A simple and rapid evaluation of boar sperm quality using the resazurin colorimetric assay

Ensuring boar sperm quality before insemination is crucial for maximizing field fertility and efficient pig production. The computer-assisted sperm analysis (CASA) and fluorescence probes combined with flow cytometry (FC) are commonly used techniques for evaluating sperm kinematics and functions, closely related to animal fertility. However, their high cost and complex operations make it challenging to apply them in laboratories or pig breeding farms with limited resources. Here, our aim was to develop a new protocol using a resazurin redox dye to assess boar sperm quality for practical application. We first created simulated semen samples with different levels of sperm quality (0, 20, 40, 60, 80, 100%) at concentrations of 300 and 150 × 10⁶ cells/mL. Subsequently, the simulated semen was used to establish an optimal standard protocol based on the results of the resazurin colorimetric assay. Finally, the condition that showed the strongest correlation between resazurin redox rate and sperm parameters was selected to perform a linear regression test. Two optimal working conditions were identified, involved incubating 10 µL of resazurin reagent with 100 µL of semen for either 20 or 40 min, depending on the sperm concentration of either 300 or 150 × 10⁶ cells/mL. We subsequently conducted a linear regression analysis using data that included the resazurin reaction rate and measurements of sperm parameters. Finally, we obtained two sets of five equations, allowing directly convert the absorbance of the resazurin assay into values for sperm quality parameters. These parameters include total motility, progressive motility, viability, acrosome integrity, and mitochondrial activity. This new protocol is valuable for sperm evaluation because it is cost-effective, time-efficient, and labor-saving.