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

The research article studies the efficacy of anti-freeze protein (AFP) in the cooling and freezing of horse embryos, detailing the processes used and the results obtained, which showed AFP alone, or together with glycerol, could significantly reduce cell death during cooling and freezing.

Methodology

• The research team collected equine embryos on the sixth day after ovulation.
• These embryos, measuring between 140-200 microm in diameter, were cooled to +4 degrees Celsius. This cooling was done with the anti-freeze protein (AFP) alone or in conjunction with glycerol.
• A total of twenty embryos were arbitrarily divided into six different treatment groups.
• For the first three groups, the embryos underwent cooling from room temperature to +4 degrees Celsius. This cooling process had a rate of 3 degrees Celsius per minute. Warming also took place, clocking in at a rate of 32 degrees Celsius per minute. A programmable freezer facilitated this technique.
• The remaining three groups had embryos that were frozen using a customary protocol. These were then stored in liquid nitrogen for a period spanning five to seven days. The embryos were then thawed in a 37 degrees Celsius water bath.
• After the embryos were cooled and heated again or frozen and defrosted later, they were all stained with DAPI (a nucleic acid stain).

Findings

• The research yielded notable results relating to the impact of cooling and freezing on equine embryos.
• The percentage of dead cell area was considerably reduced in the cooling groups in comparison to the freezing ones.
• Moreover, it was discovered that there were no discernible differences between the cryoprotectants used in the study.
• From this, it can be inferred that using the anti-freeze protein (AFP) either alone or alongside glycerol can play a crucial role in minimizing cell death during the freezing and cooling of equine embryos.