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 North African houbara bustard (Chlamydotis undulata). Ejaculates were stained with two nuclear fluorescent stains [Hoechst 33342 (HBlue) and NUCLEAR-ID® Red DNA (NRed)] and a mitochondrial stain [MitoTracker™ Red FM (MRed)], and motility and velocity of stained and unstained sperm were compared. Given that NRed impaired sperm motility and velocity, HBlue and MRed were retained for in vivo experiments. In a first in vivo experiment, females were inseminated with HBlue-stained sperm and the sperm stored in tubules were counted at 24, 48 and 72 h post-insemination. The number of sperm counted at the three timings was not significantly different, suggesting no sperm loss over 72 h post-insemination. In a second in vivo experiment, we performed sequential inseminations where females were either first inseminated with HBlue-stained sperm and subsequently with MRed-stained sperm, or vice versa. 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%) contained both HBlue and MRed sperm. However, tubules contained more sperm from the second insemination, and they were located significantly closer to the bottom of the tubule. We discuss these results in light of the possible mechanisms underlying the LMP.