134 CYSTEINE AND CATALASE SUPPLEMENTATION FOR 7 DAYS DURING IN VITRO CULTURE IMPAIRS BOVINE EMBRYO DEVELOPMENT

Abstract

The production of reactive oxygen species (ROS), such as superoxide anion (O₂^–), hydroxyl radical (OH^–), hydrogen peroxide (H₂O₂) and organic peroxide, is a normal process that occur in the cellular mitochondrial respiratory chain (Morado et al. 2009 Reprod. Fert. Dev. 21, 608–614). Supplementation with antioxidants during in vitro culture (IVC) appears to increase the resistance of bovine embryos to the oxidative stress, and consequently improve embryo development and cryotolerance (Rocha et al. 2011 Reprod. Fert. Dev. 23 157–158). This study was conducted to evaluate the effects of period of supplementation with intra (cysteine, CIST) or extracellular (catalase, CAT) antioxidants during IVC on embryo development and cryotolerance. Cumulus–oocyte complexes (n = 1132) were maturated for 24 h in B199 medium, at 38.5°C and 5% CO₂ in air. After fertilization (Day 0), zygotes were IVC for 7 days in SOF medium (0.5% BSA + 2.5% FCS) in 7% O₂, 5% CO₂ e 88% N₂ atmosphere, at 38.5°C. The antioxidant supplementation was performed during all of the culture period (from Day 1 to Day 7) or during the first 72 h (from Day 1 to Day 3), with 0.6 mM CIST, 100 UI CAT or without antioxidants (CONTR). The cleavage and blastocyst rates were evaluated, respectively, at 72 and 168 h post-insemination, when expanded blastocysts grade I were vitrified (n = 91) by Vitri-Ingá^® protocol (Ingámed^®, Maringá, PR, Brazil). Then, they were thawed and cultured for 24 h to evaluate re-expansion rates. The differences between groups were analyzed by ANOVA followed by Tukey’s test, and re-expansion rates by chi-square test (P ≤ 0.05). The cleavage and blastocyst rates were, respectively, 83.52 ± 4.52^a/36.19 ± 3.21^a (CONTR), 79.16 ± 4.52^a/38.08 ± 3.21^a (CIST Day 3), 77.74 ± 4.52^a/42.09 ± 3.21^a (CAT Day 3), 73.57 ± 4.05^a/11.15 ± 2.87^b (CIST Day 7), 71.83 ± 4.05^a/15.07 ± 2.87^b (CAT Day 7). The embryo re-expansion rates were 90.00%^a (CONTR), 93.33%^a (CIST Day 3), 75.00%^a (CIST Day 7), 63.64%^a (CAT Day 3) and 75.00%^a (CAT Day 7). Supplementation with antioxidants for 7 days of IVC impaired embryo development, compared with addition up to Day 3 (P ≤ 0.05). However, it did not affect in vitro embryo cryotolerance (P ≥ 0.05). Supplementation with antioxidants throughout all the IVC significantly impaired blastocyst rate, probably by exerting a toxic effect leading to an arrest of embryonic development. It is believed that prolonged culture in the presence of antioxidants results in excessive reduction of ROS leading to an imbalance of the cellular redox status. It is known that ROS, particularly H₂O₂, act on signaling pathways involved in the cellular proliferation and differentiation, in gene expression and metabolism during embryo development. Supplementation with antioxidants up to Day 3 did not differ from CONTR, probably due to low O₂ tension, and the presence of antioxidants in FBS and BSA. In conclusion, supplementation with cysteine and catalase during all of the culture period impaired embryo development, however this reduction did not affect embryo survival after vitrification.

Financial support was provided by FAPESP (#2011/18257-2). The authors acknowledge Ingámed, Alta Genetics Brazil.