title: Optimization of protocols for Iberian red deer (Cervus elaphus hispanicus) sperm handling before sex sorting by flow cytometry.
abstract: Currently, sperm reproductive biotechnologies such as sex sorting and cryopreservation are undoubtedly valuable tools for improving the economic and biological efficiency of red deer production systems. In this context, and because of the particular characteristics of this species (extensive exploitation typically far from laboratory facilities), a key goal is to optimize the design of an adequate handling protocol of sperm samples before samples are subjected to sex sorting and cryopreservation procedures to obtain better outputs from the application of these technologies. The main aim of this paper was to design an adequate protocol for Iberian red deer sperm handling before sex sorting by flow cytometry to obtain optimal yields when sex sorting is used in this species. Semen samples from 11 adult males were obtained by electroejaculation during the breeding season. In this study, we tested different protocols for the handling of Iberian red deer spermatozoa before sorting by using different concentrations of sperm (400 or 800 × 106) and adding or not Hoechst 33342 before the transport of samples to the sorting facilities. Based on the results, the most adequate method used to handle samples before sorting was transportation at a high sperm concentration (800 × 106/mL) without Hoechst 33342. These transportation conditions in combination with Hoechst 33342 staining at 5.2 μL/mL once at the flow cytometry laboratory resulted in better (P < 0.05) sorting efficiency (99.9% of the samples showing split) than both, those samples transported at 400 × 106sperm/mL (between 51.2 and 55.2% of the samples showing split) and those samples stained before transport at a sperm concentration of 400 × 106sperm/mL (between 15.4 and 75.7% of the samples showing split). Sorting rates and sperm quality after sorting and cryopreservation was not affected (P > 0.05) by sperm handling before sorting. Moreover, the sorting yields were compatible with the practical application of these reproductive biotechnologies.
authors.affiliation: SaBio IREC (CSIC-UCLM- JCCM), Albacete, Spain.
authors.forename: Luis
authors.initials: L
authors.lastname: Anel-López
authors.affiliation: SaBio IREC (CSIC-UCLM- JCCM), Albacete, Spain.
authors.forename: Olga
authors.initials: O
authors.lastname: Garcia-Álvarez
authors.affiliation: SaBio IREC (CSIC-UCLM- JCCM), Albacete, Spain.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Maroto-Morales
authors.affiliation: Department of Animal Medicine and Surgery, University of Murcia, Murcia, Spain.
authors.forename: Tatiana
authors.initials: T
authors.lastname: Tarantini
authors.affiliation: Department of Animal Medicine and Surgery, University of Murcia, Murcia, Spain.
authors.forename: David
authors.initials: D
authors.lastname: Del Olmo
authors.affiliation: Medianilla S.L. Finca Las Lomas, Vejer de la Frontera, Cádiz, Spain.
authors.forename: Jose Antonio
authors.initials: JA
authors.lastname: Ortiz
authors.affiliation: Department of Animal Medicine and Surgery, University of Murcia, Murcia, Spain.
authors.forename: Emilio Arsenio
authors.initials: EA
authors.lastname: Martinez
authors.affiliation: Department of Animal Medicine and Surgery, University of Murcia, Murcia, Spain.
authors.forename: Jordi
authors.initials: J
authors.lastname: Roca
authors.affiliation: Department of Animal Medicine and Surgery, University of Murcia, Murcia, Spain.
authors.forename: Juan Maria
authors.initials: JM
authors.lastname: Vazquez
authors.affiliation: SaBio IREC (CSIC-UCLM- JCCM), Albacete, Spain.
authors.forename: Jose Julian
authors.initials: JJ
authors.lastname: Garde
authors.affiliation: Department of Animal Medicine and Surgery, University of Murcia, Murcia, Spain. Electronic address: parrilla@um.es.
authors.forename: Inmaculada
authors.initials: I
authors.lastname: Parrilla
mesh_terms: D000818:Animals; D015925:Cryopreservation / Q000662:veterinary; D003670:Deer / Q000502:physiology*; D005434:Flow Cytometry / Q000662:veterinary*; D008297:Male; D055101:Semen Analysis / Q000662:veterinary; D012662:Semen Preservation / Q000662:veterinary; D012743:Sex Preselection / Q000379:methods / Q000662:veterinary*; D013048:Specimen Handling; D013094:Spermatozoa / Q000502:physiology*; D013194:Staining and Labeling
keywords: Cryopreservation; Red deer; Sex sorting; Sperm