254 EFFECTS OF OXYGEN TENSION AND OOCYTE DENSITY DURING IN VITRO MATURATION ON THE LEVELS OF REACTIVE OXYGEN SPECIES IN BOVINE OOCYTES AND MATURATION MEDIUM

The reactive oxygen species (ROS) produced by animal cells and at physiological levels are responsible for several cellular functions. However, when there is an imbalance between ROS production and the antioxidant system in the cell, oxidative stress occurs and causes severe cell damage. In oocytes, ROS can affect the dynamics of maturation and early embryo development processes. Oxygen tension and the density of oocytes by medium volume during in vitro maturation (IVM) can influence ROS production. The aim of this study was to evaluate the influence of the association between oxygen tension (5 or 20%) and different oocyte densities during IVM (1 : 10 or 1 : 20 oocytes µL–1 of medium) on the ROS levels in oocytes and medium. Bovine oocytes (n = 420) were obtained from slaughterhouse ovaries by aspiration of 2- to 8-mm follicles. Quality I and II oocytes (De Loss et al. 1989 Gamete Res. 24, 197–204) were homogeneously distributed into groups of 15 oocytes per treatment: Treatment (T) 1 = 1 : 10 in 5% of O2; T2 = 1 : 10 in 20% of O2; T3 = 1 : 20 in 5% of O2; and T4 = 1 : 20 in 20% of O2. The oocytes were matured in TCM-199 supplemented with 10% oestrous mare serum, 100 µg mL–1 of epidermal growth factor, 50 µg mL–1 of LH, 5 µg mL–1 of FSH, and 22 µg mL–1 of pyruvate for 22 to 24 h at 39°C, in 5% CO2 and saturated humidity. To assay ROS production, denuded oocytes and 60-µL samples of IVM medium were evaluated by the spectrofluorometric method with 2′7′-dichlorofluorescein-diacetate, in which the fluorescence intensity emission was considered an indicator of ROS production and measured by a spectrofluorophotometer. The ROS production in oocytes and in IVM medium was expressed as units of fluorescence (UF); data were analysed by ANOVA and Duncan’s test with a 5% level of significance. Seven replications were performed. In treatment groups T1 and T3, the ROS production in oocytes was higher (P < 0.05) than in oocytes of treatment groups T2 and T4 (13.53 and 18.78 UF v. 7.92 and 6.15 UF, respectively). The ROS production in IVM medium was higher in the T1 (23.86 UF) and T2 (24.12 UF) treatment groups than in the T3 (18.78 UF) and T4 (18.57 UF) treatment groups. These results suggest an increase in ROS production in IVM oocytes under a 5% O2 atmosphere in relation to a 20% O2 atmosphere, irrespective of the oocyte density by volume of IVM medium. On the other hand, the accumulation of ROS in IVM medium seemed higher when the oocyte density was 1 oocyte to 10 µL of IVM medium, independent of the oxygen tension used. A higher level of ROS in 5% O2 tension may be caused by competition for O2 between oocyte and cumulus cells, causing a reduction in O2 levels and changing the availability of O2 to energy generation in oocytes and consequently increasing ROS generation. In this respect, 5% O2 during IVM may contribute to the onset of oxidative stress in oocytes, which may compromise fertilization and early embryo development. Further research is necessary to clarify esterase activity in oocytes and the addition of exogenous peroxidase to validate the assay. Financial support: FAPERGS (1011575) and CNPq (501763/2009).