P-100: Improved Efficiency of Somatic Cell Nuclear Transfer in Sheep by Persecution of Recipient Oocytes with Brilliant Cresyl Blue Staining

Background: The most important factor affecting the efficiency of somatic cell nuclear transfer (SCNT) is the initial quality of the oocyte. Since the abattoir-derived oocytes are heterogeneous in quality, selection of cohort fully grown/competent oocytes is necessary for proper development of SCNT embryos. Materials and Methods: Glucose-6-phosphate dehydrogenase (G6PDH) is actively expressed in growing oocytes, but decreased in activity as oocytes developing to fully grown state. We selected growing and fully grown sheep oocytes based on G6PDH reaction with brilliant cresyl blue (BCB); growing oocytes (unfinished growth phase with abundant G6PDH) degrade BCB and remain a colorless cytoplasm (BCB-) while fully grown oocytes (with reduced G6PDH activity) are unable to degrade BCB leaving a blue cytoplasm (BCB+) after incubation with 26 μM BCB for 45 minutes. BCB- and BCB+ oocytes were separately matured in vitro and their reprogramming capacity and developmental competence were compared after SCNT by using a handmade method. Results: The percentage of cleavage was significantly higher in cloned embryos reconstituted with BCB+ vs. BCB- oocytes (99.33 ± 0.66% vs. 86 ± 1.15%, respectively). Notably, cloned blastocyst development rate was significantly higher in BCB+ vs. BCB- oocytes (15.66 ± 1.2% vs. 6.33 ± 0.88%, respectively). Conclusion: BCB staining test of immature oocytes is a useful method for selecting competent oocytes and improve SCNT in sheep through increasing the reprogramming ability of the cytoplasm.