335 EFFECT OF MEM VITAMINS AND FORSKOLIN ON IN VITRO EMBRYO PRODUCTION AND SOPS-VITRIFICATION ABILITY OF IN VITRO DERIVED PORCINE BLASTOCYSTS

Abstract

The aims of this study were, first to investigate the effect of minimum essential medium (MEM) vitamins (VITs) during IVM of porcine oocytes on in vitro embryo production of porcine embryos and second, to determine if the addition of VITs during IVM and the chemical delipidation with forskolin improve the vitrification ability of in vitro-derived porcine blastocysts. COCs were divided in two groups and matured in NCSU-23 for 44 h with 0.05% VITs (V group) or without VITs (NV group). Matured oocytes were inseminated with frozen-thawed spermatozoa. Presumptive zygotes were cultured for 16 h to assess IVF parameters (N = 767) or for 6 days (N = 2858) to evaluate the in vitro embryo development (Day 0 = day of IVF). On Day 5, some embryos from NV and V groups were cultured for 24 h with 10 μM forskolin (NVF and VF groups). The remaining embryos were cultured without forskolin (NVNF and VNF groups). On Day 6, embryos from the four experimental groups were assessed for blastocysts formation and some blastocysts (N = 414) were vitrified using superfine open pulled straws (SOPS). Vitrified blastocysts were warmed (one-step dilution method) and cultured for 24 h to assess their viability. Blastocysts with totally or partially reformed blastocoel cavity and normal or excellent morphology were considered viable. Five replicates of a 2 × 2 factorial design were conducted. Data was analyzed with the MIXED procedure. The threshold for significance was set at P < 0.05. Results are expressed as least squares means ± SEM. No differences were observed in the maturation of COCs treated with VITs (92.7 ± 1.3%) and non-treated COCs (94.3 ± 1.3%). The NV and V maturation groups showed similar penetration (83.0 ± 3% and 82.6 ± 3%, respectively) and monospermy (48.9 ± 6%, and 48.3 ± 6%, respectively) rates. The rate of monosermic oocytes related to the total of analyzed oocytes was similar for NV (38.1 ± 3.7%) and V (38.4 ± 3.7%) groups. The values of cleavage rate on Day 2 were similar for NV (66.7 ± 1.5%) and V (69.6 ± 1.6%) embryos. The addition of VITs to IVM medium improved (P < 0.01) up to 10 points the blastocysts formation rate, but the addition of forskolin at Day 5 did not affect this parameter. The V group showed a higher (P < 0.01) blastocysts rate (45.1 ± 2.5%) than the NV group (38.4 ± 2.3%).The addition of VITs did not affect the survival of in vitro-derived blastocysts after SOPS-vitrification on Day 6. However, the blastocysts cultured for 24 h with forskolin showed higher (P < 0.05) viability (44.0 ± 7.9%) after vitrification and warming than those embryos cultured without forskolin (34.1 ± 7.8%). In conclusion, the addition of MEM vitamins to IVM medium improves the blastocysts formation rate and the chemical delipidation with forskolin improve the cryosurvival of SOPS-vitrified porcine in vitro-derived blastocysts. This study was supported by the Seneca foundation of Murcia (GERM 04543/07), MICINN (AGL2009-12091 and RC-2007), and CARM (2I05SU0012).