SummaryThis study examined the effects of antioxidant supplementation and O₂tension on embryo development, cryotolerance and intracellular reactive oxygen species (ROS) levels. The antioxidant supplementation consisted of 0.6 mM cysteine (CYST); 0.6 mM cysteine + 100 μM cysteamine (C+C); 100 IU catalase (CAT) or 100 μM β-mercaptoethanol (β-ME) for 3 or 7 days ofin vitroculture (IVC). Two O₂tensions (20% O₂[5% CO₂in air] or 7% O₂, 5% CO₂and 88% N₂[gaseous mixture]) were examined. After 7 days of antioxidant supplementation, the blastocyst frequencies were adversely affected (P< 0.05) by CYST (11.2%) and C+C (1.44%), as well as by low O₂tension (17.2% and 11.11% for 20% and 7% O₂, respectively) compared with the control (26.6%). The blastocyst re-expansion rates were not affected (P> 0.05) by the treatments (range, 66–100%). After 3 days of antioxidant supplementation, the blastocyst frequencies were not affected (P> 0.05) by any of the antioxidants (range, 43.6–48.5%), but they were reduced by low O₂tension (P< 0.05) (52.1% and 38.4% for 20% and 7% O₂, respectively). The intracellular ROS levels, demonstrated as arbitrary fluorescence units, were not affected (P> 0.05) by antioxidant treatment (range, 0.78 to 0.95) or by O₂tension (0.86 and 0.88 for 20% and 7% O₂, respectively). The re-expansion rates were not affected (P> 0.05) by any of the treatments (range, 63.6–93.3%). In conclusion, intracellular antioxidant supplementation and low O₂tension throughout the entire IVC period were deleterious to embryo development. However, antioxidant supplementation up to day 3 of IVC did not affect the blastocyst frequencies or intracellular ROS levels.