Temporal effects of alpha-tocopherol and L-ascorbic acid on in vitro fertilized porcine embryo development

The susceptibility of embryos to reactive oxygen species (ROS) varies in different stages of embryo development. The present study evaluated temporal effects of alpha-tocopherol and L-ascorbic acid on the porcine embryo development, and investigated whether a single or twice supplements of these two antioxidants at a divided concentrations favors the embryo development. In order to determine temporal effects of alpha-tocopherol and/or L-ascorbic acid, 100 microM alpha-tocopherol or 200 microM L-ascorbic acid were supplemented to the North Carolina State University (NCSU)-23 embryo culture media at 0, 48, 96 and 120 h of culture. In another set of experiments, the concentration was divided into two equal halves, i.e., 50 microM alpha-tocopherol and 100 microM L-ascorbic acid, and supplemented twice at 0 and 48, 0 and 96, or 48 and 96 h of culture. Supplementing culture media with 100 microM alpha-tocopherol for the entire culture period of 168 h or starting from the 48 h of culture yielded higher blastocyst percentage compared with the control or starting from the 96 or 120 h of culture. L-Ascorbic acid (200 microM) alone or together with alpha-tocopherol (100 microM) with a single supplement did not affect the frequency of blastocyst formation or number of cells in blastocyst. L-ascorbic acid with a divided supplements yielded higher blastocyst percentage compared with the control. No synergistic effect was observed on embryo development at a single supplement of these antioxidants. Although, at divided supplements higher blastocyst percentage was observed compared with control group, no further beneficial effect was observed compared with alpha-tocopherol or L-ascorbic acid alone. Our results demonstrated that the embryotrophic effects of alpha-tocopherol and/or L-ascorbic acid, in terms of frequency of blastocyst formation and number of cells in blastocyst, depends on the concentration and supplementation timing.