title: Effect of overnight staining on the quality of flow cytometric sorted stallion sperm: comparison with tradtitional protocols.
abstract: Flow cytometry is considered the only reliable method for the separation of X and Y chromosome bearing spermatozoa in equines. The MoFlo SX DP sorter is highly efficient, allowing the production of foals of the desired sex. However, to achieve acceptable pregnancy rates the currently used protocol requires working with fresh semen obtained close to, or at, the sorting facility. An alternative protocol was tested during two consecutive breeding seasons. Fresh stallion semen was cooled for 20 h, during which staining with Hoechst 33342 took place. On the following day, this sample was flow sorted and compared with spermatozoa from the same ejaculate that had been sexed on the previous day. All sperm parameters evaluated remained unchanged when fresh sorted and refrigerated sorted semen were compared. Pre-sorting storage at 5°C did not alter sperm velocities nor kinetics, viability or membrane permeability, production of reactive oxygen species, mitochondrial membrane potential or DNA fragmentation index of the sorted sample. The findings open for the possibility of using semen from stallions housed far from the sorting facilities. Processed and stained sperm could be shipped refrigerated on the previous day, sorted and inseminated on the next day.
authors.affiliation: Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
authors.forename: C M
authors.initials: CM
authors.lastname: Balao da Silva
authors.forename: C
authors.initials: C
authors.lastname: Ortega Ferrusola
authors.forename: J M
authors.initials: JM
authors.lastname: Gallardo Bolaños
authors.forename: M
authors.initials: M
authors.lastname: Plaza Dávila
authors.forename: P
authors.initials: P
authors.lastname: Martín-Muñoz
authors.forename: J M
authors.initials: JM
authors.lastname: Morrell
authors.forename: H
authors.initials: H
authors.lastname: Rodriguez Martínez
authors.forename: F J
authors.initials: FJ
authors.lastname: Peña
mesh_terms: D000818:Animals; D001562:Benzimidazoles / Q000494:pharmacology*; D005434:Flow Cytometry / Q000662:veterinary*; D006736:Horses / Q000502:physiology*; D008297:Male; D053078:Membrane Potential, Mitochondrial; D017382:Reactive Oxygen Species; D012743:Sex Preselection / Q000662:veterinary*; D013081:Sperm Motility; D013094:Spermatozoa / Q000166:cytology* / Q000502:physiology; D013194:Staining and Labeling / Q000379:methods / Q000662:veterinary*; D013696:Temperature; D013997:Time Factors
chemical_list: D001562:Benzimidazoles; D017382:Reactive Oxygen Species; C017807:bisbenzimide ethoxide trihydrochloride