Antioxidant favors the developmental competence of porcine parthenogenotes by reducing reactive oxygen species

Abstract

Reactive oxygen species (ROS) generate during electrical activation of oocytes which has detrimental effects on embryo survival when overwhelmed. The present study was designed to investigate the ability of L-ascorbic acid, a novel water soluble antioxidant, to reduce the ROS level in developing embryos and their subsequent effects on embryo development in vitro. The compact cumulus oocyte complexes (COCs) were cultured in tissue culture medium (TCM)-199 supplemented with 10 ng/ml epidermal growth factor, 4 IU/ml pregnant mare serum gonadotropin (PMSG), and human chorionic gonadotropin (hCG) and 10% (v/v) porcine follicular fluid (pFF) for 44 h. After maturation culture, the denuded oocytes were activated with a single DC pulse of 2.0 kV/cm in 0.3 M mannitol solution containing 0.5 mM of HEPES, 0.1 mM of CaCl2 and 0.1 mM of MgCl2 for 30 μs using a BTX Electro-cell Manipulator. The activated oocytes were cultured in modified North Carolina State University-23 (mNSCU-23) medium for 168 h. The level of H2O2 in each embryo was measured by the dichlorohydrofluorescein diacetate (DCHFDA) method at 48 h after activation. The blastocyst formation rate was significantly higher when culture medium was supplemented with 50 and 100 μM L-ascorbic acid (31.2 and 38.7%, respectively) compared to non-supplemented (16.1%) group. Accordingly, significantly more cells in blastocyst were found for 50 and 100 μM L-ascorbic acid (50.0 and 56.4, respectively) compared to 0 and 200 μM L-ascorbic acid (36.5 and 39.8, respectively). L-ascorbic acid reduces the H2O2 level in developing embryos in a dose-dependant manner. The H2O2 level (pixels/embryos) was 191.5, 141.0, 124.0 and 163.3 for 0, 50, 100 and 200 μM L-ascorbic acid, respectively. So, we recommend to supplement 50 or 100 μM L-ascorbic acid in porcine in vitro culture medium.