DETERMINATION OF REDOX STATE IN VITRIFIED PORCINE OOCYTES

AILÉN APARICIO; DANIELA PINCHETTI; PABLO CETICA; SERGIO MORADO

Buenos Aires

Jornada; V Jornadas INITRA; 2017

The suboptimal results observed in porcine oocyte vitrification could bedue to changes in their redox state. In our previous studies, we showed thatvitrification causes an increase in the mitocondrial activity of porcineoocyte. The aim of this study was to evaluate redox state in in vitro matured porcine oocytes aftervitrification-warming by the level of reduction/oxidation of NADH and FAD coenzymes,respectively. Immature cumulus-oocye complexes (COCs) were obtained byaspiration of antral ovarian follicles and those with a complete, intact anddense cumulus were selected by stereoscopic magnifying glass. They were maturedin 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 =100) were denuded and vitrified using permeable cryoprotectants(ethylenglycol, dimethylsulfoxide) and osmotically active components(trehalose) and a thin sheet as support to directly enter liquid nitrogen(Cryotech method®). Trehalose solutions in decreasing concentrations were usedfor warming in successive passages reaching isotonic solutions. Vitrified oocyteswere evaluated at 3 different time points (0, 3 and 21h) after warming for NADHand FAD autoflorescence intensity. Matured non-vitrified porcine oocytes wereused as a control group at the same time points. Digital microphotographs wereobtained with an epifluorescence microscope and images were analyzed using IMAGEJ software. FAD levels remained constant at all studied times, both on thevitrified and control group. There were also no significant differences betweenboth groups? levels of this coenzyme. NADH levels didn?t present significantdifferences inside each group overtime, however the vitrified-warmed group hada lower value when compared with its control (p