INTERRELATIONSHIPS OF CERTAIN THERMAL AMD ENDOCRINE PHENOMENA AND REPRODUCTIVE FUNCTION IN THE FEMALE BOVINE BY . FRANCIS CHARLES GWAZDAUSKAS A DISSERTATION PRESENTED TO THE GRADUATE COUNCIl OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1974 ACKNOWLEDGEMENTS The author is sincerely grateful to Dr. W. W. Thatcher, Chairman of the Supervisory Committee, for his guidance, assistance, encourage- ment, patience and friendship during the study. Gratitude is expressed to Dr. C. J. Wilcox for his invaluable assistance with statistical analyses and preparation of this manuscript. The author is grateful to Dr. R. M. Abrams for the close association and assistance throughout this endeavor. A word of thanks is due Drs. D. H>Jarron, F. W. Bazer, D. Caton and H. H. Head for suggestions and moreover for their assistance in projects and the authors' increase in knowledge as members of the Supervisory Committee. A special thanks goes out to Dr. R. B. Becker for his encourage- ment and friendship throughout the entire study. The writer is indebted to Drs. P. S. Kalra, C. A. Kiddy and M. J. Paape for their assistance . during different phases of the experiments. Thanks are given to Mr. J. P. Boggs, Mr. A. L. Green and Mr. J. E. Lindsey for their help in the barn and with cattle handling; to Mr. M. Casey, Mrs. D. Clark, Mrs. L. Owens and Miss N. Baldwin for laboratory assistance and Miss L. Buzzerd for clerical assistance. Gratitude is expressed to R. W. Adkinson, J. R. Chenault, H. Roman, L. C. Fernandez, R. Eley, J. M. Knight, E. Muljono, E. G. Bonya, L. W. Whitlow, S. Chakriyarat and J. L. Kratz who as fellow graduate - n students assisted technically and encouraged the author academically. The author wishes to express his gratitude and appreciation to his wife, Judy, for her constant understanding and encouragement during the course of his studies. - m - TABLE OF CONTENTS Page No. ACKNOWLEDGEMENTS 1i LIST OF TABLES vi LIST OF FIGURES vii ABSTRACT ix INTRODUCTION 1 SECTION I 3 REVIEW OF LITERATURE 3 Influences of Thermal Stress on Reproductive Performance 3 Prostaglandins 10 Hormone Relationships of Uterine Blood Flov; and Temperature 18 SECTION II 21 HORMONAL PATTERNS DURING HEAT STRESS FROM PGF2a INJECTION THROUGH ESTRUS AND OVULATION AND FOLLOWING ADRENAL STIMULATION BY ACTH IN HEIFERS 21 Introduction 21 Materials and Methods 23 Results and Discussion 27 SECTION III 60 EXPERIMENT 1 : THERMAL CHANGES OF THE BOVINE UTERUS FOLLOWING ADMINISTRATION OF ESTRADIOL-! 7p 60 Introduction 60 - IV Table of Contents (continued); Page No. Materials and Methods 61 Thermocouple Preparation and Calibration 61 Surgical Techniques and Experimental Protocol ■ 62 Results and Discussion 64 EXPERIMENT- 2: THERMAL CHANGES OF THE BOVINE UTERUS FOLLOWING PGF2ct INJECTION THROUGH ESTRUS AND OVULATION 72 Introduction 72 Materials and Methods 72 Results and Discussion 74 SECTION IV 89 SUMMARY AND CONCLUSIONS ' 89 APPENDIX 95 LIST OF REFERENCES 108 BIOGRAPHICAL SKETCH 118 V - LIST OF TABLES Table Page No. 1 Physiological parameters of heifers in environmental 28 chambers at 21.3 C and 32.0 C. 2 Simple correlations between hormone measurements. 37 3 Physical characteristics of plasma in heifers at ^9 21.3 C and 32.0 C. 4 Overall least squares analyses of variance for 96 hormones in heifers at 21.3 C and 32.0 C. 5 Plasma progestins (ng/ml) following PGF^ injection. 97 6 Plasma estradiol (pg/ml) following PGFp injection. 98 7 Plasma estrone (pg/ml) following PGFp injection. 99 8 Plasma LH (ng/ml) following PGFp injection. 100 9 Plasma prolactin (ng/ml) following PGFp injection. 101 10 Plasma corticoids (ng/ml) following PGFp injection. 102 11 Plasma corticoids (ng/ml) prior to and following 103 200 lU ACTH. 12 Plasma progestins (ng/ml) prior to ACTH injection. 104 13 Simple correlations betv;een hormones and temperatures. 105 14 Analysis of variance for aortic and uterine temperatures. 106 15 Hormonal and temperature measurements for 6665 (6) and 107 JN15 (J). VI LIST OF FIGURES Figure PageJio, 1 Evaluation of thermal stress on transitory hormonal 24 changes in the bovine during the period of luteal regression, estrus and ovulation: Experimental design. 2 Sequential changes in plasma progestins in heifers 32 at 21,3 C or 32.0 C synchronized to the time of the LH peak. 3 Sequential changes in plasma estradiol in heifers 35 at 21.3 C or 32.0 C synchronized to the time of the LH peak. 4 Sequential changes in plasma estrone in heifers 38 at 21.3 C or 32.0 C synchronized to the time of PGFo injection. CO. 5 Sequential changes in plasma LH in heifers at 40 21.3 C or 32.0 C. 6 Sequential changes in plasma prolactin in heifers 43 at 21.3 C or 32.0 C synchronized to the time of PGFo injection. 7 Sequential changes in plasma corticoids synchronized 46 to the time of the LH peak using pooled means of heifers at 21.3 C and 32.0 C. 8 Transitory changes in plasma corticoids following 53 injection of 200 lU ACTH in heifers at 21.3 C and 32.0 C. 9 Uterine and aortic temperature prior to and following 65 IV injection of 12 ml sterile physiological saline. 10 Uterine and aortic temperature prior to and following 67 IV injection of 3 mg estradiol -17e. 11 ATuterus-aorta P'^'o'' to and following either 12 ml 69 saline or 3 mg estradiol--17B. vn - List of Figures (continued); Figure Page No. 12 Uterine and aortic temperature prior to and after 70 injection of estradiol -17e from continuous recording. 13 Uterine and aortic temperatures prior to and following 75 PGFo injections in G665. CO. 14 Uterine and aortic temperatures prior to and follov/ing 76 PGFo injections in JN15. ca 15 Changes in aT, , following PGF, injections. 77 u-a CO. 16 Uterine and aortic temperatures, LH and estradiol in 82 G655 and air temperatures. 17 Uterine and aortic temperatures, LH and estradiol in 83 JN15 and air temperatures. 18 Circadian uterine, aortic and air temperature changes. 85 19 Changes in AT associated with endogenous LH and 87 estradiol concentrations. vm - Abstract of Dissertation Presented to the Graduate Council of the University of Florida in Partial Fulfillment of the Requirein.nts for the Degree of Doctor of Philosophy INTERRELATIONSHIPS OF CERTAIN THERMAL AND ENDOCRINE PHENOMENA AND REPRODUCTIVE FUNCTION IN THE FEMALE BOVINE By Francis Charles Gwazdauskas December, 1974 Chairman: W. W. Thatcher Major Department: Animal Science Ten normally cycling Hoi stein heifers were assigned to one of two environmental treatment groups (21.3 C, 59% RH or 32.0 C, 67% RH). PGF^^ was used to cause luteal regression and synchronize estrus. Least-squares analyses were conducted to characterize treatment, animal and within- animal time trends in plasma progestins, estradiol, estrone, LH, prolactin and corticoids. Environmental treatment (32.0 C) evoked a 1.49 C increase in rectal temperature and a 3.59 C increase in skin temperatures. Length of estrus was shorter (P<.10) for the 32.0 C heifers. Two of four heifers at 21.3 C inseminated were pregnant at 40 days compared to none of five at 32.0 C. Average progestin concentration between treatments were not differ- ent (P>.10; .53 ng/ml at 21.3 C compared to .65 ng/ml at 32.0 C). Mean estradiol concentrations were significantly (P<.10) lower in 32.0 C heifers (3.45 pg/ml compared to 2.96 pg/ml). There was a significant elevation (P<.05) of estrone due to heat stress (1.55 pg/ml compared to 1.85). No significant differences (P>.10) were found in mean LH con- centrations between heifers at 21.3 C or 32.0 C. Preovulatory peak LH - ix - concentrations were 32.2 and 33.2 ng/ml plasma, respectively. All animals had a preovulatory LH surge, suggesting that hyperthermia did not prevent the triggering mechanism for LH release. Mean prolactin (14.51 ng/ml at 21.3 C compared to 14.78 ng/ml at 32.0 C) and corticoid (8.01 ng/ml at 21.3 C compared to 7.76 ng/ml at 32.0 C) concentrations were not different between temperature treatments (P>.10). In an attempt to determine if plasma dilution may have occurred, total protein concentration and osmolality were measured. There v/as no difference (P>.10) in total protein concentration or osmolality between treatment groups. The affinity (K ) of Cortisol for CBG was not a different between treatments (P>.10); however, the binding capacity of CBG for Cortisol was reduced (P<.05) in the 32.0 C heifers. Results of this experiment showed only subtle thermal effects on estradiol and estrone plasma concentrations and no effects on LH, pro- gestins, corticoids and prolactin. Apart from possible hormonal involve- ment with duration of estrus, heat stress did not appear to affect the hormonal mileau in peripheral plasma associated with corpus luteum re- gression, follicle growth and ovulation. Eight days following ovulation in the last heifer, 200 ID ACTH was injected, IV, into the 10 heifers. The 32.0 C heifers responded with significantly lower (P<.10) corticoid concentrations. The 6th order re- gression response curves were not parallel (P<.01) suggesting that the hot group response was earlier to reach a peak (75 compared to 105 min.), had a lower magnitude (73.5 compared to 100.2 ng/ml corticoids) and was of shorter duration (4 compared to 5 hr.). Because the first experiment did not specifically consider environ- mental and hormonal effects on uterine temperature it was necessary to - X - document possible estrogen induced uterine thermal changes. In the second experiment thermocouples were placed into the uterine serosa and saphenous artery of four dairy heifers. Injection of 3 mg estradiol-176 caused a .25 C decrease (P<.01) in the difference between uterine and aortic temperature (aT, ) by 2,5 hr. postinjection. In contrast, there was no U~a significant change (P>.10) in the AT,, , after injection of saline. u~a The final experiment was an attempt to document and evaluate changes in uterine temperature during the period of luteal regression, follicle growth and ovulation induced by PGFp under conditions of a mild heat stress. Thermocouples were placed into the uterine serosa and aortic blood vessel of four dairy cattle. PGF^ caused an immediate drop in uterine and aortic temperatures, and a decrease in the AT of almost u-a .4 C at 45 min. postinjection. The two cows, in which thermocouples remained operational for the duration of the study, had monophasic daily uterine and aortic temperature rhythms. However, both temperatures lagged about 6 hr. behind air temperature changes. Uterine temperatures reached 40 C for periods of up to 6 hr. Failure to detect an association between AT and hormonal measurements may have been due to a time lag association. Not until the preovulatory surge of LH was there an appreciable rise in aT^_^ (P<.01), and this occurred at a time when estradiol was decreasing. The mild environmental heat stress may have contributed to the high uterine and aortic blood temperatures. V '/ / -.*. 1 'U Y tt^^^-x Y ..... ^r "—//-^^^f^-U' Chairman - XI INTRODUCTION Reduced reproductive efficiency occurs during the hot seasons of the year in many parts of the United States. Lov/ered conception rate due to heat stress occurs over a prolonged period of the year in Florida and represents a major production problem to dairymen. A 12 year study in the University of Florida dairy herd revealed a con- ception rate per service of less than 40% (Gwazdauskas, Thatcher and Wilcox, 1975). Economically, poor reproductive performance under con- ditions of thermal stress decreases heifer replacement availability and long term milk production and increases calving interval and culling rate. Before systems for reproductive management can be developed to counter these adverse effects of heat stress, several fundamental questions must be answered. Among these are: 1. How does a standard heat stress alter hormonal and phy- siological responses during the normal estrous cycle? The objective of the first experiment (Section II) was to characterize hormonal changes (progestins, estradiol, estrone, LH, prolactin and corticoids), rectal temperature, plasma protein concentration and osmolality and plasma Cortisol binding capacity (CBC) in heifers subjected to a standard heat stress (21.3 compared to 32.0 C). In addition, plasma 1 - - 2 - corticoids in response to ACTH were measured to evaluate possible thermal stress effects on adrenal responsiveness. 2. What are the factors influencing uterine temperature? Can estradiol, which is known to have a marked effect on uterine blood flow and metabolism, alter uterine temperature? What are the changes in uterine temperature during the period of luteal regression, follicle growth and ovulation under conditions of mild heat stress? In Section III a series of experiments were designed in an attempt to answer these questions. Prostaglandin Fo (PGFp ) causes luteal regression in the bovine and has enabled the researcher to use it efficiently to control endo- crine and physiological changes near the time of estrus and ovulation. Such a compound maximizes use of experimental facilities over short periods of time without adverse chronic alterations of normal bovine physiology. In answering questions one and two above, PGFp was used to synchronize the hormonal events associated with corpus luteum re- gression, estrus and ovulation. SECTION I REVIEW OF LITERATURE Stress is defined as a condition harmful to an organism, which results from inability of the organism to maintain a constant internal environment (Taber, 1961). Factors involved in altering homeostasis Include trauma, surgical operations, restraint, extreme cold or heat, Intense solar radiation, social stress due to peck order, nutritional stress and internal stress caused by pathogens or toxins (Hafez, 1968; Guyton, 1966). The purpose of the initial review section i? to report on the effects of thermal stress on reproductive performance with major emphasis on hormonal or endocrine aspects. Influence of Thermal Stress on Reproductive Performance Hot environments may exert their depressive effect on fertility via the gonads,' accessory sex glands, uterine environment, gametes or endocrine system (Hafez, 1959; Ulberg and Burfening, 1967). Reproductive behavior has been shown to be altered by heat stress, in that estrous duration was shorter (Branton et aj_. , 1957; Gangwar, Branton and Evans, 1965; Hall et al_. , 1959), there v/as an increased frequency of quiet ovulations (Labhsetwar et^ al. , 1963) and anestrus (Bond and McDowell, 1972) and a reduction in estrous intensity (Gangv/ar, Branton and Evans, 1965), -3- High temperatures exert direct effects on fertilized ova grown in vitro (Alliston et al. , 1965), Fertilized ova grown through first cell division at 40 C in vitro had a lower rate of embryo survival than those grown at 38 C when returned to synchronized pseudopregnant recipient rabbits. There were no morphological differences between ova in dif- ferent media. As the period of culture at 40 C was delayed to second cell division, differences in post-implantation death losses disappeared. An environment of 32.2 C and 65% Relative Humidity (RH) did not inhibit estrus or alter ovulation rate in sheep (Alliston and Ulberg, 1961). However, an increase in embryo death was detected when embryos (2-32 cell stage) were transferred from donor ewes kept at 32.2 C to recipient ewes at 21.1 C ambient temperature. Embryo survival was highest when both donor and recipient ewes were maintained at 21.1 C, indicating that damage to the early embryo was most likely to occur in uteri of ewes kept at 32.2 C. Heat stressing one or both parents of a mouse embryo affected the rate of thymidine, uridine and guanine incorporation into nucleic acids during pre-implantation development, which may lead to altered DNA and RNA synthesis and subsequent embryonic mortality (Sheean, Durrant and Ulberg, 1974). Due to limitations of facilities and methodology, most investi- gations of the effects of heat stress on hormonal balance and their relationships to reproductive performance have monitored only one or two hormonal responses. Therefore pooling results from different laboratories by combining data from different animals within and between species can be misleading when an effort is made to develop a hypothesis on how thermal stress affects reproduction. Stott, Thomas and Glenn - 5 - (1967) found progesterone to be elevated on the day of estrus in thermally stressed cows. Heifers maintained at 32 C and 21 C for 72 hr. beginning at the onset of estrus had conception rates of 0 and 48%, respectively, according to a report by Dunlap and Vincent (1971). Associated with the decreased fertility was an elevated plasma progestin concentration of only ,42 ng/nil plasma (Mills et a^. , 1972). In con- trast, Stott and Wiersma (1973) reported depressed plasma progestin levels during chronic heat stress in the bovine. Evidence of detrimental progesterone effects on embryo cleavage stages has been reported by Dickman (1970). Fertilized ova were transferred on day 4 of pregnancy to pseudopregnant rats which had been ovariectomized on day 2. When transfer was preceded by 2, 3, 4, 5 or 6 days of progesterone treatment in pseudopregnant rats, 49, 38, 13, 2.5 and 2% of the transferred morula developed into fetuses. However, when blastocysts were transferred, there was a 62.5% fetal survival. Overstimulation with progesterone apparently interfered with embryonic development. Johnsson et al_. (1974) reported a 60 to 75% reduction in fertility in ewes receiving a single injection of progesterone on days 0, 1, 2 or 3 or daily injections on days 1 to 4. The progesterone given before day 4 may have affected embryo transport through the oviduct or directly altered it and therefore inhibited or abolished its ability to cppe with the 'luteolysin' and prevent corpus luteum regression during pregnancy. Progesterone, superimposed on estradiol administration in ewes, caused a prompt decrease in uterine blood flow (Greiss and Anderson, 1970). Extreme or prolonged limitation of blood flow to the vicinity - 6 - of the embryo can result in fetal death. Blockage of blood supply to the uterus one day post coitum in the mouse was most detrimental to implantation rate (Senger et al. , 1967). These observations may have been the result of the uterine coagulation procedure since necrosis of the tissue was detected (F. W. Bazer, personal communication). However, coagulation of blood vessels to one uterine horn resulted in 51% fewer embryos migrating to the uterus by 4 days after mating in mice. At 10 days post mating there were fewer live fetuses on the coagulated side compared to the control side (57% compared to 73%). Therefore, reduced blood flow may be responsible for failure of embryo transport to the uterus and increased fetal death rate (Bazer, Ulberg and LeMunyan, 1969). Thus, if heat stress increased plasma progesterone levels, an altered blood flow may be a factor associated with reduced fertility. Heat stress has been shown to cause elevated plasma corticoid levels in the bovine within 4 hr. of exposure which suggests increased adrenal activity (Christison et al. , 1970), Other workers reported that plasma corticoids decreased during chronic heat stress in cattle (Alvarez and Johnson, 1973; Christison and Johnson, 1972; Rhynes and Ewing, 1973) and that corticoid turnover rates decreased (Christison and Johnson, 1972). However, chronic hyperthermia resulted in elevated epinephrine and norepinephrine, though corticoids were depressed, which suggested a decreased sensitivity to physiological actions of catecholamines (Alvarez and Johnson, 1973). Shayanfar (1973) compared adrenal responsiveness to adrenocorticotropin (ACTH) in cows exposed to ambient temperatures of greater or less than 21.1 C. At ambient temperatures above 21.1 C, plasma corticoid response to ACTH was slower. peak levels were lower and the response was of shorter duration. Yousef and Johnson (1967) reported a 30 to 40% increase in heat production fol- lowing injections of hydrocortisone acetate to cattle at 35 C ambient temperature. Thus, during prolonged thermal stress, depressed plasma corticoids and lowered adrenal responsiveness to ACTH may be indicative of altered adrenal function. Madan and Johnson (1971) have reported that the preovulatory peak of plasma LH and basal plasma LH concentrations were lovyer in heifers maintained at 33.5 C and 55% RH compared to those at 18,2 C and 55% RH. These results support the hypothesis that thermal stress may alter secretion or metabolism of various hormones associated with reproductive function. However, Riggs, Al listen and Wilson (1974) detected a breed difference in response of the pre-ovulatory surge of LH to heat stress in gilts. Heat stressed pigs of the Duroc breed had no difference in magnitude of the preovulatory plasma LH surge compared to controls, whereas pigs of the Hampshire breed had a three to sixfold increase in the preovulatory peak of LH compared to their controls. It appears that species and breeds may, therefore, respond differently to thermal stress and that inferences among species and breeds must be reviewed with caution. Koprowski and Tucker (1973), Schams and Reinhart (1974) and Thatcher (1974) found elevated peripheral plasma prolactin concentrations during the hot months of the year, suggesting that photoperiod and temperature modulate prolactin release. At a constant day length, calves exposed to 27 C had significantly higher plasma prolactin con- centrations than those exposed to 10 C, whereas at 27 C prolactin levels - 8 - were only slightly higher than at 21 C (Wetteman and Tucker, 1974). However, Karg and Shams (1974) found a positive correlation between day length and basal prolactin concentrations in male and female cattle. Relkin (1972) demonstrated that changes in light:dark ratios for rats altered pituitary content and plasma concentrations of prolactin. It would appear that this question has yet to be resolved in cattle, i.e. whether photoperiod or temperature is the primary factor affecting plasma prolactin concentrations. These various studies suggest that thermal stress may alter circulating plasma concentrations of certain pituitary, adrenal and ovarian hormones. Such excesses or deficiencies of these hormones may influence certain reproductive phenomena and account for lowered fertility. Environmental factors play an important role in bovine fertility. Seasonal depressions in conception rate due to heat stress effects on the male can be eliminated through A.I. (artificial insemination) in which semen from bulls can be collected and frozen during cooler times of the year. Under these circumstances Stott (1961) still found a seasonal depression in breeding efficiency of cows which paralleled high climatic temperatures in Arizona and California. Thus, this experiment indicated that altered reproductive efficiency in the female was the major contributor to summer depression of fertility. In Florida, a year-long study was conducted to relate climatic, rectal and uterine temperatures, plasma corticoid and progesterone concentrations, breed, service number, time of service, sire and age to conception rate (Gwazdauskas, Thatcher and Wilcox, 1973). Significant - 9 - effects due to environmental temperature on the day after insemination, rectal and uterine temperatures at insemination, sire and days post- partum were detected on conception rate. Deleting environmental temperature from the statistical model revealed significant effects of uterine temperature the day after insemination on fertility. Relationships between uterine temperatures at insemination or the day after insemination with fertility were intriguing. Uterine temperature the day after in- semination appeared to be positively associated with environmental temperature on that day. Their inverse associations with fertility may reflect direct detrimental thermal effects on early cleavage and development of the embryo. In contrast, the association of uterine temperature at insemination with fertility might be related to certain physiological (uterine blood flow and vaginal thermal conductance) and hormonal changes occurring at estrus that may be associated with proper timing of insemination to achieve maximal fertility. In a subsequent study with 12 years of data, effects on conception rate of age of cow, inseminator, service sire, month, year, breed and 21 climatological variables were evaluated (Gwazdauskas, Wilcox and Thatcher, 1975). Age, inseminator, sire and breed had significant effects on conception. Maximum temperature the day after insemination, rainfall the day of insemination, minimum temperature the day of insem- ination, solar radiation the day of insemination and minimum temperature the day after insemination were the five highest ranking climatological variables associated with fertility. The most potent environmental variable, maximum temperature the day after insemination, had a significant curvilinear relationship with fertility. As maximum - 10 - temperature increased from 21.1 to 35 C, conception rates declined from 40 to 31% Month effects were found to have a significant relationship with fertility when climatological measurements were deleted from statistical models. This agreed with most previous research (Stott, 1961; Hafez, 1959). In no case were month effects significant when climatological n.easurements were included in the model, suggesting that month effects may have represented climatological factors to a greater degree than nutritional and management factors. This work clearly showed the importance of ambient environmental conditions at the time of insemination and fertilization on bovine fertility. Alterations of peripheral plasma estrogen concentrations during periods of thermal stress have not been reported previously. Therefore, speculation as to estrogenic effects on uterine blood flow and estrogen participation in pre-ovulatory LH release cannot be reviewed here. Physiological and endocrine factors controlling thermal properties of the uterus at estrus and ovulation need clarification. In addition, effects of stressful ambient temperatures on these factors and uterine temperature need further study as they relate to fertility. Prostaglandins Prostaglandins (PG), unsaturated 20 carbon fatty acids containing a cyclopentane ring and two alipahtic side chains, were first dis- covered in extracts of human and sheep seminal vesicles during the 1930's. The first report of their activity before identification v/as shown v/hen fresh semen was placed into the human uterus and caused the - n - uterus to contract or relax (Kurzok and Lieb, 1930). The luteolytic effects of PGFp were reviewed extensively by Inskeep (1973), and effects in cattle v/ell documented by Hafs et al_. (1974) and Chenault (1973). Injection of PGF2^-Tham Salt in the bovine does not drastically alter the normal sequential hormonal patterns leading to estrus and ovulation. In addition, fertility of cattle to the PGFo induced ovulation is apparently the same as in a normal spontaneous ovulation of control cattle (Lauderdale et al , , 1974). Therefore, PGFo can be CO. used as an experimental tool to synchronize estrus and investigate physiological and hormonal changes under conditions the researcher wishes to impose. This would be beneficial in large animal research where animal numbers may be few and biological events (the estrous cycle and pregnancy) of long duration. The intention of this portion of the review on prostaglandins is to recapitulate various effects of prosta- glandins on the circulatory system and reproductive tract. Such knowledge is essential in evaluating effects of PGFp in the following experiments. The role of the autonomic nervous system in controlling uterine contractility and blood flow has been discussed by Shabanah et al . (1964). The parasympathetic system generates uterine contractions and causes vasodilation. The excitatory (a) action of the catecholamines is manifested chiefly on the circular fibers whereas the inhibitory (3) action influences the whole myometrium. Acetylcholine causes vasodilation, especially of smaller blood vessels (Koelle, 1970). Epinephrine is a vasopressor. Vasoconstriction occurs markedly in the venous system, as well as smaller arterioles and precapillary sphincters. - 12 - Norepinephrine increases peripheral vascular resistance due to veno- constriction (Innes and Nickerson, 1970). Estrogens govern the parasympathetic (acetylcholine) activity and are responsible for the basic contractile mechanism of the uterus (Shabanah et aj_. , 1964), whereas progesterone influences the s^onpathetic activity (epinephrine and norepinephrine). Morris (1967) reviewed the sympathetic vaso- constrictor action on the uterine vascular bed. Epinephrine and nore- pinephrine reportedly cause a decrease in uterine blood flow with a concomitant increase in arterial pressure, suggesting increased vascular resistance in the uterus. Isoproterenol also acts as a vasoconstrictor. These vasoconstrictor actions appeared to be due to increased vascular resistance because myometrial tension changes v.'ere negligible. CI egg (1966) reported that prostaglandins produce two types of effects on smooth muscle. They produce direct short-lived actions such as stimulation of the isolated uterus or relaxation of the isolated tracheal chain preparation. Alternately, they potentiated long-term effects of other stimulants when given in low doses. An example of an indirect long-term effect of prostaglandins (PGF and PGE series) is depression of responses of various isolated smooth muscle preparations to sympathomimetic substances (epinephrine, norepinephrine, phenylephrine and isopropyl noradrenaline). Different classes of prostaglandins have various effects on smooth muscle and blood pressure and have been reviewed by Bergstrom et al . (1968). PGE's and PGFp cause contraction of uterine myometrium in rats c^nd guinea pigs. However, in humans, the PGE's decrease tonus, frequency 13 - and amplitude of spontaneous contractions of the myometrium. There is an increase in sensitivity of the rat uterus to PGF^ following estrogen treatment (Anggard and Bergstrom, 1963). PGFp also has been shown to stimulate and increase tone of the rabbit fallopian tube in vivo (Bergstrom et al_. , 1968; Horton and Main, 1965), whereas PGE, causes relaxation. Isolated strips of human myometrium have a regular motility pattern. This motility in the nonpregnant myometrium was inhibited by PG's A, B and E; however, PG's F, and F^ stimulated contractions. The la ^a sensitivity of the myometrium was highest late in the menstrual cycle and during pregnancy. PGF2 also increased motility of the human oviduct in vivo. Intra-uterine application of PGF^ or intravenous infusion increased the motility of the non-pregnant human uterus (Eliasson, 1973). Following intramuscular injection of 10 or 20 mg PGFp in non- pregnant women, no cardiovascular changes were observed but there was pain at the injection site and increased uterine activity within minutes. The uterine contractility lasted 2 to 3 hr. (Karim et al. , 1971). Within 1 to 6 hr. after vaginal insertion of PGEp or PGFp , 10 to 12 women had menstrual -like uterine bleeding. This bleeding was preceded by a marked increase in uterine contractions which started within 10 min., peaked betv/een 60 to 90 min., and lasted about 4 hr. PGE and PGF^ induced uterine activity that was similar to that recorded for the non-pregnant uterus during the time of the menstrual flow. Contractions measured between 50 to 200 mm Hg and occurred every 1 to 2 min. (Karim, 1971). In non-pregnant dogs, PGE-, infused into the uterine artery reduced perfusion pressure. The dilator effect of PGE, was seen at doses as - H - little as 20 pg/ml blood. Such potent vasodilatory effects of PGE^ were not seen in pregnant dogs near term even with large doses. PGF2^, on the other hand, had little effect on vascular smooth muscle in dogs, but potentiated responses to sympathetic nerve stimulation, occasionally in parallel with increased responses to norepinephrine. PGF2^ appears to work primarily on nerve terminals in the dog uterus since there was a greater effect on neurogenically induced vasoconstrictor responses than to responses of norepinephrine itself (Clark et a_l . , 1972). In a review by Brody (1973) PGF2^ was reported to influence effector response to sympathetic nerve stimulation. Vasoconstrictor action in cutaneous and muscle vessels was facilitated by PGF2^ without any change In responsiveness of the vessels to norepinephrine, suggesting that PGF2^ facilitated liberation of the adrenergic transmitter. This specificity was not found in venous smooth muscle when PGF2^ facilitated responses to both sympathetic nerve stimulation and to norepinephrine. Thus, PGF^ venoconstrictor action was dependent upon integrity of sympathetic 2a innervation. No changes in cholinergic vasodilator nerves were noted in the presence of prostaglandins (Brody and Kadowitz, 1974). Responses of uterine vessels to norepinephrine were potentiated at PGFg^^ concentra- tions which had no effect on uterine vascular resistance. Recently, Ryan et al. (1974) showed that, in the dog.PGE^ redistributed the blood flow from the myometrium to the endometrium. Therefore, PGE^ may be a vasodilator intermediate in an estrogen induced uterine hyperemic response. To test this hypothesis estrogen was in- jected into rats causing a visible intense hyperemia and a doubling of - 15 - uterine blood volume. In comparison, rats pre-treated with indomethacin, a prostaglandin inhibitor, failed to show a large increase in uterine blood volume. In conflict with the observation that PGFo was a vaso- constrictor was the finding of elevated uterine PGF content following estrogen treatment which could be inhibited by indomethacin pre- treatment. Except for this latter observation, the PGF series appears to be associated with vasoconstrictor actions and reduced blood flow to the uterus. The cardiovascular actions of PGF^ also are complicated because da of quantitative species variation. Anggard and Bergstrom (1963) reported that intravenous injection of P6F2 into cats caused increased right ventricular pressure and a decreased systemic blood pressure. Intra- arterial injections into muscles caused increased blood flow through that area, i.e. vasodilation. PGF^ perfused into rabbit hindquarters also caused tissue vasodilation. Horton and Main (1965) reported that PGFp or PGE, injected intravenously in rabbits caused a fall in arterial blood pressure. A review by Bergstrom et^ al_. (1968) contrasts these re- sults with the pressor action of prostaglandins in the rat, dog and spinal chick. In dogs the pressor action of PGF^ is accompanied by an increase in cardiac output and right atrial pressure, but the calculated peripheral resistance was unchanged. It appeared as if there were a decrease in venous capacitance because when a pressure stabilizer was put into the venous side, it caused a shift of blood into the stabilizer reservoir. Ducharme et al_. (1968) reported similar results and also found that, in the dog, PGFp had little effect on femoral arterial pressure or small artery pressure but caused an increase in small vein - 16 - pressure when administered to an innervated limb. Abolishing the sympathetic chain to the limb eliminated the venoconstrictor activity of PGFp . They found no real change in myocardial contractility. Thus the pressor action was due to an increased venous return. Horton (1969) found PGF to be weakly dilatory on arterioles. In some species (rat and dog) they act as a venoconstrictor, thus in- creasing venous return and cardiac output. Neither PGE, or PGF, injected close-arterially released catecholamines from the adrenals of anesthetized cats, but PGE-, did so in dogs. PGF^^ injected intra- arterially caused no constant change in blood pressure or in baroreceptor discharge frequency. Moreover, intravenous injections caused a transient rise in arterial pressure which was associated with an increase in baroreceptor discharge. It appeared that the increased discharge fre- quency was secondary to the pressure rise because in animals where the blood pressure fell slightly, so did discharge frequency. PGF2^ in- jections into the carotid artery resulted in a variable response on chemoreceptor discharges. Intravenously injected PGF2^ caused a small increase, decrease or no change at all in blood pressure (McQueen and Belmonte, 1974). Therefore, the authors suggested that direct action was on pressure changes not by v/ay of baroreceptors. These actions may be related to the rapid disappearance of prostaglandins as only 5 to 10% of the injected PGFp was detected 1 min. later and negligible PGFo was found at 90 sec. (Raz, 1972). Also, more than 95;^ of injected prostaglandins were removed during one circulation through the pulmonary vascular bed (Ferreira and Vane, 1967), Various investigators observed actions of prostaglandins on 17 respiratory smooth muscle. Main (1964) has shown that PG's E, , E^, E- and F^ relaxed tracheal muscle in vitro in rabbit, guinea pig (also Puglisi, 1972), ferret, pig, sheep, cat and monkey preparations. Except fn the cat, they decreased lung resistance to inflation in vivo (also Anggard and Bergstrom, 1963). PGFp has similar biological activity to PGFi , so these observations should hold for its actions. This con- elusion was confirmed in a cat-trachea preparation by Morton and Main (1965), in which PGF2^ inhibited acetylcholine produced contractions. In the dog the action of PGF^ was a reduction in dynamic lung compliance and alveolar ventilation (Horton, 1969). Investigations on systemic actions of prostaglandin in the bovine are very limited to date. Lewis and Eyre (1972) reported that PGE, and l^ T wared systemic blood pressure in calves, but PGF^ caused a pressor response. Furthermore, pulmonary arterial pressure and abdominal venous pressure were raised by the three substances. PEF^ caused con- traction of the pulmonary artery and vein and produced an increase in heart rate. Also noted v/as an increase in respiratory volume produced by PGF^^. Anderson et aj_. (1972) also concurred that PGFp increased pulmonary arterial pressure, but they found a drop in cardiac output and essentially no change in femoral arterial pressure, left ventricle and diastolic pressure, heart rate, blood gases and pH. There is a definite need for more study on actions of prostaglandins to determine their roles in physiological functions in the bovine. Additional work is needed because of the contradictory results obtained among and within species. - 18 - Hormone Relationships of Uterine Blood Flow and Temperature The uterus responds to cyclic hormonal changes during the estrous or menstrual cycles. Blood levels of estrogen and progesterone are in- volved with this phenomena. In the human, the first half of the cycle is associated with rapid grov/th of the uterine vascular elements and is under estrogenic control. This is a period of tissue repair and pro- liferation. The latter half of the cycle is characterized by glandular secretory activity and elaboration of vascular elements under the con- trol of estrogen and progesterone (Reynolds, 1949). One of the principal characteristics of the uterus following estrogen administration is its bright red color. The degree of redness suggests a high level of oxygen saturation of the blood and there is a high rate of blood flow. In the presence of an active corpus luteum (progestational influence), the uterus is bluish in color. Oxygen consumption is low and blood flow is sluggish. Oxytocin causes intense muscular spasms within the uterus without affecting the rate of blood flow, whereas vasopressin causes relaxation of uterine musculature but a constriction of its vasculature (Reynolds, 1949). Uterine hyperemia following injection of estrogen has been estimated in ewes by direct collection of uterine venous blood (Huckabee et^ al_. , 1970), by flow meters (Greiss and Anderson, 1970; Rosenfeld et^ al . , 1973) and microspheres (Rosenfeld et al_. , 1973). Endogenous estrogens produced during the estrous cycle appear to have similar effects on uterine blood flow. Patterns of change in plasma estradiol concentrations (Scaramuzzi , Caldwell and Moor, 1970) are very similar to records of - 19 - uterine blood flow changes in the ewe (Greiss and Anderson, 1970; Huckabee e;t al. , 1968, 1970). Specificity of estrogen actions on uterine blood flow have been shown by local injection of estrogen into one uterine horn artery. An increase in blood flow was measured only in that uterine artery (Resnik et al. , 1974). Progesterone injected into ovariectomized ewes did not alter uterine blood flow, whereas progesterone superimposed on estradiol in- jections caused a decrease in uterine blood flow rates (Greiss and Anderson, 1970). Estrogens did not appear to affect systemic blood pressure (Huckabee et al. , 1968, 1970; Resnik et al. , 1974), but caused a fall in the coefficient of oxygen utilization i Jl^^ X 100] in the uterus. However, due to the higher uterine blood flow there was essentially no change in oxygen uptake of the uterus (Huckabee el al. , 1968, 1970). Thus a dissociation between uterine metabolic rate and the rate of blood flow might be reflected in temperature differences between the uterus and aortic blood. In sheep a decrease in the temperature difference between the uterine cavity and aortic blood provided a con- venient method for monitoring increased uterine blood flow changes following estrogen injection. A rise in blood flow resulted in a lowered uterine temperature (Abrams el al. , 1970a, 1971).. The actions of estrogen to lower uterine temperature may be mediated through its inter- action with acetylcholine to cause vasodilation (Shabanah et al. , 1964) or through the release of uterine histamine which was shown to be involved in a rapid onset of hyperemia and water imbibition , ( Jensen and DeSombre, 1972). Lowering the rate of uterine heat production is unlikely because of the many metabolic activities induced by estrogens - 20 - (Talwar and Segal, 1971; Jensen and DeSombre, 1972). In cattle, plasma estrogens increased prior to estrus and declined precipitously during estrus (Chenault et al_. , 1973; Henricks, Dickey and Hill, 1971). These changes may have distinct thermal effects on the uterus. Greiss and Anderson (1969) reported increased uterine blood flow associated with the onset of estrus in sheep, which could cause a drop in uterine temperature (Abrams et_ al_. , 1970a, 1971; Caton et al . , 1974) and thus be related to an optimal time for insemination to achieve maximal fertility. However, the thermal response of the bovine uterus to estrogen has not been documented. Although several hormonal changes due to hyperthermia have been documented in the bovine there is a sparcity of results related to a multiplicity of hormonal responses to a controlled thermal stress in which such sources of variation due to breed, age, animal and time responses are evaluated. Uterine blood flow and temperature relation- ships have been reported in sheep in response to estrogen injections, but have not been reported in the bovine. Also, changes in uterine temperature during phases of the estrous cycle under conditions of mild heat stress have not been reported. SECTION II HORMONAL PATTERNS DURING HEAT STRESS FROM PGF2a INJECTION THROUGH ESTRUS AND OVULATION AND FOLLOWING ADRENAL STIMULATION BY ACTH IN HEIFERS Introduction Lowered conception rate due to heat stress occurs over a pro- longed period of the 'year in tropical and subtropical climates. Before systems for reproductive management can be developed to counter these adverse effects of heat stress, a more complete understanding of endocrine and physiological changes within the same animals must be made. We need to know how a standard heat stress alters hormonal and physiological responses during the normal estrous cycle, and determine if chronic heat stress alters adrenal responsiveness to an IV injection of ACTH. Due to limitations of facilities and methodology, most inves- tigations of heat stress effects on hormonal balance and their relationships to reproductive performance have monitored only one or two hormonal responses. Therefore, pooling results from different laboratories and from different animals within and between species can be misleading when an effort is made to develop a hypothesis on how thermal stress affects reproduction. - 21 - 22 - Reduced fertility in hot environments was associated vyith elevated body temperature (Dunlap and Vincent, 1971; Gwazdauskas, Thatcher and Wilcox, 1973). Hot climates may exert their depressive effects on fertility by acting on the gonads, uterine environment, endocrine system or gametes (Hafez, 1959; Ulberg and Burfening, 1967). Seasonal Infertility has been attributed primarily to the bovine female (Stott, 1961). Studies on hormonal alterations due to thermal stress have failed to document interrelationships of more than two different hormones in the same animals. Plasma progestin changes have been documented by Mills Gt al. (1972), Abilay and Johnson (1973) and Abilay, Johnson and Seif (1973); changes in plasma corticoid levels have been reported by Lee, Roussel and Beatty (1973), Christison and Johnson (1972), Abilay c\nd Johnson (1973), Shayanfar (1973) and Miller and Alliston (1974a). Plasma LH changes have been reported by Madan and Johnson (1971) and Miller and Alliston (1973) and seasonal changes in prolactin have been detected by Koprowski and Tucker (1973), Schams and Reinhart (1974) and Thatcher (1974). Such hormonal alterations may be causative agents contributing to suppressed estrous manifestation and depressed fertility under hot climatic conditions. There are essentially no studies designed to test specific effects of heat stress environments on a multiplicity of hormonal responses with- in the same animal. Such a study is needed in which variations due to breed, age, animal (among and within) and hormonal interrelationships are considered in evaluating thermal stress effects. Objectives of this study were to characterize changes in peripheral plasma concentrations of LH, progestins, estradiol, estrone, prolactin 23 - and cor.ticoids after an intramuscular (IM) injection of PGF2 -Tham Salt (PGF^ ) under controlled environmental temperatures (21,3 C compared to 32,0 C), and to determine if chronic heat stress alters adrenal responsive- ness to an intravenous (IV) injection of ACTH (200 lU). Materials and Methods Ten normally cycling Hoi stein heifers at the USDA, Agricultural Research Center, Beltsville, Maryland, were assigned alternately, based on age, to one of two treatment groups (figure 1). All heifers were placed in one of two environmental chambers at 21.3 C, 59% RH for 2 weeks. On day 9 of this adaptation period, 8 of 10 heifers in the luteal phase of the estrous cycle received 30 mg PGFp ^ (IM) to cause luteal regression. This injection allowed all heifers to be in the luteal phase of the cycle when PGFp v/as injected 12 days later. PGFp effectively regresses the bovine corpus luteum and synchronizes estrus. Lauderdale et al. (1973, 1974), Louis et al, (1974) and Chenault et al_. (1974) reported that fertility at the synchronized estrus, and induced hormonal changes resulting in estrus and ovulation appeared normal in PGFo treated cattle. Thus, it was felt that PGF^ treatment could be 2a 2a . utilized as a tool to best control reproductive status of the heifers and maximize efficient use of the chambers. On day 14, the environment of one chamber was adjusted to 32,0 C, 67.2% RH. On day 20, all heifers were fitted with indwelling polyvinyl jugular catheters (V-7; Bolab Inc., Derry, N.H.) and PGFp^ (30 mg, IM) ^PGF2a-Tham Salt was graciously supplied by Dr. J. Vi. Lauderdale, Upjohn Co., Kalamazoo, Michigan. 24 - 10 Heifers 30 mg 5 Hei fers (21 3 0 at 21.3 C 2a 1 ( 5 Hei fers H — (32 0 C) Day 1 Day 9 Day 14 Catheterize sample ewery 6 hr. \ Day 20 30 mg P^^2a I 6 hr. samples- Day 21 4 hr. samples Day 23 ovulation 200 lU ACTH 1 8 Days post ovulation Figure 1. Evaluation of thermal stress on transitory hormonal changes in the bovine during the period of luteal regression, estrus and ovulation: Experimental design. - 25 - was given on day 21. Such treatment would allow for monitoring of hormonal responses associated with corpus luteum regression, follicle growth and ovulation under two different environments (21.3 C compared to 32.0 C). Blood samples (50 ml) were collected from jugular catheters at -18, -12 and 0 hr. pre-PGF2^ (day 21), at 6 hr. intervals for 48 hr. post-injection, every 4 hr. thereafter until ovulation, and then twice daily until the last heifer ovulated (figure 1). All blood was col- lected into heparinized syringes, placed immediately into an ice bath, centrifuged at 12,000 g for 10 min. at 4 C, and plasma stored at -20 C until analyzed for progestins, LH, estradiol, estrone, corticoids, pro- lactin, protein concentration, osmolarity and Cortisol binding capacity. Beginning 48 hr.post-PGFp injection, animals were checked visually for estrus at 4 hr. intervals. Heifers were artifically inseminated 12 hr. after onset of estrus, and ovulation determined by rectal palpation of an ovarian ovulatory crypt. Palpations were performed at 4 hr. intervals following cessation of estrus. Chamber temperatures and relative humidities were recorded con- tinuously (Honeywell Recorder, Washington, Pa.), and temperature of each chamber verified daily with a tele-thermometer [Model 46 TUC, Yellow Springs Instrument Co., Inc. (YSI), Yellow Springs, Ohio; air temperature probe T 2620 (YSI)]. Rectal temperatures were monitored daily (tele-thermometer probe T 2600, YSI). Skin temperatures taken on the shoulder, rump and approximately 5 to 8 cm lateral to the vulva (surface temperature probe T 2530, YSI) also were monitored daily during the serial blood collection period. Thermistor probes were calibrated - 26 - against a Bureau of Standards Certified Thermometer in a well-stirred, insulated water bath held at 35 to 40 C. Data collected from the various probes were corrected for constant temperature differences above and below the certified thermometer readings. Plasma samples were pooled within heifers (after the drop of the preovulatory LH peak to basal levels and having less than 3 pg/ml estradiol) to determine total protein concentration (Lowry method), osmolality, (freezing point depression, Fiske Osmometer, Model G-61 , Fiske Ass., Inc., Bethel, Conn.), Cortisol bindi;.g capacity and Cortisol association constants (Pegg and Keane, 1969; Shayanfar, 1973) for each heifer. In the second phase of the trial adrenal responsiveness to ACTH was tested. Eight days after the last heifer ovulated, all heifers received 200 lU ACTH (Porcine ACTH, Sigma Chemical Co., St. Louis, Mo.). Blood samples (50 ml) were collected from indwelling jugular catheters at -2, -1, 0 hr. pre-injection, 15, 30, 45, 60 min. and hourly thereafter up to 12 hr. postinjection. LH and prolactin were assayed in plasma at tm dilutions using the double antibody radioimmunoassay (RIA) of Niswender et_ al. (1969). Guinea pig antibovine LH serum (Oxender, Hafs and Edgerton, 1972) was supplied by Dr. H. D. Hafs of Michigan State University and revalidated with NIH-LH-B7 for measuring plasma LH in our laboratory (Troconiz, 1973). Guinea pig anti-bovine prolactin serum (Koprowski and Tucker, 1971) was donated by Dr. H. A. Tucker of Michigan State University and revalidated for measuring plasma prolactin with NIH-P-B3 prolactin (Chakriyarat, 1974 personal communication). Plasma progestins, - 27 - estradiol and estrone were measured by RIA procedures described by Abraham et aT_. (1971) and Hotchkiss et al_. (1971), respectively. The anti progesterone antibody was a gift from Dr. K. Kirton of the Upjohn Co., and the estrogen antibody was donated by Dr. V. L. Estergreen of Washington State University. Extraction, purification and quantita- tive procedures were validated in our laboratory by Chenault et al. (1973, 1974, 1975). Plasma corticoids were extracted, isolated and quantified by competitive protein binding (Gv/azdauskas, Thatcher and Wilcox, 1972, 1973). The only modification was use of a ,2 ml dextran coated charcoal suspension (100 mg Dextran, Type 60 C, Sigma Chemical Co,, St, Louis, Mo.; 1 gm Norit A, Sigma Chemical Co. and 100 ml deionized water) instead of 80 mg florisil for adsorption of free steroid in the competitive protein binding assay. An extensive series of least-squares analyses was conducted to characterize treatment, animal and within-animal time trends in plasma LH, progestins, estradiol, estrone, prolactin and corticoids. Other response variables v/ere analyzed by analysis of variance. Results and Discussion Averages and standard deviations for chamber conditions, rectal and skin temperatures, and events associated with estrus are shown in table 1. Based on Christison and Johnson's (1972) criteria for a moderate heat stress condition (rectal temperature increase of .5 C) climatic conditions of our study exerted a greater than moderate heat stress since rectal temperatures of heifers in the 32.0 C chamber were - 28 - Table 1. Physiological parameters of heifers in environmental chambers at 21.3 C and 32.0 C. >a ^ -jr-c ^ .« . . ..d 38.75 + .23^ C 40.24 + ,33^ C** 32.60 + .97^ C 36.54 + .47^ C** 33.68 + .71^ C 36,83 + .68^ C** Air temperature 21.3^+ .75^ C 32.0 + ,48 C Relative humidity 58,9 + 3.3^ % 67.2 + 3.5^ % Rectal temperature Shoulder temperature Vulval temperature Rump temperature ' 33.29 + .89*^ C 36.97 + ,45^ C** PGF2^ to4fi peak 94.4 +26,2^ hr. 72.8 +23.2^ hr. PGFo to ovulation 118.4 + 23,9^ hr. 96.0 + 24.8^ hr. Estrus length 21.0 + 3,8^ hr. 16.0 + 3.7^ hk ^(X + SD) *'-(P<.01) ■^{P<.10) ^ . c(n=26), d(n=23), ^(n=5), ■r(n=4) - 29 - 1.49 C greater than heifers in the 21.3 C chamber. Skin temperatures also were significantly (P<.01) elevated in the hot chamber. Visual appraisal of the data showed there was no tendency for skin or rectal temperatures to decline during chronic exposure to the heat stress of our experiment which would have suggested adaptation. Indices of physiological response to PGF^ (duration of time between PGFp injection and the LH peak and time between PGFp injection and ovulation) v/ere not different between treatments (P>.10). The interval from the LH peak to ovulation was approximately 24 hr. for both groups (24 hr. in 21.3 C; 23.2 hr. in 32.0 C). This interval is similar to that reported by Chenault et al. (1973, 1975). Arije, Wiltbank and Hopwood (1974), and Christenson, Echternkamp and Laster (1974) for unsynchronized animals and the PGFp induced interval reported by Chenault et^ al_. (1974) and Hafs et al_. (1974). If the trend for the heat stress group to have shorter intervals from PGF^ injection to LH peak and ovulation is real ca (table 1), we may have failed to detect differences due to small numbers (n=5 each) and appreciable variation. In this study, if hyperthermia affected endocrine-physiological interactions, it did not appear to alter time between the LH peak and ovulation. Length of estrus was significantly shorter (P<.10.) for the heat stressed heifers and was comparable to the 14 hr. duration of estrus re- ported by Gangwar, Branton and Evans (1965) for Hoi stein heifers in hot natural summer climatic conditions of Louisiana. Two of four heifers inseminated in the 21.3 C chamber were pregnant at 40 days compared to none of 5 in the 32,0 C chamber. Though there were small numbers of animals inseminated in this trial, the percentage of successful pregnancies - 30 - was comparable to that of the Dunlap and Vincent (1971) environmental chamber study. This related well to observations that thermal stress in this study interfered with the overall reproductive process in heifers. Thus, the environmental condition did affect body temperature, duration of estrus and probably overall fertility. Whether hormonal re- sponse under these conditions varied v;as of utmost interest. Pre-PGFp injection plasma samples were analyzed by analysis of variance to detect possible differences in progestins, estradiol, estrone, LH, prolactin and corticoids due to temperature, sampling time, temper- ature X sampling time interaction and animals within temperature treatment. Progestins (X = 3.21 ng/ml , 21.3 C; X - 3.16 ng/ml , 32.0 C) were not in- fluenced by temperature or sampling time. However, a significant (P<.05) temperature by time interaction suggested different progestin concentra- tions at different times of sampling in each treatment chamber. Plasma progestins appeared to decline in the heat stressed group with pro- gressive sampling, whereas in the cool group they did not change (Appendix, table 12). Also, there was significant (P<.01) variability in progestin concentrations among heifers in each chamber. This would suggest that there is considerable variation in progestin secretion from heifers during the luteal phase of the cycle. Animal variability in pre-injection plasma estradiol concentrations (X = 3.02 pg/ml , 21.3 C; 2.32 pg/ml, 32.0 C) was significant (P<.01). Estrone (X = 3.04 pg/ml, 21.3 Ci X = 3.25 pg/ml, 32.0 C), LH (X = .53 ng/ml. 21.3 C; X = 182 ng/ml, 32.0 C) and prolactin (X = 12.73 ng/ml, 21.3 C; X = 15.32 ng/ml, 32.0 C) pre-PGFp plasma concentrations were not influenced by temperature, time of sampling, temperature X time interaction or animals within temperature - 31 - treatment (P>.10). Variability in plasma corticoids was significant due to sampling time (P<.01) and treatment X sample time interaction (P<.01). However, there was no difference due to main effects of temperature (9.0 ng/ml , 21,3 C; 9.55 ng/ml , 32.0 C). A logical physiological reference to analyze the data is the preovulatory surge of LH. All animals had a preovulatory LH surge, and each hormone was analysed initially to determine time relationships with the LH surge. Least squares statistical models were selected based on tests of significance of higher order terms (time) in the regression analyses and visual appraisal of the graphs. Figure 2 shows the progestin responses at 21.3 C and 32.0 C, Data for the regression analyses have been synchronized to the LH peak for analysis. The statistical model included treatment, heifer within treatment and time trends (Appendix, table 4). The progestin time trend for heifers at 21.3 C was best described by the equation Y (progestin, ng/ml) = 1.533 + 1.160X - .344X^ + .034X^ - .0014X^ + ,00002X^ (P<.01) where X = .1 hr., whereas the time trend for the 32.0 C heifers shov/ed a significant (P<.(lil) curvilinear relationship best described by a third order equation: Y = 7.923 - 1.123X + .061X^ - .OOIOX^. Tests for heterogeneity of regression were significant (P<.01), suggesting that the 5th order regression curves for each treatment were not parallel. This observation implies that there was a different time response to treatments. We feel that this difference was probably due to heifers in the 21,3 C chamber having their LH peak approximately 22 hr. later from the start of blood sampling than the heifers main- tained at 32,0 C (table 1), Therefore, on the average cool heifers had 32 0.5 e ID CO n o O to O CM CVJ 1 A^svid is^^/or^) s^^iissoo^d CD ex: CD <_> CNJ LU OO 1— o_ OO LU rr: CCS 1 CD oc: LU OO LU CD OO UJ CD — ^".** rn CD U'J cry I — LU C3 LU OO CD OO CNJ UJ CD 33 a longer plateau of low progestins prior to the LH peak. PGF2 caused a drop in progestin concentration by 18 hr. after injection in all heifers (Appendix, table 5). Apparently, factors controlling the pre- ovulatory release of LH in cool heifers were slightly delayed (~ 24 hr.) due either to treatment effects or chance. Thus, due to a shorter interval between PGF2q^ and the LH peak for the 32.0 C heifers, one would expect higher progestins because of shorter trough duration. Also, the number of observations earlier than -120 hr. was \?ery small. Significant (P<.01) among heifer differences were detected within treatments, but differences among treatments were not significant [(P>;i0) Appendix, table 4). This finding is in direct conflict with Stott, Thomas and Glen (1967), who reported elevated progesterone on day of estrus in thermally stressed cows. However, Stott and Wiersma (1973) more recent- ly reported a depression in plasma progestins due to chronic thermal stress. The progestin RIA has a sensitivity of 25 pg or .025 ng/ml plasma. Coefficients of Variation (C.V.) for progestins after accounting for variability due to treatment, heifer in treatment and time trends v/ere 115% (cool) and 99% (hot). Acute Increases of 2.5 (Gwazdauskas, Thatcher and Wilcox, 1972) and 1.5 ng progesterone per ml plasma (VJagner, Strohbehn and Harris, 1972) for a period of 2 hr. were detected follow- ing 200 ID and 100 ID ACTH injections, respectively. If thermal stress had elicited an adrenal release of progesterone we would have been able to detect it. Mills et^ £L. (1972) detected a significant elevation of only .47 ng/ml progestins in heifers thermally stressed for 72 hr. at the onset of estrus. Therefore, with overall progestin levels of - 34 - .147 + .095 ng/ml {J ± SD) the day of estrus and day after estrus and no differences due to temperature, we cannot conclude that chronic heat stress caused adrenal hyperprogesterone response. .Our findings support Miller and Al listen (1974b), who found no difference in plasma progesterone during the bovine estrous cycle v/hen twice daily measure- ments were made in control (17-21 C) or heat stress (21-34 C) environments. Progestins do not appear to be elevated during the period between luteal regression and ovulation. Data for regression analyses of estradiol were separated into two periods and independently analyzed to characterize time trends. The two periods were from -144 hr. to time 6f the LH peak (including LH peak time) and from the LH peak to +96 hr. (also including the Lfl peak time). Pre and post LH peak time trends for estradiol were best described by Y (estradiol, pg/ml ) = 4.53 - .482X - .045X^ + .005SX^ (•P<.05) and ? ^^^ = 45860.38 - 12165. 711X + 1285. 829X^ - 67.674X-^ + 1,773X^ - .019X^ (P<,05) for the cool heifers and 9 ^^ = 5.67 - 1.279X pre + .092X^ (P<.01) and Y ^^^ = 490.168 - 73.503X + 3.647X^ - .060X"^ (P<.01) for the hot heifers (figure 3). Plasma estradiol was depressed (P<,10) in the 32.0 C chamber (peak estradiol :10. 4 pg/ml p'lasma for 21.3 C heifers compared to 7.2 pg/ml plasma for 32.0 C heifers; Appendix, tables 4 and 6) . Lov/er plasma estradiol may have contributed to the shorter periods of estrus seen in the 32.0 C heifers. Hov/ever, these lower concentra- tions of estradiol were adequate to elicit estrous behavior and LH release causing a subsequent ovulation. The lower estradiol may reflect altered production, secretion, clearance or receptor binding under 35 O G> CO !«^ O ^ I CD CNI r-H CNJ CO LU Li_ Oc2 I -7-h- UJ re CT) CD I LjJ LU 00 -c: LU I — CO r;^ CD <=C I— <_) c:^ UJ — I r-vj ■=a: ' — ' • — • . ::: I — CD ~:z c^ LU rr: rr5 CD CD r:r Lj-J >- c/j) CO 101QV^1S3 UJ -10) suggesting that in both treatments estrone followed the same decline post-PGF2^. However, estrone levels were higher through 72 hr. in heat stressed heifers. The slight rise in estrone prior to PGF^^ injection to +12 hr. may be related to a luteolytic action as suggested by Hansel (1971), although heifers were only day 9 of the estrous cycle at time of P(^^2a ■'"J^^''^^*°"- 37 Table 2. Simple correlations between hormone measurements' Progestins Estradiol Estrone Corticoids Prolactin LH -.14* .45** .03 -.02 -.01 Progestins -.14* .40** .14* -.07 Estradiol .00 .11* .02 Estrone .09 -.06 Corticoids .22** ^n=291 *(P<.05), >.ll **(P<.01), >.15 - OO OO UJ CD (VlAJSVld I'/^/Od) 3N0^1S3 - 39 - ChenauU et aT_. (1974) and Henn'cks et a^. (1974) reported estrone to vary greatly within and among animals after PGFp injections. The C.V.'s for estrone in this study after accounting for heifer and time variability were 65.3 and 74.0% for cool and hot groups, respectively. Plasma estrone concentrations were less than 5 pg/ml and agree with the work of Echternkamp and Hansel (1973). However, they reported that estrone v/as slightly elevated at estrus in one cow. This observation was difficult to support since there was no statistical analysis or re- port of estrone variability. Because LH increases above basal levels for only about 10 hr. (Chenault et_ al_. , 1975), LH data were separated into four time periods to analyze, independently, bot[i basal and preovulatory peak concentra- tions. These periods were: (1) from -148 to 8 hr. prior to the LH peak (-8 hr.); (2) -8 hr. to the peak of LH; (3) LH peak to +8 hr. and (4) +8 hr. to +96 hr. (figure 5). Average LH concentrations for period (1) were 1.80+1.21 ng/ml plasma (I + SD; n=102) for the cool heifers (21.1 C) compared to 1.75 il.27 ng/ml (n=74) for the heat stressed heifers (32.0 C). A linear increase (P<.01) in LH occurred between -8 hr. to the LH peak (peak LH 32.19 ng/ml for cool heifers compared to 33,17 ng/ml for hot heifers), and then dropped linearly (P<.01) to basal levels by +8 hr. (.43 ng/ml - cool compared to .49 ng/ml - hot). Basal levels were defined as any LH concentration within three standard deviations of the mean for all samples (n=169) up to -8 hr. (1.75 j^l.22 ng/ml). During the initial period, two heifers in the cool chamber had sporadic peaks (>3 S.D.) of LH that occurred between -72 and -12 hr. prior to the LH peak (Appendix, table 8). - 40 O o CD t_5 f—\ CVl (XI UJ rrr \— UJ CO 0:C UJ !d IIAI/ON) HI Cl- co LU CD C_5 LU UJ CO u-^ UJ e3 - 41 - The preovulatory surge of LH remained above basal levels for 10.4 and 9.6 hr. for the 21.3 C and 32.0 C groups, respectively. LH concentra- tions in this study agree with those of Henri cks, Dickey and Niswender (1970) and Snook, Saatman and Hansel (1971). Unlike results of Madan and Johnson (1971) and Miller and Alliston (1973), v/e found no significant difference in LH levels in response to heat stress (Appendix, table 4). Our contradictory results under conditions of thermal stress may be due to frequency of sampling (twice daily; Miller and Alliston, 1973), sensitivity of experimental design in which among animal variability was considered in the present study, duration of the LH peak (~ 10 hr.) or breed differences (Madan and Johnson, 1971). Riggs, Alliston and Wilson (1974) detected a difference in the preovulatory LH surge during heat stress between Hampshire and Duroc gilts. Such differences may exist between the study of Madan and Johnson (1971) in which Guernsey cattle were used and in our study where only Hoi stein heifers were used. All animals had a preovulatory LH surge, suggesting that hyper- thermia did not prevent the triggering mechanism for LH release. Although estradiol levels in peripheral plasma were slightly depressed in the 32.0 C heifers, it does not appear that these lowered estradiol concentrations altered LH release. Plasma prolactin was analyzed initially in the same manner as estradiol (pre- and post peak of LH). However, there was no change in prolactin associated with estrus or the LH peak as previously reported by Swanson and Hafs (1971). Absence of any association between prolactin and estrus is supported by work of Hoffman et aj_. (1974) in which an - 42 - inhibitor of prolactin secretion caused no estrous cycle disorders. Also, Wetteman and Hafs (1973) were unable to find elevated prolactin on the day of estrus. Since there were no detectable changes in prolactin associated with the LH peak, data v/ere analyzed further relative to time of PGFo injections. Hafs et al_. (1974) reported that prolactin increased immediately following PGF^ injection (within 1 hr. and lasting for 4 hr.). However, an increase in our study was not detected because the first blood samples were not taken until 6 hr. after injection. No differences were detected between the 21.3 C and 32.0 C treatment groups (P>.10; Appendix, tables 4 and 9). Time trends of prolactin for the 21.3 C and 32.0 C treatment groups were described by the following equations: Y (prolactin, ng/ml ) = 14.50 - 1.707X + .371X^ - .OlSX-^ and Y = 14.28 + 2.738X - .733X^ + .066X^ - .002X^ (3?.0 C) (figure 6). The 4th order time curves were not parallel (P<.005). Prolactin C.V. after accounting for heifers and the above time equations was 49.4% (21.3 C) and 26.3% (32.0 C). During the initial 42 hr. (-18 to 24 hr.), the prolactin response in the cool chamber appeared to decline. This observation may be due to a lowering of stress-induced prolactin secretion with more sampling (Tucker, 1971). Apparently heifers in the 32.0 C chamber could not adjust to sampling as quickly since prolactin increased and remained elevated until 24 hr. after PGFo . However, this is 2a questionable since trends are very s'jbtle and the curves account for little of the variability (figure 6). An increase in plasma prolactin due to heat stress was antici- pated in the present study based on reports of Koprowski and Tucker (1973), r o CM / \ 43 - \ \ lO .10) between treatment means (Appendix, tables 4 and 10). Furthermore, we were unable to detect any individual treatment time trends after looking at regressions up to the 5th order. After account- ing for corticoid variability due to treatment, heifers within treatment and time trends (up to the 5th order), there was a 65% C.V. for plasma corticoid concentrations. Our data, with blood samples taken at 4 hr. intervals at least 2 days prior to estrus, do not sup- port Miller and Alliston's (1974a) finding of increased corticoids early the day of estrus (twice daily sampling). Nor does it support a report which showed lower plasma corticoids in dairy cows during summer months in Arizona (Stott and Wiersma, 1973). However, Arizona climatic conditions of high temperature and low humidity may be dif- ferent from our study with high humidity and high temperature. Our results show numerous episodic peaks during the day. Wagner and Oxenreider (1972) also reported episodic peaks of plasma corticoids when measured at 30 min. intervals throughout the day. They also noted diurnal corticoid variation, but we were unable to detect any time of day differences (P>olO) when data were analysed at 4 hr. intervals. Due to large variability in plasma corticoid levels, a large treatment difference in corticoid concentrations v/ould be needed to detect a - 46 - UJ CD C7> CM CVJ I CD \ O T T re <_> I — cr> CD - I — CNI 00 K> t I ZED AND ^ CD C_> CJ SYKCHR 21.3 o 1— en CO (_> 1 • 1 1 — (It OJ O 1— — ' 1 X ct: UJ O -J- C_) CO M •=SZ CD 1 oo oo (vl^'sv■1cJ i'y\]/9N) saiooii^oo G!] — (=a LU C/) — ! UJ CD CD CD ^^ D_ nr CD CD zsr 1 oo UJ UJ ZD Q_ CD UJ rr: .oo — I UJ CD - 47 - significant difference. We failed to detect any differences in plasma corticoid associated with estrus, ovulation or heat stress when monitoring plasma concentrations at 4 hr. intervals. When time was removed from the model and each hormone considered as a dependent variable, plasma LH had a negative association with progestins (r = -.14, P<.05; table 2). Progestins also were negatively related to estradiol (r = -.14, P<.05). In the overall model LH was positively related to estradiol (r = .45, P.10) in total protein concentration or plasma osmolality between heifers at 21.3 C and 32.0 C (table 3). These re- sults suggest that no appreciable plasma dilution had occurred. However, we have no measurement of total plasma volume of heifers for this study. A Corticosteroid Binding Globulin (CBG) of plasm.a has been re- ported for various species (Seal and Doe, 1965) and also is present in the bovine (Lindner, 1964). Such a protein acts as a corticoid carrier molecule through the blood. Although total plasma protein concentration (table 3) did not vary between treatments, certain alterations of pro- tein composition may have occurred. Although plasma corticoid concentrations did not differ between treatments, their potential biological effectiveness would be appreciably altered if the concentra- tion of plasma CBG differed. Utilizing the procedure of Pegg and Keane (1969), the association constant (K ) and Cortisol binding capacity of CBG were determined on a pooled samples (within heifer) of each experimental heifer. The association constant did not vary due to treatment (P>.10; table 3). An average experimental K^ of 1.86 X 10' M"^ was indicative of a protein with an intermediate affinity for the Cortisol ligand. It is a protein - 49 Table 3. Physical characteristics of plasma in heifers at 21.3 C and 32.0 C. 21.3 C 32.0 C ^•"^^^l" 76.38 + 8.52^ 75.50 + 9.05 (mg/ml) - - Osmolality (milliosmoles/kg H2O) Corticoids (ng/ml ) 259.65+11.65 268.70+10.41 6.7 + 1.2 5.9 + 1.0 Cortisol Binding Capacity ^^3^3 ^ ^^j^^ gggg + ^^ .,g* (ng/ml ) Association Constant (K X 10' M"M a ^(X + SD) *(P<.05) 1.52 + ,72 2.20 + .69 50 - with an association constant higher than a low affinity protein such as human serum albumin (K = 1 X 10" M'M but lower than the K^ for a d tissue receptor protein such as the Cortisol mammary gland receptor (K = 5 X 10^ M"^; Tucker, Larson and Gorski, 1971). It was not expect- a ed that thermal stress would alter the physical chemical properties of the CBG protein (K^) but perhaps may alter the amount (capacity) of CBG per ml of plasma. Indeed there was a significant difference (P<.05) in Cortisol binding capacity (ng/ml) between treatments (table 3). Thus under experimental conditions for quantifying Cortisol binding capacity at 4 C, plasma of heat stressed heifers had a 53% lower capacity to bind Cortisol. This suggested that under such conditions, plasma from hyperthermic heifers contained a decreased concentration of CBG. Therefore, under environmental temperatures of 32.0 C at a body tempera- ture of 40.24 C both the concentration of plasma CBG and Cortisol bound CBG (product) would be less if the rate constant for the forward reaction was not different at an elevated body temperature of 1.5 C (table 1). With this reasoning, heifers exposed to a thermal stress characteristic of our experiment would have a greater percent free Cortisol compared to CBG bound Cortisol at a constant total Cortisol concentration. As previously described, total plasma corticoid concentrations did not vary between treatments (6.7 compared to 5.9 ng/ml; table 3). Under other stressful conditions a lowered corticoid binding capacity has been reported for various species. In human burn patients a slightly lowered Cortisol binding capacity has been reported (Mortensen et al., 1972), in which decreased capacity was inversely related to burn area. By analogy, lactation can be considered a stress in the sense - 51 - that reproductive efficiency is lower during this period. Lactation inhibits the onset of estrous cycling in rats nursing 6 or 12 pups compared to post-parturient rats which do not lactate (Tucker and Thatcher, 1968). Early weaning of calves from their dams increased the occurrence of estrus and increased pregnancy rates in beef and dairy cattle (Laster, Glimp and Gregory, 1973). Troconiz (1973) has reviewed the cystic ovary condition in dairy cattle. High milk producing cows had a greater incidence of cystic ovaries and therefore e greater fre- quency of reproductive problems. In rats nursing 12 pups, CBG activity was lower in comparison to rats nursing only four pups (Westphal, 1970). Such a nursing intensity will delay occurrence of normal estrous cycles (Tucker and Thatcher, 1968). Thus under conditions of lactational stress (relative to reproductive performance) CBG activity was depressed. The liver is the reported source of CBG (Guyton, 1966) and the thyroid gland is reported to exert a controlling influence on CBG activity. Gala and Westphal (1966) showed that TSH stimulated CBG activity in hypophysectomized rats and was primarily responsible for regulation of CBG levels. In cattle under conditions of high environ- mental temperatures, thyroid activity v/as depressed (Johnson and Yousef, 1966). If the hormonal control of CBG production is grossly comparable between rats and cattle, then lower CBG binding capacity of heifers detected in our study would be expected. Hypothyroid patients have a slower turnover of Cortisol. Both bound and free steroid fraction disappearance rates were slower than normal or hyperthyroid patients (Beisel et al_. , 1964). In our study the amount of free hormone would have a greater biological role in the - 52 - heat stress group due to the lower binding capacity. This may result in a lower level of ACTH secretion due to greater negative feedback inhibi- tion. In the bovine, corticoid turnover rates were depressed during chronic heat stress (Christison and Johnson, 1972). This also suggests a longer biological life for the circulating corticoid allowing a greater ACTH negative feedback since less corticoid is also bound to transcortin. However, the amount of free Cortisol in our study, estimated by extra- polation at 4 C, was not different (P>.10) between the 21.3 C heifers (1.39 ng/ml) and 32„0 C heifers (1.43 ng/ml). Clarification is needed in this area as to the physiological role of bound and free steroids because of conflicting reports between species and stress situations. Our finding that corticoids were not elevated during chronic heat stress, irrespective of the binding capacities, may be advantageous to the cow in that heat production has been shown to increase 30 to 40% at 35 C when hydrocortisone acetate was administered (Yousef and Johnson, 1967). In the second phase of the experiment, 8 days following ovulation in the last heifer, 200 lU ACTH was injected, IV, into 10 heifers. The ACTH was given while heifers were in the luteal phase of the estrous cycle or at a time when a progesterone increases in peripheral plasma due to ACTH injection (Gwazdauskas, Thatcher and Wilcox, 1972; Wagner, Strohbehn and Harris, 1972) may not have a detrimental effect on the developing embryo (Johnsson et al- , 1974). Figure 8 shows the corticoid response curves following ACTH injection. The 32.0 C group responded with significantly lower (P<.10) corticoid concentrations. The 6th order regression curves were not parallel (P<.01) suggesting that the - 53 - o r- • • It ti r o o o o CO 1" o "7" o CD o o o to (viAisvid m/DH) saiooiiHoo •X -•^ o o OJ h— t_> CD CD — CD CD CO 1^ CD cr LO 3 O X CD CD LjlJ - OO CD L-lJ CO LU OO LU o:: CD - 54 - hot group response was earlier to reach a peak (75 min. compared to 105 min.), had a lower magnitude (73.5 compared to 100.2 ng/ml corticoid) and was of shorter duration (4 hr. compared to 5 hr.; Appendix, table 11). this response is comparable to that reported by Shayanfar (1973) in which lactating cows exposed to environmental temperctures ^bove 21.1 C responded the same way. The cool heifer response was best described by: V (corticoids, ng/ml) = -521,387 + 4648. 999X - 12253. 643X^ + 13509. 684X^ - 5942. 539X^ + 4.326X^ + 464.541X^ (P<.01), whereas the hot heifer re- sponse was best characterized by Y = -613.342 + 6525. 433X - 23576. 092X^ + 41551. 725X^ - 38756. 632X'^ + 18359. 263X^ - 3477. 023X^ (P<.01). The apparent reduced ability of the adrenal to secrete and/or synthesize corticoids following ACTH stimulation during heat stress may be related to a chronic lower level of endogenous ACTH secretion. The lower plasma Cortisol binding capacity in the 32.0 C heifers may provide a greater amount of free corticoid to exert a feedback inhibition on endogenous ACTH secretion. In addition, there is also a lower level of corticoid turnover and secretion during chronic heat stress (Christison and Johnson, 1972). As a result the degree of chronic endogenous ACTH secretion may be less, causing a reduction of responsive adreno-cortical tissue. These conditions may result in a lower adrenal corticoid increase in response to a pharmacological challenge with ACTH. A reduced level of adrenal function during heat stress would be advantageous to the animal calorigenically. Corticoid secretion did not appear to be higher in the heat stressed group since resting corticoid levels were not greater than controls. However, a possible decreased adrenal secretion rate was not reflected by a lower plasma corticoid concentration. It was not until - 55 - a response to ACTH was evaluated that adrenocortical function appeared to be depressed. To determine the significance of this apparent reduction in adrenal responsiveness to ACTH due to hyperthermia plasma ACTH levels need to be determined in the bovine under different physiological stress situations. Other possibilities include determining effects of stress on ACTH receptors, more definitive studies on corticoid-CBG binding properties in relation to thermal stress and possible steroidogenic- enzyme alterations in the adrenal. Pre-ACTH plasma progestin and corticoid concentrations were test- ed to detect differences in levels due to heat stress and pregnancy status (Appendix, tables 11 and 12). The analyses include temperature, pregnancy status and heifers nested in temperature-pregnancy status. There were no statistically significant differences (P>.10) either in hdrmone concentrations due to temperature or pregnancy status. However, significant among animal variability (P<.05) was found in progestin levels. These results agree with the pre-PGFp treatment hormonal values in the first phase of this experiment (Page 30). Our observa- tions conflict with a summer seasonal depression in corticoid and progestin concentrations reported by Stott and Wiersma (1973). The present study also did not confirm their finding of higher progestins in fertile cows on day 15 of pregnancy or the estrous cycle. This period of corpus luteum function is comparable to our study (heifers were between estrous cycle days 9-13). In summary, environmental treatment of 32.0 C evoked a 1.49 C increase in rectal temperature and a 3 to 4 C increase in skin - 56 - temperatures. The time durations between PGFp injection and the LH peak and the period between PGF^ and ovulation were not different (P>.10) between treatments. Length of estrus was shorter (P<.10) for the heat stressed heifers. Two of four heifers inseminated in the 21.3 C chamber were pregnant at 40 days compared to none of five in the 32.0 C chamber. Thus, the environmental condition did affect body temperature, duration of estrus and overall fertility. Preinjection plasma samples showed no differences {P>.10) in any of the hormonal measurements due to the main effect of temperature. Average progestin concentration between treatments was not different (P>.10). However the 5th order response curves were not parallel (P<.01) Indicating a different time response between treatments. Progestin concentrations declined in a similar manner in both groups following PGFp injection. Heifers in the 21.3 C group, on the average, had a LH surge about 24 hr. later than heifers in the 32.0 C group. This 24 hr. time lag would account for the difference in time responses when data were synchronized to time of LH peak. Mean estradiol concentra- tions were significantly (P<.10) lower in the heat stressed heifers. The lower plasma estradiol may have contributed to the shorter estrous periods seen in the 32.0 C heifers. However, these lower concentrations of estradiol were adequate enough to elicit estrous behavior and trigger LH release causing a subsequent ovulation. Estrone showed no apparent association with the onset of estrus or LH peak when the data were synchronized to the time of the LH peak. There was a significant elevation (P<.05) of estrone due to heat stress but there was no evidence that estrone time trends following PGFp^ were - 57 - not parallel (P>.10) suggesting that in both treatments estrone follow- ed a similar decline postinjcction. No significant di-rfe:'ences (P>.10) were found in mean LH concentrations between heifers at 21.3 C or 32.0 C. Preovulatory peak LH concentrations were 32.2 ng/ml and 33.2 ng/ml plasma for the 21.3 C and 32.0 C heifers, respectively. All animals had a preovulatory LH surge, suggesting that hyperthermia did not pre- vent the triggering mechanism for LH release. There was no change in prolactin associated with estrus or the LH peak, therefore prolactin was analyzed relative to time of PGF^ injection. Mean prolactin concentrations were not different between treatments (P>.10)o The 4th order time curves were not parallel (P<.005), Heifers in the 21,3 C chamber had a decline in plasma prolactin after the initial sampling as compared to increased prolactin concentrations in the 32.0 C heifers during this early blood sampling period. The summer seasonal increase in plasma prolactin reported by various researchers may be more related to photoperiod effects. There was no difference (P>.10) between treatment means in plasma corticoid concentra- tions. Furthermore, we were unable to detect any individual treatment time trends after looking at regressions up to the 5th order. Plasma corticoid C.V. was 65% after accounting for variability due to treat- ment, heifers within treatment and time trends up to the 5th order. In an attempt to determine if plasma dilution may have occurred, total protein concentration and osmolality were measured. There was no difference (P>,10) in total protein concentration or osmolality between treatment groups. However, no measurement of total plasma volume was made. Cortisol binding capacity of CBG and its association constants - 58 - (K ) were determined. The affinity (K ) of Cortisol for CBG was not different between treatments (P>.10)5 however, the binding capacity of CBG for Cortisol was significantly (P<.05) reduced in the 32.0 C heifers. This observation suggested that under experimental conditions (4 C) for determining the binding capacity of Cortisol, the hyperthermic heifers may have had a decreased concentration of CBG. ACTH (200 lU) was injected, IV, into 10 heifers. The 32.0 C heifers responded with a significantly lower (P<.10) corticoid concen-- tration. The 6th ord-.:r regression response curves were not parallel (P<.01) suggesting that the hot group response was earlier to reach a peak (75 min. compared to 105 mi n.), had a lower magnitude (73.5 compared to 100.2 ng/ml corticoids) and was of shorter duration (4 hr. compared to 5 hr.). Adrenal responsiveness was significantly less in heifers maintained at 32 C. .^ Results of this experiment show only subtle thermal effects on \r/ plasma concentrations of estradiol and estrone and no effects on LH, progestins, corticoids and prolactin. Apart from possible hormonal involvement with duration of estrus, heat stress does not appear to af- fect the hormonal milieu associated with corpus luteum regression, follicle growth and ovulation. The significance of possible lowered adrenal response in hot environments may be related to a state of lowered heat production. Since corticoids are known to be calorigenic (Yousef and Johnson, 1967) a lowered adrenal responsiveness in hyper- thermic heifers might be physiologically advantageous. The experiment described in this section has not specifically considered the possible environmental and hormonal effects on uterine - 59 - temperature. It was of prime importance to characterize uterine thermal changes during the period of luteal regression, follicle growth and ovulation under conditions of a mild heat stress, and to document possible estrogen induced uterine thermal changes. SECTION III EXPERIMENT I: THERMAL CHANGES OF THE BOVINE UTERUS FOLLOWING ADMINISTRATION OF ESTRADI0L-17B Introduction The first experiment (Section II) indicated that a thermal stress increased body temperature, suppressed fertility and caused a slight de- crease in endogenous estradiol secretion. Furthermore, we reported previously that uterine temperatures both on day of and day after insemination were inversely related to fertility (Gwazdauskas, Thatcher and Wilcox, 1973). This directly indicated that temperature of the uterus was closely associated with fertility. Other factors in addition to environmental temperature may in- fluence uterine temperatures. For example estrogen administration was shown to increase uterine blood flow in sheep (Huckabee e^ al_. , 1970; Greiss and Anderson, 1970; Rosenfeld et al. , 1973; and Resnik et al. , 1974). This uterine hyperemia may have caused heat to be dissipated from the uterus, thus cooling the uterine cavity (Abrams. et al- , 1970a). Uterine blood flow changes in sheep were monitored following estrogen injections by looking at differences in temperature between the uterus and aorta. A rise in blood flow rate resulted in a lower uterine temperature (Abrams et^ al_. , 1970a). - 60 - - 61 - Although uterine temperature and estrogen relationships have been found in sheep, this phenomena has not been examined in the bovine. Objectives of this study were to determine if uterine-aortic temperature differences exist in the bovine, and if such differences change following injection of Estradiol -17g. Materials and Methods Thermocouple Preparation and Calibration Lengths of 35 gauge, nylon coated, copper constantan wire (Revere Corp., Wallingford, Conn.) were pulled through polyvinyl tubing (V5-V7; Bolab Inc., Derry, N. H.) for measurements of uterine and blood temperatures. The terminal thermo junctions to be placed in the saphenous artery then were pulled through a larger polyvinyl tube (V-12) for additional support. The ends of all thennojunctions v/ere heat-sealed in the polyvinyl by pushing them through a siliconized, narrowbore glass tubing which was being heated on a soldering iron. After sealing, ends were coated with liquid tygon (U. S. Stoneware Co.). Stranded, untinned copper extension wires (Leads and Northrup, #27-32-36, Philadelphia, Pa.) were soldered to divided copper wires leading to the thennojunctions. All extension wires led either to a millivolt potentiometer (#8686, Leads and Northrup, Philadelphia, Pa.; limits of error of recording system +_ .075 C) or to a strip chart recorder (Hewlett-Packard, M 7100B; limits of error of recording system j^ .03 C). Most, but not all of the potentials from the aortic-ice water thermo- couple were suppressed by knovrn amounts before being amplified and recorded. - 62 - Calibration of the thermocouples was made routinely by use of a Bureau of Standards Certified Thermometer in a well-stirred, insulated water bath held at intervals between 36 to 40 C. The thermocouple readings were 0.05 to 0.075 C above the certified thermometer reading, so all data collected were corrected for these constants. Surgical Techniques and Experimental Protocol Four 2-year-old heifers with histories of regular estrous cycles were used in these experiments. Prior to surgery, heifers v/ere placed on a 48 to 72 hr. feed and water fast. Heifers were anesthetized with 2 to 4 g sodium thiopental (Abbott Laboratories, North Chicago, 111.) dissolved in saline (2 g/20 ml) while standing and restrained. They were placed onto a portable operating table, tracheotomized and maintained under surgical anesthesia with methoxyfluorane (Pitman-Moore, Washington Crossing, N. J.). After removal of hair, the abdominal and inguinal regions were scrubbed thoroughly with germicidal soap and rinsed with 70% alcohol. A 15 cm longitudinal midventral incision was made through the abdominal wall at the cranial margin of the mammary gland. A sharpened stainless steel cannula was carried into the abdominal cavity through this midventral incision and pressed through the abdominal v/all in the flank area. All thermojunctions and approximately 2.5 m of extension wires were drawn through the cannula leaving the remainder of the 3 m of extension wire and connectors coiled up in a canvas pack. The cannula was removed from the abdominal cavity by sliding it over the thermojunctions and withdrawing it through the midline incision. The - 63 - pack subsequently was attached to the flank with one or two stainless steel pins passed through a flap of skin. The uterus was elevated so that the junction of the uterine horns with the uterine body could be visualized. Using small scissors and straight forceps, a 3 to 4 cm tunnel was made under the serosa in the medial aspect of one uterine horn about 1 cm from the bifurcation. A thermojunction was inserted into this tunnel and tied in place with 000 silk thread. The extension wires were secured by two to three additional ties through the serosa along the uterine horn. Thermojunctions for aortic blood temperatures were routed through the midventral incision, tunneled under the skin to the inguinal area where the saphenous artery was exposed. These thermojunctions then were inserted into the saphenous artery, passed 70 to 75 cm upward to the abdominal aorta and extension wires fixed with silk suture at the point of entry into the vessel. Incisions were closed in layers. Thermocouple placements were confirmed prior to their surgical removal 7 to 10 days after completion of the experiment. Twenty-four hr, prior to intravenous (IV) injection either of 3 mg Estradiol-173 (Progynon-Schering Corp., Bioomfield, N. J.) or 12 ml of .9% sterile saline, heifers were fitted with polyvinyl catheters (V-7) by jugular venipuncture. Catheters were filled with heparin solution (15 U/ml of .9% saline), capped with a brad and the external catheter placed in an adhesive tape pouch glued to the neck with branding cement (Electro Cote Co., Minneapolis, Minn.). On the day of injection heifers were placed in a stanchion barn on rubber comfort mats at least 2 hr. prior to recording temperatures. - 64 - Each of the four heifers received an estradiol injection, and two of the heifers also received two saline injections each. Thus there were a total of four estradiol and four saline experiments. All heifers received treatment during the luteal phase of the cycle. Recordings were made from the millivolt potentiometer at 15-min. intervals beginning 1 hr. prior to IV injection either of Estradiol-173 or saline and ending 6 hr. after the injections. A pre-experimental control period of 1 hr. was used to determine a steady state level of uterine temperature. Repeatability of triplicate measurements at each time was 0.92 for aortic temperature (mV) with a C.V. of 0.07% (n=66). Repeatability and C.V. for AT . (uV) were 0.99 and 6.44%, respectively, uterus-aorta Initially, an additional thermocouple was placed in the uterine lumen as well as in the uterine serosa. Prior to and following estrogen injection the temperatures at both reference points were identical. To avoid any possible complication due to presence of an intrauterine object, all subsequent animals were fitted only with a uterine serosa thermo- junction. In a separate experiment, temperatures were recorded continuously before and after an injection of Estradiol-173. The major statistical technique to analyre time changes was least squares as de- scribed by Harvey (1960). Statistical models were selected based on tests of significance of the higher order terms in the regression analyses and visual appraisal of the graphs. Results and Discussion The uterine and aortic temperature response following intravenous injection of 12 ml saline is shown in figure 9. The slight increase in 65 c\i - 66 - both mean temperatures (~ 0,2 C) v/hich occurred during the 7 hr. experi- ment may be related to the normal rhythmic rise in body temperature in cattle during the day (Bianca, 1968). The greater variability in both temperatures 4 to 6 hr., after saline injection could have been because of blood temperature changes induced by some restlessness due to long confinement in the stanchions. In spite of these changes in uterine and aortic temperatures, temperature differences between the two were quite stable during the experiment, indicating that the ratio between ■ uterine heat production and uterine heat loss had remained unchanged. Relationships of time (X) and uterine (Y^) and aortic (Y^) temperatures are shown in figure 9, There was no evidence of curvilinearity; fitting the two equations accounted for 36 and 37% of the within-heifer vari- ability in Y and Y , respectively. There was no evidence that the two slopes were not parallel, which suggested that the saline vehicle had no depressive effect either on uterine or aortic temperature. Effects of Estradiol -173 on uterine and aortic temperatures are illustrated in figure 10. The initial fall in uterine temperature of slightly more than 0.3 C compares favorably with the response noted previously in sheep (Abrams et a]_. , 1970a). The slight rise in uterine temperature between 4 to 6 hr. post injection was undoubtedly due to the rise in blood temperatures as noted in control experiments. Uterine changes were curvilinear (P<.01) as indicated by the equation p 2 (R = 0.18). A significant quadratic equation (P<.01; R = 0.04) best describes the aortic temperature response. Why aortic temperature fell initially is not known. Increased respiratory evaporative heat loss or sweating may have been responsible. Estrogens are known to be potent - 67 - h- OJ X lO X o I ro lO 00 o < v> c o E o >«— o o II o CO ro CD CO CD CD to -^ CD CD _J I CD ca ro 1=1 CO ^ t- CD f > — ■ Q_ J__ UJ O LU CD o CD _ r— i -— », 1 •=3: 1 CD ijj »■- •■« ^^ C=3 <=!:. Qi CcT CO . (0,) viyov-snd3in J.V CD ZD CD - 68 - vasodilators of skin blood vessels (Reynolds and Foster, 1940), and to the extent that heat loss was promoted by this increased skin blood flov/, a lowered temperature may result. One may propose that estrogens could have had a subtle effect of the thermoregulatory "set point" (Hammel et al_. , 1963) which resulted in activating one or more heat loss mechanisms. However, the decrease in aortic temperature was only about .1 C. When the difference in temperature between uterine serosa and aorta was examined the result of Estradiol-173 administration was obvious (figure 11). The decrease in AT^terus-aorta ^^Va^ °^ ^'^^ ^ "^^ ^^" scribed by a highly significant (P<.01) curvilinear trend over time. The AT beoan to plateau at approximately 2.5 hr. post estrogen U-a " r injection and remained depressed for the duration of the recording period, although both uterine and aortic temperature started to rise 4 to 5 hr. post-injection. There was no significant change (P^.IO) in AT^_g follow- ing saline injection. Figure 12 is a plot in 30 sec. intervals taken from a continuous recording of temperatures of one heifer prior to and following Estradiol- 173 injection. In this animal the estrogen effect on uterine temperature was noted within 1 hr. Rapid oscillations in temperature of the uterine tracing were considerably dampened by the heat capacity of the uterine tissue. A consistent finding in the estrogen experiments was the decrease in the temperature difference between the uterus, as represented by the subserosal temperature and the blood of the abdominal aorta. Such a decrease in AT could occur as a result of a lowered rate of uterine 69 (Do) ViyOV snd3in J.V CXI r— I I —J CD — • SZ C=! UJ Cc:: I — CD CD CD UJ ►— • Qi f— cc: CO CD z=> •cC CD ZD Q :"- ^ t- UJ o 2:: CD UJ CD f— o::: ID U_ CSI »-H UJ ZZD CD - 71 - heat production, a possibility which appears remote in viev/ of the many cellular metabolic activities induced by estrogens (Talwar and Segal, 1971). A more reasonable explanation for the lowered AT is the u~a augmented rate of heat loss resulting from the marked estrogen induced elevation in uterine blood flow. Endogenous estrogens released during the estrous cycle in ewes are known to be associated with elevated uterine blood flow rate (Greiss and Anderson, 1970) and increased vaginal blood flow as inferred from a significant rise in vaginal thermal conductance in cattle (Abrams et al_. , 1973). Thus, there is reason to believe that comparable cyclic, blood flow-induced changes in temperature of the reproductive tract may occur during the estrous cycle in the bovine. High uterine temperatures at the time of artificial insemination are associated with diminished fertility (Gwazdauskas, Thatcher and Wilcox, 1973). Elevated environmental temperature is thought to suppress fertility by acting directly on the developing embryo and/or through altering maternal endocrine function (Vincent, 1972). Findings in the first experiment indicated that plasma estradiol of heat stressed heifers was lower during the pre-estrous period. Results of the present study indicate that a pharmacological injection of Estradiol-173 can significantly decrease uterine temperatures. In the final experiment, attempts were made to evaluate changes in uterine temperature during the period of luteal regression (decreasing progesterone) follicle growth (increasing estradiol) and ovulation under conditions of a mild heat stress. EXPERIMENT 2: THERMAL CHANGES IN THE BOVINE UTERUS FOLLOWING ~PGFo INJECTION THROUGH ESTRUS AND OVULATION 2a Introduction The first experiment (Section II) indicated that a thermal stress increased body temperature, suppressed fertility and caused a slight , decrease in endogenous estradiol secretion. Next, an effect of exogenous Estradiol-173 on uterine temperature was documented. In this final experiment, estrus was synchronized by PGF^^ and an attempt was made to evaluate changes in uterine temperature and aortic blood temperature with plasma estradiol and LH under conditions of mild heat stress. Such an experiment would closely mimic responses of animals under normal field conditions and provide additional insight into factors controlling uterine temperature under conditions of poor reproductive efficiency. Materials and Methods Thermocouple preparation and calibration were the same as described in the previous experiment with the exception that all thermocouples were made in triplicate for each location. During the experiment, extension wires led to a recording potentiometer (9835 A, D-C Microvolt Amplifier and Speedomax G, Model S6000 Recorder, Leeds and Northrup, Philadelphia, Pa.; limits of error of the recording system + .0125 C). Surgical 72 - 73 - techniques were identical except cattle v;ere anesthetized with 3 g sodium thiamylal (Surital-Park Davis, Detroit, Michigan) dissolved in saline (3 g/20 ml) and were maintained under surgical anesthesia with halothane (Fluothane-Ayerst Laboratories, Inc., New York, N. Y.). Blood samples were collected prior to PGFp injection (0 hr.), at 6 hr. intervals for 48 hr. and every 4 hr. until 24 hr. after visual detection of estrus. Measurements of LH and estradiol were by methods previously cited. Three cycling first lactation dairy cows between 60 to 90 days postpartum and one cycling heifer were given 30 mg PGFp - Tham Salt (IM). All animals v/ere between days 9 to 15 of the estrous cycle at the time of injection. Each animal had a functional corpus luteum at the time of surgery, 4 to 5 days earlier. At the time of PGF^ injection each animal maintained a uterine-aortic temperature difference (AT ) greater than .3 C during the previous 2 days and a palpable corpus luteum. A second injection of PGF^ (10 mg) was given to 3 of the 4 cows at 21 hr. after the first injection. This was done to insure complete luteal regression. Cow aortic temperatures and AT were monitored continually from 5 hr. prior to the initial PGF^ injection until 24 hr. after the detection of estrus. Twice daily, recordings were temporarily interrupted for 90 min. (0800 and 2000 hr.) for estrous checks and exercise. Temperatures in the heifer were re- corded continually for 15 min. prior to PGF2 injection until 6 hr. postinjection. Rectal palpations were made 2 days postinjection to confirm corpus luteum regression, and again approximately 24 hr. after visual appraisal of estrous behavior to detect occurrence of ovulation. - 74 - Thermocouple placement was verified 4 days after estrus by surgical examination of the reproductive tract. At ovariectomy confirmation of luteal regression, ovulation and new corpus luteum formation was verified by disection. Results and Discussion Regression of the corpus luteum, as determined by rectal palpation, occurred in all four animals. The three cows, at the time of ovariectomy, had newly formed corpora lutea near the area where the old corpus luteum had regressed. Two of the cows were detected in estrus while the thermo- couples remained functional. The usual life span of thermocouples was about 2 weeks. However, due to mechanical failure the thermocouples in one cow lasted only 7 days, and the heifer was not detected in estrus during this period. At the time of ovariectomy and recovery of thermo- couples, confirmation of ovulation was maide on the basis of a newly formed corpus luteum. The immediate effects of PGF^^ on uterine and aortic temperatures of two cows and AT of all four animals are shown in figures 13, 14 u-a and 15. To simplify and assimilate the continuous recordings, points at 15 min. intervals were taken to describe the data. Following the 30 mg injection of PGF2^, the AT^_^ dropped .4 C (P<.01) from approximately .54 C to .16 C at 45 min. postinjection (figure 15). A similar drop (Pc^.lO) in AT occurred following the 10 mg PGF. injection. However, u-a ^^ the magnitude of the decline was only about .15 C which occurred 30 min. postinjection. The lower AT^_^ before injection of PGF^^ (10 mg) and - 75 ,-^ 1. co ^ o »— < f— c_> LU —3 o o s: (X) r—-~ £3 CM U_ CD ri o o «=3- CD o o o o o o >- CD I I CD U_ CD CD »■ — « -I g o UJ o ^=5 8 S= <_> o ^^ TO O o o <3- O koXWM » dneMBaKIMiMMJ C=5 UJ Lr>» • — I (J3 01: CJD LU N^ CVJ lU 76 - CJ ^ o o CO CD I— C_3 8 ex. CD C=) o I— « CL. O Di lI3 c_> o o CD CD CD o cC o ^ 'd- I 1,1 LH LU LU CD 7-7 f fO H K 2 z »-' Va« » « UPJ U?^ o w 0. a (5 v> S 2 o O #U CD » CM U_ CD , V viHov-snjJHin v^ - 78 - smaller decline may be related both to time and hormonal status after first injection and also dose of PGFp • These observations were not anticipated because various researchers (Bergstrom et al_. , 1968; Brody and Kodowitz, 1974; Clark et al_. , (1972) have reported a vasoconstrictor effect of PGFo • A vasoconstrictor action would tend to decrease blood flow through the uterus and therefore elevate the AT, ,. The marked ^ u-a drop in blood temperature might be attributed to an increased respiratory evaporative heat loss. Indeed, an increased respiratory rate was de- tected shortly after FGFp injection but not quantified. Sweating is negligible in cattle, so in order for heat to be eliminated by way of respiratory evaporative heat loss there has to be a tremendous increase in lung ventilation (Brody, 1945). Also, Lewis and Eyre (1972) report- ed increased respiratory volume following PGFp administration to calves. However, if aortic temperature did fall, a decrease in the AT would not occur unless there was selective PGF^ action on the u-a ^a uterus to increase heat loss or decrease heat production. If one uses the thermal balance equation, Q=FcaT, then theoretical heat production, Q, and uterine blood flow, F, can be calculated based on the aT _ changes. Q = rate of uterine heat production (cal/gm tissue-min.) F = rate of uterine blood flow (gm blood/gm tissue-min.), density of blood taken as 1 gm/ml c = specific heat of blood (.87 cal/gm blood-C) AT = temperature difference between the uterus and u-a aortic blood (C); (adapted from Abrams et al_. , 197Cb). - 79 - The major assumption is that all heat loss from uterine tissue is by v;ay of the uterine veins. Therefore, in order to calculate Q, uterine blood flow during the luteal phase of the estrous cycle needs to be obtained. Assuming no species differences, then. we can use for cattle the value of 119 ml blood/kg-min. for uterine blood flow in sheep (Huckabee et^ al_. , 1968). Theoretically, at AT, ^ = .55 C just prior to PGF„ injection u-a "^ CO. in the present experiment: Q = FcAT Q = .119 gm blood/gm tissue-min. X .87 cal/gm blood-C X .55 C = .057 cal/gm-min. By contrast, the uterine heat production rate, Q, calculated 45 min. post-PGF2j^ injection when AT^__^ = .16 C was determined to be: Q = .119 X .87 X .16 = .017 cal/gm-min. This theoretical calculation would suggest a 3.4 fold decrease in uterine heat production in response to PGF^ . Lowered AT in response to PGFp could also be explained by an increase in heat loss. One mechanism of heat loss v/ould be an increase in uterine blood flow. Be rearranging the equation, the theoretical blood flow, before and after PGF^ , can be calculated based on oxygen consumption data for a 350 kg Jersey cow (oxygen consumption 3.26 ml/ kg-min.; Brody, 1945). Assuming no differences between oxygen con- sumption (per kg) of various organs of the body (in sheep the oxygen consumption of the total body as well as the uterus is approximately 5 ml/kg-min.), oxygen consumption of uterine tissue of 3.26 ml/kg-min. - 80 - multiplied by a calorific value of 4.8 cal/ml oxygen (based on an assumed R.Q. of .8; Brody, 1945) would give a calculated heat production of 3.26 ml/kg-min. X 4<,8 cal/nil = .0156 cal/gm-min. Rearranging the original Equation: CAT then at AT = .55 C (pre-PGFo injection): u-a \r . 2a ' F - -OlSe cal/gm-min, .87 cal/gm blood-C X .55 C .0326 gm/gm-min. or 32.6 ml blood/kg--min. and at AT ^ = .16 C (post-PGF., injection): u-a ^^ ^a ' ^ .87 X .16 .122 gm/gni-min. or 112 ml blood/kg-min. The 3.4 fold increase in theoretical uterine blood flow calculated above would be comparable to changes reported by various researchers (Huckabee et a^l_. , 1970; Abrams et al_. , 1970a) following estrogen in- jections. However, the time course of maximum PGFp response (45 min.) was of shorter duration and had a more rapid onset than an estrogen in- duced decrease in AT _ (Section III, Experiment 1). These observations indicate the need for more definitive experiments in the bovine to pin- point the cause of the lowered AT in response to PGFr, . A basic u-a "^ 2a question is whether there is an increase in uterine blood flow or a - 81 - decrease in heat production. Figures 13, 14, 16 and 17 show individual cow aortic and uterine temperature changes pre- and post PGFp injection (figures 13 and 14) and at the time of the LH peak (figures 16 and 17). Both cows appeared to exhibit circadian changes in aortic and uterine temperatures. The range of aortic temperatures (37.9 to 41.0 C) within the two cows is comparable to body temperatures reported by Bligh and Harthoorn (1965) in African cattle. They reported that maximum and minimum body tempera- tures were closely associated with sunset and sunrise, respectively. In the latter study thermistors were implanted 8 cm into the dorsal caudal neck region to record deep body temperature. Aortic temperature patterns in our study showed that maximum daily deep body temperature occurred close to midnight, whereas minimal body temperature occurred between 0800 and 1200 hr. Although cows v,/ere turned out to exercise at 0800 and 2000 hr., their aortic temperatures returned to pre- turnout baselines within 2 hr. after their return to the barn. Also, there appears to be a 4 to 6 hr. lag behind barn air temperature in maximum and minimum body temperature. The uterine and aortic temperatures v^ere highly correlated (Appendix, table 13) but no correlation between AT ^ and either uterine u-a or aortic temperature was detected. This might suggest that there was no change in uterine blood flow and uterine heat production. However, these correlations were based on data throughout the entire experiment and any possible increases in AT ^ at higher body temperatures (figures u-a 16 and 17) may have been undetected statistically. Visual appraisal of figures 16 and 17 do show a widening of the AT at maximum daily ^ u-a ■^ 82 40.5 r 25.0 PGF 2a E "^40 a. k •2 30 •o S20 10 PGF i 2a Estradiol Estrus 1 2400 1200 2400 1200 2400 1200 2400 1200 "' ■ HOURS OF DAY . FIGURE 15. UTERINE AND AORTIC TEMPERATURES. LH AND ESTRADIOL IN G555 AND AIR TEMPERATURES. - 83 - 40.5 r 40.0 39.5 39,0 38.5 25.01- PGF 2 40 o •-5 30 s_ I 2a 20 10 en PGF 2a i Estrus I Estradiol A n>e /\iv.H\ \ / T T T. — IW««^^-TT- =V*3 I-— -!SJS5=5rj35aH III \ ^ 1 1 2400 1200 2400 1200 2400 1200 2400 1200 2400 HOURS OF DAY FIGURE 17. UTERINE AND AORTIC TEMPERATURES. LH AND ESTRADIOL IN JN15 AND AIR TEMPERATURES. 84 uterine and aortic temperatures. Figure 18 shows the significant (P<.01) curvilinear (2nd order) time trends for uterine and aortic temperatures when data were pooled across days for each time of blood sampling. The data representing each individual sampling point is the average of individual 15 min. points + 2 or 3 hr. from time of the blood sample. Also only tempera- tures preceding turn out of cows (0800 and 2000 hr.) were used in obtaining an average for the 0800 and 2000 hr. blood sampling times. The air temperature plot is comprised of average values across the individual days. Uterine temperature and aortic temperature were not influenced by barn air temperature (P>.10; Appendix, table 13). The uterine temperature trend throughout the day was best described by Y .^ = 40.04 - .143X + .007X^ (P<.01) where X = hr., (uterine temperature, C) whereas aortic temperature was best characterized by Y^^q^^^-j, tgmpera- ^ = 39.50 - .125X + .005X^ (P<.01; Appendix, table 14). The ture, C) body temperature lag of about 6 hr. behind air temperature (1600 hr.- peak air temperature compared to 2400 hr. body temperature peak) is best seen in figure 18. Possible explanations for the time delay could be: 1) Thermal inertia of the cow. For example, ambient temperature will increase more rapidly than body temperature because of the mass and heat capacity of the large mammal. 2) Inherent circadian rhythm of body temperature which may be independent of environmental temperature and cued to external events such as light-dark cycles, feeding regimen, presence of barn personnel and other factors which were uncontrolled in this experiment. - 85 o ^ o CM oo LU m Ci3 ':^^ >=C ni c_> LlJ o rv^ o o LU UJ Di: >- o ^r CD (Alliston et al. , 1965). In JN15 (figure 17) this increased uterine temperature v^as at the time when artificial insemination would normally have been performed. We failed to detect an association between concurrent measurements of AT with estradiol or LH (Table 13). Based upon Experiment 1 there was a 2.5 hr. delay between injection of a pharmacological dose of estradiol and the minimum AT. ^. From PGF, injection through the u-a co, LH surge (figure 19) estradiol concentrations fluctuated considerably as did the AT (figures 16 and 17). Not until the massive LH dis- u-a ^ ^ charge was there an appreciable rise in AT ^ at a time when estradiol u-a was decreased (figure 19). Findings of this experiment indicate that uterine and aortic temperatures followed a daily circadian rhythm, and.because of a time lag in these temperatures behind air temperatures, correlations between body temperatures and ambient temperature were negligible. Failure to detect an association between AT , and hormonal measurements may be u-a due to the time lag, also. The mild heat stress (which by definition occurs in cattle anytime ambient temperatures exceed 30 C), to which these cows were subjected to, may have contributed to the high uterine and aortic blood temperatures. Uterine temperatures periodically 87 CO CD LH (ng/ml . ) o en CO LU CD ir> o ID CO OO r— in ( — * [Lu/5d) [0!.pejiiS3 LU CJ3 CD . <}■ C5 C\J ZIP^" 1 to LU en ZD V£) o re 1— • CO 1 II 1 1 — . CO OC «:*• o o CO oo o tj3 - 88 - exceeded 40 C (Appendix, table 15). Since survival rate of fertilized ova exposed to 40 C for 3 hr. is seriously decreased, these observations may be of some practical significance in improving reproductive per- formance in a hot climate. SECTION IV SUMMARY AND CONCLUSIONS Ten normally cycling Hoi stein heifers at the USDA, Agricultural Research Center, Beltsville, Maryland, were assigned to one of two en- vironmental treatment groups (21.3 C, 59% RH or 32.0 C, 67% RH). PGFp -Tham Salt (PGFp ) was used to cause corpus luteum regression and synchronize estrus. Blood samples were collected prior to PGFp injection and at 6 or 4 hr. intervals following injection through ovulation. Plasma samples were analysed to determine concentrations of progestins, estradiol, estrone, LH, prolactin, corticoids, total protein concenti^ation, osmolality, Cortisol binding capacity and Cortisol association constants. In the second phase of this first experiment adrenal responsiveness to ACTH (200 lU) was tested by quantification 'of corticoid concentrations in plasma prior to and up to 12 hr. following injection of ACTH. Least-squares analyses were conducted to characterize treatment, animal and within-animal time trends in plasma progestins, estra- diol, estrone, LH, prolactin and corticoids. Other response variables were analyzed by analysis of variance. Environmental treatment of 32.0 C evoked a 1.49 C increase in rectal temperature and a 3.59 C increase in skin temperatures. Time durations between PGFp injection to LH peak and ovulation were not different (P>.10) betv/een treatments. Length of estrus was shorter 89 - - 90 - (P<.10) for heat stressed heifers (21 compared to 16 hr.). Tv.'O of four heifers inseminated at 21.3 C were pregnant at 40 days compared to none of five at 32.0 C, Thus, the environmental thermal stress did affect body temperature, duration of estrus and overall fertility. No differences (P>.10) in plasma samples, prior to injection of PGFj, , v/ere detected due to main effects of temperature. Hormonal responses post-injection between treatments were detected. Average progestin concentration between treatments v;as not different (P>.10), however, 5th order regression curves v/ere not parallel (P<.01) indicating different time responses between treatments. Progestin concentrations declined in a similar manner in both groups immediately following PGFo injection. Heifers in the 21.3 C group had an LH surge about 24 hr. later than heifers in the 32.0 C group. This 24 hr. time lag accounted for the difference in tim.e responses when data were synchronized to time of LH peak for analysis. Mean estradiol concentration was significantly (P<.10) lower in heat stressed heifers. The lower plasma estradiol may have contributed to the shorter periods of estrus in the 32.0 C heifers. Hov/ever, these lov/er concentrations of estradiol were adequate enough to elicit estrous behavior and trigger a preovulatory surge of LH to cause ovulation. . Estrone shov/ed no apparent association with either onset of estrus or peak of LH when the data were synchronized to time of the LH peak. There was a significant elevation (P<.05) of estrone due to heat stress, but there was no evidence that estrone time trends following PGFp were not parallel (P>.10). In both treatments, estrone follov/ed a similar decline postinjection. No differences (P>.10) were found in mean - 91 - LH concentrations between heifers at 21.3 C or 32.0 C. Plasma pre- ovulatory peak of LH concentrations were 32.2 and 33.2 ng/ml for 21.3 C and 32.0 C treated heifers, respectively. All animals h.ad a pre- ovulatory LH surge indicating that hyperthermia did not prevent the triggering mechanism for LH release. There was no change in prolactin associated with estrus or peak of LH, therefore, prolactin was analyzed relative to time of PGF^ in- jection. Mean prolactin concentrations v/ere not different between treatments (P>.10), but 4th order response time curves were not parallel (P<.005). Heifers in the 21.3 C chamber had a decline in plasma prolactin associated with sequential blood sampling, whereas prolactin increased in 32.0 C treated heifers during the early blood sampling period. The decline of prolactin in cooler heifers may be related to a lowering of stress-induced prolactin secretion associated with initial sampling. Apparently heifers in the 32.0 C chamber could not adjust to sampling as quickly since prolactin remained elevated until 24 hr. after PGFp (42 hr. after the initial sample). The summer seasonal increase in plasma prolactin, reported by various researchers, may be more related to photoperiod. There was no difference (P>.10) between treatments in plasma corticoid concentrations. The coefficient of variation for plasma corticoid v/as 65% after accounting for variability due to treat- ment, heifers within treatment and time trends up to the 5th order. In an attempt to determine if plasma dilution may have occurred, total protein concentration and osmolality were measured. There was no difference (P>.10) in total protein concentration or osmolality between treatment groups. However, no measurement of total plasma volume was - 92 - made. Cortisol binding capacity of CBG and its association constant (K ) a were determined. The affinity (K^) of Cortisol for CBG v/as not different a between treatments (P<.10); however, the binding capacity of CBG for Cortisol was reduced (P<.05) in the 32.0 C heifers. This observation suggested that, under experimental conditions (4 C) for determining Cortisol binding capacity, hyperthermic heifers had a decreased plasma concentration of CBG. Results of this first experiment show only subtle thermal effects on plasma estradiol and estrone concentrations, and no effects on plasma LH, progestins, corticoids and prolactin. Apart from possible hormonal involvement with duration of estrus, heat stress does not appear to affect drastically the peripheral plasma hormonal milieu associated with corpus luteum regression, follicle growth and ovulation. At 8 days following ovulation in the last heifer of the first experiment, the 10 heifers were injected with 200 lU ACTH (IV). The 32.0 C heifers responded with significantly lower (P<.10) corticoid concentra- tions. The 5th order regression response curves were not parallel (P<.01) suggesting that the hot group response v/as earlier to reach a . peak (75 min. compared to 105 min.), had a lower magnitude (73.5 compared to 100.2 ng/ml corticoids) and was of shorter duration (4 hr. compared to 5 hr.). The significance of a possible lowered adrenal function in hot environments may be related to state of lowered heat production. Since corticoids are known to be calorigenic, a lowered adrenal responsiveness in hyperthermic heifers might be physiologically advantageous. However, a possibly lowered adrenal responsiveness at 32 C was detected only after stimulation of - 93 - the adrenal with ACTH. Because the first experiment did not specifically consider pos- sible environmental and hormonal effects on uterine temperature, it was necessary to document possible hormonal and environmental effects on uterine thermal changes. Estrogen induced thermal changes were documented and an attempt was made to characterize uterine thermal changes during the period of luteal regression, follicle growth and ovulation under conditions of mild heat stress. In experiment two, thermocouples were placed into the uterine serosa and aortic blood vessel of four dairy heifers. Injection of 3 mg estradiol-17s caused a .25 C decrease (P<.01) in the difference between uterine and aortic temperature (aT, ) by 2.5 hr. postinjection. In u~a contrast there was no significant change (P>.10) in aT , after injection u-a of saline. An augmented rate of heat loss resulting from a marked estradiol -17b induced elevation in uterine blood flow may account for the decreased difference between uterine and aortic temperature after estradiol-178 injection. The final experiment was an attempt to document and evaluate changes in uterine temperature during the period of luteal regression (decreasing progesterone), follicle growth (increasing estradiol) and ovulation induced by PGFo under conditions of a mild heat stress. Thermocouples v/ere placed into the uterine serosa and aortic blood vessel of four dairy cattle. Blood samples were collected at 6 to 4 hr. intervals following PGFp injection in order to monitor endogenous peripheral plasma concentrations of estradiol and LH. PGF^ caused an immediate drop in uterine and aortic temperatures, and a decrease in the AT of almost - 94 - .4 C at 45 min. postinjection. This effect of PGF2 on the uterus may be due to a selective vasodilatory response at the uterus. The PGF2 induced transient decline in AT in the bovine needs further u~a clarification. Two cows, in which thermocouples remained operational for the duration of the study, had monophasic daily uterine and aortic tempera- ture rhythms. However, both temperatures lagged about 6 hr. behind air temperature changes. Thermal inertia or inherent circadian rhythms of the body may be involved in the time delay. Uterine temperatures reached 40 C for periods of up to 6 hr. During this time ambient temperature fluctuated around 30 C (mild heat stress). Failure to detect an overall association between concurrent AT , and hormonal measurements u-a might have been due to a time lag association and estradiol variability following PGFp injection. Not until the preovulatory surge of LH was there an appreciable rise in AT ^ (P<.01), and this occurred at a time u-a when plasma estradiol was decreasing. The mild environmental heat stress may have contributed to the high uterine and aortic blood temperatures. Since survival rate of fertilized ova exposed to 40 C for 3 hr. is seriously decreased, interrelationships in the last experiment between ambient, uterine, aortic temperatures and hormonal status may have major practical implications. In conclusion, under experimental conditions of the above study, heat stress caused only subtle effects on peripheral plasma hormonal concentrations during the preovulatory period. Adrenal responsiveness to ACTH appears to be altered under conditions of thermal stress. Uterine temperatures are sensitive to estradiol, PGFp and ambient temperatures. APPENDIX - 96 - c: CO CO >> e: CM o CO 0) X5 J3 Q I — I O 00 O O 5s OO to LU o cc to «4- * in 00 tn CJ 2: * * 1 — t * ■)c 4-> f - OO O 00 • » <: to ^ r-. o t/> —i *^ OO S_ o CM vc O) CtT <+- a_ 'r— o j= o c LU •1 — 2: * t/1 cc to * LO • r-s * «=J c_> c_> OO o TD ^ CM +J CM OO t= ^»^ ^v. 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CO <^ c «JD • • • o 1 1 I •1 — •t-> o o CO r^ CO •'—> CM LO 00 0 a • <^ • • • •1 — O r— t— CM oc I— _i C_) cC (/> »£5 0 UD 0) 00 cr> 0 C3 cr> V£) • • • o CM CM CM s_ o_ nj ID 0 r— e n 1— 0 CO (/> UD • • • 03 CM «* U^ 1 — Ci_ CM 4-> -PiH OS ■ CM r— 0 c HOURS AC TM.lFr > CD $- »— ^ Cl. 105 Table 13. Simple correlations between hormones and temperatures, LH .16 Air Temperature -.24 ATu-a Aortic Temperature .23 Uterine Temperature Estradiol^ .02 .23 LH^ .11 -.11 -.03 -.06 Air temperature -.38** .15 .05 iT " u-a -.08 .17 Aortic temperature .97** ®n=34; ^n=49 ** {P<.01) 106 - Table 14. Analysis of variance for aortic and uterine temperatures. Source d.f. Aort ic Temperature MS Uti erine Temperature MS Cow 1 3.69** 1.79** Time of Day 1 .01 .12 Time of Day (Q) 1 2.61** 3.20** Error 45 .17 .21 **(P<.01) 107 CI in V£) ID (3 s_ o ^- c cu E S- v> ro S_ (U Q. E o i- o in — ' <_> ir> r— f« 2: +J •-D S- o tD rc c ^- CD *iD 00 "^ CO r— ( c\j c\j CO in (X) I es C3 t:5 to s- CD CD CD C— - "I— • 3: i- O SI o e5 W- CM E D- ijdcocoi — looiOt— incMOr^i — ir>t-^Oi— r-^o^i — < — uimini — 1 — <\jr^oocooc\iro(x;>roroLncoooi — i~^r~»LOLf>ooor^ioc3CMCDi^mLnoi — i — r^voi£)Oc\ji — cococo cjCMC\jCMooc\jc\jc\jcocococ\jc\joJoorooocvicMC\ioorococ\jcvJc\j «if-r^coooc\jcsjr-.«^CMO<>oijDr^ un coor— ooiSjOr— ojomi— ro cocvjCM'^mr^iocO'^-coojr — r^ofouncMOCMLniOi — incocvjin (X)0^cricr>criC5-(CTicO(Ticr>oocriCTio-io-icriooocyiCDOOoo o'joororoo-)corooororO'c3-.^-roooroooro«;j-«d-^d-ro ■* «;3- «=r ^ ;;j-corooororocococoropOfO ^co^«d-i— cy>i£)r-«*00^:j-f— «:a-oouD«^ooLr)Oir>cr>r-coir)oo ^-oocooiCMOocoiOr— Lncr>cO';rr-~r-c\jcr>r^r-,C)cor-.ocot^c^ o3COCorocr^cricTi(X)a^c^a^cricr>oocricrioocriCTiOo~)Cr>ocrvcT>cri rooooorooocorocooororococororooororoc>o«:i-roco«;j-moTro Lor^cocoir. '-.oo^ovDcocococncnr— C)Ocrtcnt^r-.r— o^ c\jCMOuD«a-'rLr)CM>jDCvJoou.or^i — ojujOi — r~~c\icMaDCM O^O^C^l(X)OOCOCX^COCT)O^O^COCOCOCOC^lCT^OOOOCOCo'cTI cococofocococooofo^oo^coco^cv:lC^JC>Jcooo^•■)roo^c^lC\JC\Joo>v^LOC\J^o«^>vJ-LnLn «ac>sJalco>^<^^0'v^«^^oun«:J-LnLn^o«:d-"^a^^ooooo^>-Lft tnmLr)Ln«^ir)mLn«^<^ir>iovomcMo-)^j-vDr~^<£><*Ln(£i i i i ooi — t^cvj c>di — r~.i — in i — cyicy^r--i — cr>crv loi — •***l ••II •••! •II ••••••! ••! ror^r^r~» r — o cn^xjcvj o oocMiDcvicriLn voi — I — i — OJ C\Ji — 1 — 1 — I — C\JCM r— ooo o ocor-^ i£)CO(£>ojco ro cocMr-^ ••III •! •••I ......| .| •••III CTico r^ oocMi->. «:i-^CMLOOrv. co incooj r— r— I— I— CSJ CM I— I— I— I— ,— CMCM^^CMC^^^ocMlDC^JCM^JOC^JC^JC^J<;^mLn^o^v.coc^JC^JcvlCMC^JC\J 0C5:^cocM^oc^«^cocM^oO'5^ I — r— c\jroro«=j-«^LnLf)Lnix)ior^r^r^cococDcrvcr>oOi — r — LIST OF REFERENCES Abilay, T. A. and H. D. Johnson. 1973. Influence of high environ- mental temperature (33.5 C) on plasma progesterone and Cortisol. J. Dairy Sci . 56:642. Abilay, T. A., H. D. Johnson and S. Seif 1973. Heat effect on plasma steroids in Zebu and Highlands. J. Amm. Sci . 37.298. Abraham, G. E., R. Swerdloff, D. Tulchinsky and WD Odell. 1971 Radioimmunoassay of plasma progestin. J. Clin. Endocr. 6d.b\J. Abrams, R. M., D. Caton, J. F. Clapp III and D. H. Barron. 1970a. Thermal aspects of uterine blood flow in non-pregnant sheep. Amer. J. Obstet. Gynecol. 108:919. Abrams, R. M., D. Caton, J. Clapp and D. H ^^^^^J!; . J^^°^: J^^;;S'^ and metabolic features of life in utero. Clin. Obstet. and Gyncc. 13:549. Abrams, R. M., D, Caton, J. Clapp and D. H Barron. ^^Jl temperature differences in reproductive tract of non-pregnant ewe. Amer. J. Obstet. Gynecol. 110:370. Abrams R M , W. W. Thatcher, F. W. Bazer and C. J. Wilcox. ■1973. Effect of Estradiol-173 on vaginal thermal conductance in cattle. J. Dairy Sci. 56:1058. Alliston, C. W., B. Howarth and L. C. Ulberg. 1965. 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Calorigenesis of dairy cattle as influenced by Hydrocortisone and environmental temperature. J. Anim. Scio 26:1087. BIOGRAPHICAL SKETCH r Francis C. Gwazdauskas was born July 25, 1943, at Waterbury, Connecticut. In June, 1961, he was graduated from Crosby High School, Waterbury, Connecticut. He received the degree of Bachelor of Science with a major in Animal Science from the University of Connecticut in June, 1956. From February, 1966, until January, 1967, he was employed at the USDA-C&.MS as a meat grader. From January, 1967, until November, 1968, he served as a Veterinary Specialist in the United States Army and was stationed in Viet Nam. Since September, 1969, he has been enrolled in the Graduate School of the University of Florida and has worked as a graduate research assistant in the Dairy Science Department. In March, 1972, he received a Master of Science degree in Dairy Science. The author married Judy Keller in 1971, and they have a daughter, Jennifer and a son, James. He is a member of Gamma Sigma Delta, Alpha Zeta, Sigma Xi, the American Dairy Science Association, the American Society of Animal Science and the Society for the Study of Reproduction, - 118 I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy, / / William W. Thatcher, Chairman Associate Professor (Associate Animal Physiologist) I certify that I have read. this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. /i...i.///^<- Donald H. Barron Professor in Obstetrics and Gynecology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Charles J. Wilcox ' Professor (Dairy Geneticist) I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Robert M. Abrams Associate Professor in Obstetrics and Gynecology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. c til Fuller W. Bazer Associate Professor (Associate Animal Physiologist) I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Donald Caton Associate Professor of Anestliesiology and Associate Professor in Obstetrics and Gynecology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. % Herbert Head Associate Professor (Associate Animal Physiologist) This dissertation was submitted to the Graduate Faculty of the College of Agriculture and to the Graduate Council, and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. December, 1974 'iOLkiLiM. Dean, College of Agricultur Dean, Graduate School A