44 REACTIVE OXYGEN SPECIES IN VITRIFIED BOVINE IN VITRO-MATURED OOCYTES

Abstract

Vitrification of in vitro-matured oocytes has important applications in fertility preservation and management of genetic resources. However, despite the increasing interest, the efficiency of oocyte vitrification needs to be improved. It was demonstrated that under stressful conditions of cryopreserving pig oocytes accumulate reactive oxygen species (ROS; Gupta et al. 2010 Fertility and Sterility 93, 2602–2607). Reactive oxygen species are known to exert harmful effects such as mitochondrial damage, ATP (ATP) depletion, altered calcium oscillation during fertilization and consequently their developmental ability may be compromised (Takahashi et al. 2003 Mol. Reprod. Dev. 66, 143–152). The aim of the present study was to evaluate whether the exposure to cryoprotectants and vitrification procedure affect ROS production in bovine in vitro-matured oocytes. Abattoir-derived bovine (n = 360, over 6 replicates) cumulus oocyte complexes (COCs), were in vitro-matured. COCs were mechanically stripped of their cumulus cells by gentle pipetting, washed and divided into 3 groups: control (C; i.e. fresh non treated oocytes), toxicity (T) and vitrification (V) groups. In group V, oocytes were exposed to 10% ethylene glycol (EG) + 10% DMSO for 3 min, then to 20% EG + 20% DMSO and 0.5 M sucrose, loaded on cryotops and plunged into liquid nitrogen within 25 s. Oocytes were warmed into a 1.25 M sucrose solution for 1 min and then to decreasing concentrations of sucrose (0.625 M, 0.42 M and 0.31 M) for 30 s each. In group T, oocytes were simply exposed to the vitrification and warming solutions. ROS determination was carried out by a spectrofluorometer at 495 nm excitation and 525 nm emission. Frozen oocytes were thawed and incubated in 500 μL of TRIS-HCl 40 mM, pH 7.0 in the presence of 5 μmol L^–1 of 2′,7′-dichlorfluorescein-diacetate, for 20 min at 37°C into a shaker. After incubation, the extraction was obtained by a syringe and the samples were centrifuged at 3000 rpm for 10 min at 4°C. Data were expressed as arbitrary ROS units per oocyte per min (U) and analysed by ANOVA. The results of this study showed that in bovine oocytes ROS levels tend to increase in the T and V groups compared to group C (76.0 ± 6.4, 249.9 ± 87.3 and 147.6 ± 42.6 in C, T and V groups, respectively). However, there were no statistical differences among groups and this was mainly due to the high variability recorded in both treated groups. In conclusion, these results suggest that both exposure to cryoprotectants and vitrification of in vitro-matured oocytes may influence ROS generation. However, the high variability recorded among replicates recommends further investigations.