Introduction
Sperm motility is a key determinant of fertilization success in externally fertilizing fishes. While dilution is inevitable through activation, its effects on motility are not fully understood. In various fish species, we observed a paradoxical pattern: increasing sperm concentration was associated with reduced sperm velocity. This raised critical questions about the biological underpinnings of sperm behaviour and the reliability of motility measurements under varying conditions. We therefore experimentally tested the effect of cell density on sperm motility.
Materials and Methods
Semen samples were collected from 12 Eurasian perch Perca fluviatilis and measured after activation under 5 different dilution ratios (1:25 to 1:200), under constant or variable osmolarity (i.e. 60 treatments), to disentangle effects from cell density and osmolarity variation (typical with dilution). To disentangle potential physical (cell collisions) and biological effects, we conducted an experiment comparing velocity patterns of the same samples in controlled proportion (0, 12.5, 25, 50 and 100%) of live and dead sperm. Motility parameters were quantified using Qualisperm CASA on a total of 180 scenario. Linear mixed-effects models were used to assess the effects of dilution, osmolarity, and live/dead ratio on sperm velocity and progressive motility.
Results
Sperm motility and dilution were inversely correlated regardless of osmolarity. When looking closer, we found that it was due to the activation of slow sperm (5-25 µm/s) at lower sperm dilution / higher cell densities. Indeed, neither fast sperm (> 25 µm/s) nor non progressive sperms (1-5 µm/s) proportions were affected by dilution. The live-dead sperm experiment revealed that at constant cell density, the velocity was significantly negatively affected by the proportion of live sperm. Together, these results suggest an active, biologically mediated mechanism rather than passive physical interference, which modulates activation of slower sperm in the context of higher live cell densities.
Discussion
These findings challenge the assumption that sperm motility measurements are concentration-independent and call into question the comparability of published data without standardized protocols. Our results highlight the importance of live-cell-dependent density effects, possibly involving chemical signalling, ionic feedback, or metabolic interactions among spermatozoa. Such mechanisms may reflect evolved strategies of sperm competition, where motility is modulated in response to perceived gametic density. These insights have direct relevance for artificial fertilization practices in aquaculture and offer a new lens for understanding reproductive strategies in externally fertilizing species.