Application of Ovum Pick-Up, Intracytoplasmic Sperm Injection and Embryo Culture in Equine Practice

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1 REPRODUCTIO Application of Ovum Pick-Up, Intracytoplasmic Sperm Injection and Embryo Culture in Equine Practice Silvia Colleoni, PhD; Sandro Barbacini, DVM*; Denis ecchi, DVM, Diplomate ECAR; Roberto Duchi, DVM; Giovanna Lazzari, DVM; and Cesare Galli, DVM Ovum pick-up, intracytoplasmic sperm injection and embryo culture applied to subfertile mares and stallions are safe and reliable techniques for the production of offspring; often, they are the only options available. Embryos produced outside the breeding season can be frozen for later transfer, which makes these techniques very versatile. Authors addresses: Laboratorio di Tecnologie della Riproduzione (LTR), Istituto Sperimentale Italiano Lazzaro Spallanzani, CIZ srl, Via Porcellasco 7, Cremona, Italy (Colleoni, Galli, Duchi, Lazzari); Select Breeders Services Italia, Via Argine 39, San Daniele Po (CR), Italy (Barbacini, ecchi); and Dipartimento Clinico Veterinario, Università di Bologna, Via Tolara di Sopra 50, Ozzano Emilia, Italy (Galli); AAEP. *Presenting author. 1. Introduction For many years, artificial insemination was the most widely used assisted reproductive technique (ART) in the horse-breeding industry. 1 However, other procedures based on the in vivo and in vitro production of equine embryos have emerged in recent years. Reasons for the delayed development of in vivo and in vitro production of equine embryos include the scarce availability of abattoir ovaries and the lack of interest from horse breeders and registries. Moreover, in spite of the early success of equine oocyte in vitro maturation, 2 no relevant research on the issue has been reported in the horse for some time. Instead, many efforts concentrated on conventional in vitro fertilization (IVF). This method was successful in other species but not in horses; only two foals were reported to be born from IVF, and both were derived from in vivo matured oocytes collected by ovum pick-up (OPU) from gonadotrophin-stimulated mares. 3,4 The application of intracytoplasmatic sperm injection (ICSI) to the horse has overcome the barrier of inefficient IVF; ICSI produced the first pregnancy derived from an in vitro matured oocyte, 5 and it was successfully carried to term. In recent years, a few other scientific reports showed that it is possible to obtain pregnancies and live foals after collection of immature oocytes by OPU with in vitro maturation, ICSI, and immediate transfer 6,7 or with in vitro embryo culture (IVC) and non-surgical transfer of blastocyst-stage embryos. 8 Today, the most practical use of OPU or transabdominal follicle aspiration is to recover in vivo ma- OTES Vol. 53 AAEP PROCEEDIGS

2 REPRODUCTIO tured oocytes 9 for oocyte transfer. It has been shown that the use of this procedure results in satisfactory pregnancy rates, except when intrinsically compromised oocytes are collected from geriatric mares. 10,11 OPU has been proven safe and repeatable in mares. 8,12,13 When used in combination with in vitro maturation and ICSI, OPU has the considerable advantage of not requiring any hormonal stimulation of the donor; this aspect is of particular importance in mares, because superovulation still gives unsatisfactory and inconsistent results. Another advantage of using OPU, ICSI, and IVC is the possibility to widen the choice of stallions to be used, including those with poor sperm motility and reproductive performance in vivo. 14 Finally, an important application of OPU, ICSI, and IVC is rescuing the fertility of aged donors. It is well known 10,15,16 that mare fertility decreases after yr of age. In fact, mature mares can be more easily affected by many reproductive problems that can arise from abnormalities of the external genitalia, altered neuroendocrine system functionality, ovarian or uterine pathologies, or anomalies related to aging. It has also been reported 17 that endometritis has more deleterious effects on aged mares than on young animals. A previous study conducted in Italy on five experimental mares provided the first large dataset on the application of OPU, ICSI, and IVC for in vitro embryo production. 8 A total of 54 consecutive OPU sessions were carried out at 10- to 15-day intervals. An average of 6.6 follicles per mare per session were aspirated, and 3.2 oocytes were retrieved (48% recovery rate). Overall, 141 oocytes were cultured, and 101 (71.6%) reached metaphase II after in vitro maturation. The matured oocytes were fertilized by ICSI. There were 74 (52%) cleaved embryos that were either transferred into a sheep oviduct after cleavage and recovered on day 7 (day 0 fertilization day) or cultured in vitro. During that study, a total of 32 blastocysts (0.71/OPU) were obtained. On the basis of these preliminary data, the objective of this study was to investigate the possible practical applications of OPU, ICSI, and IVC in the mare. We present here the results from our study in a commercial setting over a 4-yr period. 2. Materials and Methods Donor Animals This paper analyses data obtained from 47 commercial OPU sessions performed from 2004 to 2007 on 30 donor mares aged 3 24 yr. The majority of the mares (21) were Warmblood, but there were also 4 Quarter Horses, 1 Paint Horse, 2 Standardbred, and 2 Arabian Thoroughbreds. Frozen-thawed semen from 27 stallions of varying quality and fertility was used. During the breeding season, mares were subjected to OPU in diestrus in the absence of a dominant follicle if at all possible. OPU, ICSI, and Embryo Culture Oocytes were collected by transvaginal ultrasoundguided follicular aspiration (OPU) from donor mares as previously described. 8 Briefly, all ovarian follicles ranging from 0.5 to 4.0 cm in diameter were aspirated using a 12-gauge coaxial double lumen needle connected to an aspiration pump. a The recovered oocytes were then matured in vitro by culturing them in TCM199- or DMEM-F12 based medium as described by Galli et al. 18 The oocytes reaching the metaphase II stage were in vitro fertilized by ICSI and in vitro cultured up to the blastocyst stage as described. 18 The blastocysts were either transferred fresh or frozen in 10% glycerol and subsequently, were stored in liquid nitrogen. Embryos were frozen at day 6, 7, 8, or 9 (day 0 is the day of ICSI) when the blastocyst stage was achieved. Embryos were transferred non-surgically to recipient mares 4 6 days (preferably at 5 days) after spontaneous ovulation Results A total of 808 follicles were aspirated, and 474 oocytes were recovered with a mean recovery rate of 58.19% (10.08 oocytes per OPU). Of these, 66.05% reached metaphase II stage and were fertilized by ICSI, which resulted in 199 cleaved embryos and 40 blastocysts (0.85 per OPU, ICSI, and IVC session). To date, 18 embryos were non-surgically transferred and 15 recipient mares were detected pregnant by ultrasonography performed 8 days posttransfer. Three pregnancies resulted in early embryonic losses before 30 days of gestation, and two more were lost before 60 days of gestation. Four pregnancies are ongoing. Six foals were born. One foal was lost at birth, and one foal died at 10 days after a neonatal septicemia (Table 1). The results were also evaluated in more detail by focusing on several aspects that could have influenced the outcome of the study: Age of donor mares Impact of reproductive disorders Use of frozen semen with poor post-thaw quality and/or with low in vivo fertility Use of transitional versus cycling mares Use of performance mares versus non-performance mares. Moreover, results achieved by OPU, ICSI, and IVC were compared with those obtained by conventional embryo transfer when data were available for individual mares for direct comparison. Age of Donor Mares In this study, donor mares were divided in two age groups: group 1 included mares 14 yr (n 17), and group 2 included mares 14 yr (n 13). Thirty-two OPU sessions were performed on group 1, and 15 were performed on group 2; 319 (10 oocytes per OPU) and 155 oocytes (10.3 per OPU), respec- AAEP PROCEEDIGS Vol

3 REPRODUCTIO Table 1. Summary Data on the Application of OPU-ICSI In Vitro Embryo Culture to a Group of 30 Mares Belonging to a Commercial OPU Program Donor o. Age (yr) OPUs follicles oocytes Recovery Rate Injected Percent MII Cleaved Cleavage rate (%) Blastocysts Percent Blastocysts/ Injected Blastocysts Transferred Recipients Pregnant at 14 Days Foals Born T T Due in T T T T Due in 2007 Due in 2007 Total Per OPU Per mare T, not transferred Vol. 53 AAEP PROCEEDIGS

4 REPRODUCTIO Table 2. Embryo Production by OPU-ICSI From Performance and on-performance Mares Donors OPUs Follicles Oocytes Recovery (%) MII MII (%) Cleaved Cleavage (%) Blastocysts Percent Blastocysts/ Injected Performance mares a a a on-performance mares b b b 2 test, number within columns with different letters differ (p 0.05). tively, were collected from the two groups of mares. o difference in oocyte morphology was observed at collection between the two groups. At the end of in vitro maturation, 210 oocytes of the 319 (65.83%) collected from group 1 reached metaphase II stage (MII stage). They were injected and produced 27 blastocysts (12.85%). In group 2, 103 oocytes from the aged mares reached MII stage (66.45%) and produced 12 blastocysts (11.65%). Impact of Reproductive Disorders The data were analyzed by dividing the animals subjected to ICSI into groups showing or lacking reproductive problems. Eight of the thirty donor mares included in the program had reproductive disorders that limited their ability to establish or carry a pregnancy to term. One mare (donor 21) presented an irreparable cervical laceration, two mares (donors 3 and 7) had degenerative endometriosis, one mare (donor 29) had partial uterine aplasia, two mares (donors 15 and 19) produced abnormal oocytes that could not even reach the MII stage, and two mares (donor number 6 and 23) were repeat breeders (Table 1). Fifteen OPU sessions were performed on mares with reproductive problems, and 32 were performed on reproductively sound subjects. When OPU was performed on the mares with reproductive problems, 234 follicles were aspirated, and 122 oocytes were collected and matured (8.13 per OPU). In the reproductively sound group, 574 follicles were aspirated, and 352 oocytes were collected and matured (11 per OPU). In the first group, 73 oocytes were injected and 55 (73.34%) cleaved, whereas 240 were injected and 144 (60%) cleaved in the second group. Surprisingly, cleavage rate was significantly higher when OPU and ICSI were carried out on mares showing reproductive disorders (p 0.017). onetheless, there was no significant difference in percentage of cleaved oocytes reaching the blastocyst stage between the two groups (15.06% vs % corresponding to 0.73 vs blastocysts per OPU; p 0.44). Use of Frozen Semen With Poor Post-Thaw Quality and/or Low In Vivo Fertility We analyzed the results by dividing the semen used for ICSI into two groups: a subfertile category (5 stallions) that included frozen semen with post-thaw motility 15% or a history of no pregnancies obtained, and a fertile group that included semen of good quality and fertility (22 stallions). Cleavage rate was 62.96% when semen of poor post-thaw motility and/or fertility was used and 63.79% when semen of good quality and fertility was used. The embryo development rates (12.34% vs %) were similar (p 0.89). Transitional Versus Cycling Mares In this study, we considered three periods: reproductive season (from March to September), fall transition (from October to December) in which reproductive activity decreases, and spring transition (January and February) in which reproductive activity gradually increases. Our data suggest that in vitro embryo production can be performed both during the breeding season and during the fall- and spring-transition periods without affecting oocyte maturation (65.86%, 61.32%, and 73.07%, respectively), cleavage rate (64.39%, 56.92%, and 68.42%, respectively), or embryo development (11.51%, 12.30%, and 17.54%, respectively). Performance Mares Versus on-performance Mares In our study (Table 2), we found a large difference between the mares engaged in intense sporting activities (n 8) and the mares at rest (n 22). Statistically significant differences were observed in oocyte maturation (57.03% vs %; p 0.009), cleavage rate (51.94% vs %; p 0.01), and embryo development (3.89% vs % for sporting and resting mares, respectively; p 0.008). These results suggest that the intense training and stressful conditions experienced by sporting mares may have a detrimental effect on their reproductive performance. Comparison Between OPU and Conventional Embryo Transfer Five donor mares included in this study previously underwent conventional flushing before the OPU, ICSI, and IVC method was used. The data are shown in Table 3. In total, six embryos were obtained from 30 flushes, and seven embryos were obtained from the OPU, ICSI, and IVC method. In particular, two mares (donors 23 and 26) were subjected to a total of 22 flushes, but they produced only two embryos. By contrast, the same donors produced 3 blastocysts after three OPU and ICSI embryo-culture sessions. AAEP PROCEEDIGS Vol

5 REPRODUCTIO Table 3. Comparison Between Embryo Production by Conventional Flushing or OPU-ICSI From Five Donor Mares Donor o. Age (yr) Technique Flushings or OPUs Recovered or Produced Embryos 4 20 Flushing 1 1 OPU-ICSI Flushing 1 0 OPU-ICSI Flushing 10 0 OPU-ICSI Flushing 6 3 OPU-ICSI Flushing 12 2 OPU-ICSI Discussion In this study, the techniques of OPU, ICSI, and IVC were applied to a group of mares of different ages, reproductive soundness, and physical activity using stallion semen of varying in vivo fertility. The analysis of the results has taken into account all these aspects. Our results show that in vitro oocyte maturation, cleavage rate, and subsequent embryo development are not influenced by the age of donor mares. This suggests that the application of in vitro embryo production can resolve most of the problems related to aging of the reproductive tract. Another conclusion from this study is that in vitro embryo production is a helpful technique in obtaining offspring from subfertile mares with reproductive disorders. We found no relation between semen quality and the rate of embryo production after ICSI. This finding is in agreement with previous studies 14,19 in which the use of a stallion s semen of low sperm motility and/or low in vivo fertility did not influence the in vitro cleavage rate or the development to the blastocyst stage. The horse is a seasonally polyestrous breeder, and in the northern hemisphere, mares enter into anestrus during the late fall and start the breeding season in the early spring when daylight and temperature increase. 20 The effect of the season on in vitro maturation was previously analyzed, and that study showed that meiotic competence does not change between seasons. 21 In this study, we found no relation between season and embryo production. This finding is of enormous importance in an applied context, because it allows the production of embryos that can be frozen for later transfer. In summary, although the number of donors was limited, the parameters used to categorize the donor mares and stallions did not seem to have an effect on the outcome. An exception worth noting is the finding that the less productive donors were young performance mares. A previously published technique by Carnevale 9 and Carnevale and Maclellan 22 allows achievement of similar results. However, there are several advantages in the approach described in this paper. First, the production of embryos at the blastocyst stage that can be transferred non-surgically to a recipient mare is more acceptable from an animal welfare point of view. 23 Second, the embryos produced in vitro can be successfully frozen. This is particularly important, because it allows production of embryos outside the breeding season when recipients are not cycling. Third, it is possible to use stallions who have low-quality semen or semen that is available in very small amounts, which were previously insufficient for insemination. Fourth, there is no need for a herd of recipients, because the embryos, when frozen, can be returned to the owner and transferred into his/her recipients. Finally, there is a real opportunity to develop a market for frozen equine embryos like there is for other domestic species. We can conclude that OPU, ICSI, and embryoculture techniques applied to a variety of subfertile mares and stallions are safe; they provide a viable option for the production of offspring and in many cases, represent the only option available. Mares can be repeatedly subjected to consecutive OPU sessions without any side effects. 8 Embryos produced outside the breeding season are frozen for later transfer, which makes these techniques very flexible and versatile in a clinical setting. Pregnancy rates after non-surgical embryo transfer have been similar to those obtained by conventional embryos collected after uterine flushing. Overall, this study shows that the OPU, ICSI, and IVC method offers a real opportunity for breeding valuable stock in the equine practice. The authors are grateful to Gabriella Crotti Paola Turini and Massimo Iazzi for excellent technical assistance and to Gaetano Mari for helpful discussions. Part of this work was funded by the Italian Ministry for Education, Universities and Research (FIRS grant TECLA). References and Footnote 1. Squires EL. Integration of future biotechnologies into equine industry. Anim Reprod Sci 2005;89: Zhang JJ, Boyle MS, Allen WR, et al. Recent studies on in vivo fertilization of in vitro matured horse oocytes. Equine Vet J 1989;8(Suppl): Palmer E, Bezard J, Magistrini M, et al. In vitro fertilization in the horse. A retrospective study. J Reprod Fertil 1991;44(Suppl): Bezard J. In vitro fertilization in the mare, in Proceedings. International Scientific Conference on Biotechnics in Horse Reproduction Squires EL, Wilson JM, Kato H, et al. A pregnancy after intracytoplasmic sperm injection into equine oocytes matured in vitro. Theriogenology 1996;45: Cochran R, Meintjes M, Reggio B, et al. Live foals produced from sperm-injected oocytes derived from pregnant mares. J Equine Vet Sci 1998;18: McKinnon AO, Lacham-Kaplan O, Trounson AO. Pregnancies produced from fertile and infertile stallions by intracytoplasmic sperm injection (ICSI) of single frozen-thawed spermatozoa into in vivo matured mare oocytes. J Reprod Fertil 2000;56(Suppl): Vol. 53 AAEP PROCEEDIGS

6 REPRODUCTIO 8. Galli C, Crotti G, Turini P, et al. Frozen-thawed embryos produced by ovum pick up of immature oocytes and ICSI are capable to establish pregnancies in the horse. Theriogenology 2002;58: Carnevale EM. Oocyte transfer and gamete intrafallopian transfer in the mare. Anim Reprod Sci 2004;82: Carnevale EM, Ginther OJ. Defective oocytes as a cause of subfertility in old mares. Biol Reprod Monogr 1995;1: Carnevale EM, Coutinho da Silva MA, Panzani D, et al. Factors affecting the success of oocyte transfer in a clinical program for subfertile mares. Theriogenology 2005;64: Bogh IB, Brink P, Jensen HE, et al. Ovarian function and morphology in the mare after multiple follicular puncture. Equine Vet J 2003;35: Vanderwall DK, Hyde KJ, Woods GL. Effect of repeated transvaginal ultrasound-guided follicle aspiration on fertility in mares. J Am Vet Med Assoc 2006;228: Lazzari G, Crotti G, Turini P, et al. Equine embryos at the compacted morula and blastocyst stage can be obtained by intracytoplasmic sperm injection (ICSI) of in vitro matured oocytes with frozen-thawed spermatozoa from semen of different fertilities. Theriogenology 2002;58: Hemberg E, Lundehim, Einarsonn S. Reproductive performance of Thoroughbred mares in Sweden. Reprod Domest Anim 2004;39: Madill S. Reproductive consideration: mare and stallion. Vet Clin orth Am [Equine Pract] 2002;18: Hurtgen JP. Pathogenesis and treatment of endometritis in the mare: a review. Theriogenology 2006;66: Galli C, Colleoni S, Duchi R, et al. Developmental competence of equine oocytes and embryos obtained by in vitro procedures ranging from in vitro maturation and ICSI to embryo culture, cryopreservation and somatic cell nuclear transfer. Anim Reprod Sci 2007;98: Choi YH, Love CC, Varner DD, et al. Equine blastocyst development after intracytoplasmic injection of sperm subjected to two freeze-thaw cycles. Theriogenology 2006;6: agy P, Guillaume D, Daels P. Seasonality in mares. Anim Reprod Sci 2000;60 61: Bruck I, Grondahl C, Host T, et al. In vitro maturation of equine oocytes: effect of follicular size, cyclic stage and season. Theriogenology 1996;46: Carnevale EM, Maclellan LJ. Collection, evaluation, and use of oocytes in equine assisted reproduction. Vet Clin orth Am [Equine Pract] 2006;22: Hinrichs K. Update on equine ICSI and cloning. Theriogenology 2005;64: a Cook Veterinary Products, Queensland 4113, Australia. Vol. 53, o. 1 AAEP PROCEEDIGS Vol