Pre-selection by double layer density gradient centrifugation improves the fertilising capacity of frozen-thawed, capacitated stallion sperm.

This study investigates the impact of double layer density gradient centrifugation (DL-DGC) accompanied by various capacitation treatments on the fertilizing ability of frozen-thawed stallion sperm. The findings reveal that combining DL-DGC with ionomycin capacitation enhances the fertilization potential of stallion sperm after freezing and thawing.

Objective of the Research

• The primary objective of this research was to determine the effect of combining DL-DGC with varying capacitation treatments on the fertilizing capacity of frozen-thawed stallion sperm.
• The study involved an in-vitro method of testing on bovine oocytes that had matured and were missing their outer ‘zona pellucida’ layer.

Methodology

• The study was implemented in two stages to track changes in fertilization capacity. In the first part of the experiment, aliquots of frozen-thawed stallion sperm were treated with one of five capacitation treatments without DL-DGC.
• These treatments included ionomycin at different concentrations or caffeine. The fertilizing capacity of this semen was then assessed by staining the prepared oocytes with DAPI and observing sperm penetration, the count of penetrated spermatozoa per oocyte, and the formation of the male pronucleus.
• In the second part of the experiment, aliquots of frozen-thawed stallion sperm were either subjected to DL-DGC selection or not, then capacitated using the two optimal ionomycin treatments (0.1μM and 0.05μM) from the first experiment. After this step, the fertilizing capacity of the semen was calculated using the same methods as in the initial experiment.

Results and Conclusion

• The sperm samples that underwent both DL-DGC and capacitation demonstrated the highest average penetration rates (24.16% and 12.21% for the two ionomycin treatments respectively), compared to a significantly lower performance in the capacitated but non-DL-DGC-selected sperm samples (6.26% and 7.02% respectively).
• This signifies that combining DL-DGC selection with ionomycin capacitation significantly enhances the fertilising capacity of frozen-thawed stallion sperm and may be beneficial for improved conception rates in assisted reproductive technologies.