Effects of extender, cryoprotectants and thawing protocol on motility of frozen-thawed stallion sperm that were refrozen for intracytoplasmic sperm injection doses.

The research investigates how the mixture used to preserve stallion sperm, the protective agents included, and the temperature at which semen is first thawed can influence the movement of the sperm when it is unfrozen, re-frozen, and then thawed again, aiming to find the most effective method for intracytoplasmic sperm injection (ICSI) doses.

Objective of the Study

• The research aimed to understand how different freezing extenders, cryoprotectant agents (CPAs), and initial thawing temperatures affect the motility (movement capabilities) of refrozen stallion sperm, particularly for use in intracytoplasmic sperm injection (ICSI) procedures.

Methodology

• The study used single ejaculations from twelve stallions, which were then frozen in a lactose-EDTA-egg yolk extender (LE) with 5% glycerol.
• Two main experiments were conducted. In the first, sperm were initially thawed to either 5°C or 37°C, then diluted in LE or skim milk-egg yolk extender (SMEY) containing either 5% glycerol (GLY), 5% methylformamide (MF), or 5% of both combined (GMF).
• In the second experiment, frozen sperm were thawed to 5°C, then diluted and refrozen in SMEY containing varying concentrations (2%, 4%, 6%, or 8%) of GLY or GMF.

Results

• The first experiment concluded that sperm motility was significantly reduced after each cycle of freezing and thawing. The type of extender did not significantly affect motility, but sperm initially thawed to 5°C had higher motility than those thawed to 37°C. Furthermore, sperm that were refrozen in SMEY with MF or GMF had higher motility than sperm refrozen with GLY only.
• The second experiment found an interaction between the choice of CPA and its concentration. Sperm refrozen with GMF had higher post-thaw motility than those refrozen with just GLY. Also, sperm refrozen with higher concentrations of GMF (6% or 8%) showed the highest post-thaw motility.

Conclusion

• The research concluded that for ICSI doses, the best method to maintain sperm motility is to initially thaw the sperm at 5°C and dilute it in a freezing extender with a concentration of 8% GMF before refreezing.