Equine spermatozoa stored in the epididymis for up to 96h at 4°C can be successfully cryopreserved and maintain their fertilization capacity.

The research presents evidence that horse sperm stored in the epididymis for up to 96 hours at 4°C can be successfully freeze-stored and still retain their ability to fertilize.

Research Objective and Methodology

The study primarily aimed at understanding the possible impacts of storing horse (equine) sperm within the epididymis under cooled conditions (at 4°C) for up to 96 hours. This was performed in the context of male horse castration. The researchers were interested in examining three aspects:

• Impact of sperm storage on their viability and chromatin (DNA-protein complex) condensation.
• Effect of pre-incubation of recovered sperm in a freezing extender (a solution that helps preserve the sperm during cryopreservation) before freezing on sperm viability and chromatin condensation.
• Effect of freezing-thawing process on sperm viability, chromatin condensation, reactive oxygen species (ROS) generation, protein tyrosine phosphorylation (a process critical for sperm function), and in vitro fertilization rate.

Key Findings

The researchers analysed the volume and concentration of sperm recovered from the epididymis and found these factors unaffected by storage. Notably, sperm cells remained viable after being refrigerated at 4°C for up to 72 hours.

Furthermore, pre-incubation of the sperm cells in the freezing medium did not negatively impact their viability up to 48 hours. Sperm viability was maintained even after 72 hours with only a minimal drop.

The freezing and thawing process did not significantly affect the sperm cells’ viability at different storage times. However, incubation for 30 minutes in the freezing medium and the subsequent freezing-thawing process resulted in increased chromatin decondensation (a process where DNA unwraps from the protein complex, making it more accessible for transcription).

Moreover, the researchers found no effects on the production of reactive oxygen species (molecules that can lead to cellular damage if produced in excess) up to 96 hours of storage. Furthermore, sperm protein phosphorylation patterns remained unchanged with prolonged storage.

Critically, the research also highlighted successful in vitro (lab-based) fertilization reaffirming that stored and cryopreserved sperm maintained their fertilizing capacity.

Conclusion

The study concludes that horse sperm can be successfully stored within the epididymis at a cooled temperature (4°C) for up to 72 hours and still retain their viability and ability to fertilize after being cryopreserved. This research has significant implications in the preservation of valuable genetic material, especially in instances where injury or death of a valuable male horse occurs.