Method: Cold-storage temperature and dilution effects on kinematics of Brahman bull sperm patterns assessed with a Computer-Assisted Semen Analysis portable device

Beef cattle profitability depends on bull fertility. To replace traditional subjective bull quality ratings, a portable Computer-Assisted Semen Analysis (CASA) device can be used to generate objective results. The device requires careful control of temperature, dilution rate, and other methodological variables. This work provides a bovine semen analysis adapted methodology using a CASA portable device. It considers the effects of dilution, cooling times, and temperature in farms, where CASA evaluations of sperm quality, motility, and kinematics are frequently carried out. Fifteen ejaculates from five Brahman bulls were collected by electro-ejaculation. Aliquots were extended in Optixcell at 1:1 or 1:40 (v:v), stored at 5 °C and 17 °C, and evaluated after 2, 4, 8, 24, 48, and 72 h. Motility and kinematics were measured with iSperm®, and viability with an eosin–nigrosine stain. An ANOVA determined the effects of dilution rate, temperature, and cooling times, as well as interactions of the same. When effects proved statistically significant, mean comparisons ± SEM were carried out using the Bonferroni test (P < 0.05). The 1:1 dilution precluded CASA tracking, whereas the 1:40 dilution (∼90 × 10⁶ mL⁻¹) produced reliable outputs. Total and progressive motility declined with storage time (P < 0.05), occurring after 8 h at 17 °C and 6 h at 5 °C. Cooling reduced curvilinear, average-path, and straight-line velocities; yet at 5 °C, the highest absolute speeds were observed, whereas 17 °C yielded the most progressive trajectories, with higher LIN and STR indices. Membrane integrity dropped sharply between 48 and 72 h. Dilution to 1:40 and storage at 17 °C conserved more progressive motility, viability, and membrane integrity for up to 24 h than 5 °C. The protocol is useful for developing field guidelines to preserve sperm quality traits that could be associated with fertility during short-term storage in beef systems using fresh artificial insemination.