title: Single-layer centrifugation separates spermatozoa from diploid cells in epididymal samples from gray wolves, Canis lupus (L.).
abstract: Sperm samples may be used for assisted reproductive technologies (e.g., farmed or endangered species) or as a source of haploid DNA or sperm-specific RNA. When ejaculated spermatozoa are not available or are very difficult to obtain, as is the case for most wild endangered species, the epididymides of dead animals (e.g., animals that have been found dead, shot by hunters or poachers, or that that require euthanasia in zoological collections) can be used as a source of sperm. Such epididymal sperm samples are usually contaminated with cellular debris, erythrocytes, leukocytes, and sometimes also bacteria. These contaminants may be sources of reactive oxygen species that damage spermatozoa during freezing or contribute undesired genetic material from diploid cells. We used single-layer centrifugation through a colloid formulation, Androcoll-C, to successfully separate wolf epididymal spermatozoa from contaminating cells and cellular debris in epididymal samples harvested from carcasses. Such a procedure may potentially be applied to epididymal sperm samples from other species.
authors.affiliation: Department of Population Biology, Uppsala University, Uppsala, Sweden; Conservation and Evolutionary Genetics Group, Estación Biológica de Doñana (EBD-CSIC), Sevilla, Spain; Senckenberg Research Institutes and Natural History Museums, Conservation Research Section, Gelnhausen, Germany. Electronic address: vmfuentes@senckenberg.de.
authors.forename: Violeta
authors.initials: V
authors.lastname: Muñoz-Fuentes
authors.affiliation: Division of Reproduction, Swedish University of Agricultural Sciences, Uppsala, Sweden.
authors.forename: Catharina
authors.initials: C
authors.lastname: Linde Forsberg
authors.affiliation: Conservation and Evolutionary Genetics Group, Estación Biológica de Doñana (EBD-CSIC), Sevilla, Spain.
authors.forename: Carles
authors.initials: C
authors.lastname: Vilà
authors.affiliation: Division of Reproduction, Swedish University of Agricultural Sciences, Uppsala, Sweden.
authors.forename: Jane M
authors.initials: JM
authors.lastname: Morrell
mesh_terms: D000818:Animals; D002498:Centrifugation / Q000379:methods / Q000662:veterinary*; D003102:Colloids / Q000737:chemistry; D004171:Diploidy; D004822:Epididymis / Q000166:cytology* / Q000502:physiology; D004847:Epithelial Cells / Q000166:cytology; D008297:Male; D055101:Semen Analysis / Q000662:veterinary; D013094:Spermatozoa / Q000166:cytology*; D019596:Wolves / Q000502:physiology*
chemical_list: D003102:Colloids
keywords: Androcoll-C; Assisted reproductive technologies; Colloid; RNA; Single-cell DNA; Sperm genotyping