Data from: Exploring the mechanisms underlying the last male precedence in the North African houbara bustard

The last male precedence (LMP) effect refers to the competitive advantage of the last male in the mating sequence. We conducted in vitro and in vivo experiments to explore the mechanisms underlying LMP in the polyandrous North African Houbara Bustard (Chlamydotis undulata). Ejaculates were stained with two nuclear fluorescent stains [Hoechst 33342 (BLUE) and NUCLEAR-ID® Red DNA (NRED)] and a mitochondrial stain [MitoTracker™ Red FM (MRED)], and the motility and velocity of stained and unstained sperm were compared. Given that NRED impaired sperm motility and velocity, BLUE and MRED were retained for in vivo experiments. First, females were inseminated with the sperm of a single male stained with BLUE, and the number of sperm stored in tubules was counted at 24, 48, and 72 hours post-insemination. Similar sperm counts indicated no loss of sperm over 72 hours. Second, we performed sequential inseminations (48-hour delay) in which females either were first inseminated with BLUE and subsequently with MRED-stained sperm, or were first inseminated with MRED and then with BLUE-stained sperm. Twenty-four hours after the second insemination, we counted the number of stored sperm and assessed their location within the tubules and their distribution across the tubules. The majority of tubules (79.1 %) were filled with BLUE- and MRED-stained sperm. However, tubules contained more sperm from the second insemination, and they were located closer to the bottom of the tubule. Therefore, active sperm displacement during successive mating is likely to be the principal mechanism underlying the LMP in this species.