Matsuura Koji, Wang Wen-Hsin, Ching Alex, Chen Yu, Cheng Chao-Min
Department of Biomedical Engineering, Faculty of Engineering, Okayama University of Science, 1-1 Ridai-Cho Kita-Ku, Okayama 700-0005, Japan.
Institute of Biomedical Engineering, National Tsing Hua University, Hsinchu 30013, Taiwan.
Micromachines (Basel). 2019 Jul 25;10(8):495. doi: 10.3390/mi10080495.
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 ( < 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.
通过使用我们自己设计的一种新型纸质检测方法，利用刃天青还原导致的精子细胞颜色变化来评估猪精子活力。我们将刃天青溶液和猪精液的混合物滴加到纸质检测装置上的亲水性测试圆片上，并研究产生的还原反应，该反应以红色和蓝色强度（RBCI）表示。我们使用来自8×3 = 24位RGB彩色图像的蓝/粉颜色比例对该反应进行定量。为了检测精子细胞中的酶活性，我们使用了两种抑制剂：3-硝基丙酸（3-NPA）和3-溴丙酮酸（3-BP）。3-NPA抑制线粒体中的柠檬酸循环和电子传递反应，但在我们的测试中并没有显著降低精子活力。3-BP降低了线粒体电子传递和细胞质中糖酵解酶的活性，这显著降低了猪精子的活力。与未处理的对照相比，经3-NPA和3-BP处理的样品的RBCI显著更低（<0.025）。基于这些结果，我们认为刃天青可用于评估有无抑制剂处理时还原剂的量。为了继续研究评估猪精子中刃天青还原的分子机制，一种使用嵌入我们纸质检测装置中的两种或更多种氧化还原指示剂（例如刃天青和噻唑蓝四唑溴盐（MTT））的联合检测方法可能会加深我们对精子细胞生物能量学的理解。
