Cryopreservation of equine embryos with glycerol plus sucrose and glycerol plus 1,2-propanediol.

The research article investigates the effectiveness of two different cryoprotectant mixtures, glycerol plus sucrose and glycerol plus 1,2-propanediol, in the cryopreservation of equine embryos. The study found that neither mixture was able to adequately protect the embryos from damage during the freezing and thawing process.

Research Methods and Groups

• The study involved testing on four groups of six or seven-day-old equine embryos: Group 1 (15 embryos due for immediate transfer), Group 2 (15 embryos for deep-freezing using glycerol + sucrose), Group 3 (10 embryos for deep-freezing using glycerol + 1,2-propanediol), and Group 4 (3 fresh embryos for ultrastructural analysis).
• All embryos, except three from Group 3 used for ultrastructural analysis, were subjected to the freezing and thawing process and then transferred to recipient mares.

Cryoprotection Removal and Pregnancy Determination

• After thawing, cryoprotectants were removed through progressive dilution in a solution of PBS (buffer used to maintain cell health) + 15% fetal calf serum (FCS) featuring decreasing concentrations of cryo-protectants.
• Pregnancies were then determined ultrasonographically, revealing differing success rates across the groups: 53.3% (Group 1), 13.3% (Group 2), and 0% (Group 3).

Ultrastructural Analysis and Results

• The ultrastructural analysis undertaken on the frozen/thawed embryos and fresh embryos disclosed significant differences.
• In the frozen/thawed embryos, embryonic cells appeared malformed, demonstrating dilation of internal and perivitelline spaces (the space enveloping the embryo), fewer desmosomes (junctions between cells), minimal microvilli (microscopic cellular protrusions that increase surface area for absorption), and a nearly total absence of pinocytotic vesicles (small vesicles that ‘drink’ in extracellular fluid).
• Most mitochondria presented irregularities and dilation on the cristae (folds in the mitochondria), appearing electron-dense.
• The empirical data suggest that the chosen cryoprotectants were inadequate in safeguarding embryos against damage during freeze-and-thaw cycles, specifically highlighting the ultrastructural alterations detected in Group 3 embryos as the reason for zero pregnancies in the group.