Evaluation of fluorescent stains and flow cytometry for viability assessment of gilthead sea bream (Sparus aurata) spermatozoa

Abstract

Viability evaluation using flow cytometry is a very important tool for sperm quality assessment. Fluorochromes and flow cytometry have been used in several fish studies, but staining protocols have not been thoroughly adapted to teleost species. The objective of this study was to evaluate viability fluorochromes in gilthead sea bream sperm, analyzing the stain dynamics and its repeatability. Cryopreserved spermatozoa were thawed, diluted in PBS (106/mL) and stained with 1.5 µM propidium iodide (PI), alone or combined with either 5 µM Hoechst 33342 (H342) or 0.1 µM SYBR-14. Tubes were incubated at room temperature and analyzed by flow cytometry at 1, 5, 10, 15, 30, and 60 min. The experiment was performed in triplicate. Fluorescence intensity and proportion of spermatozoa stained with PI (%PI+, dead) were fitted to asymptotic curves (vs. time), CV% of triplicates were obtained for each time, and % of dead spermatozoa were compared among methods (Bland-Altman agreement coefficients). For PI and PI/H342, fluorescence and %PI+ increased quickly, reaching a plateau by 15 min (~45% viability, CV% ~10%). CV% were higher for 1 and 5 min, implying less repeatability at short incubation times. PI/SYBR-14 was more dissimilar and showed higher CV% (22% for 15 min). This study contributes with basic knowledge for the use of fluorochromes in teleost species.