P-101: Developmental Competence of Bovine Vitrified GV Oocyte Selected by BCB

Background: Cryopreservation of farm animal embryos is nowadays a widely used routine technique. Success in cryopreservation of form animal depends on many factors including: freezing medium, types of cryoproctants, melting process, lipid content in ooplasm and oocyte quality It has been found that immature Oocytes with reduced reproductive performance or slaughtered at the end of their use are heterogeneous in quality. It is generally believed that glucose-6-phosphate dehydrogenase (G6PDH) protein is active in the growing oocyte, but its activity is decreased in oocytes that have finished their growth phase. Then, to have achieved developmental competence. It seems the enzyme G6PDH can degrade brilliant cresyl blue (BCB). Thus, oocytes yielding decreased G6PDH (finished growth phase) showing a blue cytoplasm (BCB+) after BCB staining, while growing oocytes (unfinished growth phase) have abundant G6PDH and a colorless cytoplasm (BCB-). Materials and Methods: The oocytes were exposed to 26μM BCB and classified according to their cytoplasm coloration. After selection of Oocytes from each group, they were then vitrified by using a crytop. Cumulus-enclosed oocytes (3-5) were suspended in equilibration solution (HM containing) ethylene glycol (EG) and dimethylsulfoxide (DMSO)) for 10 minutes to allow initial shrinkage and recovery. Following equilibration, they were transferred to the vitrification solution (HM containing EG, DMSO and 0.5 M sucrose) for 45-60 seconds. After thawing, Oocytes were then matured, fertilised, and cultured in vitro for 7 days. Viability following vitrifiaction and warming, fertilisation events following IVF and subsequent preimplantation embryo development were evaluated Results: Significant differences were observed in survival rates between BCB+ and BCB- oocytes in both vitrified. Cleavage was significantly higher BCB+ vitrified oocyte (p<0.05) in in compared to BCB- oocytes (65.38 ± 3.8 vs. 34.5 ± 3.2% respectively). Blastocyst development rate was significantly higher in BCB+ vs. BCB- oocytes (13.76 ± 1.2 vs. 2.4 ± 0.88%, respectively). Conclusion: Therefore, the BCB test is a useful method for selection of more competent immature bovine oocytes for oocyte crypreservation.