Abstract

Simple SummaryGood 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.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.

Highlights

  • Assisted reproductive technology (ART) including artificial insemination (AI) has been utilized by the swine industry to improve reproductive performance for more than 50 years [1]

  • The aim of the current study is to establish a standard protocol and working procedure for the WST-8 assay for boar semen quality assessment, and to correlate the WST-8 reduction rate with various sperm quality-related parameters measured from flow cytometry and Computer-assisted sperm analysis (CASA), in order to provide a rapid, reliable and affordable assessment for boar semen quality evaluation

  • We demonstrate for the first time that the WST-8 assay could be used as a reliable chromogenic indicator for semen quality in mammals

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Summary

Introduction

Assisted reproductive technology (ART) including artificial insemination (AI) has been utilized by the swine industry to improve reproductive performance for more than 50 years [1]. Due to breed variations and post-ejaculation semen handling procedures, comprehensive and precise evaluation of sperm quality before AI is essential to correlate boar fertilizing potential and to increase fertilization success [2,3,4,5]. Spermatozoon is a specialized type of cell with polarized domains, including the sperm head, mid-piece, and tail. The sperm head, in addition to chromatin, contains a large. Golgi-derived acrosome with various hydrolytic enzymes that are essential for sperm-zona pellucida penetration [6]. The sperm mid-piece on the other hand is packed with mitochondria that are critical for sperm motility [7]. Functional integrity of all the above-mentioned compartments is essential for successful fertilization

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