Effects of hesperidin, thymol, rosmarinic acid and their combined effect on growth performance, intestinal barrier function and cecal microbiota in broilers

Abstract

This study aims to investigate the effects of hesperidin (Hes), thymol (Thy), rosmarinic acid (RA) and their combined effect on broiler growth performance, intestinal barrier function, and cecal microbiota. A total of 240 newly hatched Arbor Acres broiler chicks were randomly divided into 5 treatments with 6 replicates of 8 chickens. The birds were fed a basal diet (Con group), a basal diet supplemented with 40 mg/kg Hes (Hes group), a basal diet supplemented with 40 mg/kg Thy (Thy group), a basal diet supplemented with 20 mg/kg RA (RA group), or a basal diet supplemented with 40 mg/kg Hes + 40 mg/kg Thy + 20 mg/kg RA (HTR group) for 42 d. The results indicated that dietary Hes and HTR supplementation enhanced average daily gain, final body weight, and eviscerated yield of broilers compared with the Con group (P < 0.05). Notably, the HTR treatment showed a decrease in abdominal fat yield and ratio of feed to weight gain (P < 0.05). HTR treatment increased ileal villus height, villus height/crypt depth, and number of goblet cells, decreased the crypt depth (P < 0.05), up-regulated the mRNA expression of tight junction proteins (ZO-1, Claudin-1, Occludin) and MUC2 (P < 0.05). Hes, Thy, RA, HTR treatment decreased the concentrations of pro-inflammatory factors (IL-8, IFN-γ and TNF-α), and down-regulated the mRNA expression of TLR4/MyD88/NF-κB (P < 0.05). Importantly, the supplementation of HTR increased the relative abundance of beneficial bacteria (Parabacteroides, Lachnosiraceae NK4A136 and Turicbacter) and significantly decreased the relative abundance of opportunistic pathogenic bacteria such as Colidextribacter (P < 0.05). Additionally, the concentrations of propionate and butyrate in the cecum were elevated in the HTR group (P < 0.05). These findings indicate that the diet supplemented with HTR improved the growth performance and intestinal barrier function in broilers by modulating the cecal microbiota and its metabolites.

Keywords: broiler; hesperidin; intestinal barrier; rosmarinic acid; thymol.

Figure 1

Effect of dietary supplementation with Hes, Thy, RA and their combination on ileal morphology and the number of goblet cells of broilers. (A) Representative image of the ileal morphological structure, which was observed at 40× magnification. (B) Representative image of goblet cells in the ileum, which was observed at 200× magnification. (C) The villus height of ileum. (D) The crypt depth of ileum. (E) The villus to crypt ratio of ileum. (F) The ileal goblet cell count. (G) The relative mRNA expression of MUC2. Con: feed with the corn-soybean basic diet; Hes: feed with basic diet and 40 mg/kg hesperidin; Thy: feed with basic diet and 40 mg/kg thymol; RA: feed with basic diet and 20 mg/kg rosmarinic acid; HTR: feed with basic diet and 40 mg/kg hesperidin, 40 mg/kg thymol and 20 mg/kg rosmarinic acid. Values at the same index with no common superscript differ significantly (n = 6; P  <  0.05).

Figure 2

Effect of dietary supplementation with Hes, Thy, RA and their combination on ileal barrier function and inflammatory response of broilers. (A) The content of interleukin 1β (IL-1β) in the ileal mucosa. (B) The content of interleukin 6 (IL-6) in the ileal mucosa. (C) The content of interleukin 8 (IL-8) in the ileal mucosa. (D) The content of interferon gamma (IFN-γ) in the ileal mucosa. (E) The content of tumor necrosis factor alpha (TNF-α) in the ileal mucosa. (F) The mRNA expression levels of tight junction. (G) TLR4 signaling pathway-related genes. Con: feed with the corn-soybean basic diet; Hes: feed with basic diet and 40 mg/kg hesperidin; Thy: feed with basic diet and 40 mg/kg thymol; RA: feed with basic diet and 20 mg/kg rosmarinic acid; HTR: feed with basic diet and 40 mg/kg hesperidin, 40 mg/kg thymol and 20 mg/kg rosmarinic acid. Values at the same index with no common superscript differ significantly (n = 6; P  <  0.05).

Figure 3

Effect of dietary supplementation with Hes, Thy, RA and their combination on diversity of the cecal microbiota in broilers. (A) A Venn diagram based on the OTU level. (B) Principal coordinate analysis (PCoA) based on Bray-Curtis. The alpha diversity indices observed species (C) Ace index, (D) Chao 1 index, (E) Shannon index. Con: feed with the corn-soybean basic diet; Hes: feed with basic diet and 40 mg/kg hesperidin; Thy: feed with basic diet and 40 mg/kg thymol; RA: feed with basic diet and 20 mg/kg rosmarinic acid; HTR: feed with basic diet and 40 mg/kg hesperidin, 40 mg/kg thymol and 20 mg/kg rosmarinic acid. Values at the same index with no common superscript differ significantly (n = 6; P  <  0.05).

Figure 4

Effect of dietary supplementation with Hes, Thy, RA and their combination on the cecal microbiota of broilers. (A) Microbial composition at the phylum level. (B) The ratio of Firmicutes / Bacteroidetes. (C) Microbial composition at the genus level. (D) The changes of the intestinal microbiota at the genus level. Con: feed with the corn-soybean basic diet; Hes: feed with basic diet and 40 mg/kg hesperidin; Thy: feed with basic diet and 40 mg/kg thymol; RA: feed with basic diet and 20 mg/kg rosmarinic acid; HTR: feed with basic diet and 40 mg/kg hesperidin, 40 mg/kg thymol and 20 mg/kg rosmarinic acid. Values at the same index with no common superscript differ significantly (n = 6; P  <  0.05).

Figure 5

Linear discriminant analysis effect (LEfSe) size of intestinal microbiota (LDA > 3, P < 0.05). Con: feed with the corn-soybean basic diet; Hes: feed with basic diet and 40 mg/kg hesperidin; Thy: feed with basic diet and 40 mg/kg thymol; RA: feed with basic diet and 20 mg/kg rosmarinic acid; HTR: feed with basic diet and 40 mg/kg hesperidin, 40 mg/kg thymol and 20 mg/kg rosmarinic acid.

Figure 6

Effect of dietary supplementation with Hes, Thy, RA and their combination on the correlation analysis between intestinal microbiota and intestinal inflammatory indicators. Con: feed with the corn-soybean basic diet; Hes: feed with basic diet and 40 mg/kg hesperidin; Thy: feed with basic diet and 40 mg/kg thymol; RA: feed with basic diet and 20 mg/kg rosmarinic acid; HTR: feed with basic diet and 40 mg/kg hesperidin, 40 mg/kg thymol and 20 mg/kg rosmarinic acid. Significant correlations are noted by 0.01 ≤ P < 0.05 *, 0.001 ≤ P < 0.01**, and P < 0.001***; Values at the same index with no common superscript differs significantly (n = 6; P < 0.05).