Effect of anti-freeze protein (AFP) on the cooling and freezing of equine embryos as measured by DAPI-staining.

Equine embryos recovered on Day 6 after ovulation were cooled to +4 degrees C, or frozen with AFP alone or together with glycerol. Twenty embryos (140-200 microm in diameter) were randomly assigned to 6 treatment groups. In the first 3 groups, the embryos were cooled from room temperature to +4 degrees C at a rate of 3 degrees C/min and warmed again at a rate of 32 degrees C/min in a programmable freezer. In the second 3 groups, the embryos were frozen using a standard protocol, stored in liquid nitrogen for 5-7 days and then thawed in a 37 degrees C waterbath. After cooling/warming or freezing/thawing all the embryos were stained with DAPI. The percentage of dead cell area was significantly lower in the cooling groups than in the freezing groups and no significant differences were apparent between the cryoprotectants used in the study.