EFFECT OF RESVERATROL AND TROLOX SUPPLEMENTATION ON ACTIVE MITOCHONDRIA OF VITRIFIED AND WARMED PORCINE OOCYTES

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

Buenos Aires

Jornada; XXIV Jornadas de la Sociedad Argentina de Biología; 2022

The increase in the use of reproductive biotechnologies has generated the need to develop reliable techniques for the conservation of gametes. Porcine oocytes present a major challenge for the success of these techniques due to their high susceptibility to cold injury. Mitochondria are one of the most metabolically involved organelles in this process. One of the alternatives to increase the efficiency of cryopreservation techniques is the use of antioxidants. The aim of this research was to evaluate the effect of the antioxidants trolox and/or resveratrol on active mitochondria of vitrified and warmed porcine oocytes. Immature cumulus-oocyte complexes (COCs) were matured in vitro in 199 medium supplemented with porcine follicular fluid (FFP), cysteine, FSH and LH, at a 39°C, 5% CO2 in humidified air for 44 h. Then, the COCs were incubated with hyaluronidase for 5 minutes at 37°C and denuded with a fine Pasteur pipette. The oocytes were vitrified and warmed by the Cryotech® minimum volume method. To evaluate the effect of the antioxidants, the vitrification and warming solutions were supplemented, or not, 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, the evaluation of active mitochondria was carried out by staining the oocytes with 0.5 µM of MitoTracker Green for 45 minutes in the dark. Then, they were washed three times in PBS-PVA medium 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. Data were analyzed by ANOVA (p