Poster Abstracts

Trabajo presentado en la 15th Annual Conference of the European Society for Domestic Animal Reproduction (ESDAR), celebrada en Ankara del 15 al 17 de septiembre de 2011.-- Resumen publicado en Reproduction in Domestic Animals 46(s3): 127 (2011).Special Issue: The 15th Annual Conference of the European Society for Domestic Animal Reproduction (ESDAR).10.1111/j.1439-0531.2011.01839.xViability 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.This work was supported by Fundación Ramón Areces and Ramón y Cajal program (RYC-2008-02339 and RYC-2008-02560, MICINN, Spain).Peer Reviewe