LAPARosceptc STUD-:53 0F evumnon ‘ ' . man PREQNANCY WGNGSiSJN SHEEP-__ I ' a inesés'mrme Mme 9? M. S. ' I MéCi-ikfifiifé .STA'EE UNEVERSW DMD Afii‘éRE‘fl SEWER '_ m4? ' LIBRARY “W Michigan St; :5. University p ,5 Emma“ ‘3'- uona & sous" 300K amnsnv W0". Imam: i Mi '5. ‘A 1l . ,..;-__: 14‘- Trmoqm"F e ‘1’ ’3’...“- ux ; “M y. ABSTRACT LAPAROSCOPIC STUDIES OF OVULATION AND PREGNANCY DIAGNOSIS IN SHEEP By David Andrew Snyder A technique for laparosc0pic ovarian examination was developed for the sheep. Ewes under general anesthesia were placed in a dorsal recumbent position, head down, on a 45 degree sloped table. Introduc- tion of a 135 degree pediatric laparoscope, 5 mm in diameter, was made through a laparosc0pic cannula which was inserted with the aid of a round pointed trochar through a skin incision and the abdominal wall along the ventral mid-line l to 2 cm anterior to the mammae. Insufla- tion of the abdominal cavity with 5% 002 in air and the use of a tactile probe, inserted 5 to 6 cm lateral to the laparoscope incision, facilitated examination. A total of 170 laparoscopies were performed on 19 ewes with one ewe being laparoscoped 31 times. Laparoscopies were performed as often as every other day for ll days. The laparosc0pic technique was used for ovulation detection in gonadotropin stimulated anestrous ewes, pregnancy diagnosis, and aspiration of ovarian follicles. Six ewes which had not ovulated for at least 27 days and one ewe with existing corpora lutea were placed on gonadotropin treatment Q5 David Andrew Snyder regimes beginning in late June. Intravenous administration of HCG was considered as time 0. FSH was administered intramuscularly in doses of 5 mg per day with the last dose 48 hours before HCG. Progesterone, where administered, was injected subcutaneously in oil at a rate of 10 mg per day for 3 days with the last injection occurring 48 hours before the first FSH. Eight treatments, consisting of 5 to 10 mg of FSH and 100 to 2000 iu of HCG were used. Thirty mg of progesterone was administered as a pre-treatment in one regime. Ovulation resulted from all regimes used. Six ewes were used to study the use of laparoscopy in pregnancy diagnosis. Three criteria were found to be extremely useful in the diagnosis. Corpora lutea maintenance or regression gave the most positive indication of the state of pregnancy. Maintained corpora lutea changed from a red color and mushroom shape to a reddish-yellow color and round shape by day 11 to 13. In nonpregnant ewes corpora lutea regression began by this time. New ovulation, evidenced by pre— ovulatory follicles, could sometimes be seen as early as day 15 and was taken as an indication of nonpregnant state. The pregnant uterus remained light with evident vascularity. Increases in uterine size could be seen as early as day 17. Using these criteria, 100% accuracy was achieved in predicting the state of pregnancy in 6 ewes. LAPAROSCOPIC STUDIES OF OVULATION AND PREGNANCY DIAGNOSIS IN SHEEP By David Andrew Snyder A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Animal Husbandry 1974 ACKNOWLEDGMENTS Completion of this study would not have been possible without the assistance of many people. Special recognition is due to Dr. w. Richard Dukelow, not only for his assistance and sound advice but also for his expression of confidence, and to Dr. Gabel H. Conner for his advice and encouragement. Appreciation is expressed to Dr. Ronald Nelson, Chairman of the Department of Animal Husbandry, and to Dr. Harold A. Henneman and George C. Good for providing the research animals. Recognition is also due to Clifford B. Morcom, David E. Wildt, and the other members of the Endocrine Research Unit for their assistance and cooperation during the course of this study. Finally, greatest appreciation is expressed to my parents, Mr. and Mrs. Andrew J. Snyder, for their ongoing encouragement and understanding, and especially to my wife Renate, without whose assistance, both psychological and physical, this study and this manuscript would not have been possible. ii TABLE OF CONTENTS Page LIST OF TABLES ........................... iv LIST OF FIGURES .......................... v INTRODUCTION' ............................ 1 LITERATURE REVIEW ......................... 2 Laparoscopy in Domestic Animals ............ 2 Gonadotropin Stimulated Estrus and Ovulation in Anestrus Ewes .................... 7 Laparoscopic Pregnancy Diagnosis in Ewes ........ 12 SECTION I. LAPAROSCOPIC TECHNIQUE IN ENES .............. 17 Anesthesia and Positioning ............... 17 Instrumentation and Procedure ............. 19 Examination and Conclusions .............. 23 II. GONADOTROPIN STIMULATION IN ANESTROUS ENES ........ 29 Materials and Methods ................. 29 Results ........................ 32 Discussion ....................... 34 III. LAPAROSCOPIC PREGNANCY DIAGNOSIS IN ENES ......... 35 Materials and Methods ................. 35 Results ........................ 36 Discussion ....................... 38 GENERAL SUMMARY AND CONCLUSIONS .................. 39 APPENDIX--PAPERS BY THE AUTHOR ................... 42 BIBLIOGRAPHY ............................ 43 VITA ................................ 47 LIST OF TABLES TABLE I Page 1. Summary of Gonadotropin Treatments in Anestrous Ewes . . . l3 2. Pregnancy Diagnosis in Ewes ................ l6 3. Anesthesia for Laparoscopy ................ l8 4. Numbers of Laparoscopies ................. 25 5. Gonadotropin Stimulation Regimes ............. 30 6. Results of Gonadotropin Stimulation ............ 33 7. Laparoscopic Pregnancy Diagnosis in Ewes ......... 37 iv LIST OF FIGURES Figure Page 1. Laparoscopy table ..................... 20 2. Laparoscopy equipment ................... 22 3. Ewe on table ....................... 22 4. Laparoscopic instruments in place ............. 24 5. Laparoscopic examination ................. 24 6. Sheep ovary: inactive .................. 27 7. Sheep ovary: early post-ovulatory follicle ........ 27 8. Sheep ovary: early corpus luteum, pre-ovulatory follicle in background .................. 28 9. Sheep ovary: early corpus luteum, showing vascularity . . 28 INTRODUCTION In the past, ovulation studies in sheep have involved the use of major surgical procedures. A technique for rapid and efficient ovarian examination was needed. The use of the laparoscope for such examina- tions has been described for several species, including the domestic sheep. Repeated examinations over a short period of time were needed to evaluate ovarian activity in the sheep. The domestic sheep, at latitudes away from the equator, undergo a period of anestrus and anovulation during each year. This is season-. ally oriented and, in Michigan, corresponds roughly with the late spring and sUmmer months. Because of this season, concentrated lambing regimes have been severely hampered. It was one purpose of this study to discover if ovulation could be induced with low doses of gonadotropins during the anestrous season. In the late anestrous period the induction of estrus and the ability of stimulated ewes to conceive was also evaluated. Ewes which showed estrus and were bred to a fertile ram Were used to test the usefulness of the laparoscopic technique in pregnancy diagnosis. Both ovarian and uterine characteristics were used for this diagnosis. LITERATURE REVIEW Laparoscopy of Domestic Animals Observation of ovarian_changes jn_vjyg_has been greatly facilitated through use of laparoscopy (endoscopy). This involves the introduction of a viewing apparatus equipped with a light source, usually remote, into the abdominal cavity of the subject. It is associated with less surgical trauma than laparotomy procedures and allows repeated observations. The laparosc0pic technique for ovarian examination has been most highly developed for use in human medicine (Semm, 1969; Balin, Wan, and Rajan, 1969) but has also been adapted for use in nonhuman primates and domestic animals. Dukelow, Jarosz, Jewett, and Harrison (1971) described a laparoscopic technique in goats and nonhuman primates utilizing a remote light source and a mid-ventral site of entrance into the periteneal cavity. Ovarian changes as seen through the laparoscope were further described for the cynomologus and squirrel monkey (Jewett and Dukelow, 1972; Rawson and Dukelow, 1973; Harrison, Rawson, and Dukelow, 1974). Fujimoto, Rawson, and Dukelow (1974) have modifed this technique for use in the rabbit and have described ovarian changes in response to hormonal stimulation. Lamond and Holmes (1965) examined ovarian changes in cattle using a laparoscope inserted through a chronically implanted cannula in the paralumbar fossa and with a distal light source. Megale, Fincher and McEntee (1956) performed laparoscopy in the cow through the vaginal fornix and via the paralumbar fossa with and without a retained cannula. Their route of choice was through the paralumbar fossa which was further described by Megale (1967) and also adapted to the sheep and goat. The light source was a miniature incandescent lamp powered by a 6 volt pocket battery. Betteridge and Raeside (1962) described a technique for ovarian observation in the pig through a cannula chronically implanted in the flank region. Laparoscopy in the pig through a mid-ventral incision was described by Nildt, Fujimoto, Spencer, and Dukelow (1973). Pigs, under general anesthesia, were placed in a dorsal recumbent position, head down, on a 30 degree sloped table. The use of a manipulatory probe inserted about 10 cm lateral to the mid-line incision, and abdominal insuflation with 5% CO2 in air facilitated examination. A 135 degree laparoscope, 5 mm in diameter was used, with light transmitted by a fiber optic cable. Nitherspoon and Talbot (1970), using fiber Optic light trans- mission and both a 180 and 135 degree laparoscope, 9 mm in diameter, described ovarian changes in the mare. Entry into the peritoneal cavity was made through both the paralumbar fossa and the vagina. Heinze, Klug, and von Lepel (1972) have described a laparoscopic technique in both the horse and donkey. Inserting the laparoscope just caudal to the last rib, they entered the muscle layer. Under visual guidance they inserted a cannula for abdominal insuflation before complete insertion of the laparoscope. This reduced the danger of cecal puncture. Intra-abdominal pressure was monitored and regulated at about 15 mm Hg. Because of the size of the animals, examination of both ovaries was only possible in donkeys. During laparosc0pic examination, ovaries could be held stationary, via the rectum, for aspiration of follicles. Rapid laparotomy procedures have been developed in the sheep. Lamond and Urguhart (1961) developed a sheep laparotomy table and procedure which involved a 2 inch incision just anterior to the mammae and lateral to the mid-line. This allowed rapid examination with a low degree of trauma. Hulet and Foote (1968) modified the table and proce- dure, using a plastic speculum and long spoon to facilitate examination. Although lower than conventional laparotomy methods, the surgical trauma involved in these procedures was still a limiting factor for repeated examinations. Megale (1967) showed a picture of the uterine horn of a ewe apparently taken through a laparoscope inserted through the paralumbar fossa as described in the cow. Dierschke and Hyatt (1969) using a chronically implanted cannula in the right paralumbar fossa were able to perform periodic peritonescopic examination for as long as 424 days in one animal. Corticosteroid and enzyme treatments were necessary to prevent granuloma and adhesion formation and maintain a clear route of examination. Roberts (1968) employed Lamond and Urguhart's laparotomy table and a laparoscope with a proximal light source to examine ovaries of sheep through a mid-ventral approach. His incision sites were 6 cm cranial to the mammae and 4 cm on either side of the mid-line for the laparoscope and a pair of manipulatory forceps. Ewes were fasted and water withheld prior to examination. Two percent procaine was used for local anesthesia, then the animals were placed at a 45 degree angle. The cavity was insuflated with air through a hand operated squeeze bulb. One Michel clip was used to close the laparoscope incision but the forceps incision was not sutured. Thimonier and Mauleon (1969) also used the mid-ventral approach. Insertion was 15 cm cranial to the mammae through a 1 cm mid-line inci- sion. Air was insuflated to facilitate viewing. The muscle layer was sutured following each examination and although intestinal damage did sometimes occur, animals recovered uneventfully with antibiotic therapy. This procedure allowed as many as 40 examinations of one animal, over a 2 year period, to detect ovulation for characterization of the estrous cycles and for assessment of hormonal treatments in ewes (Pelletier and Thimonier, 1973; Land, Pelletier, Thimonier, and Mauleon, 1973). Phillippo, Swapp, Robinson, and Gill (1971) used laparoscopy through a mid-ventral approach to diagnose early pregnancy in sheep. Ewes were placed head down at a 30 degree angle under general anesthesia with intravenous thiopentone sodium and inhalation of an oxygen-fluethane mixture. Entrance was through a 15 mm skin incision located 5 to 6 cm anterior to the mammae and 3 cm lateral to the mid-line. Approximately 4 liters of C02 was infused to facilitate viewing. Michel clips were used to close the incision. Recovery was within 10 minutes. Boyd and Ducker (1973) used a mid-ventral approach in sheep under general anesthesia with etorphine hydrochloride and acepromazine. Animals were placed on a sectional table allowing the abdominal region to be placed at a 45 degree angle while the thoracic and cranial region was almost horizontal, thus facilitating respiration. Insertions for the laparoscope and a manipulating probe were made 4 cm on either side of the mid-line about 10 to 12 cm cranial to the mammae. If bladder distension was a problem it was catheterized with a Nielsen catheter. After examination incisions were sutured and ewes were given 1,000,000 iu penicillin and then diprenorphine hydrochloride to counteract the anesthesia. Recovery was usually within 5 minutes. Seeger (1973) utilized Hulet and Foote's laparatomy table for laparoscopy in the sheep. Although local anesthesia had been used, general anesthesia was preferred because of the anesthetic effect on internal organ movement. This was accomplished with prepionylpromazine and methadone plus parasympatholytikum (Polamivet) followed 15 to 20 minutes later with sodium pentobarbital. This could be reversed with levallorphan (Lorfan) if necessary. Entrance into the peritoneal cavity was made through a skin incision about 1.5 cm cranial to the mammae and 2 cm lateral to the mid-line. Fasting for 24 hours reduced the possibility of intestinal damage. When damage did occur, high doses of antibiotics were sufficient for recovery. Seeger and Humke (1974) used this technique to monitor ovulation in sheep stimulated with gonadotropin releasing hormone. Gonadotropin Stimulated Estrus and Ovulation in Anestrus Ewes The anestrous period in ewes is a seasonally and lactationally controlled period of time when estrus and ovulatory activity are not exhibited on a regular basis. The major external contributing factor to anestrous activity is the variation in day length that occurs with seasonal changes. MacFarlane (1969) has observed that ewes raised near ‘the equator or mature ewes transferred to equatorial regions, where day length is relatively constant, are not subject to anestrous periods. Rather they exhibit normal cyclic activity on a year around basis. Investigators have employed various light control studies and found that a constant six to seven hours of daylight per day will reverse the anestrous condition (Rowson, 1968; Fraser and Laing, 1969). The rapidity of response of ewes to this stimulus is dependent upon lactational status, depth of anestrus at the start of treatment and the breed of ewe. , Endogenously, the anestrus period has been attributed to abnormal secretion of follicle stimulating hormone (FSH) and lutenizing hormone (LH) through some hypothalamic mechanism. Various hormone treatments have been employed to induce both ovulation and estrus during the anestrous period. Hunter (1968) reviewed work completed prior to 1968. .. It should be noted that some cases of extreme variation have been attributed to ram variability (Leman, Dziuk, and Doane, 1970; Norman, 1970; Cooper, Wallace, Nishart, and Hoskin, 1971). In one study, Cooper et a1. (1971) have employed the use of artificial insemination in an attempt to avoid this problem. The most common source of gonadotropin that has been used to replace or enhance the endogenous FSH and LH is pregnant mares serum (PMS). The high FSH like properties of this compound were described by Cole and Hart (1930). Some investigators have also used human chorionic gonadotropin (HCG) (described by Aschheim and Zondek, 1927) as an additional source of gonadotropin high in LH like activity. Several early workers injected PMS in two consecutive admin- istrations one cycle length apart in the hopes that the ovulations produced by the first injection would produce enough endogenous progesterone to induce estrus simultaneously with the ovulations caused by the second PMS injection (Hunter, 1968). Results were poor because of rapid regression of the corpus luteum and the uterine endometreum after the first ovulation. The use of a progestin regime prior to the gonadotropin administration has met with a higher incidence of ovulation and coincident estrus (Hunter, 1968; Tweed, 1969; Burfening and Van Horn, 1970; Norman, 1970; Leman et al., 1970; Ross, 1971; Cooper et al., 1971; Hulet and Stormshak, 1972; Pelletier and Thimonier, 1973; Keane, 1973; Shelton and Klindt, 1974). MAP (6 alpha-methyl-l7 alpha- acetoxyprogesterone) fed at rates of 50 to 60 mg per day or CAP (6-chloro-delta 6-l7 alpha-acetoxyprogesterone) fed at rates of 0.5 to 1.0 mg per day for periods ranging from 3 to 17 days or 10 to 30 mg fluorogesterone acetate (FGA) implants or sponges for up to 19 days each followed by 500 to 1000 iu PMS produces ovulation and estrus comparable to the normal breeding season (Hunter, 1968). Burfening and Van Horn (1970) fed 1 mg CAP per day for 13 days with 800 iu PMS given on -1, O, l, or 2 days from the last feeding day of CAP. They found a higher ovulation rate with earlier PMS adminis- tration (4.0, 2.3, 2.0, and 2.8, respectively) but higher conception rate with later PMS administration (20%, 30%, 50% and 50%, respectively). The total percentage of ewes in estrus was 68, conception was 58, and the ovulation rate was 2.6. Several investigators have assessed the value of a sponge containing 60 mg of melangesteral acetate (Veramix, Upjohn, Ldt.) with 750 iu PMS for breeding of ewes during the anestrous period (Tweed, 1969; Norman, 1970; Ross, 1971). Inserting the sponge for 13 days with PMS injected on the day of removal, Tweed (1969) got 100% of 40 ewes to exhibit estrus with a subsequent 90% pregnancy rate and 200% lambing rate in ewes lambing. Norman (1970), inserting the sponge for 14 days with PMS given 48 hours before removal, got 82% estrus with 74% con— ception and 2.14 lambs per ewe lambing in 60 ewes. Reducing the time of sponge insertion to 13 days, but still administering PMS 48 hours before removal, Ross (1971) got only 69% conception with 1.58 lambs per ewe lambing in a flock of 100 ewes. 10 Leman, Dziuk, and Doane (1970), using a subcutaneous progesterone implant for 12 to 14 days followed by 750 or 500 iu PMS at the time of removal, got 82% of 1,251 ewes in estrus but only 34% conception and 1.3 lambs per ewe lambing. They saw definite breed differences in response with Rambouillet ewes giving the best response. Cooper et a1. (1971) fed FGA (syncro-mate) for 12 days with 750 iu PMS on the day of removal and combined with a 14 day flushing regime. In two separate studies, both utilizing artificial insemination, they got 68.1% and 76.2% conception and 1.74 and 1.85 lambs per ewe lambing in 204 and 383 ewes, respectively. Hulet and Stormshak (1972), using a 375 mg progesterone implant for 16 days with 750 iu PMS on the day of removal, got 20% conception with 1.5 lambs per ewe lambing in 20 ewes. Increasing the PMS dose to 800 iu and administering a second dose 16 days later in 100 ewes, divided evenly and placed on high or low total digestible nutrient rations, they got 28% conception with 1.35 lambs per ewe and 44% conception with 1.00 lambs per ewe lambing, respectively. Keane (1973) separated ewes by adequate and restricted diets, based on total digestible nutrients, and inserted 60 mg MAP pessaries for 12 days with 500 iu PMS at the time of removal. Evaluating percent ewes in heat, percent conceived, and fetuses per ewe pregnant, he found 84.5, 78.5, and 1.95, respectively, for the adequate diet and 66.7, 65, and 1.54, respectively, for the restricted diet. In a further study, using 400 iu or 800 iu PMS and evaluating only ewes in estrus and ovulation rate, he found 100% with 2.77 and 89% with 2.0 for adequate 11 and restricted ewes receiving 800 iu PMS and 80% with 1.75 and 82% with 1.53 for adequate and restricted ewes receiving 400 iu PMS. Pelletier and Thimonier (1973) inserted 40 mg FGA sponges for 12 days with 600 iu PMS at the time of removal. In dry ewes this regime produced 3.8 ovulations while it produced only 3.0 ovulations in lactating ewes. Shelton and Klindt (1974) compared 20 mg FGA pessaries and 375 mg subcutaneous progesterone implants each with 750 iu of PMS, HCG, or PMS-+HCG in a flock of 137 ewes. No difference was seen between the progestin treatments. Evaluating the control, PMS, HCG, AND PMS-FHCG treatments for percent ewes in estrus, ovulation rate per ewe, percent conception, and embryos per ewe they found: 2.63, 1.33, 29.1, and 1.57; 91.9, 3.76, 51.9, and 1.79; 63.6, 2.00, 13.0, and 1.00; and 79.3, 2.50, 5.0, and 1.00, respectively. A second dose of gonadotropin given at about one cycle length after the first progestin-gonadotropin regime has been evaluated by some investigators and shown to produce higher conception rates (Hunter, 1968; 'Burfening and Van Horn, 1970; Hulet and Stormshak, 1972). Other workers have shown an equal or greater conception and ovulation rate in subse- quent cycles following a single progestin-PMS regime without further gonadotropins being administered (Tweed, 1969; Norman, 1970). Estrogen, usually in the form of estradiol-l7 beta, was com- bined with the progestin-PMS regime by several investigators resulting in improved expression of estrus and conception rates but generally decreased ovulation rates (Hunter, 1968; Burfening and Van Horn, 1970; Hulet and Stormshak, 1972; Pelletier and Thimonier, 1973). 12 Burfening and Van Horn (1970) administered 1 mg estradiol on the first day of progestin. Evaluating percent of ewes in estrus, conception rate, and ovulation rate, for 42 ewes, they got 98, 73, and 2.2, respectively, as compared to 68, 58, and 2.6 for 38 ewes not receiving estradiol. Hulet and Stormshak (1972) administered 2 mg estradiol, also on the first day of progestin. Evaluating percent of ewes lambing and lambs per ewe, they got 44 and 1.0 without estradiol and 70 and 1.23 with estradiol in one study with 50 ewes receiving each treatment and 20 and 1.5 as compared to 30 and 1.17 in another study with 20 ewes receiving each treatment. Administering 0.025 mg estradiol in 112 ewes, on the day of PMS, they got much lower conception rate (7% vs 27%). Pelletier and Thimonier (1973) administered 50 ug estradiol 24 hours after 400 iu PMS getting a lower ovulation rate and avoiding excessive superovulation (3.4 vs 2.0). A comparison of some of the successful gonadotropin regimes is shown in Table l. Laparoscopic Pregnancy Diagnosis in Ewes Accurate diagnosis of pregnancy in the ewe is valuable to both the commercial breeder and the research scientist. A fast and reliable method would be of extreme usefulness as an aid in culling, feeding regime planning, and choosing animals to fit a particular protocol. Richardson (1972b) has published a review of the subject in which she identifies several methods used by various researchers and ranks them according to their usefulness in her flock. 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