EFFECT OF RESVERATROL AND TROLOX SUPPLEMENTATION ON REACTIVE OXYGEN SPECIES PRODUCTION IN VITRIFIED AND WARMED PORCINE OOCYTES

STEPHANIA MADRID GAVIRIA; SERGIO MORADO; MARIANA CÓRDOBA; PABLO CETICA

Congreso; Reunión Conjunta de Sociedades Biomédicas; 2023

The aim of this research was to evaluate the effect of the antioxidants trolox and/or resveratrol on reactive oxygen species (ROS) production in vitrified and warmed porcine oocytes. Immature cumulus-oocyte complexes (COCs) were matured in vitro in 199 medium added with porcine follicular fluid (FFP), cysteine, FSH and LH, at a 39°C, 5% CO2 in humidified air for 44 h. Then, oocytes were denuded by incubation with hyaluronidase for 5 minutes at 37°C. Oocytes were vitrified and warmed by a minimum volume method. To evaluate the effect of the antioxidants, the vitrification and warming solutions were supplemented with resveratrol and/or trolox at a concentration of 2 µM and 50 µM, respectively. After warming, oocytes were cultured in 199 medium + FFP for 3 h to allow their recovery. After this time, oocytes were stained with 10 µM of DCH2FDA for 30 minutes in dark. A small group of oocytes from each treatment was stained with 0.12 µM of FDA for 15 minutes in dark. Then, they were washed three times in PBS-PVA and placed on a slide for their observation under an epifluorescence microscope. The exhibited fluorescence by each oocyte was analyzed using the IMAGEJ software from the images obtained. As fluorescence levels detected by DCH2FDA are dependent on esterase activity, the ratio between the fluorescence intensity for each oocyte measured by DCH2FDA and the mean fluorescence detected by FDA for each treatment was considered a better indicator of ROS levels. Data were analyzed by ANOVA (p