Sperm vitrification as alternative to conventional freezing is increasing in popularity in many species. It has been achieved by direct exposure of diluted semen to liquid nitrogen in spheres or straws. Both techniques have been successfully developed, but they had not been compared yet in donkeys. The aim of this study was to compare these two methods of vitrification for donkey semen. Ejaculates from six Andalusian donkeys were collected and extended in Gent without glycerol supplemented with sucrose 0.1 M (Molar). Samples were slowly cooled at 5°C. For vitrification, 30 μl suspensions (spheres) were dropped directly into liquid nitrogen (LN2 ) or filled in covered 0.25 ml straws and then plunged into the LN2 (straws). For warming, straws and spheres were directly immersed in 3 ml of INRA-96 at 43°C. Total (TM, %) and progressive motility (PM, %) were objectively evaluated by computer-assisted sperm analysis and plasma membrane integrity (PMI, %) by epifluorescence microscopy. Results showed the straw method resulted in significantly higher values than spheres for: TM (54.7% ± 10.1 vs. 28.6% ± 6.5) and PM (44.2% ± 9.4 vs. 17.7% ± 6.4), but no significant differences were found between straws or spheres for PMI (31.5 ± 10.7 vs. 41.6 ± 14.3) respectively. In conclusion, donkey sperm could be vitrified in straws obtaining better sperm motility parameters after warming in comparison to the sphere method.
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