data indicate that the maximum dose (100 pg) of TRH did not maximally stimulate the pituitary to release GH. This submaximal stimulation of GH release from the pituitary following TRH administration is probably related to smaller amounts of GH, as compared with prolactin, that are released to a give dose of TRH. Thus, a larger dose of TRH is necessary for maximal stimulation of GH release. Experiment 3. Effects of various doses of TRH via different routes of administration on serum prolactin and GH This experiment was designed to determine the effects of TRH administrated via IV, IM or SC injection on serum prolactin and GH concentrations using different doses of TRH. Serum prolactin concentrations averaged 22 ng/ml prior to administration of TRH. There was no prolactin response to the IV, IM or SC administration of NaCl (P>.05). Maximum serum prolactin concen- trations after IV administration of 10 or 25 pg TRH occurred within 7 and 9 minutes, and averaged 145 and 174 ng/ml, respectively (table 3). Following IM or SC administration of TRH the peak heights were lower (48 to 107 ng/ml) and the time required to reach the peaks were longer (19 to 22 min) than those listed above for IV injections. Schams (1972) reported a longer lasting peak of serum prolactin concentrations follow- ing IM injections of TRH when compared with IV injections of TRH in cattle. He also reported a tendency for enhanced serum prolactin con- centrations following increasing doses of TRH via IV administration. 37 Table 3.--Serum prolactin (PRL) responses to doses of thyrotropin releasing hormone (TRH) and routes of administrationa Peak Dose of TRH Route of Concentration- Time to: Areg? (pg) Administration (ng/ml) (min) (ng ml min) 0 IV --- --- -34 :_105 (.851; NaCl) IM --- --- 23 i 228 SC --- --- 142 :_106 10 IV 145 7 1544 :_398 IM 48 19 167 :_l45 SC 64 22 533 :_309 25 IV 174 9 2170 :_477 IM 105 22 1322 i_552 SC 107 22 798 :_253 aOverall serum PRL concentrations (ng/ml) before TRH averaged 22. bIntegrated area of plot of hormone concentrations for 30 min after NaC1 or TRH minus baselines before NaCl or TRH, respectively. Values are means :_standard errors (n=4 for heifers receiving NaC1; =6 for heifers receiving 10 or 25 pg TRH). In total, these results suggest that IM and SC injected TRH is retained within'the depot sites and released from these stores relatively slowly over a period of time. Therefore, serum concentrations of TRH immedi- ately following IM and SC injections of TRH are less than those after IV injection, but these concentrations are maintained for a longer of time. Doses of TRH, routes of administration and heifers affected (P<.Ol) areas under the serum prolactin response curve. In addition, there was a dose x route interaction (P<.05). In comparison with NaC1 controls, the serum prolactin response was greater after IV administration of 10 (P<.05) or 25 (P<.01) U9 TRH. 38 Although mean prolactin response curve areas after 10 or 25 pg TRH administered IM or SC were numerically greater than control response areas, they were not significantly differently (P>.05). Areas under the prolactin response curve after 10 pg TRH injected IV were greater (P<.05) than that after IM administration of TRH. On the other hand, prolactin response areas after 10 pg TRH administered IM or SC were not different (P>.05) from each other. Nor were the areas under the pro- lactin response curve different (P>.05) after IV, IM or SC injections of 25 pg TRH (Figure 1.). Again, these data point out that more prolactin is released, although not always significantly greater, following IV administration of TRH, as compared with IM or SC injection, during a 30-minute post-TRH sampling period. Overall, serum GH baseline concentrations averaged 7 ng/ml before TRH treatment. Injection of NaC1 via any of the routes tested did not increase serum GH (P>.05). The means of maximum GH concentra- tions following IV, IM and SC injections of 10 pg TRH were 24, 14 and 11 ng/ml and they were attained at 7, l9 and 18 minutes, respectively (table 4). Following 25 pg TRH, serum GH peaks averaged 35, 25 and 14 ng/ml and occurred at 12, 14 and 22 minutes, respectively. Doses of TRH, routes of administration, heifers (P<.01) and interaction of doses and routes (P<.05) affected areas under the serum GH response curves. Intravenous administration of 10 or 25 pg TRH increased serum GH response areas above controls (P=.05); P<.05, respec- tively). However, neither IM or SC administration of 10 or 25 pg TRH increased (P>.05) the GH response curve area over that of NaCl controls. In addition, GH response areas after IV, IM or SC administration of 25 pg TRH did not differ from each other (Figure 2). These data show 39 .mummwm: Hmuuonsmnmum ca mma on mm mo coflumpumHGHEpm Um no 2H .>H ocwzoaaom cauomHoum Eduom . a $de 4O L |230 I220 IV I IZIO l200 Time of Day II50 i 1 H40 I O O Q' N snauuoo- aunt—rs um; obunqo (lm/Ouwuoolmd [Lllllll _ O O O o o- l O O a: to st N 41 Table 4.--Serum growth hormone (GH) responses to doses of thyrotropin releasing hormone (TRH) and routes of administrationa Peak Dose of TRH Route of Concentration Time to: Area: (pg) Administration (ng/ml) (min) (ng ml min) 0 IV --- --- -34 :_38 (.85% NaC1) IM --- --- 12 :.50 SC --- --- 102 i'74 10 IV 24 7 232 :_47 IM l4 19 82 :.42 SC 11 18 -23 :_51 25 IV 35 12 326 :_99 IM 25 14 147 i'87 SC 14 22 115 :_17 aOverall serum GH concentrations (ng/ml) before TRH averaged seven. bIntegrated area of plot of hormone concentrations for 30 min after NaCl or TRH minus baselines before NaC1 or TRH, respectively. values are means i_standard errors (n=4 for heifers receiving NaC1; =6 for heifers receiving 10 or 25 pg TRH). that more GB is released, although not always significantly greater, from the pituitary following IV administration of TRH when compared with IM or SC administration. As discussed previously in this section concerning release, IV administered TRH reaches the pituitary in greater concentrations earlier and results in a greater initial release of GH than after IM or SC injections of TRH. The fact that serum GH response curve areas following IV administration of 25 pg TRH, as compared with IM or SC administration, were numerically but not statistically different can partially be attributed to heifer variation since heifers affected (P<.01) areas under the response curve. 42 Figure 2. Serum growth hormone following IV, IM or SC administration of 25 pg TRH in prepubertal heifers. N m l N .b I N O r 5 I a) I Growth hormone Ina/ml) change from saline control RS l .h I ”:30 ”240 43 IV ":50 l2:0 l2:l0 Time of day \ I2:20 A I2230 44 Experiment 4. Effects of an increased dose of TRH via different routes of administration on serumxprolactin and GH This experiment was designed to determine if increasing the dose of TRH to obtain near maximum hormone releases, as determined in a previous experiment, affected the total amount of prolactin and GH released over a 2-hour post-TRH sampling period. Serum prolactin concentrations averaged 17 ng/ml before injec- tion of 50 pg TRH. Maximum prolactin concentrations after IV and SC injections of TRH averaged 119 and 81 ng/ml, respectively, and these peaks were attained 7 and 46 minutes after TRH administration, respectively. Administration of TRH by IV or SC routes increased (P<.01) magnitude of prolactin release above that of NaCl controls (table 5), but there was no difference in area under the prolactin response curve (P>.05) between IV and SC routes of TRH administration. As in experiment 3, route of administration of TRH affected maximum serum prolactin concentrations and time required to achieve these peaks. However, when sufficient time is allowed following TRH administration for serum prolactin to return to pretreatment concentrations, the total prolactin response curve areas were not different after IV or SC administration of TRH. These results suggest that while route of administration affected the magnitude of response and time required to reach maximal concentrations of prolactin, the total amount of prolac- tin released following TRH injection is unaffected by route of administration. Therefore, when a prolonged release of prolactin is desired, the IM or SC route of administration of TRH should be the method of choice. 45 Table 5.--Serum prolactin (PRL) and growth hormone (GH) responses to doses of thyrotropin releasing hormone (TRH) and routes of administration Route of Administration PRL GH Treatment IV SC IV SC -1 . ng ml min .85% NaCl -249 :_252 464 :_ 182 122 i 92 45 :_139 50 pg TRH 3639 :_638 4198 :_1390 504 :_93 218 :_247 aArea of plot of hormone concentrations for 120 min after NaCl or TRH minus baselines before NaCl or TRH, respectively. Values are means :_standard errors (n=4, except for GE after IV administration of 50 pg TRH where n=3). Serum GH concentrations increased from 4 ng/ml before TRH to peaks of 28 and 17 ng/ml after IV or SC injections of 50 pg TRH, respectively. The time required to achieve these peaks was 8 and 28 minutes, respectively. One heifer did not reach maximal GH concentra- tions (16 ng/ml) until 70 minutes after IV injection of TRH. In the author's opinion this release was not associated with the TRH injection; thus, GH data after IV administration of 50 pg TRH for this heifer were excluded. The area under the GH response curves after IV injection of 50 pg TRH was greater (P<.05) than that for NaC1 controls (table 5); whereas, the response to SC injection of 50 pg TRH was not different from controls (P>.05). These data suggest that high serum concentra- tions of exogenous TRH following IV administration will result in a greater total release of GH from the pituitary over a 2-hour period, even though a more sustained release of GH is obtained following SC administration of TRH. 46 Overall, results from experiments 3 and 4 demonstrate that serum prolactin and GH concentrations increase more rapidly and decline more precipitously following IV administration of TRH when compared with either IM or SC administration of TRH. In addition, maximum serum pro- lactin and GH concentrations are greater after IV administration of TRH, but increased serum prolactin and GH concentrations are sustained for longer periods of time following IM or SC administration of TRH. Experiment 5. Effect of TRH on serum prolactin and GH during the estrous cyple This experiment was designed to determine if different stages of the estrous cycle would affect prolactin and GH release from the pituitary following IV administration of TRH. Overall, baseline concentrations of prolactin in serum before TRH averaged 6 ng/ml. Maximum serum prolactin concentrations after TRH ranged from 52 to 81 ng/ml and time required to attain these maximal values ranged from 10 to 17 minutes (table 6). Days of the estrous cycle did not affect (P>.05) the quantity of prolactin released, as measured by area under the response curve. Although prolactin released in response to injections of TRH at various days of the estrous cycle were not significantly different, maximum concentrations after TRH, pre-TRH concentrations and areas under the prolactin response curve at days 2 and 4 of the estrous cycle tended to be less than those at other stages of the estrous cycle. These suppressed prolactin responses may be related to low serum estrogen and progesterone concentrations. Serum estrogen and progesterone concentrations are known to be decreased at this time when compared with other stages of the estrous cycle (Wettemann et al., 1972). The concept that low serum estrogen and/or 47 Table 6.--Serum prolactin response to thyrotropin releasing hormone (TRH) during the estrous cycle Pre-TRH Peak prolactin Time to Prolactin release Day of prolactin after TRH peak after TRHa estrous cycle (ng/ml) (ng/ml) (min) (ng ml'lmin) o 8 73 13 1191 i 408 2 3 61 12 812 i 301 4 3 52 10 820 i 179 7 9 77 12 1166 :_169 15 5 74 11 1368 i_225 18 6 81 17 1560 i 247 aIntegrated area of plot of prolactin concentrations for 30 min after TRH minus baselines before TRH. Values are means i standard errors (n=6). progesterone may be related to low serum prolactin is supported by the study of Reece and Bivens (1942) who reported that pituitary prolactin content was increased in ovariectomized rats following estrogen, pro- gesterone or estrogen plus progesterone injections. In addition, there is another "tendency" for serum prolactin response curve areas and peak prolactin values after TRH to be larger at day 18 (proestrus) than at other days of the estrous cycle. This gives support to the concept that serum estrogen concentrations may affect serum prolactin concen- trations since serum estrogen concentrations are increased at this time (Wettemann et al., 1972). Although infusions of estradiol-17B in cows (Schams and Karg, 1972) and progesterone in bulls (Schams et al., 1974) increased serum prolactin concentrations, serum estrogen and progesterone concentrations 48 probably are of minor importance in the regulation of serum prolactin concentrations during the estrous cycle. This concept is supported by the study of Beck et a1. (1976) who found that there were no differences in serum prolactin concentrations in ovariectomized heifers bearing estradiol-17B implants and/or progesterone pessaries. These authors suggested that neither estrogen nor progesterone alone or in combination influenced serum prolactin concentrations when present in blood at con- centrations comparable with those found during the normal estrous cycle. In addition, Koprowski and Tucker (1973) found no differences in serum prolactin concentrations during the estrous cycle in lactating cows. In total, these data suggest that estrogen and progesterone are of minor importance in the regulation of circulating prolactin concentra- tions during the estrous cycle in heifers. Serum GH concentrations before TRH administration averaged 6 ng/ml. Serum GH concentrations increased to maximum values of 18 to 24 ng/ml following injections of TRH (table 7). These maximal concen- trations were attained 11 to 15 minutes after TRH was administered. Days of the estrous cycle did not affect (P>.05) the quantity of GH released, as measured by area under the response curve. These data are in agreement with Beck et a1. (1976) who reported no differences in serum GH concentrations between ovariectomized control heifers and those heifers that had estradiol-l7B implants and/or progesterone pessaries. These authors suggested, as with prolactin, that neither estrogen nor progesterone alone or in combination influenced serum GH when present in blood at concentrations comparable to those found during the normal estrous cycle. 49 Table 7.--Serum growth hormone (GH) response to thyrotropin releasing hormone (TRH) during the estrous cycle Pre-TRH Peak GH Time to GH release Day of GH after TRH peak after TRHa estrous cycle (ng/ml) (ng/ml) (min) (ng ml-lmin) O 5 24 12 283 :.43 2 6 21 11 200 :_96 4 6 18 11 187 1.56 7 7 20 11 206 i’46 15 7 20 14 193 i-44 18 5 24 15 270 :_40 aIntegrated area of plot of growth hormone concentrations for 30 min after TRH, minus baseline before TRH. Values are means :_ standard errors (n=6). On the other hand, Koprowski and Tucker (1973) reported a small, but significant (P<.05), increase in serum GH concentrations around estrus as compared with the luteal phase of the estrous cycle in lactating cows. In addition, administration of diethylstilbesterol increased serum GH in steers (Trenkle, 1970) and injection of proges- terone appeared to enhance the secretion of GH in ovariectomized ewes (Davis and Borger, 1974). In summary, although exogenous administra- tion of estrogen or progesterone may increase serum GH concentrations, it is my opinion that estrogen and progesterone are of minor importance in the regulation of circulating GH concentrations during the normal estrous cycle in heifers. 50 Experiment 6. Effect of TRH on serum prolactin and GH during re anc This experiment was designed to determine the effects of TRH on serum prolactin and GH concentrations at 3, 6 and 9 months of pregnancy at various seasons of the year. Serum prolactin concentrations, prior to TRH administration, averaged 14 ng/ml. After TRH maximum serum prolactin concentrations ranged from 125 to 184 ng/ml and time required to attain these maximal values were 11 to 12 minutes (table 8). Stage of pregnancy did not affect (P>.05) the quantity of prolactin released, as measured by area under the response curve. However, pre-TRH concentrations, maximum concentrations after TRH and response curve areas for prolactin followb ing TRH administration tended to be greater with advancing pregnancy. Month of year affected (P<.01) the quantity of prolactin released, as measured by area under the response curve (table 9). Prolactin response curve areas were greater (P<.01) in July when compared with response curve areas in November and April. However, response curve areas in November and April were not significantly different (P>.05) from each other. The "tendency" for increased pre-TRH values, peak concentrations after TRH and response curve areas for prolactin with advancing preg- nancy agrees with the data of Oxender et a1. (1972) in heifers. Also, serum prolactin concentrations increased during late pregnancy in cows (Schams and Karg, 1970 and Johke et al., 1971) and ewes (McNeilly, 1971). In addition, Bates et a1. (1935) and Reece and Turner (1937a) reported that pituitary prolactin content increased during late preg- nancy in cattle. 51 Table 8.--Serum prolactin response to thyrotropin releasing hormone (TRH) during pregnancy Pre-TRH Peak prolactin Time to Prolactin release Month of prolactin after TRH peak after TRHa pregnancy (ng/ml) (ng/ml) (min) (ng ml’lmin) 3 12 125 12 2205 i 1309 6 14 148 12 2831 :_1037 9 15 184 11 3155 :_1210 aIntegrated area of plot of prolactin concentrations for 30 min after TRH, minus baselines before TRH. Values are means :_standard errors (n=8 for heifers at 3 month stage of pregnancy; n=9 for heifers at 6 and 9 month stages of pregnancy). Table 9.--Serum prolactin and growth hormone responses in pregnant heifers to thyrotropin releasing hormone (TRH) during seasons Prolactin release Growth hormone release Month of after TRHa after TRHa Year (ng ml'lmin) (ng ml'lmin) November 455 :_ 115 121 i_24 April 1193 :_ 270 132 :'27 July 7087 :_1010 134 + 21 aIntegrated area of plot of hormone concentrations for 30 min after TRH, minus baselines before TRH. Values are means :_standard errors (n=9 for heifers during November and April; n=8 for heifers during July). 52 In contrast, Wettemannn and Hafs (1971) found that serum pro- lactin concentrations did not change significantly during the first 75 days of pregnancy in heifers. Arai and Lee (1967) reported a gradual decline in serum prolactin concentrations with advancing pregnancy in ewes. Davis et a1. (1971) also found decreasing or low serum prolactin concentrations in ewes during advancing pregnancy, but serum prolactin concentrations began to increase gradually 3 to 5 weeks prior to parturition. In total, these studies indicate that serum prolactin concentrations remain relatively stable throughout most of pregnancy, but probably increase in late pregnancy. This increase may be impor- tant in the biochemical and anatomical changes that occur in the mammary gland prior to the initiation of lactation. Month of year affected (P<.01) the quantity of prolactin released following TRH administration. This agrees with Koprowski and Tucker (1973) findings in lactating cows that serum prolactin concen- trations were higher during April to September (74 ng/ml) than during October to March (35 ng/ml). Similarly, Schams and Reinhardt (1974) reported that serum prolactin concentrations were higher in summer, lower in winter in cattle. Furthermore, they found a significant correlation (r=.9; P<.001) between serum prolactin concentrations and the number of daylight hours. In addition, Tucker et a1. (1974) reported that in bulls, blood serum collected in July contained more than twice as much prolactin as serum collected in January. These data demonstrate the existence of a seasonal effect on serum prolactin concentrations. Daily illumination and temperature are important factors in the seasonal cyclicity of serum prolactin concentrations. Increasing or decreasing the length of photoperiod increased or 53 decreased serum prolactin concentrations, respectively (Pelletier, 1973 and Bourne and Tucker, 1975). Similarly, increasing or decreasing temperature increased or decreased serum prolactin concentrations, respectively (Wettemann and Tucker, 1974). These effects are though to be mediated via the hypothalamus, but the exact mechanisms involved in this regulation are still unknown. Furthermore, the physiological significance, if any, of increased serum prolactin concentrations dur- ing summer, as compared with winter, remains a mystery. Overall, baseline concentrations of GH in serum before TRH administration averaged 4 ng/ml. Maximum.serum GH concentrations after TRH administration were 10 to 12 ng/ml and time required to attain these maximal values were 9 to 13 minutes (table 10). Neither stage of pregnancy (table 10) nor month of year (table 9) affected (P>.05) the quantity of GH released, as measured by area under the response curve. Lack of effects of stage of pregnancy on serum GH concentrations are in agreement with Oxender et a1. (1972) who reported that serum GH concen- trations did not change significantly during pregnancy in cows. Ingalls et a1. (1973) observed no changes in serum GH concentrations between 26 and 9 days before parturition in heifers. However, serum GH increased as parturition neared and peaked at parturition. In contrast, Koprowski and Tucker (1973) found a small linear increase in serum GH concentrations with advancing pregnancy in lactating cows. In total, these data suggest that serum GH concentrations remain relatively stable throughout pregnancy, but probably increase just prior to parturition. This increase in serum GH concentrations just prior to parturition may be essential for the initiation of lactation since exogenous GH has been shown to be galactopoietic when given to lactating cows. 54 Table lO.--Serum growth hormone (GH) response to thyrotropin releasing hormone (TRH) during pregnancy Pre-TRH Peak GH Time to GH release Month of GH after TRH peak after TRH pregnancy (ng/ml) (ng/ml) (min) (ng ml'hmin) 3 4 11 13 132 i 19 6 4 10 12 112 :_24 9 4 12 9 142 + 28 aIntegrated area of plot of growth hormone concentrations for 30 min after TRH, minus baselines before TRH. Values are means 1 standard errors (n=8 for heifers at 3 month stage of pregnancy; n=9 for heifers at 6 and 9 month stages of pregnancy). In contrast to the prolactin data, serum GH concentrations following TRH administration were not affected (P>.05) by month of year. These results agree with Koprowski and Tucker's (1973) findings that serum GH concentrations were not affected by season of year in lactating cows. Tucker et al. (1974) observed that serum GH concentra- tions in bulls were not different in blood collected in July, as com- pared with blood collected in January. In addition, no significant differences were noted in serum GH concentrations in heifer calves exposed to 4.5, 21 or 32C for 9 days although GH concentrations tended to increase with increasing temperature (Tucker and Wettemann, 1976). These data suggest that seasonal effects on serum GH concentrations are minimal, and probably are of little importance in the regulation of serum GH concentrations. 55 Experiment 7. Effect of TRH on serum prolactin and GH during lactation This experiment was designed to determine if serum prolactin and GH concentrations following TRH administration are affected at various stages of lactation within different months of the year and stages of pregnancy. Each cow received TRH on two consecutive days at each stage of lactation to determine possible carryover effects on serum prolactin and/or GH concentrations. However, day of injection did not affect (P>.05) the quantity of prolactin released, as measured by area under the response curve (table 11). Therefore, data for days 1 and 2 were pooled for further analysis. Prior to TRH administration, serum prolactin concentrations averaged 16 ng/ml. Maximum serum prolactin concentrations after administration of TRH ranged from 202 to 282 ng/ml at the five stages of lactation and time required to attain these maximal values were 12 to 16 minutes (table 12). Stage of lactation did not affect (P>.05) quantity of prolactin released. However, prolactin response curve areas following TRH administration tended to be greater at the 2-month stage of lactation (table 12). Analysis of variance showed that stage of pregnancy did not affect (P>.05) quantity of prolactin released, as measured by area under the response curve. Season of year affected (P<.01) the quantity of prolactin released, as measured by area under the response curve (table 13). Prolactin response curve areas were greater (P<.01) at March-May, June-August and September-November when compared with response curve areas at December-February. 56 Table ll.--Serum prolactin and growth hormone (GH) response to thyro- tropin releasing hormone (TRH) on two consecutive days during lactation Stage of Pre-TRH Prolactin release Pre-TRH GH release lactation Day of prolactin after TRHa GH after TRHa (months) injection (ng/ml) (ng ml‘lmin) (ng/ml) (ng md'lmin) 2 1 24 5341 :_870 6 259 :_43 2 20 5056 :_734 6 269 :_43 4 1 17 3694 :_614 4 152 i_42 2 18 3348 :_472 4 116 :_23 6 l 12 3321 :_582 5 144 i'28 2 14 4165 :_709 5 143 i_35 8 1 13 4132 i 502 4 157 i 50 2 12 3448 :_592 4 137 :_33 10 l 16 4113 i_633 4 138 :_38 2 17 5335 :_686 4 134 :.47 aIntegrated area of plot of hormone concentrations for 30 min after TRH, minus baselines before TRH. Values are means :_standard errors (n=l6). 57 Table 12.--Serum prolactin response to thyrotropin releasing hormone (TRH) during lactation Stage of Pre-TRH Peak prolactin Time to Prolactin reéease lactation prolactin after TRH peak after TRH (months) (ng/ml) (ng/ml) (min) (ng ml-lmin) 2 22 282 16 5199 :_792 4 18 202 15 3521 i_521 6 13 210 12 3743 :_575 8 12 234 12 3790 :_361 10 16 241 13 4724 + 606 a . . Integrated area of plot of prolactin concentrations for 30 min after TRH, minus baselines before TRH. Values are means : standard errors (n=l6). Table l3.--Serum prolactin and growth hormone (GH) responses to thyrotropin releasing hormone (TRH) during seasons Prolactin release Growth hormone gelease Seasons after TRH after TRH (ng ml'lmin) (ng ml'lmin) Mar-May 4274 i 368 156 1.29 June-Aug 5836 i 525 139 :_36 Sept-Nov 5009 :_516 213 :_31 Dec-Feb 1782 + 244 131 + 29 aIntegrated area of plot of hormone concentrations for 30 min after TRH, minus baselines before TRH. Values are means i_standard errors. 58 Analysis of covariance was performed to determine what effects were attributable to either stage of lactation, stage of pregnancy or season (month) of year. Each of these factors was used in turn as a main effect with the other two factors and their squared terms serving as the covariates. Squared terms were used as covariates because the raw means indicated that the response curves over time were curvilinear. As with analysis of unadjusted data, analysis of covariance showed that neither stage of lactation nor stage of pregnancy affected (P>.05) quantity of prolactin released following TRH administration (table 14). However, analysis of covariance showed, like analysis of unadjusted data, that month of year affected (P<.01) quantity of prolactin released following TRH administration (table 14). Koprowski and Tucker (1973) reported that serum prolactin concentrations increased in response to stimuli associated with milking and this response was largest at 8 weeks of lactation, but then qradually decreased as lactation advanced until at 32 weeks prolactin was no longer released in response to milking associated stimuli. These results agree with the "tendency" I found for increased prolactin response to TRH during early lactation. In addition, Johke (1970) reported a rapid increase in serum prolactin concentrations associated with the milking stimulus during early lactation in cows and goats. In total, these data indicate that serum prolactin concentrations are increased during early lactation following either TRH or milking stimuli. I speculate that this increased pituitary responsiveness during early lactation is associated with lactational performance since milk yields are maximum during early lactation. 59 Table 14.-—Serum prolactin (PRL) response to thyrotropin releasing hormone (TRH) during lactation, pregnancy and month of year Lactation Pregnancy Month of Year PRL rele e PRL rele e PRL rele e Month after TRH Month after RH Month after REE (ng ml‘lmin) (ng ml- min) (ng ml. min) 2 4622 0 3771 Jan. 1559 l 5275 Feb. 1883 4 4177 2 4623 Mar. 5031 3 4287 Apr. 3602 6 4234 4 4822 May 4122 5 4011 June 6558 8 3304 6 4065 July 5916 7 1602 Sept. 7467 10 4193 8 3410 Oct. 4229 Nov. 3184 Dec. 2423 aEach variable used as main effect with other two variables serving as covariates. Analyses were performed as three separate analyses because of high correlation (r=.83) between stage of lactation and month of pregnancy. bIntegrated area of plot of prolactin concentrations for 30 min after TRH, minus baselines before TRH. Values are means. The correlation coefficient between average daily milk yield for the 5 days preceding TRH administration at each stage of lactation was -.01 (P>.05). Schams and Karg (1970) also found no correlation between serum prolactin concentrations and stage of lactation or milk yield. In contrast, Koprowski and Tucker (1973) reported that milk yield and serum prolactin concentrations in blood collected immediately after and 1 hour after milking were significantly (P<.01) correlated (r=.36 and .18, respectively). In total, these data suggest that serum prolactin concentrations following TRH or milking stimuli are only very 6O slightly associated with lactational performance throughout the entire lactational period, but may be more associated during early lactation. Seasonal and stage of pregnancy effects on serum prolactin concentrations confirm the results in experiment 6 and the discussion of these effects will not be repeated. Serum GH concentrations, prior to TRH administration, averaged 5 ng/ml. Maximum serum GH concentrations were 13 to 24 ng/ml at the five stages of lactation (table 15). Time required to attain these maximal values were 9 to 13 minutes. Since day of injection did not affect (P>.05) quantity of GH released, as measured by area under the response curve (table 11), data for days 1 and 2 were pooled for further analysis. One-way analysis of variance showed that stage of lactation affected (P<.05) quantity of GH released. GH response areas were greater (P<.05) at the 2-month stage of lactation when compared with 4-, 6-, 8- and lO-month stages. Stage of pregnancy did not affect (P>.05) quantity of GH released; however, stage of lactation and month of pregnancy were highly correlated (r=.83). Therefore, a least squares analysis of covariance was used to analyze effects of stage of lactation, month of pregnancy and season of year using in turn one of these variables as a main effect and the other two as covariates (table 16) with analyses of all possible combinations. Adjusting for the respective two covariates in separate analyses, neither the main effects of stage of lactation nor month of pregnancy was significant (P>.05) when the other was used as a covariate. Therefore, as reported above, the data were analysed by one-way analysis of variance for unbalanced data. 61 Table 15.--Serum growth hormone (GH) response to thyrotrOpin releasing hormone (TRH) during lactation Stage of Pre-TRH Peak GH Time to GH release lactation GH after TRH peak after RH (months) (ng/ml) (ng/ml) (min) (ng ml' min) 2 6 24 13 264 i 40 4 4 13 13 134 i 30 6 5 16 11 144 $.28 8 4 15 12 147 i 37 10 4 l4 9 136 + 34 a . Integrated area of plot of growth hormone concentrations for 30 min after TRH, minus baseline before TRH. Values are means :_ standard errors (n=l6). 62 Table l6.--Serum GH response to thyrotropin releasing hormone (TRH) during lactation, pregnancy and month of year Lactation Pregnancy Month of Year GH releas GH release GH releasg Month after TRH Month after RHD Month after RH (ng ml'lmin) (ng ml- min) (ng ml- 'n) 2 260 O 175 Jan. 124 l 125 Feb. 182 4 144 2 133 Mar. 143 3 110 Apr. 224 6 164 4 173 May 163 5 182 June 167 8 142 6 205 July 121 7 226 Sept. 215 10 110 8 161 Oct. 314 Nov. 143 Dec. 151 aEach variable used as main effect with other two variables serving as covariates. Analyses were performed as three separate analyses because of high correlation (r=.83) between stage of lactation and month of pregnancy. bIntegrated area of plot of GH concentrations for 30 min after TRH, minus baselines before TRH. Values are means. 63 Season of year did not affect (P>.05), using either one-way analysis of variance or analysis of covariance, the quantity of GH released, as measured by area under the response curve. Numerous researchers have demonstrated that administration of exogenous GH enhanced previously existing lactation in cows (Cotes et al., 1949, Donker and Peterson, 1951, Chung et al., 1953, Bullis et al., 1965 and Machlin, 1973). Since exogenous GH is galactopoietic in cows, the association of increased GH response curve areas at the 2-month stage of lactation and near maximum milking performance at this time suggests that circulating GH may be insufficient for maximum milk production during early lactation. The lack of effect of season or month of pregnancy on serum GH concentrations confirm the results in experiment 6. GENERAL DISCUSSION Serum prolactin and GH concentrations generally increase rapidly following IV injection of TRH. Peak concentrations are usually attained within 6 to 12 minutes post-TRH. When only peak hormone concentrations are used to evaluate pituitary responsiveness to TRH, the results are based only on one value per sampling period and this value may be rather variable depending upon the variation from cow to cow as to their "actual" peak and whether or not the hormone has had time to be distributed uniformily throughout all compartments of the vascular system. Therefore, to minimize this problem, a third degree least squares polynomial hormone response curve was calculated to determine total hormone release. This was usually based on 11 samples collected over a 30-minute period after TRH administration minus the baseline average of 4 to 7 samples collected 15 to 30 minutes before TRH injections. I think that this gives a more reliable estimate of hormone release following TRH administration. Season of the year has been reported to have a dynamic effect on serum prolactin, but not serum GH, concentrations in cattle (Koprowski and Tucker, 1973 and 1973a and Schams and Reinhardt, 1974). Highest serum prolactin concentrations are found in summer and lowest in winter. Illumination (Bourne and Tucker, 1975) and temperature 64 65 (Wettemann and Tucker, 1974) have been reported to be important in regulation of serum prolactin concentrations. Since increased tempera- tures and increased daylight occur concurrently during summer with the reverse true in winter, it is not possible to differentiate between the two effects except under controlled environmental conditions. Conse- quently, any effects thought to be associated with either temperature or daylight will be discussed as seasonal effects. Similarly to many other researchers, I found that season of year was very important in regulation of serum prolactin concentrations in all physiological states that were studied. Therefore, based on the results of my early experiments and on the results of other workers, subsequent experiments were designed to take into account seasonal effects during pregnancy and lactation. Even during the same season, increased serum prolactin concentrations were noticed in prepubertal heifers on days when temperatures were higher than normal for that time of year. Results from the estrous cycle experiment, which was conducted from mid-November to mid-December, clearly demonstrate the suppression of serum prolactin concentrations when compared with values obtained in other experiments during the summer. Season of year affected (P<.01) prolactin response curve areas in the pregnant heifer and lactating cow experiments. These results clearly demonstrate that season of year is a very important determinant of serum prolactin concentrations. The physiological significance of increased serum.prolactin concentrations during summer is unknown. Several stimuli that are reported to be "stressful" to cattle result in increased serum prolactin concentrations. Whether increased ambient temperatures and/or increased 66 daylight during summer can be considered "stressful" must await further investigation. Another plausable explanation may be associated with water metabolism. Prolactin is known to be associated with water metabolism in certain lower species (Nicoll and Bern, 1972). Increased water consumption is known to occur in cattle during summer (Winchester and Morris, 1956). Therefore, increased serum prolactin concentrations may be associated with the increased water load that occurs. More research is needed in this area to explain the physiological signifi- cance of increased serum prolactin concentrations during summer. Overall, neither serum prolactin nor GH concentrations were greatly affected by different physiological states. Pre-TRH serum prolactin concentrations averaged from 6 to 39 ng/ml in the seven experiments. However, if the experiment in prepubertal heifers, where increased serum prolactin concentrations were attributed to increased environmental temperature, and the estrous cycle experiment (conducted in Nov-Dec) were eliminated, pre-TRH serum prolactin concentrations were 13 to 22 ng/ml in virgin, pregnant or lactating cattle. These data suggest that different physiological states do not readily affect baseline prolactin concentrations. Similarly, pre-TRH GH concentrations (4 to 8 ng/ml) are not greatly different at the various physiological states that were studied. These data also suggest that baseline GH concentrations are not readily affected by different physiological states. Routes of administration (IV, IM and SC) of TRH affected (P<.01) the quantities (response curve areas) of prolactin and GH released during a 30-minute post-TRH sampling period. In contrast, during a 2-hr post-TRH sampling period, neither prolactin nor GH 67 response curve areas were different (P’.05) following IV or SC adminis- tration of TRH. Following IV administration of TRH, serum prolactin and GH concentrations reach peak values more rapidly and decline more precipitously than following IM or SC administration of TRH. In total these results suggest that IM or SC injected TRH is retained within the depot sites and released from these stores relatively slowly over a period of time. Also, when time was allowed for serum prolactin and GH concentrations to return to baseline concentrations following TRH administration, hormone response curve areas were not different. Therefore, when a prolonged hormonal response is desirable, the SC or IM route of administration of TRH should be the method of choice. Times required to reach maximum serum prolactin concentrations after IV administration of TRH in prepubertal heifers and postpubertal cattle were 7 to 10 minutes and 10 to 17 minutes, respectively. I speculate that these differences may be related to increased serum estrogen and/or progesterone concentrations in the postpubertal animals. These steroids may act to suppress TRH effects on the pituitary and thus delay prolactin release, but not affect the total amount of prolactin released. Dose of TRH affected prolactin and GH response curve areas. The quantities (response curve areas) of prolactin and GH released increased linearly (P<.01) with increases in the log of the dose (5 to 100 pg) of TRH. Fell et a1. (1973) and Noel et a1. (1974) reported a dose-response increase in serum prolactin concentrations following TRH administration in ewes and humans. Maximal prolactin response curve areas were attained using 50 pg TRH. Convey (1973) reported a dose- response increase in serum GH concentrations following TRH 68 administration in lactating cows. Unlike prolactin, the largest GH response curve areas were attained using 100 pg TRH. However, it is possible that a larger dose of TRH would result in still greater GH response curve areas. Peak GH concentrations and response curve areas following IV administration of TRH in pregnant heifers were slightly suppressed when compared with the responses in heifers during the estrous cycle and lactating cows. I speculate that these differences might possibly be related to estrogen and/or progesterone serum concentrations. Whether these differences are real and whether estrogen and/or progesterone are acting as moderators of TRH at the pituitary must await further research. Johke (1970) and Koprowski and Tucker (1973) reported increased serum prolactin concentrations associated with milking stimuli. Correlation coefficients between serum prolactin concentrations prior to milking, immediately after milking and 1 hour after milking and milk yield were -.03 (P>.05), .36 (P<.01) and .18 (P<.01), respectively (Koprowski and Tucker, 1973). I found correlation coefficients (P>.05) of -.01 and .15 between milk yield throughout lactation (average of daily production for 5 days prior to TRH) and prolactin and GH response curve areas following TRH administration, respectively. Although both stimuli released prolactin, they probably act through different mechanisms and therefore it is not surprising to find a significant correlation associated with milking stimuli and not TRH. Johke (1970) and Koprowski and Tucker (1973) found a diminished prolactin response to milking stimuli in late lactation. In contrast, I found that serum prolactin concentrations increased in response to TRH at all stages of lactation that were tested. In particular, prolactin response curve 69 areas at the 10-month stage of lactation was 4,193 ng ml-lmin (covariate adjusted mean). This value is as large as the average of serum prolactin response curve areas (4,084 ng ml-1min) at 2-, 4-, 6- and 8-month stages of lactation. These results indicate that the pituitary is equally capable of releasing prolactin in late lactation as in other stages of lactation, although there was a "tendency" for greater prolactin release at the 2-month stage of lactation. The decreased prolactin release associated with milking stimuli in late lactation may possibly be related to increased hypothalamic PIF or biogenic amine concentrations. If this is so, even though the pitui- tary is capable of releasing prolactin, the pituitary may be under near constant inhibition and thus, the diminished prolactin release. Nearly without exception, serum prolactin concentrations increased following TRH administration. In contrast, there were several animals that did not always have increased serum GH concentra- tions following TRH administration. This phenomenon has also been reported in humans (Saito et al., 1971). The GH response following TRH is not nearly as reliable as is the prolactin response. I have no explanation for this occurrence. The understanding of this phenomenon awaits further investigation. In overview, season of the year is more important in determin- ing serum prolactin, but not serum GH, concentrations than are any of the different physiological states that I studied. Furthermore, physiological states which I examined have relatively little effect on serum prolactin or GH concentrations. SUMMARY AND CONCLUS IONS Changes in serum prolactin and GH concentrations, following administration of TRH, were determined in prepubertal heifer calves, heifers during the estrous cycle and pregnancy, and in cows lactating 2 to 10 months. Parameters measured were: pre-TRH hormone concentra- tions, peak hormone concentrations after TRH, times required to achieve maximum hormone concentrations and integrated areas of plot of hormone concentrations for 30 minutes after TRH minus baselines before TRH. Overall serum prolactin concentrations increased from pre-TRH concentrations of 16 ng/ml to 81 ng/ml following IV administration of 10 pg TRH in prepubertal heifers. Prolactin response curve areas ranged from 1,381 to 1,585 ng ml-lmin (P>.05). Repeatability coefficients for day to day baseline prolactin concentrations and prolactin response curve areas following TRH administration were .27 and .61, respectively. These results suggest that there are great differences from day to day in baseline prolactin concentrations and that there is less day to day variation in prolactin response curve areas following TRH administration. However, the correlation coefficient, within days, between baseline serum prolactin concentrations and maximum serum prolactin concentra- tions following TRH administration was .64 (P<.01). These results 70 71 suggest that on a given day there is a close relationship between base- line and peak prolactin concentrations. Serum GH concentrations in prepubertal heifers prior to TRH averaged 8 ng/ml and increased to a maximum of 21 ng/ml after TRH. GH response curve areas ranged from 102 to 148 ng m1-1min between days 1 and 8 (P>.05). Based on averaged GH response curve areas, there was little day to day variation in pituitary responsiveness to TRH. However, because of animal variation, averaged GH response curve areas are misleading. This is demonstrated by the low repeatability coefficient (.35) for GB response curve areas following TRH. Similarly, the day to day repeatability coefficient for baseline GH concentration was low (.37). Correlation coefficients between baseline GH concentra- tions and post-TRH maximum GH concentrations was .60 (P<.01). Similar to prolactin, post-TRH maximum GH concentrations on a given day are very dependent on baseline GH concentrations. The quantity (response curve areas) of prolactin,re1eased increased linearly (P<.01) with increases in log of the dose (5 to 100 pg) of IV administered TRH. Peak serum prolactin concentrations increased from an averaged pre-TRH concentrations of 39 ng/ml to 143 to 277 ng/ml after TRH. Maximum quantities of prolactin released were achieved with 50 pg TRH. Areas under the GH response curve increased linearly (P<.01) with increasing log of the TRH dose (5 to 100 pg). Pre-TRH GH concen- trations averaged 5 ng/ml and increased to values of 11 to 46 ng/ml following TRH administration. The greatest dose of TRH tested (100 pg) resulted in the largest GH response curve areas, but may not have maxi- mally stimulated the pituitary to release GH. 72 Following IV administration of TRH, serum prolactin concentra- tions increased to a greater peak (174 ng/ml) more rapidly and declined more precipitously when compared with either IN (105 ng/ml) or SC (107 ng/ml) administration of TRH with times required to attain those peak values being 9, 22 and 22 minutes, respectively. Prolactin response curve areas were 2,170, 1,322 and 798 ng ml-lmin following IV, IM and SC administration of 25 pg TRH, respectively, during a 30-minute post-TRH sampling period. In contrast, during a 2-hr post-TRH sampling period, prolactin response curve areas following IV or SC injections of TRH were 3,639 and 4,198 ng ml-lmin (P>.05), respectively. I concluded that route of administration affected peak serum prolactin concentra- tions and time required to attain these peaks, but not the total amount of prolactin released when time is allowed for serum prolactin concen- trations to return to pre-TRH concentrations. Maximum GH concentrations were 35, 25 and 14 ng/ml following IV, IM and SC administration of 25 pg TRH, respectively. Times required to attain these peak values were 12, 14 and 22 minutes follow- ing IV, IM and SC administration of 25 pg TRH, respectively. GH response curve areas were 326, 147 and 115 ng ml—lmin following IV, IM and SC administration of 25 pg TRH, respectively, during a 30-minute post-TRH sampling period. During a 2-hr post-TRH sampling period, GH response curve areas were 504 and 218 ng md-lmdn following IV and SC injections of 50 pg TRH, respectively. It was concluded that, similar to prolactin, route of administration affected maximum serum GH concen- trations and times required to attain these peaks, but not the total amount of GH released when time is allowed for serum GH concentrations to return to pre-TRH concentrations. Therefore, when a prolonged 73 hormonal response is desirable, the SC or IM route of administration of TRH should be the method of choice. Times required to reach maximum serum prolactin concentrations after IV administration of TRH in prepubertal heifers and postpubertal cattle were 7 to 10 minutes and 10 to 17 minutes, respectively. I speculate that these differences may be related to increased serum estrogen and/or progesterone concentrations in the postpubertal animals. They may act to suppress TRH effects on the pituitary and thus delay prolactin release, but not affect the total amount of prolactin released. Serum prolactin and GH response curve areas did not differ (P>.05) following administration of TRH on days 0 (estrus), 2, 4, 7, 15 and 18 of the estrous cycle in heifers. However, there was a "tendency" for prolactin response curve areas, pre-TRH concentrations and maximum post-TRH concentrations at days 2 and 4 of the estrous cycle to be less than those at other stages of the estrous cycle. In contrast, there were no detectable effects of days of the estrous cycle on GH response curve areas. It was concluded that suppression of prolactin release from the pituitary at days 2 and 4 may be related to low serum estrogen and/or progesterone concentrations, but overall, neither serum estrogen nor progesterone plays a major role in regulation of serum.prolactin or CH concentrations during the estrous cycle in heifers. Neither prolactin nor GH response curve areas following TRH were affected (P>.05) by stage of pregnancy (3, 6 and 9 months). However, pre-TRH prolactin concentrations, maximum post-TRH prolactin concentrations and prolactin response curve areas after TRH "tended" to increase with advancing pregnancy. Whether this numerically greater 74 increase in prolactin is related to initiation of lactation awaits further study. Season of year did not affect (P>.05) the GH response curve areas following TRH administration. In contrast, prolactin response curve areas were affected (P<.01) by season of year being 6 to 15 X greater in summer than in fall and spring. I concluded that season of the year was probably of greater importance in determining serum prolactin concentrations than was pregnancy stage. Neither stage of lactation (2, 4, 6, 8 and 10 months) nor month of pregnancy (concurrent with lactation) affected (P>.05) the quantity (response curve areas) of prolactin released following TRH administra- tion. However, there was a "tendency" for greater prolactin release at the 2-month stage of lactation. In addition, month of year affected (P<.01) prolactin response curve areas following TRH administration. It was concluded from these data that the pituitary is more responsive to TRH during early lactation and that this increased responsiveness may be associated with increased milk yields at this early stage of lactation. Yet, when lactation was considered in its entirety, there was no correlation between prolactin and milk production (r=-.Ol). Neither month of pregnancy nor month of year affected (P>.05) GH response curve areas following TRH administration. In contrast, stage of lactation affected (P<.05) the GH response curve areas following TRH administration. I concluded from these results that the pituitary is more responsive to TRH during early lactation and that circulating GH concentrations may not be sufficient for maximum milk production. 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