The effectiveness of ovarian fluid on spermatozoa performances in Mediterranean brown trout wild population (Molise region - Italy)

Knowledge of gamete quality is a prerequisite to fertilize eggs in an artificial environment, preserve gametes in gene banks, forecast recruitment variability for aquatic stocks, and understand natural mating processes. For externally fertilizing fishes, the ovarian fluid (OF) that surrounds the eggs is possibly a mediator of sperm selection. OF affects various sperm traits (sperm activation, chemotaxis, longevity, swimming performance and trajectory across) by modifying spermatozoa behaviours and fertilization outcomes, however, does not seem to affect sperm of all males in the same way, but varies depending on male and female identity, indicating cryptic female choice. Here, we evaluated for the first time in Mediterranean brown trout (Salmo cettii) the effect of the OF on the sperm performance variability according to specific combinations of males and females. In particular, the study was conducted on a threated wild population of Mediterranean brown trout inhabiting the Biferno river (Molise region, Italy), where few breeding sites are reached by most of the migrating spawners resulting in high levels of reproductive competition. Spermatozoa from eight males were activated in river water (RW) and OF from five females at 20% concentration. Sperm motility parameters were measured using computer-assisted sperm analysis (CASA). The mean of two independent activations was used for statistical analyses. Sperm motility parameters in RW and in OF were compared using two-tailed t-test. A generalized linear model (GLM) procedure was used to determine both the fixed effects of the male and female identity and their interaction on the sperm motility traits. In comparison to RW, OF significantly (p < 0.05) increased the sperm motility (85.1 vs 92.1%), sperm velocity parameters (curvilinear velocity [VCL], angular path velocity [VAP] and straight line velocity [VSL]) and prolonged the movement duration (23.1 vs 32 s). A significant male effect for all motility traits was found, whilst the female effect was significant only for VSL, straightness (STR), linearity (LIN), beat cross frequency (BCF) and duration of sperm movement. Interestingly, we found a significant interaction between male and female for all traits considered, indicating that OF from distinct females differently affected the sperm from specific males. In agreement with other studies, we can assume that trout spermatozoa with higher velocity and motility have the advantage of reaching the micropyle within a shorter time, which is crucial for fish that spawn in highly competitive environments. The male-female interactions observed in our in vitro study suggests that females could favour specific male genotypes or phenotypes, as a result of an evolutionary mechanism for sexual selection in an environment characterized by a high degree of reproductive competition.