In the present work, the effects of the activation media with different osmolality and storage conditions on sperm motility and kinetic parameters over time after activation, using CASA system (ISAS®) in Danio rerio were analysed. The effect of pooled and individual ejaculate samples was also studied. Spermatozoids activated with high osmolality medium showed higher values in motility parameters and kinematic parameters in comparison with low osmolality medium. Respect to the individual ejaculates or the pool, although significant differences were observed in some parameters, no relevant changes were observed in total motility neither in kinetic parameter. Total motility and velocity of sperm showed significant lower values after sperm storage for 24 h at 4 ºC.