90 INTRACELLULAR REACTIVE OXYGEN SPECIES IN BOVINE EMBRYOS CULTURED IN VITRO WITH CATALASE UNDER VARIOUS OXYGEN TENSIONS

Abstract

The production of reactive oxygen species (ROS), such as superoxide anion (O₂^–), hydroxyl radical (OH^–) hydrogen peroxide (H₂O₂) and organic peroxides, is a normal process that occurs in the cellular mitochondrial respiratory chain. The high oxygen tension in in vitro culture (IVC) conditions is believed to induce oxidative stress, as a result of increase in ROS intracellular production, that can be correlated with embryonic developmental failure. Supplementation with antioxidants during IVC appears to increase the resistance of bovine embryos to the oxidative stress and consequently improve embryo development. The aim of this study was to evaluate the effects of antioxidant (catalase) and oxygen tensions during IVC on the embryonic development and quantification of intracellular ROS. Cumulus–oocyte complexes (COC; n = 337) were in vitro matured (IVM) in TCM-199 supplemented with 0.2 mM pyruvate, 25 mM sodium bicarbonate, 75 μg mL^–1 gentamicin, 10% FCS and hormones for 24 h at 38.5°C and 5% CO₂ in air. Then they were fertilized and the presumptive zygotes were cultured in SOFaa medium without (control) or with 100 UI catalase (CAT) for 7 days at 38.5°C in one of 2 types of humified atmosphere: 5% CO₂ in air (≈20% O₂) or in gaseous mixture (7% O₂, 5% CO₂ and 88% N₂). The cleavage rate was evaluated at 72 hours post-insemination (hpi) and the embryonic development at 168 hpi. At this time, the level of intracellular ROS was measured using the fluorescent probe 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA; Molecular Probes, Invitrogen, Oakville, Canada), at 5 μM (Bain et al. 2011 Reprod. Fertil. Dev. 23, 561–575). Stained embryos were imaged immediately using an inverted microscope and analysed by Q-Capture Pro image software (QImaging, Surrey, BC, Canada). The signal intensity values of embryos were subtracted by the average of backgrounds in the images. Embryo development was analysed by chi-squared test and means of the intensity of fluorescence were compared by ANOVA followed by Tukey's test (P < 0.05). The cleavage rates were 84.04%^a (control 20% O₂), 77.55%^a (CAT 20% O₂), 77.03%^a (control 7% O₂) and 71.83%^a (CAT 7% O₂). The embryonic development rates were 40.43%^a (control 20% O₂), 33.67%^a (CAT 20% O₂), 20.27%^b (control 7% O₂) and 16.90%^b (CAT 7% O₂). The fluorescent intensity were 3.9 ± 0.4^a (control 20% O₂), 1.8 ± 0.2^b (CAT 20% O₂), 2.7 ± 0.2^ab (control 7% O₂) and 2.8 ± 0.2^ab (CAT 7% O₂). Although catalase did not significantly affect blastocyst frequencies (P > 0.05), embryo development was adversely affected by reduced O₂ tension (P < 0.05). H2DCFDA staining indicated a significant (P < 0.05) reduction in the levels of intracellular ROS within embryos cultured with catalase under 20% O₂ compared with the control group in the same O₂ tension. Additionally, a consistent but insignificant reduction in intracellular ROS within embryos cultured under 7% O₂ was found. We can conclude that supplementation with catalase to IVC medium at 20% O₂ is suitable for lowering intracellular ROS levels in IVP bovine embryos, without lowering the rates of blastocysts production. This finding corroborates with theory that antioxidants are beneficial to embryo quality.

Alta Genetics Brazil, Deoxi Biotecnologia.