Effects of gaseous atmosphere and antioxidants on the development and cryotolerance of bovine embryos at different periods of in vitro culture

Abstract

This study examined the effects of antioxidant supplementation and O-2 tension 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 mu M cysteamine (C+C); 100 IU catalase (CAT) or 100 mu M beta-mercaptoethanol (beta-ME) for 3 or 7 days of in vitro culture (IVC). Two O-2 tensions (20% O-2 [5% CO2 in air] or 7% O-2, 5% CO2 and 88% N-2 [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 O-2 tension (17.2% and 11.11% for 20% and 7% O-2, 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 O-2 tension (P<0.05) (52.1% and 38.4% for 20% and 7% O-2, 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 O-2 tension (0.86 and 0.88 for 20% and 7% O-2, 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 O-2 tension 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.