Synergistic Effects of Fermented Bile Acids and Cholesterol on Growth Performance, Immune Response, and Intestinal Microbiota of <i>Litopenaeus vannamei</i> in Freshwater Environment

Cholesterol (CHO) is an expensive essential nutrient for crustaceans. Bile acids (BAs), which function as emulsifiers facilitating lipid absorption in vertebrates, play a crucial role in the growth and sterol metabolism. This study conducted a 2‐month feeding experiment and aimed to investigate both the individual and interactive effects of dietary CHO and fermented BAs (FBAs; a novel type of FBAs) on growth performance, immune response, and intestinal health in Litopenaeus vannamei in freshwater environment. A total of 12 isonitrogenous and isolipidic diets were formulated. These diets were formulated based on a basal diet by separately adding FBAs at a level of 0.04% (A3), CHO at levels of 0.05% (C1), 0.10% (C2), 0.20% (C3), 0.30% (C4), and 0.40% (C5) as well as combinations of FBAs and CHO at levels of A3C1, A3C2, A3C3, A3C4, and A3C5. A control group (N) without the addition of either FBAs or CHO was also included. Considering that the basic diet contained 0.08% CHO (from fish meal and other ingredients) and no detectable FBAs, the actual levels of CHO were adjusted to 0.08% (N), 0.13% (C1), 0.18% (C2), 0.28% (C3), 0.38% (C4), and 0.48% (C5). After 60 days, both FBAs, CHO, and their combination could improve the growth performance of shrimp, as indicated by final weight (FW), weight gain (WG), and specific growth rate (SGR). The best promoting effect was found in A3C2 and A3C3 groups. Two‐way analysis of variance (ANOVA) analysis revealed significant synergistic effects between FBAs and CHO ( p &lt; 0.05). These results suggest that CHO is more efficient than FBAs in promoting growth, but its efficiency can be significantly enhanced when combined with FBAs. Hemolymph biochemical parameters, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), α‐amylase (α‐AMS), triglycerides (TGs), and acid phosphatase, were significant affected by different treatments ( p &lt; 0.05). Gene expression levels in the hepatopancreas showed significantly lower levels of anti‐lipopolysaccharride factor ( ALF ), prophenoloxidase ( proPO ), and alpha‐2‐macroglobulin ( α2M ) and significantly higher levels of alkaline phosphatase (AKP) in different treated groups compared to the control groups ( p &lt; 0.05). The addition of FBAs, CHO, and their combination to the diet increased gut microbiota diversity in L. vannamei . At the phylum level, there was a significant decrease in Proteobacteria abundance and a significant increase in Firmicutes, Tenericutes, and Cyanobacteria compared to the control group. At the genus level, Pseudoalteromonadaceae Vibrio , Vibrionaceae vibrio , Shewanella , and Synechococcus were found to be more abundant in the FBAs and CHO treatment group compared to the control group. In conclusion, a combination of 0.18%–0.28% CHO and 0.04% FBAs into feed formulations demonstrated synergistic effects on L. vannamei under freshwater conditions, significantly enhancing their growth performance, hepatopancreatic and intestinal health, and gut microbiota. This study provides a novel approach for improving the efficiency of L. vannamei breeding in freshwater environments by optimizing the ratio of FBAs and CHO. Furthermore, it provides a potential strategy to reduce dietary CHO content, thereby lowering feeding costs.