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

Abstract

The objective of this study was to determine how the extender and dilution ratio used during centrifugation affect bear spermatozoa quality before and after freezingthawing. Semen was collected from 15 brown bears by electroejaculation. In experiment 1, 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 Triscitric-glucose. In both experiments, one aliquot was left undiluted and it was used as a control. All the aliquots were centrifuged at 600×g for 6 min and frozen. Samples were analysed by post-thawing for 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.

Salvaging urospermic ejaculates from brown bear (Ursus arctos)

“…Centrifugation is a necessary step to increase the concentration of brown bear semen obtained by electroejaculation and particularly in urospermic ejaculates that usually have lesser cell concentrations. Previous studies assessing the deleterious effects of centrifugation indicated that it is not a harmful procedure for brown bear spermatozoa (Nicolas et al, 2011;2012a). In the present study, the Control sample treated only with centrifugation was amongst those with the most desirable values for sperm motility, viability and acrosomal status, and the toxic effect of urine was not evident.…”

“…Immediately after collection, fresh semen was divided into six aliquots; five were diluted with each previously described washing extender (1 fresh semen: 2 extender, v/v) and centrifuged at 600 × g for 6 min (Nicolas et al, 2011); and the other was centrifuged without dilution and was used as the Control. After centrifugation, the supernatant was removed and the pellet was re-suspended in TTF-ULE extender at 20-22 • C and cryopreserved (Section 2.4).…”

“…Immediately after collection, fresh urospermic semen was divided into two aliquots: one aliquot (Centrifugation Control; C-Control) was diluted with the same volume of TCG (Tris, Citric acid, glucose extender) (Nicolas et al, 2011) and centrifuged at 600 × g for 6 min; the other was treated with PureSperm ® (Nidacon Laboratories AB, Goteborg, Sweden).…”

Spermatozoa recovery and post-thawing quality of brown bear ejaculates is affected for centrifugation regimes

“…The effects of centrifugation on spermatozoa may be influenced by many factors, such as centrifugation time, centrifugal forces, dilution rate, semen extender and individual variability. Several authors working (Okano et al 2006), 200×g for 8 min (Spindler et al 2004), and we have used 600×g for 6 min (Nicolas et al 2011). A variety of centrifugation regimes for dog has been reported: 400×g for 5 min (Kawakami et al 2002), 700×g for 5 min (Schafer-Somi et al 2006), 700×g for 10 min (Okano et al 2004a), and 700×g for 6 min (Rota et al 1995).…”

Effect of colloid (Androcoll-Bear, Percoll, and PureSperm) selection on the freezability of brown bear (Ursus arctos) sperm

“…Thus, for the ejaculates of some species, centrifugation of semen before cryopreservation is necessary to reach an appropriate cellular concentration (dog [12], stallion [13,14], and brown bear [2,6]) or even to remove seminal plasma (goat [15]) and clean urinecontaminated samples (horse [16] and brown bear [17]). Despite previous studies assessing the deleterious effects of centrifugation, this procedure does not appear to be harmful for brown bear spermatozoa [18,19]. Regarding seminal plasma, some studies have shown beneficial effects of its addition during semen processing and cryopreservation (deer [20] and ram [21]), whereas other authors have reported deleterious effects (dog [12], goat [22], ram [23], stallion [13,14], and bull [24]).…”