The isoform lactate dehydrogenase C (LDHC) has a major role in improving stallion spermatozoa incubated in pyruvate-based media

Stallion spermatozoa use different energy sources; while oxidative phosphorylation predominates, glycolysis and beta-oxidation of fatty acids are also important. Moreover, interactions among different pathways are of major importance. Glycolysis depends on the availability of NAD+ electron acceptor, which is reduced to NADH, that in the complex I of the electron transport chain (ETC) donates an electron regenerating NAD+. On the other hand, if mitochondria are damaged, regeneration of NAD+ may be reduced leading to reduced glycolysis further altering sperm metabolism. However, alternative ways to regenerate NAD+ may be present. We hypothesized that aerobic glycolysis is present in the stallion spermatozoa, constituting a backup mechanism to regenerate NAD+. To test this hypothesis, we incubated stallion spermatozoa in different media, including a high glucose media of 67 mM with 1 mM pyruvate and 10 mM glucose with 10 mM pyruvate. The addition of 10 mM pyruvate to the 67mM glucose media improved sperm motility (P<0.001) Aliquots extended in the high glucose media (67 mM glucose) after 3 h of incubation at 37°C experienced a significant drop in motility respect initial values (58.1 ± 1.8% vs 81.2 ± 1.8%; P<0.0001), while aliquots incubated in the 67mM glucose 10 mM pyruvate media, maintained the motility all along the incubation period (77.1± 1.4%), viability and mitochondrial membrane potential were also improved (P<0.001). We studied the metabolic proteome and metabolome using UHPLC/MS/M and identified for the first time three different isoforms of the enzyme lactate dehydrogenase (LDH), LDHA (cytosolic), LDHB (mitochondrial, with higher affinity for pyruvate), and LDHC (cytosol, motile cilium), the latter was the predominant isoform detected. We performed a custom analysis of the stallion sperm proteome using human orthologs in Metascape (https://metascape.org/gp/index.html#/main/step1), using the terms “pyruvate” and “lactate” to study its enrichment. Both terms were highly enriched in the stallion spermatozoa; LACTATE p= 1.7 × 1e-13 and PYRUVATE p=4.2 xe-13. Metabolic analysis showed that including 10 mM pyruvate, induced aerobic glycolysis as increased amounts of Lactate and NAD+ were detected. We concluded that activation of aerobic glycolysis in a high glucose media improves sperm survival through the regeneration of NAD+.