EFFECT OF VITRIFICATION ON FUNCTIONAL PARAMETERS OF IN VITRO MATURED PORCINE OOCYTES

SERGIO MORADO; CECILIA ARRAZTOA; GABRIEL ÁLVAREZ; DEBORAH NEILD; GABRIEL DALVIT; PABLO CETICA

Ciudad Autónoma de Buenos Aires

Jornada; IV Reunión Conjunta de Sociedades de Biología de la República Argentina; 2020

Our aim was to evaluate the effect of the vitrification of porcine oocytes using a minimum volume system (Cryotech® method) on functional parameters throughout different recovery times after warming. Immature cumulus-oocyte complexes (COCs) were obtained by aspiration of antral ovarian follicles and were selected under a stereomicroscope. After 44h of maturation, oocytes were partially denuded by gentle pipetting and then were vitrified using the Cryotech® vitrification method. Metaphase II plate recovery time analysis, in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) were carried out as functional studies to establish whether vitrification-warming affected oocyte nuclear and cytoplasmic competence. To study metaphase plate recovery time, vitrified-warmed oocytes were incubated for 2, 3 or 4h in the same medium used for oocyte maturation. After incubation, oocytes were stained with Hoechst 33342 fluorochrome to establish the time point when most of the metaphase plates appear. IVF was performed using fresh semen from a Yorkshire boar of proven fertility and sperm head decondensation and/or pronuclear formation at 18h post-insemination was evaluated. ICSI was carried out using an inverted Leica® DMIL microscope equipped with Narishige® micromanipulators. Briefly, the oocyte was fixed by the holding pipette and the injection pipette with an immobilized spermatozoon was pushed through the zona pellucida and subsequently through the oolemma into the cytoplasm. Presumed fertilized oocytes from IVF or ICSI were fixed on a glass slide with Carnoy?s fixing solution for at least 24h, incubated in a 10 mgL-1 Hoechst 33342 fluorochrome aqueous solution for 15 min at room temperature and observed under an epifluorescence microscope. Oocyte morphology was not affected by the vitrification-warming procedure. The metaphase II configuration recovered 3h after warming, but only 75% of the matured oocytes were able to recover the metaphase II plate configuration. No further improvement was found after 4h, while 2h of incubation post warming proved to be insufficient. Although IVF and ICSI fertilization rates did not differ from the controls, a significant decrease was found in the cleavage rate. Possibly vitrification-warming alters the cytoskeleton involved in the processes of metaphase II spindle formation and cleavage, thus reducing porcine oocyte competence. Therefore, improvements should be included in the vitrification protocols to minimize these alterations.