Paper-Based Resazurin Assay of Inhibitor-Treated Porcine Sperm.

Koji Matsuura,Yu Chen,Alex Ching,Chao-Min Cheng,Wen-Hsin Wang

doi:10.3390/mi10080495

Abstract

Porcine sperm motility was assessed via resazurin reduction color change in sperm cells using a novel paper-based assay of our own design. We applied mixtures of resazurin solution and porcine semen onto hydrophilic test circles on our paper-based device and investigated the resulting reduction reaction expressed as red and blue color intensity (RBCI). We quantified this reaction using a blue/pink color ratio from our 8 × 3 = 24 bit RGB color image. To examine enzymatic reactivity in sperm cells, we used two inhibitors: 3-Nitropropanoic acid (3-NPA) and 3-Bromopyruvic acid (3-BP). 3-NPA inhibits the citric acid cycle and electron transfer reaction in mitochondria, but did not strongly reduce sperm motility in our tests. 3-BP decreases reactivity of both mitochondrial electron transfer and glycolytic enzymes in cytosol, which significantly lowers porcine sperm motility. RBCIs of 3-NPA- and 3-BP-treated samples were significantly lower compared to our untreated control (p < 0.025). Based on these results, we feel that resazurin can be used to estimate the amount of reductants with and without inhibitor treatment. For continued research assessing the molecular mechanisms of resazurin reduction in porcine sperm, a combination assay using two or more redox indicators (e.g., resazurin and Thiazolyl Blue Tetrazolium Bromide (MTT)) embedded into our paper-based device could further our understanding of sperm cell bioenergetics.

Highlights

Resazurin (IUPAC name: 7-hydroxy-10-oxidophenoxazin-10-ium-3-one) is a redox indicator that irreversibly changes from its blue oxidized form to its pink reduced form resofurin
We examined the effects of these inhibitors using a paper-based, colorimetric, resazurin assay
Resazurin color change on paper-based devices can be used for porcine sperm motility analyses

Summary

Introduction

Resazurin (IUPAC name: 7-hydroxy-10-oxidophenoxazin-10-ium-3-one) is a redox indicator that irreversibly changes from its blue oxidized form to its pink reduced form resofurin. This redox reaction can be analyzed using optical absorption or fluorescence. The correlation between resazurin reduction and sperm motility is relatively lower than that between resazurin reduction and sperm concentration, suggesting that a redox reaction of resazurin can be used to detect reductants such as nicotinamide adenine dinucleotide (NADH) in sperm cells [11,14] Correlations between resazurin reduction within mammalian sperm and sperm parameters such as concentration, motility, or acrosomal integrity have been investigated [12,13,14,15,16,17,18,19].

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Discussion

Conclusion