Standardization of CASA sperm motility assessment in the ram

This study was designed to evaluate the effect of different viewing chambers or slides, drop sample volume, and number of sperm per field on CASA sperm motility parameters in the ram. In the first trial, the effect of two different drop sample volumes, 5 and 10 μl, on the parameters of sperm motility were compared using slide-coverslip and Makler chamber. No differences between the two drop sample volumes in the two devices were found in the different sperm motility parameters. In the second experiment, significant differences (P < 0.001) were observed for all the sperm motility parameters studied among three devices evaluated: slide-coverslip, Makler and ISAS chambers. A higher proportion of motile spermatozoa was observed in Makler chamber than in other devices, while the ISAS chamber caused an important decrease in the percentage of motile and progressive sperm. Sperm velocity variables were different in the three systems with slide-coverslip > Makler > ISAS. Similar results were recorded in Makler and ISAS chambers, but lower than in the slide-coverslip in the parameters related to the quality of movement. Makler chamber showed to be less variable, whereas the greatest variability was recorded in slide-coverslip, where significant differences between central and peripheral fields were found. In the third trial, fresh semen samples were mixed with different volumes of forced dead semen to determine proportions of non-motile spermatozoa in the samples, which were evaluated for CASA sperm motility and membrane integrity. Slide-coverslip was more accurate, as the percentages of motile sperm were more correlated to the sperm viability results (r = 0.95, 0.90, 0.92 for slide-coverslip, Makler and ISAS chamber, respectively, P < 0.001) and to the theoretical values of sperm motility after adding known percentages of dead sperm (R2 = 0.84, 0.79. 0.79, respectively, P < 0.001). It was concluded that the type of viewing chamber used for CASA sperm motility analysis in the ram had a considerable influence on results, precision and accuracy, whereas the drop volume had little effect. The final goal of sperm motility assessment is the evaluation of male fertility, the rigorous standardization and validation of the CASA protocol being a pre-requisite to obtain reliable results. Results of the present study contribute to this standardization and validation process in the ram.