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 usi ng glycerol and ethylene glycol as cryoprotectants. Embryos were produ ced in a low-phosphate culture system (medium VT1 + 10% foetal calf se rum) and vitrified at day 7 post-insemination either in a mixture of 2 5% glycerol-25% ethylene glycol or a mixture of 10% glycerol-40% ethyl ene 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 secon d one (n = 65) only 5% survived. The mean number of cells of the survi ving blastocysts was correlated with the rate of survival (R-2 = 0.47; P = 0.0024). Embryo size(diameter < or > to 180 mu m) did not influen ce blastocyst survival or cell number, but hatching rate was higher fo r embryos > 180 mu m. Embryo survival, hatching rate and cell number 7 2 h post-warming were not affected by the mode of vitrification (direc t 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 diluen t (0 to 0.85 M). Only one calf was born after transfer of 22 vitrified blastocysts. These results confirm the apparent lack of correlation f or cryopreserved embryos between in vitro survival or hatching and via bility after transfer. (C) 1998 Elsevier Science B.V. All rights reser ved.