Strategies for minimizing oxidative stress in in vitro production system of bovine embryos destined for vitrification

The study aimed to evaluate different strategies to minimize the effects of oxidative stress induced by menadione during in vitro culture on the potential for in vitro embryonic development. Therefore, in Experiment I, the oocytes were IVM in medium supplemented with 0.6 mM cysteine and 100 mM cysteamine (C+C), 100 IU catalase (CAT), 0.6 mM cysteine associated with 100 uM cysteamine and 100 IU catalase (C+C+CAT) or without antioxidants supplementation (Control). Then, were fertilized and the presumptive zygotes were in vitro cultured for 7 days. Immature oocytes (0h) and oocytes after 22 h of IVM were evaluated for intracellular levels of GSH and ROS, mitochondrial membrane potential, nuclear maturation and apoptosis. In Experiment II, oocytes were IVM in medium supplemented with C+C+ CAT (ATX) and the presumptive zygotes were IVC in SOF medium. From Day 6 to the end of the IVC, embryos were cultured in the presence or absence of 5 uM menadione (MD). On day 7, embryo development to the blastocyst rate was evaluated. Blastocysts were subjected to evaluations for intracellular GSH and ROS content, caspases 3 and 7 activity and apoptosis. Finally, Experiment III, oocytes were IVM and the presumptive zygotes were IVC in SOF medium supplemented with 100 ng/mL IGF-I (IGF). From Day 6 to the end of the IVC, embryos were cultured in the presence or absence of 5 uM menadione (MD). On day 7, embryo development to the blastocyst rate was evaluated. Blastocysts were subjected to evaluations for intracellular GSH and ROS content, caspases 3 and 7 activity, apoptosis, re-expansion rates after vitrification/thawing and expression of CASP3, GPX-1 and SOD-1 genes. In the first experiment, nuclear maturation rate was not affected by treatments (P>0.05). The percentage of apoptosis in C+C+CAT group was higher (P<0.05) compared to CAT group, but the Control and C+C groups did not differ from other groups (P>0.05). Intracellular ROS levels in C+C and C+C+CAT groups were similar to that observed in immature oocytes (0h) and higher (P<0.05) ROS levels were found in Control group. The intracellular GSH stocks was reduced (P<0.05) in all groups compared to immature oocytes, however the Control, CAT and C+C+CAT groups had lower (P<0.05) GSH concentrations compared to C+C group. The mitochondrial membrane potential was increased in all groups (P<0.05) when compared to immature oocytes (0h), with the only exception of C+C group. The blastocyst rate was higher (P<0.05) in the C+C+CAT group compared to the other groups. In the second experiment, the intracellular GSH levels was similar (P>0.05) between the experimental groups. The intracellular ROS levels found in the MD group was higher (P<0.05) than Control and Control ATX groups, ATX MD group did not differ (P>0.05) from the groups mentioned above. The caspases 3 and 7 activity did not differ (P>0.05) between groups. However, the percentage of apoptosis found in MD and MD ATX groups were higher (P<0.05) compared to Control and Control ATX groups. The blastocyst rate was lower (P<0.05) in the MD group compared to Control and Control ATX groups, but the ATX MD group did not differ (P>0.05) from the other experimental groups. In Experiment III, the intracellular GSH levels was similar (P>0.05) between the experimental groups. The intracellular ROS levels found in the MD group was higher (P<0.05) than observed in the Control group, however IGF and IGF MD groups did not differ (P>0.05) from the groups mentioned above. Caspases 3 and 7 activity and the total number of blastomeres did not differ (P>0.05) between experimental groups. However, the highest apoptotic percentages were found in the MD group and were higher (P<0.05) than Control, IGF and IGF MD groups. Intermediate apoptosis percentage levels were found in Control and IGF MD groups, which did not differ (P>0.05), but these groups were higher (P<0.05) compared to IGF group, which had the lowest percentages (P<0.05). The blastocyst rate was lower (P<0.05) in the MD group compared to Control and IGF groups, but IGF MD group did not differ (P>0.05) from the other experimental groups. The re-expansion rates was lower (P<0.05) in the MD group compared to Control and IGF groups, which did not differ (P>0.05). The IGF MD group did not differ (P>0.05) of the IGF and MD groups, but was lower (P<0.05) when compared to the Control group. There was no difference (P>0.05) in the relative expression of CASP3 and GPX-1 genes between experimental groups. However, expression of the SOD-1 gene was higher (P<0.05) in the IGF MD compared to IGF group, however these groups did not differ (P>0.05) from Control and MD groups. In conclusion, supplementation with intra- and extracellular antioxidants during IVM improved oocyte competence for embryonic development and minimized the deleterious effects of oxidative stress induced by menadione. Furthermore, the addition of IGF-1 for the IVC increased resistance to oxidative stress induced by menadione, whereas increased the expression of the antioxidant gene SOD-1, which resulted in reduction of apoptosis and improvement of blastocyst development and cryotolerance. (AU)