The Combined Supplementation of Azomite and Citric Acid Promoted the Growth,Immunity, Antioxidant and Resistance Against Aeromonas Hydrophila for Largemouth Bass, Micropterus Salmoides

Citric acid is an organic acid extensively used in feed industry, and AZOMITE is a hydrated aluminosilicate compound rich in rare earth elements and trace mineral elements. This study investigated the supplemental effects of AZOMITE and citric acid individual or in combination on the growth performance, intestinal microbiota, morphology, digestive enzyme activity, serum antioxidant and immune indexes, and disease resistance of juvenile largemouth bass. Six diets were designed, including the control diet (CON) and the five additive-supplemented diets with the addition of 4 g/kg or 8 g/kg citric acid (CA4, CA8), 3g /kg AZOMITE (A3), and their combined addition as 4 g/kg citric acid + 1.5 g/kg AZOMITE) (C4A1.5) and 8 g/kg citric acid +3 g/kg AZOMITE (C8A3). Juvenile largemouth bass with initial body weight of 22.01±0.04 g were fed the six diets for 56 days. The results revealed that the combined addition of 4 g/kg citric acid and 1.5 g/kg AZOMITE (C4A1.5) enhanced the weight gain by 7.99% (P<0.05), and decreased feed conversion ratio by 0.07 (P<0.05). However, the weight gain and feed conversion ratio were just numerically affected by the other supplements (P>0.05). The whole fish crude lipid content in the CA8、A3 and C4A1.5 groups, the protein retention in the C4A1.5 group and the lipid retention in all additive-supplemented groups were significantly higher than those in the control group (P<0.05), but no significant differences were observed in total crude protein and crude ash content among all the groups (P>0.05). In serum biochemical indices, all additive-supplemented groups showed significantly higher glutathione peroxidase (GSH-PX) activity than the control group (P<0.05). The activities of superoxide dismutase (SOD) and catalase (CAT) in the CA8, A3, C4A1.5, and C8A3 groups were significantly higher (P<0.05), while the concentration of malondialdehyde (MDA) was significantly lower than those in the control group (P<0.05). Moreover, the total antioxidant capacity in the A3 and C4A1.5 groups, acid phosphatase activity in the CA4 and CA8 groups, and lysozyme activity in the A3, C4A1.5, and C8A3 groups were significantly increased when compared to the control group (P<0.05). In digestive enzyme, the protease activity in the A3, C4A1.5 groups, and amylase activity in the CA4, CA8, C4A1.5 groups were significantly higher than those in the control group (P<0.05). In intestinal microbiota, all groups presented the similar predominant phyla such as Firmicutes, Fusobacteriota, and Proteobacteria. Compared to the control group, Firmicutes abundance was elevated in all additive groups, while the Fusobacteriota and Plesiomonas shigelloides abundance were decreased. In the intestinal histology, there was no significant difference in villus width and muscle thickness among all the groups. However, the CA8, A3, and C4A1.5 groups showed significantly higher villus height than the control group (P<0.05).After the infection with Aeromonas hydrophila, the cumulative mortality of all additive-supplemented groups was significantly lower than that of the control (P<0.05), and the C4A1.5 group demonstrated the lowest mortality. In conclusion, the combined supplementation of citric acid and AZOMITE promoted the growth, antioxidant, immune capacity, improved the intestinal morphology and microbial flora, and raised the disease resistance against Aeromonas hydrophila infection. A dietary dosage of 4 g/kg citric acid +1.5 g/kg AZOMITE is recommended for juven