Development and ultrastructure of bovine matured oocytes vitrified using electron microscopy grids

The aim of this study was to establish a methodology of cryopreservation of cattle oocytes and the quality assessment of oocytes and subsequent embryos produced in vitro under our laboratory conditions. Previously in vitro matured (IVM) oocytes were vitrified in minimum volume by ultra-rapid cooling technique. The oocytes were put into the equilibration solution (3% ethylene glycol in M199-HEPES + 10% foetal bovine serum) for 12 min, transferred to vitrification solution (30% ethylene glycol + 1 M sucrose in M199-HEPES + 10% foetal bovine serum) at room temperature for 25 s, then placed onto nickel electron microscopy grids and plunged into liquid nitrogen. After warming 75% of the oocytes were assessed as viable. Part of viable oocytes was taken for electron microscopy, the remaining oocytes were fertilized in vitro, and the presumptive zygotes were cultured until the blastocyst stage. Embryo cleavage and blastocyst rates in vitrified group after warming were 64.98% and 17.3%, resp. versus 70.72% and 25.54% in the control group (P < 0.05). No significant differences were found in the blastocyst total cell number, TUNEL and dead cell indexes between both groups. Ultrastructure of vitrified oocytes showed damages in smooth endoplasmic reticulum (SER) vesicles and lipid droplets as well as irregular arrangement of solitary cortical granules. Several mitochondria were damaged and the microtubules around the chromosomes were less occurred compared to the control group. However, the extent of injuries was lower than reported by other authors studying the ultrastructure of vitrified bovine oocytes, what is also supported by the better development of our oocytes after IVF. In conclusion, the designed oocyte vitrification technique ensures obtaining the blastocysts of the quality comparable to the fresh oocytes.