Dietary vitamin B6 supplementation alleviates heat stress-induced intestinal barrier impairment by regulating the gut microbiota and metabolites in broilers

Abstract

Heat stress (HS) brings great challenges to the poultry industry. Vitamin B6 (VB6) is an essential micro-nutrient for animals to maintain normal physiological functions and possesses antioxidant and anti-inflammatory properties. This study aimed to explore the effect of VB6 on alleviating HS-induced intestinal barrier impairment in broilers. A total of 250 broilers (609.76 ± 0.34 g) were randomly allocated to 5 groups with 5 replicate cages of 10 birds each. The broilers in thermoneutral (TN) group were raised in thermoneutral conditions (23 ± 1°C) and fed with a basal diet. The birds in other four groups were housed under cycle high temperature (34 ± 1°C for 8 h/d) from d 21 to 35 and fed with the basal diet (HS group) or basal diet supplemented with 6, 12, or 24 mg/kg VB6 (HB-6, HB-12, HB-24 groups). The results showed that HS reduced the growth performance, increased ileum inflammatory cytokines levels, and impaired the gut barrier function (P < 0.05). Compared to the HS group, final body weight, average daily gain, and average daily feed intake, and the feed conversion ratio were improved by VB6 supplementation. The diamine oxidase, interleukin (IL)-1β, tumor necrosis factor-α, IL-18, IL-10, and interferon-γ levels were reduced by VB6 supplementation (P < 0.05). Moreover, VB6 supplementation linearly or quadratically enhanced villus height and villus height-to-crypt depth ratio of duodenum and jejunum, and decreased crypt depth of duodenum and ileum. The mRNA expression of Occlaudin, ZO1, Mucin2, Mucin4, E-cadhein, and β-catenin were increased by VB6 treatment (P < 0.05). Furthermore, dietary VB6 altered the diversity and community of gut microbiota (P < 0.05). A total of 83 differential metabolites associated with the amelioration of VB6 were identified, which were primarily enriched in glycerophospholipid metabolism, caffeine metabolism, and glutathione metabolism pathway. Collectively, VB6 may improve the growth performance and intestinal barrier function of heat-stressed broilers by regulating the ileal microbiota and metabolic homeostasis.

Keywords: gut barrier; gut microbiota; heat stress; metabolite; vitamin B6.

Figure 1

Effect of vitamin B6 on serum intestinal permeability biomarkers of heat-stressed broilers. Data are presented as mean ± SEM (n = 10). Mean values of the TN group and HS group with * differ significantly (P < 0.05). Mean values of the HS group, HS + 6 mg/kg VB6 group, HS + 12 mg/kg VB6 group and HS + 24 mg/kg VB6 group without common superscript letters differ significantly (P < 0.05). Abbreviations: DAO, diamine oxidase; LPS, lipopolysaccharide; D-LA, D-lactic acid. Treatments: TN, thermoneutral group; HS, heat stress group; HB-6, 12, 24, heat stress + 6, 12, 24 mg/kg vitamin B6 group.

Figure 2

Effect of vitamin B6 on ileum cytokines levels of heat-stressed broilers. Data are presented as mean ± SEM (n = 10). Mean values of the TN group and HS group with * differ significantly (P < 0.05). Mean values of the HS group, HS + 6 mg/kg VB6 group, HS + 12 mg/kg VB6 group and HS + 24 mg/kg VB6 group without common superscript letters differ significantly (P < 0.05). Abbreviations: IL, interleukin; TNF-α, tumor necrosis factor-α; IFN-γ, interferon-γ. Treatments: TN, thermoneutral group; HS, heat stress group; HB-6, 12, 24, heat stress + 6, 12, 24 mg/kg vitamin B6 group.

Figure 3

Effect of vitamin B6 on the mRNA expression of intestinal barrier function of heat-stressed broilers. Data are presented as mean ± SEM (n = 10). Mean values of the TN group and HS group with * differ significantly (P < 0.05). Mean values of the HS group, HS + 6 mg/kg VB6 group, HS + 12 mg/kg VB6 group and HS + 24 mg/kg VB6 group without common superscript letters differ significantly (P < 0.05). Abbreviations: ZO1, zonula occludens1. Treatments: TN, thermoneutral group; HS, heat stress group; HB-6, 12, 24, heat stress + 6, 12, 24 mg/kg vitamin B6 group.

Figure 4

Effect of vitamin B6 on the composition of gut microbiota in the ileum mucosa of heat-stressed broilers. (A) α diversity indices (Chao1, Shannon, Simpson). (B) Non-metric multidimensional scaling (NMDS) plot. (C) Venn plot. * Represents significant difference between TN and HS, HS and HB-12 (P < 0.05). Treatments: TN, thermoneutral group; HS, heat stress group; HB-12, heat stress + 12 mg/kg vitamin B6 group.

Figure 5

Differences in the bacterial communities of ileum mucosa determined by LEfSe analysis and MetaStat analysis. (A) The microbial community at the phylum level. (B) The microbial community at the genus level. (C) The significant differential microbiota at the phylum level by MetaStat analysis. (D) The significant differential microbiota at the Top 30 genus level by MetaStat analysis. (E) The LEfSe analysis (LDA score ≥ 2.5). * Represents significant difference between TN and HS, HS and HB-12 (P < 0.05). Treatments: TN, thermoneutral group; HS, heat stress group; HB-12, heat stress + 12 mg/kg vitamin B6 group.

Figure 6

Effect of vitamin B6 on the ileum mucosa metabolites of heat-stressed broilers. (A-B) Partial least squares-discriminant analysis (PLS-DA) score plot. (C-D) PLS-DA valid plot. (E) Pie chart of identified metabolites classification. (F) Venn plot. (G) KEGG pathway enrichment analysis of differential metabolites. Treatments: TN, thermoneutral group; HS, heat stress group; HB-12, heat stress + 12 mg/kg vitamin B6 group.

Figure 7

Spearman correlation among ileal differential metabolites with FC > 2, differential microbiota at the phylum and genus levels, differential gut barrier parameters and inflammatory cytokines. The red circle represents a positive correlation and blue circle represents a negative correlation. * Represents significant difference (P < 0.05).