Tetrazolium Salt WST-8 as a Novel and Reliable Chromogenic Indicator for the Assessment of Boar Semen Quality

Abstract

A tetrazolium salt, 2-[2-methoxy-4-nitrophenyl]-3-[4-nitrophenyl]-5-[2,4-disulfophenyl]-2H-tetrazolium (WST-8), has been used widely to determine cell viability; however, its application in the field of reproduction is still limited due to this assay merely providing information regarding cell viability. The aim of this study was to correlate the WST-8 reduction rate with various sperm quality-related parameters (i.e., sperm viability, motility, progressive motility, acrosome integrity and mitochondria integrity) in order to provide a rapid, reliable and affordable assessment for boar semen quality evaluation. Using different ratios of active/damaged sperm cells, we first validated our sample preparations by standard flow cytometry and computer-assisted sperm analysis. Further analyses demonstrated that the most efficient experimental condition for obtaining a reliable prediction model was when sperm concentration reached 300 × 10⁶ cells/mL with the semen/cell-counting kit-8 (CCK-8^®) ratio of 200/10 and incubated time of 20 min. Under this set up, the WST-8 reduction rate (differences on optic density reading value, ΔOD at 450 nm) and sperm parameters were highly correlated (p < 0.01) for all sperm parameters evaluated. In the case of limited semen samples, a minimal semen concentration at 150 × 10⁶ cells/mL with the semen/CCK-8^® ratio of 200/20 and incubation time for 30 min could still provide reliable prediction of sperm parameters using the WST-8 assay. Our data provide strong evidence for the first time that the WST-8 assay could be used to evaluate boar semen quality with great potential to be applied to different mammalian species.

Keywords: WST-8; boar; computer-assisted sperm analysis (CASA); flow cytometry; semen quality.

Figure 1

Linear regression and prediction models for sperm concentration of 300 × 10⁶ cells/mL. Prediction model and formula between the WST-8 reduction rate (ΔOD) and boar sperm viability (%), sperm motility (%), progressive sperm (%), acrosome integrity (%) and mitochondrial integrity (%), at a sperm concentration of 300 × 10⁶ cells/mL after 10 min incubation at 37 °C with a semen/CCK-8^® ratio of 200/10.

Figure 2

Linear regression and prediction models for sperm concentration of 150 × 10⁶ cells/mL. Prediction model and formula between the WST-8 reduction rate (ΔOD) and boar sperm viability (%), sperm motility (%), progressive sperm (%), acrosome integrity (%) and mitochondrial integrity (%), at a sperm concentration of 150 × 10⁶ cells/mL after 30 min incubation at 37 °C with a semen/CCK-8^® ratio of 200/20.