To evaluate the effect of acquisition rate in the computer-assisted sperm analysis (CASA) of sperm motility with different kinetic characteristics (linearity and track speed) belonging to different species.Prospective research study.Andrology laboratory of department of applied biology.Semen samples from human fertile donors, rabbit, bull, and ram.Semen analysis by optical microscopy and sperm motility evaluation with the use of CASA.Analysis of sperm tracks with a CASA system acquired at different frame rates (FRs) (12–200 Hz).The FR had no effect on the motility rate or on the sperm track reconstruction, whereas all the other kinetic values were affected by sampling frequency. Sperm with low speed (category b) and/or linear trajectories (category a) are less affected by sampling frequency and do not require particularly high FR to be correctly evaluated. On the contrary, fast nonlinear sperm (category c) would require a very high frequency to fully describe the path.Because all the commercial CASA systems acquire at an FR of 30–60 Hz, an increase in FR is required to render the estimation results closer to the “real path” mainly for fast nonlinear sperm.