title: Tolerance of brown bear spermatozoa to conditions of pre-freezing cooling rate and equilibration time.
abstract: Specific protocols for the cryopreservation of endangered Cantabrian brown bear spermatozoa are critical to create a genetic resource bank. The aim of this study was to assess the effect of cooling rates and equilibration time before freezing on post-thawed brown bear spermatozoa quality. Electroejaculates from 11 mature bears were extended to 100 × 10(6) spermatozoa/mL in a TES-Tris-Fructose-based extender, cryopreserved following performance of the respective cooling/equilibration protocol each sample was assigned to, and stored at -196 °C for further assessment. Before freezing, after thawing, and after 1 hour's incubation post-thawing at 37 °C (thermal stress test), the quality of the samples was assessed for motility by computer-assisted semen analysis, and for viability (SYBR-14/propidium iodide), acrosomal status (peanut agglutinin-fluorescein isothiocyanate /propidium iodide), and sperm chromatin stability (SCSA) by flow cytometry. In experiment 1, three cooling rates (0.25 °C/min, 1 °C/min, and 4 °C/min) to 5 °C were assessed. After thawing, total motility (%TM) was higher and percentage of damaged acrosomes (%dACR) was lower (P < 0.05) for 0.25 °C/min than for 4 °C/min. The thermal stress test data indicated equally poor quality (P < 0.05) for the 4 °C/min cooled samples in viability (%VIAB), %dACR, %TM, and progressive motility (%PM). In experiment 2, the effect of a pre-freezing equilibration period at 5 °C for 1 hour (cooling at 0.25 °C/min) was evaluated. Samples kept at 5 °C for 1 hour showed higher (P < 0.05) values than the nonequilibrated ones for both thawing (%dACR) and thermal stress test (%VIAB, %TM, and %PM). In experiment 3, samples stored without cooling and equilibration (direct freezing) were compared with the samples cooled at 0.25 °C/min and equilibrated for 1 hour (control freezing). Using thermal stress test, we observed that direct freezing causes damage in viability, acrosomal status, and motility of spermatozoa compared with the control group (P < 0.05). In conclusion, our results suggest that slow cooling rates to 5 °C and at least 1 hour equilibration time are necessary for the effective cryopreservation of brown bear sperm.
authors.affiliation: ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
authors.forename: E
authors.initials: E
authors.lastname: López-Urueña
authors.affiliation: ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
authors.forename: M
authors.initials: M
authors.lastname: Alvarez
authors.affiliation: ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
authors.forename: S
authors.initials: S
authors.lastname: Gomes-Alves
authors.affiliation: ITRA-ULE, INDEGSAL, University of León, León, Spain; Molecular Biology (Cell Biology), University of León, León, Spain.
authors.forename: C
authors.initials: C
authors.lastname: Martínez-Rodríguez
authors.affiliation: Cabárceno Park, Cantabria, Spain.
authors.forename: S
authors.initials: S
authors.lastname: Borragan
authors.affiliation: ITRA-ULE, INDEGSAL, University of León, León, Spain; SaBio IREC (CSIC-UCLM-JCCM), Campus Universitario, Albacete, Spain.
authors.forename: L
authors.initials: L
authors.lastname: Anel-López
authors.affiliation: ITRA-ULE, INDEGSAL, University of León, León, Spain; Molecular Biology (Cell Biology), University of León, 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, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
authors.forename: L
authors.initials: L
authors.lastname: Anel
mesh_terms: D000818:Animals; D015925:Cryopreservation / Q000379:methods / Q000662:veterinary; D005615:Freezing; D008297:Male; D012662:Semen Preservation / Q000379:methods*; D013081:Sperm Motility; D013094:Spermatozoa / Q000502:physiology*; D013997:Time Factors; D001503:Ursidae / Q000502:physiology*
keywords: Brown bear; Cooling rates; Direct freezing; Equilibration time; Sperm cryopreservation