129 EFFECT OF DEVELOPMENTAL STAGE OF ICSI-PRODUCED EQUINE EMBRYOS ON PREGNANCY RATES

Abstract

Intracytoplasmic sperm injection (ICSI) can be used to produce offspring from mares or stallions with fertility problems. Early embryos can be transferred into recipients’ oviducts or embryos can be cultured for nonsurgical transfer into recipients’ uteri. The aim of this research was to evaluate the optimal time to transfer ICSI-produced embryos into recipients’ uteri. The objective was to compare pregnancy rates after the nonsurgical transfer of early morulae, compact morulae, and blastocysts. Oocytes were collected by transvaginal, ultrasound-guided follicular aspirations between 20 and 24 h after administration of deslorelin (1.5 mg, i.v., Francks Pharmacy, Ocala, FL, USA) to donors. Oocytes were cultured for 16 to 18 h in M199 (Invitrogen, San Jose, CA, USA) with 10% FCS (HyClone, Logan, UT, USA), 0.2 mm pyruvate (Sigma, St. Louis, MO, USA) and 25 μg mL^–1 gentamycin (Sigma, St. Louis, MO, USA) at 38.5°C and 6% CO₂. Cumulus cells were denuded by gentle pipetting, after oocytes were placed into hyaluronidase (500 U mg^–1, Sigma, St. Louis, MO, USA). Oocytes were injected with a single sperm from one of two stallions, with sperm being frozen or sex-sorted and refrozen (Squires EL et al. 2008). Forty-six of 62 (74%) injected oocytes cleaved. The presumptive zygotes were cultured in DMEM/F12 (Sigma, St. Louis, MO, USA) with 10% FCS at 38.5°C and an atmosphere of 5% CO₂, 5% O₂ and 90% N₂. Embryos were placed in fresh medium every 3 days. Injected oocytes were observed for cleavage at 2 days, and embryos were assigned to a transfer group. Embryos were transferred as early morulae (EM, 8-cell to precompaction stage, n = 14), compact morulae (CM, postcompaction, n = 10) or blastocysts (B, observed blastocoele, n = 9) into recipients at 3 to 5 days (EM), 3 to 6 days (CM) or 5 to 6 days (B) after the recipient’s ovulation or follicle aspiration. Pregnancy scans were performed on Day 12, 14, and 16 after ICSI, and pregnant recipients were examined until 30 days to detect the embryo proper and heartbeat. Number of embryonic vesicles detected per transferred embryo was determined by Fisher’s Exact Test. Pregnancy rates differed (P = 0.0017) among groups (EM, 1/14, 7%; CM, 4/10, 40%; B, 7/9, 78%), with fewer (P = 0.001) EM than B resulting in embryonic vesicles; however, pregnancy rates were not significantly different between CM and other embryo stages. An embryo proper with heartbeat was observed for all pregnancies, with the exception of one pregnancy resulting from the transfer of a blastocyst. In this study, all blastocysts were transferred prior to the embryo attempting to hatch from the zona pellucida, but after the appearance of a distinct blastoceole. In our study, pregnancy rates were higher after the transfer of later v. earlier stages of embryo development.