SURVIVAL RATE AND ULTRASTRUCTURE OF VITRIFIED BOVINE IN-VITRO AND IN-VIVO DEVELOPED EMBRYOS

The capacity of different vitrification media and methods was tested o nto in vivo and in vitro produced bovine morula/blastocysts and their ultrastructure and survival studied post-thawing. Two vitrification so lutions were finally selected, named 40 ES (40% ethylene glycol in PBS containing 0.5 M sucrose) and 35 EFS (composed of 35% (v/v) ethylene glycol in PBS containing 0.5 M/1 sucrose and 30% (w/v) Ficoll 70). The straws were either precooled or not precooled in nitrogen vapour, plu nged and stored in LN, for 10-25 days, and then thawed in a 20 degrees C waterbath. The content of the straws was rediluted in 1M sucrose so lution in PBS and later cocultured with BOEC for 48 h. The overall sur vival rates for in vitro and in vivo embryos were 36% (12 of 33) and 2 0% (3 of 15) after 24 h and 21% (7 of 33) and 33% (5 of 15) after 48 h . The survival rates for precooled embryos were significantly higher t han for not precooled (48% vs 13% after 24 h and 44% vs 4% after 48 h) when tested across vitrification media. The in vitro-produced embryos presented an ultrastructure similar to the pre-freeze state, irrespec tive of the vitrification media used. The in vivo developed embryos sh owed a rather modified post-thaw ultrastructure, with clear signs of o smotic changes at both the trophoblastic and embryonic cells. The resu lts Indicated that in vitro and in vivo developed bovine embryos can s urvive vitrification using ethylene glycol as a cryoprotectant.