COMPARISON OF 2-STEP VITRIFICATION VERSUS CONTROLLED FREEZING ON SURVIVAL OF IN-VITRO PRODUCED CATTLE EMBRYOS

Grade 1 in vitro produced bovine day 7 embryos at the compact morula-e arly blastocyst, blastocyst and expanded blastocyst stage were selecte d for cryopreservation. From 7 replicates, 572 embryos were randomly d ivided into two soups. One soup was cryopreserved by controlled freezi ng after 20 minutes equilibration in 10% v/v glycerol and slow cooling (0.3 degrees C / minute) from -7 degrees C to -30 degrees C. The othe r group was vitrified after 3 minutes exposure to 20% v/v ethylene gly col and 30 to 45 seconds exposure to a vitrification solution consisti ng of 40% v/v ethylene glycol, 18% w/v ficoll and 10.26% w/v sucrose. Embryos from both soups were thawed in a water bath at 20+/-1 degrees C. Frozen-thawed embryos were diluted in three successive solutions co nsisting of 10.26% w/v sucrose and 6.6%, 3.3% and 0% glycerol, respect ively, during 5 minutes for each seep. Vitrified-warmed embryos were d iluted in a 8.58 w/v sucrose solution during 5 minutes, All diluted em bryos were cultured in Menezo-B2 medium supplemented with bovine ovidu ct epithelial cells. The survival rate of all developmental stages of embryos was significantly higher after vitrification than after contro lled freezing (P<0.001). It was 39.4% and 11.2% for compact morulae-ea rly blastocysts, 81.9% and 34.9% for blastocysts and 96.7% and 72.3% f or expanded blastocysts, respectively. The overall hatching rate follo wing the two cryopreservation methods was not significantly different. However, the hatching rate of vitrified expanded blastocysts was sign ificantly higher than the hatching rate of frozen expanded blastocysts (71.38 and 43.3%, respectively, P<0.001). It is concluded that the ch illing sensitivity of embryos is significantly reduced by vitrificatio n. A high proportion of blastocysts and expanded blastocysts can be su ccessfully cryopreserved with a two-step vitrification procedure.