The correlates and alleged biochemical background of the resazurin reduction test in semen

Reduction of the blue dye resazurin to pink resorufin is used to estimate the concentration of metabolically active spermatozoa in semen samples. In order to quantify the reduction of resazurin, a spectrophotometric method was developed measuring the change from blue to pink in the butanol extracted colour. The biochemical mechanisms involved in the reduction of resazurin by motile spermatozoa and seminal plasma were investigated. Addition of NADH + H+ to sperm suspension or seminal plasma increased the reduction of resazurin. The reduction reaction was inhibited by high concentrations of dicoumarol, a specific inhibitor of the diaphorase enzyme, in a dose‐dependent manner. It is suggested that the sperm diaphorase enzyme transfers electrons from NADH + H+ to resazurin, reducing it to resorufin. The degree of resazurin reduction was strongly correlated with the concentration of motile spermatozoa recovered from the 90% Percoll fraction (r = 0.98, p