Vitrification of in vitro produced bovine blastocysts: methodological studies and developmental capacity

Methodological studies were undertaken to test the validity of a three-step vitrification procedure for bovine in vitro produced embryos using glycerol and ethylene glycol as cryoprotectants. Embryos were produced in a low-phosphate culture system (medium VT1+10% foetal calf serum) and vitrified at day 7 post-insemination either in a mixture of 25% glycerol—25% ethylene glycol or a mixture of 10% glycerol—40% ethylene glycol. In the first mixture 67% (n=283) of blastocysts were re-expanded after 72 h of culture and 53% were hatched while in the second one (n=65) only 5% survived. The mean number of cells of the surviving blastocysts was correlated with the rate of survival (R2=0.47; P=0.0024). Embryo size (diameter < or > to 180 μm) did not influence blastocyst survival or cell number, but hatching rate was higher for embryos >180 μm. Embryo survival, hatching rate and cell number 72 h post-warming were not affected by the mode of vitrification (direct plunging into nitrogen liquid or vitrification into nitrogen liquid vapour), the mode of preparation of the vitrification solutions (molar or molal basis) or by the concentration of galactose used as a diluent (0 to 0.85 M). Only one calf was born after transfer of 22 vitrified blastocysts. These results confirm the apparent lack of correlation for cryopreserved embryos between in vitro survival or hatching and viability after transfer.