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Gamete Intrafallopian Transfer
REPRODUCT1VE TECHNOLOGY
0749- 0739/96 $0.00 + .20
GAMETE INTRAFALLOPIAN TRANSFER Elaine M. Carnevale, DVM, MS, PhD
Since the first successful equine embryo collections and transfers were reported in 1972/· 2H embryo transfer has been accepted by many breed registries within the horse industry. One of the predominant uses of embryo transfer has been to obtain offspring from valuable mares that are incapable of maintaining a pregnancy to term. However, the collection of embryos for transfer has been more successful in fertile than subfertile mares (e.g., 80 'X, vs 28°;;), respectively)35 and in recently foaling than sub fertile mares (53% vs 29%).'7 Failure to collect embryos from subfertile mares could be the result of altered gamete or embryo transport, failure or defects of fertilization, or early embryo death. Potentially, fertility could be enhanced by collecting the donor's oocyte before ovulation, thus preventing the oocyte from contacting an inadequate or deleterious tubular genitalia. Gamete intrafallopian transfer (GIFT) involves placement of a donor's oocyte into a recipient's oviduct. Fertilization and embryo development occur within the reproductive tract of the recipient. GIFT has been successful in the human,' bovid,16 equid,23 and porcid." 1 In women, oocytes and spermatozoa are placed into the oviduct during GIFT. In the mare, spermatozoa were provided by natural cover or artificial insemination of recipients and only the oocyte has been transferred into the recipient's oviduct. Currently, GIFT has been used in equine research programs, but further refinement should make GIFT a viable procedure for commercial use.
From the Depa rtment of Anima l Science, Food, and Nutrition, College of Agriculture, Southern Illinois University, Carbondale, Illinois
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INDICATIONS FOR GIFT
Mares from which viable embryos cannot be obtained for transfer are potential oocyte donors for GIFT. A number of factors could affect embryo collection rates or embryo viability before the time that the embryo would be recovered from the uterus (days 6 to 8 after ovulation). Ovulatory Failure
Two types of potential ovulatory failure have been described in mares. Hemorrhagic follicles (HF)1 2 occurred more frequently in older mares and during autumn. 7 The follicles do not seem to ovulate and enlarge to diameters of more than 70 mm as blood enters the antrum. When imaged by ultrasonography, fibrin strands or floating debris are often present within the antrum. A second type of apparent ovulatory failure has been reported to occur more often in older mares than in younger mares (2"20 years, 10 of 68; 15-19 years, 4 of 78; 5-7 years, 1 of 91 atypical ovulations per total ovulations).9 In contrast to HF, diameter of the follicle does not increase. The preovulatory follicle becomes flaccid and demonstrates ultrasonographic characteristics associated with imminent ovulation,?· 25 including an irregular shape and an echogenic and thickened border. A central area of nonechogenic fluid remains in the follicle even after luteinization occurs. Ovulatory sites with similar characteristics were described previously as luteinized, unruptured follicles. 25 Aspiration of contents from similar structures resulted in the recovery of mature or atretic oocytes and bloody fluid ? Limited attempts to obtain pregnancies or recover oviductal embryos from mares with follicles that appeared to luteinize without rupturing have been unsuccessfuJ.7,25 Mares that have abnormal ovulations during successive cycles could provide oocytes for GIFT. However, the oocyte must be removed from the follicle before contamination of follicular fluid with blood and before the oocyte ages. The decision to collect an oocyte should be based on a history of repeated abnormal ovulations imaged by ultrasound. After an atypical appearance to the follicle has been imaged, the oocyte has probably undergone degenerative changes. If abnormal follicles are due to hormonal alterations, viability of the oocyte could be compromised. Oocyte Pickup
After ovulation, the viscous cumulus complex surrounding the oocyte contacts the fimbriae and is transported to the ampulla, partly, through ciliary activity and peristalsis. Ability of the infundibulum to collect and transport oocytes could be altered by infundibular adhesions or infundibulitis, which were found to be more frequent in older mares than in younger maresY Collection rates of recentl y ovulated oocytes or oviductal embryos per ovulation were significantly lower in older mares
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than in younger mares on day 1.5 (day 0 = day of ovulation; 4-7 years, 17 of 18; 15-19 years, 10 of 14; =2:20 years, 11 of 20) and on day 3 (2-9 years, 9 of 9 and =2:20 years, 6 of 9). J() Ball et al 4 also recovered fewer oocytes or embryos from oviducts of sub fertile, aged (mean age, 19 years) mares than normal, young (mean age, 6 years) mares (24 of 41 and 28 of 30, respectively). The findings suggest that failure of oocytes to gain entry into the oviduct could account for some cases of subfertility, especially in older mares. Oviductal Pathology
Obstruction of the oviductal lumen would prevent passage of spermatozoa and the embryo. Various anomalies and lesions of mares' oviducts recently have been reviewed 1H; however, their incidence is low. Globular masses, composed of type I collagen, frequently are found in the oviducts of mares. 21 Masses were recovered more often from oviducts of older (8-26 years) mares than younger (2-7 years) mares (13 of 22 vs 5 of 21, respectively). In another study,36 masses were found in 16 of 22 (67%) oviducts from mares of different ages (2-22 years). The collagenous masses occupied and distended the entire luminal diameter in a small number of mares (3 of 43), potentially occluding the lumen and resulting in delayed or interrupted oviductal transport of the oocyte or embryo.21 Surgical procedures have been developed to flush oviducts 39 with the intention of removing blockages. However, the importance of oviductal masses in reducing fertility has not been proven. Developing embryos remain in the oviduct for 5 or 6 days before entering the uterus. Oviducts must provide an environment suitable for gamete survival and transport, fertilization, and early embryo development. An inadequate or deleterious oviductal milieu could have harmful effects on gametes and embryos. Despite the oviduct's importance during early pregnancy, minimal clinical attention or research has been devoted to studying oviductal function and pathology. Findings from slaughterhouse studies suggest a role of oviductal pathology in reduced fertility. Inflammatory changes within the oviduct occurred twice as often in mares with endometritis than mares without endometritis,!'\ and lymphatic infiltration was more frequent in the infundibular-ampullar region of nonpregnant than pregnant mares."3 The infiltration of inflammatory cells could have a d etrimental effect on fertility; however, sufficient data have not been collected to directly correlate oviductal inflammation with pregnancy failure. Uterine Pathology
Historically, the uterus has been considered the primary cause of reproductive failure in mares. Increasing inflammatory changes, detected with uterine histology, were associated with a concomitant de-
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crease in fertilityY When murine embryos were cultured in uterine flushings from mares with different histological scores for inflammation,14 the quantity of lymphocyte infiltration was inversely proportional to the rate of embryo development. Intrauterine fluid collections imaged with ultrasonography represent an inflammatory process. Diestrous fluid collections were associated with lower pregnancy rates and higher embryo loss rates. 1 Motility was suppressed immediately when spermatozoa were cultured with fluid from intrauterine collections."4 Exposure of spermatozoa or embryos to inflammatory products of the uterus was detrimental to their survival. Using GIFT, the effects of inflammatory or infectious problems in the donor's uterus would be eliminated.
Miscellaneous Pathology
Miscellaneous reproductive pathology, such as cervical lacerations or urine pooling, can prevent pregnancy or the successful collection of embryos. Mares in which surgical or medical treatments are unsuccessful would be acceptable oocyte donors. In humans, GIFT has been used to treat immunologic infertilities. 22 Antisperm antibodies have been produced experimentally in mares. 20 However, the importance of antisperm antibodies in equine reproduction has not been established. Problems associated with the production of antibodies to spermatozoa by the donor would be eliminated using GIFT.
TRANSFER OF OOCYTES Oocyte Donors
Mares from which pregnancies have not been obtained after embryo transfer are potential oocyte donors. Oocyte collection techniques are more invasive than nonsurgical embryo flushes, because the abdominal cavity is penetrated. Bracher et al 6 monitored mares after repeated transvaginal ultrasound-guided follicular aspirations during a period of 3 months. The procedure was well tolerated by mares, and no clinical side effects were observed. One mare, slaughtered 6 weeks after the last follicular puncture, had fine serosal adhesions on the ovarian surface and small hemorrhagic areas within the ovarian stroma. Data have not been collected to determine if repeated follicular aspirations impair fertility in normal mares. Requirements for oocyte donors are minimal. Donors must have regular estrous cycles and viable oocytes. The uterus should be cultured to identify potential pathogens and treated as indicated. Because a needle is punctured through the vaginal wall during transvaginal oocyte aspirations, microbes could potentially be introduced into the abdomen. After oocyte collections, prostaglandins are used to short cycle the donor
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once the corpus luteum has matured (approximately 6 days after expected ovulation date or 8 days after hCG administration). Recipients
Selection of suitable recipients is imperative to the success of GIFT. Reproductive tracts of oocyte recipients must provide the proper milieu for sperm survival and transport, fertilization, and embryo development. Because recipients will be inseminated or bred, often at the approximate time of ovulation, they must be resistant to uterine infections. Recipient requirements were previously described for embryo transfer. 2-1 Adequate exposure of oviducts during standing flank laparotomies can be difficult, but problems can be minimized through the careful selection of recipients. Exposure of the oviduct is easier in mares with no excess fat and with a longer coupling (distance between the last rib and tuber cocci). Mares that have had at least one foal are preferred to maidens, because additional laxity of the broad ligament will facilitate oviductal exposure. A recipient's suitability for transfer can be evaluated by grasping the ovary per rectum and manually moving it toward the flank. Laxity of the broad ligament and ovarian mobility can be evaluated, and the most advantageous placement of an incision site can be determined. Synchronization of Donors and Recipients
A critical window for synchronization between donors and recipients has not been established. Oocytes have been transferred between 44 to 48 hours after the administration of hCG;il between the third day of estrus and just before ovulation,,2 or within 24 hours of ovulation." Because the recipient will be inseminated, the oocyte from the recipient's dominant follicle must be removed to prevent fertilization. Removal of the recipient's oocyte can be accomplished by transvaginal or flank aspiration before GIFT. Aspiration of the follicle also can be attempted at the time of transfer; however, the preovulatory follicle is often large and flaccid and may rupture before exposure. Collapse of the recipient's follicle does not assure that the recipient's oocyte will not gain access to the oviduct and result in fertilization and embryo development. 2h Oocyte Evaluation
On completion of follicular aspiration, recovered fluid is transferred immediately into a petri dish and searched to recover the oocyte. Changes in temperature are minimized. The mature cumulus complex surrounding the oocyte can be more than 2 or 3 mm in diameter. The extensive investment of cumulus and granulosa cells is often identifiable
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with the naked eye, appearing as a light mass of cells with a nondistinct border. Oocyte maturation can be evaluated roughly by imaging the cumulus complex and granulosa cells through a dissecting scope. Immature follicles typically result in collection of yellow follicular fluid with little blood contamination. Granulosa cells are present in compact sheets. The cumulus cells form tight layers around the zona pellucida. Immature oocytes can be dissociated from the bulk of the cumulus mass during collection with only a few layers of cells surrounding the zona pellucida. Oocytes collected approximately 24 hours after the injection of hCG (12 hours before expected ovulation) usually are surrounded with a moderately expanded cumulus complex and attached granulosa cells. The cumulus is distinct, but "fluffy" in appearance, and the granulosa cells form large, slightly grainy sheets. The aspirate is often bloody when the follicle is lavaged. On completion of maturation (in vivo or in vitro), cells from the cumulus complex are arranged in a diffuse array extending from the oocyte. The cumulus becomes light in appearance and tenacious. Morphology of the oocyte is difficult to evaluate because of the surrounding cellular investment. Mature oocytes often have a heterogeneous appearance due to the unequal distribution of lipid vesicles. Del Campo et al ll evaluated the preculture morphology of oocytes collected from a slaughterhouse. Cytoplasmic characteristics of oocytes did not correspond to differences in postculture results, with the exception that most (67%) of the oocytes classified as heterogeneous / fragmented (4% of all oocytes) degenerated after culture. Extrusion of a polar body is difficult to evaluate through the cumulus investment surrounding equine oocytes. Occasionally during aspiration, the oocyte is fragmented. The damaged oocyte may appear normal, slightly oval, or as a dark string of ooplasm extending through the cumulus complex. Imaging the oocyte through a dissecting microscope while pulling the cumulus complex into a glass pipet will confirm damage. The suctioning effect of the pipet will accentuate breaks within the zona pellucida causing the fragmented sections to separate. The author has not experienced iatrogenic damage to intact oocytes when pulling them into a pipet or catheter. Because of the large and tenacious investment of cells surrounding the oocyte, care should be taken to move the oocyte slowly and to have a pipet of adequate diameter. Fragmented oocytes are probably nonviable and do not justify transfer. Timing of Oocyte Collection
Detailed methods for collection and culture of oocytes are described elsewhere in this volume. Depending on the time of oocyte collection relative to anticipated ovulation, oocytes could require further maturation (cytoplasmic and meiotic) before transfer. Maturation of the equine
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oocyte begins approximately 36 hours before ovulation with breakdown of the nuclear membrane. When the oocyte is released into the oviduct, meiosis has progressed to metaphase II, the first polar body has been extruded, and the oocyte is capable of fertilization. Fertilization of the oocyte must occur after maturation is complete but before the oocyte has aged. Maturation of oocytes in vivo would eliminate the need of equipment and expertise for in vitro cultures. Completion of maturation occurs at approximately the time of ovulation. In two studies,23,32 oocytes were collected from preovulatory follicles and transferred into recipients within 24 to 36 hours after the injection of hCG (3300 IV intravenous [IV]) or when ovulation was predicted to be imminent. Before transfer, oocytes were held in follicular fluid «1 hour)2:l or in medium (Tissue Culture Medium [TCMj-199 supplemented with 10%, fetal calf serum or estrous mare serum; 5% CO2; 0.5 to 3 hours).32 An in vivo-matured oocyte resulted in the first foal from GIFT,23 but success rates have been relatively low. Zhang et al 40 used GIFT to evaluate the viability of in vitro-matured oocytes. For the study, ovaries were collected from an abattoir. The oocytes were recovered and placed in culture (37.5°C, 5% COb TCM-199 supplemented with estrous mare serum or fetal calf serum and exogenous hormones) within 9 hours after slaughter. Before transfer into recipients' oviducts, oocytes were cultured for 24 or 30 hours, with the length of culture depending on the extent of cumulus expansion. Some of the oocytes developed into blastocysts before recovery from the recipients' uteri 8 days after GIFT. Hunter et al 16 used a similar method to determine viability of bovine oocytes collected from slaughterhouse ovaries. However, the culture medium was considered inadequate to support oocyte maturation beyond the first metaphase, so oocytes were transferred into the oviducts of estrous heifers. From 55 transferred oocytes, cultured between 24 and 26 hours in vitro, 14 ova at some stage of fertilization were later recovered from the recipients' oviducts. Apparently, oocyte maturation was completed within the oviduct. The extent that equine oocytes would continue to mature within the oviduct is not known. Carnevale and GintherH combined in vivo and in vitro maturation of oocytes. Oocytes were collected from donors between 21 to 26 hours after an intravenous injection of hCG (2000 IU) or approximately 12 hours before ovulation. Before transfer, oocytes were cultured for 16 to 20 hours in medium (TCM-199 supplemented with 10% fetal calf serum and pyruvate, 5'}'0 CO 2 , and air). The study resulted in the highest success rate to date from transferred oocytes. Oocyte Transfer
Oocytes have been transferred into oviducts ipsilateral and contralateral to the side of ovulation/ aspiration in mares. Retrospective analy-
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sis of data in women' l demonstrated significantly more pregnancies developed after GIFT ipsilateral versus contralateral transfer to the dominant ovary (the ovary with the greater number of dominant follicles). In mares, pregnancies have resulted after oocyte transfers ipsilateral or contralateral to the side of the recipient's ovulatory follicle; however, data were insufficient to determine if side of transfer affected pregnancy results. With few exceptions/"29 more than one oocyte has been transferred into each mare's oviduct during GIFT. Multiple oocytes are often transferred in women to increase the chance of at least one fetus developing. 27 Because multiple pregnancies are undesirable in the mare, GIFT on a commercial basis would likely involve the transfer of only one oocyte. A potential positive effect of transferring more than one oocyte per recipient n eeds to be investigated. GIFT in the equid requires surgical exposure of the oviduct. General anesthesia and a mid ventral incision has been used for successful transfers/ o but most GIFT procedures have been performed through a standing flank laparotomy. Tranquilization, preparation, closure, and aftercare of recipients are similar to that previously described for embryo transfer. 2~ Exposure of the ovary and oviduct is facilitated if the flank incision is made slightly more dorsal and posterior to the preferred incision for embryo transfer. The ovary is located and gently exteriorized through the incision without gripping the ovarian stroma or oviduct. If required to facilitate relaxation and ovarian exposure, a gauze square soaked with lidocaine can be held against the broad ligament while maintaining light pressure for retraction. The exposed ovary is rotated gently to allow maximum exposure of the oviduct. Oocytes have been successfully transferred using a polished glass pipet/ 21 a "Tom Cat" catheter,.Jo or a 16 g X 8.9 cm IV cathe ter (containing oocyte) inserted through a 14 g X 5.7 cm catheter (threaded into the oviduct).12 The oviductal os is located by following the outline of the oviduct along the external surface of the infundibulum. When the end of the tubal structure is id entified, the fimbriae can be lifted to observe the opening. The catheter or pipet containing the oocyte is inserted into the os and carefully advanced between 2 and 3 cm. The ovary may need to be rotated to facilitate threading the pipet into the oviduct. The oocyte with a minimal amount of medium «0.5 mL) can then be expressed into the ampullar region of the oviduct. After transfer of the oocyte, the ovary and oviduct are returned carefully to the proper position within the abdominal cavity before surgical closure. Insemination of Recipient The equine oocyte retains maximal viability for approximately 12 hours after ovulation .12 Because of the limited lifespan of the mature oocyte, recipients must be bred b efore and / or directly after oocyte transfers. Potentially, capacitation and migration of spermatozoa already
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within the oviduct could be affected by manipulation of the oviduct and exposure to medium during GIFT. Inseminations after GIFT must allow time for movement of spermatozoa through the reproductive tract and for capacitation before aging of the oocyte. In the studies resulting in pregnancies with GIFT, recipients were bred in a variety of methods. For fertilization of in vitro-matured oocytes, recipients were mated naturally or artificially inseminated with at least 1 billion motile sperm at 7 hours before and 1 hour after transfer.· ll Recipients of in vivomatured oocytes were artificially inseminated with 500 million progressively motile spermatozoa within 3 to 0.5 hours before oocyte transfer 32 or within 1 hour after transfer?' Carnevale and Ginther 8 inseminated recipients approximately 12 hours before GIFT and immediately after the procedure. The future development of techniques to transfer spermatozoa into the oviduct during GIFT would eliminate insults to the recipients' uterus occurring from insemination.
Luteal Function and Progesterone Supplementation During oocyte collection, granulosa cells are removed from the follicle. After follicular aspirations in monkeys, a strong inverse correlation was noted between number of granulosa cells recovered from preovulatory follicles and subsequent circulating concentrations of progesterone. I '! Effects of follicular aspiration on circulating progesterone concentrations have been studied in hCG-treated mares. After administration of hCG and vigorous aspiration of the follicle, circulating concentrations of progesterone were reduced in the early luteal phase (days 3 and 5) but adequate later in the cycle. 23 Luteinization and production of progesterone was related to time of hCG administration rather than time of follicular aspiration.'s Aspiration of the dominant follicle from mares treated with hCG generally did not result in luteal failure; however, to assure sufficient progesterone concentrations after GIFT, exogenous progesterone has been provided. Ray et ap2 administered Altrenogest orally (0.044 mg/kg) to recipients beginning 24 or 48 hours after oocyte transfer. Carnevale and Ginther" injected recipients with progesterone in oil after GIFT (50 mg on day 1 and 100 mg on subsequent days) . Additional studies are needed to develop methods that will minimize the risk of fertilization of the recipient's oocyte while maintaining luteal integrity.
SUCCESS OF DIFFERENT PROCEDURES Success of GIFT in the mare has been variable. In a preliminary study by McKinnon et aV 6 six oocytes were collected from preovulatory follicles and transferred back into the oviducts of previously inseminated mares. No embryos were collected by uterine lavage eight days after transfer. Palmer et aF" injected mares with crude equine pituitary gonadotrophin preparation to induce follicular maturation. Oocytes were col-
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lected from three mares and transferred back into the donor's oviduct within 4 to 6 hours after collection. Mares were inseminated with 200 million spermatozoa, but no pregnancies developed. A second study by McKinnon et aF3 resulted in the first foal born after GIFT. Fifteen oocytes were collected from preovulatory follicles and inserted into oviducts of recipients. Two days after GIFT, recipients' oviducts were removed and flushed to collect unfertilized oocytes or developing embryos. Three oviductal embryos were collected and transferred into uteri of different recipients. Two pregnancies were detected after embryo transfer, and one pregnancy was carried to term. In a recent experiment,32 26 oocytes were collected from preovulatory follicles and transferred into the oviducts of eight recipients. Two singleton pregnancies resulted. DNA testing of the developing fetuses confirmed parentage. Although the transfer of in vivo-matured oocytes has resulted in some success, pregnancy rates have been low. Zhang et al 40 used GIFT to evaluate the viability of in vitro-matured oocytes. Twenty-nine oocytes were transferred into four recipients. On day 8 after GIFT, recipients were flushed for embryo recovery. Approximately 24% of the transferred oocytes (7 of 29) resulted in blastocysts. One blastocyst was transferred into a recipient, but no pregnancy developed. The highest embryo-development rates after GIFT resulted when oocytes were collected from mares approximately 24 hours after the administration of hCG and cultured for an additional 16 to 20 hours in vitro.HTwelve oocytes were collected from young (6 to 10 years) donors and transferred into five recipients. More than 90% (11 of 12) of the oocytes developed into embryonic vesicles as detected by ultrasonography on day 14. Oocyte viability did not seem to be affected adversely during GIFT, because all recipients maintained pregnancies through day 30. FACTORS AFFECTING OOCYTE VIABILITY
Defective oocytes as a cause of reduced fertility in mares has been widely postulated; however, few studies have been designed to study fertility associated with oocytes. Chromosomal abnormalities (intrinsic or induced), endocrine abnormalities, age of the donor, or postmaturation aging could result in oocyte defects and unsuccessful GIFT. In women and domestic animals, fertility decreases with increasing maternal age. In mares older than 15 years of age, pregnancy rates are reduced and embryo-loss rates are increased in comparison to younger maresY A recent study used GIFT to evaluate viability of oocytes from young and old mares. 8 Oocytes collected from old (2:20 years) mares and young (6-10 years) mares, were transferred into oviducts of young (3-7 years) recipients. More oocytes from young than old mares resulted in embryonic vesicles on day 14 (11 of 12, 92'Yo vs 8 of 26, 31 'X" respectively). Results of the study indicated that oocytes from old mares are
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not as viable as oocytes from young mares. From the experiment, intrinsic defects of oocytes could not be differentiated from defects caused by an inadequate or a harmful follicular environment. Potentially, if oocytes from older mares are not defective intrinsically, methods to salvage the oocytes can be devised. FUTURE CONSIDERATIONS Research Potentials
Reproducible techniques for GIFT would provide a method for evaluating fertility before the time that the embryo enters the uterus. The technique could facilitate the study of oocyte viability, maturation, fertilization, and early embryo development. Pregnancies from Subfertile Stallions
In humans, male infertility affects more than 40% of infertile couples; and in vitro fertilization or GIFT are the best options for successful pregnancy.22 Following injuries or age-associated degenerative changes, some stallions may produce too few progressively motile spermatozoa to result in pregnancies by insemination or natural cover. Development of techniques for transfer of spermatozoa into the oviduct during GIFT could result in pregnancies if limited numbers of spermatozoa were available. Potentially, techniques will eventually be developed to obtain low numbers of spermatozoa that have been gender selected; the spermatozoa could be used with GIFT to select the sex of offspring for commercial and experimental purposes. With further refinement, GIFT could prove a valuable technique to augment and study fertility in stallions. Oocyte Transportation and Cryopreservation
Portable containers with internal heat control (Minitub GMBH, Tiefenbach, Germany) have been used for the transportation of oocytes in other species. This system could prove to be suitable for transportation of equine oocytes from farms to a centralized facility for transfer. Although the preservation of mammalian oocytes through freezing or vitrification has been limited in success, live offspring have been obtained from some species (cow, human, rabbit, mouse) after freezing and thawing oocytes. High rates of oocyte survival and fertilization after vitrification have been reported for mice. 5 Immature equine oocytes were capable of in vitro maturation to metaphase II after freezing and thawing in either ethylene glycol or 1,2-propanediol. 15 Capabilities to freeze 00cytes and later transfer them into recipients would provide a valuable
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store of genetic material in the horse and could facilitate international transport of genetic lines.
Commercial Uses of GIFT
Establishment of techniques to efficiently collect oocytes through transvaginal ultrasound-guided follicular aspirations has enhanced greatly the feasibility of GIFT on a commercial basis. Further refinement of procedures used in GIFT should result in acceptable pregnancy rates for commercial programs. Gamete intrafallopian transfer has the potential to become an invaluable tool to obtain offspring from horses that are infertile using currently available treatment and assisted reproductive techniques.
SUMMARY
GIFT involves placement of a donor's oocyte into a surrogate's oviduct. Fertilization and embryo development occur within the recipient's reproductive tract. GIFT provides a viable method to obtain pregnancies from subfertile mares for which embryo transfer has been nonproductive. Currently, pregnancy rates after GIFT have been variable, although high success rates have been reported recently. Further refinement of techniques should allow GIFT to be used in research and commercial programs.
References 1. Adams GP, Kastelic JP, Bergfelt DR, et al: Effect of uterine inflammation and ultrasoni-
2.
3. 4.
5. 6. 7. 8. 9.
cally-detected uterine pathology on fertility in the mare. J Reprod Fertil 35(suppl):445454, 1987 Allen WR, Rowson LEA: Surgical and non-surgical egg transfer in horses. III Proceedings of the 7th International C'ongress on Animal Reproduction and Artificiallnsemination, Munich, Germany, 1972, pp. 484-487 Asch RH, Ellsworth JP, Balmaceda, et al: Pregnancy after translaparoscopic gamete intrafallopian transfer. Lancet 2:1034-1035, 1984 Ball BA, Little JA, Weber JA, et al: Survival of Day-4 embryos from young normal and aged, subfertile mares after transfer to normal recipient mares. J Reprod Ferti! 85:187-194, 1989 Bos-Mikich A, Wood MJ, Candy CJ, et al: Cytogenetical analysis and developmental potential of vitrified mouse oocytes. BioI Reprod 53:780-785, 1995 Bracher V, Parlevliet J, Fazeli AR, et al: Repeated transvaginal ultrasound-guided follicle aspiration in the mare. Equine Vet J 15(suppl):75-78, 1993 Carnevale EM: Folliculogenesis and Ovulation. Til Rantanen NW, McKinnon AO (eds): Equine Diagnostic Ultrasound, Williams & Wilkins, Media, PA (in press) Carnevale EM, Ginther OJ: Defective oocytes as a cause of subfertility in old mares. Bioi Reprod Mono 1:209-214, 1995 Carnevale EM, Bergfelt DR, Cinther OJ: Follicular activity and concentrations of FSH and LH associated with senescence in mares. Anim Reprod Sci 35:231-246, 1993
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10. Carnevale EM, Griffin PC, Ginther OJ: Age-associated subfertility before entry of embryos into the ute rus in mares. Equine Vet J 15(suppl):31- 35, 1993 11. Del Campo MR, Oonoso X, Parrish JJ, et al: Selection of follicles, preculture oocyte evaluation, and duration of cu Iture for in vitro maturation of equine oocytes. Theriogenology 43:1141-1153, 1995 12. Ginther OJ: Reproductive Biology of the Marc, cd 2. Cross Plains, WI, Equiservices, 1992 13. Henry M, Vandeplassche M: Pathology of the oviduct in mares. Vlaams Diergeneeskundig Tijdschrift 50:301-325, 1981 14. Heuer JA, King SS, Gardiner CS, et al: Uterine secretions from different endometrial classifications affect the viability of early murine embryos cultured in vitro. J Equine Vet Sci 13:494-497, 1993 15. Hochi S, Fujimoto T, Choi YH, et al: Cryopreservation of equine oocytes by 2-step freezing. Theriogenology 42:1085-1094 16. Hunter RHF, Lawson RAS, Rowson LEA: Maturation, transplantation and fertilization of ovarian oocytes in cattle. J Reprod Fertil 30:325-328, 1972 17. Kenny RM: Cyclic and pathologic changes of the mare endo metrium as detected by biopsy, with a note on early embryonic death. J Am Vet Med Assoc 172:241-262, 1978 18. Kenny RM: A review of the pathology of the equine oviduct. Equine Vet J 15(suppl):4246, 1993 19. Kreitmann 0, Nixon WE, Hodgen GD: Induced corpus luteum dysfunction after aspiration of the preovulatory follicle in monkeys. Fertil Steril 35:671-675, 1981 20. Lee C, Nie Cf, Joo HS, et al: An enzyme-linked immunosorbent assay (ELISA) for the detection of antisperm antibodies in horse serum. Theriogeno logy 40:1117-1126, 1993 21. Liu IKM, Lantz KC, Schlafke S, et al: Clinical observations of oviductal masses in the mare. 111 Proceedings of the 36th Annual Convention of the American Association of Equine Practitioners, Lex ington, KY, 1990, pp. 41-45 22. Mastroyannis C: Gam ete intrafallopian transfer: ethical considerations, historical development of the procedure, and comparison with other advanced reproductive technologies. Ferti I Steril 60:389-402, 1993 23. McKinnon AO, Carnevale EM, Squires EL, et al: Heterogenous and xenogenous fertilization of ill vivo matured equine oocytes. J Equine Vet Sci 8:143-147, 1988 24. McKinnon AO, Squires EL: Equine embryo transfer. Vet Clin North Am: Equine Pract 4:305-333, 1988 25. McKinnon AO, Squires EL, Pickett BW: Equine reproductive ultrasonography. Anim Reprod Lab Bull 4:1988 26. McKinnon AO, Wheeler MB, Carnevale EM, et al: Oocyte transfer in the mare: Preliminary observations. Equine Vet J 6:206- 309, 1987 27. Nelson JR, Corson SL, Batzer FR, et al: Predicting success of gamete intra fallopian transfer. Fertil Steril 60:116-122, 1993 28. Oguri N, Tsutsumi Y: Nonsurgical recov ery of equine eggs, and an attempt at nonsurgical egg transfer in horses. J Reprod Fertil 31:187-195, 1972 29. Palmer E, Bezard J, Magistrini M, et al: III l'ill'O fertilization in the horse: A retrospective study. J Reprod Fertil 44(suppl):375- 384, 1991 30. Rath 0, Johnson LA, Welch CR, et al: Successful gamete intra fallopian transfer (GIFT) in the porcine. Theriogenology 41:1173- 1179 31. Ransom MX, Corsan CH, Carcia AJ, et al: Tubal selection for gamete intrafallopian transfer. Fertil Steril 61:386- 389, 1994 32. Ray BS, Squires EL, Cook NL, et al: Pregnancy following gamete intrafallopian transfer in the mare. J Equine Vet Sci 14:27-30, 1994 33. Saltiel A, Paramo R, Murcia C, ct al: Pathological findings in the oviducts of mares. Am J Vet Res 47:594-597, 1986 34. Squires EL, Barnes CK, Rowley HS, et al: Effect of uterine fluid and volume of extender on Fertility. 111 Proceedings of the 35th Annual Convention of the American Association of Equine Practitioners, Boston, MA, 1989, pp. 25-30 35. Squires EL, Imel KJ, Iuliano MP, et al: Factors affecting reproductive efficiency in an equine embryo transfer programme. J Reprod Fertil 32(suppl):409-414, 1982
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36. Tsutsumi Y, Suzuki H, Takeda T, et al: Evidence of the origin of the gelatinous masses in the oviducts of mares. J Reprod Fertil 57:287-290, 1979 37. Vogelsang SC, Vogelsang MM: Influence of donor parity and age on th e success of commercia l equine embryo transfer. Equine Vet J 8(suppl): 71 - 72, 19R9 38. Watson ED, Sertich PL: Effect of aspiration of follicular fluid on s ubsequent luteal function in the mare. Theriogenology 33:1263- 1268,1990 39. Zent WW, Liu IKM, Spirito MA: Oviduct flushing as a treatme nt for infertility in the mare. Equine Vet J 15(suppl):47-48, 1993 40. Zhang JJ, Boyle MS, Ga lli C, et a l: Recent stud ies on ill vivo fertilisation of in vitro matured horse oocytes. Equine Vet J 8(suppl):101- 104, 1989
Address reprillt requests to Elaine M. Carneva le, DVM, MS, PhD Department of Anima l Science, Food, and Nutrition Southern Illinois Un iversity Carbonda le, IL 62901-4417
0749- 0739/96 $0.00 + .20
GAMETE INTRAFALLOPIAN TRANSFER Elaine M. Carnevale, DVM, MS, PhD
Since the first successful equine embryo collections and transfers were reported in 1972/· 2H embryo transfer has been accepted by many breed registries within the horse industry. One of the predominant uses of embryo transfer has been to obtain offspring from valuable mares that are incapable of maintaining a pregnancy to term. However, the collection of embryos for transfer has been more successful in fertile than subfertile mares (e.g., 80 'X, vs 28°;;), respectively)35 and in recently foaling than sub fertile mares (53% vs 29%).'7 Failure to collect embryos from subfertile mares could be the result of altered gamete or embryo transport, failure or defects of fertilization, or early embryo death. Potentially, fertility could be enhanced by collecting the donor's oocyte before ovulation, thus preventing the oocyte from contacting an inadequate or deleterious tubular genitalia. Gamete intrafallopian transfer (GIFT) involves placement of a donor's oocyte into a recipient's oviduct. Fertilization and embryo development occur within the reproductive tract of the recipient. GIFT has been successful in the human,' bovid,16 equid,23 and porcid." 1 In women, oocytes and spermatozoa are placed into the oviduct during GIFT. In the mare, spermatozoa were provided by natural cover or artificial insemination of recipients and only the oocyte has been transferred into the recipient's oviduct. Currently, GIFT has been used in equine research programs, but further refinement should make GIFT a viable procedure for commercial use.
From the Depa rtment of Anima l Science, Food, and Nutrition, College of Agriculture, Southern Illinois University, Carbondale, Illinois
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INDICATIONS FOR GIFT
Mares from which viable embryos cannot be obtained for transfer are potential oocyte donors for GIFT. A number of factors could affect embryo collection rates or embryo viability before the time that the embryo would be recovered from the uterus (days 6 to 8 after ovulation). Ovulatory Failure
Two types of potential ovulatory failure have been described in mares. Hemorrhagic follicles (HF)1 2 occurred more frequently in older mares and during autumn. 7 The follicles do not seem to ovulate and enlarge to diameters of more than 70 mm as blood enters the antrum. When imaged by ultrasonography, fibrin strands or floating debris are often present within the antrum. A second type of apparent ovulatory failure has been reported to occur more often in older mares than in younger mares (2"20 years, 10 of 68; 15-19 years, 4 of 78; 5-7 years, 1 of 91 atypical ovulations per total ovulations).9 In contrast to HF, diameter of the follicle does not increase. The preovulatory follicle becomes flaccid and demonstrates ultrasonographic characteristics associated with imminent ovulation,?· 25 including an irregular shape and an echogenic and thickened border. A central area of nonechogenic fluid remains in the follicle even after luteinization occurs. Ovulatory sites with similar characteristics were described previously as luteinized, unruptured follicles. 25 Aspiration of contents from similar structures resulted in the recovery of mature or atretic oocytes and bloody fluid ? Limited attempts to obtain pregnancies or recover oviductal embryos from mares with follicles that appeared to luteinize without rupturing have been unsuccessfuJ.7,25 Mares that have abnormal ovulations during successive cycles could provide oocytes for GIFT. However, the oocyte must be removed from the follicle before contamination of follicular fluid with blood and before the oocyte ages. The decision to collect an oocyte should be based on a history of repeated abnormal ovulations imaged by ultrasound. After an atypical appearance to the follicle has been imaged, the oocyte has probably undergone degenerative changes. If abnormal follicles are due to hormonal alterations, viability of the oocyte could be compromised. Oocyte Pickup
After ovulation, the viscous cumulus complex surrounding the oocyte contacts the fimbriae and is transported to the ampulla, partly, through ciliary activity and peristalsis. Ability of the infundibulum to collect and transport oocytes could be altered by infundibular adhesions or infundibulitis, which were found to be more frequent in older mares than in younger maresY Collection rates of recentl y ovulated oocytes or oviductal embryos per ovulation were significantly lower in older mares
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than in younger mares on day 1.5 (day 0 = day of ovulation; 4-7 years, 17 of 18; 15-19 years, 10 of 14; =2:20 years, 11 of 20) and on day 3 (2-9 years, 9 of 9 and =2:20 years, 6 of 9). J() Ball et al 4 also recovered fewer oocytes or embryos from oviducts of sub fertile, aged (mean age, 19 years) mares than normal, young (mean age, 6 years) mares (24 of 41 and 28 of 30, respectively). The findings suggest that failure of oocytes to gain entry into the oviduct could account for some cases of subfertility, especially in older mares. Oviductal Pathology
Obstruction of the oviductal lumen would prevent passage of spermatozoa and the embryo. Various anomalies and lesions of mares' oviducts recently have been reviewed 1H; however, their incidence is low. Globular masses, composed of type I collagen, frequently are found in the oviducts of mares. 21 Masses were recovered more often from oviducts of older (8-26 years) mares than younger (2-7 years) mares (13 of 22 vs 5 of 21, respectively). In another study,36 masses were found in 16 of 22 (67%) oviducts from mares of different ages (2-22 years). The collagenous masses occupied and distended the entire luminal diameter in a small number of mares (3 of 43), potentially occluding the lumen and resulting in delayed or interrupted oviductal transport of the oocyte or embryo.21 Surgical procedures have been developed to flush oviducts 39 with the intention of removing blockages. However, the importance of oviductal masses in reducing fertility has not been proven. Developing embryos remain in the oviduct for 5 or 6 days before entering the uterus. Oviducts must provide an environment suitable for gamete survival and transport, fertilization, and early embryo development. An inadequate or deleterious oviductal milieu could have harmful effects on gametes and embryos. Despite the oviduct's importance during early pregnancy, minimal clinical attention or research has been devoted to studying oviductal function and pathology. Findings from slaughterhouse studies suggest a role of oviductal pathology in reduced fertility. Inflammatory changes within the oviduct occurred twice as often in mares with endometritis than mares without endometritis,!'\ and lymphatic infiltration was more frequent in the infundibular-ampullar region of nonpregnant than pregnant mares."3 The infiltration of inflammatory cells could have a d etrimental effect on fertility; however, sufficient data have not been collected to directly correlate oviductal inflammation with pregnancy failure. Uterine Pathology
Historically, the uterus has been considered the primary cause of reproductive failure in mares. Increasing inflammatory changes, detected with uterine histology, were associated with a concomitant de-
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crease in fertilityY When murine embryos were cultured in uterine flushings from mares with different histological scores for inflammation,14 the quantity of lymphocyte infiltration was inversely proportional to the rate of embryo development. Intrauterine fluid collections imaged with ultrasonography represent an inflammatory process. Diestrous fluid collections were associated with lower pregnancy rates and higher embryo loss rates. 1 Motility was suppressed immediately when spermatozoa were cultured with fluid from intrauterine collections."4 Exposure of spermatozoa or embryos to inflammatory products of the uterus was detrimental to their survival. Using GIFT, the effects of inflammatory or infectious problems in the donor's uterus would be eliminated.
Miscellaneous Pathology
Miscellaneous reproductive pathology, such as cervical lacerations or urine pooling, can prevent pregnancy or the successful collection of embryos. Mares in which surgical or medical treatments are unsuccessful would be acceptable oocyte donors. In humans, GIFT has been used to treat immunologic infertilities. 22 Antisperm antibodies have been produced experimentally in mares. 20 However, the importance of antisperm antibodies in equine reproduction has not been established. Problems associated with the production of antibodies to spermatozoa by the donor would be eliminated using GIFT.
TRANSFER OF OOCYTES Oocyte Donors
Mares from which pregnancies have not been obtained after embryo transfer are potential oocyte donors. Oocyte collection techniques are more invasive than nonsurgical embryo flushes, because the abdominal cavity is penetrated. Bracher et al 6 monitored mares after repeated transvaginal ultrasound-guided follicular aspirations during a period of 3 months. The procedure was well tolerated by mares, and no clinical side effects were observed. One mare, slaughtered 6 weeks after the last follicular puncture, had fine serosal adhesions on the ovarian surface and small hemorrhagic areas within the ovarian stroma. Data have not been collected to determine if repeated follicular aspirations impair fertility in normal mares. Requirements for oocyte donors are minimal. Donors must have regular estrous cycles and viable oocytes. The uterus should be cultured to identify potential pathogens and treated as indicated. Because a needle is punctured through the vaginal wall during transvaginal oocyte aspirations, microbes could potentially be introduced into the abdomen. After oocyte collections, prostaglandins are used to short cycle the donor
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once the corpus luteum has matured (approximately 6 days after expected ovulation date or 8 days after hCG administration). Recipients
Selection of suitable recipients is imperative to the success of GIFT. Reproductive tracts of oocyte recipients must provide the proper milieu for sperm survival and transport, fertilization, and embryo development. Because recipients will be inseminated or bred, often at the approximate time of ovulation, they must be resistant to uterine infections. Recipient requirements were previously described for embryo transfer. 2-1 Adequate exposure of oviducts during standing flank laparotomies can be difficult, but problems can be minimized through the careful selection of recipients. Exposure of the oviduct is easier in mares with no excess fat and with a longer coupling (distance between the last rib and tuber cocci). Mares that have had at least one foal are preferred to maidens, because additional laxity of the broad ligament will facilitate oviductal exposure. A recipient's suitability for transfer can be evaluated by grasping the ovary per rectum and manually moving it toward the flank. Laxity of the broad ligament and ovarian mobility can be evaluated, and the most advantageous placement of an incision site can be determined. Synchronization of Donors and Recipients
A critical window for synchronization between donors and recipients has not been established. Oocytes have been transferred between 44 to 48 hours after the administration of hCG;il between the third day of estrus and just before ovulation,,2 or within 24 hours of ovulation." Because the recipient will be inseminated, the oocyte from the recipient's dominant follicle must be removed to prevent fertilization. Removal of the recipient's oocyte can be accomplished by transvaginal or flank aspiration before GIFT. Aspiration of the follicle also can be attempted at the time of transfer; however, the preovulatory follicle is often large and flaccid and may rupture before exposure. Collapse of the recipient's follicle does not assure that the recipient's oocyte will not gain access to the oviduct and result in fertilization and embryo development. 2h Oocyte Evaluation
On completion of follicular aspiration, recovered fluid is transferred immediately into a petri dish and searched to recover the oocyte. Changes in temperature are minimized. The mature cumulus complex surrounding the oocyte can be more than 2 or 3 mm in diameter. The extensive investment of cumulus and granulosa cells is often identifiable
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with the naked eye, appearing as a light mass of cells with a nondistinct border. Oocyte maturation can be evaluated roughly by imaging the cumulus complex and granulosa cells through a dissecting scope. Immature follicles typically result in collection of yellow follicular fluid with little blood contamination. Granulosa cells are present in compact sheets. The cumulus cells form tight layers around the zona pellucida. Immature oocytes can be dissociated from the bulk of the cumulus mass during collection with only a few layers of cells surrounding the zona pellucida. Oocytes collected approximately 24 hours after the injection of hCG (12 hours before expected ovulation) usually are surrounded with a moderately expanded cumulus complex and attached granulosa cells. The cumulus is distinct, but "fluffy" in appearance, and the granulosa cells form large, slightly grainy sheets. The aspirate is often bloody when the follicle is lavaged. On completion of maturation (in vivo or in vitro), cells from the cumulus complex are arranged in a diffuse array extending from the oocyte. The cumulus becomes light in appearance and tenacious. Morphology of the oocyte is difficult to evaluate because of the surrounding cellular investment. Mature oocytes often have a heterogeneous appearance due to the unequal distribution of lipid vesicles. Del Campo et al ll evaluated the preculture morphology of oocytes collected from a slaughterhouse. Cytoplasmic characteristics of oocytes did not correspond to differences in postculture results, with the exception that most (67%) of the oocytes classified as heterogeneous / fragmented (4% of all oocytes) degenerated after culture. Extrusion of a polar body is difficult to evaluate through the cumulus investment surrounding equine oocytes. Occasionally during aspiration, the oocyte is fragmented. The damaged oocyte may appear normal, slightly oval, or as a dark string of ooplasm extending through the cumulus complex. Imaging the oocyte through a dissecting microscope while pulling the cumulus complex into a glass pipet will confirm damage. The suctioning effect of the pipet will accentuate breaks within the zona pellucida causing the fragmented sections to separate. The author has not experienced iatrogenic damage to intact oocytes when pulling them into a pipet or catheter. Because of the large and tenacious investment of cells surrounding the oocyte, care should be taken to move the oocyte slowly and to have a pipet of adequate diameter. Fragmented oocytes are probably nonviable and do not justify transfer. Timing of Oocyte Collection
Detailed methods for collection and culture of oocytes are described elsewhere in this volume. Depending on the time of oocyte collection relative to anticipated ovulation, oocytes could require further maturation (cytoplasmic and meiotic) before transfer. Maturation of the equine
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oocyte begins approximately 36 hours before ovulation with breakdown of the nuclear membrane. When the oocyte is released into the oviduct, meiosis has progressed to metaphase II, the first polar body has been extruded, and the oocyte is capable of fertilization. Fertilization of the oocyte must occur after maturation is complete but before the oocyte has aged. Maturation of oocytes in vivo would eliminate the need of equipment and expertise for in vitro cultures. Completion of maturation occurs at approximately the time of ovulation. In two studies,23,32 oocytes were collected from preovulatory follicles and transferred into recipients within 24 to 36 hours after the injection of hCG (3300 IV intravenous [IV]) or when ovulation was predicted to be imminent. Before transfer, oocytes were held in follicular fluid «1 hour)2:l or in medium (Tissue Culture Medium [TCMj-199 supplemented with 10%, fetal calf serum or estrous mare serum; 5% CO2; 0.5 to 3 hours).32 An in vivo-matured oocyte resulted in the first foal from GIFT,23 but success rates have been relatively low. Zhang et al 40 used GIFT to evaluate the viability of in vitro-matured oocytes. For the study, ovaries were collected from an abattoir. The oocytes were recovered and placed in culture (37.5°C, 5% COb TCM-199 supplemented with estrous mare serum or fetal calf serum and exogenous hormones) within 9 hours after slaughter. Before transfer into recipients' oviducts, oocytes were cultured for 24 or 30 hours, with the length of culture depending on the extent of cumulus expansion. Some of the oocytes developed into blastocysts before recovery from the recipients' uteri 8 days after GIFT. Hunter et al 16 used a similar method to determine viability of bovine oocytes collected from slaughterhouse ovaries. However, the culture medium was considered inadequate to support oocyte maturation beyond the first metaphase, so oocytes were transferred into the oviducts of estrous heifers. From 55 transferred oocytes, cultured between 24 and 26 hours in vitro, 14 ova at some stage of fertilization were later recovered from the recipients' oviducts. Apparently, oocyte maturation was completed within the oviduct. The extent that equine oocytes would continue to mature within the oviduct is not known. Carnevale and GintherH combined in vivo and in vitro maturation of oocytes. Oocytes were collected from donors between 21 to 26 hours after an intravenous injection of hCG (2000 IU) or approximately 12 hours before ovulation. Before transfer, oocytes were cultured for 16 to 20 hours in medium (TCM-199 supplemented with 10% fetal calf serum and pyruvate, 5'}'0 CO 2 , and air). The study resulted in the highest success rate to date from transferred oocytes. Oocyte Transfer
Oocytes have been transferred into oviducts ipsilateral and contralateral to the side of ovulation/ aspiration in mares. Retrospective analy-
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sis of data in women' l demonstrated significantly more pregnancies developed after GIFT ipsilateral versus contralateral transfer to the dominant ovary (the ovary with the greater number of dominant follicles). In mares, pregnancies have resulted after oocyte transfers ipsilateral or contralateral to the side of the recipient's ovulatory follicle; however, data were insufficient to determine if side of transfer affected pregnancy results. With few exceptions/"29 more than one oocyte has been transferred into each mare's oviduct during GIFT. Multiple oocytes are often transferred in women to increase the chance of at least one fetus developing. 27 Because multiple pregnancies are undesirable in the mare, GIFT on a commercial basis would likely involve the transfer of only one oocyte. A potential positive effect of transferring more than one oocyte per recipient n eeds to be investigated. GIFT in the equid requires surgical exposure of the oviduct. General anesthesia and a mid ventral incision has been used for successful transfers/ o but most GIFT procedures have been performed through a standing flank laparotomy. Tranquilization, preparation, closure, and aftercare of recipients are similar to that previously described for embryo transfer. 2~ Exposure of the ovary and oviduct is facilitated if the flank incision is made slightly more dorsal and posterior to the preferred incision for embryo transfer. The ovary is located and gently exteriorized through the incision without gripping the ovarian stroma or oviduct. If required to facilitate relaxation and ovarian exposure, a gauze square soaked with lidocaine can be held against the broad ligament while maintaining light pressure for retraction. The exposed ovary is rotated gently to allow maximum exposure of the oviduct. Oocytes have been successfully transferred using a polished glass pipet/ 21 a "Tom Cat" catheter,.Jo or a 16 g X 8.9 cm IV cathe ter (containing oocyte) inserted through a 14 g X 5.7 cm catheter (threaded into the oviduct).12 The oviductal os is located by following the outline of the oviduct along the external surface of the infundibulum. When the end of the tubal structure is id entified, the fimbriae can be lifted to observe the opening. The catheter or pipet containing the oocyte is inserted into the os and carefully advanced between 2 and 3 cm. The ovary may need to be rotated to facilitate threading the pipet into the oviduct. The oocyte with a minimal amount of medium «0.5 mL) can then be expressed into the ampullar region of the oviduct. After transfer of the oocyte, the ovary and oviduct are returned carefully to the proper position within the abdominal cavity before surgical closure. Insemination of Recipient The equine oocyte retains maximal viability for approximately 12 hours after ovulation .12 Because of the limited lifespan of the mature oocyte, recipients must be bred b efore and / or directly after oocyte transfers. Potentially, capacitation and migration of spermatozoa already
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within the oviduct could be affected by manipulation of the oviduct and exposure to medium during GIFT. Inseminations after GIFT must allow time for movement of spermatozoa through the reproductive tract and for capacitation before aging of the oocyte. In the studies resulting in pregnancies with GIFT, recipients were bred in a variety of methods. For fertilization of in vitro-matured oocytes, recipients were mated naturally or artificially inseminated with at least 1 billion motile sperm at 7 hours before and 1 hour after transfer.· ll Recipients of in vivomatured oocytes were artificially inseminated with 500 million progressively motile spermatozoa within 3 to 0.5 hours before oocyte transfer 32 or within 1 hour after transfer?' Carnevale and Ginther 8 inseminated recipients approximately 12 hours before GIFT and immediately after the procedure. The future development of techniques to transfer spermatozoa into the oviduct during GIFT would eliminate insults to the recipients' uterus occurring from insemination.
Luteal Function and Progesterone Supplementation During oocyte collection, granulosa cells are removed from the follicle. After follicular aspirations in monkeys, a strong inverse correlation was noted between number of granulosa cells recovered from preovulatory follicles and subsequent circulating concentrations of progesterone. I '! Effects of follicular aspiration on circulating progesterone concentrations have been studied in hCG-treated mares. After administration of hCG and vigorous aspiration of the follicle, circulating concentrations of progesterone were reduced in the early luteal phase (days 3 and 5) but adequate later in the cycle. 23 Luteinization and production of progesterone was related to time of hCG administration rather than time of follicular aspiration.'s Aspiration of the dominant follicle from mares treated with hCG generally did not result in luteal failure; however, to assure sufficient progesterone concentrations after GIFT, exogenous progesterone has been provided. Ray et ap2 administered Altrenogest orally (0.044 mg/kg) to recipients beginning 24 or 48 hours after oocyte transfer. Carnevale and Ginther" injected recipients with progesterone in oil after GIFT (50 mg on day 1 and 100 mg on subsequent days) . Additional studies are needed to develop methods that will minimize the risk of fertilization of the recipient's oocyte while maintaining luteal integrity.
SUCCESS OF DIFFERENT PROCEDURES Success of GIFT in the mare has been variable. In a preliminary study by McKinnon et aV 6 six oocytes were collected from preovulatory follicles and transferred back into the oviducts of previously inseminated mares. No embryos were collected by uterine lavage eight days after transfer. Palmer et aF" injected mares with crude equine pituitary gonadotrophin preparation to induce follicular maturation. Oocytes were col-
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lected from three mares and transferred back into the donor's oviduct within 4 to 6 hours after collection. Mares were inseminated with 200 million spermatozoa, but no pregnancies developed. A second study by McKinnon et aF3 resulted in the first foal born after GIFT. Fifteen oocytes were collected from preovulatory follicles and inserted into oviducts of recipients. Two days after GIFT, recipients' oviducts were removed and flushed to collect unfertilized oocytes or developing embryos. Three oviductal embryos were collected and transferred into uteri of different recipients. Two pregnancies were detected after embryo transfer, and one pregnancy was carried to term. In a recent experiment,32 26 oocytes were collected from preovulatory follicles and transferred into the oviducts of eight recipients. Two singleton pregnancies resulted. DNA testing of the developing fetuses confirmed parentage. Although the transfer of in vivo-matured oocytes has resulted in some success, pregnancy rates have been low. Zhang et al 40 used GIFT to evaluate the viability of in vitro-matured oocytes. Twenty-nine oocytes were transferred into four recipients. On day 8 after GIFT, recipients were flushed for embryo recovery. Approximately 24% of the transferred oocytes (7 of 29) resulted in blastocysts. One blastocyst was transferred into a recipient, but no pregnancy developed. The highest embryo-development rates after GIFT resulted when oocytes were collected from mares approximately 24 hours after the administration of hCG and cultured for an additional 16 to 20 hours in vitro.HTwelve oocytes were collected from young (6 to 10 years) donors and transferred into five recipients. More than 90% (11 of 12) of the oocytes developed into embryonic vesicles as detected by ultrasonography on day 14. Oocyte viability did not seem to be affected adversely during GIFT, because all recipients maintained pregnancies through day 30. FACTORS AFFECTING OOCYTE VIABILITY
Defective oocytes as a cause of reduced fertility in mares has been widely postulated; however, few studies have been designed to study fertility associated with oocytes. Chromosomal abnormalities (intrinsic or induced), endocrine abnormalities, age of the donor, or postmaturation aging could result in oocyte defects and unsuccessful GIFT. In women and domestic animals, fertility decreases with increasing maternal age. In mares older than 15 years of age, pregnancy rates are reduced and embryo-loss rates are increased in comparison to younger maresY A recent study used GIFT to evaluate viability of oocytes from young and old mares. 8 Oocytes collected from old (2:20 years) mares and young (6-10 years) mares, were transferred into oviducts of young (3-7 years) recipients. More oocytes from young than old mares resulted in embryonic vesicles on day 14 (11 of 12, 92'Yo vs 8 of 26, 31 'X" respectively). Results of the study indicated that oocytes from old mares are
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not as viable as oocytes from young mares. From the experiment, intrinsic defects of oocytes could not be differentiated from defects caused by an inadequate or a harmful follicular environment. Potentially, if oocytes from older mares are not defective intrinsically, methods to salvage the oocytes can be devised. FUTURE CONSIDERATIONS Research Potentials
Reproducible techniques for GIFT would provide a method for evaluating fertility before the time that the embryo enters the uterus. The technique could facilitate the study of oocyte viability, maturation, fertilization, and early embryo development. Pregnancies from Subfertile Stallions
In humans, male infertility affects more than 40% of infertile couples; and in vitro fertilization or GIFT are the best options for successful pregnancy.22 Following injuries or age-associated degenerative changes, some stallions may produce too few progressively motile spermatozoa to result in pregnancies by insemination or natural cover. Development of techniques for transfer of spermatozoa into the oviduct during GIFT could result in pregnancies if limited numbers of spermatozoa were available. Potentially, techniques will eventually be developed to obtain low numbers of spermatozoa that have been gender selected; the spermatozoa could be used with GIFT to select the sex of offspring for commercial and experimental purposes. With further refinement, GIFT could prove a valuable technique to augment and study fertility in stallions. Oocyte Transportation and Cryopreservation
Portable containers with internal heat control (Minitub GMBH, Tiefenbach, Germany) have been used for the transportation of oocytes in other species. This system could prove to be suitable for transportation of equine oocytes from farms to a centralized facility for transfer. Although the preservation of mammalian oocytes through freezing or vitrification has been limited in success, live offspring have been obtained from some species (cow, human, rabbit, mouse) after freezing and thawing oocytes. High rates of oocyte survival and fertilization after vitrification have been reported for mice. 5 Immature equine oocytes were capable of in vitro maturation to metaphase II after freezing and thawing in either ethylene glycol or 1,2-propanediol. 15 Capabilities to freeze 00cytes and later transfer them into recipients would provide a valuable
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store of genetic material in the horse and could facilitate international transport of genetic lines.
Commercial Uses of GIFT
Establishment of techniques to efficiently collect oocytes through transvaginal ultrasound-guided follicular aspirations has enhanced greatly the feasibility of GIFT on a commercial basis. Further refinement of procedures used in GIFT should result in acceptable pregnancy rates for commercial programs. Gamete intrafallopian transfer has the potential to become an invaluable tool to obtain offspring from horses that are infertile using currently available treatment and assisted reproductive techniques.
SUMMARY
GIFT involves placement of a donor's oocyte into a surrogate's oviduct. Fertilization and embryo development occur within the recipient's reproductive tract. GIFT provides a viable method to obtain pregnancies from subfertile mares for which embryo transfer has been nonproductive. Currently, pregnancy rates after GIFT have been variable, although high success rates have been reported recently. Further refinement of techniques should allow GIFT to be used in research and commercial programs.
References 1. Adams GP, Kastelic JP, Bergfelt DR, et al: Effect of uterine inflammation and ultrasoni-
2.
3. 4.
5. 6. 7. 8. 9.
cally-detected uterine pathology on fertility in the mare. J Reprod Fertil 35(suppl):445454, 1987 Allen WR, Rowson LEA: Surgical and non-surgical egg transfer in horses. III Proceedings of the 7th International C'ongress on Animal Reproduction and Artificiallnsemination, Munich, Germany, 1972, pp. 484-487 Asch RH, Ellsworth JP, Balmaceda, et al: Pregnancy after translaparoscopic gamete intrafallopian transfer. Lancet 2:1034-1035, 1984 Ball BA, Little JA, Weber JA, et al: Survival of Day-4 embryos from young normal and aged, subfertile mares after transfer to normal recipient mares. J Reprod Ferti! 85:187-194, 1989 Bos-Mikich A, Wood MJ, Candy CJ, et al: Cytogenetical analysis and developmental potential of vitrified mouse oocytes. BioI Reprod 53:780-785, 1995 Bracher V, Parlevliet J, Fazeli AR, et al: Repeated transvaginal ultrasound-guided follicle aspiration in the mare. Equine Vet J 15(suppl):75-78, 1993 Carnevale EM: Folliculogenesis and Ovulation. Til Rantanen NW, McKinnon AO (eds): Equine Diagnostic Ultrasound, Williams & Wilkins, Media, PA (in press) Carnevale EM, Ginther OJ: Defective oocytes as a cause of subfertility in old mares. Bioi Reprod Mono 1:209-214, 1995 Carnevale EM, Bergfelt DR, Cinther OJ: Follicular activity and concentrations of FSH and LH associated with senescence in mares. Anim Reprod Sci 35:231-246, 1993
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Address reprillt requests to Elaine M. Carneva le, DVM, MS, PhD Department of Anima l Science, Food, and Nutrition Southern Illinois Un iversity Carbonda le, IL 62901-4417