84 Effect of Embryo Stage and Cryopreservation Method on Pregnancy Rates Obtained Following the Transfer of In Vivo-Derived Ovine Embryos on Small-Scale Farms in Thailand

Abstract

Assisted reproductive technologies including superovulation, laparoscopic AI (LAI), and embryo transfer (ET) are important tools for genetic improvement in the sheep industry. The present study aimed to determine the effects of embryo stage and cryopreservation method on field trial outcomes of embryo transfer on small-scale farms in Thailand. Black Dorper ewes (n = 16) were used as donors and mixed breed ewes (n = 21) were used as recipients. Donors were superovulated as previously described (Tríbulo et al. 2012 Theriogenology 77, 1679-1685, 10.1016/j.theriogenology.2011.12.013) and inseminated by LAI within 22 to 24 h after standing heat (Day 0). Donors females were flushed on Day 2 to recover 2- to 8-cell embryos (n = 8) or on Day 6 to recover blastocyst-stage embryos (n = 8). Recovered embryos were randomly cryopreserved by either slow freezing or vitrification methods. Into 21 recipients was inserted an intravaginal device impregnated with CIDR® that was left in place for 10 days. Those received 300 IU of pregnant mare serum gonadotropin (PMSG) on Day 9 and randomly assigned to receive embryos on either Day 2 or 6 after oestrus. Two- to 8-cell embryos were thawed and transferred into the ipsilateral oviduct (n = 1-5 embryos/recipient) on Day 2. Five recipients received 15 slow-frozen embryos and 5 received 13 vitrified embryos. Blastocyst stage embryos were thawed and transferred into the ipsilateral uterine horn (n = 1-4 embryos/recipient) on Day 6. Five recipients received 11 slow-frozen embryos and 4 received 7 embryos. Pregnancy diagnosis was determined by ultrasonography 45 days after embryo transfer. Pregnancy rate was calculated as the proportion of ewes with at least one pregnancy out of the total number of ewes that received embryos. Chi-squared analysis was used to determine the effects of embryo freezing technique and embryo stage on pregnancy rate (SAS 9.2, SAS Institute Inc., Cary, NC, USA). All donor ewes responded to the superovulation program (donor had 3 to 6 corpora lutea). The mean number of viable embryos recovered was 4.3 ± 2.4 and 3.1 ± 3.7 for Day 2 and Day 6, respectively. Nineteen of the 21 recipient ewes responded to the synchronization program and received embryos in the study. There was no effect (P > 0.05) of embryo stage (5/10 = 50% v. 3/9 = 33.3% for 2- to 8-cell v. blastocyst, respectively) or cryopreservation method (4/10 = 40% v. 4/9 = 44.4% for slow-freezing and vitrification, respectively) on pregnancy rate following embryo transfer. Results of the present study suggest that similar pregnancy rates following embryo transfer in sheep under tropical conditions in Thailand can be obtained using either 2- to 8-cell embryos or blastocyst-stage embryos and with embryos that have been cryopreserved by slow-freezing or vitrification. Further research with larger numbers of animals is necessary to confirm the preliminary results of the present study.