Semen quality assessment requires accurate, reliable and objective methods for examination of sperm variables including sperm motility. For preparation of semen samples for artificial insemination, as a genetic resource, samples that are used for insemination need to have the capacity to result in a highly acceptable fertility rate. Several methods have been developed for evaluation of bull sperm in laboratory conditions and for preparation of doses for artificial insemination. Computer-assisted semen analyses can provide objective information on various sperm variables. Nevertheless, this equipment requires fine-calibrations considering differences among species, breeds and conditions for sample evaluation and data analyses. In the present study, there was examination of the interaction between factors such as image frame rate and type and depth of counting chamber in which sperm were evaluated, together with differences between bulls of four breeds. The use of the Spermtrack® reusable 10 μm-depth chamber provided more reliable results than results obtained using disposable chambers (10 and 20 μm depth). A capture rate of at least 90 fps is required for assessment of sperm motility percentage, whereas a rate of 250 fps is needed for obtaining consistent kinematic data. Differences among breeds in the present study indicate conditions for sperm analyses should include specific equipment calibrations for each breed. These results contribute to development of more precise conditions for assessments of bull sperm quality taking into account breed differences and the requirement each breed has for the adequate evaluation and preparation of samples for artificial insemination.