Abstract
Aqueous Chlorella extract (CE) is a functional feed additive derived from widely cultivated microalgae. It is rich in proteins, polysaccharides, pigments, polyunsaturated fatty acids, vitamins and other bioactive compounds. CE possesses great potential to improve the health status and physiological performance of aquatic animals. This study investigated the effects of dietary CE on inflammatory response, non-specific immunity, antioxidant capacity, lipid metabolism, immune gene expression, intestinal histomorphology and intestinal microbiota in Litopenaeus vannamei. Shrimp were fed diets containing 0%, 1%, or 5% CE for 60 days. The 1% CE treatment produced stronger responses in several non-specific immune and antioxidant-related indicators and induced broader changes in immune-related gene expression. In contrast, the 5% CE treatment showed more pronounced changes in inflammation-related factors, lipid metabolism, and intestinal morphology. CE supplementation also altered intestinal microbial composition and predicted microbial functions, with different response patterns between the two inclusion levels. A distinctive feature of this study is the integrated identification of inclusion-level-specific response patterns across host physiological and intestinal microbial endpoints, rather than a simple increase in effects with increasing CE inclusion. Overall, the 1% treatment was more closely associated with immune and antioxidant-related responses, whereas the 5% treatment was more closely associated with anti-inflammatory, lipid-regulatory, and intestinal effects. These findings demonstrate the potential of CE as a functional aquafeed additive for improving aquatic animal health and provide a scientific basis for its application in sustainable shrimp aquaculture.