Comparison of two-step vitrification versus controlled freezing on survival of in vitro produced cattle embryos

Abstract

Abstract Grade 1 in vitro produced bovine day 7 embryos at the compact morula-early blastocyst, blastocyst and expanded blastocyst stage were selected for cryopreservation. From 7 replicates, 572 embryos were randomly divided into two groups. One group was cryopreserved by controlled freezing after 20 minutes equilibration in 10% v/v glycerol and slow cooling (0.3 °C / minute) from −7 °C to −30 °C. The other group was vitrified after 3 minutes exposure to 20% v/v ethylene glycol and 30 to 45 seconds exposure to a vitrification solution consisting of 40% v/v ethylene glycol, 18% w/v ficoll and 10.26% w/v sucrose. Embryos from both groups were thawed in a water bath at 20±1 °C. Frozen-thawed embryos were diluted in three successive solutions consisting of 10.26% w/v sucrose and 6.6%, 3.3% and 0% glycerol, respectively, during 5 minutes for each step. Vitrified-warmed embryos were diluted in a 8.5% w/v sucrose solution during 5 minutes. All diluted embryos were cultured in Menezo-B2 medium supplemented with bovine oviduct epithelial cells. The survival rate of all developmental stages of embryos was significantly higher after vitrification than after controlled freezing (P