Comparison of different cryopreservation methods for horse and donkey embryos.

The study evaluates the effectiveness of two methods of cryopreservation – slow freezing and vitrification – on horse and donkey embryos, with the aim of identifying the least damaging protocol. Results indicated that while both methods caused some damage, vitrified and slow frozen embryos did not show significantly different rates of cell damage, with donkey embryos exhibiting less susceptibility to vitrification than horse embryos.

Study Design and Methodology

• The study followed a randomized control experiment design, using horse (n=19) and donkey (n=16) embryos that were collected between the sixth and seventh day post ovulation.
• Embryos were subjected to either of two cryopreservation methods – slow freezing or vitrification – or left untreated as a control. The slow freezing method used a solution of 1.5 mol/L Ethylene Glycol (EG).
• The vitrification process involved exposing horse embryos to various EG-glycerol solutions and implementing either a Fibreplug or a 0.25mL straw. Donkey embryos underwent a similar process, but with the addition of a 7.0 mol/L EG solution.
• After thawing or warming, the embryos were assessed for quality. Variables studied included the number of dead cells, incidence of apoptosis (programmed cell death), presence of fragmented nuclei and the quality of the cytoskeleton (cellular structure).

Results and Findings

• Ideally, cryopreservation should not alter embryo quality. However, the study found a significant decrease in the quality amongst the embryos subjected to cryopreservation, regardless of the method used, compared to their non-frozen controls.
• (No significant difference was unearthed between the slow freezing and vitrification methods used on the horse and donkey embryos, in terms of dead cell percentages.
• Meanwhile, the rate of apoptosis varied with the species and method used. For horse embryos, the apoptosis rate was unaffected by the method of cryopreservation. However, donkey embryos vitrified using EG-glycerol in a Fibreplug showed a higher incidence of cell apoptosis.
• Both the methods resulted in a higher percentage of nonviable cells for horse embryos compared to donkey embryos.
• Across every method, the quality of the actin cytoskeleton, an indication of embryo health and developmental potential, remained unchanged.

Conclusions

• The study concluded that vitrified horse and donkey embryos were not more susceptible to cell damage than those preserved by slow freezing, irrespective of whether straws or Fibreplug devices were used.
• However, donkey embryos appeared less susceptible to damage during vitrification than horse embryos, with fewer nonviable and apoptotic cells when vitrified with an EG solution of 7 mol/L.
• While the number of embryos per group was low due to difficulties in sourcing, this still represents key insights into slow freezing and vitrification as cryopreservation methods for horse and donkey embryos.