RESUMO
PURPOSE: To determine the most optimal stage for antioxidant supplementation of culture medium to improve developmental competence, cryotolerance and DNA-fragmentation of bovine embryos. METHODS: Presumptive zygotes were first cultured in presence or absence of beta-mercaptoethanol (beta-ME), for 8 days. Subsequently, half of the expanded blastocysts developed in both groups were vitrified, warmed within 30 min and post-warming embryos along with their corresponding non-vitrified embryos were cultured for two further days in presence or absence of (100 microM) betaME. RESULTS: For vitrified and non-vitrified embryos, the best effect was found when betaME was added from day 1 of in vitro culture in continuation with post-warming culture period. Day 1-8 supplementation significantly increased the rates of cleavage, day 7 and day 8 blastocyst production. For non-vitrified embryos, betaME addition during day 1-8 and/or 9-10 of embryo culture improved both hatching rate and quality of hatched embryos. For vitrified embryos, however, the percentage of DNA-fragmentation (18.5%) was significantly higher (p < or = 0.05) than that of embryos developed in absence of betaME but supplemented with betaME during post-warming period (13.5%). CONCLUSIONS: Exogenous antioxidant increases the chance of embryos, even those of fair-quality, to develop to blastocyst. However, antioxidant inclusion during in vitro embryo development is not sufficient to maintain the redox state of these embryos during the critical period of post-warming embryo culture, and therefore, there should be a surplus source of exogenous antioxidant during post-warming embryo culture.