Combinations of Trolox and ascorbic acid have a beneficial effect on in vitro maturation of pig oocytes

The poor in vitro development of pig oocytes and embryos has been blamed on oxidative stress. We sought to find out if combinations of Trolox (T), a synthetic and cell-permeable derivative of vitamin E, and ascorbic acid (AA) could improve the maturation rates of in vitro cultured pig oocytes. Pig oocytes underwent maturation for 44-45 h in medium M 199 supplemented with 0 μM T + 0 μM AA, 100 μM T + 250 μM AA, 300 μM T + 250 μM AA, 100 μM T + 750 μM AA or 300 μM T + 750 μM AA. These combinations were chosen based on previous research conducted in our laboratory and on the available literature. After maturation, several parameters were assessed: cumulus oophorus expansion, oocyte viability (based on the presence of metabolic activity versus membrane damage), extrusion of the first polar body, mitochondrial membrane potential (MMP), pronucleus formation, and embryo development after fertilization. All antioxidant combinations significantly improved cumulus expansion and formation of the first polar body. The best was 300 μM T + 250 μM AA for the first characteristic and 300 μM T + 750 μM AA for the second. Antioxidant presence in the maturation media increased the percentages of viable oocytes but not significantly. MMP was not significantly modified by the addition of antioxidant combinations. We also found that a low concentration of T (100 µM) mixed with a high concentration of AA (750 µM) in the oocyte maturation media led to significantly higher rates of both female and male pronuclei formation and also enhanced embryo development to the morula stage. Therefore, we recommend this combination to improve the in vitro maturation media of pig oocytes.