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.

Development of resazurin microtiter assay for drug sensibility testing of Trypanosoma cruzi epimastigotes

“…The resazurin test has been used since the 1950s to assess bacterial or yeast contamination in biological fluids (Erb and Ehlers 1950). It is also used to measure the semen quality (Wang et al 1998;Zrimsek et al 2004), corneal viability (Perrot et al 2003), mitochondrial function (Zhang et al 2004), quantity/proliferation of living organism (Mann and Markham 1998;Guerin et al 2001;Gabrielson et al 2002;Palomino et al 2002;Martin et al 2003), and different types of cells for its cytotoxicity, toxicology, and apoptosis (O'Brien et al 2000;Evans et al 2001;Putnam et al 2002;Batchelor and Zhou, 2004;Benavides et al 2004).…”

Growth kinetics of Mycobacterium tuberculosis measured by quantitative resazurin reduction assay: a tool for fitness studies

“…In M. tuberculosis, resazurin has been used successfully to test the susceptibility of active and dormant bacilli to anti-tuberculosis drugs by the resazurin microtiter assay (REMA) plate (6, 10, 13). Reduction of resazurin can be measured using a spectrophotometer due to its change in colour from blue (oxidized form) to pink (reduced form) (18). …”

Semen quality assessments and their significance in reproductive technology

“…Studies have shown that there were positive relationships between ATP measurements and the percentage of spermatozoa with functional mitochondria following preservation of boar and canine semen (Fraser et al 2007, Dziekońska et al 2009, Kordan et al 2010). These methods can be complemented with the spectrophotometric application of the resazurin reduction test (RRT) to evaluate sperm quality (Zrimšek et al 2004). The RRT is based on the ability of metabolically active sperm cells to reduce the resazurin redox dye to resorufin, and was correlated with sperm characteristics, such as motility and viability (Dart et al 1994, Zrimšek et al 2004).…”

“…These methods can be complemented with the spectrophotometric application of the resazurin reduction test (RRT) to evaluate sperm quality (Zrimšek et al 2004). The RRT is based on the ability of metabolically active sperm cells to reduce the resazurin redox dye to resorufin, and was correlated with sperm characteristics, such as motility and viability (Dart et al 1994, Zrimšek et al 2004). …”