Teratospermia (ejaculation of ≥ 60% structurally abnormal spermatozoa)is common in certain felid species or populations, especiallyin the presence of low genetic diversity. We recently determinedthat (1) sperm cellular rates of pyruvate uptake and lactateproduction are reduced in ejaculates from teratospermic cheetahsand inbred domestic cats and (2) spermatozoa from both speciesminimally utilize exogenous glucose regardless of ejaculatephenotype. Sperm metabolism of endogenous glucose (i.e., glycogen)has been documented in the domestic dog, and also could occurin the domestic cat and cheetah. However, there is no informationto indicate which metabolic pathways support motility and viabilityin felid spermatozoa. In this study, we tested the hypothesesthat oxidative phosphorylation, rather than glycolysis, is theprimary energy source in felid spermatozoa and is impaired interatospermic ejaculates. Our objective was to comparativelyassess glycolytic and oxidative sperm metabolism in the domesticcat and cheetah in relation to ejaculate phenotype (normospermicvs. teratospermic). Electroejaculates (n = 31 total, 9-13 pergroup) were collected from: 1) normospermic domestic cats (3males); 2) teratospermic domestic cats (4 males); and 3) cheetahs(13 males). Washed sperm samples were incubated (37°C) ina chemically-defined mouse tubal fluid medium (cMTF) in thepresence/absence of exogenous 1 mM glucose and 1 mM pyruvate.A second set of ejaculates was incubated in cMTF containingboth substrates as well with an inhibitor of glycolysis (50mM alpha-chlorohydrin) or of oxidative phosphorylation (160nM myxothiazol) to control for endogenous substrate metabolism.Cellular lactate production, percent sperm motility (%M), forwardprogression (FPS) and acrosomal integrity (correlated with spermviability in felids) were assessed in all samples at 0, 1, 3and 7 h of incubation. Overall, the absence of exogenous glucose/pyruvatedid not (P > 0.05) influence sperm motility (%M and FPS)or acrosomal integrity in either domestic cat group or in cheetahs.Unexpectedly, spermatozoa produced lactate in the absence ofglucose, but only when pyruvate was present. The influence ofmetabolic inhibition on sperm function was consistent acrossanimal groups. Glycolytic inhibition resulted in decreased (P< 0.05) lactate production, %M and FPS after 1 h, but acrosomalintegrity did not decline (P < 0.05) until 7 h of incubation.In contrast, oxidative inhibition severely (~70%) decreased(P < 0.05) %M and FPS and caused a ~20% loss (P < 0.05)of acrosomal integrity by 1 h of incubation. A 100x higher concentrationof oxidative inhibitor was required to alter (P < 0.05) spermfunction in the domestic cat (normospermic and teratospermic)compared to the cheetah. Results indicate that both oxidativeand glycolytic pathways are required to maintain motility andviability of domestic cat and cheetah spermatozoa. However,a greater proportion of the energy supporting these processesis obtained from the more efficient oxidative pathway. The sensitivityof cheetah spermatozoa to extremely low concentrations of theoxidative inhibitor suggests that mitochondrial load/functionis reduced in these cells compared to domestic cats. Importantly,comparison with the teratospermic domestic cat model revealsthat this metabolic defect is related to species-specific physiologyrather than ejaculate phenotype.