IN BRIEF: Three isoforms of lactate dehydrogenase (LDH) - LDHA (cytoplasmic), LDHB (mitochondrial), and LDHC (flagellar) - have been identified and localized in stallion spermatozoa. Functional inhibition assays indicate that these three isoforms constitute a lactate shuttle of crucial importance for sperm function. ABSTRACT: Stallion spermatozoa use different energy sources; while oxidative phosphorylation predominates, glycolysis and beta-oxidation of fatty acids are also present. Glycolysis depends on the availability of NAD+ as an electron acceptor. During glycolysis, NAD+ is reduced to NADH. To ensure glycolysis can continue, NAD+ must be regenerated. This regeneration typically occurs when NADH donates its electrons to the electron transport chain (specifically at Complex I), where it is oxidized back to NAD+. If mitochondria are damaged, the regeneration of NAD+ may be compromised, leading to reduced glycolysis and altering sperm metabolism. However, alternative ways to regenerate NAD+ may be present. We hypothesized that aerobic glycolysis is present in the stallion spermatozoa as a backup mechanism to regenerate NAD+. We incubated spermatozoa in two Tyrode's modified media with either 67 mM glucose and 1 mM pyruvate or 67 mM glucose and 10 mM pyruvate. The addition of 10 mM pyruvate improved sperm motility (P < 0.001). Spermatozoa incubated in 67 mM glucose and 1 mM pyruvate for 3 h at 37°C showed a significant decrease in motility (58.1 ± 1.8% vs 81.2 ± 1.8%, P < 0.0001). In contrast, spermatozoa incubated in 67 mM glucose and 10 mM pyruvate retained motility (77.1 ± 1.4%), viability, and mitochondrial membrane potential. We studied the metabolic proteome and metabolome and identified three different isoforms of the enzyme lactate dehydrogenase (LDH), LDHA (cytosolic), LDHB (mitochondrial, with higher affinity for pyruvate), and LDHC (cytosol, motile cilium). Functional experiments using a specific inhibitor of LDHC demonstrated that this isoform may be essential for sperm function. We concluded that activation of aerobic glycolysis in a high-glucose medium improves sperm survival through the regeneration of NAD+.