Effect of L-ascorbic acid supplementation at different gaseous environments on in vitro development of preimplantation sheep embryos to the blastocyst stage

Abstract

Ascorbate is an important water-soluble antioxidant. Oxidative stress during in vitro gamete and embryo culture leads to defective development of gametes and embryos. In this context, the present study aimed to evaluate the effects of L-ascorbic acid supplementation to oocyte maturation media and embryo culture media and subsequent culture at different gaseous environments on the overall yield of ovine embryos. Rates of cleavage, morula and blastocyst, blastocyst total cell count and comet assay were taken as indicators of developmental competence of embryos. In experiments 1 and 2, L-ascorbic acid at concentrations of 0, 50, 100, 200, 400 and 500 µm was supplemented to oocyte maturation medium and cultured in an environment of 5% or 20% oxygen. L-ascorbic acid supplementation to oocyte maturation medium did not have any significant effect on yield of mature oocytes or embryos. In experiments 3 and 4, L-ascorbic acid at the aforesaid concentrations was supplemented to embryo culture media followed by culture in 5% or 20% oxygen environment. It was observed that 50 μM L-ascorbic acid in embryo culture medium at 5% oxygen levels significantly increased the rates of morulae (P < 0.001), blastocysts (P < 0.01) and blastocyst total cell number (P < 0.05) when compared to control. 100 μM Lascorbic acid in embryo culture medium at 20% oxygen levels significantly increased rates of cleavage (P < 0.01), morulae (P < 0.05), blastocyst (P < 0.01) and blastocyst total cell number (P < 0.01) compared to control. Supplementation of embryo culture media with 400 μM and 500 μM L-ascorbic acid significantly retarded the rates of embryo formation and development. We conclude that optimal L-ascorbic acid supplementation may enhance the in vitro developmental competence of ovine embryos by protecting them from oxidative damage.