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.