The improvement of freezing extenders is critical when defining sperm cryopreservation
protocols for wild species, in order to create germplasm banks. The aim of this study
was to evaluate the effect of additives (Equex Paste and EDTA) supplementation, egg-yolk
(10 and 20%) and glycerol (4 and 8%) concentrations and extender osmolality (300 and
320 mOsm/kg) on the post-thawing quality of brown bear semen. Semen was obtained from
20 adult males by electroejaculation, and centrifugated individually (600 x g for
6 min). The pellets were diluted 1:1 in the corresponding extender TTF (TES-Tris-Fructose
with the aforementioned variants) and cooled to 5 degrees C. Then, it was diluted
down to 100 x 10(6) spz/mL, loaded in 0.25 mL straws and frozen at -20 degrees C/min.
After thawing (in water at 65 degrees C for 6s), the semen samples were assessed for
motility (CASA), viability (SYBR-14 with propidium iodide), acrosomal status (PNA-FITC
with propidium iodide) and mitochondrial activity (JC-1). Extender supplementation
with additives rendered significantly higher results for these sperm parameters. Comparing
the two percentages of egg yolk, 20% egg yolk showed the highest motility results,
percentages of viable spermatozoa and viable spermatozoa with intact acrosome. No
differences were detected among samples frozen using 4 or 8% glycerol. For extender
osmolality, 300 mOsm/kg showed higher values of VAP, VCL, VSL, and ALH than 320 mOsm/kg.
Based on the best performance of sperm motility, viability and acrosome status, we
conclude that the most suitable extender to cryopreserve brown bear spermatozoa was
TTF adjusted to 300 mOsm/kg, supplemented with 20% egg yolk, 4-8% glycerol, and the
additives 1% Equex paste and 2% EDTA.