CONICET | Buscador de Institutos y Recursos Humanos

Successful in vitro embryo production is determined amongst other factors by sustaining a balanced redox state during oocyte maturation. In vitro matured oocytes are subject to higher oxygen levels (20%) than those in vivo (7%). Manipulation of oocytes also contributes to high levels of reactive oxygen species (ROS) production and resulting oxidative stress. It is known that sperm chromatin decondensation and later male pronuclear (MPN) formation is related to oxidative stress and glutathione (GSH) levels in oocytes during in vitro maturation (IVM). We previously reported that the antioxidant dimethylthiourea (DMTU) reduces ROS level in oocytes. This study aimed to evaluate the effects of different concentrations of DMTU on matured oocyte GSH concentrations and MPN formation. We obtained pig COC through follicular aspiration from slaughterhouse ovaries. Three treatments were applied during aspiration and further search: PBS + 10% porcine follicular fluid (control group); or supplemented with 1mM of DMTU (1 mM treatment); or 10 mM of DMTU (10 mM treatment). Oocytes were matured in vitro in wells containing supplemented Medium 199, under 5% of CO2 in humidified air at 39ºC, during 44 h. COC were denuded with hyaluronidase and oocytes (30 per tube, 8 tubes per treatment) were homogenized in PBS on ice and centrifuged at 4500 x g for 10 minutes. Total GSH was measured through colorimetric assay adapted to 96 well microplates, and it was relativized to protein level (measured by Bradford technique). IVF was performed with fresh boar semen (1x106 sperm/mL) in modified Medium 199, under 5% of CO2 in humidified air at 39°C, during 6 h. MPN formation was evaluated through Hoechst stain after 20 h of IVF (n control= 77; 1 mM treatment=96, and 10 mM treatment=87). There was a significant decrease in GSH levels in the 10 mM treatment in comparison with control and 1 mM treatment (p

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