Mitochondrially active boar sperm are essential for fertilization but evade pelleting by centrifugation – implications for in-vitro sperm enrichment and antioxidant enzyme analysis

The significance of sperm properties in fertility prediction remains a subject of ongoing research. A previous study based on data from 130 boars, each of which provided two ejaculates, found a correlation between the percentage of sperm with active mitochondria and fertility. Because mitochondrial activity is necessary for sperm to maintain motility, gametes require a finely tuned antioxidant defense system against the associated formation of radical oxygen species. In the current study, we examined the intracellular activity of the antioxidant enzymes superoxide dismutase and glutathione peroxidase in washed sperm pellets from one of the ejaculates from our previous study that were stored at -80°C. We investigated whether antioxidant enzyme activity contributes to fertility prediction. To relate enzyme activity to the respective sperm counts in the pellets, we first determined the sperm concentration in the pellets. After comparing the sperm counts in the pellets with the original sperm counts used for centrifugation, we found that the conventional washing protocol resulted in sperm losses of 37–87%. Losses were greater in samples with higher sperm quality. The antioxidant enzyme activity in a sperm pellet was therefore not representative of the entire sperm population in the sample and was not meaningful for fertility prediction. The percentage of sperm with active mitochondria still correlated with fertility, although only one ejaculate per boar was evaluated. These results should generally be considered when analyzing intracellular sperm properties. • Mitochondrial activity is essential for boar sperm fertilization ability • Mitochondrially active motile sperm evade enrichment by centrifugation • Antioxidant enzyme activity in the subpopulation of pelleted sperm is not indicative of sample fertility • Optical density proved suitable to determine pellet sperm concentration after storage at -80°C