P-81: Distinct Epigenetic Status of Fully Grown and Growing Oocytes Selected by BCB Staining

Background: Since the sheep ‘Dolly’ was successfully cloned by somatic cell nuclear transfer (SCNT), the inefficiency of SCNT cloning and abnormality of cloned animals have been a focus of considerable controversy. Oocyte quality has been shown to contribute to poor somatic cloning and cell reprogramming efficiency with many factors affecting oocyte quality and developmental potential. It has been found that immature oocytes with reduced reproductive performance or slaughtered at the end of their use are heterogeneous in quality. Then selecting immature oocyte is necessary and important for embryos laboratory. 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 likely to have achieved developmental competence. The enzyme G6PDH can degrade brilliant cresyl blue (BCB). Thus, oocytes yielding decreased G6PDH (finished growth phase) show 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. However, to address the effects of oocyte selection by BCB staining on the nuclear reprogramming capacity of oocytes, embryo quality, we analyze developmental competence, status of epigenetic MII oocyte modifications, embryo quality assessment. Results: Levels of DNA Methylation, H3K9ac and H3K4me3 in BCB + group was significantly higher (89.19±1.49), (90.08±2.18), (98.53±2.62) in compare to the BCB- group (81.95±1.27), (78.73±2.16), (80.32±3.82). The percentage of Parthenogenetic blastocyst development was higher in BCB+ and was significantly different between BCB+ (39.25±9.03) and BCB- (23±7.48) oocytes. Total cell number, TE cell number, ICM cell number: TE ratio of blastocysts were significantly higher in the BCB+ group compared with the BCB- group. Conclusion: Therefore, the BCB test is a useful method for selecting more competent immature sheep oocytes and it made improve Scnt method.