title: 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.
abstract: 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.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, State University of Ghent, Belgium.
authors.forename: A R
authors.initials: AR
authors.lastname: Mahmoudzadeh
authors.forename: A
authors.initials: A
authors.lastname: Van Soom
authors.forename: P
authors.initials: P
authors.lastname: Bols
authors.forename: M T
authors.initials: MT
authors.lastname: Ysebaert
authors.forename: A
authors.initials: A
authors.lastname: de Kruif
mesh_terms: D000818:Animals; D001755:Blastocyst; D002417:Cattle / Q000502:physiology*; D002470:Cell Survival; D003470:Culture Media; D005314:Embryonic and Fetal Development; D005026:Ethylene Glycols; D005307:Fertilization in Vitro; D017110:Freeze Substitution / Q000379:methods*; D009028:Morula; D013395:Sucrose
chemical_list: D003470:Culture Media; D005026:Ethylene Glycols; D013395:Sucrose