THE TECHNIQUE FOR CRYOPRESERVATION OF CATTLE EGGS
Keywords:
bovine, oocyte, embryo, blastocyst rate, vitrificationAbstract
The aim of this investigation was to establish a methodology of cryopreservation of cattle eggs (oocytes) under our laboratory conditions. For cryopreservation of in vitro matured oocytes, the freezing in minimum volume by ultra-rapid cooling technique was used. Oocytes with at least three layers of cumulus cell were placed into the equilibration solution (ES: 3 % ethylene glycol in M199-HEPES, supplemented with 10 % foetal calf serum) for 12 min. Following equilibration, the oocytes were transferred to vitrification solution (30 % EG + 1M sucrose in M199-HEPES with 10 % foetal bovine serum) at room temperature for 25 sec. Then the oocytes were placed onto nickel grid (electron microscopy grade) and plunged into liquid nitrogen. After thawing the oocytes were fertilized in vitro. Development of produced embryos (cleavage on day 2, and blastocyst yield on day 7) and total cell number of blastocysts after DAPI staining were determined. We obtained a relatively high cleavage rate (55.81 %) of fertilized oocytes after thawing; of them 11.24 % developed to the blastocyst stage. The quality of blastocysts obtained from vitrified oocytes was similar to the control blastocysts, as evidenced by a comparable total cell number (84.45 vs 97.29, resp.). In conclusion, the designed freezing technique proved to be suitable for cryopreservation of cattle oocytes, nevertheless further optimization is required.
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