The aim of this study is to validate and optimize the computer-assisted semen analysis system Sperm Class Analyzer® (SCA®, Microptic S.L., Barcelona, Spain) for the assessment of sperm quality in C57BL/6N mice. The focus is on the analysis of sperm motility under different experimental conditions to investigate the effects of technical and biological confounders as well as cryopreservation on CASA measurements. The first experiment compares sperm motility in two media, Ham's F-10 and TYH. Ham's F- 10 results in significantly higher values for all measured parameters except beat cross frequency (BCF). This is due to the lower viscosity of the medium, which allows a more accurate assessment of sperm movement. In contrast, TYH is primarily used for sperm capacitation prior to in vitro fertilization (IVF). The second experiment examines the variability of sperm motility in fresh samples. Significant differences are observed between individuals, samples, and video fields analyzed, despite the fact that all animals are genetically identical and kept under standardized laboratory conditions. Variance component analysis reveals that these factors together account for only about 2–5% of the total variance, indicating a high proportion of unexplained variation. A statistically sufficient number of mice, samples, and video fields is therefore essential to ensure reliable and representative results. The third experiment investigates the effects of cryopreservation on sperm motility. After freezing and thawing, motility is drastically reduced, especially the proportions of motile and progressively motile sperm (a reduction of 77–84%). Velocity and direction of movement are less affected. These results confirm previous studies and show that cryopreservation can significantly affect sperm integrity. However, fertility is maintained by improved IVF protocols, such as the use of antioxidants. In summary, this study highlights the importance of media selection, accounting for biological variability, and the effects of cryopreservation. The results provide reference values for CASA measurements in C57BL/6N mice and serve as a basis for future studies investigating the effects of genetic or environmental factors on sperm motility.
Lara Paar (Fri,) studied this question.