Spectrophotometric application of resazurin reduction assay to evaluate boar semen quality.

Abstract

The resazurin reduction assay depends on the ability of metabolically active cells to reduce the resazurin redox dye to resorufin. In the present study we applied and made a diagnostic evaluation of a spectrophotometric application of the resazurin reduction assay to assess the colour change of resazurin reduction in butanol extracted colour to evaluate boar semen quality. Forty-one samples of boar semen from various breeds were included in the study. The absorption peaks for resazurin and resorufin were found to be 610 and 575 nm, respectively. Absorbance at 610 nm, where the minimum overlap of the two peaks was observed, was used in further analysis. Spearman rank correlation analysis was used to determine the correlation between the resazurin reduction assay and various semen parameters. The highest correlations were observed with the concentration of motile spermatozoa (r = -0.841; p < 0.001), sperm concentration (r = -0.833; p < 0.001), sperm index (-0.826; p < 0.001) and concentration of viable spermatozoa (r = -0.763; p < 0.001). Sensitivity and specificity, at 94.1 and 91.7%, respectively, indicate that the present test is highly accurate in discriminating between the samples according to the sperm index. When motile sperm concentration was used to distinguish between good and poor samples, high sensitivity (93.6%) was also found, whereas the test was only moderately, 80%, specific. The stability of butanol extracts in terms of A610 at different times of measurement confirmed that the resazurin reduction could be spectrophotometrically measured within 7 days from the time of assay performance, making the assay much more useful. Based on these results, the assay could be used as an additional tool for evaluating the quality of boar semen.