Optimization of a simple vitrification procedure for bovine embryos produced in vitro: effect of developmental stage, two-step addition of cryoprotectant and sucrose dilution on embryonic survival

摘要

Experiments were designed to determine optimal conditions for the cryopreservation of bovine embryos produced in vitro. In Expt 1, embryos were exposed for 1, 3 or 5 min to a vitrification solution consisting of 40% (v/v) ethylene glycol, 18% (w/v) Ficoll and 10.26% (w/v) sucrose (EFS) and were subsequently vitrified. After warming in water at room temperature and diluting in a solution of 0.25 mol sucrose l?1, the in vitro survival rate in Ménézo-B2 medium was highest after exposure to EFS for 1 min. In Expt 2, embryos at day 7 and day 8 were vitrified after exposure to EFS for 1 min. The survival rate of embryos at day 7 was significantly improved, especially at the blastocyst and expanded blastocyst stage, when the Ménézo-B2 medium was supplemented with bovine oviduct epithelial cells (BOEC). Embryos at day 8 exhibited a significantly lower survival rate than did embryos at day 7 in both culture media. In Expt 3, one-step exposure of embryos to EFS for 1 min was compared with two-step exposure to 20% ethylene glycol for 3 min and EFS for 30–45 s. Embryos exhibited significantly higher survival and hatching rates after two-step vitrification, especially at the expanded blastocyst (89% and 69%, respectively) and the blastocyst stage (75% and 38%, respectively). In Expt 4, embryos were diluted in solutions of 0, 0.25 or 0.5 mol sucrose l?1 after two-step vitrification. There were no significant differences in the survival rates between the three dilution treatments. It can be concluded that (i) the optimal exposure time to EFS for one-step vitrification is 1 min; (ii) embryonic survival depends on the developmental stage; (iii) the addition of BOEC to culture medium after warming is beneficial for culture of vitrified embryos in vitro; (iv) two-step addition of EFS improves the survival rate and (v) vitrified embryos can be diluted from EFS in a single step without the use of sucrose as an osmotic buffer.