Resazurin reduction test by spectrophotometer for boar semen

The aim of the present study was to develop an objective method for evaluating semen characteristics. Using various boar semen samples of different sperm concentrations, and samples stored at 15 deg C in liquid state, we compared the results of the rezasurin reduction test and sperm concentration, and sperm motility. Semen was collected from 14 boars. The seminal plasma was removed, and diluted with Modena to obtain 3x 10**8 sperm cells per ml. The rezasurin reduction test was performed as follows. One ml of a semen sample was added to 25,micro l of 2 mM resazurin solution and mixed. The samples were cultured at 38 deg C for 1 hour, and then measured by spectrophotometer. In experiment-I, resazurin and resorufin solutions were scanned, respectively, to examine the peak absorbance at specific wavelengths by spectrophotometer. In experiment-II, we measured the relative absorbance of different sperm concentration samples. In experiment-Ill, we compared sperm motility and the relative absorbance of semen stored at 15 deg C. In experiment-I, the resazurin solution and the resorufin solution peaked at 600 nm and 570 nm, respectively. Based on the results of experiment-I, in experiment-II and -III, absorbance of the samples was measured at 570 nm and 600 nm. In experiment-II, the absorbance of samples measured at 600 nm (resazurin) decreased as sperm concentration increased, whereas the absorbance of samples measured at 570 nm (resorufin) increased as sperm concentration increased. In experiment-III, sperm motility decreased as semen storage periods increased, the absorbance measured at 570 nm (resorufin) also decreased, whereas the absorbance of samples measured at 600 nm (resazurin) decreased as storage periods increased. In conclusion, the resazurin reduction test used in the present study reflected the sperm metabolic state, concentration and motility. Therefore, the resazurin reduction test may be useful for objectively evaluating boar semen characteristics.