‘. flm— -7- "ullllllllnlmllllllllfl THESIS ,_._.3129.3 10714 8193_ l _ I'!i"1-IQ”£§_ This is to certify that the thesis entitled Superovulation of Crossbred Targhee Ewes With Follicle Stimulating Hormone and Prostaglandin F The Effect of Varying FSH Dosage. 20.: presented by Dr. Cynthia L. Smith has been accepted towards fulfillment of the requirements for M.S. degreein Large Animal Clinical Sciences / . lr"“-—___ I Major professor QW WAD Charles D. Gibson Date August 7, l98ll 0-7639 MSU LIBRARIES ” RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. -[f r“? r" / Wfld .' " SUPEROVULATION OF CROSSBRED TARGHEE EWES WITH FOLLICLE STIMULATING HORMONE AND PROSTAGLANDIN F20: THE EFFECT OF VARYING FSH DOSAGE By Cynthia Lynne Smith A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Large Animal Clinical Sciences 1984 COPYRIGHT BY CYNTHIA L. SMITH 1984 ABSTRACT SUPEROVULATION OF CROSSBRED TARGHEE EWES WITH FOLLICLE STIMULATING HORMONE AND PROSTAGLANDIN an: THE EFFECT OF VARYING FSH DOSAGE BY Cynthia Lynne Smith Six doses of Follicle Stimulating Hormone (FSH), combined with Prostaglandin F2 (PGF2 ) were evaluated for their effect on superovu- lation in 79 crossbred Targhee ewes. An effect of the timing of FSH treatment in midcycle of the estrous cycle was tested for four days using PGF2 to induce estrus. Dependent variables evaluated were numbers of corpora lutea, follicles, embryos, degenerated embryos and unfertilized ova. A significant effect of FSH dose was seen on corpora lutea (CL) response. A significant (p<.05) cubic regression curve for FSH dose effect for CL response and a significant (p<.05i linear regression curve for FSH dose effect for embryo response were produced. The dose of FSH had no effect on any other variables. The timing of FSH treat- ment in midcycle had no effect. An optimum dose range of 22 mg to 30 mg FSH was estimated from the analysis. ii Dedicated to Kit whose steadfast devotion and unwavering cheerful presence were given freely in this and all my endeavors. jilflhnanifl“%1 iii acmowmmn'rs I wish to express my sincere thanks to Dr. Clyde R. Anderson for the many hours of advice and instruction in the scientific method and for his constructive criticism throughout this investigation and preparation of the thesis. I extend special thanks to Dr. John B. Kaneene whose invaluable guidance, support and criticism were inspira- tional during the course of the study. I thank also my advisor, Dr. Charles D. Gibson for his advice and support throughout my post- graduate research and study. My appreciation is extended to Dr. Richard VL.Dukelow for serving on my guidance committee. Special thanks and mention are given to the following people whose assistance, encouragement and friendship made completion of this investigation possible: Dr. Denise A. Simpson for her unwavering friendship and assistance. Judy Weed, Marianne Vaccaro and Charles McClure for their technical assistance. Also Dr. C. N. Murphy and D. McCalla for participating in the origination of this study and for training in embryo collection methods. I extend my sincere appreciation to Sheila.M. Wing-Proctor and the members of the 1983 and 1984 classes of Michigan State University College of Veterinary Medicine for their technical assistance. Special mention and thanks are extended to Dr. Cynthia A. Berardi and Mike L. Mirky for their patience, suggestions and criticisms during the final preparation of the thesis. My thanks are also extended to iv Maggie Hofmann‘ for preparation of the graphs and diagrams and Martha Devlin for assistance in typing of the thesis. I also thank Dr. J. H. Kirk for his support in making time available to complete this study, and Dr. E. C. Mather and Dr. J. R. welser for obtaining funds which made completion of this project possible. TABLE OF CONTENTS Page LIST or TABLES . . . . . . . . . . . . . . . . . . . . . . ix LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . xi GLOSSARY or ABBREVIATIONS . . . . . . . . . . . . . . . . xii INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . 1 LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . 4 Reproductive Cycle of the Ewe . . . . . . . . . . . . . The Estrous Cycle . . . . . . . . . . . . . . . . . . Follicular Development and Ovulation . . . . . . . . . Other Factors Affecting Ovulation Rates and Estrus . . . . . . . . . . . . . . . . . . . . ll Superovulation . . . . . . . . . . . . . . . . . . . . 12 Gonadotropins Used to Cause Superovulation . . . . . . . l4 Pregnant Mares Serum Gonadotropin . . . . . . . . . . l4 Anterior Pituitary Preparations . . . . . . . . . . . 18 Ebrse Anterior Pituitary . . . . . . . . . . . . . . . 19 Follicle Stimulating Hormone-p . . . . . . . . . . . . 20 Human Chorionic Gonadotropin . ... . . . . . . . . . . 21 Control of the Cycle: Estrous Synchronization . . . . . 22 Other Problems Associated with Superovulation . . . . . 26 Effect of Breed, Season, Plane of Nutrition, Age . . . . 27 MEEERIALS AND METHODS . . . . . . . . . . . . . . . . . . 29 Animals . . . . . . . . . . . . . . . . . . . . . . . . 29 Estrous Detection . . . . . . . . . . . . . . . . . . . 29 Breeding . . . . . . . . . . . . . . . . . . . . . . . 29 Estrous Synchronization . . . . . . . . . . . . . . . . 30 Treatment Methods . . . . . . . . . . . . . . . . . . . 30 vi Experimental Design . . . . . . . . . . . . . . . . 1981 Experiment . . . . . . . . . . . . . . . . Part I . . . . . . . . . . . . . . . . . . . . PartII.................... 1982 Experiment . . . . . . . . . . . . . . . . . Hormones Used . . . . . . . . . . . . . . . . . . . Follicle Stimulating Hormone . . . . . . . . . . Prostaglandin an . . . . . . . . . . . . . . . . Data Collection . . . . . . . . . . . . . . . . . . Iaparotomy Technique . . . . . . . . . . . . . . The Ovarian Structures . . . . . . . . . . . . . The Uterine Flush . . . . . . . . . . . . . . . . Embryo Counting . . . . . . . . . . . . . . . . . Statistical Analysis . . . . . . . . . . . . . . . RESULTS AND DI swss ION C O O O O O O O O O O O O O 0 Effect of Treatments on Corpora Lutea . . . . . . Effect of Treatments on Embryos . . . . . . . . . . Effect of Treatments on Follicles . . . . . . . . . Effect of Treatments on Unfertilized Ova . . . . . Effect of Treatments on Degenerated Embryos . . . . Prostaglandin E a . . . . . . . . . . . . . . . . . 2 0% meowry Mte O O O O O O O O O O O O O I O O O SUMMARYANDCOMZLUSIONS............... LITERATURE CITED 0 O C O O O O O O O C O O O O I O I APPENDICES Appendix A Table A1 Descending dose regimes for FSH doses: 0 m 30 m. 0 O O O O O C O O O O O O O O O O 0 Table A2 The frequency of observations within the treatment cells for the 1981 experiment . . . . Table A3 The frequency of observations within the treatment cells fbr the 1982 experiment . . . . Appendix B Schematic diagram of‘ovine uterine flush technique vii 30 30 31 31 32 33 33 34 34 34 34 35 36 36 38 38 44 50 52 54 S6 58 61 66 75 76 76 78 Appendix C Table Cl Results of superovulation: total CL, follicles, embryos, unfertilized ova, and degenerated embryos by FSH dose . . . . . . . . . 80 Table C2 Ova recovery rate in % (Total ova/corpora lutea) by FSH @Se 0 O O O C O O O O O O O O O O 82 viii 10. 11. 12. 13. 14. A1. A2. LIST OF TABLES Summary of the ANOVA for year effect on corpora lutea response . . . . . . . . . . . . . . . . Summary of the ANOVA of the treatment effects on corpora lutea . . . . . . . . . . . . . . . . Main effects of FSH dose on corpora lutea . . . Summary of the ANOVA for year effect on embryos Summary of the ANOVA for treatment effect on mtyos O O O O O O O O O O O O O O O O O O O 0 Main effects of FSH dose on embryos . . . . . . . Summary of the ANOVA of treatment effects on follicles . . . . . . . . . . . . . . . . . . . Main effects of FSH dose on follicles . . . . . . Summary of the ANOVA of treatment effects on unfertilized ova . . . . . . . . . . . . . . . Main effects of FSH dose on unfertilized ova . . Summary of the ANOVA of treatment effects on degenerated embryos . . . . . . . . . . . . . Main effects of FSH dose on degenerated embryos . Estrous synchronization with PGF a: interval from treatment to estrus . . . . . . . . . . . Sumary of mean ova recovery rate in % by FSH ase O O O O O O O O O O I O O O I O O I O O O Descending dose regimes for FSH doses: 0 to 3 0 mg 0 O O I O O O O 0 O O O O O O I O O The frequency of observations within the treatment cells for the 1981 experiment . . . . . . . . . ix Page 38 39 41 44 45 47 50 51 52 53 54 55 57 59 75 76 A3. The frequency of observations within the treatment cells for the 1982 experiment . . . . . . . . . . 76 C1. Results of superovulation: total CL, follicles, embryos, unfertilized ova, and degenerated embryos by FSH dose . . . . . . . . . . . . . . 80 C2. Ova recovery rate in % (Total ova/corpora lutea) by FSH dose 0 O O O O O O O O O O O O O O O O O 8 2 Bl. LIST OF FIGURES Schematic representation of the 16-day estrous cycle of the ewe . . . . . . . . . . . . . . . Graph of cubic regression curve and mean corpora lutea/ewe by FSH mg . . . . . . . . . . . . . Graph of adjusted mean corpora lutea/ewe by dose Of FSH O O O O O O O O O O O O O O O O O O 0 Graph of adjusted mean embryos/ewe by dose of FSH O O O C O D O O O O O O O O O O O O O O 0 Graph of linear regression curve and mean embryos/ewe by FSH mg . . . . . . . . . . . . Graph of the distribution of ewes with low, medium, or high ova recovery rates (%) . . . . Schematic diagram of ovine uterine flush technique . . . . . . . . . . . . . . . . . . xi 40 43 46 48 6O 78 Clo DOCI DOSE FSH FSHp GnRH hCG IU PMSG ugm GLOSSARY OF ABBREVIATIONS Anterior pituitary preparations Corpus luteum or corpora lutea Cloprostenol, a synthetic prostaglandin an Day of the cycle the FSH was initiated Dose of FSH in milligrams Follicle stimulating hormone Follicle Stimulating Hormone-p, a commercial FSH made from pituitaries of porcine species Gonadotrophin releasing hormone Horse anterior pituitary extract Human chorionic gonadotropin International units Intravenous Luteinizing hormone Milligrams Milliliters Prostaglandin F a 2 Purified luteinizing hormone extract from ovine pituitaries Pregnant mares serum gonadotropin Sheep anterior pituitary extract Micrograms xii INTRODUCTI ON Superovulation is the use of exogenous gonadotropins to increase the number of ovulations at a given estrus. The gonadotropins used are unpurified or semipurified preparations that contain follicle stimulating hormone (FSH) and luteinizing hormone (LB) or have FSH and LH activity. The mechanism by which these gonadotropins cause increased follicular growth, maturation of the oocytes within the follicles and ovulation is not understood. The superovulated animals are bred and their embryos are recovered by a uterine flush technique. These embryos can then be placed in a surrogate dam's uterus to be carried to term. The limitations of superovulation treatments are many. The dif- ferent gonadotropins have varying degrees and duration of FSH and LH activities resulting in varying effect between gonadotropins. Incon- sistency of ovulation rates among individuals occurs with any of the gonadotropins. Degenerated embryos and unfertilized ova are observed when ewes are superovulated. Additionally, unovulated follicles and prematurely luteinized follicles are sometimes observed in high numbers on ovaries of superovulated ewes and this may contribute to a reduced number of fertile embryos collected. Pregnant mares serum gonadotropin (PMSG) has been the most widely used gonadotropin to cause superovulation in the ovine. Along with PMSG, progesterone treatment has been used to synchronize the onset of estrus with peak follicular growth. Both of these products have 1 been is t' The fhs envi and obse obse This pre: dew Syn tio fer Dru A c: been shown to cause fertilization failure in the ewe. Progesterone is thought to interfere with sperm transport through the cervix.85 The mechanism by which PMSG depresses fertilization is not known. Degeneration of early embryos and lack of recovery from the uterine flush are thought to be due to alteration of the normal endocrine environment in the oviduct and uterine horn. Chromosomal abnormalities and altered substrate utilization of the early embryos have been observed in PMSG stimulated cattle.7O Follicle stimulating hormone has been widely used to superovulate cattle. This is because a lower incidence of embryonic loss is observed in cattle at superovulatory dose levels of FSH than to PMSG. This may be due to FSH's lower LH activity and short half life.70 At present a dose regimen to cause superovulation with FSHp has not been developed for the ovine. Along with FSH treatments, prostaglandin F may be used to 2 synchronize estrus in ewes. One research trial with PGF in conjunc- 2! tion with superovulation with PMSG in ewes, did not show an altered fertilization rate.121 Studies where PGF has been used at the time 2 of artificial insemination have shown increased fertility in ewes.111 Controversy involving the fertilization rates following estrous 44,111 synchronization with PCP exists. Reports of normal and sub- 2 37,47 normal fertility can be found in the literature. The purpose of this study was to evaluate the effect of FSHp and PGF2 in superovulated ewes. Three objectives were proposed to evalu- ate this effect: first, to determine if FSHp and PCP would super- 2 ovulate ewes; second, to determine if the dosage of FSHp had an effect 3 on the superovulation response in ewes: and third, to determine if a dose of 22 mg FSH or above would maximize embryo response in super- ovulated ewes. "h 'r1 LITERATURE REVIEW Reproductive Cycle of the Ewe The normal reproductive cycle of the ewe will be reviewed to facilitate a discussion of exogenous hormones used to control repro- duction and fecundity. It should be pointed out, however, that the interrelations of the endocrine, anatomical, and behavioral events that occur in the cycle of the ewe are complex and not completely under- stood. Sheep are seasonally polyestrous. The estrous season begins in the late sunmer to early fall, during which estrous cycles, 16—17 days in length, occur in succession until late winter.- At this time, the anestrous season begins, characterized by reduction of both ovarian 28’117 The transition from the estrous activity and behavioral estrus. season to the anestrous season and vice verse is controlled by the change in the photoperiod. The progressive shortening of daylight hours initiates the onset of the estrous season in the fall. The opposite, lengthening daylight hours, initiates the onset of the anestrous season in the spring.60 The Estrous Cycle The 16-day ovine estrous cycle is divided into the luteal and follicular phases. Throughout the luteal phase, 12-13 days in duration, progesterone is the predominant steroid secreted from the corpus luteum on the ovary. During the fbllicular phase, 3-4 days in duration, serum inc: leve the sio: Uta] red of fur. uh: '9 S progesterone levels drop rapidly and estrogen begins to rise. Large increases in secretion of LH and FSH follow the increased estrogen 28,43,83 levels, see Figure 1. Regression of the corpus luteum (CL) is responsible for the sudden drop in serum progesterone.80 Initiation of this regres- sion is controlled by PGFZ, a luteolysin, secreted by the nongravid uterus beginning on day 12-14 and reaching maximum levels just 6,11,41,60,94 prior to the onset of estrus. There are two stages of luteal regression. The first stage is functional regression, during which progesterone production by luteal cells is significantly reduced, but no anatomical cell changes have occurred. The beginning of serum.progesterone decline on day 12-14 corresponds with the functional regression. This regression is triggered by PGF2 which is released by the uterus after 7-10 days of progesterone influence. It is independent of preovulatory estradiol concentration and is reversible. The second stage is structural regression, charac- terized by irreversible morphological change of the luteal cells which occurs by day 16 due to continued PGF2 influence. Estradiol secretion and the decline of progesterone enhance PGF2 secretion from the uterus during structural luteolysis.6 Estradiol begins to rise rapidly (14-27 pg/ml) at the time of luteolysis just prior to estrus. This rapid estradiol rise is respon- sible for the initiation of behavioral estrus. Additionally, when serum progesterone is at a low level (0.5 ng/ml), estradiol has a positive feedback effect on the hypothalamal-pituitary axis to 0f. ‘0‘ T I I I I I 1 r l 1 I l l I I 1 r ESTR US Periovulatory A Pe_r_lo_d ’2 I I I j‘“ I l Pnosssrsnone ' l i I I . I . l I . I I l . I I l ° I L . \ I.——. . [V‘l- \ FSH / ’I V .\ l K I OLLICLE L” | SIZE (mm). I i 12 ,I‘. ESTRADIOL ' ,I‘. V I '5‘ ’ | 9 , ’ “ ’o"~‘ I ’4’ ‘ I :74' n I n n 1‘1'1 1 1 1'7 1 44"" DAY OF ESTROUS CYCLE FIG. 1. Schematic representation of the 16-day estrous cycle of the ewe. Adapted from 5.117 LEGAN and F. J. KARSCH (1979)60 and WARD (1980) 7 increase frequency of tonic LH release (LH pulses).6 The effect of estradiol is to increase the sensitivity of the pituitary to gonado- tropin releasing hormone and increase the frequency of GnRH release from the hypothalamus.54 The increasing pulses of LH over a 48-hour period result in a five-fold rise in LH above basal levels. The increasing LH has a positive feedback effect on the ovarian follicleIs) to increase estradiol secretion.6 When the estradiol concentration reaches a threshold level for the hypothalamal-pituitary axis, surges 28,83,117 of LH (up to 80 ng/ml) and FSH (up to 170 ng/ml)43 are released from the pituitary. These gonadotropins acting in concert and in the proper critical ratio trigger ovulation of the preovulatory follic1e(s).83 The LH and FSH surges occur 8-12 hours after the onset of behavioral estrus. Ovulation occurs 12-14 hours after the LH and FSH peaks or 24 hours after the onset of behavioral estrus.117 During the luteal phase of the cycle, LH remains at a low level 6,28,83 (2-3 ng/ml) due to negative feedback of progesterone on 59'“ A second FSH peak. occurring 24 hours after pituitary secretion. the first, has been reported.by one group of researchers.83 Other researchers have not measured this second FSH peak.28 The relationship of this FSH rise to ovulation, if any, is not known. Some workers propose that this second FSH rise is the result of an increase in tonic secretion when inhibition by the preovulatory follicle(s) is removed.44 Others speculate that the FSH rise initiates a second wave of follicular growth resulting in the increased serum estradiol seen at 3-4 days post 6,28,83,11? estrus. However, FSH is also elevated up to 80 ng/ml at day 3 and again at days 8-12 of the estrous cycle, then declines to 40 ng/ml until the next preovulatory surge. These two modest rises in FSH in the luteal phase are proposed to cause the two waves of follic- ular growth observed in sheep. These waves occur at days 3-4 and 6-9 . . 69,117 of the cycle and correspond to undulating estradiol levels. Other unknown factors may be responsible since the FSH rises do not 28'117 The decline of serum directly precede the growth of follic1e(s). FSH just prior to estrus and ovulation may reflect an increase in FSH utilization by the follicle, rather than increased FSH secretion, pro- moting follicular growth. Other investigators suggest that the FSH surge may be more important in ovulation than in last-minute follicular growth.83 After ovulation has occurred, granulosa cells under the influence of LH106 proliferate, hypertrophy, and begin to secrete progesterone66 and thus, the CL of the next estrous cycle is formed and progesterone rises from 0.5 ng/ml to 4 .. 5 ng/ml by the third day post estrus.83 This level of progesterone is maintained to day 12-13 when luteolysis begins and the estrous cycle repeats.28’60'83 Follicular Development and Ovulation Superovulation involves the use of exogenous hormones to influence the complex hormonal environment that controls growth of follicles, oocyte maturation or resumption of meiosis, and ovulation. Undesirable effects have been observed as evidenced by increased chromosomal abnormalities in superovulated rabbits,39 cattle,68 and an increase in abnormal embryos from repeat breeder heifers.61 Some of the aspects of follicular growth and deve10pment that apply to superovulation will be reviewed. 9 In sheep and cattle, waves of follicular growth have been documented throughout the estrous cycle. During a wave of follicular growth, several preantral follicles grow to large antral follicles and then undergo atresia.65'101 If an antral fOllicle(s) is at a critical size at the time of the regression of the CL and the fall in progesterone, then its secretion of estradiol will have a positive feedback effect on the hypothalamus.7’8'101 Gonadotropin releasing hormone (GnRH) will be released causing the increase in tonic LH release from the pituitary.77 The tonic LH acts on the antral follicle(s) to promote growth and estradiol secretion which proceeds until an LH surge is achieved.7 Shortly after the LH surge, the oocyte is observed to undergo resumption of meiosis (maturation) and ovulation follows.7O These two events are reviewed by Hansel and Convey54 and are believed to be under LH control which affect the pre- ovulatory size follicle and have no effect on small antral follicles. At this time the increased estradiol output promotes follicle vascular- ization and may thereby increase gonadotropin uptake into the follicle in the bovine. Additional observations indicate that the most active follicle stimulates its own growth while simultaneously inhibiting growth and differentiation of other follicles.65 Two waves of follicle growth have been observed,7'101 corres- ponding with moderate increases in estradiol69 during the luteal phase of the sheep's estrous cycle,7'101 see Figure 1. It has been proposed for the bovine, that endogenous FSH is sufficient to stimulate follicle growth but is unable to sustain the large follicle resulting in atresia and growth of other small follicles.55 The moderate increases seen in 10 the luteal phase of the ovine estrous cycle are believed to stimulate the growth of small antral follicles.7'83 The mechanism by which preantral follicles become antral is not 65,101 wn. kno An intrafollicular polypeptide hormone called inhibin has been observed to suppress the release of FSH from the pituitary in the 29,114 bovine and rat, and inhibit oocyte maturation in the mouse, rat, 105,113,114 pig, and sheep. The suppression of oocyte maturation can be reversed by LH (5 mg/ml) ig.vitro.26’53 LH binding inhibitory substance, that prevents LH binding to receptors on the granulosa cell, is thought to be inhibin. The proposed course of events suggests that as the follicle grows and matures, it releases less of the LH binding inhibitor and thus enables more LH to bind to granulosa cells. The LH bound to the granulosa cells in turn reduces the release of inhibitory substances and resumption of oocyte meiosis occurs.23 Hansel and Convey,55 in their review, discuss the changes in LH and FSH receptors in preovulatory follicles. LH receptors and binding of LH in the theca and granulosa cells increase prior to ovulation in large follicles. These follicles become more responsive to LH and increase their estradiol secretion. In contrast, the numbers of FSH receptors decrease in ovulatory follicles just prior to ovulation.54 Additionally, an apparent effect of the preovulatory follicle to inhibit growth of other follicles has been observed.65 The control of this inhibition is not known. In summary, many factors play a role in the control of follicular growth. Follicle stimulating hormone causes increased growth of antral follicles while the LH surge initiates oocyte maturation and is believed 11 to cause ovulation. Three waves of follicular growth occur in the cycle of the ewe, but only one gives rise to follicles destined to ovulate. Inhibin may be the controlling hormone that inhibits preantral follicular development and luteal follicle development by regulating gonadotropin release and receptor binding in the follicle. Other Factors Affecting Ovulation Rates and Estrus In the anestrous season, follicles grow and some ovulate, but in only a small percentage of sheep does behavioral estrus accompany ovulation.21'6o'117 Also, greater numbers of antral and atretic follicles have been observed in ovaries in the estrous as compared to the anestrous season.21 Corpora lutea formed from induced ovulation by exogenousCHuuiin the anestrous season, secrete inadequate proges- terone even though LH receptors are in normal numbers.67 Estradiol becomes a negative feedback hormone on the pituitary in the anestrous season.42 Ewes in the anestrous season were observed to undergo luteal regression and show estrus but not develop mature follicles.65 All of the above reports indicate an alteration in folliculogenesis in the 27,119 anestrous season. Diminished success of superovulation as ewes approach or enter anestrus may be a reflection of this altered follic- ulogenesis. Variation between breeds has been reported for normal ovulation 14,15,87 rates and superovulation response. Other breed differences include, height of LH peak in response to GnRH,52 timing of the spontaneous LH peak,86 and duration of estrus.14 Plasma progesterone has been shown to be higher in Finnish Landrace ewes compared to 12 breeds with lower fecundity.86 Differences of fecundity have been observed within breeds when individuals are selected for litter size. One proposal for the mechanism for the observed breed difference is a genetically predetermined difference in sensitivity of the hypothalamal- PituitarY-ovarian axis.15'52 A positive correlation between body weight and ovulation rate has 26,38,90 been well documented. Feeding high-energy rations prior to the 65,117 The breeding season to increase ovulation rate is routinely done. mechanism. by which this occurs is not understood.88 The role of FSH or LH in this phenomenon has been investigated and no differences in secretion or ovarian uptake were observed.26'38'62 One proposed mechanism is that undernutrition changes the responsiveness of the ovary to gonadotropins.62 Superovulation The aim of superovulation is to enhance folliculogenesis and ovulation by giving exogenous hormone preparations that have FSH and LH activity. TWO commonly used gonadotropins, reviewed by Betteridge13 and Seidel and Seidel,100 are PMSG and FSH. Also, desiccated pituitaries 25.51.72.73 or sheep (SAP)22 have been used. The of horses (HAP) gonadotropin preparations either contain FSH and LB, or they have mole- cules with FSH and LH activity. The combination of FSH and LH activity, reviewed by Moor,7O affects antral follicles in the ovary causing growth, oocyte maturation, and ovulation of greater than the normal number of ova. The gonadotropin activity can be divided into two effects on the ovary. The FSH portion acts to increase the growth 13 rate of medium to large antral follicles and also rescues follicles undergoing early atresia resulting in increased population of large antral follicles. The LH portion acts to activate the oocyte to resume meiosis and to cause ovulation.70 Betteridge,13 in his review, cites the use of Human Chorionic Gonadotropin (hCG), in combination with PMSG, FSHp or HAP to imitate or heighten the preovulatory IH surge and increase the number of ovulating follicles.13 The importance of timing of the gonadotropin treatment within 79,103,116 the cycle has been demonstrated. The FSH and LH activity are thought to act optimally to cause superovulation when given during a wave of follicular growth.79 Gonadotropin treatment during a wave of follicular growth is accomplished by two methods. One method is to initiate treatment in the animal's follicular phase. On the average, the individual's CL regression and resulting estrus and ovulation will coincide with the folliculogenic effect of the treatment; however, due to individual variation of cycle length, this does not always occur. The second method is to artificially control progesterone withdrawal so the ensuing estrus will coincide with the effects of the treatment. This is accomplished with either prolonged progesterone treatment 32,63,91 followed by sudden withdrawal, or by giving a luteolytic preparation such as PGF2.1'SO'96'102'119" Both treatments have been shown to cause a rapid drop in plasma progesterone fbllowed by an LH surge, estrus, and ovulation in a majority of animals treated.1'31'32'36'94 Many factors are involved and interact in superovulation schemes to effect the results. These will be discussed in detail in the following sections. 14 Gonadotropins Used to Cause Superovulation Pregnant Mares Serum Gonadotropin Pregnant Mares Serum Gonadotropin (PMSG) is widely used to superovulate domestic species, and its use has been reviewed by Betteridge13 and Seidel and Seidel.100 Folliculogenic properties of PMSG were first used to cause ovulation in anestrous sheep in 1933, before its use for superovulation.108 Dosage recommendations vary greatly within the literature from 100 in used in the Rhesus monkey12 to 3000 in used to superovulate cattle.19 Wide dose ranges have also been reported within species. The recommended dose range of PMSG for 88,118 superovulation in swine is 750 in - 1000 iu, in cattle 500 iu - 19,97 35,92,116 3000 iu and in sheep 500 in - 2500 iu. The most commonly reported dosages used for cattle range between 1500 in - 2000 iuz'97 17,121 and for sheep range between 1000 in - 1500 iu A positive linear dose-response effect on follicular growth has been demonstrated in 34,40,92,116 cattle and sheep for PMSG. The low dose of 500 iu - 600 in is used in sheep to produce 2-4 lambs, and high doses, 1000 in or . . 34,116. greater, result in up to 30 ovulations per ewe. Advantages of using PMSG as a superovulation gonadotropin are as follows: PMSG can be used fresh, freeze-dried for storage and later use, or purified to increase the concentration of FSH/LB activity.79 The entire superovulatory dose can be given as a single injection because PMSG has a long half life.79'92 84,92 One molecule of PMSG has 2 subunits with FSH and LH activity and an interaction of these two gonadotropins is believed to be necessary for ovulation in sheep.43'92 15 Radioreceptor analysis of FSH and LH in several lots of PMSG showed a ratio that varied from 177.1:1 - 2:1 and a 5:1 ratio is considered to be close to optimal for follicular growth and ovulation.7s Immunoassay measurements of FSH and LH content along with amino acid sequence indicate the ratio of FSH to LH may be 1:12.84 The LH activity or the long half life (t5=40-50 hrs and 118-123 hrs for the rapid and slow component respectively84) may account for some of the undesirable effects of PMSG as a superovulatory hormone. A large degree of individual variation has been reported in cattle and l9,20,34,121 sheep, ranging from zero to 30 ovulations. This variation may account in part for the large dose ranges recommended in the litera- ture. Significant variations in activity have been demonstrated between the different PMSG preparations, between harvested batches and between individual mares.75 The dose reponse relationship of PMSG for total follicular development does not always reflect ovulation rate. At doses of 800 in PMSG and above in sheep, the ovulation rate does not linearly increase and the percent of ovulations to total follicles decreases. In sheep and cattle, lack of ovulation of large 19,30,34,115,121 34,71,73 follicles, premature luteinization of follicles, 92,93,115 a and reduction of recovery rate of ovulated ova, 3 well as 3,34,92,115,121 account for decreased conception rate of recovered ova the decrease in percent ovulation. Many mechanisms for these effects of PMSG have been proposed. High estradiol levels from the multiple follicles may feedback on the hypothalamus causing release of LH from the pituitary and result in 34,93 premature luteinization of follicles before estrus. A more 16 plausible explanation is a direct effect of the . LH activity in PMSG.34'70'75 The long-acting LH activity in PMSG has been suggested to inhibit ovulation of large follicles while FSH activity acts simultaneously to recruit more antral follicles to grow with a net result of many large unovulated follicles.79 A comparison done in cattle where superovulation with PMSG or when purified LH (PLH) was added to FSHp superovulation treatments compared to FSHp alone showed that ovarian response (CL number) and ova number recovered was reduced by the former two treatments. The addition of PLH to FSHp also reduced the fertilization rate compared to FSHp alone.70 Oocyte activation in sheep was altered by PMSG. A trial com- paring superovulation by PMSG or FSHp showed that both stimulated follicular development, but that PMSG caused premature activation in 33% of the oocytes. This resulted in retention of the oocyte in luteinized follicles or ovulation of aged eggs which contributed to the increased number of abnormal embryos and fertilization failure. These researchers concluded that a reduction of LH content of gonadotropin treatment may decrease the premature activation of oocytes during superovulation.7o The hormonal environment for capacitation, fertilization, and ova transport has been thought to be altered by excessive gonado- 19,92,115 tropins. Moor recently demonstrated grossly altered nuclear and cytoplasmic substrate uptake in oocytes in_vitro due to PMSG but not to FSHp.7o Premature zona pellucida shedding has been reported in PMSG superovulated cattle and is speculated to be the effect of high 19,91 progesterone and estrogens on the early embryo. Rapid transport 17 of ova through the reproductive tract has been documented in sheep. This has been thought to be a result of increased oviduct motility due to 92,93,121 high estrogen levels from the superovulated ovaries. The rapid transport places the ova beyond the ampular-isthmal junction of . . . . . . . . 24,92,93 the ov1duct and in this way interferes with fertilization. This phenomenon may also contribute to low recovery rates of ova seen with high PMSG dosages. Several authors suggested that the ova are either transported through the oviduct into the uterus rapidly and oviduct flushes do not recover them or retrograde movement of ova l9,92,93,121 Another occurs and they are lost in the abdominal cavity. contributing factor to fertilization failure was the occurrence of silent estrus observed in PMSG treated sheep.92 The animals ovulated but did not show'behavioral estrus, making natural breeding difficult and timing of artificial insemination inaccurate. One explanation for silent estrus was that progesterone from prematurely luteinized follicles inhibits estradiol's positive feedback on the hypothalamus and pituitary. Tonic LH did not increase rapidly and the rapid rise of estradiol, responsible for triggering expression of estrus, did not occur.92 Progesterone has also directly interfered with sperm transport through the cervix in the ewe thereby interfering with fertilization.85 Some ovulated ova that are not capable of fertilization may be due to either lack of maturation or premature activation of maturation prior to being 70,92 19,93 ovulated. PMSG has been reported to lengthen the estrous cycle 93,120 and hasten the onset of estrus post treatment. High progesterone levels from the multiple CLs has been implicated in lengthening the cycle.19 18 Estradiol levels have been examined as a predictor of ovulation rate in cattle. Estradiol and progesterone levels were higher in animals with high ovulation rates, however, there was too much individual variation to use them to predict ovulation rate.107 Estradiol levels of superovulated prepuberal heifers remained high after treatment. High estradiol may deleteriously affect the ova and explain the high numbers of unfertilized and degenerated ova seen in these animals.97'107 Timing of the PMSG treatment to coincide with a wave of follic- ular growth affects follicular response and conception rate.116 In earlier studies, PMSG was given in the follicular phase, day 16-17 in cattle.78 and days 12-14 in sheep.92 PMSG alone given on days 9, 10, and 11 resulted in significantly reduced ovulation and conception rates in sheep.116 PMSG has been given during midcycle in cattle to coincide with one of the midcycle waves of follicular growth.79 This treatment was combined with estrous synchronization which will be discussed later. The timing of PMSG treatment in midcycle also has an effect on follic- ular growth. In cattle, treatment at days 9, 10, and 11 generally 78'79 This was thought to correspond with maximum population of medium size antral follicles.7o yields optimum ovulation rates. Anterior Pituitary Preparations Several different anterior pituitary (AP) preparations have been used to superovulate domestic species. These preparations are desiccated anterior pituitary collected from sheep (SAP)32 or horses (HAP)25'51'72 and a commercial FSH, called Follicle Stimulating Hormone-p.a These aBurns Biotec Company, Chromally Pharmaceutical Inc., Omaha NE. 19 products contain a combination of FSH and LH and have a shorter half life than PMSG. These preparations will be discussed in the following sections and their properties described in comparison to those of PMSG. Horse Anterior Pituitary Horse anterior pituitary (HAP) was the most frequently used AP preparation from 1937 to 1955. Researchers harvested pituitaries from horses at the slaughter plant and prepared their own HAP. Lack of commercial availability and the labor to prepare the hormone are the 25,51,72,73,73 main disadvantages of HAP. The short half life of all the AP preparations is a disadvantage in that they must be given twice daily for several days. Anterior pituitary preparations are given for 16,73,73 3-4 days in sheep. The interval from the last treatment to onset of estrus is correlated to ovulation and fertility. The shorter the interval the higher the ovulation rate and fertilization rate.74 A linear dose-response for ovulation rate has been documented for 51,74 25,73,74 HAP Doses have varied from 45 - 200 mg Decreased ovulation rates were reported at 200 mg HAP.25 The wide variation in reported doses may reflect variation in the preparation and standard- ization of the HAP between research laboratories. The advantages of HAP to be discussed will be in comparison to the previously reported effects of PMSG. Less individual variation of ovulation rates has been reported with HAP than with PMSG at their respective superovulation dosage levels. Stimulation of follicular growth is as good as with PMSG and ovulation rate is greater and more 92,115 consistent. Unruptured fbllicles, premature luteinization or 20 overstimulation of ovaries reported with PMSG occur less frequently . 7 . . . . . . with HAP. 4 Betteridge,l3 in his reView, pOints out that Simultaneous superovulation trials comparing PMSG to HAP or FSHp have not confirmed better ovulation rates, fertility or recovery rates for the anterior pituitary preparations.13 Later, a comparative trial comparing PMSG to FSHp resulted in better ovulation and fertilization rates for FSHp.70 Decreased recovery rates have been reported for HAP at 200 mg, but superovulation can be accomplished at a much lower dose and thereby avoid this.25 Fertilization rates reported for HAP (65-90%)16’72'73 are higher than for PMSG, 33-60%.91 Anterior pituitary preparations shortened the onset of estrus and this may have a positive effect on fertilization.74 As with PMSG, the timing of HAP treatment is con- sidered important. Those days reported for optimal effect with PMSG are also used when HAP is given.74 A survey of optimal days during midcycle to institute HAP treatment has not been reported for sheep. Follicle Stimulating Hormone-p Follicle Stimulating Hormone-p (FSHp) is prepared commercially and the dosages reported in the literature are less varied than with 3,4,5 HAP. Doses for sheep and goats are 17 - 25 mg, and cattle doses 13,21 are 35 - 45 mg FSHp contains molecules of both FSH and LB with the half lives of 30 and 110 minutes for LH and FSH respectively,104 requiring the drug be given twice a day for several days. The total superovulatory dose is divided over a 3-4 day period in sheep17 and a 4-5 day period for cattle.76'79'99 In cattle and sheep, several authors suggest that higher ovulation rates, higher fertility rates, less individual variation, higher recovery rates, and fewer unruptured 21 3'17'79 Premature activation of oocytes, observed with PMSG treatment, has not been seen with FSHp.7O fellicles occur with FSHp than PMSG. Studies using FSHp noted lower estradiol serum levels around the time of ovulation than seen with PMSG, and high estradiol is implicated in I fertilization failure. Researchers recently observed that gonado- tropins with high LH activity tended to adversely affect ovulation and fertilization to a greater degree than FSHp.7O'75 The timing of FSHp treatment as observed for HAP and PMSG may have an effect on follicular growth rate in cattle. The days in the cycle that FSHp treatment is given are the same as enumerated for HAP.99 The time in the cycle of the ewe to give FSHp has not been studied. Additionally, silent estrus has been reported in the ewe with FSHp alone or in combination with progesterone pretreatment.17 Human Chorionic Gonadotropin Human chorionic gonadotropin (hOG) contains primarily LH activity and some FSH activity.93 In sheep, hCG in combination with PMSG or HAP increases follicular growth but significantly reduces ovu- 51’115 The usefulness of hCG to create an exogenous LH lation rate. surge at the end of superovulation treatments of PMSG or FSH to enhance Iovulation rate has been examined in sheep. The use of Prolanb with PMSG caused follicle development but ovulation rate was reduced due to .premature luteinization attributed to the LH activity. Production of luteinized follicles by the LH activity and the progesterone secretion from them.are believed to cause decreased fertility rates, recovery bCommercial hCG available in Britain. 22 rates and prolonged estrous cycles (25 days) in sheep.93 In FSH super- ovulated sheep, the addition of purified luteinizing hormone (PLH) on the last day of FSH treatment did not increase ovulation rates. The addition of hCG or LH to superovulation regimens was not beneficial and may be detrimental in the ovine. Control of the Cycle: Estrous Synchronization The interval between gonadotropin treatment and onset of estrus and ovulation has an effect on ovulation and fertilization rate. In the case of PMSG, the greater the interval the greater the number of 40,93 fertilized ova. For the anterior pituitary preparations, the shorter the interval the higher the ovulation and fertilization rates.74 The average interval, cited by Betteridge,13 when FSH is used for superovulation is 48 hours for cattle. The observed optimum interval to estrus in HAP superovulated sheep is 24-48 hours.74 A great deal of individual variation in estrous cycle length exists and treatment With a gonadotropin on day 12-13 of the cycle of sheep can result in a variation from the end of treatment to onset of estrus93 and therefore reduced ovulation rates.34 Efforts to control the onset of estrus and ovulation have been attempted to reduce this variation. The methods 'used attempt to control the rapid drop in serum progesterone because the fall in progesterone removes the inhibition of progesterone on the estradiol induced LH release. The events that lead to estrus and ovu- lation as previously reviewed are then allowed to take place.6'56 An early method used was enucleation of the corpus luteum. This was done by removing the structure from the ovarian stroma by 23 rectal manipulation in cattle. This has proven unsatisfactory because all of the progesterone secreting tissue was not always removed in this manner and resulting hemorrhage would sometimes result in adhe- sions that would interfere with subsequent ovulations.78 The next method employed was progesterone treatment which has been reviewed by Thimonier.109 The forms of progesterone that have 17,32 been used are daily injections of progestogen, daily oral feeding of progesterone salts,32 intravaginal pessaries of progesterone salts,32'34'63'91 and subcutaneous silastic implants containing progesterone.28 These treatments are given or left in place for 10-14 days (average of 12 days), and when removed, estrus and ovulation occur in 36-48 hours in sheep.91'109 The continuous progesterone inhibits the positive feedback of estradiol and blocks LH discharge and formation of a new CL. If a CL is present at the time of proges- terone treatment, it regresses and secretes very little progesterone. Removal of the exogenous progesterone results in the sudden drop of serum progesterone and loss of inhibition of the estradiol-LH discharge.32'56 Although progesterone control of the cycle is an effective method to synchronize estrus and ovulation, fertility can be reduced.110 Progesterone has been thought to cause faulty transport 57'85'110 reduced cervical mucus, and of sperm through the cervix, decreased duration of estrus in the ewe.32 Surgical artificial insemination is described as one method to bypass the cervix. Placing fresh semen directly into the uterus of the ewe did improve fertility of superovulated ewes; however, recovery rate of ovulated ova was 24 reduced by the technique so the overall numbers of fertilized ova obtained were similar to naturally bred ewes.110 Progesterone is known to inhibit LH release by blocking the estradiol induced release of GnRH.56 The LH rise observed following removal of progesterone treatment is delayed so that it does not coincide with the estradiol rise. Uterine contractions are also inhibited.32 These effects may alter the uterine environment necessary for sperm transport, fertilization and development of the early embryo. Progesterone has been used in anestrous sheep to induce estrus with superovulation treatments. This remains the most effective method to induce estrus and ovulation in anestrous sheep. However, there is an even greater degree of variation in response to gonadotropin treatments 27,34,123 than seen in sheep during the estrous season. This will not be reviewed here as this thesis involves cycling ewes. The third method of controlling the estrous cycle has been by using a PGF called Lutalysec or a synthetic prostaglandin, 2 Cloprostenol sodiumé (Clo). Both of these products are known to be 11,41,50,82,94 luteolytic in cattle, sheep, and goats. The hormonal changes fOllowing PGF or Clo luteolysis are not different from those 7,11 2 of naturally occurring luteolysis. Prostaglandin F and Clo 2 reduced progesterone secretion (functional luteolysis) within 2 hours 36,93 of administration. This is believed to act intracellularly on cLutalyse - Upjohn Company, Kalamazoo, MI. dEstrumate - ICI 80996, ICI Pharmaceutical, Division At Kemix Inc., Mississauga, Ontario . 25 steroid synthesis since LH receptor numbers and activity are unchanged 31,35,36 . is until structural luteolysis occurs. 2 Prostaglandin F thought to be the luteolysin of sheep and is secreted from the uterus into the uterine veins. A counter current exchange between juxtaposed ovarian artery and uterine veins transfers PGF from the uterus to the 2 . . . . . 41 ovary without reaching the systemic Circulation. Prostaglandin F and Clo have been used to synchronize estrus in 2 cattle where superovulation was instituted in the luteal phase of the cycle.2 Within the cycle of the sheep, PGF2 has been an effective luteo- lytic drug between days 4-13. Prostaglandin F does not improve ovula- 2 tion, fertilization or synchronization when given on day 14 or later in the ovine estrous cycle.121 Favorable ovulation and fertility rates as 19,102 an well as heat synchronization have been reported fbr cattle d 1'49'122 when used with superovulation. When PMSG was used, PGF sheep 2 or Clo was given 48 hours after treatment in cattle and 1-2 days after treatment in sheep.122 The doses reported for cattle are 500 - 19,20,96 O 2,19,20,102 1000 ugm C10 r 30 - 50 mg PGF2 . Doses reported for sheep are 100 ugm Clo or 15-- 20 mg pep2 ,50'122 has been reported in Seasonal variation in response to PGF2 sheep. In the mid-anestrous season, if a CL was present, it regressed following PGF treatment; however estradiol and LH peaks were very 2 low or absent and therefbre no ovulation or estrus occurred. The use of PGF to induce ovulation during anestrus has been limited.119 2 There have been reports of low fertilization rates from.breed- 37,47 ings subsequent to PGF estrous synchronization. One researcher 2 measured the number of motile sperm in the anterior reproductive tract 26 and found sperm numbers in PGF treated sheep to be lower than in 2 untreated estrous ewes.57 The majority of these studies compared a single appointed breeding time in the PGF treated sheep to breeding 2 of the untreated ewes at estrus, which may account for lower fertility if not all ewes were bred at estrus.18 In other reports of PGF2 estrous synchronization and subsequent fertilization rates, no reduc— 44,111 tion in lambing and fertilization rates were observed. One group of researchers demonstrated increased transport of fresh and frozen ram spermatozoa, and correspondingly higher fertilization rates, with the addition of 100 ugm of Clo to semen or 2.5 mg Clo given intramuscularly to the ewe at the time of artificial insemination.33'45'46 A controlled trial where PGF2 treatment combined with progesterone synchronized was compared to non-PGF2 treated ewes have shown no reduc- tion in fertility48 and no adverse effect on fertilization rate when used along with superovulation treatments.121 Other Problems Associated withfiSuperovulation A progressive reduction in ovulation rate occurs with repeated 64,95 treatment with any of the superovulatory hormones. This reduc- tion was not observed in one study where cattle were superovulated 5 successive times at increased FSHp dosages each time.70 Two explana- tions for the reduced response are currently being investigated. First, the hormones, as foreign protein, may induce an antibody response and become less effective as antibody titers increase. Efforts to produce and measure the antibody response to these gonadotropins have 12,68 been unsuccessful. Second, studies involving repeated 27 superovulation revealed that a rest period between treatments of one to two cycles would regain the original ovulatory response. Investi- gators proposed the reduced ovulation rate was due to either too short a time between treatments for follicular development,64 or exhaustion of the proliferating follicular pool.95 In sheep, repeated super- ovulation has had limited success because these animals entered their anestrous period. Poor results obtained during this time were reversed with the fbllowing breeding season.34 Large follicles in the ovary may have an influence on super- ovulation response. Occasionally, the gonadotropin will be given when one large fbllicle is present in the ovary during midcycle. This follicle is stimulated by treatment to ovulate and luteinize, and then inhibits growth of other fbllicles, either by a direct intraovarian influence, or because the progesterone secretion inhibits LH release and prevents ovulation of those follicles that do grow.93 Effect of Breed, Season, Plane of Nutrition, Age As previously discussed, ovulation in the ewe is affected by breed, nutrition, age, and season. These factors are also thought to have an effect on ovulatory response to superovulation in ewes. Breeds that have high lambing rates tend to respond with greater ovu- lation rates than less prolific breeds, for example Finnish Landrace versus Suffolk breeds. English breeds that have long, deep anestrous seasons have a poor response to superovulation when in anestrus and 34,112 during the transitional periods of their breeding season. The level of nutrition in sheep has been shown to delay onset of cycling 28 by a low plane of nutrition as well as reduced ovulatory rates in severely thin animals.116’117 However, the effect of nutrition on superovulatory response has not been demonstrated by a well controlled study at the present time. Factors that reduce the number of fertilized embryos recovered from superovulated ewes have been attributed to undesirable effects of PMSG and progesterone. Because of these effects, this study was begun to determine the effects of FSHp and PGFZ, as an alternative for superovulation of ewes. The objectives of this research were: first, to determine if FSHp and PGF would superovulate ewes; second, to 2 determine if dosage of FSHp had an effect on the superovulation response in ewes; and third, to determine if a dose of 22 mg FSH or above would maximize embryo response in superovulated ewes. MATERIALS AND METHODS Animals Seventy-nine crossbred Targhee ewes, between 2 and 4 years of age, and 100-110 lbs were used in these experiments. Thirty ewes were used from September to December 1981 and 49 ewes were used from September to December 1982. The ewes were kept on pasture and fed alfalfa hay and corn as a maintenance diet. Three weeks prior to being used in the experiment, the ewes were fed an increased energy ration of corn (3/4 lb per ewe per day). Estrous Detection All ewes were detected for estrus prior to initiation of treatments to determine the day of estrus. A painted vasectomized marker ram was turned in with the ewes twice a day and observed for 30 minutes. Ewes were defined as being in estrus when they would stand stationary when mounted by the marker ram. Breeding Rams used for breeding underwent a breeding soundness exam and 10'98 Based semen testing as outlined by the Society of Theriogenology. on this exam and previous fertile breedings in each year used, the rams were determined to be fertile. Two and 4 rams were used for breeding in 1981 and 1982, respectively. All ewes were exposed to two painted fertile rams from the time of the last hormone treatment to the end of 29 30 estrus. These rams were painted different colors to facilitate confirma- tion and identification of the two rams that bred each ewe. Estrous Synchronization In 1981, all ewes were allowed to cycle naturally and were estrous detected and selected in groups of 2 to 4 for treatment. In 1982, as a larger number of animals was used, the ewes were estrous synchronized in groups of 10 to 15 with 15 mg PGFZ.C Those ewes not showing estrus within 3 days of the PGF were given 15 mg .PGF 5 to 7 days 2 2 after the first treatment. Treatment Methods All ewes were given injections of either FSH or saline intra- muscularly. The total dose was divided into a descending mg dose schedule and given at 12 hour intervals for 3.5 days. The divisions of FSH dosages are listed in Appendix Table A1. Ten mg PGF2 was given intramuscularly in conjunction with both treatments on the third day of this schedule. Ewes were bred at the ensuing induced estrus 12 to 36 hours after the last FSH or saline treatment. All treatments were initiated on an assigned day of the ewe's cycle. This was based on the previous estrus assigned to be day 0. Experimental Design 1981 Experiment Prior to testing for effect of FSH dosage on superovulation response we were interested in testing 4 days of the estrous cycle for the effect of initiating FSH treatment on these days. This is based on cLutalyse - Prostaglandin F '- Upjohn Company, Kalamazoo, MI. 2 a the 4 d 17 ass 31 observations in cattle where the days of the cycle that FSH is given has 78,79,99 an effect on ovulatory response. In cattle, the range of days when optimum ovulatory response occurs is thought to correspond to a _ 65,79,99 . . midcycle follicular growth wave. When PMSG is given outSide the range of optimum days immediately preceding estrus in cattle, ovula- 7 tion rates are reduced.8'79'103'116 An optimal range of days in midcycle of the ewe has not been tested. The 4 days tested were selected from a period corresponding to a rise in the ewe's estradial levels (Figure l - literature review) which is to occur when a number of antral follicles . . ; ,65'101 would be growing (a follicular growth wave)60 ' and are thought to be susceptible to superovulation treatment?0 Part I This experiment was designed to evaluate the effect of the day of the cycle when the FSH treatment is initiated (DOCI) on response, for 4 days of the estrous cycle. This was done by determining the effect of 17 mg FSH initiated on days 7, 8, 9, and 10 of the estrous cycle. we assumed that the effect of DOCI was the same for all FSH dosages and that 17 mg FSH would produce a representative superovulation response for the effect of DOCI. Three ewes were assigned to each treatment group. The statistical model used was Y.. = u + DOCI. + E . i i ij j . . . . . .t where Y is the dependent variable, u is a constant, DOCIi is the i h 13 DOCI effect, and E i j is random experimental error. Part II This experiment was designed to evaluate the effect of FSH dosage from 12 mg to 22 mg. This was done by determining the effect 32 of 6 FSH dosages: 0.0 mg, 12.0 mg, 14.5 mg, 17.0 mg, 19.5 mg, and 22.0 mg. All treatments were initiated on day 8 of the estrous cycle. Day 8 was selected because no difference based on visual observation of the data from.Part I was observed, and it was intermediate in the 7-10 day interval. Three ewes were assigned to each treatment group. The treatment cells tested are listed in Appendix Table A2. The statistical model used was: = + + Yijk u DOCIi FSHk + Eikj where Yijk is the dependent variable, u is a constant, DOCIi is the ith DOCI effect, FSHk is the kth FSH dose effect, and Eikj is the random experimental error. 1982 Experiment An apparent positive dose response relationship was seen from the 1981 experiment. Based on this, the 1982 experiment was designed to evaluate the effect of FSH dosage from 19.5 mg to 30 mg. Additionally, two days of the cycle were evaluated at each dosage level. The reason for testing FSH dosage at two days of the cycle was that the effect of FSH may vary for different days of the estrous cycle and this inter- action was not tested in the 1981 experiment. This was done by deter- mining the effect of 4 FSH dosages: 0.0 mg, 19.5 mg, 22.0 mg, and 30.0 mg, initiated on day 8 and day 10 of the estrous cycle. Four ewes were assigned to treatment with 0.0 mg, with 2 of the ewes on treatment day 8 and 2 ewes on treatment day 10. Fifteen ewes were assigned to each of the remaining FSH treatments, with S of the ewes on treatment day 8 and 10 ewes on treatment day 10. The treatment cells tested are listed in Appendix Table A3. The statistical model used was: where 1 IDCI e: action error. done tx FSH do: include OfEs Where 1 and x : where E the FSE 1here I list}. 33 Y..""- 'I' ,+ + - .4. .. 1k] U DOCI1 FSHk DOCI FSH1k E 1k 3 where Yikj is the dependent variable, u is a constant, DOCIi is the ith DOCI effect, FSHk is the kth FSH dose effect, DOCI-FSHik is the inter- ik j is the random experimental error. Using this data and a second model, a multiple regression was action of DOCIi and FSH dosek , and E done to calculate a polynomial or line of best fit for the effect of FSH dosage on corpora lutea and embryo number. This second model included linear, quadratic, and cubic regression to determine the line of best fit. These models are, first: Y = a + bx where Y is the dependent variable, a is a constant, b is a constant, and x is the FSH dosage. Second: Y = a + bx + cx2 where Y is the dependent variable, a, b, and c are constants, and x is the FSH dosage. Third: Y + a + bx + cx2 + dx3 where Y is the dependent variable, a, b, c, and d are constants, and x is the FSH dosage. Hormones Used Follicle Stimulating Hormone Follicle Stimulating hormone-P was used which consists of porcine pituitaries that are desiccated and freeze-dried by the manufacturer and FSH content is expressed in milligram equivalents of the Armour Standard as described by Steelman and Pohley.104 Follicle stimulating hormone-P is aFSHp--lot number 550C81 - Burns Biotec Company, Chromally Pharmaceutical Inc., Omaha, NE. 34 sold in 50 mg vials of freeze-dried product to be diluted with 10 ml sterile water to a 5 mg/ml concentration. In these experiments the 50 mg vial was diluted with 50 cc of sterile water to a 1 mg/ml concentration to facilitate more accurate delivery of dosages. Prostaglandin F 2 The PGF2 used was Lutalysec which contains 5 mg/ml of clinoprost tromethamine, a natural PGF In these experiments 20 mg (4 ml) of 2. clinoprost tromethamine was given intramuscularly to the ewes during the FSH treatment to synchronize estrus. In the 1982 experiment 15 mg (3 ml) of clinoprost tromethamine was also given to the ewes to synchronize estrus prior to the FSH treatments. Data Collection Laparotomy Technique The sheep were given 1300 mg (13 ml) Ketamine Hle and 10 mg (0.5 g, intramuscularly. Following shear- ml of 20 mg/ml concentration) Xylazine ing and clipping of wool of the ventral abdomen, the sheep were placed in dorsal recumbency on a table slanted at a 300 angle with the ewe's head faced downhill. A surgical scrub was performed on the skin of the ventral abdomen and aseptic technique was maintained throughout the procedures. A ventral midline laparotomy was done inmediately anterior to the ewe's udder. The uterus and ovaries were elevated out of the laparotomy inci- sion and examination of the ovaries, followed by flushing of the uterine horns was done. cLutalyse - lot numbers OlOJH and 572JH - Upjohn Company, Kalamazoo, MI. fVetalar - Parke Davis, Warner Lambart, Morris Plains, NJ. gRompun - Haver Lockard, Miles Laboratory Inc., Shawnee, KS. 1'8 obs tis str fil 35 The Ovarian Structures The ovarian structures were counted, diameter measured in milli- meters and recorded for right and left ovary, separately. The structures recorded were corpora lutea and follicles. Any abnormal findings such as poorly luteinized Cls, edema of the bursa or oviduct, and adhesions were recorded. Corpora lutea.were defined as those structures that upon gross observation were greater than 5 mm in diameter and were comprised of luteal tissue with or without an ovulatory papilla. Follicles were defined as structures that upon gross observation were greater than 5 mm and fluid- filled with no evidence of an ovulation point. The Uterine Flush An antegrade uterine flush was done to collect ova and embryos from each horn (Appendix B). The technique is described below. An intravenous (IV) catheter was placed in the tip of the horn and directed toward the oviduct. A l4-gauge, Foley catheter was placed into the uterine lumen and the cuff inflated to occlude the lumen and hold the Foley catheter in place. A sterile syringe, containing 60 cc of Dulbecco's phosphate- buffered saline with 2% ovine serum and 5% glucose, was attached to the IV catheter and media were infused. The outflow of media, through the Foley catheter, was collected into separate, labeled petri dishes. The flushing procedure was done on each uterine horn. The uterus was then rinsed with sterile saline solution and replaced into the abdomen and the abdomen was closed. The ewes were given 1,000,000 in Benzathine Penicillineh and 150 iu of Tetanus Antitoxinl intramuscularly after surgery. g h . . . - Benzathine Penicilline - Beecham Inc., Bristol, TN. 1 . . . . Tetanus Antitoxin - Jensen Salsbery Laboratory; Div. Richardson Merrell, Inc., Kansas City, MO. 36 In order to determine the volume of media to use for the uterine flush, 120 ml was used in 30 ewes. This volume was divided equally into two petri dishes. No ova were found in the second 60 ml volume. Sequen- tially, a 60 ml flush volume was used and divided equally into 2 petri dishes. Ova were found in the second petri dish from 3 of the 11 ewes. Based on these findings, a 60 ml flush volume was used for the remaining ewes in these experiments. Because adhesions or damage to the reproductive tract has been reportedg'68 the uteri of 50 of the ewes were examined. A laparotomy was done at 30-60 days after the uterine flush. Also, 30 of the ewes were given prostaglandin F2 immediately after the flush and housed with fertile rams for 30 days. This was done to assess adverse effects of the uterine flush technique on fertility based on gross anatomy of the reproductive tract and subsequent pregnancy rate in the ewes. Embryo Counting The petri dishes containing the uterine flushings were allowed to settle at 250 C for 30 minutes. Using a stereoscope at 40x magnification, the petri dishes were systematically scanned three times to locate the ova. The ova were then examined for fertilization, stage of development and degeneration, and the number recorded by horn for each ewe. Evalua- tion of enbryos was done according to the criteria described by Seidel and Seidel. 100 Statistical Analysis An analysis of variance was used to determine the treatment effects on corpora lutea, embryos, follicles, unfertilized embryos, and degenerated embryos.j The treatments tested were FSH dosage, day of the 3Version SAS 79.5 of the GLM series of Statistical Analysis System,58 Wayne State University, Detroit, MI. 37 cycle the FSH was initiated (DOCI) and interaction between FSH dosage and DOCI. The statistical model used was Y. = + + + - , + , . ikj u DOCIi FSHk DOCI FSH1k E(lk)j where Yikj is the dependent variable, u is a constant, DOCIi is the ith DOCI effect, FSHk is the kth FSH dosage effect, DOCI-FSHik is the effect of the interaction between DOCIi and FSH dose k, and E(ik)j is the random experimental error. A one-way analysis of variance was done to determine the effect of year of the experiment on corpora lutea and embryosk. The model used was = + +an Yij “ A1 j/Ai where Y is the dependent variable, u is a constant, A1 is the ith year effect, andanj/Ai is the jth animal within year and used as the year error term. In order to determine a trend for the effects of FSH dosage on corpora lutea and embryos, multiple regression of the data was donej. The statistical models used were Y = a + bx, Y = a + bx + cxz, and Y = a + bx + cx2 + dx3 where Y is the dependent variable, a, b, c, and d are constants, and x is the FSH dosage. Significant (P<.05) polynomial regression equations were determined for the change in corpora lutea and embryos response over increasing FSH dosage. In order to estimate the optimum FSH dosage at which maximum CL response would occur, the derivative of the polynomial regression equation for CL response by FSH dose was solved. An estimate for the optimum.FSH dosage at which maximum embryo response would occur was not done because the regression equation was linear and therefore had no optimum. kVersion SAS 82.3 of the GLM series of Statistical Analysis System?9 wayne State University, Detroit, MI. RESULTS AND DISCUSSION Effect of Treatments on Corpora Lutea To determine whether the year of the experiment had an effect on the CL response, a one-way analysis of variance was done and the results are summarized in Table l. A significant effect of the year of the experiment on the CL response was not demonstrated (P>.05), therefore the data of both years were pooled and an analysis of variance was done across years. TABLE 1 Summary of the ANOVA for year effect on corpora lutea response Source of variation df ss ms Year 1 127.3 127.3 Animals within year 77 2977.3 38.7 The results of the analysis of variance for the effect of treatments on day of the cycle the FSH is.initiated (DOCI), FSH dosage, and the inter- action of DOCI and FSH dosage are listed in Table 2. From the analysis, three conclusions regarding the effect of the treatment can be made. First, no significant (P>.5) effect of DOCI on corpora lutea response was seen. This indicates that for 17 mg FSH the effect of initiating FSH on days 7, 8, 9, and 10 of the cycle had no significant (P>.5) effect on CL response. Therefore it appears that any one of these 38 39 TABLE 2 Summary of the ANOVA of the treatment effects on corpora lutea Source of Variation df ss ms DOCI 3 19.4 6.3 DOSE 6 991.5 165.3 DOCI-DOSE 4 259.0 64.8 Error 65 2845.5 43.8 4 days of the cycle would be equally suitable to do the FSH dosage testing on. Secondly, a significant effect (P<.01) of FSH dosage on number of corpora lutea was found, The main effects of FSH dosage on CL number are summarized in Table 3. The treatment means are: 1.86 i 2.50, 6.67 i 3.82, 6.67 i 3.82, 8.40 i 1.71, 12.06 i 1.56, 14.39 i 1.56, and 10.73 i 1.71 CL/ewe for FSH dosage 0.0, 12.0, 14.5, 17.0, 19.5, 22.0, 30.0 mg FSH/ewe, respectively. A multiple regression of the data was done to determine a regression equation that best fit the effect of FSH dose on CL numbers. A significant (P<.05) cubic regression equation was calculated. The equation was: Y = 218.088 - 36.354x + 1.937x2 - 0.0319x3 where Ywas CL numbers and xwas FSH dOSage. The coefficient of deter— mination is r2 = .31. Taking the derivative of the regression equation and solving for zero estimates the optimum dose of FSH to maximize CL response. This dose is 25.8 mg FSH. A graph of the cubic regression along with the treatment means is shown in Figure 2. The cubic regression is the best fit of the data 4O ’ O 30- .. 0 25b 0 Mean Corpus luteum per Ewe ~/ I l I I l I 12 14.5 17 19.5 22 25 MG FSH O O O 4 30 Figure 2. Graph of cubic regression curve and mean corpora lutea/ewe by FSH mg. ------- Cubic regression of mean corpora lutea/ewe by dose. Mean corpora lutea/ewe by dose. I Treatment means. . CL per individual ewe 41 TABLE 3 Main Effects of FSH Dose on Corpora Luteaa FSH mg Mean Adjusted CL*/ewe iSE Total CL No. Ewes 0.0 1.86 i 2.50 13 7 12.0 6.67 i 3.82 20 3 14.5 6.67 i 3.82 20 3 17.0 8.40 i 1.71 126 15 19.5 12.06 i 1.56 217 18 22.0 14.39 i 1.56 259 18 30.0 10.73 i 1.71 161 15 *CL = Corpora lutea aSignificant (P<.05) cubic regression for change in number of corpora lutea over change in mg of FSH. and a polynomial estimate of a dose-response curve. The calculated optimum dose 25.8 mg is relatively close to the observed optimum of 22 mg. From this, we can conclude that an optimum CL response will occur between 22 - 30 mg FSH and appears to be near 25.8 mg. The 25.8 mg dose is an extrapolated optimum and the best estimate based on the change in CL response from 22 mg to 30 mg. The real optimum may or may not be near the estimate, and more data between 22 - 30 mg are needed to determine this. The positive dose-response effect seen in these experiments is similar to those seen with other gonadotrophins. Positive linear dose- 74,93 1: response curves have been determined for both Bus? and 35,40,93,116 . PMSG" ‘ effect on follicular growth. Decreased ovulation 25 93 35,51,93 rates were observed at high doses of HAP ' and PMSG. The results of these experiments are similar in that CL response decreased at 30 mg versus 22 mg. This may indicate that an increased CL response to 42 FSH dose is seen up to apoint, after which more FSH may decrease CL response. The cubic regression may reflect the way the ovary responds to exogenous gonadotrophins. The gradual slope of the curve at 14.5 mg FSH may reflect a response threshold for the follicle population to FSH. Below a certain threshold dose the amount of FSH may not be sufficient to stimulate FSH receptors, bind to them and effect follicular growth and ovulation in greater than 2 - 4 follicles. The steep slope between 14.5 - 25.8 mg may reflect the optimal dose range to stimulate these events. Higher levels of gonadotrophins, as reported in other studiegs'lg'28 and as seen in these experiments at 30 mg, may alter an optimal relationship between receptor binding, FSH present, and FSH utilized to stimulate follicle growth. The result of excess FSH or LH activity is thought to be overstimulation of follicular growth and is reflected as unovulated follicles and prematurely luteinized fbllicles and thereby decreased corpora lutea numbers. More investiga- tion toward understanding the mechanism and control of follicular growth is necessary to determine the action of these gonadotrophins. A large degree of individual animal variation has been reported.93 In these experiments, the coefficient of determination (r2 = .31) reflects the wide degree of individual variation seen within all animals. The graph of adjusted treatment means with the 95% confi- dence intervals shows the variation around each mean (Figure 3). These experiments also have observed a wide degree of variation within animals as has been previously reported. Thirdly, no significant (P>.2) interaction of DOCI and FSH dose on CL response was seen in these experiments. Two days, 8 and 10, of 43 ZOI— Adjusted Mean CL Per Ewe o I I I l I o 12 14.5 17 19.5 22 25 30 MG FSH Figure 3. Graph of adjusted mean corpora lutea/ewe by dose of FSH. Each vertical bar represents the 95% confidence interval around the adjusted treatment means. 44 the estrous cycle were tested over four doses to determine whether an interaction existed. This had not been tested previously when DOCI testing was done, so the possibility of an interaction was evaluated by testing these data. Because no significant (P>.2) interaction was seen, the conclusion was that the effect of the FSH dosage did not differ between days 8 and 10 of the cycle. Effect of Treatments on Embrygs A one-way analysis of variance was done to determine the effect of year of the experiment on embryo response and the results are summa- rized in Table 4. A significant interaction was not found (P>.2) and the data from both years were pooled and an analysis of variance was done across years. The results of the analysis of variance for treatments, day of the cycle the FSH was initiated (DOCI), FSH dosage and the interaction of DOCI and FSH dosage are listed in Table 5. From the analysis, three conclusions regarding the effects of the treatments can be made. First, no significant (P>.5) effect of DOCI on embryo response was seen. This indicates as seen for CL in the previous section, that for days 7, 8, 9 and 10 of the cycle the embryo response was not significantly (P>.5) dif- ferent between days. The rationale for testing the effect of DOCI on embryo response is the same as discussed for CL. TABLE 4 Summary of the ANOVA for year effect on embryos Source of Variation df ss ms Year 1 59.0 59.0 Animals within year 77 2920.4 37.9 45 TABLE 5 Summary of the ANOVA for treatment effect on embryos Source of variation df ss ms DOCI 3 23.5 7.8 DOSE 6 425.7 71.0 DOCI-DOSE 4 59.3 14.8 Error 65 2920.2 44.9 Second, a significant (P<.05) linear regression for the effect of FSH dosage on embryo response was determined. The main effects of FSH dosage on embryos are listed in Table 6. The overall FSH dose effect was not significant (P>.05) and this may be because of the wide degree of individual variation in each treatment group. More ewes per treatment group may demonstrate a FSH dosage effect. The linear relationship of FSH dosage on embryo response was significant (P<.05) however; and this is the best fit of the treatment effects. The regression model used linear, quadratic and cubic regression to determine the line of best fit. Because the coefficients of determination (r2) of the quadratic and cubic polynomials did not improve the fit of the regression to the data, the linear model was selected as the polynomial of best fit. The regression equation was: Y = 0.708x - 6.213 where Y is embryo response, and x is FSH dosage. The coefficient of determination is r2 = .15. This again reflects a wide degree of individual animal variation and is illustrated by the 95% confidence intervals in Figure 4. 46 20 - 15 #- Adjusted Mean Embryos Per 10 - Ewe 5 I- of I l I I o 12 14.5 17 19.5 22 25 so MG FSH 1”Ii-911.1% 4. Graph of adjusted mean embryos/ewe by dose of FSH. Each vertical bar represents 95% confidence intervals around the adjusted treatment means. 47 TABLE6 Main effects of a FSH dose on embryos FSH mg Mean Adjusted embryos/ewe t SE Total embryos No. of ewes 0 1.14 i 2.53 8 7 12.0 1.33 i 3.87 14.5 4.33 i 3.87 13 3 17.0 5.87 i 1.73 88 15 19.5 7.72 i 1.58 139 18 22.0 8.44 i 1.58 152 18 30.0 7.93 i 1.73 119 15 aA. significant (P<.05) linear regression for change in number of embryos over change in mg of FSH A graph of the linear regression and treatment means is shown in Figure 5. A positive linear trend exists for both curves at dose 19.5 mg and below. Above the 19.5 mg level the treatment means and regression curve diverge. Because a greater number of ewes (N = 64) have a strong linear relationship at 22 and below, their data have more weight. Likewise, the fewer ewes (N = 15) at dose 30 mg do not have enough weight to alter the slope of the regression. Therefore, a linear regression remains the best estimate from the data, although the treat- ment means level off. An optimum FSH dose for embryo numbers cannot be eStjJnated from these data. Because the number of embryos is a function of the number of CLs (ovulations) , the maximum number of embryos should not exceed the number of CLs. One would expect then that the optimum dose of FSHp for embryos would be equal to or less than the optimum dose for (31.3. However, a divergence from the linear trend cannot be proven from theSe data and may be due to the amount of variation around the treatment means. 48 O 30- . 25.— e 2OI- Mean Embryos . per 0.0 Ewe 151 J. I" :. ' I so 0 ,x’ .’ e .I e 10" ” .’°’ . . . .. ,e . eee ' - e e s- e eee .. O. O” 0 e co - ' u 0 " / e 10 ee 0 9 1' e— l M I ee 0 12 14.5 17 19.5 22 25 30 MG FSH Figure 5. Graph of linear regression curve and mean embryos/ewe by FSH mg. ------- Linear regression of mean embryos/ewe by dose of FSH. Mean embryos/ewe by dose of FSH. I Treatment means. . Embryos for individual ewes. 49 More data in the dose range of 19. 5 - 30 mg are needed to demonstrate a leveling of the linear dose response curve to determine the optimum dose for embryos. Reduction of embryos/corpora lutea (ovulation ratio) has been documented by others at high doses of PMSG. Reduction of the embryo/CL ratio has been attributed to several factors. At high doses . . . . 3,92,121 of PMSG, fertilization failure has been observed. Progesterone pretreatment as a synchronizing method has resulted in fertilization failure due to interference with sperm transport.84 Fertilization fail- ure was not observed to a great degree in this experiment and would not explain the reduction of embryo/CL ratio seen at 22 mg and 30 mg. Highly stimulated superovulated ovaries produce high levels of 96,97,107 estradiol. The high estradiol is thought to increase the motil- ity of the oviduct, thereby rapidly moving ova out of the oviduct. Rapid 19,24,34,92 The ova transit of ova has been reported in cattle and sheep. are then missed by oviduct flush techniques. Recovery rates reported for uterine flush techniques are lower (38-6O%)9 than those for oviduct flush (60-70%).110 With the uterine flush method the ova are thought to be "lost" within the uterus, degenerate due to an unfavorable environment for the early embryo, or be flushed retrograde into the oviduct, prevent- ing retrieval.78 The observation that embryo numbers did not increase above 19.5 mg FSH cannot be attributed solely to fertilization failure because the fertilization rates (reported as unfertilized ova/total ova) did not differ between treatment groups. Tests for FSH dosage effect on unfertilized ova are discussed later in this thesis (page 52). Also, recovery rates did not appear to be different between FSH treatment groups (See Table 14). These effects were not tested for significant differences. 50 Factors other than either fertilization rates or recovery rates may play a role in embryo response but have not been identified in these experiments. The third conclusion is, no significant (P>.5) effect of the interaction of DOCI and FSH dosage on embryo response was seen. As discussed previously, the effect of the interaction of FSH dosage and DOCI was considered. In these experiments a significant interaction was not seen (P>.5), indicating that the effect of FSH dosage on embryo response was equivalent for days 8 and 10 of the cycle. Effect of Treatments on Follicles The results of the analysis of variance for treatments, day of the cycle the FSH was initiated(DOCI), FSH dosage and the interaction of DOCI and FSH dosage are listed in Table 7. From the analysis three conclusions regarding the effect of the treatments can be made. First, no significant (P>.5) effect of DOCI on follicle response was seen. Second, no significant (P>.5)effect of FSH dosage on follicles was TABLE 7 Summary of the ANOVA of treatment effects on follicles Source of Variation df ss ms DOCI 3 7.3 2.4 DOSE 6 10.1 1.7 DOSE-DOCI 4 9.8 2.5 Error 65 252.1 3.9 51 seen. Table 8 shows the main effects of FSH dosage on follicles and a lack of difference between the treatment means can be seen. Thirdly, no significant (P>.5) effect of the interaction of DOCI and FSH dosage was seen. The mean number of follicles observed was 1.90 1(122 and the treatment means ranged 1 i 1.12 - 2.67 i 1.12 (Table 8). Control ewes had an average of 2.14 i 0.73 follicles. Seven ewes had 5 to 10 follicles on their ovaries (Appendix Table C1). These individuals appeared to be evenly distributed between treatment groups but statis- tical testing for homogeneous distribution was not done. TABLE 8 Main effects of FSH dose on follicles FSH mg Mean Follicles t SE Total Follicles No. of Ewes 0.0 2.14 i 0.73 15 7 12.0 2.67 i 1.12 8 3 14.5 1.0 i 1.12 3 3 17.0 ' 2.13 i 0.50 32 15 19.5 1.83 i 0.46 33 18 22.0 2.11 i 0.46 38 18 30.0 1.4 i 0.50 21 15 The reason for interest in a treatment effect on follicles is based on reports of increased numbers of unovulated follicles and luteinized follicles with superovulatory dosages of PMSG.2'4’34 High LH activity of PMSG due to a long half life, may be responsible 52 for this effect on the follicle.70 Follicle stimulating hormones used in these experiments has a reported LH:FSH ratio of 1:3.75 Perhaps at higher doses of FSHp, an increase in number of unovulated follicles would be seen. In these experiments, the number of follicles was not significant (P>.5) between treatment groups. Effect of Treatments on Unfertilized Ova The results of the analysis of variance for treatments, day of the cycle the FSH was initiated (DOCI), FSH dosage and the interaction of DOCI and FSH dosage are listed in Table 9. From the analysis three conclusions regarding the effect of the treatments can be made. First, no significant (P>.5) effect of DOCI on unfertilized ova response was seen. Second, no significant (P>.5) effect of FSH dosage on unfertilized TABLE 9 Summary of the ANOVA of treatment effects on unfertilized ova Source of Variation df ss ms DOCI 3 4.6 1.5 DOSE 6 10.7 1.8 DOSE -DOCI 4 13.4 3.4 Error 65 190.3 2.9 ova response was seen. Table 10 shows the main effects of FSH dosage on unfertilized embryos and a lack of difference between the treatment means can be seen. Thirdly, no significant (P>.2) effect of the inter- action of DOCI and FSH dosage on unfertilized ova was seen. 53 TABLE 10 Main effects of FSH dose on unfertilized ova FSH mg Mean Unfertilized Total Unfertilized No. of va 1 SE Ova Ewes 0.0 0.00 i * 0 7 12.0 1.00 i 1.02 3 3 14.5 0.33 i 1.02 1 3 17.0 0.66 1' 0.46 10 15 19.5 0.83 t 0.42 15 18 22.0 0.94 i 0.42 17 18 30.0 0.06 i 0.46 1 15 *Standard error was not applicable. The mean number of unfertilized ova was 0.60 i 0. 20 and the treatment neans ranged 0.00 * - 1.00 i 1.021(Table 10). Two ewes showed high numbers of unfertilized ova, 6 unfertile ova out of 10 ova and 12 unfertile ova out of 12 ova recovered (Appendix Table C1). The ewe with 12 ova did not exhibit estrus and did not allow breeding by the ram. The reason for 6 unfertile ova out of the other ewe is not known. Fifteen ewes showed 3 or less unfertilized ova and appeared to be evenly distributed through treatment groups, although statistical testing for even distribution was not done. The control group (0.0 mg FSH) had 0 unfertilized ova, however no significant (P>.5) difference between other treatments was seen. Decreased fertilization (%) rate of recovered ova is reported at 34'92'121 Higher fertilization rates 22,115,122 have been reported for HAP (75 to 95%). Data from these superovulatory doses of PMSG. 54 experiments show a high fertilization rate (total fertilized embryos/ total ova = 91.75% i 3.00) corresponding with other reports using anterior pituitary preparations. The depressed fertilization rates observed with PMSG indicate some adverse effect on those events leading to fertilization. The mechanism of action by which PMSG exerts this effect has been discussed by many authors but has not been determined. The difference in effect on fertilization rate between FSH, HAP, and PMSG also has not been determined. The high LH activity of PMSG has been implicated in depression of fertilization by causing premature activation of the oocyte. This may result in ovulation of aged ova that are not capable of fertilization?0 Also, PMSG elevates eStradiol and progesterone serum levels above normal and this may affect the uterotubal environment and interfere with sperm transport, capacitation, or fertilization. Effect of Treatments on Degenerated Embryos The results of the analysis of variance for treatments, day of the cycle the FSH was initiated (DOCI), FSH dosage, and the inter- action of DOCI and FSH dosage are listed in Table 11. From the analysis TABLE 11 Summary of the ANOVA of treatment effects on degenerated embryos Source of Variation df ss ms DOCI 3 1.1 0.4 DOSE 6 5.2 0.9 DOCI-DOSE 4 5.5 1.4 Error 65 77.0 1.2 thes Firs resp on d effe betw effe was The ; is m aPPEa 55 these conclusions regarding the effect of treatments can be made. First, no significant (P>.5) effect of DOCI on degenerated embryo response was seen. Second, no significant (P>.5) effect of FSH dosage on degenerated embryo response was seen. Table 12 shows the main effects of FSH dosage on degenerated embryos and a lack of difference between the treatment means can be seen. Thirdly, no significant (P>.2) effect of the interaction of DOCI and FSH dosage on degenerated embryos was seen. TABLE 12 Main effects of FSH dose on degenerated embryos FSH Mean Degenerated Total Degenerated No. of mg Embryos 1 SE ‘ Embryos ' Ewes 0 0.00 i * 0 7 12.0 0.00 i *\ 0 3 14.5 0.00 i * 0 3 17.0 0.33 i 0.28 5 15 19.5 0.22 i 0.25 4 18 22.0 0.72 i 0.25 13 18 30.0 0.60 i 0.28 9 15 1Standard error was not applicable. One ewe had 8 degenerated embryos out of 29 (Appendix Table C1). The reason for this animal having a high number of degenerated embryos is not known. Seventeen ewes had 3 or less degenerated embryos and appeared to be evenly distributed throughout the treatment groups; however, they were not statistically tested for even distribution. 56 No degenerated embryos were observed in treatments of 14.5 mg FSH or less; however this difference was not statistically significant (P>.5). Degenerated embryos have been observed by many workers with PMSG and FSHp used in certain cows. The high LH activity of PMSG and the high estrogen and progesterone levels seen in superovulated cows are thought to be involved in embryo degeneration. Moor et a1 observed altered substrate uptake into the nucleus and cytoplasm of embryos from PMSG but not FSHp superovulated cows?O There appears to be an effect on the metabolism of the early embryo due to PMSG, which may account for the reports of greater numbers of degenerated embryos seen. Neither a large number of degenerated embryos or a dose effect on number of degenerated embryos was seen in these experiments. The appearance of all the degenerated embryos above dose 17 mg may reflect a relationship between degree of superovulation on the ovary and degeneration of embryo. Prostaglandin an No significant difference (P>.05) of FSH dose or DOCI effect was seen between ewes given prostaglandin F in 1982 to synchronize 2 estrus prior to FSH treatments and those allowed to cycle naturally in 1981. This was tested by a one-way analysis of variance to deter- mine if a significant difference between years on CL or embryo response existed. (pp 38,44) The results of the estrous synchronization are summarized in Table 13. The average onset of estrus following one treatment with PGF was 44 hours and within a 24-72 hour range. 2 Thirty-eight of 45 ewes (84%) came into estrus after one PGF2 treat- ment. Five of 8 of the remaining ewes came into estrus after a second 57 TABLE 13 Estrous synchronization with PGF : interval from treatment to estrus Interval from PGF Treatment Number of to Estrus Ewes 24 hours 1 30 hours 13 43 hours 14 52 hours 13 65 hours 2 72 hours 2 PGF2 injection given 5 to 7 days after the first injection. All 3 of the remaining 3 ewes came into heat after a third PGF injection 5 days 2 after the second PGF2 treatment. Five ewes came into estrus naturally and are not included in these data. Forty of 45 ewes (89%) came into estrus within a range from 30 to 50 hours after PGF2 treatment. (See Figure 6.) Analysis for the effect of multiple PGF injections was 2 not done. Prostaglandin F2 was selected to synchronize ewes in this study for several reasons. Prostaglandin F2 is considered the luteolysin of the ewe and has been shown to cause similar luteal regression and result- ing hormone profiles as normal regression. Prostaglandin F2 causes rapid luteal regression with estrus occurring in 24-72 hours and in combination with previous superovulation regimes has not had an adverse . 5 effect on ovulatory response or fertility. 0 However, progesterone, an . 4 84' alternate synchronizing agent, has produced fertilization failure.33’6 ' 58 In these experiments, one hundred percent of ewes (pre-FSH- treatment) were synchronized into 24 to 72 hour periods. Seventy-eight of 79 of FSH-treated ewes came into estrus within 48 hours of the last FSH treatment. Close synchrony of the last FSH treatment and estrus is desirable since higher ovulation rates and fertilization rates are seen when this interval is short.74 Finally, since the fertilization rate was 91.75% i 3.00 and no apparent effect on the number of unfertilized ova was observed, PGF did not appear to reduce the fertilization rate. 2 Ova Recovery Rate The ova recovery rates defined as total ova/corpora lutea are listed in Appendix Table C2. The recovery rates are summarized by FSH dose in Table 14. The arithmetic mean ova recovery rate was 65.3% i 3.7. The mean recovery rate at 12 mg FSH appears to be much lower than the rest of the treatment means. This is because 1 of the 3 sheep had a recovery rate of 0% and in a treatment group of 3 ewes this lowered the mean recovery rate. The treatment mean recovery rates appear to be similar over the remainder of the treatment group; however data were not statistically tested for significant differences. Distribution of ewes with low (0-33%), medium (34-660C) and high recovery rates (67—100%) are illustrated in Figure 6. Forty-eight ewes had recovery rates above 67%, 17 ewes had recovery rates between 34-66%, and 14 ewes had recovery rates of 33% or less. The ova recovery rate in this report is higher than previously reported for uterine flush techniques. Reports of higher ova recovery rates are seen if the ovi- duct is flushed, but this may lead to adhesions or obstructions of the «oviduct and adjacent structure.16 In this experiment, based on I. 59 TABLE 14 Summary of mean ova recovery rate in % by FSH dose FSH Dose mg Ova Recovery % 0 71.4 i 12.4 12.0 36.7 i 18.9 14.5 63.3 i 18.9 17.0 69.5 i 8.5 19.5 62.3 i 7.7 22.0 63.6 i 7.7 30.0 66.1 i 8.5 laparotomy examination, no adhesions of oviducts, fimbriae or ovaries were seen and a subsequent 100% pregnancy rate in 30 ewes bred after the flush technique indicates patent and functional oviducts, fimbriae and ovaries. Pregnancy was determined either at lambing or at the time of the laparotomy examination. 60 45 ' g7, 40- 35~ No. of Ewes 25 '- M 0-33 34-66 67-100 OVA Recovery Rate (%) 1"igure 6. Graph of the distribution of ewes with low, medium, or high ova recovery rates (%). SUMMARY AND CONCLUSIONS The effect of FSHp and PGF to induce superovulation in 79 2 crossbred Targhee ewes was determined. The variables measured were number of follicles, corpora lutea, embryos, unfertilized ova and degenerated embryos. A total of 570 ova were collected, of which 523 were fertilized and 31 degenerated. The fertilization rate was equal to 91.75% i 3.0%. A total of 816 corpora lutea and 149 follicles were observed. The experiments were doneixlthe fall of 1981 and 1982. The 1981 experiment was designed to determine the effects of the time FSHp is initiated within 4 days of the estrous cycle of the ewe. Following this, a second experiment was done to determine the effect of 6 different doses of FSHp. The 1982 experiment evaluated the interaction of the day of the cycle FSHp was initiated and 3 FSHp doses based on results of the 1981 experiment. The effect of the day of the cycle FSHp was initiated (DOCI) was examined for days 7, 8, 9, and 10 of the estrous cycle. Interaction between DOCI and FSHp dose was evaluated for days 8 and 10 and doses 0 mg, 19.5 mg, 22 mg, and 30 mg FSHp . No significant (P>.9) effect of DOCI was found and no significant (P>.2) interaction of DOCI with dose was determined. Therefore, initiation of FSHp treatment on day 7, 8, 9, or 10 of the cycle of the ewe has produced equivalent results. Seven doses of FSHp were evaluated, 0 mg, 12 mg, 14.5 mg, 17 mg, 19.5 mg, 22 mg, and 30 mg FSH. A positive effect of dose on 61 62 number of corpora lutea was determined. A cubic regression curve was calculated by method of least squares. The regression curve is the ~dose response curve for FSHp dose on CL number and can be used to predict response in areas of the curve not tested in these experiments. An optimum dose for maximum number of CLs was calculated to be 25.8 mg FSH. This dosage is close to the observed optimum of 22 mg, beyond which the response decreased. Therefore, an optimum dose to produce the maximum CLs (ovulations) was estimated to be between 22 and 30 mg. In addition, FSHp in excess of this dose may be detrimental to ovulation rate. Large variations in response existed within all treatments, and the coefficient of determination (r2 = .30) for the regression curve reflected this degree of error. Large variation in individual response to superovulation have been reported frequently for other gonadotrophins. The variation in these experiments is similar to previous reports. A linear regression curve for number of embryos by dose of FSHp was calculated by method of least squares. This curve showed a positive dose effect of FSH on embryo number. The regression curve and actual data closely agreed up to 19.5 mg FSHp. At this dose and beyond, the actual measured data leveled off while the regression curve continued to increase because the strong linear relationship below 22.0 mg exerts more influence on the curve than do data above 22.0 mg. This regression curve is the best predictor of embryo response from the data. Decrease in the embryo/CL ratio at high doses of PMSG and HAP has been docu- mented by other researchers. The observation that embryos do not increase beyond 19.5 mg may or may not reflect a lower optimal dose than fer CL numbers. More data at doses above 19.5 mg are needed to determine 63 the optimum FSHp dose for embryo response. Three additional observations were made from the data of these experiments. First, PGF was used for estrous synchronization prior 2 to and in conjunction with the FSHp treatment with satisfactory induction of estrus and fertilization rates. Second, a modified antegrade uterine flush technique was employed which resulted in satisfactory ova recovery rates and minimal trauma to the reproductive tract. Third, high fertilization rates of the ova were seen. In conclusion, FSHp and PGF in combination induced superovula- 2 tion in crossbred Targhee ewes. A significant positive dose response curve was determined for corpora lutea and embryos. An optimum dose for CL numbers was between 22 - 30 mg FSHp. No optimum dose for embryo number can be estimated from the data. The fertilization rate in this study indicates that FSHp and PGF did not adversely affect fertiliza- 2 tion to the degree reported for PMSG and progesterone. Although a small number of FSHp doses were evaluated, the information gained from this study may aid future research on superovulation of the ewe. 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APPENDICES APPENDIX A 74 75 TABLE A1 Descending dose regimes for FSH doses: 0 to 30 mg Total 1st 2nd 3rd 4th FSH mg Treatment Treatment Treatment Treatment AM 5 ml 4 m1 ioml “‘PGF 2 m1 Control ”9 2 Saline 3 ml + PM 5 m1 4 ml 10 mg PGF2 AM 3 mg FSH 1.5 mg FSH 10mggpggs+ 1 mg FSH 2 PM 3 mg FSH 1.5 mg FSH 10mggFggF+ 2 1.5 mg FSH + AM 3 mg FSH 2 mg FSH 10 mgVPGF 1.5 mg FSH 2 14.5 mg PM 3 mg FSH 2 mg FSH iésmggpgifi + 2 AM 4 mg FSH 3 mg FSH iomggpggp+ 1 mg FSH 2 I10 mg PM 4 mg FSH 3 mg FSH iomggFggF+ 2 AM 4 mg FSH 3 mg FSH iongFggr+ 1.5 mg FSH 19.5 mg 2 PM 4 mg FSH 3 mg FSH iomfigF§§F+ 2 2 mg FSH + AM 5 mg FSH 3 mg FSH 10 mg PGF2 2 mg FSH 22.0 mg PM 5 mg FSH 3 mg FSH logggFggF+ 2 3 mg FSH + AM 6 mg FSH 5 mg FSH 10 mg PGF2 2 mg FSH 30.0 mg PM 6 mg FSH 5 mg FSH iomggFggF+ 2 76 TABLE A2 The frequency of observations within the treatment cells for the 1981 experiment Total mg FSH Day of Cycle Treatment Initiated (DOCI) 7 8 9 10 0-0 3 ewes 12.0 3 ewes 14-5 3 ewes 17.0 3 ewes 6 ewes 3 ewes 3 ewes 19.5 3 ewes 22.0 3 ewes Total: 30 ewes TABLE A3 The frequency of observations within the treatment cells for the 1982 experiment Total mg FSH Day of Cycle Treatment Initiated (DOCI) Day 8 Day 10 0.0 2 ewes 2 ewes 19.5 5 ewes 10 ewes 22.0 5 ewes 10 ewes 30.0 5 ewes 10 ewes Tbtal: 49 ewes APPENDIX B 77 78 "mm“ .-:7?':"1"..5.=1-.=.5§-:: cu" “.25.:- ; 'tl-I-rgg. Internal ( .. =3.:?-."7.f..':-:,':. Bifurcation '\ . IV of Uterus? ‘ Occtud/ ' " Catheter Puncture H . 9 ' - in Base of 0'. Ovuduct terine Horn Foley Catheter Cervix Figure B1. Schematic diagram of ovine uterine flush technique. *Cutaway view of position of catheters in lumen of uterine horn APPENDIX C 79 80 TABLE C1 Results of superovulation: total CL, follicles, embryos, unfertilized ova, and degenerated embryos by FSH dose Dose Day Total Total mg Sheep of ToCEal Fofigles Eistals Unfertilized Degenerated FSH Cycle ryo Ova Embryos 0. 0 619 8 1 1 1 0 0 0. 0 627 8 2 2 3 0 0 0. 0 632 8 2 3 0 0 0 0. 0 710 8 2 2 2 0 0 0. 0 722 8 1 1 1 0 0 0. 0 728 10 1 2 1 0 0 0. 0 717 10 4 4 0 0 0 12. 0 602 8 7 3 0 0 0 12. 0 613 8 7 3 3 0 0 12. 0 614 8 6 2 1 0 14 . 5 629 8 10 1 0 0 14 . 5 669 8 5 1 1 1 0 14 . 5 670 8 5 1 3 0 0 17 . 0 649 7 5 2 1 0 0 17 . 0 642 7 6 0 1 3 0 17.0 639 7 13 2 13 0 2 l7 . 0 611 8 5 5 2 3 0 17. 0 622 8 9 0 8 0 1 17. 0 620 8 17 1 13 0 0 17. 0 607 8 9 0 7 2 1 17.0 . 625 8 3 10 2 0 0 17. 0 636 8 10 0 8 0 1 17. 0 635 9 6 6 3 2 0 17. 0 644 9 4 2 3 0 0 17. 0 633 9 9 2 8 0 0 17. 0 643 10 14 1 9 0 0 17. 0 640 10 10 0 5 0 0 17. 0 648 10 6 1 5 0 0 19. 5 605 8 4 2 0 0 0 19 . 5 628 8 8 4 7 0 0 19 . 5 674 8 14 0 0 12 0 19 . 5 718 8 20 3 10 0 0 19 . 5 709 8 18 2 8 0 0 19 . 5 706 8 ll 1 10 0 1 l9. 5 725 8 21 0 17 0 1 19 . 5 719 8 25 ‘ 0 11 3 0 19 . 5 704 10 13 2 4 0 0 19. 5 707 10 1 0 0 0 0 19 . 5 723 10 5 3 0 0 0 19. 5 739 10 1 5 1 0 0 81 TABLE C1 (continued) Dose Day Tbtal Tbtal mg Sheep of Dgial Foiiizies E:::;:s Unfertilized Degenerated FSH Cycle Ova Embryos 19.5 735 10 32 0 32 0 1 19.5 714 10 21 1 21 0 0 19.5 747 10 9 3 8 0 0 19.5 726 10 6 5 5 0 0 19.5 750 10 2 2 1 0 0 19.5 702 10 6 0 4 0 0 22.0 612 8 7 6 5 0 0 22.0 617 8 11 2 10 0 0 22.0 630 8 9 4 4 0 0 22.0 749 8 24 4 17 3 1 22.0 729 8 10 0 7 0 0 22.0 738 8 12 1 0 0 0 22.0 732 8 11 1 7 1 0 22.0 716 8 8 1 0 2 0 22.0 721 10 11 2 9 0 0 22.0 724 10 13 0 1 1 1 22.0 743 10 3 7 2 1 0 22.0 736 10 25 2 17 0 0 22.0 734 10 17 3 17 0 0 22.0 737 10 31 0 29 1 8 22.0 730 10 9 1 5 0 0 22.0 731 10 14 3 5 2 2 22.0 742 10 29 1 13 0 0 22.0 727 10 15 0 4 6 l 30-0 711 8 20 1 15 0 0 30.0 701 8 2 0 2 0 0 30.0 720 8 7 2 4 0 2 30.0 715 8 10 0 7 0 0 30.0 744 8 14 4 14 0 2 30.0 712 10 15 0 8 0 3 30.0 708 10 10 1 10 0 1 30. 0 713 10 1 1 0 0 0 30.0 748 10 3 3 1 0 1 30.0 705 10 15 0 15 0 0 30.0 740 10 14 2 14 0 0 30-0 703 10 17 0 16 0 0 30.0 746 10 13 1 2 0 0 30- 0 733 10 8 2 0 0 0 30- 0 74 1 10 12 4 11 1 0 82 TABLE C2 Ova recovery rate in % (Total ova/corpora lutea) by FSH dose FSH Dose mg Sheep Recovery % 0.0 632 0 0.0 619 100 0.0 627 100 0.0 722 100 0.0 717 0 0.0 710 100 0.0 728 100 12.0 614 67 12.0 602 0 12.0 613 43 14.5 670 60 14.5 629 90 14.5 669 40 17.0 635 83 17.0 620 77 17.0 622 78 17.0 611 100 17.0 639 85 17.0 642 67 17.0 649 20 17.0 648 83 17.0 640 50 17.0 643 64 17.0 633 67 17.0 644 75 17.0 625 67 17.0 607 67 17.0 636 60 19.5 674 86 19.5 605 0 19.5 628 86 19.5 735 94 19.5 739 100 19.5 704 44 19.5 718 50 19.5 707 0 19.5 725 81 19.5 706 91 19.5 709 44 83 TABLE C2 (continued) FSH Dose mg Sheep Recovery % 19.5 723 0 19.5 719 56 19.5 702 67 19.5 750 50 19.5 726 83 19.5 747 89 19.5 714 100 22.0 617 91 22.0 630 44 22.0 612 72 22.0 716 29 22.0 732 73 22.0 738 0 22.0 727 67 22.0 729 70 22.0 742 45 22.0 730 56 22.0 749 83 22.0 731 50 22.0 736 68 22.0 734 100 22.0 737 100 22.0 743 100 22.0 724 15 22.0 721 82 30.0 748 33 30.0 715 70 30.0 720 57 30.0 713 0 30.0 741 100 30.0 733 0 30.0 746 15 30.0 744 100 30.0 708 100 30.0 701 100 30.0 703 94 30.0 740 100 30.0 705 100 30.0 712 53 30.0 711 70