The effect of high concentrations of cryoprotectants on the passage of bovine viral diarrhea virus through the zona pellucida of in vitro fertilized embryos

The effect of high concentrations of cryoprotectants on the passage of bovine viral diarrhea virus (BVDV) through the zona pellucida (ZP) of intact bovine embryos during the pre-freezing step of cryopreservation was investigated in a series of experiments. In vitro fertilized (IVF) embryos at the blastocyst stage were exposed to 106 TCID50 BVDV (non-cytopathic NY-1 strain) in a 30% suspension of either ethylene glycol, glycerol, DMSO, or 2 M sucrose in physiological saline for 10 min at 20°C. Subsequently, the embryos were washed free of residual unbound viral particles, and the ZP of some embryos were removed by micromanipulation. Groups of ZP-intact embryos, ZP-free embryonic cells and their respective ZP were then tested separately for the presence of virus. The infectious virus was detected in association with 81% (17/21) of samples containing non-micromanipulated ZP-intact embryos which were exposed to the virus and cryoprotectants and then washed 10 times and in 83% (43/53) of the samples containing only ZP from micromanipulated embryos (P>0.05). The virus was not found in the samples containing the corresponding embryonic cells of embryos exposed previously to the virus and cryoprotectants. It was concluded that the transfer of embryos from the isotonic PBS solution into a highly hypertonic cryoprotectant solution did not cause the passage of BVDV through ZP and its entry to embryonic cells.