Simple SummaryIn mammals, sperm acquire fertilization ability after capacitation in vitro or when in the female reproductive tract. Adenosine triphosphate (ATP) is required for sperm capacitation through two main metabolic processes, oxidative phosphorylation (OXPHOS) and glycolysis. This study incubated dairy goat sperm with different concentrations of ROT, FCCP, TIG, and AMPK inhibitors. Sperm motility attributes, ATP content, pyruvate and lactate levels, mitochondrial permeability transition pore fluorescence intensity, MMP, protein synthesis, and ferroptosis were analyzed. The results showed that glucose starvation inhibited ferroptosis by activating the LKB1/AMPK signaling pathway and promoted the motility and linear motility of dairy goat sperm, thereby promoting fertilization.AbstractIn mammals, sperm acquire fertilization ability after capacitation in vitro or when in the female reproductive tract. The motility patterns of sperm undergo continuous changes from the moment of ejaculation until fertilization in the female reproductive tract. In vitro, hyperactivated motility can be induced through high glucose mediums, while in vivo, it is induced by oviduct fluids. Conversely, sperm maintain linear motility in seminal plasma or uterine fluids that contain low glucose levels. In dairy goat sperm, energy metabolism associated with capacitation depends on the energy sources in vitro, seminal plasma, or the female reproductive tract, especially the glucose levels. However, there is little experimental knowledge that glucose levels affect sperm energy metabolism in dairy goats. To clarify these hypotheses, we incubated dairy goat spermatozoa with different concentrations of rotenone-glucose (ROT), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), and tigecycline (TIG) in vitro. Sperm motility attributes, ATP content, pyruvate and lactate levels, mitochondrial permeability transition pore fluorescence intensity, mitochondrial membrane potential (MMP), and protein synthesis were analyzed. Sperm motility patterns changed from circular to linear under low glucose conditions compared with those in high glucose conditions and showed a significant improvement in progressive motility and straight line speed, whereas lactate and pyruvate levels and MMP decreased remarkably. Incubation of spermatozoa with ROT, FCCP, and TIG inhibited sperm mitochondrial activity, protein synthesis, oxidative phosphorylation, and ATP levels, thereby reducing sperm motility, including the progressive motility, straight line speed, and total motility. Simultaneously, incubation of spermatozoa with Compound C under low glucose conditions significantly decreased the ATP levels and MMP, as well as liver kinase B1 and AMPK protein expression. Under low glucose conditions, sperm mainly produce ATP through mitochondrial OXPHOS to achieve high speed linear movement, inhibit ferroptosis through the LKB1/AMPK signaling pathway, and further maintain energy metabolism homeostasis.