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

The effect of combining double layer density gradient centrifugation (DL-DGC) with different capacitation treatments on the fertilising capacity of frozen–thawed stallion sperm was examined via a heterologous assay involving in vitro-matured, zona pellucida-free bovine oocytes. In a first experiment, aliquots of frozen–thawed stallion sperm were subjected to one of five capacitation treatments without DL-DGC – ionomycin at 1.0 μM, 0.1 μM, 0.05 μM or 0.01 μM, or caffeine at 200 μg/mL. The fertilising capacity of the semen was then assessed at 18 h by staining the above oocytes with 4,6-diamidino-2-phenylindole (DAPI) and examining for sperm penetration, the number of penetrated spermatozoa per oocyte, and male pronucleus formation. In a second experiment, aliquots of frozen–thawed stallion sperm were subjected to DL-DGC selection – or not – and then further subjected to the two best capacitation treatments (0.1 μM and 0.05 μM ionomycin). The fertilising capacity of the semen was then determined as above. The DL-DGC/capacitated sperm samples showed the highest mean penetration rates: 24.16% following capacitation with 0.1 μM ionomycin, and 12.21% following capacitation with 0.05 μM ionomycin. The capacitated but non-DL-DGC-selected sperm returned significantly lower values: 6.26% and 7.02% for the same ionomycin treatments respectively. These findings suggest that combining DL-DGC selection with ionomycin capacitation improves the fertilising capacity of frozen–thawed stallion sperm.