The effect of trehalose on post-thaw viability and fertility of European brown hare (Lepus europaeus Pallas, 1778) spermatozoa

The aim of the study was to assess the effect of a different trehalose concentration on the post-thaw viability and fertility of European brown hare spermatozoa. The semen was collected under general anaesthesia with electroejaculation method from 4 males. Immediately after collection, the semen was diluted with an extender of the following composition: Tris 250 mM, citric acid 80 mM, glucose 70 mM, DMSO 1.0 M, egg yolk (17%, v/v), and kanamycin (80 mg/l)—Protocol I. In Protocols II and III, respectively, 50 mM and 100 mM of trehalose were added to the extender. Immediately after thawing and after 3 h incubation at 37 °C, motility characteristics of frozen/thawed semen were assessed with computer-assisted semen analysis system, and a percentage of viable, acrosome intact spermatozoa was evaluated using flow cytometry. Immediately after thawing spermatozoa motility (MOT), average path velocity (VAP), straight velocity (VSL) and curvilinear velocity (VCL) were the highest in the semen frozen without addition of trehalose (P < 0.01). After 3 h of incubation, MOT and spermatozoa with progressive motility (PMOT) were the lowest in the semen frozen with supplementation of 100 mM of trehalose (P < 0.01) and VAP, VSL, VCL and amplitude of lateral head displacement (ALH) were significantly lower in the semen frozen with supplementation of 50 mM and 100 mM of trehalose compared to the semen frozen without the addition of trehalose (P < 0.01), which indicates an unfavourable effect of trehalose on the motility characteristics of European brown hare spermatozoa. However, the effect of trehalose on a percentage of viable, acrosome intact spermatozoa was not observed. As a result of artificial insemination, 54.55% females became pregnant after insemination with the semen frozen according to Protocol I, 72.73% and 50% females became pregnant after insemination with the semen supplemented with 50 mM and 100 mM of trehalose, respectively. The number of young born was 1.67 ± 0.52, 1.75 ± 1.04 and 1.60 ± 0.55, in each group, respectively. There were not any significant differences in the results of artificial insemination between groups. Summing up, it should be stated that the addition of trehalose to the extender did not have a favourable effect on post-thaw viability of European brown hare spermatozoa and an influence of trehalose on the results of artificial insemination was not found, either.