We have applied a statistical protocol based on principal component analysis, clustering methods, and discriminant analysis for the identification of

Computer-assisted sperm analysis (CASA) allows assessing the motility of individual spermatozoa, generating huge datasets. These datasets can be analyzed using data mining techniques such as cluster analysis, to group the spermatozoa in subpopulations with biological meaning. This review considers the use of statistical techniques for clustering CASA data, their challenges and possibilities. There are many clustering approaches potentially useful for grouping sperm motility data, but some options may be more appropriate than others. Future development should focus not only in improvements of subpopulation analysis, but also in finding consistent biological meanings for these subpopulations.

Antioxidants could improve sperm media, extending the viability of spermatozoa and protecting their DNA. The protective ability of lipoic acid, melatonin, Trolox and crocin was tested on red deer spermatozoa incubated at 37°C. Cryopreserved spermatozoa were thawed and incubated with 1 mM or 0.1 mM of each antioxidant, with or without oxidative stress (100 µM Fe2+). Motility (CASA), viability, mitochondrial membrane potential and acrosomal status were assessed. Lipoperoxidation (malondialdehyde production), intracellular reactive oxygen species (ROS) and DNA status (TUNEL) were checked at 4 h. Incubation alone increased ROS and decreased motility. Oxidative stress intensified these effects, increasing lipoperoxidation and DNA damage. Lipoic acid had little protective effect, whereas 1 mM melatonin showed limited protection. Trolox lowered ROS and lipoperoxidation both in oxidised and non-oxidised samples. In oxidised samples, Trolox prevented DNA and acrosomal damage, and ameliorated motility. Crocin at 1 mM showed similar results to Trolox, but noticeably stimulated motility and had no effect on lipoperoxidation. In a second experiment, a broader range of crocin and melatonin concentrations were tested, confirming the effects of crocin (positive effects noticeable at 0.5–0.75 mM), but showing an increase in lipoperoxidation at 2 mM. Melatonin was increasingly effective at 2.5 and 5 mM (ROS, lipoperoxidation and DNA status). Crocin seems a promising new antioxidant, but its particular effects on sperm physiology must be further studied, especially the consequences of motility stimulation and confirming its effect on lipoperoxidation. Melatonin might be useful at relatively high concentrations, compared to Trolox.