title: Flow Cytometric Chromosomal Sex Sorting of Stallion Spermatozoa Induces Oxidative Stress on Mitochondria and Genomic DNA.
abstract: To date, the only repeatable method to select spermatozoa for chromosomal sex is the Beltsville sorting technology using flow cytometry. Improvement of this technology in the equine species requires increasing awareness of the modifications that the sorting procedure induces on sperm intactness. Oxidative stress is regarded as the major damaging phenomenon, and increasing evidence regards handling of spermatozoa - including sex sorting - as basic ground for oxidative damage. The aim of this study was to disclose whether the flow cytometric sorting procedure increases the production of reactive oxygen species (ROS), and to identify if ROS production relates to DNA damage in sorted spermatozoa using specific flow cytometry-based assays. After sorting, oxidative stress increased from 26% to 33% in pre- and post-incubation controls, to 46% after sex sorting (p < 0.05). Proportions of DNA fragmentation index post-sorting were approximately 10% higher (31.3%); an effect apparently conduced via oxidative DNA damage as revealed by the oxyDNA assay. The probable origin of this increased oxidative stress owes the removal of enough seminal plasma due to the unphysiological sperm extension, alongside a deleterious effect of high pressure on mitochondria during the sorting procedure.
authors.affiliation: Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, Caceres, Spain.
authors.forename: C M
authors.initials: CM
authors.lastname: Balao da Silva
authors.affiliation: Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, Caceres, Spain.
authors.forename: C
authors.initials: C
authors.lastname: Ortega-Ferrusola
authors.affiliation: Division of Reproduction, Faculty of Veterinary Medicine and Animal Sciences, Swedish University of Agricultural Sciences, Uppsala, Sweden.
authors.forename: J M
authors.initials: JM
authors.lastname: Morrell
authors.affiliation: Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, Linköping, Sweden.
authors.forename: H
authors.initials: H
authors.lastname: Rodriguez Martínez
authors.affiliation: Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, Caceres, Spain.
authors.forename: F J
authors.initials: FJ
authors.lastname: Peña
mesh_terms: D000818:Animals; D002469:Cell Separation / Q000379:methods / Q000662:veterinary*; D004249:DNA Damage; D053938:DNA Fragmentation; D004272:DNA, Mitochondrial / Q000235:genetics; D005434:Flow Cytometry / Q000662:veterinary*; D006736:Horses / Q000235:genetics; D008297:Male; D053078:Membrane Potential, Mitochondrial; D018384:Oxidative Stress / Q000235:genetics*; D012661:Semen / Q000502:physiology; D012743:Sex Preselection / Q000379:methods / Q000662:veterinary*; D013081:Sperm Motility; D013094:Spermatozoa / Q000502:physiology
chemical_list: D004272:DNA, Mitochondrial