Effects of equilibration media and co-culture on vitrification of sheep blastocysts derived in vitro

The effects of two vitrification protocols on the survival of sheep blastocysts were examined in embryos produced in vitro with sheep oviduct epithelial cells co-culture (CC) or without co-culture (C). Oocytes collected from slaughtered ewes were matured for 24 h, fertilized with fresh ram semen for 20 h and cultured in SOF medium for up to 9 days in vitro. For vitrification, blastocyst stage embryos were assigned to two equilibration groups (20% ethylen glycol (EG) or 10% glycerol (G) for the first equilibration), and as the second equilibration they were kept in 20% ethylen glycol plus 10% glycerol for 5 minutes. After 30 sec in vitrification solution (25% ethylen glycol + 25% glycerol), they were immediately immersed into liquid nitrogen. After thawing procedure, embryos were transferred into 0.25 M sucrose for 5 min, washed in Hepes buffered synthetic oviduct fluid (HSOF) and cultured in SOF medium for 24 h. Cleavage rates were 75.2% in C and 74.2% in CC groups, and blastocyst rates were 14.4% in C and 17.1% in CC groups. After in vitro culture of vitrified-thawed blastocysts, survival rates were 62.1, 38.4, 30.2, and 39.3% in C-EG, CC-EG, C-G and CC-G groups, respectively. This study shows that vitrification of sheep embryos using ethylene glycol instead of glycerol as a first equilibration cryoprotectant could give reasonable survival rates and that co-culture of embryos with sheep oviduct epithelial cell has no beneficial effect on vitrification of embryos.