FUNCTIONAL STUDIES TO EVALUATE BOVINE OOCYTE VITRIFICATION

CYNTHIA GUTNISKY; PABLO CETICA; GABRIEL ÁLVAREZ; SERGIO MORADO

Buenos Aires

Jornada; XX JORNADAS ANUALES DE LA SOCIEDAD ARGENTINA DE BIOLOGÍA (SAB), XVII JORNADAS DE LA SOCIEDAD URUGUAYA DE BIOCIENCIAS (SUB)-Segundas Jornadas Rioplatenses de Biología; 2018

Our main aim was to evaluate the effect of the vitrification/warming of matured bovine oocytes by a minimum volume system (Cryotech® method) on functional parameters evaluating metaphase II recovery, in vitro fertilization, parthenogenetic activation and embryo development. Partially denuded matured oocytes were vitrified and warmed using the Cryotech Vitrification Method®. To study the time of recovery of the metaphasic plate II, vitrified/warmed oocytes were incubated for 2, 3 and 4 h. After incubation, oocytes were stained with fluorochrome Hoechst 33342 to establish the time point at which the majority of the metaphasic plates appear. Three groups of in vitro matured oocytes were fertilized: oocytes surrounded by cumulus cells (IVF control), partially denuded oocytes (control) and vitrified/warmed oocytes (treatment). All the groups were incubated for 3 additional hours before insemination to let the metaphase II recover in vitrified oocytes. IVF was performed using frozen?thawed semen from a Holstein bull of proven fertility. Semen was thawed at 37ºC in mSOF. The proportion of cleaved oocytes was determined by evaluating the number of embryos that presented two or more blastomeres. In vitro embryo development was performed in IVC-mSOF, under mineral oil. The proportion of blastocysts produced was determined at day 7 following insemination. Parthenogenetic activation was performed in TALP plus ionomycin for 4 minutes and then oocytes were incubated in IVF-mSOF with 6-dimethylaminopurine for 3h, before being incubated for 21h in IVF-mSOF. Metaphase II configuration recovered between 3 and 4 h after warming (65.4 %; n=72), being 2h-incubation post warming insufficient for recovery (P