Effects of ovarian fluid on sperm traits and its implications for cryptic female choice in zebrafish

In polyandrous mating systems, females maintain the opportunity to bias male fertilization success after mating in a process known as cryptic female choice. Mechanisms of cryptic female choice have been described both in internal and external fertilizers, and may affect fertilization processes at different stages before, during, and after fertilization. In internal fertilizers, females have substantial control over sperm storage and fertilization, whereas in external fertilizers, female control is limited. A key factor proposed to mediate cryptic female choice is the fluid surrounding the eggs, the ovarian fluid, as it may directly affect sperm performance. Here, we studied the role of ovarian fluid in post-mating sexual selection using the zebrafish, Danio rerio. Firstly, we assessed how ovarian fluid affects sperm swimming performance compared with freshwater. We focused on sperm motility, velocity, swimming trajectory, and longevity, all traits associated with competitive fertilization success in externally fertilizing fish. In a second step, we used a North Carolina II design to explore female, male, and female x male effects by testing sperm motility of 2 males in the ovarian fluid of 2 females in a total of 11 blocks. Our results indicate that the ovarian fluid affects sperm performance differently from freshwater. Specifically, sperm velocity, motility, and longevity were higher in the ovarian fluid than in freshwater, whereas sperm linearity and beat cross frequency showed the opposite pattern. Moreover, these effects varied according to male, female, and male x female identities, supporting the potential for cryptic female choice mediated by ovarian fluid in this species. When females mate multiply, they can potentially use the fluid that surrounds the eggs, the ovarian fluid, to select sperm of the preferred male. Using a controlled experimental design, we demonstrated that the ovarian fluid of certain females selectively increases sperm performance of certain males in zebrafish. Though this effect, females can potentially bias the fertilization success of competing ejaculates and hence the paternity outcome in this species.