A comparative study of the effect of one-step addition of different vitrification solutions on in vitro survival of vitrified bovine embryos

Abstract

Three experiments were conducted to study the effect of vitrifying in vivo-produced bovine embryos using one-step addition of different vitrification solutions. In Experiment 1, 23 compact morulae to early blastocyst stage embryos were vitrified using a solution consisting of DMSO (2.6 M), acetamide (2.6 M), propylene glycol (1.3 M), and polyethylene glycol (7.5 mM). Only 1 embryo expanded after a 30-second exposure period. In Experiment 2, 11 compact morulae to early blastocysts were exposed for 2 minutes to a vitrification solution containing glycerol (3.4 M) and propylene glycol (3.4 M). None of the embryos survived after vitrification and post-thaw in vitro culture. Dilution of the cryoprotectants in experiments 1 and 2 were carried out in 1 M sucrose for 10 minutes. In Experiment 3, 20 compact morulae-early blastocysts were vitrified after 3 minutes of exposure to a vitrification solution consisting of 7.15 M ethylene glycol, 2.5 mM ficoll and 0.3 M sucrose prepared in embryo transfer freezing medium. As recommended for mouse and rabbit embryos, the cryoprotectant in Experiment 3 was diluted in 0.5 M sucrose. Fifteen compact morulae-early blastocysts expanded or hatched after 48 hours post-thaw in the in vitro culture. It is concluded that ethylene glycol is less toxic following one-step addition of vitrification solution to in vivo-produced bovine compact morulae-early blastocysts than the other vitrification solutions tested. A low concentration of sucrose for dilution of ethylene glycol was also found to reduce the chance of possible osmotic injuries due to dehydration.