Animal Reproduction Science 8283 (2004) 617624

Oocyte transfer and gamete intrafallopian transfer in the mare

E.M. Carnevale
Department of Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, USA

Abstract Methods for the collection and transfer of equine oocytes have been developed, and uses of these techniques have resulted in new clinical and research possibilities. Because oocyte transfer avoids reproductive problems associated with the oviduct, uterus, and cervix, pregnancies can be produced from many mares that cannot carry a pregnancy or produce embryos. Oocytes for clinical transfers are usually collected from preovulatory follicles and cultured for a short interval or transferred directly into a recipients oviduct. For oocyte transfer, the recipient is inseminated within the uterus. A large number (1109 to 2109 ) of motile sperms are preferred for inseminations. In contrast, sperm and oocyte are transferred into the oviduct during gamete intrafallopian transfer (GIFT). Therefore, a lower number (1105 to 2105 ) of sperm can be used. Potentially, GIFT could be used in situations where sperm numbers are limited. Use of oocyte transfer and GIFT in clinical and research settings will aid us in understanding the interactions between oocyte, sperm, and oviduct in the equine. 2004 Published by Elsevier B.V.
Keywords: Equine; Oocyte; Assisted reproduction; Oocyte transfer; GIFT

1. Introduction During oocyte transfer, a donors oocyte is placed in a recipients oviduct, and the recipient is inseminated within the uterus. When sperm are placed within the oviduct with the oocyte, the procedure is called gamete intrafallopian transfer (GIFT). For oocyte transfer and GIFT, fertilization and development of the embryo and fetus occur within the recipient. Technologies to collect and transfer oocytes have provided new methods to study the interaction of oocyte, sperm, and oviduct for research purposes. Oocyte transfer has also been used as a clinical procedure for subfertile mares that cannot provide viable embryos for transfer because of reproductive failure associated with abnormalities of ovulation, oviduct function, uterine health, or cervical function. GIFT has the potential to be used for subfertile
Tel.: +1-970-491-8626; fax: +1-970-491-7005. E-mail address: emc@colostate.edu (E.M. Carnevale).

0378-4320/$ see front matter 2004 Published by Elsevier B.V. doi:10.1016/j.anireprosci.2004.04.002

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stallions with low sperm production because a low number of sperm are placed within the oviduct. Development of procedures for the collection and transfer of oocytes has resulted in new clinical and research possibilities for the equine. 2. Clinical use of oocyte transfer The rst foal was born after an oocyte transfer in the late 1980s (McKinnon et al., 1988). However, the procedure was not efcient. Fifteen oocytes were transferred into inseminated recipients oviduct, three oviductal embryos were collected, two pregnancies were established, and one foal (1/15, 7%) was born. The rst report of a high success rate with oocyte transfer was in 1995 (Carnevale and Ginther, 1995). At that time, the transfer of oocytes from young donor mares into inseminated recipients resulted in an embryo development rate of 92% (11/12). The procedure was not used for commercial transfers until the late 1990s (Hinrichs et al., 2000a; Carnevale et al., 2001b). Oocyte transfer has since been proven to be a useful method to obtain offspring from mares with various reproductive problems that prevent successful pregnancy or embryo donation (Carnevale et al., 2001b). Reproductive problems include ovulatory failure, uterine infections, cervical tears, and undiagnosed anomalies. For older mares and during the autumn months, the incidence of ovulatory failure is increased (Carnevale et al., 1994; Carnevale, 1998). Some mares, especially older mares, appear to repeatedly fail to ovulate. These mares have been used as successful oocyte donors, if oocytes are collected before deleterious changes occur within the follicle (Carnevale et al., 2001b). Oviductal abnormalities are difcult to diagnose; however, results of studies have suggested that sperm selection and collection of the oocyte by the oviduct can be impaired in some mares (Carnevale et al., 1993; Scott et al., 1995). Oviductal masses have been described in the oviducts of mares (Tsutsumi et al., 1979; Liu et al., 1990). The potential of oviductal masses to affect fertility is not known. While oviductal pathology may be uncommon and is difcult to diagnose, uterine pathology is commonly diagnosed in mares. Treatment of uterine infections can be expensive and unsuccessful for some mares, resulting in their failure as embryo donors. Other pathology, such as cervical lacerations, cervical or uterine adhesions, or urine pooling, will affect a mares potential as an embryo donor. However in many of these cases, oocyte transfer provides a viable method to obtain offspring. One major factor, affecting success of oocyte transfer, is age of the donor. Pregnancy rates after transfer of oocytes from young mares are better than those for oocytes from old mares. When oocytes were collected from the follicles of young mares (610 years) and old mares (2026 years) and transferred into the oviducts of young recipients (37 years), signicantly more oocytes from young than old donors developed into embryonic vesicles (11/12, 92% versus 8/26, 31%) (Carnevale and Ginther, 1995). More morphological anomalies were observed in oocytes from old than young mares when evaluated with light and electron microscopy (Carnevale et al., 1999b). When donors for clinical transfers were old, pregnancies were obtained; however, more transfers were required to produce a viable pregnancy when oocytes were collected from old versus young donors. During a breeding season, pregnancies were established for three of four donors 16 years after only one transfer. In contrast, a pregnancy was established for only one of six donors 20 years after

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one transfer, and a pregnancy was not obtained for one old donor after repeated transfers. Between two and four transfers were required to establish pregnancies in the other old mares (Carnevale et al., 2001b). Pregnancy rates for commercial transfers, using older donors and semen of variable quality, ranged from 27 to 40% per transfer (Carnevale et al., 2001b,c). In contrast, experimental transfers done under similar conditions using oocytes from young mares and fertile stallions resulted in pregnancy rates between 54 and 83% per transfer (Carnevale et al., 2001c). However, one or more pregnancies were obtained for at least 80% of the oocyte donors during the breeding season in a clinical program, with donors in the program having long (mean of 7 years) histories of reproductive failure in breeding and embryo transfer programs (Carnevale et al., 2001b).

3. Oocyte collection Most oocytes that are transferred for clinical or research purposes are collected from preovulatory follicles. Therefore, oocyte maturation occurs in vivo; and the in vitro effects of follicle selection and oocyte maturation are minimized. Oocyte collections are scheduled based on the time of administration of hCG or a GnRH analog to the donor. The criteria that are used for hCG or GnRH administration are: (i) a follicle >35 mm in diameter, (ii) relaxed cervical and uterine tone, and (iii) uterine edema or estrous behavior for at least 2 days. In our laboratory, oocytes are collected from preovulatory follicles between 24 and 36 h after the administration of hCG (15002500 IU, i.v.) to the donor or between 14 and 0 h before anticipated ovulation, respectively. Oocytes are probably collected at metaphase I or II (Bezard et al., 1997). Oocytes have been collected from the follicles of mares using laparotomies (Vogelsang et al., 1986), colpotomies (Hinrichs and Kenney, 1987), ank punctures (Vogelsang et al., 1983; Palmer et al., 1986), and ultrasound-guided follicular aspirations (Bruck et al., 1992; Carnevale and Ginther, 1993; Cook et al., 1993). Flank or ultrasound-guided punctures are the most common methods of oocyte collection. Oocyte collection by ank puncture is done by manual palpation without the aid of an ultrasound machine. The technique requires that a trocar be placed through the ank musculature, ipsilateral to the preovulatory follicle and at the approximate position of the ovary. The ovary is manipulated per rectum, so that the preovulatory follicle is positioned against the end of the cannula. A needle (1215 gauge) is placed through the cannula and into the follicular antrum, and the follicular uid and oocyte are removed by gentle suction and lavage of the follicle. An ultrasound machine is used to view the procedure during transvaginal, ultrasoundguided follicular aspirations. A linear, curvilinear, or sector transducer can be used. The transducer is placed in a casing containing a needle guide and positioned within the anterior vagina, lateral to the posterior cervix and ipsilateral to the ovary containing the preovulatory follicle. Transrectal manipulations are used to position the ovary against the ultrasound transducer and needle guide. A needle (12-gauge, double lumen) is advanced within the needle guide until it punctures the vaginal and follicular walls. Follicular uid is aspirated from the follicle using a pump set at 150 mm Hg or large syringes. After removal of follicular uid, the lumen can be lavaged with between 50 and 100 ml of media, typically modied

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Dulbeccos phosphate-buffered solution or an embryo ush solution containing a serum source and heparin (10 IU/ml) to prevent coagulation. Oocyte recovery rates will vary with experience of the clinician. During the last 3 years in our laboratory, collection rates of oocytes from commercial donors were 98% (331/339) per cycle and 76% (331/434) per follicle (Carnevale et al., 2003a). Media and equipment for handling the oocyte are maintained at 3839 C. Because the oocyte is surrounded by expanding cumulus cells, the cumulus oocyte complex is usually large (>1 mm), translucent, and easy to identify under a stereomicroscope. Critical evaluation of the oocyte is difcult because of the surrounding cells.

4. Oocyte culture Oocytes collected 36 h after hCG administration to the donor are ready for immediate transfer into a recipients oviduct. Oocytes collected 24 h after administration to the donor are often cultured in vitro for between 12 and 16 h before transfer. Tissue Culture Medium 199 with additions of 10% fetal calf serum, 0.2 mM pyruvate, and 50 g/ml gentamicin has been established as an acceptable medium for culture of the maturing oocyte (Carnevale and Ginther, 1995; Hinrichs et al., 1998). Oocytes have been cultured at 38.239 C in atmospheres of 5 or 6% CO2 and air. Time of oocyte collection (24h versus 36 h after administration of hCG to donors) did not affect pregnancy rates (Hinrichs et al., 2000b).

5. Oocyte transfer Cyclic and noncyclic mares have been used as oocyte recipients. When cyclic mares are used, recipients are synchronized with the donor, and the recipients own oocyte is removed by aspiration to prevent its fertilization. Noncyclic mares or mares with reduced follicular activity can be used as oocyte recipients, eliminating the need to remove the recipients oocyte. Synchronization is obtained by the administration of exogenous hormones. During the spring, anestrus and early transitional mares were used as recipients (Carnevale et al., 1999a; Carnevale et al., 2001b). During the breeding season, a high dose of a GnRH agonist (4.2 mg, deslorelin acetate) (Carnevale et al., 2001a) or a period of progesterone and estrogen (150 mg P4 and 10 mg E2 ) daily for up to 10 days followed by withdrawal (Hinrichs et al., 2000a) were used to reduce follicular development. Estradiol (25 mg daily for 37 days) was administered before insemination and oocyte transfer, and progesterone (150200 mg daily) was administered after transfer. Exogenous progesterone or progestin was required to maintain pregnancies (Carnevale et al., 2001b). Pregnancy rates after commercial transfers were similar for cyclic and noncyclic recipients (8/15, 53% and 37/93, 39%) (Carnevale et al., 2001c). Oocyte transfers are performed through standing ank laparotomies with surgical procedures similar to previously described methods for surgical embryo transfer (Squires and Seidel, 1995). After the peritoneum is punctured, the ovary is located and gently exteriorized through the incision. The oocyte is loaded into a re-polished, glass pipette with a

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small volume of medium (<0.05 ml), and gently deposited within the oviduct through the infundibular os. The equine oocyte retains high fertility for approximately 12 h after a natural ovulation (Ginther, 1992). Therefore, recipients are inseminated before or directly after oocyte transfer. Pregnancies have occurred when recipients were inseminated only before (Scott et al., 2001; Maclellan et al., 2002) or after (Carnevale et al., 2000) transfer of oocytes. For most experimental transfers, recipients were inseminated approximately 12 h before and 2 h after transfer with a total of 2 109 motile sperms (Carnevale et al., 2001c). In a recent study (Carnevale et al., 2002), oocyte recipients were inseminated with semen from different stallions before and after transfer. Embryonic vesicles were collected from recipients uteri on day 16 and submitted for parentage testing. Signicantly, more transferred oocytes were fertilized by semen inseminated before versus after transfer (29/31 versus 2/31). Therefore, insemination before transfer was adequate when using semen from fertile stallions.

6. Transfer of oocytes matured in vitro In vitro fertilization and embryo culture are not very successful for the equine. Therefore, oocyte transfer has been used to determine the ability of oocytes, matured in vitro, to undergo fertilization and embryo development. In a study by Scott et al. (2001), oocytes were collected from small, diestrus follicles and from preovulatory follicles. Oocytes, collected from small follicles, were matured in vitro for 3638 h before transfer, while oocytes collected from preovulatory follicles were transferred immediately into a recipients oviduct. Embryo development rates after transfers were signicantly lower for oocytes matured in vitro versus in vivo, 9% versus 82%, respectively. Hinrichs et al. (2002) transferred multiple oocytes, matured in vitro, into the oviducts of recipient mares. Fertilization rates of nondegenerate oocytes were high, but of 87 recovered oocytes/embryos, only 16 had cleaved to 2 cells (18%). Results of the studies suggest that oocyte transfer can be used to evaluate fertilization and developmental competence of oocytes matured in vitro. Oocyte transfer has also been used for the transfer of oocytes collected from the ovaries of valuable mares that have been euthanized, or that have died. This procedure was initially attempted in 1999 (Carnevale et al., 2001b), but the rst foal, from ovaries transported across the country, was not produced until 2002 (Carnevale et al., 2003b). During the past 3 years in our laboratory, oocytes have been collected and transferred from the ovaries of 21 mares, with three healthy foals born and four ongoing, late-term pregnancies. 7. Gamete intrafallopian transfer (GIFT) Gamete intrafallopian transfer involves transfer of sperm, in addition to an oocyte, into a recipients oviduct. For oocyte transfer, intrauterine inseminations of high numbers of sperm are desirable (Carnevale et al., 2001c). However, for GIFT, substantially lower numbers of sperm can be used because the sperms are placed directly into the oviduct. Because low numbers of sperm are used and because sperms are placed near the site of fertilization,

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GIFT could theoretically be used to produce pregnancies from subfertile stallions, frozen semen, and sex-sorted sperm. The rst successful GIFT in the horse was achieved in 1998 (Carnevale et al., 1999a). Since that time, GIFT has been attempted with fresh, frozen, and cooled sperm. Typically, a density gradient has been used to select a population of sperms with a high percentage of motility, and to minimize debris and seminal plasma. Between 2 and 5 105 motile sperms are pulled into a transfer pipette containing the oocyte and placed into the oviduct of a recipient mare. Using fresh semen and young oocyte donors, embryo development rates after GIFT ranged from 27 to 82% (Carnevale et al., 2000; Coutinho da Silva et al., 2001). In comparison to results using fresh semen, semen that has been cooled or frozen for GIFT was less successful in establishing pregnancies. Recent studies in our laboratory using cooled and frozen semen for GIFT resulted in pregnancy rates of 25 and 8%, respectively (Coutinho da Silva et al., 2002). Projects are ongoing to investigate the causes of limited success.

8. Conclusions The minimal success of in vitro fertilization in the equine has hindered clinical and research investigations. Methods involving transfer and fertilization in vivo, however, have proven valuable. Using oocyte transfer, interactions between oocyte, sperm, and oviduct can be studied, and factors affecting oocyte viability can be determined. Oocyte transfer has also been used to produce offspring from mares that are unsuccessful oocyte donors in clinical practices. Transfer of oocyte and sperm (GIFT) into a recipients oviduct has resulted in high embryo development rates when fresh semen was used. Further development of GIFT could result in its use for cases in which sperm numbers are limited. Findings from oocyte transfer and GIFT will increase our understanding of equine gametes and oviductal function.

Acknowledgements Research in oocyte transfer and GIFT was funded by benefactors of Preservation of Equine Gametes and the Colorado Equine Racing Commission through the Research Council of the College of Veterinary Medicine and Biomedical Sciences at Colorado State University. References
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