Dietary Tannic Acid Promotes Growth Performance and Resistance Against Aeromonas hydrophila Infection by Improving the Antioxidative Capacity and Intestinal Health in the Chinese Soft-Shelled Turtle (Pelodiscus sinensis)

Abstract

To investigate the effect of tannic acid (TA) on the growth, disease resistance, and intestinal health of Chinese soft-shelled turtles, individual turtles were fed with 0 g/kg (CG), 0.5 g/kg, 1 g/kg, 2 g/kg, and 4 g/kg TA diets for 98 days. Afterwards, the turtles' disease resistance was tested using Aeromonas hydrophila. The results showed that 0.5-4 g/kg of dietary TA increased the growth performance and feed utilization (p < 0.05), with 2.38 g/kg being the optimal level for the specific growth rate (SGR). The addition of 0.5-4 g/kg of TA in diets increased the mucosal fold height and submucosa thickness of the small intestine, which reached a maximum of 2 g/kg. The addition of 0.5-2 g/kg of TA effectively reduced the cumulative mortality that had been induced by A. hydrophila, with the 2 g/kg dosage leading to the lowest mortality. Additionally, 1-4 g/kg of TA improved the T-SOD, CAT, and GSH-Px activities during infection, while 2 g/kg of dietary TA enhanced the richness and diversity of the microbiota, for example, by increasing Actinobacteria but inhibiting Firmicutes. The transcriptome demonstrated that the predominant differentially expressed genes (DEGs) in TA2 were mainly enriched in the PPAR signaling pathway (Acsl5, Apoa2, Apoa5, Fabp1, Fabp2, and Fabp6); in glycine, serine, and threonine metabolism (Chdh, Gatm, and Shmt1); and in steroid biosynthesis (Cel, Hsd17b7, Soat2, and Sqle). The main differentially expressed metabolites (DEMs) that were discovered by means of metabolome analysis included cholylhistidine, calcipotriol, 13-O-tetradecanoylphorbol 12-acetate, and hexahomomethionine in CG vs. TA2. Integrative analyses of two omics revealed that 2 g/kg of TA mitigated inflammation by activating the PPAR signaling pathway and regulating the lipid metabolism via multiple pathways, such as steroid biosynthesis and α-linolenic acid metabolism. In general, the inclusion of 2 g/kg of TA in turtle diets can optimally promote growth and bacterial resistance by maintaining intestinal health and improving antioxidant capacity.

Keywords: Chinese soft-shelled turtle; antioxidant; intestinal microbiota; metabolome; signaling pathways; tannin; transcriptome.

Figure 1

Experimental flowchart. CG indicates the control group. TA0.5 indicates the group supplemented with 0.5 g/kg TA. TA1 indicates the group supplemented with 1 g/kg TA. TA2 indicates the group supplemented with 2 g/kg TA. TA4 indicates the group supplemented with 4 g/kg TA.

Figure 2

Quadratic regression analysis of the SGR (A) and WGR (B) of Chinese soft-shelled turtles who were fed a diet with graded levels of TA for 98 days. (C) The cumulative mortality of Chinese soft-shelled turtles who were infected with Aeromonas hydrophila. Different letters indicate significant differences among groups at the same point (p < 0.05). CG indicates the control group. TA0.5 indicates the group supplemented with 0.5 g/kg TA. TA1 indicates the group supplemented with 1 g/kg TA. TA2 indicates the group supplemented with 2 g/kg TA. TA4 indicates the group supplemented with 4 g/kg TA.

Figure 3

The effect of TA on plasma T-SOD (A), CAT (B), GSH-Px (C), ACP (D), and ALP (E) activities. All data are represented as mean ± SE (n = 3). Different letters indicate significant differences among groups at the same point (p < 0.05). T-SOD, total superoxide dismutase; CAT, catalase; GSH-Px, glutathione peroxidase; ACP, acid phosphatase; ALP, alkaline phosphatase. CG indicates the control group. TA0.5 indicates the group supplemented with 0.5 g/kg TA. TA1 indicates the group supplemented with 1 g/kg TA. TA2 indicates the group supplemented with 2 g/kg TA. TA4 indicates the group supplemented with 4 g/kg TA. The x-axis represents the number of hours post-infection with Aeromonas hydrophila.

Figure 4

Images of the small intestinal morphology, observed using an optical microscope, in Chinese soft-shelled turtles after the 98-day feeding experiment. CG (A,F) indicates the control group. TA0.5 (B,G) indicates the group supplemented with 0.5 g/kg TA. TA1 (C,H) indicates the group supplemented with 1 g/kg TA. TA2 (D,I) indicates the group supplemented with 2 g/kg TA. TA4 (E,J) indicates the group supplemented with 4 g/kg TA. Scale bar = 500 μm (A–E). Scale bar = 50 μm (F–J). MH indicates the mucosal fold height, MW represents the mucosal fold width, LP denotes the lamin propria width, and SM denotes the submucosa.

Figure 5

Analyses of the intestinal microbiome. (A) Venn diagram showing the number of OTUs that were identified in the CG and TA2 groups. (B) Comparison of the Shannon indexes of the CG and TA2 groups using Student’s t-test. (C) PCoA analysis of the intestinal microflora of the CG vs. the TA2 group. Intestinal microflora at the OTU (D) and species (E) levels. (F) LEfSe multilevel species hierarchy tree in the CG vs. the TA2 group. (G) A comparative KEGG pathway enrichment analysis of the microbial functional abundance in the CG and TA2 groups with PICRUSt2. p < 0.05 indicates a significant difference in the Shannon index. “CG” indicates the control group. “TA2” indicates the group supplemented with 2 g/kg TA.

Figure 6

Overview of the DEGs from the transcriptome in the CG vs. TA2 group comparison. (A) The results of the PCA demonstrating the difference in gene expression patterns between the CG and TA2 groups. (B) A correlation heatmap of all the gene expression patterns in the three replicates of the CG and TA2 groups. (C) A volcano plot of the DEGs in the CG vs. TA2 group comparison. (D) A KEGG enrichment analysis of the DEGs in the CG and TA2 group comparison. (E) A GO enrichment analysis of the DEGs in the CG and TA2 group comparison. “CG” indicates the control group. “TA2” indicates the group supplemented with 2 g/kg TA.

Figure 7

Identification of the DEMs and signaling pathways from the metabolome in the CG vs. TA2 group comparison. The cross-validation (A) and permutation test (B) of the metabolite profiles were carried out using orthogonal projection to latent structures–discriminant analysis (OPLS-DA). (C) The number of DEMs, filtered based on |log2 (FoldChange)| > 1 and an adjusted p-value < 0.05 in the CG vs. TA2 group comparisons. (D) Z-score plots exhibiting the top-30 DEMs from the CG vs. TA2 comparison. (E) The differential abundance score based on a pathway analysis of the metabolic changes in the CG vs. TA2 group comparison.

Figure 8

Joint analysis of the DEGs and DEMs in the CG vs. TA2 group comparison for the small intestine. (A) A nine-quadrant diagram indicating the correlation of the DEGs and DEMs in the CG vs. TA2 group comparison. The DEGs and DEMs with the absolute value of fold change ≧ 2 were marked with red color, which, with the absolute value of fold change < 2, were marked with yellow color. (B) A chord diagram exhibiting the significant association of DEGs with DEMs in the CG vs. TA2 group comparison. (C) Conjoint analyses of the DEG and DEM-enriched KEGG pathways. (D) The interactive networks of the DEGs and DEMs in the dominant KEGG pathways. “CG” indicates the control group. “TA2” indicates the group supplemented with 2 g/kg TA.