Cryptic choice of conspecific sperm controlled by the impact of ovarian fluid on sperm swimming behavior.

Despite evidence that variation in male-female reproductive compatibility exists in many fertilization systems, identifying mechanisms of cryptic female choice at the gamete level has been a challenge. Here, under risks of genetic incompatibility through hybridization, we show how salmon and trout eggs promote fertilization by conspecific sperm. Using in vitro fertilization experiments that replicate the gametic microenvironment, we find complete interfertility between both species. However, if either species' ova were presented with equivalent numbers of both sperm types, conspecific sperm gained fertilization precedence. Surprisingly, the species' identity of the eggs did not explain this cryptic female choice, which instead was primarily controlled by conspecific ovarian fluid, a semiviscous, protein-rich solution that bathes the eggs and is released at spawning. Video analyses revealed that ovarian fluid doubled sperm motile life span and straightened swimming trajectory, behaviors allowing chemoattraction up a concentration gradient. To confirm chemoattraction, cell migration tests through membranes containing pores that approximated to the egg micropyle showed that conspecific ovarian fluid attracted many more spermatozoa through the membrane, compared with heterospecific fluid or water. These combined findings together identify how cryptic female choice can evolve at the gamete level and promote reproductive isolation, mediated by a specific chemoattractive influence of ovarian fluid on sperm swimming behavior.