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

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 yviability60 = 162.8x + 104.96 (R2 = 0.9594) after 60 min of incubation and ymotility40 = 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.

Molecular analysis of an extracellular protease gene from Vibrio parahaemolyticus.

The structural gene prtVp encoding the extracellular protease of Vibrio parahaemolyticus strain 93 was cloned in Escherichia coli and sequenced. The cloned DNA fragment contained a 1761 bp ORF encoding a 587 amino acid protein. The deduced polypeptide is composed of a 25 amino acid signal peptide and a 562 amino acid extracellular polypeptide with a calculated molecular mass of 63,156 Da. Protease analysis using a gelatin-containing SDS-polyacrylamide gel detected the presence of a 62 kDa protease that was present in the culture supernatant fractions of the wild-type V. parahaemolyticus strain and of E. coli bearing a pUC119 recombinant with the prtVp DNA insert. The protease activity was inhibited by zinc- and metal-specific inhibitors such as EDTA and 1,10-phenanthroline, which suggested that it is a metalloprotease. The deduced amino acid sequence of PrtVp has 32% identity with that of the collagenase of Vibrio alginolyticus, but has no identity with those of the bacterial proteases. A conserved zinc-binding domain was also found in PrtVp from homology comparison with other metalloproteases. This PrtVp can cause weak haemolysis on blood agar.