Ooplasm cryopreservation: ovarian fragments versus oocytes alone

The cryopreservation of the farm animal ooplasm is very useful in the conservation of female gametes. Since the use of histological assay alone is insufficient to assess the freezing-thawing process, the future oocyte developmental competence need to be validated by in vitro fertilization and embryo production. The aim of the study was to find optimal method of cryostorage of bovine oocytes with preserving their viability and fertility. In the first experimental series we vitrified ovarian fragments isolated from the cortical area of the cow ovary, which contained antral follicles, using two vitrification techniques: solid-surface vitrification (SSV) and liquid vitrification (LV). After warming we isolated the oocytes by follicle aspiration and placed them into the in vitro maturation (IVM). At maximum, only 8.3% of vitrified/warmed (LV) oocytes were matured, whilst none of the oocytes were matured following SSV technique. In all frozen ovarian fragments, regardless of vitrification technique, serious damages in the oocytes were detected at the histological and ultrastructural levels, what prevented the oocyte development. In the second series of experiments, cumulus-oocyte complexes without surrounding ovarian tissue were vitrified following IVM procedure. Using this scheme we obtained more than 50% embryo cleavage rate and 4.5% of embryos reached the blastocyst stage, which proves that the cumulus-oocyte complexes after vitrification can retain their developmental ability. Our preliminary results show that cryopreservation of previously matured oocytes is more promising than the vitrification of ovarian tissue fragments.