β-sitosterol, as the most abundant phytosterol, has been shown to exert multiple biological roles in in vitro and in vivo animal studies. Largemouth bass (Micropterus salmoides) is an economical freshwater-farmed species widely cultivated in China due to its nutritious and delicious meat. This study investigated the effects of dietary β-sitosterol supplementation on largemouth bass farming. In the study, largemouth bass were fed with five levels of β-sitosterol (0, 20, 40, 80, and 160 mg/kg) supplementation in a commercial feed for four weeks. The growth performance, antioxidant ability, intestinal structure, immune-related gene expression, and resistance to pathogens such as Aeromonas hydrophila and largemouth bass virus (LMBRaV) were detected. The results revealed that β-sitosterol supplementation in feed increased the weight gain rate of largemouth bass, with 40 mg/kg supplementation displaying the best effect. The serum biochemical indices all showed an increase, including glucose and alkaline phosphatase. The intestinal villus length and muscularis propria thickness also increased, accompanied by elevated digestive enzyme activities, which promoted digestion and absorption of nutrients. Meanwhile, the levels of total cholesterol, aspartate aminotransferase, and alanine aminotransferase in serum decreased, indicating reduced liver damage. In addition, β-sitosterol supplementation effectively enhanced the antioxidant capacity of largemouth bass by decreasing malondialdehyde and elevating superoxide dismutase. Immune-related gene expression also changed. Pathogen infection tests revealed that β-sitosterol supplementation in feed had a certain protective effect against infection in largemouth bass, with the protection rate against A. hydrophila being higher than LMBRaV. The 40 mg/kg β-sitosterol supplementation group exhibited the best results. Collectively, the results revealed that the β-sitosterol can promote growth, improve enzyme activity, stimulate intestinal digestion, increase immune gene expression, and induce disease resistance in largemouth bass, with the optimal supplementation dosage being 40 mg/kg. β-sitosterol might be an excellent feed additive in aquaculture.

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