Evaluation of cockerel spermatozoa viability and motility by a novel enzyme based cell viability assay

ABSTRACT

1. The results of spermatozoa assessment by the WST-8 (2-[2-methoxy-4-nitrophenyl]-3-[4-nitrophenyl]-5-[2,4-disulfophenyl]-2H-tetrazolium, monosodium salt) assay, flow cytometry (FC) or computer-assisted sperm analysis (CASA) were compared.

2. Different live/killed ratios of cockerel semen were serially diluted to 120, 60, and 30 × 10⁶ cells/ml, and each sample was analysed by (1) WST-8 assay at 0, 10, 20, 30, 40, 50, 60 min, (2) viability with FC, and (3) motility with CASA.

3. The WST-8 reduction rate was closely correlated with spermatozoa viability and motility. The optimal semen concentration for the WST-8 assay was 120 × 10⁶ cells/ml, and the standard curves for spermatozoa viability and motility predictions, respectively, were y_viability60 = 162.8x + 104.96 (R² = 0.9594) after 60 min of incubation and y_motility40 = 225.09x + 96.299 (R² = 0.8475) after 40 min of incubation.

4. It was concluded that the WST-8 assay is useful for the practical evaluation of cockerel spermatozoa viability and motility. Compared to FC and CASA, the WST-8 assay does not require expensive and complex instrumentation in the lab. Furthermore, one well of the WST-8 reaction can be used to predict spermatozoa viability and motility at the same time, which all lead it to be efficient and economical for semen quality assessment.

KEYWORDS:

• CASA
• flow cytometry (FC)
• formazan
• MTT
• reduction rate
• standard curve

Acknowledgements

The authors would like to thank Mr Ming-Chuan Chien, Mr Yung-Yao Cheng, and Ms Wen-Tsen Chen of Animal Industry Division at LRI for assistance with animal management and semen collection.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by the Council of Agriculture, Executive Yuan [1062101011003-020501L1]