title: Alternative procedures for the cryopreservation of brown bear ejaculates depending on the flexibility of the "in cooling" period (5°C).
abstract: The adaptability of cryopreservation protocols for brown bear spermatozoa collected under field conditions and frozen in a nearby laboratory (transported for a few hours) or shipped to a reference laboratory for sex sorting (transported for a few days) was evaluated. Forty-nine electroejaculates from 15 mature brown bears were extended to 100×10(6) sperm/mL in a TES-Tris-Fructose based extender and cryopreserved (-20°C/min to -100°C and stored at -196°C). After thawing, the quality of the seminal samples was assessed for total (TM), progressive (PM) motility and kinetic parameters - by CASA -, and viability (VIAB), viable and non-apoptotic status (YOPRO-), high membrane mitochondrial potential (MIT) and intact acrosomes (iACR) - by flow cytometry -. In Experiment 1, we assessed different storage times (0, 0.5, 1 - control -, 4-5, 7-8 and 11-12 h) at 5°C from final dilution to freezing. After thawing, non-equilibrated samples (0 h) showed lower values of iACR, TM and PM. No significant differences were found for the different periods of equilibration tested. In Experiment 2, we evaluated three long-term storage times (24, 48 and 72 h) at 5°C before freezing using storage for 1h as control. The post-thawing quality of brown bear spermatozoa declined markedly after 48-72 h of pre-freezing. In conclusion, our findings suggest the possibility of extending the pre-freezing cooling period up to 24h post-collection without freezing. This knowledge should enable the adaptation of the freezing protocols for when a special handling conditions are required such as the shipment of seminal samples to technological centers for the pre-freezing application of enhancer spermatic biotechnologies.
authors.affiliation: ITRA-ULE, INDEGSAL, University of León, 24071 León, Spain; Animal Reproduction and Obstetrics, University of León, 24071 León, Spain.
authors.forename: E
authors.initials: E
authors.lastname: López-Urueña
authors.affiliation: ITRA-ULE, INDEGSAL, University of León, 24071 León, Spain; Animal Reproduction and Obstetrics, University of León, 24071 León, Spain.
authors.forename: M
authors.initials: M
authors.lastname: Alvarez
authors.affiliation: ITRA-ULE, INDEGSAL, University of León, 24071 León, Spain; Animal Reproduction and Obstetrics, University of León, 24071 León, Spain.
authors.forename: S
authors.initials: S
authors.lastname: Gomes-Alves
authors.affiliation: ITRA-ULE, INDEGSAL, University of León, 24071 León, Spain; Animal Reproduction and Obstetrics, University of León, 24071 León, Spain.
authors.forename: P
authors.initials: P
authors.lastname: Manrique
authors.affiliation: ITRA-ULE, INDEGSAL, University of León, 24071 León, Spain; SaBio IREC (CSIC-UCLM-JCCM), Campus Universitario s. n., 02071 Albacete, Spain.
authors.forename: L
authors.initials: L
authors.lastname: Anel-López
authors.affiliation: ITRA-ULE, INDEGSAL, University of León, 24071 León, Spain; Veterinary Anatomy, University of León, 24071 León, Spain.
authors.forename: C A
authors.initials: CA
authors.lastname: Chamorro
authors.affiliation: Cabárceno Park, Cantabria, Spain.
authors.forename: S
authors.initials: S
authors.lastname: Borragan
authors.affiliation: ITRA-ULE, INDEGSAL, University of León, 24071 León, Spain; Molecular Biology (Cell Biology), University of León, 24071 León, Spain. Electronic address: ppazc@unileon.es.
authors.forename: P
authors.initials: P
authors.lastname: de Paz
authors.affiliation: ITRA-ULE, INDEGSAL, University of León, 24071 León, Spain; Animal Reproduction and Obstetrics, University of León, 24071 León, Spain.
authors.forename: L
authors.initials: L
authors.lastname: Anel
mesh_terms: D000818:Animals; D015925:Cryopreservation / Q000379:methods / Q000662:veterinary*; D008297:Male; D053078:Membrane Potential, Mitochondrial; D055101:Semen Analysis; D012662:Semen Preservation / Q000379:methods / Q000662:veterinary*; D013081:Sperm Motility; D013094:Spermatozoa / Q000166:cytology* / Q000378:metabolism; D001503:Ursidae / Q000502:physiology
keywords: Brown bear; Equilibration time; Long-term storage; Spermatozoa cryopreservation