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I. . 4. . . . . .. _ . .. . . _. . . ... . . . v .. ......w:.::r.... 21..) . .lai .. 1.. ..v.) r .. ...... .... . 2‘ TA... . ...... 1.1.7... .. . . .....v. 19:12:... ....IvI...... .0). .....l: 4 ..L. ......4......;. . i... 4. _ ....»1._v...:..r.to!»| Ch... . . . . . .5 9.39.2...51. ......) it! we“ in! .. Michigan State University This is to certify that the ‘ thesis entitled " Steroid Therapy and Stress Effects on Embryonic Survival and the Adaptation of Laparoscopy to Swine presented by David E. Wild 1: has been accepted towards fulfillment of the requirements for Ph. D. degree in PhYSiOIOg y and Animal Husbandry /7 v 7?} ,7 7 W 5/ M/wza‘g g/béé 171/ Major professor Date May 7, 1975 0-7639 #5 MICHIGAN STATE UNIVERSITY CHAPTER OF THE SOCIETY 0E THE SIGMAXI TO Bavib Effllmifbt F OR MERITORIOUS RESEARCH IN Eubacrine 33%“ch 311m MK tHIGAH STATE I NIVIZRSITY CHAPTER ~/ ABSTRACT STEROID THERAPY AND STRESS EFFECTS ON EMBRYONIC SURVIVAL AND THE ADAPTATION OF LAPAROSCOPY TO SWINE BY David Edwin Wildt The influence of steroid therapy and thermal stress on reproduction in the pig were studied during the first 25 days of gestation. In addition, laparoscopy, which allows repeated direct observation of reproductive organs was adapted to this same species and tested as a diagnostic tool. In the first study, progesterone, estrone, or proges— terone-estrone in combination were administered to intact pregnant multiparous sows during various intervals of the first 23 days of gestation. In the first trial conducted at Michigan State University, the results demonstrated that 25.0 mg of progesterone and 12.5 Mg estrone injected together daily in 2 ml quantities for the 10 day interval (days 14 through 23 of pregnancy) would result in a significant increase (2.1 pigs/litter, p <.02) in both the total number of pigs born and pigs born alive when compared with litter sizes of sows receiving cottonseed oil only. This same treatment resulted in significant increases (p <.05) in plasma progesterone when compared with control sows. The David Edwin Wildt administration of the two steroids together for five days (days 14 through 18) or two days (days 16 and 17) resulted in litter size increases of 1.2 and 1.6 pigs born respec- tively, with the latter being significant (p <.05) from the control mean. The results were also examined on the basis of population distribution of sows producing specific litter sizes. Treatment groups receiving 25.0 mg proges- terone and 12.5 pg estrone together on days 14 through 23 and on days 16 and 17 of gestation demonstrated significance over controls (p <.05 and p <.01, respectively) in the pro— portion of the treatment population producing litters of 12 pigs or greater. Sows receiving the same hormone admin- istration on days 14 through 18 and on days 15 and 16 showed significant increases above controls (p <.01 and p <.O75, respectively) in the proportion of the population producing litters of greater than 13 pigs. Treatment with 25.0 mg progesterone and 12.5 pg estrone for 5 or 10 days during the pre-implantation period was ineffective in altering litter size. Increasing the dosage of steroids above 25.0 mg progesterone and 12.5 ug estrone had no effect on litter size nor did estrone or progesterone administered alone during the implantation interval. A second trial was conducted in a similar experimental design at Illinois State University. There was a mean increase of .8 and 1.5 total pigs born when 25.0 mg proges- terone and 12.5 Hg estrone were administered during implan— tation for 10 and 5 day periods, respectively. This was David Edwin Wildt not a significant increase when compared to control values. In the second study, physiological response and repro- ductive parameters were examined in mated gilts subjected to a short-term heat stress during two intervals of early gestation. Trial 1 control and treated animals experienced temperatures of 24.0 and 40.20C, respectively, during the pre-implantation interval from days 2 through 13 of preg- nancy. Trial 2 control and treated gilts were exposed to 23.3 and 40.4OC, respectively, in the implantation and post-implantation stage from days 14 through 25 of pregnancy. Both high thermal exposures caused significant increases (p <.05 or p <.Ol) in rectal temperature. At body temper- atures of 41.10C or above, animals became more active and behavioral patterns erratic. Animals in Trial 1 demon- strated more rapid and less variable increases in body temperature than animals in Trial 2. Stressed pigs in both trials exhibited adaptation to the high thermal environ- ment as treatment days progressed. Embryonic mortality in the animals stressed during pre—implantation (Trial 1) was greater than controls. The gilts of the former group tended to either retain a normal size litter or lose the entire litter by day of slaughter. Fetal weights and mean corpus luteum size were not different within trials. A significant proportion (p <.01) of litters from stressed animals con— tained degenerating fetuses at day 42 of gestation suggesting a continuous or delayed effect of thermal stress on embryo survival. David Edwin Wildt The technique and use of laparoscopy in swine is described. Gilts were anesthetized with sodium pento— barbital at a level of 10.0 to 32.0 mg/kg body weight, and placed head down on a sloped surgery table. A 130 degree pediatric laparoscope was inserted through a 1 cm midline incision with a tactile probe inserted into the abdominal cavity to one side which allowed repositioning and manipu- lation of internal organs. Documentation of observations was made using a 35 mm camera attached to the laparoscope. At the termination of each laparoscopic examination, the incisions were treated with an antibacterial ointment. Suturing was unnecessary. Results demonstrated that laparoscopy could be used frequently in studying ovarian morphology with minimal stress and trauma to the animal. Corpora lutea numbers observed with laparoscopy did not differ significantly from numbers counted after slaughter. Pregnancy was accurately confirmed in 100 percent of the gilts examined after day 12 of gestation and laparoscopy of pregnant gilts had no effect on embryonic mortality, fetal weights, or crown-rump measurements. STEROID THERAPY AND STRESS EFFECTS ON EMBRYONIC SURVIVAL AND THE ADAPTATION OF LAPAROSCOPY TO SWINE BY David Edwin Wildt A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Departments of Animal Husbandry and Physiology 1975 ACKNOWLEDGMENTS Sincerest appreciation is expressed to my advisor, Dr. W. Richard Dukelow, whose counsel, forbearance, and criticism enabled me to learn the methods of scientific research. Special thanks are also extended: To my wife, Brenda, whose enduring encouragement, advice, and aid made the completion of the requirements for this doctoral degree possible. To Dr. Ronald Nelson, Chairman, Department of Animal Husbandry, for providing facilities, equipment, and financial support that contributed to the studies. To members of my doctoral committee for their advice and criticism of the thesis manuscript. To Dr. Gail D. Riegle for his assistance with the stress study and his very competent instruction in the area of endocrinology. To Dr. A. A. Culver and Charles Alan Wildt for their willing and able cooperation in coordinating the Illinois State University field trial. To Dr. Jon M. R. Rawson, Clifford B. Morcom, David A. Snyder, and James L. Spencer not only for their counsel and help with various research projects but also for their love and understanding of country and western music. To Peterina Smith and LaVonda Cleeves for their secretarial skills and to all members of the Endocrine Research Unit for their thoughtfulness and support during the course of these studies. ii TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . . . . . . . . . . . LIST OF FIGURES . . . . . . . . . . INTRODUCTION . . . . . . . . . . . . . . . . . . . LITERATURE REVIEW . . . . . . . . . . . . . . . . Additional ova to increase litter size . . . . Superovulation . . . . . . . . . . . . . Embryo transfer . . . . . . . . . . . . . Increased energy intake . . . . . . . . . Intrauterine mechanisms controlling litter size Uterine secretions . . . . . . . . . . . . . . Steroid hormone effects . . . . . . . . . . . . Genetic influences . . . . . . . . . . . . Stress: the effect on the reproductive system and embryo survival . . . . . . Laparoscopy . . . . . . . . . . . . . . . . . INFLUENCE OF STEROID ADMINISTRATION ON SWINE LITTER SIZE . . . . . . . . . . . . . . Materials and Methods . . . . . . . . . . . . Trial 1 . . . . . . . . . . . . . . . . . Trial 2 . . . . . . . . . . . . . . . . . Trial 3 . . . . . . . . . . . . . . . . Data collection and analysis . . . . . . Results . . . . . . . . . . . . . . . . . . . . Treatment administration . . . . . . . . . Conception rate . . . . . . . . . . . . . Litter size . . . . . . . . . . . . . . . Trial 1 . . . . . . . . . . . . . . . - Trial 2 . . . . . . . . . . . . . . . Plasma progesterone . . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . INFLUENCE OF HEAT STRESS ON PHYSIOLOGICAL RESPONSE AND EMBRYONIC DEATH . . . . . . . . . . . . . . Materials and Methods . . . . . . . . . . . . iii Page vi OJ OQKOCD \lU'ln-bsb NH MN nth-H 27 27 27 30 31 32 32 32 32 34 34 38 38 41 49 49 TABLE OF CONTENTS (Continued) Page Results . . . . . . . . . . . . . . . . . . . . . 52 Discussion . . . . . . . . . . . . . . . . . . . 62 ADAPTATION OF LAPAROSCOPY FOR USE IN SWINE . . . . . . 67 Materials and Methods . . . . . . . . . . . . . . 67 Results and Discussion . . . . . . . . . . . . . 72 Anesthesia . . . . . . . . . . . . . . . . . 72 Ovarian observation . . . . . . . . . . . . 74 Barbiturate influence on ovulation . . . . . 77 Determination of the number of ovulations . . . . . . . . . . . . . . . . 78 Pregnancy diagnosis . . . . . . . . . . . . 78 Uterine and oviductal characteristics . . . 81 Laparoscopy and embryonic mortality . . . . 82 SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . . . 84 LITERATURE CITED . . . . . . . . . . . . . . . . . . . 88 APPENDIX A. PUBLICATIONS BY THE AUTHOR . . . . . . . . . 99 VITA . . . . . . . . . . . . . . . . . . . . . . . . . 108 iv 10. 11. 12. 13. LIST OF TABLES SPECIES IN WHICH LAPAROSCOPY HAS BEEN SUCCESSFULLY USED . . . . . . . . . . . . . . . NUMBER OF SOWS RETURNING TO ESTRUS FROM EACH TREATMENT GROUP IN TRIALS 1 AND 2 . . . . MEAN NUMBER OF PIGS BORN AND PIGS BORN ALIVE FROM EACH STEROID TREATMENT GROUP (MSU) . PROPORTION OF SOWS IN EACH STEROID TREATMENT GROUP PRODUCING 1 OF 5 LITTER SIZES (MSU) . . . MEAN NUMBER OF PIGS BORN AND PIGS BORN ALIVE FROM EACH STEROID TREATMENT GROUP (ISU) . . . . PROPORTION OF SOWS IN EACH STEROID TREATMENT GROUP PRODUCING 1 OF 5 LITTER SIZES (ISU) . . . ENVIRONMENTAL CHAMBER TEMPERATURES AND RELATIVE HUMIDITY FOR GROUPS IN STRESS STUDY. . MEAN RECTAL TEMPERATURE DURING THE TWO HOUR CHAMBER PERIOD BASED ON 12 DAY EXPOSURES . NUMBER OF ANIMALS RETURNING TO ESTRUS, MEAN OVULATION AND FETAL NUMBERS, AND PERCENT EMBRYO MORTALITY IN LITTERS FROM STRESS STUDY . . . . FETAL WEIGHTS ADJUSTED TO DAY 42 OF GESTATION AND NUMBER OF LITTERS WITH ONE OR MORE DEGENERATING FETUS . . . . . . . . . . . . . . COMPARISON OF CORPORA LUTEA NUMBERS QUANTIFIED AT LAPAROSCOPY AND SLAUGHTER . . . . . . . . PREGNANCY DIAGNOSIS RESULTS USING LAPAROSCOPY FETAL MORTALITY, WEIGHT, AND CROWN-RUMP MEASUREMENT OF FETUSES FROM GILTS SUBJECTED TO LAPAROSCOPY . . . . . . . . . . . . . . . . Page 26 33 35 37 39 39 51 6O 61 63 79 80 83 LIST OF FIGURES Page Plasma progesterone during early pregnancy ”in control and steroid treated sows . . . . . . 40 Average rectal temperature at 15 minute intervals (i standard error) over the 2 hour period of control and stressed animals in Trial 1 . . . . . . . . . . . . . . 54 Average rectal temperature at 15 minute intervals (i standard error) over the 2 hour period of control and stressed animals in Trial 2 . . . . . . . . . . . . . . 55 Average rectal temperature (T standard error) for each day of the 12 day chamber exposure in Trial 1 . . . . . . . . . . 57 Average rectal temperature (T standard error) for each day of the 12 day chamber exposure in Trial 2 . . . . . . . . . . 58 Laparoscopy technique in the gilt . . . . . . . 68 69 Relationship of pentobarbital administration and respiration and heart rate in a 100 kg non-pregnant gilt . . . . . . . . . . . . . . . 73 Ovarian observation by laparosc0py . . . . . . 75 vi INTRODUCTION The pig is a unique species for reproductive research because it experiences an incidence of embryonic mortality four times higher than most other species. Although loss of the fetus can occur throughout pregnancy, most of the mortality occurs during the early part of gestation. There is disagreement on the cause and time course of embryonic mortality. The inconsistencies originate from the contro- versy over the role of the intrauterine environment and the factors controlling this environment on embryo survival. Previous studies had suggested that steroid therapy or stress might influence embryo viability during the early weeks of gestation. To successfully study swine reproductive phenomena in yiyg, it has been necessary to expose animals to major abdominal surgery (laparotomy) and surgical stress. The effect of this stress on normal reproductive cyclicity, pregnancy, and intrauterine environment is unknown. Con- sequently, there has been a need for an improved technique for the examination of the reproductive tract of the pig. The present investigations had the following goals: 1. To determine the effect of exogenous steroids administered during different periods of the first 23 days of gestation on litter size. 1 2 To determine the effect of severe thermal stress imposed during two intervals of early pregnancy on embryo survival. To adapt the technique of laparoscopy for repro- ductive study in the female pig and to determine the practicality of this technique as a research tool for this species. LITERATURE REVIEW The high incidence of swine fetal mortality during the first 25 days of pregnancy is important in limiting litter size at term. Because of recent developments in mechanical feeding operations designed for the neonatal pig (Lecce, 1969), traditional female parameters (i.e. lactation, teat numbers) no longer place a severe restriction on litter size. Determining the cause of early embryonic death would solve an enigmatic reproductive problem. It is therefore important to determine the causes of early embryonic death. The following review concerns the interrelationships of physiological parameters and environmental stressors on litter size and fetal survival. It is almost exclusively limited to the domestic pig. Only a brief historical sketch on laparoscopy will be made since this subject has recently been extensively reviewed (Jewett, 1972; Rawson, 1973; Snyder, 1974). There are three possible approaches that can be used to increase litter size in the pig: I) increased ovulation rate; II) decreased embryonic mortality; or III) a combina- tion of the two. Zimmerman (1972) recently reviewed the possibilities of increasing the number of fertilized ova by superovulation, embryo transfer, and nutritional variation. 4 For this reason, only a brief mention of these techniques as well as more recent developments in this field will be discussed. A more comprehensive examination will be made of studies concerned with the factors surrounding the embryonic mortality characteristic of this species. Additional ova to increase litter size Currently there are three practical methods for increasing the number of ova available for improved litter size: I) ovulation induction (superovulation); II) embryo transfer; and III) increased energy intake ("flushing"). Superovulation Doses of 1,200 i.u. (or higher) of pregnant mare's serum (PMS) normally induce superovulation in the cycling gilt or sow or in the ovulation-inhibited gilt (Hunter, 1964; Longenecker and Day, 1968; Day and Longenecker, 1968; Polge et al., 1968). Following administration of PMS on day 15 or 16 of the estrous cycle or on the day of weaning, a luteinizing hormone source (LH) may be given 84 to 96 hours later although this hormone may not be absolutely necessary for superovulation (Zimmerman, 1972). The dose and timing of gonadotropin treatment as well as the stage of gestation when litter size is measured determines the effectiveness of superovulation techniques. At 25 to 40 days of gestation, potential fetal numbers are greater in ovulation-induced animals (Day et al., 1967; Pope et al., 1968). However, at the termination of preg- nancy litter size is not significantly improved (Pope et al., 5 1968). Apparently an increase in embryo mortality occurs under superovulatory conditions. The reasons for the greater death loss are unknown although it has been sug- gested that the additional ovulated ova may be abnormal or that normal uterine function may be altered by the hormone administration (Ulberg and Rampacek, 1974). Embryo transfer The transfer of fertilized ova to non-mated recipient gilts or the transfer of additional ova to mated gilts (superinduction) with the stages of the estrous cycle of the donor and recipient being naturally synchronized is another method to increase litter size. Dziuk (1968) has found significant increases in embryo numbers in early gestation from ova transfers into mated gilts on day 4 after breeding, but Bazer et al. (1968) observed no improve- ment in number of embryos at 25 days of gestation when the transfer was performed 2.5 days after the onset of estrus. Fenton et a1. (1969) later reported significant increases in fetal numbers when transferred ova were introduced into mated gilts at day 7 following the onset of estrus behavior. The latter two reports suggest some mechanism functioning between days 2.5 and 7 of pregnancy is controlling the number of viable embryos present at 25 days of gestation in gilts receiving embryo transfer. However, studies in the same laboratory later suggested the concept of limited uterine capacity and that mechanisms controlling uterine capacity act between days 7 and 25 to regulate litter size 6 (Fenton et al., 1972). In two of three trials, superinduc- tion on day 7 of pregnancy failed to significantly increase 25 day litter size. Apparently the primary determinant of litter size in animals undergoing superinduction occurs sometime during the first 25 days of gestation. Pope et a1. (1972) determined litter size at 25 days of pregnancy following the transfer of either 12 or 24 embryos into non-mated, hormone stimulated gilts. The average number of embryos per animal was significantly higher (p <.01) in gilts receiving 24 ova than those receiv- ing 12 ova. The former group produced a mean of 17.7 viable embryos and the latter 12.9 embryos. Embryo numbers exce- eded the number of corpora lutea (CL) in both groups. An increase in CL numbers did not seem to be a prerequisite for maintaining the additional ova introduced by embryo transfer, at least until 25 days of gestation. In addition, the results indicated that uterine crowding is not a major factor limiting litter size during early gestation. Dziuk (1968) supported the theory that embryo crowding has no effect on litter size. The uterine space available per embryo was altered by varying the number of embryos (by superovulation or superinduction) or the length of the uterus. Intrauterine space per embryo, whether greater or smaller than normal, had no effect on embryo survival near day 30 of gestation. The litter size of the mated gilts subjected to the transfer of 13 additional ova exceeded the control group by 4.1 embryos. However, embryo survival was 7 depressed in the superinduction group (52.0% vs 74.0% for controls). Bazer et a1. (1969) examined litter size at 25 and 105 days in mated gilts receiving fertilized ova and unoperated controls. There was a tendency for increased embryo numbers at day 25 of gestation for the superinduction group, but the difference from control values was not significant. In addition, the treatment group at day 25 experienced less embryo survival (50.0% vs 74.0% for controls). Litter size was similar for both groups and a further decline in pre- natal survival was observed in the gilts subjected to super- induction (42.0% vs 75.0%) at 105 days of gestation. These data help confirm the hypothesis that the primary control mechanism governing litter size occurs during the first 25 days of gestation. The lack of cyclic synchrony between the donor and recipient females may be a more feasible reason than uterine overcrowding as the cause of the increased embryo mortality in animals receiving fertilized ova. In studies in which synchronization was not controlled closely, litter size was not improved and donor embryos were less developed than native embryos (Bazer et al., 1969a). However, exact synchrony has not been found to be a necessity in transfers made into unmated recipients (Webel et al., 1970b). Increased energy intake It is well established in several species that the number of ovulated ova can be increased by increasing energy 8 intake of females at'the time of mating. Self et a1. (1955) was the first to report this practice commonly known as "flushing" in swine. Ovulation rate is consistently im- proved if a high energy intake is imposed on animals in the latter one to two weeks of the estrous cycle prior to breeding (Zimmerman et al., 1960; McGillivray et al., 1963; Naber and Zimmerman, 1971). The physiological mechanism for this phenomenon is unknown. Brooks et a1. (1972) has reported that increasing the feed intake from 1.8 kg to 3.6 kg/day either on the day of mating or from puberty to the third estrus produced significantly higher (p <.05) ovula— tion rates. Following ovulation the pituitaries were assayed for LH potency. Residual levels of LH detected in the pituitary extracts were unaffected by the nutrition treatment and remained constant. The authors suggest that the increased ovulations from higher energy intake are not the result of more LH released. Continuing the high energy intake into early gestation has adverse effects on embryo survival (Self et al., 1955; Sorensen et al., 1961). The flushing technique results in increased litter size at 25 to 40 days of pregnancy (Self et al., 1955; Short et al., 1963; Christenson and Zimmerman, 1966; Schultze et al., 1966; Moore et al., 1971) and also at term (Bazer et al., 1968). Intrauterine mechanisms controlling litter size The roles of intrauterine space and intrauterine com— petition between embryos has been mentioned in the previous 9 discussion. Bazer et a1. (1969) demonstrated that donor embryos of equal age to native embryos could successfully compete but if donor embryos were 12 to 24 hours younger than native embryos the survival of the former was drasti- cally reduced. They suggested that embryos compete with one another for some critical factor within the uterine environ- ment, i.e. there is a specific uterine mechanism for select- ing the most appropriate embryos to compose a maximal litter size. This mechanism is not associated with uterine space although that space probably is important in the later stages of gestation (Fenton et al., 1970). Both Dziuk (1968) and Fenton et a1. (1970) have surgically removed an ovary and adjacent uterine horn from one side of the repro- ductive tract and mated these unilaterally ovo-hysterecto- mized gilts. Compensation for the removed ovary and horn was observed in both studies. There was no significant difference at 25 days in the numbers of CL or embryos between treated and intact gilts. Thus uterine space is not the limiting or controlling mechanism to litter size during early gestation. The uterus and ovary are capable of responding to some controlling force to produce a com— pensatory hypertrophy. Uterine secretions Because the quantity of uterine tissue does not influ- ence litter size, it has been suggested by Bazer et al. (1969b) that 25 day litter size is limited by an insufficiency 10 of some factor required for development of the embryo. This hypothesis gains support from studies in which a specific intrauterine protein (blastokinin) was isolated from rabbits and found necessary for development of blastocysts (Krishnan and Daniel , 1967). In addition, Beier (1968) isolated a similar protein (uteroglobin) from the rabbit uterus and determined its involvement in blastocyst development and the relationship of progesterone-estrogen therapy to the level of this protein in uterine secretions. In the pig, it may be that levels of the intrauterine factor, possibly a protein, were altered by ovarian hormones and then the uterine capacity and subsequent litter size could be increased by the proper balance and quantity of these hormones (Fenton et al., 1970). The secretions of the uterus show considerable cyclic variation. Murray et a1. (1972) has determined the protein content of porcine uterine flushings throughout the estrous cycle. The average total protein remained stable from days 2 to 9 of the estrous cycle and then began to increase. The maximum value occurred on day 15 and was followed by a reduction so that on days 17, 18, and 20 of the cycle the values were similar to those observed on days 6 to 9. The total protein peak coincided with the time when the repro- ductive system was under progestational influence, i.e. during days 10 to 14 when plasma progesterone is maximal (Tillson et al., 1970; Henricks et al., 1972). In addition, the reduction of intrauterine protein coincided with the 11 time of corpora lutea regression (Masuda et al., 1967). Three protein fractions were found after gel filtration. These were designated Fractions I-III and were found throughout the estrous cycle. Their molecular weights were from 90,000 to greater than 200,000. Between days 9 and 16 of the cycle, two additional protein fractions (Fraction IV and V) were isolated with estimated molecular weights of 32,000 (Chen et al., 1973) and 20,000 (Squire et al., 1972) respectively. Fraction IV contained a single basic protein at pH 8.0 which gave the uterine flushings a characteristic lavender color. Fraction V consisted of six specific pro- teins. The secretion patterns of both Fractions IV and V suggested that functional CL are necessary for their secre- tion and that decreased production of these protein frac- tions occurred concomitantly with CL regression and a decrease in plasma progesterone. It is doubtful that either Fraction IV or V is involved in blastocyst formation as has been found for the rabbit uterine protein, blastokinin. Perry and Rowlands (1962) have established that blastulation of swine embryos occurs between days 6 and 8 after the onset of estrus before Fractions IV and V are produced. However, initial placen— tation occurs around day 11 of pregnancy followed by rapid growth of the blastocyst, particularly elongation and development of the trophoblast. It is possible that these proteins, particularly the specific Fraction IV, are related to these occurrences. 12 Chen et a1. (1973) induced the production of Fraction IV in unmated ovariectomized animals given daily doses of progesterone or progesterone and estrogen. Estrogen admin- istered alone was ineffective while gilts treated with pro- gesterone and estrogen produced significantly more uterine protein than did intact animals. This same laboratory determined the amino acid and amino sugar content of Fraction IV. The basic amino acid content was high and there were considerable amounts of aspartic and glutamic acids present as well as substantial amounts of ammonia. The protein also contained glucosamine and galactosamine in the ratio of 8:1 and approximately 12.5 percent of the molecule (by weight) was carbohydrate composed of the neutral sugars fucose, mannose, glucose, and galactose. The analysis determined that each molecule of protein contains 15 residues of neutral sugar and 9 residues of amino sugar. Lamb antiserum was prepared against the purified protein and did not cross-react with extracts of homogenized heart, lung, stomach, intestine, liver, spleen, kidney, and oviduct tissue obtained from gilts. In addition, there was no cross-reaction with pig serum obtained on day 15 of the estrous cycle. Therefore, the results suggested that Fraction IV protein was uniquely located only in the uterus. Previous discussion has indicated that unilateral hysterectomy-ovariectomy (UHOX) does not reduce litter size at 25 days of gestation. Also discussed was that although 13 litter size wasrun:affected at term, superovulation would result in increased 25 day litter size. Knight et a1. (1973) suggested that the increased numbers of embryos in UHOX and superovulated gilts may be associated with increased amounts of uterine protein secretion. The experimental design con— sisted of three groups of gilts: I) control; II) super- ovulated; and III) UHOX. Uterine protein secretions were obtained by surgical laparotomy on day 15 of the estrous cycle before treatment and again on day 15 of each animal's second post-surgery estrous cycle. The difference in total protein in control gilts at both flushings was not signifi— cant. In contrast, a highly significant increase (p <.01) in total uterine protein was detected in the post-treatment flushings in comparison to the pre-treatment flushings in the superovulated animals. In the UHOX group, the total protein content of both uterine horns in the pre-treatment surgery did not differ significantly from the post-treatment flushing of only the one remaining horn. Obviously, a compensatory uterine protein secretion occurred in both instances. In the superovulated gilts, the increase in uterine protein secretion paralleled the increase in ovu- lation rate. The correlation coefficient between CL numbers and total uterine protein was 0.65 and a significant (p <.05) correlation (r=0.52) was calculated for the relationship of CL weight and uterine protein. The results of this study suggest two important hypo- theses. Since in the UHOX group there was a compensatory 14 response in ovulation rate and total uterine secretions, and if one assumes that there is a linear relationship between CL numbers and amount of progesterone secreted, then it may be suggested that there was twice as much progesterone available per unit of uterine tissue to stimulate uterine protein secretion. In addition, the data indicate a reason for the failure of superinduction (the transfer of embryos into mated females) to significantly increase litter size. There may simply be no compensatory increase in uterine protein secretion to maintain and develop the additional embryos. Progesterone was found to be the key hormone controlling quantitative aspects of uterine protein production in the pig (Knight, et al., 1973). However, there was a synergis- tic effect if estradiol and progesterone were administered together in ovariectomized (OX) gilts. If the OX gilts received only corn oil or estradiol, only small quantities of uterine protein Fractions I, II, and III were detected. The Fraction IV as well as the other four fractions were found only in OX gilts treated with progesterone or proges- terone-estradiol indicating that progesterone was also primarily responsible for the secretion of the specific protein components from the secretory cells of the uterine endometrium. In a more recent study, Knight et al. (1974) presented more complete results on the effects of varying amounts and ratios of progesterone and estradiol on porcine protein 15 secretions. From quantitative studies in OX gilts, it was determined that at least 100.0 mg of exogenous progesterone/ 45.36 kg/day is required to produce the same degree of uterine protein secretory activity as that observed in a sham-operated gilt receiving no additional progesterone. In one trial where varying amounts of progesterone with constant amounts of estradiol was given to OX gilts, highly significant (p <.01) correlations were found between total daily administered progesterone (r=.72) and estradiol (r=.61) given and total uterine protein recovered. The correlation coefficient between progesterone administered daily and total protein recovered was .78 (p <.01) for sham-operated intact gilts. When progesterone doses exceeded 300.0 mg/ 45.36 kg body weight/day in the OX group, there was a marked decline in protein recovered indicating that the relation- ship between progesterone administered and total uterine protein recovered was curvilinear. A negative correlation (r=-.44) was recorded in one experiment of sham-operated gilts between total recoverable uterine protein and total daily estradiol. This suggested that estradiol may inhibit or delay secretions of uterine protein when the exogenous dosages surpass a certain level. Thus, this study presented evidence that both progesterone and estradiol administered above an optimal level could produce a negative response on uterine protein secretion. 16 Steroid hormone effects The discussion to this point has presented evidence for the presence of specific uterine protein secretions and their relationship to both endogenous and exogenous proges— terone and estradiol. The effects of steroid hormones as well as the effect of steroid stimulated uterine proteins on embryo survival is unclear. There are conflicting results on the effects of exogenous steroids on embryonic mortality in swine. Progesterone administered alone for extended periods during early gestation has no effect or a detrimental effect on embryo viability. Sammelwitz en: a1. (1956) administered progesterone to intact gilts at daily dosages of 50.0 to 400.0 mg for 26 days following mating. Levels of 100.0 mg or greater decreased embryonic survival and dosages of 200.0 mg daily or greater caused complete degeneration of corpora lutea. Haines et a1. (1957) reported no significant effect of 25.0 mg of progesterone administered every other day during the first 25 days of gestation on embryo survival at 25 days or 40 days post-breeding or at term. In a later study, it was reported that progesterone administered at the rate of 1.0 mg/lb. of body weight (approximately 220.0 mg daily) to mated gilts at various intervals during the first 25 days of gestation had no beneficial effects on embryo survival (Spies et al., 1959). However, embryo mortality in control gilts in this study was lower than normally reported in the literature and in some instances 17 in which exogenous progesterone was given embryo survival rates were improved. Due to the low mortality in control animals and the variability in the hormone treated groups, it is difficult to make valid conclusions from this study. Positive results of the effects of progesterone— estrogen therapy on litter size have been reported by Reddy et a1. (1958). In these studies, two ratios of progesterone to estrogen were used; in one series of exper— iments a 1000:l ratio (25.0 mg progesterone, 25.0 ug estrone daily) was used while in a second series the ratio was 2000:l (25.0 mg progesterone, 12.5 ug estrone daily). The latter treatment administered for 10 day periods from the fourth day of gestation or from the fourteenth day of gestation resulted in highly significant (p <.Ol) increases in litter size at 55 days of gestation. Day et a1. (1959) reported similar results in gilts ovariectomized at day 15 of gestation and maintained on a combination of progesterone and estradiol benzoate (ratio 2000:l) until slaughter at day 25. The average embryonic mortality rate in the pregnant gilts was 22.0 percent in comparison to the control rate of 33.0 percent. The data indicated that an estrogen-progesterone combination was more effective in promoting embryonic survival in ovariec- tomized gilts than either hormone alone. It is interesting to note that the level of progesterone administered in this study (200.0 mg/lOO kg of body weight) which improved embryo survival is a similar dosage (100.0 mg progesterone/ 18 45.36 kg or 222.0 mg/100 kg of body weight) to that reported by Knight et a1. (1974) to sustain normal uterine protein secretory activity in OX gilts. In a more recent study, Gentry et a1. (1973) reported the highest embryo survival rates in OX gilts treated with 80.0 or 160.0 mg progesterone and 0 to 1,000 pg estradiol benzoate daily. When low doses of 40.0 mg daily of proges- terone were given, increasing levels of estradiol benzoate from 125.0 to 500.0 pg had an adverse effect on embryo sur- vival. The results of this study seem to indicate that normal uterine protein secretion is not a prerequisite for optimal embryo survival since the dosage level of proges— terone reported in this study to produce highest survival rates (80.0 mg/lOO kg body weight) is well below the minimum requirement for normal secretory activity reported by Knight et al. (1974). Day et a1. (1963) altered the mode of administration by inserting subcutaneous implants containing progesterone caproate and estradiol benzoate (ratio 2000:1) at the base of the ear of intact gilts. There was a trend toward increased litter size (0.5 to 0.6 embryos per litter) in animals retaining the implant from days 11 to 25 of gesta— tion; however, the difference was not significant. There is one report available on the effect of proges- terone and estrogen therapy on uterine secretory activity and development of the porcine conceptus (Knight et al., 1974). Thirteen animals were randomly assigned to one of 19 four treatments as follows: I) sham-operation plus corn oil (SO-CO); II) sham-operation plus 330.0 mg progesterone and 55.0 pg estradiol/100 kg/day (SO-HP); III) bilateral ovar- iectomy plus 110.0 mg progesterone and 55.0 pg estradiol/ 100 kg/day (OVX-LP); and IV) bilateral ovariectomy plus 330.0 mg progesterone and 55.0 pg estradiol/day (OVX-HP). Treatment began on day 4 after the onset of estrus and was terminated at day 40 of gestation when all gilts were hys- terectomized. Steroid treatment had no effect on pregnancy rate or percent embryonic survival. The average embryonic survival percentages were high in all groups: 86.1; 88.6; 82.9; and 88.7 percent respectively. Placental length was significantly (p <.05) increased in the SO—HP and OVX-HP treated gilts compared to the SO-CO and OVX-LP treated groups. In addition, the SO-HP group had significantly (p <.05) heavier placentae than those gilts in the other treatment groups. Allantoic fluid volume was determined and the results demonstrated that both groups receiving the high doses of progesterone produced significantly greater (p <.05) allantoic fluid volume than the SO-CO and OVX—LP treated groups. The authors suggest that the combination of increased placental length and allantoic fluid volume are causing an increase in the placental areolae surface area which serve to increase the absorption area for water and other nutrients. They further speculated that the establishment and increased proliferation of the placental surface area that was observed in steroid treated gilts 20 during early gestation may be exerting a more critical role with respect to fetal growth and maturation as pregnancy nears term. Genetic influences Rampacek and Ulberg (1973) have demonstrated that certain strains of swine are more capable of supporting larger litters for the first 25 days of gestation. Follow— ing superinduction it was shown that crossbreds were carrying 4 and 2.7 more viable embryos at 25 days than Duroc and Yorkshire gilts, respectively. In addition, peripheral progesterone as determined on days 13 and 20 was not statistically different between groups. It was noted that the correlation between day 13 progestin levels and number of live embryos at 25 days was significant (p <.06). Revelle and Robison (1973) demonstrated evidence that gilts produced from larger litters have the tendency to produce small litters. In turn, F2 females tend to have large litters. The authors' explanation for this phenomenon is that the highly competitive intrauterine environment in large litters may mask certain genes including those involved with reproductive performance and especially litter size. Consequently, litter size from this female would be small but due to fewer numbers of competing embryos the reproduc— tive genes of these young would not be masked and their future litters would be larger in size. In actuality, the positive genotype of the female is exerting a negative effect on the environment of the offspring. 21 Stress: the effect on the reproductive system and embryo survival Temperature or restraint stress as well as injections of ACTH or adrenal glucocorticoids have been impliCated in impaired reproductive function in a variety of species including cattle (Stott and Williams, 1962), sheep (Alliston and Ulberg, 1961; Howarth and Hawk, 1968), and rats (Arvay, 1967; Zondek and Tamari, 1967; Yang et al., 1969; Euker and Riegle, 1973). The mechanism by which stress affects reproduction is vague and the results of stress studies are often incon- sistent. It is unclear whether stress alters the supportive endocrine environment of the pregnant female or directly affects reproductive structures or even the embryo itself. It has been suggested that stress exerts its effect indirectly and that the increased glucocorticoids produced in a stressed animal block or reduce pituitary gonadotropins, especially LH (Hagino et al., 1969). Maintenance of the corpus luteum and proper progesterone secretion could be adversely affected and embryo survival would suffer. Miller and Alliston (1974) have recently reported significant decreases (p <.05) in pre-ovulatory and basal luteinizing hormone (LH) levels in heat stressed heifers. In contrast, temperature stressed pigs experiencing estrus behavior have shown increased LH levels (Riggs et al., 1974). Stressed rats and rabbits exhibited increased luteal tissue and uterine weights which presumably reflected greater luteal secretion (Arvay, 1967; 22 Zondek and Tamari, 1967). Euker and Riegle (1973) using the rat demonstrated that a chronic restraint stress resulted in an all or none effect on embryo survival. Following exposure to stress, females either delivered litters of normal size or lost the entire litter. There is a limited amount of evidence on the effects of prolonged stress on reproductive parameters including embryo survival in the pig. Early studies by Heitman et a1. (1951) have shown that temperatures of 36.80C had no effect on the maintenance of pregnancy or fetal survival in sows in the 85th day of gestation. These studies also first demonstrated that thermal stress caused reduced feed and water consump- tion and that the critical body temperature before death occurred was 41.6OC. Prolonged periods of increased environmental temperature (31.90C) between days 3 and 25 of gestation have a detri- mental effect on embryonic survival (Warnick et al., 1965). The precise periods during early gestation when stress is more damaging were studied by Tompkins et a1. (1967) who subjected gilts to the following treatments: I) 35.00C for 24 hours on days 1, 5, and 20; II) 35.O°c for 48 hours on days 1 and 20; and III) 36.70C for 5 days beginning either on day 1 or day 20. There was a tendency for increased mortality in all stress groups with a significant increase (p <.05) in animals exposed to thermal stress from days 1 to 5 of gestation. In addition, animals stressed from 20 to 25 days of gestation tended to die from the severe 23 stress before embryonic mortality was significantly increased in the survivors of this treatment group. Teague et a1. (1968) presented a study on the effects of increased environmental temperature on reproductive per- formance including ovulation, mating, and embryonic via— bility at 25 days post-breeding. Temperatures of 26.7, 30.0, or 33.30C were imposed for one estrous cycle prior to breeding and during the first 25 days of gestation. Increas- ing the environmental temperature resulted in an increased incidence of anestrus as well as a greater prOportion of animals failing to maintain pregnancy. Although ovulation rate was significantly depressed in groups exposed to the higher temperature, the ratio of corpora lutea present to live embryos at 25 days was not related to temperature. The data suggested that once ovulation had occurred, fertil- ization and survival were not significantly affected in animals exposed to this moderate stress regime. In a similar experimental design, Edwards et a1. (1968) subjected gilts to high ambient temperatures prior to breeding and in early gestation. The control environmental chamber was maintained at 23.4OC and the stress chamber at 38.9OC for 17 hours daily and then lowered to 32.20C for the remaining 7 hours. When gilts were exposed to the high temperature daily for 21 days before breeding, the onset of estrus was delayed by more than 2 days. Groups heat stressed during the first 15 days after mating tended to have lower conception rates and fewer (p (.01) viable 24 embryos at 30 days post-breeding. Although there was a tendency for embryos from gilts stressed on days 1 to 15 to be smaller, the differences were not significant. There was no difference in any of the reproductive parameters studied between controls and gilts subjected to thermal stress 15 to 30 days post-breeding. It was concluded that gilts were more susceptible to high environmental temper- atures the first few days after mating than after implan- tation had occurred. Although variable stress regimes have been employed, the data indicate that prolonged thermal stress between 31.9 and 38.9OC generally has a detrimental effect on embryo survival when imposed during the first two weeks of pregnancy. Plasma ACTH as well as adrenal corticoid hormone levels have been shown to be markedly increased in heat stressed pigs (Marple et al., 1972; Marple et al., 1974). Studies providing evidence for the effect of these hormonal alterations on the embryo or its environment have not yet been done. Laparoscopy The use of laparoscopy or endoscopy has greatly facil- itated intra-abdominal observation in both biomedical and research situations. Jewett (1971) and Rawson (1973) have presented comprehensive reviews on the development of endoscopy from its crude beginnings in the early nineteenth century to the highly developed fiber optic systems in use today. 25 Much of the modern developments and descriptions for the use of laparoscopy came from the human medicine studies of Steptoe (1967), Fear (1968), and Semm (1969, 1970). Only more recently has the technique been adapted to laboratory and domestic animals and these studies have been recently reviewed by Snyder (1974). Table 1 presents a summary of species in which laparoscopy has been successfully used. 26 .Hema ..Hm DO Naommn “HASH ..Hm um sonxsa .qeaa .zoamxso paw umpmcm “mnma .memmm ammma .me050 can pmom “mnma .umflcoeflee can pmfiumaflma “HASH ..Hm um ommflflaflem “mesa .muumnom .msma ..Hm pm ueHAz “sema ..Hm um ueHHz “mema ..Hm Om OSHA: .vbma .cmEHDB paw omom “mmma .mmEHom paw UGOqu “mmma C.Hm um mammmz .memfi ..Hm pm mwcflmm lemma .uoname ecu coommumnuflz .qnma ..Hm um ouoafiflsm .mema ..Hm um peHHz “mesa .comfluumm wee ueHHz “asma ..Hm pm GOmHHHmm “mnma ..Hm um Emnmuw “muma .Bonxso paw c0m3mm “NASH .Hema .aonxso mam anaeme “ckma .onmxso cam comfluumm .osma .meumsem cam moudmum lemma .moma .semm “mama .ummm “noma .moudmpm umoo .m mmmrm .k mam .o 300 .m mmxcoa .mumz .v pflnnmm .m mmumfiflum amEDmucoz .m cmEDm .H mocmummmm mmflommm DmmD MQQDmmmMUUDm mem mmm meUmomdmdq SQHmB ZH mmHUmmm H mqmdB INFLUENCE OF STEROID ADMINISTRATION ON SWINE LITTER SIZE Materials and Methods The effect of steroid therapy on litter size of swine was studied in a series of trials conducted at the Michigan State University swine facility. Hormonal treatments using the MSU herd were initiated in September 1972 and terminated in early June of 1974. In the fall of 1973, a cooperative experiment using previously successful hormonal treatments was conducted on the swine herd at Illinois State University. Since obvious differences exist in facilities and manage- ment between the two universities, each will be discussed as a separate experiment. Trial 1 (Michigan State Universipy) This study included 183 multiparous sows of three genetic groups (Yorkshire, Hampshire, and Yorkshire- Hampshire crossbred type) mated to 10 sires of either the Yorkshire, Hampshire, or Duroc breeds. The experimental animals ranged from 18 to 30 months of age and 182 to 205 kg in weight. Sows were housed in groups of 6 to 8 indoors in 3.5 x 3.5 m pens until breeding and then confined for approximately 105 days of the pregnancy in individual tether stalls. One week before expected parturition sows were transferred to farrowing stalls. A complete 13.0 percent 27 28 corn-soy ration was fed at a rate of 1.8 kg/animal/day throughout the breeding and gestation periods. Steroid solution containing both progesterone and estrone was prepared by the technique described by Reddy et a1. (1958). To reduce error, quantities of oil were measured by weight. The weights of 200 and 250 m1 of oil were determined. A stock solution of estrone was prepared by dissolving 31.25 mg estrone (Al'3'5 (10)—Estratrien-3— ol-l7-one, Sigma Chemical Co., St. Louis) in 183.0g (200 m1) of cottonseed oil. Exactly 3.125 g of progesterone (A4- Pregnen-3,20-dione, Sigma Chemical Co., St. Louis) was added to a 500 m1 flask and the latter weighed. A quantity of 10 ml of the estrone stock solution was added to the 500 m1 flask. Cottonseed oil was added to the flask until the weight of the container and progesterone plus 250 ml of oil (228.89) was attained. Two ml of this solution contained 25.0 mg progesterone and 12.5 pg estrone (ratio = 2000:1 progesterone:estrone). To prepare the estrone treatment, cottonseed oil was added to 10 ml of the estrone stock solution to a total weight of 250 m1. Two ml of this solution contained 12.5 pg of estrOne. The progesterone treatment was prepared by adding 3.125 g of progesterone to the weight of 250 ml of oil giving 25.0 mg of progesterone/ 2 ml volume. Sows were mated once each day on the first two days of estrus (day l of mating = day l of gestation) and then observed daily for further evidence of estrus behavior. 29 Experimental animals were assigned to one of ten treatment groups based on three criteria: I) breed of sow; II) parity of sow; and III) boar used. Using these standards, it was possible to establish parity as a nonsignificant variable and distribute breeds and boars as evenly as practicable throughout the treatment groups. The steroids were injected intramuscularly (anterior to the scapula) at intervals of 24 hours (range 23 to 25 hours). Control animals received oil at corresponding times of treatment administration. Since no differences in mean litter size were observed in the control subgroups, these results were pooled into one control group (CO,T0). In six treatment groups, steroids were administered in combination in 2 ml quantities at the total dosage level of 25.0 mg progesterone and 12.5 pg estrone. Sows received these treatments during the following periods of gestation: days 4 through 13 (P:E,Tl); days 14 through 23 (P:E,T2); days 9 through 13 (P:E,T3); days 14 through 18 (P:E,T4); days 15 and 16 (P:E,T5); and days 16 and 17 (P:E,T6). One treatment group (2XP:E,T7) received twice the dosage level of the first six treatments or 50.0 mg progesterone and 25.0 pg estrone daily. Sows in this group were injected with 4 m1 of the stock progesterone-estrone mixture. Single hormones were administered in 2 m1 daily quantities to two groups: estrone only at 12.5 pg/day (E,T8); and proges- terone only at 25.0 mg/day (P,T9). The latter three treatments were administered from day 14 through day 23 of 30 gestation. Trial 2 (Illinois State University) Experimental animals in this trial consisted of 33 multiparous sows (16 to 48 months of age and 160 to 230 kg in weight) of the Berkshire, Chester White, Hampshire, or Chester White—Hampshire crossbred type. All animals were mated once daily on the first two days of estrus to one of four straightbred boars and then kept in drylot with access to small portable buildings. Each sow was supplemented with 1.8 kg/day of a 14.5 percent corn—soy ration. At approxi- mately 100 days of pregnancy, sows were moved into the farrowing barn and confined to individual farrowing stalls. A double blind experimental design was used with previously successful treatments screened on the MSU swine herd being retested at the ISU facility. Progesterone- estrone mixture (concentration = 12.5 mg and 6.25 pg/ml, respectively) prepared from stock solutions at MSU, and cottonseed oil were delivered to the experimenters at ISU in individual Vials referenced A through D. Two vials contained progesterone-estrone solution and two vials con- tained oil only. Sows were assigned to treatment groups based on the criteria described in Trial 1. Treatments were administered during two periods of early gestation, days 14 through 23 (P:E,T2) and days 14 through 18 (P:E,T4). Two groups of sows received cottonseed oil during respective stages of steroid administration and then the results of these two groups combined for statistical analysis. Methods 31 of administration of the treatments were similar to Trial 1 except that animals in Trial 2 were daily moved in groups to small pens to facilitate administration of injections. Trial 3-P1asma progesterone following steroid administration In Trial 3 conducted at Michigan State University, the effect of combined progesterone-estrone administration on plasma progesterone was studied. Twelve multiparous sows (Yorkshire, Hampshire, or Yorkshire—Hampshire crossbreds) individually confined in tether stalls were restrained with a nose snare and bled by anterior vena cava puncture at the following times: 5 days before mating, day of mating (day 1 of gestation), days 4, 8, 12 and at 2 day intervals through day 24 and on day 29 of pregnancy. Beginning on day 14 of gestation 25.0 mg progesterone and 12.5 pg estrone were administered together (im) to six of the sows. The remaining six sows received cottonseed oil only. Injections were made for 10 days, each day at 1400 hours. Blood samples (10 ml) were drawn, using a 7 cm, 18 gauge needle, 60 I 1 minutes later on the designated days. Samples were immediately centrifuged at 2,400 rpm and sera collected and frozen until radioimmunoassays were begun. Samples were transported to the United States Department of Agriculture, Beltsville, Maryland, where the progesterone radioimmuno- assays were performed. The progesterone concentration (ng/ml) was determined by the competitive protein assay. Assay procedures and data on accuracy and reproducibility 32 for this particular radioimmunoassay have been reported (Henricks et al., 1972; Guthrie et al., 1972). Data collection and analysis In both litter size trials, records were kept of the number of sows returning to estrus, the number carrying the litter to term, total number of pigs farrowed, and the total number of live pigs farrowed. To remove the effects of breed of dam and boar used, mean litter size was cal- culated and statistically analyzed by least squares regression analysis (Harvey, 1960). Chi square analysis was used to test litter size distribution between treat— ment populations. The Student's t test was used to analyze progesterone concentration differences. Results Treatment administration Daily intramuscular injections of 2 or 4 ml of hormone mixture or oil alone caused no abnormal clinical signs. In Trial 2, the experimenters reported some difficulty in administering the injections and observed considerable hyperactivity among animals as treatment days progressed. Conception rate The total number of sows assigned to each group and the number returning to estrus during the subsequent cycle are shown in Table 2. Conception rate was high in all treatment groups and not significantly different from controls (CO,TO) within either trial. 33 0.00 H OH vB.mum 0.0m H OH NB.mnm m.m> m MH 05.00 ”N HMHHB m.mh m VH ma.m m.mm N NH m9.m m.mh m VH he.mumxw o.Hm v HN mB.mum h.mm m HN mB.mum N.Hm m mH v9.mum n.Hm H NH me.mum N.Hm m mH NB.mum m.mm N NH HB.mum b.0m o mv 08.00 "H HMHHE msuumm umHHm mmmp HN CH manumm mBOm ucmfipmmue um COHummocoo unmoumm on mchHDumn “@9852 HMHOB N 02¢ H deHmB ZH mDOMU BzszfimmB m0¢m 20mm mDmemm OB UZHszBmm WBOm m0 mmmZDZ N mqmfia 34 Litter size Statistical analysis demonstrated no effect of breed of sow or boar used in mating on treatment results. Means of total pigs born and total pigs born alive are adjusted for unequal numbers by least squares regression. Trial 1 (Michigan State University) There was a significant increase (p <.02) in litter size when progesterone and estrone were administered together for the 10 day interval on days 14 through 23 of gestation (P:E,T2) which corresponds with the implantation period (Table 3). This treatment resulted in a 2.1 pigs/litter increase in the total number of pigs born or pigs born alive in comparison with the control group. In addition, the administration of the two steroids together for shorter intervals during the implantation period resulted in a significant increase or a tendency toward increased litter size. The five day treatment (P:E,T4) and the two day treatment (P:E,T6) resulted in increases above the control mean of 1.2 and 1.6 pigs born, respectively, with the latter value being significant (p <.05). Similar results were observed for the number of pigs born alive from sows receiving the two day treatment on days 16 and 17. This value (11.6) was significantly greater (p <.02) than the control mean (9.7). The results indicate that higher levels of the pro— gesterone-estrone mixture above 25.0 mg progesterone and 12.5 pg estrone (2XP:E,T7) had no beneficial effect on 35 mo.v m .oe.oo Soup OcOAOMMAe.SHuam0HMHcmHm e NO.v d ~onuoo Eoum DGOHDMMHO mchmoHMHcmHm o conmmumou mmHMDWm Dmme an Hones: Hmsvmcs How pmpmsnpm names mum mmsHm> b mHucmoHMHcmHm HOMMHU uoa pHp mHouucoo mo masonmnsm one 8 hm.OHm.OH NO.HH0.0H OH AmmlvH DUO OB.m Oh.OHm.m mn.oflm.m OH AmNIOH mpv ms.m N0.0HH.OH wa.oHH.OH HH Amman mpv he.mumxm Nm.OHO.HH vm.me.HH SH ASHIOH mpv OB.mum mo.oHv.OH O0.0MS.OH OH AOHImH mpv mB.mum Ow.OH0.0H O0.0HO.HH mH AmHlvH mpv v9.mum mn.oHH.m mm.me.m HH AmHIm mpv ma.mum vo.on.HH O0.0Hm.NH MH AMNIOH mpv NB.mum O0.0H0.0 N0.0Hm.OH OH AMHIO may He.mum hv.OH>.O Nv.OHN.OH mm m Oa.ou Q m>HHm anon mmHm anon mmHm mHmEHcm mo pamEummuB MO HGQEDC GMT: QED: com: Hones: Hepoa Asmzv moomo ezmzemmme oHommsm mocm 20mm m>Hq¢ zmom mon oz¢ zmom mon mo mmmzsz 24m: m mqmfia 36 litter size. In addition, estrone (E,T8) or progesterone (P,T9) alone for the 10 day interval during implantation had no effect on litter size. Since one extreme litter size can influence overall treatment group means the results were also examined on the basis of population distribution. Table 4 demonstrates the data as a function of the proportion of animals in each treatment group falling into one of five litter size groups. Group 3 (litter size 9 to 11) represents a range for the average litter size expected of total pigs born based on results of the control group (CO,TO; 10.2 total pigs born). Groups 1 and 2 and groups 4 and 5 represent arbitrary litter size groups below and above the average range of group 3. The CO,T0 group shows a normal population distribution. The data indicate that P:E,T2 resulted in a significant increase over controls in the proportion of the treatment population producing litters of 12 pigs or greater (combined groups 4 and 5; p <.05) or greater than 13 pigs (p <.01). In P:E,T4, an equal proportion of sows (38.5%) to those observed in P:E,T2 produced litters greater than 13 and this was a significantly greater value (p <.01) when com- pared with controls. The results from the two day hormone administration of P:E,TS were significant (p <.075) when compared with the control group in the proportion of sows producing litters greater than 13. The two day adminis- tration sequence of P:E,T6 resulted in a highly significant increase (p <.01) in litter size of 12 pigs or greater with 37 HO.v d .mHouucoo Eoum ucmHTMMHU wHucmoHMHcmHm p mO.v m .mHouucoo Eoum pcmHOHMHp mHucmoHMHcmHm o th.v a .mHOHucoo Eouw acmHDMMHp mHucmoHMHcmHm Q unmoumm m A~.mHO HH\~ Am.mHO HH\N Im.emv Haxm Am.hmv HH\m AH.mO HH\H ma.m Io.ov oH\o xo.omv oa\m Io.omv Ome Ao.omv oaxm Io.ov oa\o we.m AH.mO HaxH As.omc Haxs Iq.msc HH\m Io.ov HH\o AH.mO HH\H ea.mumxm AS.SHO SH\m elm.mmc SH\m Am.mmc AH\S Ao.ov AH\o Im.mv SH\H ma.mum Ao.mmv SH\4 Ao.mmv SH\S Am.nmv SH\S IN.SO SH\H A~.oc SH\H me.mum Am.mmc mH\m In.nv MH\H AH.mNO mH\m In.omv maxs Ao.oc mH\o as.mum Io.ov HH\o Im.smc HH\m As.m4v,aa\m Aa.mc HH\H Am.mHO HH\~ me.m m Am.mmv ma\m oxo.mmc ma\m Am.mmv ma\m 10.03 ma\o Ao.ov ma\o Ne.mum Io.OHO OH\H Ao.oac oaxv Ao.omv OH\N Ao.omv oa\m 10.0HO OH\H Ha.m"m AH.SO mm\m Ao.mmv mm\m Io.HSO mm\SH Av.mac mm\o mlm.mHO mm\m 09.00 mH A maumH HHum m-» A v pamaummua m msouo v maonw m msouw N msouo H msouw m N H m m m a a H q Homzv mmNHm mmequ m mo H ozHoOoomm macmo azmzemmme oHommem moam zH mzom mo onemomomm v mHmfifi 38 the greatest influence of the effect resulting from the high proportion of animals (52.9%) producing litters of 12 to 13 pigs. Similar results were obtained when examining total pigs born alive. Significant increases in the popu- lation distribution toward increased litter size above controls were observed in P:E,T2, P:E,T4, and P:E,T6. Trial 2 (Illinois State University) In this study, sows receiving hormone treatment for 10 days (P:E,T2) or 5 days (P:E,T4) showed increases of 0.8 and 1.5 respectively in total pigs born over the control mean (Table 5). The same increase (0.8) was observed in total pigs born alive in the P:E,T2 group and was 0.9 for the P:E,T4 group when compared to control values. The increases observed in the steroid treated groups were not significantly greater than control means. The population distribution for the CO,T0 group as observed in Table 6 is skewed slightly to the right. Although there is a tendency for a greater proportion of hormone treated sows to produce litters greater than 13 (22.2%), this was not significant when compared to the value of 10.0 percent for the control group. Trial 3-Plasmagprogesterone Plasma progesterone is shown in Figure 1. The control and steroid treated group had similar concentrations of progesterone during the first 14 days of gestation. Pro- gesterone was then consistently 4.3 to 6.2 ng/ml greater in the steroid treated sows than control sows through day ucmoumm m AN.NNO m\m AH.HHO m\H Is.ssc m\s Am.mmc m\~ Ao.ov m\o Se.mum x~.mmv m\~ AN.NNV m\m AN.~NO m\m I~.mmv m\m 1H.HHO m\H Ne.mnm Ao.oac OH\H Ao.omc oa\m Io.omc oa\m Io.omc oa\m HIO.OHO oaxa 09.00 mH A maumfl HHIS mun A v pamaummne m msouu v ddouw m msonw N msouw H msonw m N H m m m E B H H 39 AOmHO mmNHm mmequ m mo H ozHoanmm moomo ezmzecmme oHommem momm zH meow mo onemomomm o mqmdB muouum pumpcmum M mcmoE conmmnmmu mmumswm uwmmH mum mmsHm> m Nm.O H m.OH O0.0 H H.HH O AOHIOH mpv va.mum O0.0 H N.OH O0.0 H v.OH m AMNIOH mpv Ne.mum H0.0 H v.m mm.O H O.m OH Oe.ou m>HHm :uon mmHm canon mmHm mHmEHcm ucmeummne mo Hones: new: mo Hones: cams Hmnadc Hmuoa AOmHO moomo ezmzecmme oHommew roam 20mm m>Hua zmom mon oz¢ zmom mon mo mmmzoz z¢mz m MHMNB 40 ON ON mBOw Ompmmua OHonwpm cam Houucoo CH Hocmcmoud >Humm mcHuso mcoumwmmmoum meMHm NN ON OH OH . vH doHumummm mo moo mcoHpomncH UHoumum CHOmm Umumouu pHouwpm mHoupcoo NH .H THDOHm m O OH mH ON mN om Im/bu auoxeqsebord 41 23 of gestation, the last day of hormone administration. Progesterone levels were similar in both groups on days 24 and 29 of pregnancy. The difference in plasma progesterone between the two groups was significant at day 20 (p (.01) and day 22 (p <.05). Discussion The results of Trials 1 and 2 indicate that certain steroidal therapeutic treatments imposed during implantation can exert a beneficial effect on the litter size of swine. Progesterone-estrone treatment (ratio of 2000:1) for 10, 5, or even 2 days during implantation resulted in significant increases in mean litter size and the proportion of sows producing litters of 12 pigs or greater or litters of more than 13 pigs. The results of Trial 1 (MSU) confirm the earlier studies of Reddy et a1. (1958) who reported significant increases in litter size at day 55 of gestation following 10 days of exogenous progesterone-estrone therapy during the third and fourth week of gestation. This study also reported a slight increase in litter size from gilts treated with steroids during the first two weeks of gesta- tion which contrasts with the present results. The ISU data are similar to those reported by Day et a1. (1963) who observed a nonsignificant trend toward increased litter size at day 25 of gestation in steroid treated gilts. The results of the third trial indicate that daily injections of 25.0 mg progesterone and 12.5 pg estrone 42 during implantation affect plasma progesterone. Progesterone values were maximal on day 12 in control sows and then declined through day 29 of gestation. This agrees with previous observations (Guthrie et al., 1972). Treated sow plasma progesterone concentration was greater than controls during the hormone administration interval and greater than day 12 concentrations on days 16 and 18. Because a multiplicity of events can occur after steroid administration, it is difficult to precisely deter— mine the effect or site of action of exogenous progesterone and estrogen on the intrauterine environment or the embryo itself. Comparative histological studies of the endome- trium of nontreated and progesterone-estrone treated gilts during early gestation demonstrated a prevalence in the latter group in the proportion of relatively large and conspicuous endometrial glands. This effect was more pro- nounced in gilts treated with steroids during the implan- tation period (Reddy et al., 1958). Under normal conditions, the blastocyst is bathed in endometrial secretions rich in substrate materials assuring adequate nutrition. Progesterone and estrogen are the controllers of intrauterine secretions. It can be postu- lated that the exogenous hormone administration of the present studies is responsible for the production or the alteration in production of a substance which improves embryo viability. The extensive studies by Bazer and his associates have demonstrated the dramatic relationship of 43 ovarian hormones and uterine proteins in the pig (Murray et al., 1972; Chen et al., 1973; Knight et al., 1973; Knight et al., 1974). The affiliation of specific proteins of uterine origin and embryo survival has yet to be established. The evidence presented by Krishnan and Daniel (1967) that a specific protein fraction "blastokinin" produced by the rabbit uterus and shown to promote and regulate blastocyst development is still controversial. It is evident that the specific protein fractions isolated from the pig uterus appear sometime later than initial blastulation which occurs between days 6 and 8 (Perry and Rowlands, 1962). Thus, these proteins in the pig are not the inducers of blastocyst formation. However, it has been suggested that these specific proteins may be related to later blastocyst development (Chen et al., 1973). If indeed a specific quantity of uterine protein is required for embryo survival, in a competitive environment too little available "specific protein fraction" could result in embryo death. In the present study, it can be hypothesized that steroid therapy is influencing intrauterine protein secretion and sub- sequent embryo survival. There are several lines of evidence which lend credence to this hypothesis. 1. Steroid therapy was beneficial to litter size only when initiated 14 days after estrus and mating, a time period similar to that reported when specific fractions of uterine proteins are produced in the cycling gilt (Murray et al., 44 1972). This stage also coincides with the time of maximum production of these specific proteins. In the present study, the pregnant gilts' serum progesterone peaked at 22.6 ng/ml at day 12 and then gradually declined to 9.5 ng/ml by day 29. Specific uterine protein production is known to decline with decreasing progesterone administered to the cycling ovariectomized gilt (Knight et al., 1974). The present studies demonstrated signifi- cant increases in litter size from 10, 5, and 2 day hormone administrations during the implanta- tion interval. In addition, it was shown that the 10 day injection sequence significantly increased plasma progesterone above control values during the implantation period when progesterone concentration is normally declining. It can be suggested that the increased progesterone detected in the steroid treated sows provided the stimulus for increased uterine protein production which conceivably improved embryo survival and subsequent litter size. Doubling the daily dosage level of steroids from 25.0 mg progesterone and 12.5 pg estrone to 50.0 mg and 25.0 ug, respectively, had no effect on litter size. This agrees with Reddy et a1. (1958) who reported no improvement in 55 day 45 litter size when the steroid dosage was increased above 25.0 mg progesterone and 12.5 pg estrone. Recent studies by Knight et a1. (1974) have demonstrated the curvilinear relationship of progesterone and estradiol to uterine protein recovered. Progesterone in excess of 6.6 mg/kg body weight/day administered to ovariectomized gilts caused a marked decline in uterine protein recovered. This same study provided evidence for an inhibitory effect of estradiol on protein secretion when administered at excessive levels. 4. Both progesterone and estrone were necessary to improve litter size. Chen et a1. (1973) observed no difference in quantities of uterine proteins produced in ovariectomized gilts and ovariecto— mized gilts treated with estrogen alone. Like- wise, there was a synergistic effect in the quantitative aspects of uterine protein produc- tion if estradiol and progesterone were adminis— tered together in ovariectomized gilts in com- parison to ovariectomized animals receiving only progesterone. In the present study, there is only suggestive evidence that the treatments are affecting uterine protein produc— tion. There are alternative means by which the steroids could be exerting their effect. Steroids can affect the blastocyst or embryo directly. Estrogen can stimulate 46 uptake and incorporation of amino acids in pre-implantation mouse embryos through direct action on the blastocyst (Smith and Smith, 1971). $2 KEEEQ (Jacobson et al., 1970) and $2.3i29 (Prasad et al., 1968) studies have shown that progesterone, estradiol and estrone can stimulate blasto- cyst synthesis of RNA. Certainly if these processes are occurring in the developing pig trophoblast, the steroids could be playing a supportive role for the embryo. The role of estrogen is of particular interest. Blastocysts from ovariectomized, progesterone—treated rats or mice will not implant until estrogen is administered (Psychoyos, 1961; Humphrey, 1967). Ferrando and Nalbandov (1968) after destroying uterine mast cells, demonstrated the inability of rat embryos to implant and then demon- strated recovery of that ability with injections of estro- gen or histamine. Perry et a1. (1973) reported i§_yitrg pig blastocyst and trophoblast synthesis of estrogens and progesterone and provided biochemical evidence for the presence of enzyme systems concerned with the formation of estrogens, the interconversion of estradiol and estrone and the production of estrone from estrone sulfate. They suggest a relationship of estrogen synthesis by the pig blastocyst to the estrogen required to stimulate the endo- metrium, a prerequisite of implantation in other species such as the rat or mouse where an estrogen surge is believed to be required in order for implantation to occur (Heap and Perry, 1974). It is also possible that the local production 47 of estrogens may have a stimulatory effect on local uterine tissue vascularization and blood flow beneficial to the developing trophoblast. In the present study, a low dose of estrone was required with progesterone to improve litter size. In addition, combined progesterone and estrone therapy was only effective during implantation. It is difficult to interpret these results by speculating that daily intra— muscular injections of only 12.5 pg estrone induce a pro- longed estrogen surge necessary to prepare the endometrium for implantation. If this is true, one must consider the progestational influence since estrone administered alone had no effect on litter size. The present work has supported the hypothesis that exogenous steroid administration would improve embryo sur— vival and actual litter size of the pig. Although pig embryos in the blastocyst stage have been shown to synthe— size steroids, especially estrogens (probably from the trophoblast), the requirement for an estrogen surge at the time of implantation has not been demonstrated in this species. It may be more logical to assume that the beneficial effects of the present steroid therapy exerted its effect through the production of some essential intra- uterine substance, probably protein in nature, necessary for embryo survival at the critical implantation stage. The support of this theory lies in the voluminous amount of evidence reported on the highly correlated relationship of 48 progesterone and estrogen administration and intrauterine protein production and secretion. Future work in this field must include studies of the affiliation of these secretions to embryo viability and a more detailed examina— tion of the quantity or concentration of steroids necessary to promote maximum embryo survival. INFLUENCE OF HEAT STRESS ON PHYSIOLOGICAL RESPONSE AND EMBRYONIC DEATH Materials and Methods An insulated environmental chamber (1.1 x 1.7 x 2.2 meters) was utilized as the stress unit. Thermal neutral temperature was maintained with a thermistor temperature controller (Yellow-Springs Instrument Co., Model 71) which regulated the pumping of cooled liquid through a convective exchanger located in front of a fan inside the chamber. Temperature was increased by controlling a set of six heating coils located within the chamber also in front of an oscillating fan. Relative humidity of the chamber was controlled by a humidity controller (Hydrodynamic Inc., Model 15-3205) which circulated chamber air through a humidifier or dehumidifier apparatus. Two plexiglas sliding windows allowed observation of the animals and one to enter the chamber in order to measure rectal temperature. The experimental animals consisted of 49 mature (8 to 10 months of age) Hampshire, Yorkshire, or Hampshire— Yorkshire crossbred gilts which were mated to one of three straightbred boars. Each gilt received 1.1 kg/day of a 13.0 percent corn—soy ration. All animals were housed in an open—fronted barn during the chamber exposure period of each trial. At other times, gilts were maintained in dry lot. 49 50 None of these females could be classified as being of the stress susceptible genetic strain that has been previously described (Forrest et al., 1968). The experimental design consisted of randomly assigning the animals to one of two trials each composed of a stress subgroup and its corre— sponding control (Table 7). The thermal neutral group in Trial 1 underwent chamber exposure at an average ambient temperature of 24.00C for 12 days beginning on the day after mating or day 2 of gestation. This interval coincided with the pre-implantation stage of pregnancy. Treated animals in Trial 1 underwent a daily two hour thermal stress which averaged 40.20C for the same period. In Trial 2, each subgroup was exposed to a daily two hour period of temper— atures averaging 23.3 and 40.4OC respectively during the 12 day interval on days 14 to 25 of gestation, the implan- tation interval. The mean relative humidities for the four subgroups were 59.5, 56.6, 61.3, and 58.9 percent respec- tively. During the months of June through early September, one-half of the controls and one-half of the stressed animals in each trial were exposed to their respective treatments. The remaining 24 gilts underwent their treat- ments during the months of November through January. Animals were placed in the chamber in pairs and rectal temperatures were recorded at 15 minute intervals using a standard rectal thermometer. Individual gilts were removed from the chamber when rectal temperatures exceeded 42.20C. All gilts were sacrificed between days 38 and 45 of 51 uonum pumpcmpm + mcmoE mum mmDHm> m o.H H m.mm N.O + 4.0s ma mmmuum o.H M m.Hm a.o H m.mm mH Houpaoo eoflumummo mo mmuaH ammo "m Hmflue n.H H 0.0m N.O H N.Ov OH mmouum H.H H m.mm m N.O H O.¢N MH Houucov va muHUHEHO AOQO OHDDMHOQEOB ODOH0\mOHm coHumumww mo MHIN when "H HMHHB m>HumHmm mo Hmnasz MQDBm mmMMBm ZH mmDOMU mcm waHDHZDm m>HB¢Hmm 024 mMMDBZM h mHm¢B 52 gestation. Reproductive tracts were excised and ovaries examined for the number and size of corpora lutea. Fetuses were removed and weighed and all embryo weights adjusted to day 42 of gestation using Pomeroy's equation: W=0.l (0.2447t-4.06)3 where W represents weight and t the day of gestation (Pomeroy, 1960). Using this formula, one can calculate the expected fetal weight on each day of gestation. The increase in weight throughout pregnancy can be expressed as a percent and then this percent used to adjust weights upward or downward to day 42. Day of gestation 38 39 40 41 42 43 44 45 l ILA, JL ll EJIA J! 1111 I V * T l l I I 1 12.0 12.8 11.7 12.3 9.7 10.0 10.1 . + ¢ . Upward adjustment Downward adjustment Percent increase in mean fetal weight between days of gestation Embryonic survival was calculated by dividing fetal numbers by the number of corpora lutea. Statistical analysis of the data with one exception was performed using analysis of variance. The Chi Square test was used to analyze the results concerned with the number of degener- ating fetuses at slaughter in the combined trial subgroups. Results In three cases rectal temperatures of 43.40C were observed in gilts undergoing periodic stress. From 53 observation of the animals'behavior, most were found to tolerate the heat stress quite well for the first 60 to 75 minutes after which, in most cases, they became more active within the chamber. Generally, at rectal temperatures above 41.10C the animals behavior became erratic and included such activities as jumping at objects within the chamber. Rate and depth of respiration varied markedly between animals. The changes in deep body temperature for the gilts in Trial 1 are plotted as a function of time in Figure 2. Body temperature is shown in degrees C. Average body temperature of all control animals in this trial remained at 38.90C. In contrast, gilts subjected to heat stress began an immediate increase in body temperature which differed significantly from the control values by 30 minutes (p <.05). A linear response was observed for the entire 120 minute period. Deep body temperature at the end of the stress period averaged 40.6OC. Figure 3 shows the average rectal temperature of the animals in Trial 2. Although not as consistent as those in Trial 1, these control animals'temperatures varied over a narrow range throughout the two hour period. The stressed animals in this trial responded in a more variable manner to the high thermal temperatures as can be concluded from the finding that no significant differences between control and stress groups were found until 75 minutes after the initial thermal stress. A linear response was again Deep Body Temperature (0C) 40.5 40.0 39.5 to \D O O w m 0 U1 38.0 0 15 Figure 2. 54 Thermal stressed Thermal neutral 3O 45 60 75 '90 105 120 Minutes Average rectal temperature at 15 minute intervals (1 standard error) over the 2 hour period of control and stressed animals in Trial 1. Deep Body Temperature (0C) 40. 40.0 39. w \0 O O (A) m 0 U1 38.0 55 Thermal stressed \ I Figure 3. Thermal neutral 30 45 60 75 90 105 120 Minutes Average rectal temperature at 15 minute intervals (1 standard error) over the 2 hour period of control and stressed animals in Trial 2. 56 observed for the entire exposure period. Mean temperature in the stress subgroup was 40.1OC at the end of the stress period. To achieve some indication of the adaptation to the periodic daily exposures, an average daily rectal temperature was calculated for each animal from the nine temperature measurements made during each exposure period. Then within each subgroup an average temperature was calculated for each day of the 12 day chamber exposure. Figure 4 shows the average rectal temperature for each day of the 12 day period for thermal neutral and stressed animals in Trial 1. The average rectal temperature for the two hour period did not vary in control animals. Stressed animals did demon- strate the tendency for adaptation. All mean values on days 9 through 12 were significantly different (p <.05 or p <.01)from initial average temperatures on days 1 and 2 of chamber exposure. Similar results are observed in Trial 2 (Fig. 5). Although there was an apparent decrease in control values, the differences between days of exposure were not signifi— cant. In the stressed subgroup, there was a tendency toward adaptation with the values at days 10, 11, and 12 of chamber exposure being significantly different from initial rectal temperatures (p <.05 or p <.01). To determine if the stress regime was actually resulting in an overall increase in mean body temperature during the exposure period and thus an indirect determinant of the Deep Body Temperature (0C) 40. 40.0 b) \O 0 OJ \0 o o w CD 0 38.0 57 Thermal stressed Figure 4. Thermal neutral 4 6 8 10 12 Day of Chamber Exposure Average rectal temperature (1 standard error) for each day of the 12 day chamber exposure in Trial 1. 40. 40.0 w \0 0 U1 Deep Body Temperature (0C) 0) \0 co co 0 38.0 58 Thermal stressed Thermal neutral 2 4 6 ‘ 8 10 12 Day of Chamber Exposure Figure 5. Average rectal temperature (I standard error) for each day of the 12 day chamber exposure in Trial 2. 59 degree of stress, a final mean rectal temperature based on the entire 12 day exposure was calculated (Table 8). In Trial 1, control and stress temperatures averaged 38.8 and 39.7OC respectively. These values were significantly different (p <.01). In Trial 2, control values averaged 38.5 with stress values being 39.10C. These differences were also significant (p <.05). The number of animals failing to conceive and the percent embryonic mortality in each group as calculated from normal fetal and luteal number near day 42 of gesta— tion are depicted in Table 9. In each group with the exception of the stressed group in Trial 1, 4 of 13 females failed to conceive. Mean ovulation rate as determined by corpora lutea numbers was not significantly different between groups. Stressed gilts in the pre-implantation state of pregnancy (Trial 1) did experience a very high embryonic mortality compared to control animals, 62.6 versus 35.3 percent. In contrast, while the control gilts in Trial 2 experienced a similar death rate to controls in Trial 1, stressed animal fetal death was not greater than control values. The variability in Trial 1 was consider- able and due in part to 3 of 10 stressed females whose uteri contained only degenerating, necrotic, or abnormal fetuses and another stressed female with 93 percent embryonic mortality. In three other litters from the same group, embryonic death was 30 percent or less which is normal for this species (Wrathall, 1971). Fetal mortality differences 60 Houucoo Eoum HmO.v av pcmummeU NHucmoHMHcmHm o Houucoo Eonw AHO.v my ucmuwmmHU mHucMOHchmHm b Hound Hm scum O mammE one mm: m> p o m + m.O + H.Om H.O + m.wm N HMHHE H.O + b.mm m H.O + N.Om H HMHHB mmmuum Houucoo mMMDmOmxm wflo NH 20 ammfim DOHmmm mmm2 Q msupmw on mchHDuou mmsoqudm on pwcmHmmm mHmEHcm Hmuou mo COHpHomonm m m.aHn.n~ m.mflm.mm H.HHHO.NO hm.Hme.mm ONDHHHOHOS ONHQSO unmoumm 0.0wN.OH O.oM0.0 O.Hum.v Qm.HHm.O mmmsuom HMEHOG MO Hwflfifla COOS N.HHH.¢H m.omm.mH m.oHH.MH no.HHm.mH mumn coHumHs>o cmmz ma\e mH\H OH\o mH\e mmsuumm on meHausumn mHmEHcm mo HmQEdz msoum msoum msoum msoum mmmhflm HOHuCOU mmmhum HOHHSOU N HmHuB H HmHHB NQDBm mmmmem 20mm mmMBBHH ZH NBHHéemoz Cwmmzm Bzmummm 024 .mmmmSDZ Hdfimm 02¢ ZOHBNHD>O Zflmz ~mDmBmm OB wZHszBmm mHflSHZH m0 mmmZDZ m MHmde 62 between the control and stress groups, though considerable in Trial 1, were not statistically significant. Fetal weights adjusted to day 42 of gestation and the number of litters with at least one or more degenerating fetus within the uterus at day of slaughter are shown in Table 10. The mean fetal weight at this stage of pregnancy did not differ significantly within trials. There was a preponderance of degenerating or necrotic fetuses in both stress subgroups. Combining the results of both trials, the data indicate that 7 of 18 (38.9%) control animals and 13 of 19 (68.4%) stressed animals contained degenerating fetuses within their respective litters at slaughter. This difference was highly significant (p <.01). Mean corpus luteum size was not statistically different within trials. Discussion Short-term exposure of two hours at approximately 40.00 C was a substantial physiological stress as determined by significant increases in rectal temperature. This periodic stress regime had no effect on appetite or food consumption in contrast to that reported in pigs undergoing day long stress periods (Heitman et al., 1951; Edwards et al., 1968; Teague et al., 1968). The increased and erratic body activity observed in stressed animals was unexpected. These results indicate that at high general body temperature (41.10C) these gilts did not attempt to thermoregulate by reducing their metabolic 63 Hmmv msumm OCHQmumcmmmp OCH>MQ mmCOHOQCm ow meOHmmm mHmEHCm Hmuou mo CoHuHomoum b mEMHO CH HOHHT pnmvaam H OCHD> Com: m m\m m.HHv.OH mmmuum m\m m.on.mH Hopuaoo m HHHCH OH\H m.owm.NH mmmuum m\v m.HHH.HH Houucoo H HmHuH QHmmv mspmm OCHumumCmmmp QuH3 mumuuHH mo HwQECz w uQmHTB Hmumw mez mDBmm 02HB¢mm2m0mQ @202 mo MZO mBHS mmMBBHH mo mmmZDZ 92¢ 20HB¢BmmO mo NV N¢Q OB DmBmeD¢ mBmUHHB H¢Bmm OH WHm¢B 64 rate through inactivity. The reason for the increased activity is unknown. Alterations in the endocrine or central nervous system due to the thermal stress imposed may account for the abnormal behavior observed. Animals subjected to high temperature during the first two weeks of gestation demonstrated more rapid and less variable increases in body temperature than animals stressed during the third and fourth week of pregnancy. Stress pigs in Trial 1 showed significant elevations in rectal temper- atures above controls a full 45 minutes before significant increases were observed in Trial 2. This in part may be attributed to the greater incidence of variability between individuals in Trial 2. However, animals stressed in Trial 1 did respond more rapidly with increased body temperature than the stress animals in Trial 2 regardless of the degree of variability. Animals during the first few days of gestation have just experienced both estrus and mating. In addition, it has recently been shown that the greatest fluctuations in plasma estrogen and progesterone occur at estrus and during the first 12 days of gestation (Guthrie et al., 1972). The alterations in hormonal balance during this period play a critical role in ovum transport, blasto- cyst development, and uterine preparation for implantation. Consequently, animals at this stage of pregnancy have the potential of being more susceptible to stress than those animals exposed during the third or fourth week after con- ceiving when adrenal and ovarian hormone activity have been 65 shown to be more stabilized. A linear response of body temperature was observed in both stress trials with no indication of the ability of either subgroup to compensate for rising internal temper- ature during the daily two hour thermal exposure. Animals did demonstrate the capacity for adaptation to rthe altered environment as the number of treatment days increased. In Trial 1, accommodation occurred gradually in contrast to Trial 2 where most adaptation took place during the first five days of stress exposure. Edwards et a1. (1968) reported significantly higher rectal temperature in pigs subjected to a daily stress of 38.90C for 17 hours and 32.20C for the remaining 7 hours compared to pigs maintained at a continuous temperature of 23.4OC. There was a tendency for animals in this study to gradually adapt during the first six to eight days of exposure but to maintain body temperatures higher than controls for the remaining 22 day treatment period. Analysis of reproductive data show that the number of females returning to estrus 21 days after mating was not greater than controls for the stress subgroups. This indicated that these particular stress regimes have no effect on the ability of the animal to maintain the corpus luteum or pregnancy. Although embryonic mortality in females stressed on days 14 to 25 of pregnancy was not affected, there was a tendency for increased fetal death when thermal stress was imposed during days 2 to 13 of gestation. Because 66 40 percent of the animals had lost 90 percent or more of their entire litter while 30 percent had normal litter sizes, the data suggest that the effects of stress on pregnancy in the pig may be similar to the all or none response found in the rat (Euker and Riegle, 1973). Fetal weights did not differ within trials and this agrees with earlier reports showing no differences in crown to rump lengths in pig fetuses obtained from dams exper- iencing day long stress treatment (Edwards et al., 1968). The finding of an increased proportion of stressed litters containing degenerating fetuses suggests the possibility of either a continuous or a delayed effect of stress on embryo survival. Since there were similar numbers of litters with degenerating fetuses in both stress subgroups, it is possible these litters would have experienced a greater incidence of mortality if examined during later stages of pregnancy. ADAPTATION OF LAPAROSCOPY FOR USE IN SWINE Materials and Methods The present studies included 64 pre or post-puberal gilts used in initial adaptation procedures, ovulation site quantification, and pregnancy diagnosis studies. All gilts were of the Yorkshire, Hampshire, or Yorkshire-Hampshire crossbred type, housed in an open-fronted barn and main- tained on a 13.0 percent corn—soy ration at 1.1 kg/animal/ day. Animals ranged from 5 to 12 months of age and 82 to 145 kg in body weight. The laparoscopic apparatus consisted of a 130 degree pediatric laparoscope, 5 mm in diameter and 20 cm in length, a fiber optic cable, and a Model 4000 projector light source (Richard Wolf Co., Knittlingen, West Germany) (Fig. 6a). Manipulation of internal organs was accomplished using a 29 cm tactile probe graduated with cm markings which allowed intra-abdominal measurement of the ovaries, follicles, or corpora lutea. In cases where stabilization of reproductive structures was desired, an accessory trocar cannula and specialized laparoscopic grasping forceps were used (Fig. 6 g,h). Laparoscopy equipment was kept in benzalkonium chloride (Zephiran, Winthrop) prior to use. Morphological observations were documented with color 67 68 Figure 6. Laparoscopy technique in the gilt a. equipment necessary including pediatric laparoscope, light source, and fiber optic cable. b. site of intravenous injection of anesthetic. c. gilt on sloped surgery table. d. trocar-cannula being inserted through a 1 cm mid-line incision. 69 Figure 6.(continued) e. laparoscope being inserted through the abdominal cannula. f. observation through laparoscope. g. laparoscopic grasping forceps with accessory trocar cannula in place. h. insertion and use of forceps with laparoscope. 70 photographs taken with a Canon TL 35 mm camera (Canon Co., Tokyo, Japan) attached to the laparoscopic telescope by a 95 mm adapter. Kodak High Speed Ektachrome (EHB) film was used. All gilts were allowed access to feed and water both before and after laparoscopy. Each animal was anesthetized by an intravenous (anterior vena cava) injection of sodium pentobarbital (Halatal Solution, Jen-Sal Laboratory - 65.0 mg/ml) at a level of 10.0 to 32.0 mg/kg body weight (Fig. 6b). Initial restraint of gilts was accomplished using a nose snare and the anterior vena cava located by inserting a 7 cm, 18 gauge needle in the vicinity of the junction of the first rib and sternum. Sodium pentobarbital injections were administered in 10 ml units. The first unit produced basal narcosis and allowed the gilt to be placed in a dorsal position. The remainder of the anesthetic was administered to the animal in this position at approximately five minute intervals until the proper plane of anesthesia was achieved. The gilt was placed head down on a sloped (30°) trough surgery table and held in this position primarily by means of a leather strap encompassing the neck and attached to the anterior portion of the table (Fig. 6c). The hind legs were bound loosely allowing the greatest proportion of weight to be supported by the neck strap. After disinfecting the posterior abdominal region with benzalkonium chloride solution, a trocar cannula was inserted through a 1 cm ventral mid-line incision just 71 anterior to the position of the ovaries (Fig. 6d). The trocar cannula was passed subdermally about 2 cm and dorsally through the musculature of the abdominal wall. This procedure allowed natural closure of the incision after examination and precluded the necessity for suturing. The trocar was then withdrawn and the laparoscope inserted through the cannula (Fig. 6e). To facilitate observation, the abdominal cavity was insufflated with 5.0 percent CO2 in air through the gas connector valve on the cannula sleeve. A second incision was made 4 cm posterior and 10 cm lateral to the mid-line incision which allowed introduction of the tactile probe into the abdominal cavity. At the end of the examination, a nitrofurazone ointment or powder (Furacin, Eaton Laboratories) was applied as an antibacterial agent. Penicillin-streptomycin solution (600,000 units penicillin, 750 mg streptomycin) was administered intra— muscularly as a prophylactic measure. In the pregnancy diagnosis study, 20 mature gilts, mated twice on day 2 of estrus to one of two boars, under- went one laparoscopic examination at various stages of pregnancy. Nine pregnant gilts mated similarly served as controls. All gilts were killed between days 40 and 45 of gestation, the reproductive tracts recovered, and the fetuses measured and weighed. Laparoscopic observations included examination of the gross uterine morphology including size and coloration as well as ovarian follicular and luteal tissue development and regression. 72 The analysis of variance test was used for statistical analysis of sodium pentobarbital requirements, embryo mor— tality results, and fetal weight and crown-rump measurements. The Student's t test was used for analysis of ovulation number results. Results and Discussion Anesthesia A variable dosage of 10.0 to 32.0 mg/kg body weight of sodium pentobarbital induced surgical anesthesia. There was great variability in response to a specific dose of this anesthetic. Pentobarbital has a marked depressant effect on the medullary respiratory centers and because it is a poor analgesic, relatively high doses must be admin— istered before pain perception is inhibited. Consequently, the high dose required to achieve a plane of surgical anesthesia conducive to laparoscopy approached the lethal dose which could elicit respiratory failure. Respiratory rate was monitored as an indication of respiratory depression. Twelve respirations per minute under sodium pentobarbital influence was the critical rate immediately prior to respiratory failure. Laparoscopy was performed successfully at an Optimum respiratory rate of 15 to 18/minute. Figure 7 illustrates the relationship of sodium pentobarbital administration on respiration and heart rate in a 100 kg non-pregnant gilt. Two gilts administered sodium pentobarbital suffered respiratory failure during injection of the drug. Respiration .HHHO quCOmHmICOC ox OOH m CH mumu pume pCm COHHMHHmmmH UCM CoHumupmHCHfipm HmuHQHMQOHCmm mo mHQmCOHumHmm .n THCOHm h.¥ h R.P* OOH OO OO Oh OO om ow om ON OH O mmuCCHE HmuHQHMQOHCmm ECHUOm HE R ow mumu COHDMHHmmmm m u om n a. .m 7 / e t a r t r ONH a % mumu Qummm OOH OON OH ON om ow om aanurm/eqex uoraelrdseu 74 in both animals was restored by artificial resuscitation and the intravenous administration of respiratory stimulant (pentylenetetrazol, Am-Pent, Jen-Sal Laboratories). Unmated gilts required significantly (p <.Ol) more sodium pentobarbital/kg body weight than pregnant gilts to achieve similar planes of surgical anesthesia. Mean drug required (mg/kg body weight) was 14.8 i 0.8 ml for the non- pregnant group and 10.5 i 0.8 ml for the pregnant group. Increased blood flow in the pregnant animals probably caused more rapid distribution of plasma levels of drug and reduced the time required for inhibition of the central nervous system. Upon achieving a proper plane of anesthesia for laparoscopy, the gilt remained totally immobilized for a 30 to 50 minute period. The entire laparoscopy procedure including induction of anesthesia required 30 to 45 minutes. Recovery occurred in two to five hours and no adverSe effects were observed resulting from twice—weekly sodium pentobarbital administration. Ovarian observation Laparoscopic Visualization of the ovaries was a simple procedure usually requiring only minor manipulation of the uterine horn or oviduct with the accessory probe. Each ovary was usually located near the dorsolateral wall enclosed by the mesovarium (Fig. 8a). Slight manipulation with the probe allowed this mesentery to be removed for unobstructed observation of the ovary (Fig. 8b). Figure 8. a. b. Ovarian observation by laparoscopy ovary of a pre-pubertal gilt with mesovarium intact. same ovary with mesovarium removed, 13 days before first demonstrated estrus; extensive follicular develop— ment and absence of luteal tissue can be observed. pre-ovulatory follicle on first day of estrus; the removed fimbria is visible in the background. ovulatory follicle 36 hours after the beginning of estrus; extensive vascul- arity can be observed across the surface of the follicle. mature corpora lutea. 76 Follicular development was a gradual process with most maturation occurring during the seven day period immediately prior to ovulation. An increase in follicular activity was observed as early as day 12 of the estrous cycle (day of ovulation = day 1). With an increase in size, individual follicles appeared red or pink in color (Fig. 8c) which corresponded to an increase in vascularity over the surface of the follicle (Fig. 8d). Follicular vascularity near ovulation has been reported in other species (Jewett, 1972; Wildt et al., 1975). In the present study, follicles in which surface vascularity was not observed did not ovulate. Instead these structures re- mained follicular throughout estrus and eventually became atretic. Follicles generally matured to 12 mm in diameter just prior to ovulation. Ovulation has never been observed in the pig. Five ovarian examinations were made 36 to 40 hours after the beginning of estrus near the time of reported ovulation (Buttle and Hancock, 1967). Ovarian activity was observed in all animals for at least a two hour period. In one animal, a small quantity of cumulus cells was observed oozing from a follicle measuring 14 mm in diameter. The release of such a small quantity of cells led to doubt that actual ovulation had been observed. Dziuk (personal communication) has suggested that complete follicular collapse occurs following ovulation. The present studies in which ovarian examinations were made 77 at various periods follewing ovulation suggest that there is never a complete collapse of the follicular wall but only a slight cratering of the follicular dome which is replaced within two to three hours by the invading blood clot. The stigmal site of ovulation which forms a small orange pro- trusion on the corpus luteum surface remains visible until day 12 of the cycle. The color of the corpus luteum varies between a dark red and purple until day 11 when the corpus luteum becomes mature in size (12 mm, Fig. 8e). Luteal regression begins on day 12 and continues for the remainder of the cycle. The coloration of the corpus luteum gradually becomes blanched to a dull white corpus albicans by day 17 or 18. Barbiturate influence on ovulation Barbiturate administration in some rodent species causes inhibition of ovulation. Ovulation can be blocked in the rat with pentobarbital or phenobarbital when admin- istered near the LH surge (Schwartz, 1968). In the hamster, ovulation is not inhibited by pentobarbital but is inhibited by phenobarbital when given on the afternoon of proestrus. In the present study, pentobarbital was admin- istered to regular cycling gilts at the very first observed signs of impending estrus which is near the LH surge reported to be 36 to 40 hours prior to ovulation (Dziuk, 1973). Pentobarbital was administered also at various periods on days 17 through 20 of the estrous cycle and prior to the increase in LH. No inhibition of ovulation was observed 78 based on the presence of normal appearing maturing corpora lutea on days 2 to 6 of the cycle following pentobarbital administration. Apparently this barbiturate as used in the present study has no blocking action on ovulation in the pig. Determination of the number of ovulations In five animals CL were counted on both ovaries at laparoscopy three to six days after ovulation. All gilts were slaughtered one or two days after laparoscopy and the ovaries examined for the number of corpora lutea. The results (Table 11) demonstrate that there was no significant difference between the numbers of corpora lutea observed with laparoscopy and the number observed directly indicating that the technique can be used quite accurately to deter- mine the number of ovulations in the pig. Pregnancy diagnosis The pregnancy diagnosis results during different stages of early gestation are recorded in Table 12. Pregnancy was easily detected in the five gilts examined during the fourth and fifth week of gestation (days 28 to 33) due to the large size of the reproductive tract. However, in some cases ovarian observation was hindered due to the difficulty in manipulating the reproductive tract. High success rates were achieved between days 12 and 20 of gestation. Repro- ductive status was correctly predicted in 10 of 10 animals, accurately predicting 6 of 10 gilts pregnant. In 5 gilts examined 7 days after mating, correct predictions were made in only 2 of the gilts examined due to the immaturity 79 TABLE 11 COMPARISON OF CORPORA LUTEA NUMBERS QUANTIFIED AT LAPAROSCOPY AND SLAUGHTERa Number of corpora observed with Number of corpora observed from Animal Ovary laparoscopy slaughter data 1 Right 6 6 Left 4 4 2 Right 5 5 Left 7 8 3 Right 5 5 Left 7 7 4 Right 4 6 Left 4 4 5 Right 6 6 Left 8 9 a Difference between two techniques not significant 80 TABLE 12 PREGNANCY DIAGNOSIS RESULTS USING LAPAROSCOPY Day of gestation under- Animal going laparoscopy Reproductive statusa l 33 (P) + 2 32 (P) 3 31 (P) 4 31 (P) 5 28 (P) + 6 20 (P) + 7 19 (NP) + 8 18 (P) + 9 16 (P) + 10 15 (NP) + 11 14 (NP) + 12 14 (P) + 13 14 (P) + 14 12 (P) + 15 12 (NP) + 16 7 (NP) + 17 7 (P) + 18 7 (P) - 19 7 (P) - 20 7 (NP) — a P = Pregnant; NP = Not Pregnant; + = Positive Diagnosis; Wrong Diagnosis 81 of luteal or uterine development. There are several reports of the use of pregnancy diagnostic techniques in swine (Walker, 1967; O'Reilly, 1967; Mather et al., 1970; Lindahl et al., 1972; Diehl and Day, 1973), but these procedures are not accurate until at least 20 days after mating. The present study suggests that laparoscopy can be used as a diagnostic tool for determining pregnancy as early as 12 days after breeding. These results agree well with those of Phillippo et a1. (1971) who used laparoscopy in sheep to diagnose pregnancy and to determine the number of ovulations. Uterine and oviductal characteristics Definite uterine coloration and motility changes re— lated to endogenous hormone alterations were observed, depending on the reproductive state. In non-gravid gilts, the uterine horns were hyperemic and there were uterine peristaltic contractions presumably due to follicular development and estrogen secretion after day 15 of the cycle. Uterine contractions were observed in all mature gilts, pregnant and cyclic, during the luteal phase of the cycle and the peristalses were of less magnitude and dur- ation than those during estrogen-dominated stages of the cycle. Although contractions were observed progressing in both directions along the horn, there was a tendency for increased contractions from the cervix toward the oviducts during the estrogenic phase in cyclic gilts and the con- verse during the early luteal phase. This agrees with an 82 earlier report based on observations in sheep at laparo- tomy (Hawk and Bolt, 1974). Laparoscopy and embryonic mortality Nine pregnant untreated control gilts were compared with the gilts undergoing laparoscopy in the incidence of embryonic mortality. Fetal death was calculated from the numbers of normal fetuses and corpora lutea found at slaughter (Table 13). Laparoscoped gilts had an 8.0 percent increase in fetal mortality, but this difference was not statistically significant. Fetal death rates in both groups fall into the range normally observed in this species. Mean fetal weights and crown-rump measurements were not different from those in the control group. Thus, laparoscopy had no effect on the embryo parameters measured at least to day 45 of gestation. 83 TABLE 13 FETAL MORTALITY, WEIGHT, AND CROWN-RUMP MEASUREMENT OF FETUSES FROM GILTS SUBJECTED TO LAPAROSCOPY Gilts Control undergoing gilts laparoscopy Percent embryonic mortalitya 34.0 : 7.7b 41.9 I 10.9c Mean fetus weight (g) 6.63 t .27 6.41 f .53C Mean crown-rump length (mm) 12.17 i 1.20 11.93 i .90C a Calculated from fetal and corpora lutea numbers b Standard error c Not significant from controls SUMMARY AND CONCLUSIONS The effects of steroid administration or severe thermal stress both imposed during early gestation were studied in swine, a species known for an extraordinary high incidence of embryonic death. In addition, the technique of laparoscopy was successfully adapted and used in the female pig to directly observe and study reproductive phenomena i3 vivo. The following conclusions resulted from the data obtained: Influence of steroid therapy: 1. Administration of 25.0 mg progesterone and 12.5 pg estrone in combination to intact pregnant sows for a 10 day interval beginning on day 14 of pregnancy resulted in significant (p <.02) increases in litter size at term when compared with controls. The same dosage administered for intervals of 5 or 2 days during implantation resulted in a trend toward or a significantly larger litter size (p <.05). Doubling the daily dosage of progesterone and estrone had no effect on litter size nor did either steroid administered alone during the implantation period. 84 85 Administration of 25.0 mg progesterone and 12.5 pg estrone together for 10 days during implantation significantly increased plasma progesterone when compared with sows receiving oil only (p <.05). Steroid administration produced no overt harmful effects to the animal nor affected the ability of the animal to maintain pregnancy. Influence of heat stress: 1. Daily two hour thermal exposure at approximately 40°C is a substantial physiological stress as determined by significant increases (Trial 2, p <.05 and Trial 1, p <.01) in deep body temper- ature. When body temperatures of stressed animals equaled or exceeded 41.10C, hyperactivity and erratic behavior patterns were observed. Gilts subjected to high environmental temperature during days 2 through 13 of pregnancy demonstrated more rapid and significant increases (p <.01) and less variability in body temperature than those gilts exposed on days 14 through 23 of pregnancy. Stressed gilts in both trials demonstrated the ability to adapt to the severe thermal environment as treatment days progressed. Neither stress regime affected the ability of the animal to maintain pregnancy. 86 Only gilts stressed during the first two weeks of pregnancy demonstrated increased fetal death and this tended to be an all or none response. Although neither stress regime affected fetal weight differences, a significantly greater proportion (p <.01) of stressed litters contained degenerating fetuses at day 42 when compared to control litters. Adaptation of laparoscopy: 1. Sodium pentobarbital was an effective drug for immobilization but frequent use of this drug necessitates attentive observation of dose requirements for effective anesthesia; no inhib- ition of ovulation was observed when using pento- barbital in the late follicular phase of the cycle. Visualization of the ovaries with the laparoscope was a simple procedure requiring minor manipulation of the uterine horn or oviduct with the accessory probe. Mature ovarian follicles near ovulation developed a high degree of vascularity and only follicles in which surface vascularity was observed eventually ovulated. Data suggest that follicular collapse following ovulation does not occur in the pig. Laparoscopy was a highly effective tool for quantifying ovulation numbers following estrus 87 and for pregnancy diagnosis as early as day 12 following mating. Laparoscopy had no effect on embryo mortality rates or mean fetal weights and crown-rump measurements in pregnant gilts when compared to controls. 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L. Spencer and W. R. Dukelow. Journal 9: Reproduction and Fertility, 35:541, 1973. Nonhuman primate in vitro fertilization and embryo transfer: their relationship to birth defect research, by T. J. Kuehl, D. E. Wildt and W. R. Dukelow. Vth Cong. Intern. Primat. Sgg., August, 1974. Incidence of twinning in the nonhuman primate, by D. E. Wildt and W. R. Dukelow. Laboratory Primate Newsletter, 13:15, 1974. Laparoscopic pregnancy diagnosis and uterine fluid recovery in swine, by D. E. Wildt, C. B. Morcom and W. R. Dukelow. Journal of Reproduction and Fertility, 43:June, 1975. Induction of multiple ovulation in the cow with single injections of FSH and HCG, by D. E. Wildt, H. D. Woody and W. R. Dukelow. Journal of Reproduction and Fertility, in press, 1975. Physiological temperature response and embryonic mortality in stressed swine, by D. E. Wildt, G. D. Riegle and W. R. Dukelow. American Journal of Physiology, submitted, 1975. Correlation of laparoscopy, perineal swelling and plasma hormone levels during the baboon menstrual cycle, by D. E. Wildt, L. L. Doyle, S. Stone and R. M. Harrison. Folia Primatologica, submitted, 1975. The effect of progesterone-estrogen therapy on plasma progesterone and litter size of swine, by D. E. Wildt and W. R. Dukelow. Journal of Animal Science, submitted, 1975. Abstracts See the following pages for texts of abstracts. 99 CONTROLLED OVULATION I BEEF USING FSH AND HCG D. E. Wildt and W. R. Dukelow The objective of this study was to determine the effects of porcine-FSH and low dosages of HCG in inducing multiple ovulation in mature Angus, Angus-Hereford, and Angus-Holstein heifers. All animals were observed for two estrous cycles before treatments were begun. The number of animals and the treatments employed were as follows: (I) 7, 25 mg FSH-P, 2,000 i.u. HCG; (II) 6, 25 mg FSH-P, 1,000 i.u. HCG; (III) 6, 12.5 mg FSH—P, 1,000 i.u. HCG. The heifers were rectally palpated on day 16 of the cycle and injected with a single dose of FSH-P (im) on that day. The animals were checked for estrus twice daily. They were rectally palpated and injected (im) with HCG on the day of scheduled estrus (or earlier if signs of estrus were evident prior to the let day). The heifers were then palpated daily for 3 days. The number of ovulations, the number of animals with twin ovulations, and the number of animals excessively stimulated (>»3 ovulations) for each group were as follows: (I) 2.6, 3/7, 1/7; (II) 1.3, 1/6, 1/6; (III) 1.3, 3/6, 0/6. The above data demon- strate that controlled multiple ovulations may be produced with restricted levels of FSH-P and HCG and that the desirable twin ovulation objective may be attained without excessive ovarian stimulation. (This work supported by NIH Research Career Development Award No. l-K4-HD35, 306—01 and the Michigan Agricultural Experiment Station.) 1Presented at the Midwest. Sect. Amer. Soc. Anim. Sci. Meeting, Chicago, 111., November 24-25, 1972. 100 CONTROL OF OVULATION IN SQUIRREL MONKEYSl R. M. Harrison, D. E. Wildt, and W. R. Dukelow Ovulation induced in squirrel monkeys with progester- one-FSH-HCG can be inhibited by megestrol acetate (MA) injections. We studied MA administration by silastic implants, disposition in vivo, and rate of release. The effectiveness of MA implants depends on induction pre- treatment and time in situ. Monkeys receiving progester- one pretreatment ovulated at a lower rate (2/8; 25%) than those not receiving progesterone (12/20; 60%). Monkeys with implants 11 days prior to FSH ovulated at a rate of 62% (10/16) compared to 33% (4/12) for those with implants 29 days. Using MA-H3 implants, concentration of the pro- gestin was highest in ovaries and oviducts, less in adrenals, uterus and vagina, and not found in the pituitary. Jn_vivo release rate was determined with 40 implants in 20 monkeys. To determine in vitro release rate, each implant was incu- bated in 10 ml of TC-l99 at 37 C for 5 weeks. On m1 aliquots were removed and replaced daily and implants were transferred each week to fresh medium. The in vivo release rate was 56 i 12 ug/day, whereas the in vitro rate was 13 i 5 ug/day. J2 vitro release was ianEenced by build-up in the medium within 48 hours, and the rate was not linear over the five week period. 1Presented at the Fed. of Amer. Soc. for Exp. Biol. Meeting, Atlantic City, N.J., April 16-20, 1973. 101 OVULATION-IMPLANTATION STUDIES N SWINE AND THE USE OF LAPAROSCOPY D. E. Wildt, J. L. Spencer, and J. E. Nellor The development of the laparoscopic technique in domestic swine allows repeated direct observation before, during and after ovulation with minimal surgical stress and trauma. Using this technique, we have been able to ascer- tain ovulation, approximate the number of ova ovulated and detect the preliminary events of implantation as early as 12 days after mating. In related studies a progesterone- estrone complex (2000:1) was given to gilts at different stages of gestation to determine the effect on litter size. Five groups received either: (a) control, (b) steroids days 4-13, (c) steroids days 14-23, (d) steroids days 9-13, or (e) steroids days 14-18. The percent of pigs pregnant at 21 days for the 5 groups were, respectively, 94.1 (17 pigs); 91.7 (12 pigs); 84.6 (13 pigs); 91.7 (12 pigs), and 84.6 (13 pigs). The mean litter sizes, respectively, were: 10.0 i 0.6; 10.5 i 0.8; 12.4 t 0.7; 9.7 - 0.8, and 12.2 i 0.9. Treatments (c) and (e) resulted in a significant (p <.05) increase in litter size. (This work was supported by a cooperative Research Agreement with the USDA Animal Physiology and Genetics Institute, and an NIH Research Career Development Award No. l-K4-HD35, 306.) 1Presented at the Fed. of Amer. Soc. for Exp. Biol. Meeting, Atlantic City, N.J., April, 1974. 102 PERIODIC HEAT STRESS ON SWINE EMBRYONIC MORTALITYl D. E. Wildt, G. D. Riegle, and W. R. Dukelow Forty-four gilts were used to study the effects of periodic heat stress on early embryonic mortality. Mated gilts were randomly assigned to 2 treatment and 2 control groups. Group A was subjected to once daily, 2 hour heat stress (40.2°C, 57.4% relative humidity) in the preimplan- tation interval from days 2 through 13 of pregnancy and Group C subjected to a similar stress regime in the implan— tation and post-implantation stage of development from days 14 through 25 of pregnancy. Two control groups (B and D) were similarly exposed to the environmental chamber once daily for 2 hour intervals at ambient temper- atures (23.6°C, 60.6% relative humidity) during correspond- ing periods of early pregnancy. Rectal temperatures were monitored at 15 minute intervals during chamber exposure and animals were removed if rectal temperatures exceeded 42.6°C. Embryonic mortality was estimated from corpora lutea and fetal numbers from 40-45 day slaughter data. The number of animals pregnant at days 40—45 of gestation and the number assigned per group were as follows: Group A, 10/10; Group B (control), 8/12; Group C, 9/12; Group D (control), 8/10. Embryo loss and overall mean rectal temperatures during the 2 hour chamber period based on 12 day exposures were as follows: Group A, 62.5, 40.0 i .12; Group B, 36.0, 39.1 i .05; Group c, 30.4, 39.5 i .18; Group D, 42.7, 38.9 i .06. Average increases in rectal temperatures were significant (p <.01) during both treatment periods. 1Presented at the Amer. Soc. Anim. Sci. Meeting, College Park, Md., July-August, 1974. 103 LAPAROSCOPY FOR REPRODUCTIVE STUDY IN SWINEl D. E. Wildt, J. L. Spencer, and W. R. Dukelow Twenty-one crossbred gilts were used in the develop— ment and adaptation of laparoscopy techniques for in_vivo serial observation of reproductive phenomena in swine. Five cycling gilts were used initially in repeated obser- vation of ovarian cyclicity and 16 mated animals were used to study the efficacy of laparoscopy as a pregnancy diagnostic tool. Gilts were anesthetized with sodium pentobarbital (iv) at a level of 18—22 mg/kg body weight and placed head down on a sloped surgery table. A 135 degree pediatric laparoscope was inserted through a 1 cm midline incision with a tactile probe inserted into the abdominal cavity to one side which allowed repositioning and manipulation of internal organs. A 35 mm camera was attached to the laparoscope for photographic documentation. At the termination of each laparoscopy session, the incisions were treated with an antibacterial ointment and suturing the incisions was found to be unnecessary. Results demonstrated that laparoscopy could be used fre- quently in studying ovarian morphology (as often as 5 times in a 16 day period) with minimal stress and trauma to the animal. Pregnancy was accurately confirmed in 100 percent of the gilts examined after day 12 of gestation and corpora lutea numbers observed with laparoscopy were found not to be significant (p <.05) from slaughter data collected on days 40 to 45 of gestation. Sodium pentobarbital had no significant effect (p <.01) on embryonic mortality. 1Presented at the Amer. Soc. Anim. Sci. Meeting, College Park, Md., July-August, 1974. 104 TECHNIQUES FOR CYTOGENIC STUDIES IN BEEF CATTLE AND SWINE H. D. Woody, S. Fujimoto, H. D. Ritchie, D. E. Wildt, and W. R. Dukelow A reliable and consistent technique for cytogenetic studies in cattle and swine was developed by modification of previously reported procedures. Culturing whole blood leukocytes in a formulated culture medium in the presence of phytohemagglutinin allowed optimum leukocyte growth. Hypotonic treatment in 0.9% sodium citrate and prolonged fixations with methanol-glacial acetic acid followed by a treatment of 45% glacial acetic acid and slide preparation by air-drying provided a maximum number of well-spread metaphases. By peripheral blood leukocyte culture and metaphase slide preparation, karyotypic results of 100 cows showed one cow with an abnormal chromosome complement of 48. Three cows with histories of offspring exhibiting phenotypic disorders showed no abnormality in their chromosome complement. 105 COLLECTION OF OVA AND UTERINE SECRETIONS BY LAPAROSCOPYl C. B. Morcom, D. E. Wildt, and W. R. Dukelow The use of the laparoscope for in vivo observation of reproductive phenomena in swine has been previously reported by our laboratory. This paper deals with two techniques for the laparoscopic collection of ova and uterine secretions. Ova were collected with a 22 gauge 4 inch needle inserted via a cannula through the abdominal wall. The ovarian ligament is grasped with laparoscopic forceps and the needle is then directed into the follicle and the contents aspirated by vacuum from a syringe. The follicular contents are then examined under a dissecting microscope for ova. To collect uterine secretions a 16 gauge 1% inch needle is inserted through the abdominal wall. The uterine horn is grasped with laparoscopic forceps and brought up to the ventral abdominal wall. The needle is directed into the uterine lumen. PE90 tubing (with the end sealed and perforations along the distal axis) is then inserted through the needle such that all the openings are intra- uterine. The uterine horn is allowed to return to its normal position. Ten milliliters of 0.15M NaCl are injected into the lumen and then aspirated into a syringe. These two procedures allow the collection of samples without the major stress of surgery and permits the monitor- ing of a single animal over an extended period of time. 1Presented at the Nfidwest. Sect. Amer. Soc. Anim. Sci. Meeting, Chicago, 111., November, 1974. 106 CHARACTERIZATION OF OVARIAN MORPHOLOGY AND PLASMA STEROID LEVELS DURING NORMAL CYCLICITY OF THE BABOON1 D. E. Wildt and R. M. Harrison Perineal swelling, vaginal cytology, steroid analysis and laparoscopy were used to characterize normal cyclicity in 12 baboon females (Papio anubis and Papio cynocephalus). The average length of the menstrual cycle was 35.3 $7.76 days with a range of 25 to 48 days. Laparoscopic obser- vation and photographic documentation of follicular develop- ment was made as early as 13 days prior to ovulation with the most rapid follicular maturation occurring 24 to 48 hours before ovulation. In 38.8% of the cycles, ovulation occurred on the last day of maximal perineal tumescence with 27.7% of the ovulations occurring one day after initial detumescence. In 16.7% ovulation occurred 2 to 5 days prior to detumescence while the remaining 16.7% occurred 2 to 3 days following first observed detumescence. The mean progesterone (ng/ml) and estrogen levels (pg/ml) as quantitated by double antibody RIA were 2.5 and 11.0 respectively for the follicular phase and 7.8 and 5.7 respectively for the luteal phase. Vaginal smears were stained, observed microscopically and found to correlate with perineal swelling, hormone levels and laparoscopic observations. (Supported by PHS Grant No. 5-ROl-HD 0419-03.) 1Presented at the Fed. of Amer. Soc. for Exp. Biol. Meeting, Atlantic City, N.J., April, 1975. 107 Name: Born: Birthplace: Formal Education: Degrees Received: Experience: Member of: HOI'IOI'S: VITA David Edwin Wildt March 12, 1950 Jacksonville, Illinois Chandlerville High School Chandlerville, Illinois Illinois State University Normal, Illinois Michigan State University East Lansing, Michigan Bachelor of Science Illinois State University, 1972 Master of Science Michigan State University, 1973 _ Research Assistant, Department of Animal Husbandry Visiting Scientist, Delta Regional Primate Research Center American Society of Animal Science Society for the Study of Reproduction Society of the Sigma Xi Society of the Phi Eta Sigma Recipient of the Sigma Xi Award for Meritorious Research, 1974 108