Comparison of two CASA systems and counting chambers for the evaluation of frozen-thawed stallion semen

Semen quality assessment represents a fundamental tool for estimating stallion fertility potential and is based on the evaluation of macroscopic and microscopic parameters, including motility, concentration, and sperm morphology [1]. The introduction of Computer-Assisted Semen Analysis (CASA) systems has improved the objectivity and standardization of these evaluations, although the results may still be influenced by technical factors such as the software used, the type of counting chamber, and operating conditions [2,3]. The aim of this study was to compare two CASA systems, AndroVision® (Minitube) and Ceros II® (Hamilton Thorne), in combination with two different counting chambers, Leja® (IMV Technologies) and SpermVU® (Spermtech), for the assessment of motility, concentration, and kinematic parameters in frozen-thawed stallion semen. Semen samples from 10 stallions were analysed; for each animal, a pool of three 0.5 mL straws was prepared and diluted 1:3 with INRA96®. Evaluations were performed at 5 (T5) and 60 minutes (T60) after thawing. Sperm concentration was determined using both CASA systems and a Thoma chamber. The CASA system significantly affected (P<0.05) all evaluated parameters except VCL for total spermatozoa and STR for motile spermatozoa. The counting chamber also had a significant effect (P<0.05), with the SpermVU® chamber consistently yielding higher motility values compared to the Leja® chamber, regardless of the CASA system or incubation time. These findings confirm that semen evaluation using CASA systems is highly influenced by technical variables, highlighting the need for strict protocol standardization to ensure reliable results. Furthermore, data obtained from different instruments are not directly comparable, and this should be carefully considered for accurate clinical interpretation of stallion semen quality. [1] Kenny R.M. Manual for Clinical Fertility Evaluation of the Stallion, Hastings, NE, Society for Theriogenology: 3-100, 1983; [2] Amann, R. P. et al. Computer-assisted sperm analysis (CASA): capabilities and potential developments. Theriogenology, 81, 5–17, 2014; [3] Hoogewijs, M. K. et al. Influence of counting chamber type on CASA outcomes of equine semen analysis. Equine Veterinary Journal, 44(5), 542–549, 2012.