81 VITRIFICATION OF IMMATURE PORCINE CUMULUS–OOCYTE COMPLEXES IN A CHEMICALLY DEFINED SOLUTION

Abstract

A high concentration of serum supplements has been contained in basic vitrification solutions to protect plasma membranes. The objective of this study was to examine if vitrification of immature porcine oocytes could be achieved successfully in a chemical-defined solution containing 0.6 mg mL^–1 hydroxypropyl cellulose (HPC). Cumulus–oocyte complexes (COC) were aspirated from follicles 3 to 6 mm in diameter in abattoir-derived porcine ovaries. The COC or denuded oocytes were vitrified according to a commercial protocol of Cryotop (Kitazato BioPharma Co. Ltd., Shizuoka, Japan) with original solutions or modified ones that had serum supplement replaced with HPC. After vitrification and warming, viabilities of oocytes and cumulus cells were evaluated under a fluorescent microscope after staining with fluorescein diacetate and propidium iodide (Table 1). Statistical analyses of results from 4 replicated trials were performed by ANOVA with a Bonferroni/Dunn post hoc test (significance, P < 0.05). Although viabilities of vitrified/warmed oocytes in all groups were significantly lower (82.7–89.1%) than those of fresh controls (99.5%), there were no significant differences among vitrified groups. When COC were vitrified, viability of cumulus cells (54.3%) in HPC group was not different from that of nonvitrified controls (72.5%) but higher than that in the original solution group (48.1%). In conclusion, these results demonstrate that HPC rather than serum supplement could be a suitable chemically defined supplement for vitrification of immature porcine COC.

Table 1.  Viabilities of vitrified/warmed porcine oocytes and cumulus cells¹