14 FREEZING OF DONKEY SEMEN AFTER 24 HOURS OF COOL STORAGE: PRELIMINARY RESULTS

Several studies on sperm cooling and cryopreservation have been done in horses; however, only a few them have been developed in donkeys. In addition, no studies have been performed to freeze cooled stored donkey semen. Therefore, the aim of this study was to determine if it is possible to freeze donkey sperm after 24 h of cool storage. Semen was collected from 4 Andalusian donkeys by artificial vagina. After collection, each sample was separated into 2 aliquots; one of them was immediately frozen (t0) and the other one was cooled and stored before freezing (t24). The cryopreservation procedure consisted of a previous dilution of semen with EquiPro™. After that, semen was centrifuged and the sperm pellet resuspended with Gent® extender plus ethylene glycol (4%) to achieve a final concentration of 100 × 106 sperm mL–1. Sperm was slowly cooled to 5°C, loaded in 0.5-mL plastic straws and frozen in LN vapours. The second aliquot (t24) was diluted with Gent® extender to a final concentration of 50 × 106 sperm mL–1 and then cooled and stored at 5°C for 24 h. After that, cooled semen samples were cryopreserved following the same procedure as described above. Straws were thawed in a water bath at 37° for 30 s. Computer-assisted sperm motility analysis was performed. Total motility (TM), progressive motility (PM), and the following kinematic parameters: velocity curvilinear (VCL; µm s–1), velocity straight line (VSL; µm s–1), velocity average path (VAP; µm s–1), linearity (LIN; %), straightness (STR; %), wobble (WOB; %), amplitude of lateral head displacement (ALH; µm), and beat cross frequency (BCF; Hz) were compared between treatments by ANOVA. Results were expressed as mean ± standard error. Significant differences (P < 0.05) were found between treatments (t0 v. t24) for TM (63.76 ± 4.75 v. 51.67 ± 3.69), PM (36.01 ± 3.19 v. 27.24 ± 2.72), VCL (77.29 ± 0.65 v. 67.56 ± 0.78), VSL (58.50 ± 0.61 v. 52.11 ± 0.76), VAP (67.82 ± 0.64 v. 59.41 ± 0.79), LIN (57.90 ± 0.33 v. 59.53 ± 0.32), STR (70.39 ± 0.30 v. 72.43 ± 0.41), WOB (75.64 ± 0.22 v. 75.48 ± 0.32), ALH (1.88 ± 0.09 v. 1.69 ± 0.10), and BCF (6.28 ± 0.04 v. 6.51 ± 0.06). These preliminary results showed significant differences between cryopreservation at 0 and 24 h post-cooling; however, understanding that direct freezing is better in terms of sperm motility, cryopreservation of cooled stored semen could still be considered good according to the values obtained for sperm motility parameters after thawing. In our opinion, sperm centrifugation before cooling probably improve the results of cryopreservation 24 h post-cooling, due to the negative effect of seminal plasma on sperm viability during storage. In addition, the analysis of other sperm parameters would be useful to check more accurately differences between treatments. In conclusion, sperm motility parameters were higher in donkey semen samples immediately frozen after collection in comparison to semen samples cryopreserved after 24 h of cooling storage. Further studies are needed to improve cooling and cryopreservation procedures for freezing cooled stored donkey semen.