112 EFFECT OF THE IVM PROTOCOL OF BOVINE OOCYTES ON SURVIVAL RATES AFTER VITRIFICATION BY OPEN PULLED STRAW METHOD

Abstract

The meiotic stage and the cryopreservation protocol influence the ability of the oocytes to survive cryopreservation. The in vitro maturation (IVM) methods affect nuclear and cytoplasmic maturation and, consequently, the developmental competence of the oocytes. On the other hand, the cytoplasm of the bovine oocyte contains large amounts of lipids which, as demonstrated in the bovine embryo (Díez et al., 2001 Theriogenology 55; 923–936), can negatively affect post-thaw survival. The aim of this work was to analyze the effect of fetal calf serum (FCS) during IVM on the freezability of the bovine metaphase II oocyte. Cumulus-oocyte complexes (COCs) were recovered from slaughterhouse ovaries. Oocytes with compact cumulus cells and evenly granulated cytoplasm were matured for 22 h in TCM199, NaHCO₃, FSH, LH and 17βestradiol. Approximately half of the oocytes were allowed to mature in 10% FCS, and the remainder were matured in polyvinyl-alcohol (PVA; 0.3 g L⁻¹). For vitrification, oocytes were matured for 22 h, partially denuded of cumulus cells, and then vitrified (v-FCS and v-PVA) by the OPS system (Vajta et al. 1988 Mol. Reprod. Dev. 51; 53–58). Fresh untreated controls (c-FCS and c-PVA) were allowed to mature for 24 h and immediately fertilized in modified TALP medium with swim-up separated sperm, and cultured. After warming and dilution, vitrified oocytes were cultured in IVM medium for 2 h and then fertilized (Day 0). Presumptive zygotes with normal morphology were cultured in SOFaa + amino-acids + myo-inositol + 5% FCS (Day 3), and oocytes with a degenerated appearance were counted and discarded. Data were analyzed by ANOVA and Duncan’s test. Results are shown in the Table 1. After warming, we observed severe cryodamage in both v-FCS and v-PVA groups. Rates of degenerated oocytes were 17.8 ± 9.6 and 12.0 ± 9.6 for v-FCS and v-PVA groups, respectively (P > 0.05). The presence of PVA instead of FCS did not improve the blastocyst rates obtained from vitrified/warmed oocytes. The use of PVA during IVM (c-PVA) yielded lower (P < 0.05) blastocyst rates compared to the FCS control (c-FCS). Ultrastructural studies are in progress to analyze alterations in meiotic spindle, cytoplasmic organelles and cortical granules as possible causes of reduced oocyte competence after vitrification. Supported by CICYT, AGL2001-379.

Table 1