Resazurin reduction assay for ram sperm metabolic activity measured by spectrophotometry

Resazurin (7-hydroxy-3H-phenoxazin-3-one 10-oxide) is a redox dye that can be reduced by metabolically active spermatozoa to resorufin and manifested as visual color change from blue (resazurin) to pink (resorufin). This study combined the resazurin reduction test with spectrophotometric methods and investigated the correlation between metabolic activity and fertilization potential of spermatozoa, using ram semen as a model. The absorbance at specific wavelengths of resazurin and resorufin was determined by scanning photo spectrometer (600 nm for resazurin and 570 nm for resorufin, respectively). The absorbance at wavelengths of 600 nm (A600) and 570 nm (A570) was measured by spectrophotometry and used to evaluate sperm metabolic activity. A600 decreased and A570 increased in relationship to the increased concentrations of motile spermatozoa and increased resazurin reaction times. We observed, upon using a retrospective experimental design, that fertile rams had greater relative absorbance values than rams with lower fertility. Also, we observed a wide range variation of absorbance between the fertile rams, which is highly correlated to the sperm motility. We conclude that the spectrophotometric measurement of resazurin reduction for sperm activity might be a good assay for ram fertility.