335 EFFECT OF VITAMIN E ON THE DEVELOPMENT OF BOVINE OOCYTES AFTER CHEMICAL ACTIVATION

Abstract

Oxidative stress is known to induce apoptotic cell death by reactive oxygen species (ROS) generated from in vitro culture systems. This study was conducted to evaluate the effect of Vitamin E (VitE), as antioxidant, on development of bovine embryos activated in vitro. Bovine ovaries were collected from slaughtered cows at a local abattoir. Oocytes were aspirated from follicles 3-8 mm in diameter and transferred to maturation medium: tissue culture medium (TCM)-199 supplemented with 10% (v/v) fetal calf serum, 100 mg/mL^-1 l-cysteine, 20 mg/mL^-1 sodium pyruvate, gonadotropins (250 IU each of eCG and hCG/mL), 10 mg/mL^-1 epidermal growth factor, and 100 µM VitE. Oocytes were cultured at 38.9°C in 5% CO₂ in humidified air. After 22 hours of culture, oocytes with polar bodies were selected and subjected to activation treatments. Oocytes were exposed to calcium ionomycin (5 µM for 5 min), followed by incubation with 6-DMAP (2 mM) for 3.5 hours in medium supplemented with or without VitE (100 µM). After activation, oocytes were cultured in mSOF medium containing 0.8% BSA at 38.9°C in 5% CO₂, 5% O₂ in humidified air for 7–8 days. Cell numbers were counted by the number of nuclei of blastocysts stained with Hoechst 33342, and apoptosis was detected by TUNEL assay using a MK500 kit (Takara Bio, Inc., Otsu, Shiga, Japan). Total cell and apoptotic cell number were determined under a fluorescence microscope. Data were analyzed using Student's t-test and chi-square test. The cleavage and blastocyst rates were significantly higher (P < 0.05) after activation with VitE (78.1% and 16.3%, n = 80) than without VitE (66.7% and 11.0%, n = 60). Total cell numbers were also significantly higher (P < 0.05) in blastocysts after activation with VitE (143.0 ± 34.02, n = 21) than in those without VitE (127.63 ± 40.25, n = 20). However, the percentage of TUNEL-positive (apoptotic) cells was similar between blastocysts activated with VitE (5.38 ± 2.22) and those without VitE (6.76 ± 1.98). The results of the present study demonstrate that vitamin E added to activation medium promoted further development of activated embryos, although its role in the alleviation of apoptosis remains unclear.