Sperm counts in the reproductive tract of doubly-mated females

Assays were performed to evaluate how sperm dynamics in the reproductive tract of doubly-mated females differs when the second-to-mate experimental male is either possessing (Sdic+ ) or lacking (Sdic- ) the entire D. melanogaster-specific Sdic multigene family. Virgin females were first mated to a reference Sdic+ male and then to one of the two possible experimental males (Sdic+ or
Sdic- ) two days later. Females were randomly assigned to one of three time points after the end of the second mating period (30 min, 1 h and 45 min, and 2 days) and subsequently flash frozen. Female reproductive tracts were dissected and the sperm from the different males were counted using a fluorescent microscope. Reference and experimental sperm fluoresce red and green, respectively.Sperm counting was performed for the bursa and both sperm storage organs, i.e. the paired spermatheca and the seminal receptacle.