116 DEVELOPMENT INTO BLASTOCYSTS OF SWAMP BUFFALO OOCYTES AFTER VITRIFICATION AND NUCLEAR TRANSFER

Abstract

Oocyte cryopreservation in the domestic species is still at the experimental stage, but recent studies indicated that vitrification characterized by ultra-rapid cooling rate is promising for cryopreservation of bovine oocytes. In the present study, denuded buffalo oocytes were vitrified by minimum volume cooling procedure (Kuwayama and Kato, 2000, J Assist Reprod Genet 17, 477) after IVM or after IVM and enucleation, and developmental potential into blastocysts of the post-warm oocytes after somatic cell nuclear transplantation was examined. Cumulus-oocyte complexes were matured, denuded, and enucleated as described previously (Parnpai et al., 1999, Buffalo J 3, 371–384). The presumptive metaphase-II (M-II) oocytes before and after enucleation were first equilibrated in 7.5% DMSO + 7.5% ethylene glycol + 20% FCS in TCM199 for 10 min, and then exposed to 15% DMSO + 15% ethylene glycol + 0.5 M sucrose + 20% FCS in TCM199 for 1 min. Five oocytes were placed on a Cryotop sheet (Kitazato Supply Co., Tokyo, Japan) and vitrified in liquid nitrogen. The samples were warmed in 0.5 M sucrose solution for 5 min, directly transferred into TCM199 + 20% FCS, and kept at room temperature for 1 h before being used for a cloning experiment. The post-warm oocytes were fused with ear skin fibroblasts by two DC pulses (26 V, 17 μs) and activated with 7% ethanol for 5 min and then 10 μg/mL cycloheximide and 1.25 μg/mL cytochalasin-D for 5 h. The reconstructed embryos were cultured in mSOFaa + 0.2% BSA + 0.1% linoleic acid albumin for 2 days, and then co-cultured with bovine oviductal epithelial cells for an additional 5 days. Post-warm morphological survival of M-II oocytes (80%, 187/235) was similar to that of enucleated oocytes (75%, 158/212). Vitrified M-II oocytes were successfully enucleated (96%, 136/142) as were fresh control oocytes (88%, 143/162). Fusion rates of M-II oocytes vitrified before and after enucleation (81%, 94/116 and 78%, 106/136, respectively) were also similar to those of fresh oocytes (81%, 100/123). Percentages of reconstructed embryos developing into hatching blastocysts on Day 7 were 5% (5/91), 6% (6/103), and 8% (8/99) in the groups of oocytes vitrified before and after enucleation, and of fresh control oocytes, respectively (ANOVA tests were not significant different). These results indicate that swamp buffalo oocytes cryopreserved by ultra-rapid vitrification procedure can be used successfully for subsequent somatic cell nuclear transplantation. (Supported by Thailand Research Fund and R&D Fund of Suranaree University of Technology)