Effects on brown bear (Ursus arctos) spermatozoa freezability of different extender and dilution ratios used for pre-freezing centrifugation

摘要

The objective of this study was to determine how the extender and dilution ratio used during centrifugation affect bear spermatozoa quality before and after freezing-thawing. Semen was collected from 15 brown bears by electroejaculation. In experiment1, semen was divided into five aliquots and diluted using one of the following extenders: Tris-citric-glucose (TCG), Tris-citric-glucose-3% BSA, Tris-citric-glucose-1% egg yolk or CaninePro. In experiment 2, semen was divided into five aliquots and diluted 1:1, 1:4, 1:8 or 1:16 (semen:extender) with Tris-citric-glucose. In both experiments, one aliquot was left undiluted and it was used as a control. All the aliquots were centrifuged at 600xg for 6 min and frozen. Samples were analysed by post-thawingfor motility (CASA) and, by flow cytometry, for viability (YO-PRO-1), acrosomal status (PNA-FITC/PI) and mitochondrial status (JC-1). CaninePro rendered the highest motility with respect to the undiluted control (total motility, 53.1% vs. 38.5%, P<0.001), and CaninePro and TCG significantly increased the percentage of viable and acrosome-intact spermatozoa (43.2 and 43.4, respectively, vs. 39.4, P<0.05). In experiment 2, dilution 1:4 yielded the highest value of total motility (78.8 vs. 67.2, .P<0.05) and proportion of spermatozoa with intact membrane and acrosome (64.5 vs. 54.4, P<0.01). In general, diluting 1:4 or 1:8 brown bear semen prior to centrifugation improved the motility and acrosome status of the thawed spermatozoa.