Males and females of many species store sperm for extended periods. During storage, sperm are
predicted to undergo cellular and functional changes, especially towards glycolytic energy
metabolism because oxygen radicals derived from oxidative phosphorylation can affect sperm
motility and fertilisation ability. However, not all species can use both major energy metabolism
pathways. Here we examine the fruit fly Drosophila melanogaster and ask whether sperm
metabolism can be fuelled by both glycolysis and oxidative phosphorylation, and to what extent
metabolism changes during storage. Inhibiting glycolysis in vitro leads to a more oxidative state of
sperm, inhibition of oxidative phosphorylation increased the glycolytic component assessed by
multi-photon autofluorescence lifetime imaging (FLIM) of NAD(P)H. We further examined sperm in
male and female sperm storage organs using FLIM of NAD(P)H and FAD. In intact storage organs,
we find that, unexpectedly, i) sperm were more oxidative in females than males, and ii) oxidative
phosphorylation increased with storage duration in females. Our observation that the relative
contribution of both major energy metabolic pathways in Drosophila melanogaster sperm differs in
males and females and over storage time has important evolutionary implications.