OPTIMIZATION OF A SIMPLE VITRIFICATION PROCEDURE FOR BOVINE EMBRYOS PRODUCED IN-VITRO - EFFECT OF DEVELOPMENTAL STAGE, 2-STEP ADDITION OF CRYOPROTECTANT AND SUCROSE DILUTION ON EMBRYONIC SURVIVAL

Experiments were designed to determine optimal conditions for the cryo preservation of bovine embryos produced in vitro. In Expt 1, embryos w ere exposed for 1, 3 or 5 min to a vitrification solution consisting o f 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 Menezo-B2 medium was highest after exposure to EFS for 1 min. In Expt 2, embryos at day 7 and day 8 were vitrifie d after exposure to EFS for 1 min. The survival rate of embryos at day 7 was significantly improved, especially at the blastocyst and expand ed blastocyst stage, when the Menezo-B2, medium was supplemented with bovine oviduct epithelial cells (BOEC). Embryos at day 8 exhibited a s ignificantly lower survival rate than did embryos at day 7 in both cul ture media. In Expt 3, one-step exposure of embryos to EFS for 1 min w as compared with two-step exposure to 20% ethylene glycol for 3 min an d EFS for 30-45 s. Embryos exhibited significantly higher survival and hatching rates after two-step vitrification, especially at the expand ed 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. The re were no significant differences in the survival rates between the t hree dilution treatments. It can be concluded that (i) the optimal exp osure time to EFS for one-step vitrification is 1 min; (ii) embryonic survival depends on the;developmental stage; (iii) the addition of BOE C to culture medium after warming is beneficial for culture of vitrifi ed embryos in vitro; (iv) two-step addition of EFS improves the surviv al rate and (v) vitrified embryos can be diluted from EFS in a single step without the use of sucrose as an osmotic buffer.