Salvaging urospermic ejaculates from brown bear (Ursus arctos)

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.