The role of sodium butyrate in modulating growth, intestinal health, and antimicrobial efficacy in turbot (Scophthalmus maximus L.) fed high soy diets

Abstract

Butyrate is one of the most abundant short-chain fatty acids (SCFAs), which are important metabolites of dietary fiber by fermentation of gut commensals, and has been shown to be vital in maintaining host health. The present study mainly investigated how sodium butyrate (NaB) supplementation in the diet with high proportion of soybean meal (SBM) affected turbot. Four experimental diets were formulated: (1) fish meal (FM) based diet (control group), (2) SBM protein replacing 45% FM protein in the diet (high SBM group), (3) 0.2% NaB supplementation in the high SBM diet (high SBM + 0.2% NaB group), and (4) 0.5% NaB supplementation in the high SBM diet (high SBM + 0.5% NaB group). The fish were fed four different diets for 8 weeks. The results showed that the high SBM diet significantly suppressed growth performance, induced typical enteritis symptoms and decreased resistance to bacterial infection. However, inclusion of 0.2% and 0.5% NaB in the high SBM diet both effectively increased the growth performance of turbot. Meanwhile, dietary NaB protected the intestinal morphology, and regulated the gene expression of inflammatory cytokines to relieve the inflammation of turbot, such as TNFα, IL-1β, NFκB and IL-10. Moreover, supplementation with NaB in the high SBM diet activated HIF-1α/IL-22/Lysozyme signaling pathway to against Edwardsiella tarda (E. tarda) infection, especially 0.5% NaB supplementation exerted more effectively to defence bacterial infection under inflammatory state. In conclusion, dietary NaB significantly promoted growth and gut health of turbot. Besides, it enhanced the resistance of fish to bacterial infection, especially dietary 0.5% NaB supplementation.

Keywords: Scophthalmus maximus L.; Growth; Inflammatory cytokines; Signaling pathways; Sodium butyrate (NaB); Soybean.

Fig. 1

Schematic representation of the key steps in the experimental protocol.

Fig. 2

Effects of NaB on intestinal histomorphology. (a) The intestines of turbot in different groups were collected and sectioned. Fixation with H&E, the morphology of the distal intestines was observed. The images were representative of at least three independent experiments, and scale bar indicates 500 μm; (A) and (B) in the images manifests villus height and muscle layer thickness, respectively; lamina propria width is indicated by arrows. (b–d) The micromorphology, including villus height (b), lamina propria width (c), and muscle layer thickness (d) of the intestine, was evaluated (n = 6). Values were presented as means ± SEM. Multiple comparisons were conducted with Tukey’s test. Different superscript letters indicated significant difference (P < 0.05).

Fig. 3

Effects of dietary NaB on the gene expression of inflammatory cytokines and NF-κB in turbot intestine and kidney. The relative mRNA expressions of TNFα, IL-1β, NF-κB and IL-10 in the distal intestine (a–d) and kidney (e–h) of turbot in different groups were tested by qRT-PCR (n = 12). Values were presented as means ± SEM. Multiple comparisons were conducted with Tukey’s test. Different superscript letters indicated significant difference (P < 0.05).

Fig. 4

Effects of NaB on the anti-bacterial ability in turbot. After the feeding trial, turbot from each group were infected with E. tarda (1 × 10⁷ CFU/fish) by i.p. injection. (a) The mortality of the turbot was recorded every 6 h till 42 hpi (n = 10). (b) The viable bacteria in the spleen at 12 hpi were counted (n = 12). Values were presented as means ± SEM. Multiple comparisons were conducted with Tukey’s test. Different superscript letters indicated significant difference (P < 0.05).

Fig. 5

Effects of NaB on the gene expression of batericidal effectors in different groups. The relative mRNA expressions of HIF-1α, IL-22, and lysozyme in the distal intestine (a–c) and kidney (d–f) of turbot were analyzed by qRT-PCR (n = 12). Values were presented as means ± SEM. Multiple comparisons were conducted with Tukey’s test. Different superscript letters indicated significant difference (P < 0.05).

Fig. 6

Effects of NaB on the bactericidal activity and the production of antibacterial effectors in HKMs. After the feeding trial, HKMs were isolated and cultured. (a) The equal number of E. tarda was added and coincubated with HKMs for 2 h. Thereafter, the survival rate of ingested bacteria was calculated (n = 12). (b, c) The gene expression of HIF-1α and IL-22 was measured (n = 12). (d and e) The expression of lysozyme in mRNA level (d) and enzyme activity (c) in the cells was examined (n = 6). (f) E. tarda was added (cells: bacteria = 1:1) and coincubated for 2 h, followed by the production of ROS in the cells was analyzed (n = 12). (g) After the HKMs coincubation with E. tarda for 6 h, the production of NO in cells was investigated between different groups (n = 12). Values were presented as means ± SEM. Multiple comparisons were conducted with Tukey’s test. Different superscript letters indicated significant difference (P < 0.05).