Effects of choline chloride on intestinal microbiota and its association with liver fat accumulation in zebrafish

In this study, the effect of choline chloride on intestinal microbiota and its association with the liver lipid accumulation were tested in zebrafish (Danio rerio) as a model. We performed a trial for six weeks on zebrafish dietary with high-lipid diets containing different choline chloride levels (0%, 0.2%, 0.4% and 0.6%; labelled as C0, C2, C4 and C6 treatment, respectively). The liver samples were collected for oil red O (ORO) staining at the 2nd, 4th and 6th week. The liver fatty infiltration were formed at the 2nd week and reached maximum proportion in C0 treatment at the 6th week, while the lowest proportion was present in C6 treatment. Highthroughput sequencing was performed to characterise intestinal microbiota of fish fed C0 and C6 choline chloride. In comparison with C0 treatment, the relative abundances of Sphingomonas, Phyllobacterium and Bacteroides were increased in C6 treatment. Bacterial genes predicted that the flora of fish in C6 treatment was mainly concentrated in lipid metabolism, amino acid metabolism, xenobiotics biodegradation and metabolism. Thereafter, the gut microbiota of C0 or C6 treatment was transferred to germ-free (GF) 3 dpf (days post fertilisation) zebrafish and fed with high-lipid diet without choline. ORO revealed that the intestinal microbiota was contributed to reduce the liver lipid accumulation of fish accepted bacteria of C6 treatment (GFTC6). Transcriptome analysis showed that differentially expressed genes were mainly involved in dephosphorylation, lipid transport, lipid localisation and lipoprotein metabolic process in GFTC6 treatment. Our findings suggested a probable association between composition changes of intestinal microflora and liver fat accumulation in fish dietary with choline chloride.