Use of density centrifugation for delayed cryopreservation of stallion sperm: perform sperm selection directly after collection or after storage?

This research examined the impact of timing and techniques for stallion sperm cryopreservation, particularly the benefits of performing density centrifugation immediately after collection versus after 1 day of storage. Single and two-layer variations of centrifugation were tested and compared with standard techniques, assessing effects on sperm morphology, motility, and health. The results revealed both forms of density centrifugation yielded better results compared to traditional means, especially when performed right after the sperm collection rather than before cryopreservation.

Study Design and Methodology

• The objective was to determine whether the timing of density centrifugation, a sperm selection procedure, influences sperm survival rates during delayed cryopreservation, with testing undertaken after storing sperm for one day at 5°C.
• A two-layer method using iodixanol and a single-layer method using Androcoll for density centrifugation were both employed and compared to samples prepared with standard centrifugation
• Emphasis was placed on comparing the effects of centrifugation undertaken on the day of sperm collection with centrifugation after one-day storage in a refrigerated state.
• Sperm properties such as morphology and motility, as well as the integrity of membranes and chromatin, were evaluated before and after centrifugation. Sperm motility and membrane integrity were assessed after cryopreservation.

Results and Findings

• The research found that both single-layer and two-layer density centrifugation led to higher percentages of normal and motile sperm with better membrane and chromatin integrity than standard centrifugation or diluted samples, though the differences were only about a 5% improvement.
• The recovery rates following density centrifugation were about 30-40%.
• The study also discovered that when cryopreservation was performed after storing sperm for one day in refrigeration, conducting centrifugation immediately after collecting the semen led to a higher percentage of intact plasma membrane sperm after thawing than performing centrifugation processing of stored sperm just before cryopreservation.
• No significant variations in progressively motile sperm were observed after thawing.

Conclusion

• The study concluded that for delayed cryopreservation, it is advantageous to carry out density centrifugation immediately after semen collection, as opposed to just before cryopreservation.