REACTIVE OXYGEN SPECIES EVALUATION IN VITRIFIED PORCINE OOCYTES

DANIELA PINCHETTI; AILÉN APARICIO; PABLO CETICA; GABRIEL ÁLVAREZ; SERGIO MORADO

Buenos Aires

Jornada; V Jornadas INITRA; 2017

Reactive oxygen species (ROS) production was measured in order to evaluate the quality of porcine oocytes matured in vitro and subjected to the process of vitrification-warming by the Cryotech® minimum volume method. Ovaries were obtained from slaughtered pigs and transported in terms to the laboratory. Immature cumulus-oocye complexes (COCs) were obtained by aspiration of antral ovarian follicles and those with a complete, intact and dense cumulus were selected by stereoscopic magnifying glass. They were matured in medium 199 with gentamicin sulfate, porcine follicular fluid, FSH and LH, under mineral oil at 39 ° C, 5% CO2 in humidified air during 48hs. Matured oocytes (n =95) were denuded and vitrified using permeable cryoprotectants (ethylenglycol,dimethylsulfoxide), osmotically active components (trehalose) and placed in the Cryotech® sheet to directly enter liquid nitrogen. Trehalose solutions in decreasing concentrations were used for warming in successive passages until reaching isotonic solutions. Oocytes were evaluated at 3 times (0, 3 and 21 hs) being their respective control oocytes that were not subjected to the process of vitrification-warming. For ROS determination oocytes were incubated in PBS added with polyvinyl alcohol (PVA) and fluorescein 2',7'-dichlorodihydro fluoresceindiacetate (DCHF-DA). To evaluate esterase activity a group of oocytes was incubated in PBS added with PVA and fluorescein diacetate (FDA). Digital microphotographs were obtained with an epifluorescence microscope. Images were analyzed using IMAGE J software. Fluorescence levels of DCHF-DA depends on intracellular esterase activity, so the ratio between the brightness obtained from DCHF-DA for each oocyte and the average brightness by FDA of each treatment was considered as a measure of ROS production. ROS production was significantly higher at 0h and then decreased at 3 and 21 hs in fresh and vitrified oocytes (P