Assessment of viability of matured bovine oocytes by 3’6’-fluorescein diacetate (FDA) staining

Since in vitro maturation of oocytes is the starting point in many biotechnological procedures, it is important to have viable oocytes of good quality. This study was conducted to assess the viability of matured bovine oocytes using Fluorescein diacetate probe. Slaughterhouse derived bovine ovaries were used to obtain Cumulus Oocyte Complexes (COCs). Collected COCs were graded based on their morphological characters and high quality COCs (with three or more layers of cumulus cells and uniform granulation of ooplasm) were selected and used for in vitro maturation. Maturation was performed in freshly prepared Tissue Culture Medium-199 (TCM-199), enriched with Follicle Stimulating Hormone (FSH) – 0.5 µg/ml, Luteinising Hormone (LH) – 5 µg/ml, Oestradiol – 1 µg/ml, sodium pyruvate 0.2 mM and Foetal Calf Serum (FCS) 10%. Culture conditions set for this study were 38.5°C temperature, 5% carbondioxide tension with maximum humidity. Standard CO₂ incubator (Labline, USA) was used to ensure this culture conditions. Matured oocytes were stained with 1.7 µg/ml Fluorescein diacetate in Dulbeco's Phosphate Buffered Saline. Out of 45 matured oocytes examined 36 were viable. Viable oocytes emitted apple green fluorescence on UV excitation. It was found that 80% of oocytes were suitable for further analysis.