Comparison of different vitrification protocols on viability after transfer of ovine blastocysts in vitro produced and in vivo derived

Abstract

We compare different vitrification protocols on the pregnancy and lambing rate of in vitro produced (IVP) and in vivo derived (IVD) ovine embryos. Ovine blastocysts were produced by in vitro maturation, fertilization and culture of oocytes collected from slaughtered ewes or superovulated and inseminated animals. Embryos were cryopreserved after exposure at room temperature either for 5min in 10% glycerol (G), then for 5min in 10% G+20% ethylene glycol (EG), then for 30s in 25% G+25% EG (glycerol group), or for 3min in 10% EG+10% dimethyl sulphoxide (DMSO), then for 30s in 20% EG+20% DMSO+0.3M sucrose (DMSO group). One group of in vitro produced embryos was cryopreserved similarly to the DMSO group, but with 0.75M sucrose added to the vitrification solution (DMSO 0.75 group). Glycerol group embryos were then loaded into French straws or open pulled Straws (OPS) while the DMSO group embryos were all loaded into OPS and directly plunged into liquid nitrogen.Embryos were warmed with either a one step or three step process. In the one step process, embryos were placed in 0.5M sucrose. The three-step process was a serial dilution in 0.5, 0.25 and 0.125M sucrose. The embryos of DMSO 0.75 group were warmed directly by plunging them into tissue culture medium-199 (TCM-199)+20% foetal bovine serum (FBS) in the absence of sucrose (direct dilution). Following these manipulations, the embryos were transferred in pairs into synchronised recipient ewes and allowed to go to term. The pregnancy and the lambing rate within each group of IVP and IVD embryos indicated that there was no statistical difference among the vitrification protocols.