- Email: [email protected]
Heterogenous and xenogenous fertilization of in vivo matured equine oocytes
REFEREED PAPERS FROM THE IOTH SYMPOSIUM 2. Brazeau P: Drugs affecting uterine motility. In: The Pharmacological Basis of Therapeutics. Goodman and Gilman (eds), exl. 4, The Macmillan Co., London, pp 893-907, 1971. 3. Eiler H, Oden J, Schaub R, Sims M: Refractoriness of both uterus and mammary gland of the cowto prostaglandin F2ct administration: clinical implication. Am J Vet Res 42:314-317, 1981. 4. Goddard PJ, Allen WE: Genital tract pressures in mares. I. Norreal pressures and the effect of physiological events. Theriogenology 23:815-827, 1985. 5. Goddard PJ, Allen WE: Genital tract pressures in mares II. Changes induced by oxytoein and prostaglandin F2 a. Theriogenology 24:35-44, 1985. 6. Goddard PJ, Allen WE: The use of catheter-tipped pressure transducers for chronic measurement of genital tract pressure in the ewe. II. Effects of oxytoein and prostaglandin F2=. Theriogenology 25:693-708, 1986. 7. Kindahl H: Role of prostaglandins in uterine involution. 10th Int'l Congress on Anita Reprod and A. I., Univ. of Illinois, Urbana-Champaign, XII, pp 14-19, 1984. 8. Lauderdale JW: Hormonal therapy and residues for prostaglandin F2 alpha. 10th Int'l Congress on Anita Reprod and A. I., Uni of Illinois, Urbana-Champaign, XII, pp 14-19, 1984.
9. Lauderdale JW, Miller PA: Regulation of reproduction in mares with prostaglandins. In: Proceedings of the 21st Ann Cony of the Amer Assoc of Equine Pract Milne FJ (ed), pp 235-244, 1975. 10. Ley WB, Purswell BP, Bowen JM: The effects of prostalene and alfaprostol as uterine myotonics, and the effect on postpartum pregnancy rate in the mare following daily treatment with prostalene. Theriogenology On press) 1988. 11. Loy RG: Characteristics of post-paaum reproduction in mares. Vet Clin No Amer: Large Anim Pract 2:345-359, 1980. 12. Neely DP: Hormone Therapy in Mares. In: Equine Reproduction Neely, Liu, Hillman (eds), Vet Learning Systems Co, Inc., pp 24-35, 1983. 13. Peterson DE: Gonadotrophin releasing effects of alfaprostol in seasonally anestrous/estrous mares. J Equine Vet Sci 5:331-335, 1984. 14. Taverae MAId: Uterine motility in the post-partum female. 20th lnt'L Congress on Anita Reprod and A. I., Univ of Illinois, UrbanaChampaign, XI, ppl-8, 1984. 15. ThrelfaU WR, Carleton CL: Treatment of uterine infections in the mare. In: Current Therapy in Theriogenology, Morrow DA, (ed), ed. 2, WB Satmders Co, Saunders Co, Philadelphia, pp 730-737, 1986. 16. Vandeplassche M, Bouters R, SpincemaiUe J, Bonte P, Coryn M: Observations on involution and puerperal endometritis in mares. Irish Vet J 37:126-132, 1983.
HETEROGENOUS AND XENOGENOUS FERTILIZATION OF IN VIVO MATURED EQUINE OOCYTES A.O. McKinnon, BVSc, MSc, E.M. Carnevale, DVM, E.L. Squires, PhD, J.L. Voss, DVM, MS, G.E. Seidel Jr., PhD
SUMMARY Forty-five in vivo matured equine oocytes were recovered from 63 follicular aspiration attempts (71.4%). HCG did not improve recovery rate (65% - 24/37 for treated vs 81% 21/26 for nontreated mares). Fifteen oocytes were transferred into the oviduct of inseminated recipient mares (heterogenous fertilization) and 15 oocytes plus equine spermatozoa were transferred into rabbit oviducts (xenogenous fertilization). Ten oocytes (3 fertilized) were recovered from recipient mare oviducts following removal and flushing two days after transfer. Eight oocytes (nonfertilized) were recovered from rabbit oviducts. Oviductal transfer into separate recipient mares of three embryos produced from heterogenous fertilization resulted in two pregnancies. One mare produced a normal live foal and the other mare aborted at 20 days of gestation. Results from these studies suggest that: 1) a reliable method for collection of in vivo matured oocytes has been established, and 2) heterogenous fertilization is a technique that with refinement should be immediately applicable to obtain foals from Author's address: College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado 80523. Acknowledgement: Supported in part by the CSU Experiment Station and the Van Camp Foundation.
Volume 8, Number 2, 1988
valuable infertile mares that fail to get pregnant or produce embryos by standard methods.
INTRODUCTION Equine embryo transfer 6,18has been widely accepted and has resulted in foals from mares with a history of previous pregnancy failure. Unfortunately, embryo recovery rates from infertile mares, often older mares with the greatest genetic potential, were low (<30%) 6,7 compared to normal mares
(80%).TM Possible reasons for decreased embryo recovery from infertile mares are: 1) failure of spermatozoal transport through the uterotubaljunction and into the oviduct; 2) failure of fertilization; 3) failure of release of the fertilized egg from the oviduct into the uterus; 4) failure of the early embryo to develop because of a functionally incompetent oviduct or uterus; 5) increased early embryonic death due to genetic or other abnormalities of embryos~ from mares approaching senility; and 6) ovulation failure. Collection of oocytes prior to ovulation and fertilizing them in vivo or in vitro may be the only means of obtaining foals from some infertile mares. Three possible approaches to fertilizing these oocytes are: 1) heterogenous fertilization 143
8@¢i®IIy REFEREED PAPERS FROM THE 10TH SYMPOSIUM
fertilization in a recipient mare's oviduct; 2) xenogenous fertilization - fertilization in another species' oviduct (e.g. rabbit or sheep); and 3) in vitro fertilization (IVF) - fertilization outside the animal under controlled laboratory conditions. Transfer of gametes (oocytes and spermatozoa) to a recipient mare's oviduct is most attractive because: 1) it obviates need of sophisticated culture requirements for in vitro fertilization; 2) when oocytes are fertilized, the embryo can be maintained in the recipient mare without further manipulation; and 3) it is more physiological than both IVF and xenogenous fertilization and accordingly should be more acceptable to breed registries. Production of calves from in vitro fertilization was first reported in 19827 Since that time, numerous studies have been conducted on systems for in vitro fertilization of mammalian oocytes.3 Production of live piglets and lambs following in vitro fertilization of oocytes matured in vivo or in vitro has been reported recently.2 When fertilization and maturation of bovine oocytes has occurred in vitro, one major obstacle to embryo development is a block occurring at the 16-cell stage? This block does not occur with in vitro fertilized oocytes when resulting embryos are cultured in vivo. Cost and difficulty of obtaining equine oocytes for in vitro fertilization and maturation studies, together with the higher success rate of early embryonic development in vivo17 suggest heterogenous fertilization in the equine is the method of choice for future development. There are only a few reports on obtaining oocytes from donor mares. Vogelsang et al= attempted two methods of aspirating oocytes from preovulatory follicles in mares. Puncture of follicles that have been exposed through a flank incision resulted in seven oocytes from 17 attempts. Administration ofgonadotropin releasing hormone (GnRH) or human chorionic gonadotropin (HCG) improved oocyte recovery rate in this study (8/16 treated mares vs 3/18 untreated mares). Palmer et al~4 (1986) described a nonsurgical method for oocyte recovery in mares. Follicles were punctured 36 hours after HcG treatment. Sixteen aspirations resulted in recovery of nine oocytes. Six oocytes wemtransferred into oviducts of recipient mares but no pregnancies resulted. Preliminary observations at our laboratory revealed a 39% oocyte recovery rate from preovulatory follicles that were surgically exposed. TMRecently a 73% recovery rate was reported from non-HCG-stimulated follicles when aspiration was facilitated by colpotomy,s Luteal dysfunction following follicular aspiration of preovulatory follicles of primates and humans has been recorded,s,9,~9and decreased progesterone measurements have been incriminated in failure to achieve and maintain pregnancy with human in vitro fertilization and embryo transfer? 144
Luteal function in the mare donating the oocyte is important for two reasons: 1) abnormal luteal function may prevent the donor from returning to estrus for further collection attempts, and 2) heterogenous fertilization requires transferring a donor's ooeyte into the oviduct of a recipient mare which has been bred prior to transfer. In order to assure fertilization of the correct oocyte, the recipient' s oocyte should be removed prior to ovulation (follicular aspiration) or postovulation (oviduct removal). The purpose of this report is to describe: 1) an improved method of oocyte collection, 2) luteal function in the mare following preovulatory follicular fluid aspiration, and 3) experiences with heterogenous and xenogenous fertilization of equine gametes.
MATERIALS AND METHODS Animals Forty-three light-horse mares, kept on a dry lot and fed alfalfa hay (2.5% body weight) and grain (2.5 kg) daily, were used in this study. Procedures Preovulatory follicles were detected by frequent palpationts and ultrasonographic scanning. 13,16Changes in tone and size of the follicle, as well as ultrasonographic characteristics, were used to predict ovulation. Mares were randomly assigned to HCG-treated (n=37) or nontreated (n=26) groups. Human chorionic gonadotropin (3,300 IU) was given intravenously during estrus when a follicle >35 mm was initially detected. Time of follicular aspiration in this group was approximately 36 hours post HCG but timing was still determined by palpation and ultrasonographic criteria. Oocyte Collection Mares were tranquilized and the paralumbar fossa cfipped and aseptically prepared. A trocar and cannula" (outer diameter = 12mm) were placed through the abdominal musculature into the peritoneal cavity. The trocar was removed and the cannula identified per rectum. The preovulatory follicle was positioned per rectum against the tip of the cannula and a double-bore needle (outer diameter 9.8mm) was introduced into the follicle (through the cannula). The preovulatory follicular contents were aspirated (suction = 100 mrnHg) while concurrent flushing occurred by simultaneous infusion of Dulbecco's phosphate buffered saline + 1% fetal calf serum with 2 IU heparin/ml. Follicular fluid and medium were maintained at 37°C and examined for oocytes under a dissecting microscope within 10 minutes of collection. Re=Kad Storz, Endoscopy-AmericaInc, Culver City, CA 90232. EQUINE VETERINARY SCIENCE
REFEREED PAPERS FROM THE IOTH SYMPOSIUM -
covered oocytes were graded for maturity by degree of cumulus expansion (1 = mature; 3 = immature). In all cases, a small quantity of follicular fluid (approximately 20 ec) was retained from the initial aspirate and used to culture recovered oocytes prior to transfer (<1 hour).
Gamete Transfer- Heterogenous Fertilization Fifteen mature oocytes were transferred to individual
recipient mares through a standing flank laparotomy incision into the oviduct ipsilateral to the side of impending ovulation. A fire-polished glass pipette was placed directly into the infundibulum of the oviduct, and a small amount (<0.5 ml) of Dulbecco's phosphate buffered saline plus 10% fetal calf serum was expelled with the oocyte. Each mare was artificially inseminated with 500 million progressively motile spermatozoa from a stallion of known fertility prior to or shordy after (<1 hour) the time of transfer. In all cases, the possibility of fertilization of the recipient's own oocyte was negated by its removal with follicular aspiration, or transfer of the donor oocyte into and later removal from, the oviduct contralateral to the side of ovulation. To assess fertilization rates, ova from recipient mares were recovered by surgical excision and flushing of the oviduct two days after oocyte transfer.
Gamete Transfer - Xenogenous Fertilization Fifteen mature oocytes and spermatozoa (between 10,000-100,000) were transferred into oviducts of estrous rabbits. Rabbits were treated with GnRH (10 lag) at the rime of transfer. Both the uterotubule and fimbrial ends of the oviduct were ligated post-transfer. Spermatozoa were either from a raw ejaculate (n= 12) or centrifuged and resuspended in Ham's F10 (n=3). Embryo Transfer Transfer of early oviductal embryos was into the oviduct of recipient mares, similar to oocyte transfer in heterogenous fertilization experiments. The recipients were matched for stage of cycle (within 24 hours).
~©~©~
o f the corpora lutea determined ultrasonographically; and 3)
cyclic behavior patterns recorded from daily teasing.
RESULTS AND DISCUSSION Oocyte Collection Overall oocyte recovery rate was 71.4% (45/63). HCG treatment did not (P>0.05) improve recovery rate (65% [24/ 37] for treated versus 81% [21/26] for nontreated mares). However, the use of HCG facilitated the scheduling of follicular aspiration as most attempts were made approximately 36 hours post HCG injection (mean=35.4 hours). Of the 45 oocytes recovered, 12 (27%) were fragmented, presumably by shear forces on the cumulus complex where it attached to the zona pellucida. This may have been created by flow dynamics through a small aperture in the collection equipment. The procedure was well tolerated by mares and no complications were noted. Follicular aspiration was attempted on 27 mares one time, 12 mares 2 times and 4 mares 3 times. The technique described here is similar to that of Vogelsang et al2oand is less traumatic than most other procedures reported. ~,1°~Preliminary studies with laparoscopy and percutaneous needle puncture guided by transabdominal ultrasonography demonstrated this current procedure to be more effective. Heterogenous Fertilization Ten oocytes from 15 transferred were recovered, three of which were fertilized (Figure 1). Each fertilized oocyte was transferred into another recipient mare's oviduct that was matched for stage of cycle. Two of the three embryos developed successfully into pregnancies, however one was aborted at 20 days of pregnancy. This represents the first report of successful heterogenous fertilization and subsequent oviductal embryo transfer in the mare. ",~2The recipient mare deliv-
Pregnancy Detection Mares were examined for pregnancy by ultrasonographic examination of the uterus per rectum on Day 11, 12, 14, 20, 35 and 60 post follicular aspiration. Luteal Function Luteal function was assessed by 1) serum progesterone concentrations (by radioimmunoassay) from jugular venous samples collected on Days 3, 5, 8, 11 and 14 postaspiration (treated) or ovulation (controls); 2) morphological description Volume8, Number2, 1988
Figure 1. Equine 8-cell embryo recovered from the oviduct of a recipient mare approximately 48 hours post-transfer of an equine oocyte (embryo size 180 Ixm).
145
REFEREED PAPERS FROM THE IOTH SYMPOSIUM
6
~
=--
A
CTRL
2. 1
I 3
I 5
I 7
I 9
I 11
I 13
I 15
DAY POST OVULATION
Figure 2. Progesterone concentrations (ng/ml) In control mares (A), and mares with positive follicular aspiration (*), and negative follicular aspiration ( , ) .
Figure 3. Abnormal morphology of the corpus luteum approximately 12 hours after follicular aspiration. Hyperechogenic areas represent air.
ered a normal healthy foal without complication in July, 1987. X e n o g e n o u s Fertilization Eight unfertilized equine oocytes of 15 transferred were recovered from rabbit oviducts. It is postulated that failure of fertilization was due to inadequate sw,rm preparation. Insemination of raw equine spermatozoa into rabbit oviducts consistendy resulted in a large oviductal accumulation ofinspis sated material. Possible reasons for this are introduction of bacteria or an allergic response to foreign seminal plasma. Because of this phenomena, three oocytes were transferred into rabbit oviducts with centrifuged equine spermatozoa resuspended in Ham's F10. This resulted in recovery of all three oocytes in only a small amount of clear fluid. Regardless, fertilization did not occur and at this time it does not appear feasible to attempt xenogenous fertilization until methods of sperm treatment are improved. 146
Figure 4. Abnormal morphology of the corpus luteum approximately 48 hours after follicular aspiration. Centrally located nonechogenic areas most likely represent flush media and/or blood. Bright echogenic reflections represent air. Luteal Function Presented in Figure 2 are the mean progesterone concentrations for control and mares undergoing follicular aspiration at 3,5,8,11 and 14 days postaspiration or ovulation. Following attempts at oocyte collection, progesterone concentrations were significantly lower on Day 3 (P<0.05) and Day 5 (P<0.10) compared to control values, however the level of significance between the two groups decreased with increasing time postaspiration. Large numbers of granulosa cells recovered within the "flush" of primate follicles were closely correlated (R=0.86) with lowered progesterone secretion.9 Therefore vigorous flushing techniques are likely to increase chances of inducing hteal dysfunction. In the present study, the equine follicle was thoroughly flushed with large volumes of fluid to maximize oocyte recovery, thus a large loss of granulosa cells was anticipated. Although spontaneous luteal dysfunction cannot be eliminated, it is likely that thorough evacuation of the antral cavity with concomitant loss of granulosa cells was the direct cause of reduced progesterone concentrations. The difference in concentrations of progesterone between control mares and mares from which an oocyte aspiration was attempted was less when an oocyte was recovered. Failure to recover an oocyte may reflect relative immaturity of the follicle. Premature aspiration of follicular contents may increase removal of granulosa cells, alter granulosa cell environment, or remove cells which might function as the principal source of progesterone secretion in the early luteal phase, 9 and thus result in an apparent delay relative to the time of increasing progesterone measurements. Ultrasonographic morphologic abnormalities of corpora h t e a were noted in 33% of the mares in which follicular aspirations were performed versus none for controls. Morphologic changes upon ultrasonography included: 1) centrally located, non-echogenic areas (probably flush media or blood); EQUINE VETERINARY SCIENCE
REFEREED PAPERS FROM THE IOTH SYMPOSIUM 2) a bright, central echogenic ring within the luteal structure; 3) echogenic reflections (air) (Figure 3); or 4) a combination of these changes (Figure 4). N o r m a l appearing luteal tissue was usually present surrounding areas of detected morphologic changes. Presence o f the abnormalities observed with ultrasonography d i d not necessarily correlate with r e d u c e d luteal function. A l l mares proceeded to cycle normally postaspiration. Mares n o t given prostaglandin F2-alpha had a normal duration o f diestrus (mean=17 days). Mares short cycled with prostaglandins on D a y 8 or 9 o f diestrus returned to estrus on an average of 4.5 days. Although the majority o f m a r e s eventually d i s p l a y e d adequate progesterone concentrations after follicular aspiration, an initial delay and reduction in progesterone secretion was observed. Parameters evaluated in this experiment suggest oocyte donors could be used subsequently as either oocyte or embryo recipients, however an increased incidence o f luteal malfunction m a y be expected. Progesterone measurement and supplementation m a y be a means to alleviate this problem.
CONCLUSIONS Results from these studies suggest that 1) a reliable method for collection o f in vivo matured oocytes has been established, and 2) heterogenous fertilization is a technique that with refinement should be immediately applicable to obtain foals from valuable infertile mares that fail to g e t pregnant or produce embryos by standard methods.
REFERENCES 1. Brackett BG, Bousquet D, Boice ML, Donawick WV, Evans JF, DresselMA: Normal developmentfollowing in vitro fertilization in the cow. BiolRepro 27:147-158, 1982. 2. Cheng WTK, Moor RM, Polge CE: In vitro fertilization of pigs and sheep oocytes matured in vivo and in vitro. Theriogenology 25:146, 1986.(abstr). 3. Eppig JJ, Schroeder AC; Culture systems for mammalian oocyte
Volume 8, Number 2, 1988
development: progress and prospects. Theriogenology 25:97-106, 1986. 4. EyestoneWH, First N-L:A study of the 8-16 cell developmentblock in bovine embryos cultured in vitro. Theriogenology 25:152, 1986 (abstr). 5. Hinrichs K, Kenney RM: A eolpotomy procedure to increase oocyte recovery rates on aspiration of equine preovulatory follicles. Theriogenology 27:237, 1987 (abstr). 6. Imel KJ, Squires EL, Elsden RP, Shideler RK: Collection and transfer of embryos. J Am Vet Med Assoc 179:978-981, 1981. 7. Imel KJ: Recovery, Culture and Transfer of Equine Embryos. MS Thesis, Colorado State University, Fort Collins, CO 80523, 1981. 8. Kreitmann O, Nixon WE, Hodgen GD: Induced corpus luteum dysfunction after aspiration of the preovulatory follicle in monkeys. Fertil Steril 35:6714575, 1981. 9. Kreitmann O, Nixon WE, Hodgen GD: Ovum collection and induced luteal dysfunction in primates. In: Fertilization and Embryonic Development In Vitro. Mastroianni L, Jr and BiggersJD (eds), Plenum Press, New York, pp27-39, 1981. 10. McKinnon AO, Wheeler MB, Carnevale F_aM,Squires EL: Oocyte transfer in the mare: preliminary observations.J Eq Vet Sci 6:306-309,1987. 11. McKinnon AO, Squires EL, Camevale EM, Voss JL, Seidel GE Jr. In vitro fertilization, in vivo fertilization and embryo transfer in the horse: future prospects. Proc 48th Ann Conf Vet, Colorado Veterinary Medical Association pp 57-86, 1987. 12. McKinnon AO, Squires EL, Camevale EM, Voss JL, Seidel GE Jr: Heterogenous and xenogenous fertilization of equine oocytes. Proc Vth World Conf on In Vitro Fertilization and Embryo Transfer, Norfolk, VA, p 66, 1987. 13. McKinnon AO, Squires EL, Voss JL: Ultrasonic evaluation of the mare's reproductive tract-Part I. Compend Contin Educ 9:336-345, 1987. 14. Palmer E, Duchamp G, Bezard J, Megastrini M, King A, Bousquet D, Betteridge K: Recovery of follicular fluid and oocytes of mares by nonsurgical puncture of preovulatory follicles. Theriogenology 25:178, 1986 (abstr). 15. Parker WA: Sequential changes of the ovulating follicle in the estrous mare as determined by rectal palpation. Proc Annu ConfCollege Vet Med, Colorado State University, Fort Collins, Co, 1981. 16. PeirsonRA, Ginther OJ: Ultrasonic evaluation of the preovulatory follicle in the mare.Theriogenology 24:359-368, 1985. 17. Sirard MA, Lambert RD, Menard DP and Bedoya M: In vitro fertilization in the cow: six calves are born from surgical or nonsurgical uterine transfer to heifers. Theriogenology 25:198, 1986 (abstr). 18. SquiresEL, Cook VM, Voss JL: Collection and transfer of equine embryos. Anim Repro Lab Bull #01, Animal Reproduction Laboratory, Fort Collins, Co, 80523, 1984. 19. Stepto¢PC, Edwards RG: Laparoscopic recovery of preovulatory human oocytes after priming of ovaries with gonadotropins. Lancet 1:683689, 1970. 20. Vogelsang MM, Kraemer DC, Bowen MJ, Potter GD: Recovery of equine follicular oocytes by surgical and nonsurgical techniques. Theriogenology 25:208, 1986. (abstr).
147
9. Lauderdale JW, Miller PA: Regulation of reproduction in mares with prostaglandins. In: Proceedings of the 21st Ann Cony of the Amer Assoc of Equine Pract Milne FJ (ed), pp 235-244, 1975. 10. Ley WB, Purswell BP, Bowen JM: The effects of prostalene and alfaprostol as uterine myotonics, and the effect on postpartum pregnancy rate in the mare following daily treatment with prostalene. Theriogenology On press) 1988. 11. Loy RG: Characteristics of post-paaum reproduction in mares. Vet Clin No Amer: Large Anim Pract 2:345-359, 1980. 12. Neely DP: Hormone Therapy in Mares. In: Equine Reproduction Neely, Liu, Hillman (eds), Vet Learning Systems Co, Inc., pp 24-35, 1983. 13. Peterson DE: Gonadotrophin releasing effects of alfaprostol in seasonally anestrous/estrous mares. J Equine Vet Sci 5:331-335, 1984. 14. Taverae MAId: Uterine motility in the post-partum female. 20th lnt'L Congress on Anita Reprod and A. I., Univ of Illinois, UrbanaChampaign, XI, ppl-8, 1984. 15. ThrelfaU WR, Carleton CL: Treatment of uterine infections in the mare. In: Current Therapy in Theriogenology, Morrow DA, (ed), ed. 2, WB Satmders Co, Saunders Co, Philadelphia, pp 730-737, 1986. 16. Vandeplassche M, Bouters R, SpincemaiUe J, Bonte P, Coryn M: Observations on involution and puerperal endometritis in mares. Irish Vet J 37:126-132, 1983.
HETEROGENOUS AND XENOGENOUS FERTILIZATION OF IN VIVO MATURED EQUINE OOCYTES A.O. McKinnon, BVSc, MSc, E.M. Carnevale, DVM, E.L. Squires, PhD, J.L. Voss, DVM, MS, G.E. Seidel Jr., PhD
SUMMARY Forty-five in vivo matured equine oocytes were recovered from 63 follicular aspiration attempts (71.4%). HCG did not improve recovery rate (65% - 24/37 for treated vs 81% 21/26 for nontreated mares). Fifteen oocytes were transferred into the oviduct of inseminated recipient mares (heterogenous fertilization) and 15 oocytes plus equine spermatozoa were transferred into rabbit oviducts (xenogenous fertilization). Ten oocytes (3 fertilized) were recovered from recipient mare oviducts following removal and flushing two days after transfer. Eight oocytes (nonfertilized) were recovered from rabbit oviducts. Oviductal transfer into separate recipient mares of three embryos produced from heterogenous fertilization resulted in two pregnancies. One mare produced a normal live foal and the other mare aborted at 20 days of gestation. Results from these studies suggest that: 1) a reliable method for collection of in vivo matured oocytes has been established, and 2) heterogenous fertilization is a technique that with refinement should be immediately applicable to obtain foals from Author's address: College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado 80523. Acknowledgement: Supported in part by the CSU Experiment Station and the Van Camp Foundation.
Volume 8, Number 2, 1988
valuable infertile mares that fail to get pregnant or produce embryos by standard methods.
INTRODUCTION Equine embryo transfer 6,18has been widely accepted and has resulted in foals from mares with a history of previous pregnancy failure. Unfortunately, embryo recovery rates from infertile mares, often older mares with the greatest genetic potential, were low (<30%) 6,7 compared to normal mares
(80%).TM Possible reasons for decreased embryo recovery from infertile mares are: 1) failure of spermatozoal transport through the uterotubaljunction and into the oviduct; 2) failure of fertilization; 3) failure of release of the fertilized egg from the oviduct into the uterus; 4) failure of the early embryo to develop because of a functionally incompetent oviduct or uterus; 5) increased early embryonic death due to genetic or other abnormalities of embryos~ from mares approaching senility; and 6) ovulation failure. Collection of oocytes prior to ovulation and fertilizing them in vivo or in vitro may be the only means of obtaining foals from some infertile mares. Three possible approaches to fertilizing these oocytes are: 1) heterogenous fertilization 143
8@¢i®IIy REFEREED PAPERS FROM THE 10TH SYMPOSIUM
fertilization in a recipient mare's oviduct; 2) xenogenous fertilization - fertilization in another species' oviduct (e.g. rabbit or sheep); and 3) in vitro fertilization (IVF) - fertilization outside the animal under controlled laboratory conditions. Transfer of gametes (oocytes and spermatozoa) to a recipient mare's oviduct is most attractive because: 1) it obviates need of sophisticated culture requirements for in vitro fertilization; 2) when oocytes are fertilized, the embryo can be maintained in the recipient mare without further manipulation; and 3) it is more physiological than both IVF and xenogenous fertilization and accordingly should be more acceptable to breed registries. Production of calves from in vitro fertilization was first reported in 19827 Since that time, numerous studies have been conducted on systems for in vitro fertilization of mammalian oocytes.3 Production of live piglets and lambs following in vitro fertilization of oocytes matured in vivo or in vitro has been reported recently.2 When fertilization and maturation of bovine oocytes has occurred in vitro, one major obstacle to embryo development is a block occurring at the 16-cell stage? This block does not occur with in vitro fertilized oocytes when resulting embryos are cultured in vivo. Cost and difficulty of obtaining equine oocytes for in vitro fertilization and maturation studies, together with the higher success rate of early embryonic development in vivo17 suggest heterogenous fertilization in the equine is the method of choice for future development. There are only a few reports on obtaining oocytes from donor mares. Vogelsang et al= attempted two methods of aspirating oocytes from preovulatory follicles in mares. Puncture of follicles that have been exposed through a flank incision resulted in seven oocytes from 17 attempts. Administration ofgonadotropin releasing hormone (GnRH) or human chorionic gonadotropin (HCG) improved oocyte recovery rate in this study (8/16 treated mares vs 3/18 untreated mares). Palmer et al~4 (1986) described a nonsurgical method for oocyte recovery in mares. Follicles were punctured 36 hours after HcG treatment. Sixteen aspirations resulted in recovery of nine oocytes. Six oocytes wemtransferred into oviducts of recipient mares but no pregnancies resulted. Preliminary observations at our laboratory revealed a 39% oocyte recovery rate from preovulatory follicles that were surgically exposed. TMRecently a 73% recovery rate was reported from non-HCG-stimulated follicles when aspiration was facilitated by colpotomy,s Luteal dysfunction following follicular aspiration of preovulatory follicles of primates and humans has been recorded,s,9,~9and decreased progesterone measurements have been incriminated in failure to achieve and maintain pregnancy with human in vitro fertilization and embryo transfer? 144
Luteal function in the mare donating the oocyte is important for two reasons: 1) abnormal luteal function may prevent the donor from returning to estrus for further collection attempts, and 2) heterogenous fertilization requires transferring a donor's ooeyte into the oviduct of a recipient mare which has been bred prior to transfer. In order to assure fertilization of the correct oocyte, the recipient' s oocyte should be removed prior to ovulation (follicular aspiration) or postovulation (oviduct removal). The purpose of this report is to describe: 1) an improved method of oocyte collection, 2) luteal function in the mare following preovulatory follicular fluid aspiration, and 3) experiences with heterogenous and xenogenous fertilization of equine gametes.
MATERIALS AND METHODS Animals Forty-three light-horse mares, kept on a dry lot and fed alfalfa hay (2.5% body weight) and grain (2.5 kg) daily, were used in this study. Procedures Preovulatory follicles were detected by frequent palpationts and ultrasonographic scanning. 13,16Changes in tone and size of the follicle, as well as ultrasonographic characteristics, were used to predict ovulation. Mares were randomly assigned to HCG-treated (n=37) or nontreated (n=26) groups. Human chorionic gonadotropin (3,300 IU) was given intravenously during estrus when a follicle >35 mm was initially detected. Time of follicular aspiration in this group was approximately 36 hours post HCG but timing was still determined by palpation and ultrasonographic criteria. Oocyte Collection Mares were tranquilized and the paralumbar fossa cfipped and aseptically prepared. A trocar and cannula" (outer diameter = 12mm) were placed through the abdominal musculature into the peritoneal cavity. The trocar was removed and the cannula identified per rectum. The preovulatory follicle was positioned per rectum against the tip of the cannula and a double-bore needle (outer diameter 9.8mm) was introduced into the follicle (through the cannula). The preovulatory follicular contents were aspirated (suction = 100 mrnHg) while concurrent flushing occurred by simultaneous infusion of Dulbecco's phosphate buffered saline + 1% fetal calf serum with 2 IU heparin/ml. Follicular fluid and medium were maintained at 37°C and examined for oocytes under a dissecting microscope within 10 minutes of collection. Re=Kad Storz, Endoscopy-AmericaInc, Culver City, CA 90232. EQUINE VETERINARY SCIENCE
REFEREED PAPERS FROM THE IOTH SYMPOSIUM -
covered oocytes were graded for maturity by degree of cumulus expansion (1 = mature; 3 = immature). In all cases, a small quantity of follicular fluid (approximately 20 ec) was retained from the initial aspirate and used to culture recovered oocytes prior to transfer (<1 hour).
Gamete Transfer- Heterogenous Fertilization Fifteen mature oocytes were transferred to individual
recipient mares through a standing flank laparotomy incision into the oviduct ipsilateral to the side of impending ovulation. A fire-polished glass pipette was placed directly into the infundibulum of the oviduct, and a small amount (<0.5 ml) of Dulbecco's phosphate buffered saline plus 10% fetal calf serum was expelled with the oocyte. Each mare was artificially inseminated with 500 million progressively motile spermatozoa from a stallion of known fertility prior to or shordy after (<1 hour) the time of transfer. In all cases, the possibility of fertilization of the recipient's own oocyte was negated by its removal with follicular aspiration, or transfer of the donor oocyte into and later removal from, the oviduct contralateral to the side of ovulation. To assess fertilization rates, ova from recipient mares were recovered by surgical excision and flushing of the oviduct two days after oocyte transfer.
Gamete Transfer - Xenogenous Fertilization Fifteen mature oocytes and spermatozoa (between 10,000-100,000) were transferred into oviducts of estrous rabbits. Rabbits were treated with GnRH (10 lag) at the rime of transfer. Both the uterotubule and fimbrial ends of the oviduct were ligated post-transfer. Spermatozoa were either from a raw ejaculate (n= 12) or centrifuged and resuspended in Ham's F10 (n=3). Embryo Transfer Transfer of early oviductal embryos was into the oviduct of recipient mares, similar to oocyte transfer in heterogenous fertilization experiments. The recipients were matched for stage of cycle (within 24 hours).
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o f the corpora lutea determined ultrasonographically; and 3)
cyclic behavior patterns recorded from daily teasing.
RESULTS AND DISCUSSION Oocyte Collection Overall oocyte recovery rate was 71.4% (45/63). HCG treatment did not (P>0.05) improve recovery rate (65% [24/ 37] for treated versus 81% [21/26] for nontreated mares). However, the use of HCG facilitated the scheduling of follicular aspiration as most attempts were made approximately 36 hours post HCG injection (mean=35.4 hours). Of the 45 oocytes recovered, 12 (27%) were fragmented, presumably by shear forces on the cumulus complex where it attached to the zona pellucida. This may have been created by flow dynamics through a small aperture in the collection equipment. The procedure was well tolerated by mares and no complications were noted. Follicular aspiration was attempted on 27 mares one time, 12 mares 2 times and 4 mares 3 times. The technique described here is similar to that of Vogelsang et al2oand is less traumatic than most other procedures reported. ~,1°~Preliminary studies with laparoscopy and percutaneous needle puncture guided by transabdominal ultrasonography demonstrated this current procedure to be more effective. Heterogenous Fertilization Ten oocytes from 15 transferred were recovered, three of which were fertilized (Figure 1). Each fertilized oocyte was transferred into another recipient mare's oviduct that was matched for stage of cycle. Two of the three embryos developed successfully into pregnancies, however one was aborted at 20 days of pregnancy. This represents the first report of successful heterogenous fertilization and subsequent oviductal embryo transfer in the mare. ",~2The recipient mare deliv-
Pregnancy Detection Mares were examined for pregnancy by ultrasonographic examination of the uterus per rectum on Day 11, 12, 14, 20, 35 and 60 post follicular aspiration. Luteal Function Luteal function was assessed by 1) serum progesterone concentrations (by radioimmunoassay) from jugular venous samples collected on Days 3, 5, 8, 11 and 14 postaspiration (treated) or ovulation (controls); 2) morphological description Volume8, Number2, 1988
Figure 1. Equine 8-cell embryo recovered from the oviduct of a recipient mare approximately 48 hours post-transfer of an equine oocyte (embryo size 180 Ixm).
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Figure 2. Progesterone concentrations (ng/ml) In control mares (A), and mares with positive follicular aspiration (*), and negative follicular aspiration ( , ) .
Figure 3. Abnormal morphology of the corpus luteum approximately 12 hours after follicular aspiration. Hyperechogenic areas represent air.
ered a normal healthy foal without complication in July, 1987. X e n o g e n o u s Fertilization Eight unfertilized equine oocytes of 15 transferred were recovered from rabbit oviducts. It is postulated that failure of fertilization was due to inadequate sw,rm preparation. Insemination of raw equine spermatozoa into rabbit oviducts consistendy resulted in a large oviductal accumulation ofinspis sated material. Possible reasons for this are introduction of bacteria or an allergic response to foreign seminal plasma. Because of this phenomena, three oocytes were transferred into rabbit oviducts with centrifuged equine spermatozoa resuspended in Ham's F10. This resulted in recovery of all three oocytes in only a small amount of clear fluid. Regardless, fertilization did not occur and at this time it does not appear feasible to attempt xenogenous fertilization until methods of sperm treatment are improved. 146
Figure 4. Abnormal morphology of the corpus luteum approximately 48 hours after follicular aspiration. Centrally located nonechogenic areas most likely represent flush media and/or blood. Bright echogenic reflections represent air. Luteal Function Presented in Figure 2 are the mean progesterone concentrations for control and mares undergoing follicular aspiration at 3,5,8,11 and 14 days postaspiration or ovulation. Following attempts at oocyte collection, progesterone concentrations were significantly lower on Day 3 (P<0.05) and Day 5 (P<0.10) compared to control values, however the level of significance between the two groups decreased with increasing time postaspiration. Large numbers of granulosa cells recovered within the "flush" of primate follicles were closely correlated (R=0.86) with lowered progesterone secretion.9 Therefore vigorous flushing techniques are likely to increase chances of inducing hteal dysfunction. In the present study, the equine follicle was thoroughly flushed with large volumes of fluid to maximize oocyte recovery, thus a large loss of granulosa cells was anticipated. Although spontaneous luteal dysfunction cannot be eliminated, it is likely that thorough evacuation of the antral cavity with concomitant loss of granulosa cells was the direct cause of reduced progesterone concentrations. The difference in concentrations of progesterone between control mares and mares from which an oocyte aspiration was attempted was less when an oocyte was recovered. Failure to recover an oocyte may reflect relative immaturity of the follicle. Premature aspiration of follicular contents may increase removal of granulosa cells, alter granulosa cell environment, or remove cells which might function as the principal source of progesterone secretion in the early luteal phase, 9 and thus result in an apparent delay relative to the time of increasing progesterone measurements. Ultrasonographic morphologic abnormalities of corpora h t e a were noted in 33% of the mares in which follicular aspirations were performed versus none for controls. Morphologic changes upon ultrasonography included: 1) centrally located, non-echogenic areas (probably flush media or blood); EQUINE VETERINARY SCIENCE
REFEREED PAPERS FROM THE IOTH SYMPOSIUM 2) a bright, central echogenic ring within the luteal structure; 3) echogenic reflections (air) (Figure 3); or 4) a combination of these changes (Figure 4). N o r m a l appearing luteal tissue was usually present surrounding areas of detected morphologic changes. Presence o f the abnormalities observed with ultrasonography d i d not necessarily correlate with r e d u c e d luteal function. A l l mares proceeded to cycle normally postaspiration. Mares n o t given prostaglandin F2-alpha had a normal duration o f diestrus (mean=17 days). Mares short cycled with prostaglandins on D a y 8 or 9 o f diestrus returned to estrus on an average of 4.5 days. Although the majority o f m a r e s eventually d i s p l a y e d adequate progesterone concentrations after follicular aspiration, an initial delay and reduction in progesterone secretion was observed. Parameters evaluated in this experiment suggest oocyte donors could be used subsequently as either oocyte or embryo recipients, however an increased incidence o f luteal malfunction m a y be expected. Progesterone measurement and supplementation m a y be a means to alleviate this problem.
CONCLUSIONS Results from these studies suggest that 1) a reliable method for collection o f in vivo matured oocytes has been established, and 2) heterogenous fertilization is a technique that with refinement should be immediately applicable to obtain foals from valuable infertile mares that fail to g e t pregnant or produce embryos by standard methods.
REFERENCES 1. Brackett BG, Bousquet D, Boice ML, Donawick WV, Evans JF, DresselMA: Normal developmentfollowing in vitro fertilization in the cow. BiolRepro 27:147-158, 1982. 2. Cheng WTK, Moor RM, Polge CE: In vitro fertilization of pigs and sheep oocytes matured in vivo and in vitro. Theriogenology 25:146, 1986.(abstr). 3. Eppig JJ, Schroeder AC; Culture systems for mammalian oocyte
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development: progress and prospects. Theriogenology 25:97-106, 1986. 4. EyestoneWH, First N-L:A study of the 8-16 cell developmentblock in bovine embryos cultured in vitro. Theriogenology 25:152, 1986 (abstr). 5. Hinrichs K, Kenney RM: A eolpotomy procedure to increase oocyte recovery rates on aspiration of equine preovulatory follicles. Theriogenology 27:237, 1987 (abstr). 6. Imel KJ, Squires EL, Elsden RP, Shideler RK: Collection and transfer of embryos. J Am Vet Med Assoc 179:978-981, 1981. 7. Imel KJ: Recovery, Culture and Transfer of Equine Embryos. MS Thesis, Colorado State University, Fort Collins, CO 80523, 1981. 8. Kreitmann O, Nixon WE, Hodgen GD: Induced corpus luteum dysfunction after aspiration of the preovulatory follicle in monkeys. Fertil Steril 35:6714575, 1981. 9. Kreitmann O, Nixon WE, Hodgen GD: Ovum collection and induced luteal dysfunction in primates. In: Fertilization and Embryonic Development In Vitro. Mastroianni L, Jr and BiggersJD (eds), Plenum Press, New York, pp27-39, 1981. 10. McKinnon AO, Wheeler MB, Carnevale F_aM,Squires EL: Oocyte transfer in the mare: preliminary observations.J Eq Vet Sci 6:306-309,1987. 11. McKinnon AO, Squires EL, Camevale EM, Voss JL, Seidel GE Jr. In vitro fertilization, in vivo fertilization and embryo transfer in the horse: future prospects. Proc 48th Ann Conf Vet, Colorado Veterinary Medical Association pp 57-86, 1987. 12. McKinnon AO, Squires EL, Camevale EM, Voss JL, Seidel GE Jr: Heterogenous and xenogenous fertilization of equine oocytes. Proc Vth World Conf on In Vitro Fertilization and Embryo Transfer, Norfolk, VA, p 66, 1987. 13. McKinnon AO, Squires EL, Voss JL: Ultrasonic evaluation of the mare's reproductive tract-Part I. Compend Contin Educ 9:336-345, 1987. 14. Palmer E, Duchamp G, Bezard J, Megastrini M, King A, Bousquet D, Betteridge K: Recovery of follicular fluid and oocytes of mares by nonsurgical puncture of preovulatory follicles. Theriogenology 25:178, 1986 (abstr). 15. Parker WA: Sequential changes of the ovulating follicle in the estrous mare as determined by rectal palpation. Proc Annu ConfCollege Vet Med, Colorado State University, Fort Collins, Co, 1981. 16. PeirsonRA, Ginther OJ: Ultrasonic evaluation of the preovulatory follicle in the mare.Theriogenology 24:359-368, 1985. 17. Sirard MA, Lambert RD, Menard DP and Bedoya M: In vitro fertilization in the cow: six calves are born from surgical or nonsurgical uterine transfer to heifers. Theriogenology 25:198, 1986 (abstr). 18. SquiresEL, Cook VM, Voss JL: Collection and transfer of equine embryos. Anim Repro Lab Bull #01, Animal Reproduction Laboratory, Fort Collins, Co, 80523, 1984. 19. Stepto¢PC, Edwards RG: Laparoscopic recovery of preovulatory human oocytes after priming of ovaries with gonadotropins. Lancet 1:683689, 1970. 20. Vogelsang MM, Kraemer DC, Bowen MJ, Potter GD: Recovery of equine follicular oocytes by surgical and nonsurgical techniques. Theriogenology 25:208, 1986. (abstr).
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