title: Pre-selection by double layer density gradient centrifugation improves the fertilising capacity of frozen-thawed, capacitated stallion sperm.
abstract: 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 1.0μM, 0.1μM, 0.05μM or 0.01μM, or caffeine at 200μg/mL. The fertilising capacity of the semen was then assessed at 18h 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 (0.1μM and 0.05μM 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 0.1μM ionomycin, and 12.21% following capacitation with 0.05μM 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.
authors.affiliation: Dept. Animal Medicine and Surgery, School of Veterinary Medicine, Autonomous University of Barcelona, E-08193 Bellaterra, Spain.
authors.forename: Roser
authors.initials: R
authors.lastname: Morató
authors.forename: Juleide M De Souza
authors.initials: JM
authors.lastname: Soares
authors.forename: Guifré
authors.initials: G
authors.lastname: Orero
authors.forename: Teresa
authors.initials: T
authors.lastname: Mogas
authors.forename: Jordi
authors.initials: J
authors.lastname: Miró
mesh_terms: D000818:Animals; D002417:Cattle; D002499:Centrifugation, Density Gradient / Q000592:standards / Q000662:veterinary*; D005260:Female; D006736:Horses / Q000502:physiology*; D015759:Ionomycin / Q000008:administration & dosage / Q000494:pharmacology; D016018:Least-Squares Analysis; D008297:Male; D012662:Semen Preservation / Q000379:methods / Q000662:veterinary*; D013075:Sperm Capacitation / Q000187:drug effects / Q000502:physiology*; D013084:Sperm-Ovum Interactions / Q000502:physiology*; D013094:Spermatozoa / Q000502:physiology*
chemical_list: D015759:Ionomycin