Single-cell metabolic profiling of stallion spermatozoa by flow cytometry using NADH and FAD autofluorescence†

The metabolic activity of stallion spermatozoa was assessed using flow cytometry to detect NAD(P)H and FAD fluorescence without labels. Sperm were incubated with different energy sources-glucose, lactate, and pyruvate-individually or combined, and measurements of NAD(P)H, FAD, NAD(P)H/FAD ratio, and optical redox ratio (ORR) were taken. Additionally, a metabolic assay based on resazurin reduction and flow cytometry detection was developed. Changes in NAD(P)H and FAD fluorescence, the NAD(P)H/FAD ratio, and ORR were observed. The NAD(P)H/FAD ratio increased significantly, especially when glucose and pyruvate (P < 0.0001), glucose and lactate (P < 0.0001), or all three substrates (P < 0.0001) were present together. The mitochondrial activity index (MAI) and kinematic efficiency (KE) were also analyzed. Both indices showed positive correlations with the NADH/FAD ratio (0.6 and 0.7; P < 0.00001) and negative correlations with the ORR (-0.6 and - 0.7; P < 0.000001). Furthermore, flow cytometry was used to evaluate the spermatozoa's ability to metabolize different substrates via resazurin reduction. Results indicated that stallion sperm preferentially oxidized glucose, lactate, and oxoglutarate. In summary, we developed a straightforward flow cytometry-based redox and metabolic assay. This, combined with the resazurin reduction test, revealed a preference for glucose and lactate oxidation. This suggests specialized metabolic compartmentalization, with glycolysis occurring in the flagella and oxidative phosphorylation in mitochondria. Additionally, this assay could be useful for clinical sperm assessment, as metabolic changes often reflect physiological alterations in sperm function.