Abstract: Objective To establish a diet-induced obesity model in zebrafish larvae. Methods At 7 days post-fertilization (dpf),
200 zebrafish larvae with normal development were randomly allocated to two groups with the feeding quantity of 30 mg per
day (normal feeding group) or 180 mg per day (overfed group) for 20 days. The weight, length, BMI, triglyceride (TG) and
total cholesterol (TCH) of each group were measured. Whole-mount Oil Red O staining, frozen Oil Red O staining and
hematoxylin-eosin (HE) staining were used to estimate the rate of hepatic steatosis and liver histology of the zebrafish. The
dynamic change of hepatic lipid droplets and distribution of adipose tissue were observed with Nile Red staining in overfed
zebrafish in vivo. Results The weight, length, BMI and TG of overfed zebrafish were significantly increased compared with
those in normal feeding group. Whole-mount Oil Red O staining showed that the percent of hepatic steatosis in overfed group
(89.4%) was markedly higher than that in normal feeding group (20.7%). Macrovesicular steatosis was observed in the liver of
the overfed larvae. Nile Red staining visualized hepatic lipid droplets and the distribution of larval adipose tissue, which
increased with feeding time in the overfed zebrafish. Starving larvae showed depletion of fat and hepatic lipid, and adipose
tissue was induced after refeeding. Conclusions We successfully established an diet-induced obesity model in zebrafish larva,
in which Nile Red staining allows in vivo observation of the adipocytes and hepatic lipid droplets.