Spermatozoal methylene blue reduction: An indicator of mitochondrial function and its correlation with motility

Methylene blue reduction rates (MeBRR) were evaluated spectrophotometrically to study bovine spermatozoal mitochondrial function and its relation to motility. A chemical reaction (H 2SO 4, methylene blue solution, and zinc powder) was used to quantify methylene blue reduction. Absorbance measurements were made for 10 min at 609 nm in a narrow band spectrophotometer. In a second experiment, fresh ejaculates were assessed for concentration and motility evaluated with phase microscopy (37°C). Semen was diluted to 100 million cells/mL in a sodium citrate-glucose buffer and methylene blue. Absorbance and motility were evaluated every 30 min for 2.5 h in a water-jacketed cuvette (41°C). Methylene blue reduction rates and motility decreased at each subsequent period. Methylene blue reduction rates were correlated to sperm motility. Lastly, the methylene blue reduction rate was measured with a broad band spectrophotometer and compared with motility using similar conditions. Motility estimates were made on sperm from the cuvettes. Sperm motility was correlated to methylene blue reduction rates measured spectrophotometrically.