Dietary Clostridium butyricum and 25-Hydroxyvitamin D3 modulate bone metabolism of broilers through the gut-brain axis

Abstract

Leg disorders have become increasingly common in broilers, leading to lower meat quality and major economic losses. This study evaluated the effects of dietary supplementation with Clostridium butyricum (C. butyricum) and 25-hydroxyvitamin D₃ (25-OH-D₃) on bone development by comparing growth performance, tibial parameters, Ca and P contents of tibial ash, bone development-related indicators' level, and cecal short-chain fatty acids in Cobb broilers. All birds were divided into four treatment groups, which birds fed either a basal diet (Con), basal diet + 75 mg chlortetracycline/kg (Anti), basal diet + C. butyricum at 10⁹ CFU/kg (Cb), basal diet + C. butyricum at 10⁹ CFU/kg and 25-OH-D₃ at 25 μg/kg (CbD), or basal diet + 25-OH-D₃ at 25 μg/kg (CD). Our results suggest that the dietary supplementation in Cb, CbD, and CD significantly increased the body weight (BW) and average daily gain (ADG), and reduced the feed-to-weight ratio (F/G) at different stages of growth (P < 0.05). Dietary supplementation in Cb, CbD, and CD prolonged (P < 0.05) the behavioral responses latency-to-lie (LTL) time, reduced (P < 0.05) the levels of osteocalcin (BGP) and peptide tyrosine (PYY), and increased (P < 0.05) serotonin (5-HT) and dopamine (DA). Treatment with Cb increased (P < 0.05) the levels of acetic acid, isobutyric acid, butyric acid, and isovaleric acid compared with those in Con group. The cecal metagenome showed that Alistipes spp. were significantly more abundant in Cb, CbD, and CD groups (P < 0.05). A total of 12 metabolic pathways were significantly affected by supplementation, including the signaling pathways of glucagon, insulin, and PI3K-AKT; primary and secondary bile acid biosynthesis; and P-type Ca 2+ transporters (P < 0.05). Hence, the CbD supplementation modulates bone metabolism by regulating the mediators of gut-brain axis, which may inform strategies to prevent leg diseases and improve meat quality in broilers.

Keywords: Clostridium butyricum; bone development; broiler; gut–brain axis; vitamin D.

Figure 1

Effects of C. butyricum and 25-hydroxyvitamin D₃ on the latency-to-lie time, tibial content of calcium and phosphorus, BMD and bone-breaking strengthen of broilers. (A) LTL. (B) calcium. (C) phosphorus. (D) BMD. (E) bone-breaking strengthen. (F) bone radiograph. Con birds fed basal diet without any antibiotics and additive, Anti birds fed basal diet added with antibiotic, Cb birds fed basal diet added with Clostridium butyricum, CbD birds fed basal diet added with Clostridium butyricum and 25-OH-D₃, CD birds fed basal diet added with 25-OH-D₃. Bars represent mean ± SEM (N = 8). * represents P<0.05.

Figure 2

Effects of Clostridium butyricum and 25-hydroxyvitamin D₃ on markers of bone formation in broilers’ serum. (A) AKP level. (B) TPINP level. (C) BGP level. (D) β-crosslaps level. Con birds fed basal diet without any antibiotics and additives, Anti birds fed basal diet added with antibiotic, Cb birds fed basal diet added with Clostridium butyricum, CbD birds fed basal diet added with Clostridium butyricum and 25-OH-D₃, CD birds fed basal diet added with 25-OH-D₃. Different lowercase letters above bars represent significantly different means P < 0.05. Bars represent mean ± SEM (N = 8).

Figure 3

Effects of Clostridium butyricum and 25-hydroxyvitamin D₃ on hormones related to bone metabolism in broilers serum. (A) GLP -1 concentration. (B) PYY concentration. (C) 5-HT concentration. (D) DA concentration. (E) PTH concentration. (F) IGF-1 concentration. Con birds fed basal diet without any antibiotics and additive, Anti birds fed basal diet added with antibiotic, Cb birds fed basal diet added with Clostridium butyricum, CbD birds fed basal diet added with Clostridium butyricum and 25-OH-D₃, CD birds fed basal diet added with 25-OH-D₃. Different lowercase letters above bars represent significantly different means P< 0.05. (N = 8).

Figure 4

Effects of Clostridium butyricum and 25-hydroxyvitamin D₃ on the hypothalamic and intestinal brain-gut peptides in broilers. (A) Caecal 5-HT content. (B) Caecal DA content. (C) Caecal GLP-1 content. (D) Ileal PYY content. (E) Hypothalamic 5-HT content. (F) Hypothalamic DA content. Con birds fed basal diet without any antibiotics and additive, Anti birds fed basal diet added with antibiotic, Cb birds fed basal diet added with Clostridium butyricum, CbD birds fed basal diet added with Clostridium butyricum and 25-OH-D₃, CD birds fed basal diet added with 25-OH-D₃. Different lowercase letters above bars represent significantly different means P< 0.05. (N = 8).

Figure 5

Effects of Clostridium butyricum and 25-hydroxyvitamin D₃ on caecal SCFAs in broilers. (A) acetic acid level. (B) propionic acid level. (C) isobutyric level. (D) butyric level. (E) isovaleric level. (F) valeric level. Con birds fed basal diet without any antibiotics and additive, Anti birds fed basal diet added with antibiotic, Cb birds fed basal diet added with Clostridium butyricum, CbD birds fed basal diet added with Clostridium butyricum and 25-OH-D₃, CD birds fed basal diet added with 25-OH-D₃. Different lowercase letters above bars represent significantly different means P < 0.05. (N = 8).

Figure 6

Effects of Clostridium butyricum and 25-hydroxyvitamin D₃ on metagenome of broilers’ caecal microflora. (A) gene number venn graph. (B) core_Pan gene dilution curve. (C) species relative abundance histogram display based on genus level. (D) species relative abundance histogram display based on specie level. (E) PCA plot based on genus level. (F) PCoA plot based on specie level. Con birds fed basal diet without any antibiotics and additive, Anti birds fed basal diet added with antibiotic, Cb birds fed basal diet added with Clostridium butyricum, CbD birds fed basal diet added with Clostridium butyricum and 25-OH-D₃, CD birds fed basal diet added with 25-OH-D₃. (N = 3).

Figure 7

Effects of Clostridium butyricum and 25-hydroxyvitamin D₃ on caecal microflora on genus and species levels in broilers. (A) top 12 distinguished genus based on Kruskal_Wallis analysis; (B) top 12 distinguished species based on Kruskal_Wallis analysis; (C) LEfSe analysis in genus level. Con birds fed basal diet without any antibiotics and additive, Anti birds fed basal diet added with antibiotic, Cb birds fed basal diet added with Clostridium butyricum, CbD birds fed basal diet added with Clostridium butyricum and 25-OH-D₃, CD birds fed basal diet added with 25-OH-D₃. (N = 3).

Figure 8

Effects of Clostridium butyricum and 25-hydroxyvitamin D₃ on KEGG metabolic pathways of caecal microflora in broilers based on metagenomics. (A) Functional level PCA analysis. (B) Functional level PCoA analysis. (C) Heatmap of KEGG pathways. (D) Top 30 KEGG pathways abundance based on Kruskal–Wallis analysis. Con birds fed basal diet without any antibiotics and additive, Anti birds fed basal diet added with antibiotic, Cb birds fed basal diet added with Clostridium butyricum, CbD birds fed basal diet added with Clostridium butyricum and 25-OH-D₃, CD birds fed basal diet added with 25-OH-D₃. (N = 3).