The effects of vitrification after equilibration in different concentrations of cryoprotectants on the survival and quality of bovine blastocysts.

Abstract

This study assessed the effects of cryoprotectant concentration during equilibration on the efficiency of bovine blastocyst vitrification and the expression of selected developmentally important genes. In vitro produced bovine blastocysts were equilibrated in either 7.5% ethylene glycol (EG) + 7.5% DMSO (Va group) or in 2% EG+2% DMSO (Vb group) then vitrified on Cryotop® sheets in 16.5% EG+16.5% DMSO+0.5M sucrose. After warming, embryos were cultured for 48hr. Re-expansion, hatching, and the numbers of total and membrane damaged cells were compared among vitrified groups and a control. There was no significant difference between the vitrified groups in survival, cell numbers and the extent of membrane damage. Vitrification increased the number of membrane-damaged cells in both groups, however, in a greater extent in the Vb group. Vitrification increased (p<.05) the expression of the HSP70 gene in Va but not in Vb embryos. The expression of IGF2R, SNRPN, HDAC1, DNMT3B, BAX, OCT4, and IFN-t genes were the same in control and vitrified groups. In conclusion, the concentration of cryoprotectants during equilibration did not affect survival rates; however, normal cell numbers could be maintained only by equilibration in 15% cryoprotectants which was associated with increased HSP70 expression.