The lifespan of spermatozoa from externally fertilizing freshwater fish ranges from a few seconds to several minutes, depending on the species. External factors, such as temperature, background flows and ion composition, play an important role in fertilization success. Specific mechanisms guiding spermatozoa appear to be essential to maximize the sperm–egg encounter under these strenuous conditions. Although some existing data support the hypothesis that both the ovarian fluid and the eggs may release chemoattractants that significantly affect spermatozoa behaviour and the fertilization outcome, this hypothesis is still open to debate, as the existence of freshwater fish spermatozoa chemotaxis has yet to be demonstrated; in addition, specific mechanisms supporting spermatozoa guidance and gamete selection have not been elucidated. Is the natural selection of gametes determined by a combination of different physicochemical phenomena? Alternatively, is the natural selection of species‐specific gametes biased towards the species‐specific guidance mechanisms of their natural landscape? These questions have received more attention as new studies have revealed potential, distinct guidance mechanisms in freshwater fish reproduction. In this review, we discuss the empirical studies supporting different hypotheses about freshwater fish gamete guidance and highlight the synergistic combination of experiments and biomathematical modelling to explore these questions. Finally, we discuss the challenges in understanding the mechanisms behind sperm guidance in freshwater fish species, and we suppose that knowledge about the mechanisms that underlie spermatozoa selection and guidance in freshwater fish species may elucidate the impact of the traditional aquaculture practice of artificial fertilization on progeny quality and species sustainability.

Like seahorses, some of the closely-related pipefish species (Family Syngnathidae) incubate their eggs within a male brood pouch. This has contributed to considerable confusion about sperm transfer mechanisms to the eggs; some authors have reported that ejaculates are released directly into water before they reach the eggs, while others have suggested that eggs are fertilised using spermatozoa deposited directly into the brood pouch via an internal sperm duct. Here we present anatomical evidence from the freshwater pipefish, Syngnathus abaster, showing not only that direct sperm deposition into the pouch is impossible, but that spermatozoa must somehow travel a significant distance (>4 mm) outside the body of the male, to reach and fertilise eggs in the pouch. We have also used several putative sperm-activating solutions to identify the type of environment most conducive to sperm activation. Spermatozoa released from the testis were active for a brief period (<5 min) in water or 150 mm saline, but showed prolonged (>25 min) motility in ovarian fluid. This suggests that spermatozoa are released into a mixture of ovarian fluid and eggs while the male and female are in close contact. Our data also suggest that the fertilisation mechanism is highly efficient (sperm : egg ratio <200 : 1) even though this pipefish species produces dimorphic spermatozoa (with long and short flagellae). The shorter (<40 microm) morphotypes were not capable of motility activation, and are therefore probably incapable of fertilisation. If so, the sperm : egg ratio reported here would represent an overestimate.

Motility analysis of spermatozoa relies on the investigation of either head trajectories or flagellum characteristics. Those two sets of parameters are far from being independent, the flagellum playing the role of motor, whereas the head plays a passive role of cargo. Therefore, quantitative descriptions of head trajectories represent a simplification of the complex pattern of whole sperm cell motion, resulting from the waves developed by the flagellum. The flagellum itself responds to a large variety of signals that precisely control its axoneme to allow activation, acceleration, slowing down or reorientation of the whole spermatozoon. Thus, it is obvious that analysis of flagellum characteristics provides information on the original source of movement and orientation of the sperm cell and presents additional parameters that enrich the panoply of quantitative descriptors of sperm motility. In this review, we briefly describe the methodologies used to obtain good-quality images of fish spermatozoa (head and especially flagellum) while they move fast and the methods developed for their analysis. The paper also aims to establish a link between classical analyses by computer-aided sperm analysis (CASA) and the descriptors generated by fish sperm flagellum analysis, and emphasises the information to be gained regarding motility performance from flagellum motion data.

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