Abstract:The integrated rice-fish farming mode is an environment-friendly ecological cycle agricultural production mode, which effectively improves the utilization rate of land and water resources. Stocking density is an important factor affecting fish growth. Excessive stocking density has been found to cause eutrophication and deterioration of the water quality, leading to abnormal physiological functions in fish. It also leads to competition among cultured fish for feed and living space, which inhibits growth performance and immune function, and increases the risk of disease outbreaks. However, it remains unknown whether high stocking density affects the antioxidant status, nutritional composition, and metabolic function in the muscle of Micropterus salmoides during rice-fish integrated farming. Therefore, the aim of the study was to investigate the effects of stocking density on the antioxidant capacity, muscle nutritional composition and metabolic functions of Micropterus salmoides in integrated rice-bass farming systems. The largemouth bass with an initial body weight of 40.63±0.13 g were reared at low density (L, 40 g/m3 ) and high density (H, 120 g/m3 ) for 90 days. After culturing, muscle tissue was collected to determine biochemical indexes and perform metabolome and transcriptome sequencing. The results showed that muscle total antioxidant capacity (T-AOC) in the H group was significantly lower than that in the L group. The catalase (CAT) activity decreased and malondialdehyde (MDA) content increased, but the differences were not significant, indicating that a high stocking density caused moderate oxidative stress, and its effects may be within the tolerance range of muscle antioxidant systems. The content of alanine and histidine free amino acids decreased significantly, whereas the content of glycine, aspartic acid, glutamic acid, and umami amino acids increased significantly. With respect to fatty acids, the content of ∑SFA, ∑MUFA, ∑PUFA, and n-6 PUFA increased significantly, whereas the ratio of n-3 PUFA to n-6 PUFA decreased. These results indicate that the nutritional quality of largemouth bass muscle decreased under high density, and the flavor was affected. Metabolomic results showed that 186 significantly different metabolites were detected between the L and H groups, including 136 up-regulated and 50 down-regulated metabolites. These metabolites were mainly related to the biosynthesis of unsaturated fatty acids, phenylalanine metabolism, nicotinate and nicotinamide metabolism, pentose and glucuronate interconversions, which indicated that high density stocking may cause metabolic disorders. Transcriptomic results showed that 688 significantly differentially expressed genes (DEGs) were detected between the L and H groups, including 508 up-regulated and 180 down-regulated genes, which were mainly enriched in cellular processes, metabolic processes, and protein digestion and absorption. Meanwhile, the key signaling pathways, including ECM-receptor interaction and PI3K-Akt, were up-regulated in the H group. In conclusion, high stocking density in integrated rice-bass farming systems affected antioxidant capacity, muscle nutrition and flavor and metabolic functions of lipids, carbohydrates, and proteins in M. salmoides. Additionally, M. salmoides could adapt to environmental stress by metabolic function regulation and intracellular signaling pathway activation. The results of this study provide a new premise for the molecular mechanism for the response of farmed fish to environmental stress in rice-fish integrated farming system.