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Background: The Asian redtail catfish Hemibagrus nemurus is a promising commercial aquaculture freshwater big-sized Bagridae catfish across Asian countries such as the Mekong, Malay Peninsula, and Indonesia. This study analysed the effect of eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) supplementation in diets on changes in fatty acid compositions in feed and fish meat, lipid quality (atherogenic index and thrombogenic index), growth rate, body indicators, and serum metabolites of Hemibagrus nemurus juveniles. Methods: A total of 180 Asian redtail catfish (initial weight 54.80 ± 2.72 g) were fed four levels (0, 3,150, 6,300, and 9,450 mg of EPA+DHA/kg feed) sourced from fish oil. Diets were fed in triplicate in freshwater tarpaulin ponds, with 15 fish per tarpaulin pond. During the experiment, fish were fed 3% per day of the biomass weight. Results: Categorically, there were significant differences in the composition of fatty acids in the feed and fish meat. The atherogenic index was between 1.76 and 1.84, and the thrombogenic index was between 0.81 and 0.89 in all fish meat. Growth performance was significantly different between diets, while body indices did not make a significant difference between diets. The fish meat EPA and DHA showed positive linear relationships with diet EPA (p |