title: Salvaging urospermic ejaculates from brown bear (Ursus arctos).
abstract: The objective of this study was to reverse the osmotic stress of sperm in urine contaminated bear ejaculates that were obtained by electroejaculation using pre-freezing washing or density gradient centrifugation isolation. In Experiment 1, ejaculates were divided into six aliquots, five were diluted in each washing extender: 200, 300, 400, 500 and 700 mOsm/kg (prepared from a Tes-Tris-Fructose base, adding water or fructose as corresponds), at a 1:2 ratio (raw semen: washing solution, v/v); and the other aliquot was handled without washing (Control group). Samples were centrifuged at 600 × g for 6 min prior to freezing. In Experiment 2, ejaculates were divided into two aliquots: one was diluted 1:1 with TCG (Tris-Citric acid-Glucose) and centrifuged at 600 × g for 6 min (Centrifugation Control; C-Control); the other was treated with PureSperm density gradient column. After treatments, samples were cryopreserved. Sperm motility, viability (SYBR-14/propidium iodide (PI)) and acrosomal status (peanut agglutinin-fluorescein isothiocyanate (PNA-FITC)/PI) were analyzed before and after freezing. Ejaculates with an initial osmolality of less than 120 mOsm/kg treated with pre-freezing washing, and the Control sample had greater pre-freezing sperm motility than the raw ejaculate, but sperm viability was not different among these groups. The samples washed with 700 mOsm/kg solutions had the least pre-freezing viability. In the post-thawing evaluation, pre-freezing washing treatments did not provide any improvement in comparison with the Control sample, and treatment with 700 mOsm/kg extender had deleterious effects in all urospermic samples. PureSperm density gradient centrifugation applied to urospermic raw semen was suitable for improving sperm motility and viability of pre-freezing samples and the selected spermatozoa had greater freezing capacity.
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: 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: M
authors.initials: M
authors.lastname: Nicolas
authors.affiliation: ITRA-ULE, INDEGSAL, University of León, 24071 León, Spain; Cell Biology, University of León, 24071 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: Borragán
authors.affiliation: ITRA-ULE, INDEGSAL, University of León, 24071 León, Spain.
authors.forename: C A
authors.initials: CA
authors.lastname: Chamorro
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
authors.affiliation: ITRA-ULE, INDEGSAL, University of León, 24071 León, Spain; Cell Biology, University of León, 24071 León, Spain. Electronic address: ppazc@unileon.es.
authors.forename: P
authors.initials: P
authors.lastname: de Paz
mesh_terms: D000818:Animals; D002499:Centrifugation, Density Gradient / Q000662:veterinary; D015925:Cryopreservation / Q000662:veterinary; D004542:Ejaculation; D004558:Electric Stimulation; D006863:Hydrogen-Ion Concentration; D009997:Osmotic Pressure; D012661:Semen / Q000502:physiology*; D055101:Semen Analysis; D012662:Semen Preservation / Q000379:methods; D001503:Ursidae / Q000502:physiology* / Q000652:urine*
keywords: Brown bear; Osmotic stress reversion; PureSperm(®); Spermatozoa; Urospermic ejaculates