216 PRONUCLEAR FORMATION AND DEVELOPMENTAL COMPETENCE OF MATURE PORCINE OOCYTES DERIVED FROM SMALL- AND MEDIUM-SIZED FOLLICLES

Abstract

The maturation rate of oocytes derived from small follicles (SF) is known to be lower than that of oocytes from medium follicles (MF). The objective of this study was to assess the fertilizability and developmental competence of mature SF oocytes that were selected by the presence of the first polar body. Cumulus–oocyte complexes (COC) were aspirated from SF (1 to 2 mm in diameter) or MF (3 to 6 mm in diameter) of prepuberal ovaries. The COC were cultured in modified porcine oocyte medium supplemented with gonadotropins and dibutyryl cAMP for the first 20-h period and then in gonadotropin-free and dibutyryl cAMP-free porcine oocyte medium for another 24 h. Following IVM culture, mature oocytes with the first polar body were selected under a stereomicroscope, co-incubated with spermatozoa in a drop of modified TCM-199 containing 0.4% BSA and 5 mM caffeine for 6 h, and then incubated in porcine zygote medium-5 for 7 days. Sperm penetration, cleavage, and early development of the oocytes were examined before culture in porcine zygote medium-5 on Days 2 and 7 of culture. To analyse the fertilizability and developmental competence of oocytes from the SF and MF groups, sperm penetration, pronuclear formation, cleavage, blastocyst formation, and mean cell number in a blastocyst (as determined by fluorescence observation following Hoechst 33342 staining) were examined. Statistical analysis was performed by ANOVA with a Bonferroni-Dunn post-hoc test (P < 0.05). The percentages of oocytes in which the first polar body could be observed were 51.0 ± 4.5% and 78.5 ± 2.8% for SF- and MF-oocytes, respectively, whereas the maturation rates were 83.8 ± 4.0% and 62.8 ± 4.4% following fixation and staining. When only mature oocytes were co-cultured with sperm for 6 and 9 h, sperm penetration, monospermic penetration, and pronuclear formation were not different (P > 0.33) between mature SF- and MF-oocytes. Although there was no difference in cleavage rates between the mature SF- and MF-oocyte groups, blastocyst formation rate and mean cell number in the blastocyst were higher in mature MF-oocytes (31.0 ± 3.6% and 38.7 ± 1.9 cells, respectively) than in mature SF-oocytes (14.7 ± 3.2% and 31.2 ± 2.0 cells). From these results, we conclude that mature oocytes derived from SF have a similar fertilizability when compared with mature MF-oocytes, but the developmental competence to the blastocyst stage following IVF is significantly lower in mature SF-oocytes than in mature MF-oocytes.