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.

Beneficial Influence of Soybean Lecithin Nanoparticles on Rooster Frozen–Thawed Semen Quality and Fertility

“…Sperm mitochondria represent the regulatory center of energy metabolism and oxidative stress, and are the hub of signaling pathways that regulate cell survival and apoptosis [42]. In particular, mitochondrial functionality is critical for sperm motility.…”

A microplate-based DCFH-DA assay for the evaluation of oxidative stress in whole semen

“…Sperm viability was analyzed by the WST-8 reagent [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] (Sigma-Aldrich), which has been recently reported as an affordable and reliable method for the evaluation of sperm viability [ 39 ]. The assay was based on the cleavage of the tetrazolium salt WST-8 by cellular dehydrogenases in viable cells [ 40 ].…”

Viability Improvement of Three‐Dimensional Human Skin Substitutes by Photobiomodulation during Cultivation

“…The WST‐8 assay is a colorimetric assay based on cellular metabolic activity, enabling high‐throughput assessments. There is a linear correlation between the absorbance of WST‐8 formazan at 450 nm and the number of living cells (34). The LbL‐3D skins were transferred to new wells on a 24‐well plate containing 0.5 mL of DMEM/EpiLife mixed medium with CCK‐8 solution (10 vol%) and incubated for 3 h at 37°C.…”