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 bovi ne viral diarrhea virus (BVDV) through the zona pellucida (ZP) of intact bo vine embryos during the pre-freezing step of cryopreservation was investiga ted in a series of experiments. In vitro fertilized (IVF) embryos at the bl astocyst stage were exposed to 10(6) TCID50 BVDV (non-cytopathic NY-1 strai n) in a 30% suspension of either ethylene glycol, glycerol, DMSO, or 2 M su crose in physiological saline for 10 min at 20 degrees C. Subsequently, the embryos were washed free of residual unbound viral particles, and the ZP o f some embryos were removed by micromanipulation. Groups of ZP-intact embry os, ZP-free embryonic cells and their respective ZP were then tested separa tely for the presence of virus. The infectious virus was detected in associ ation with 81% (17/21) of samples containing non-micromanipulated ZP-intact embryos which were exposed to the virus and cryoprotectants and then washe d 10 times and in 83% (43/53) of the samples containing only ZP from microm anipulated embryos (P > 0.05). The virus was not found in the samples conta ining the corresponding embryonic cells of embryos exposed previously to th e virus and cryoprotectants. It was concluded that the transfer of embryos from the isotonic PBS solution into a highly hypertonic cryoprotectant solu tion did not cause the passage of BVDV through ZP and its entry to embryoni c cells. (C) 1999 Elsevier Science B.V. All rights reserved.