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

Abstract

Simple Summary

Good semen quality is an essential factor for breeding success when artificial insemination is performed. However, semen quality can not fully be evaluated by merely sperm viability or motility. Standard semen quality evaluation requires costly automated computer-assisted sperm analysis or specific and laborious labeling procedures before particular parameters can be assessed by flow cytometry. In the current study, we examined whether the 2-[2-methoxy-4-nitrophenyl]-3-[4-nitrophenyl]-5-[2,4-disulfophenyl]-2H-tetrazolium (WST-8) assay, which is widely used in the cell biology field, can be applied to evaluate sperm viability, and moreover, whether the WST-8 reduction rate can correlate with multiple sperm parameters that are related to sperm quality. We demonstrated in this study that the WST-8 assay can be used as a rapid, affordable, and reliable method for the prediction of semen quality in boar.

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 × 10 ⁶ 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 × 10 ⁶ 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.