Effects of compound probiotics on intestinal barrier function and caecum microbiota composition of broilers

Figures & data

Table 1. The composition levels of basal diets.

Ingredients, %	Day 1–21	Day 21–60
Corn	58.17	64.26
Soybean meal, 43%	30.44	24.05
Corn gluten meal	2.00	2.50
Fish meal, 64.6%	2.00	2.00
Soybean oil	3.38	3.60
Dicalcium phosphate	1.50	1.04
Limestone	1.30	1.35
Salt	0.30	0.30
L-lysine HCl, 78%	0.01	0.08
DL-Methionine, 98%	0.14	0.04
L-Threonine, 98%	0.01	0.03
Chromic oxide	0.25	0.25
Vitamin-mineral premix^1	0.50	0.50
Total	100.00	100.00

¹ Vitamin A, 11,000 IU; vitamin D, 3025 IU; vitamin E, 22 mg; vitamin K3, 2.2 mg; thiamine, 1.65 mg; riboflavin, 6.6 mg; pyridoxine, 3.3 mg; cobalamin, 17.6 µg; nicotinic acid, 22 mg; pantothenic acid, 13.2 mg; folic acid, 0.33 mg; biotin, 88 µg; choline chloride, 500 mg; Fe, 48 mg; Zn, 96.6 mg; Mn, 101.76 mg; Cu, 10 mg; Se, 0.05 mg; I, 0.96 mg; Co, 0.3 mg.

Table 2. PCR primer sequences.

Genes	Primer sequence (5’−3’)
ZO-1	F:5'-GAGCTCACAAGCTACGCAAA-3'
	R:5'-ACTTGTAGCACCATCTGCCT-3
Occludin	F:5'-CCTCATCGTCATCCTGCTCT-3'
	R:5-GGTCCCAGTAGATGTTGGCT-3'
Claudin-1	F:5'-TACAGCCCTTGGCCAATACA-3'
	R:5'-CCAAGAAACAACCACCAGCA-3'
GAPDH	F:5'-AGTCGGAGTCAACGGATTTGG-3'
	R:5'-AAGATAGTGATGGCGTGCCC-3'

Figure 1. Effects of compound probiotics on growth performance of broilers. (A) Average daily gain, (B) Average daily feed intake, (C) Feed conversion ratio. Data are expressed as means ± SEM (n = 6). * indicates significant difference compared to the corresponding control (*P < 0.05, **P < 0.01). # indicates statistically significant difference between corresponding groups (# P < 0.05, ## P < 0.01).

Figure 2. Histomorphometric analysis of the broiler jejunum. (A) Villus length, (B) Crypt depth, (C) Villus/crypt, (D) Haematoxylin and eosin (H&E) staining, 100 × magnification, scale bar: 100 µm. * indicates significant difference compared to the corresponding control (*P < 0.05, **P < 0.01). # indicates statistically significant difference between corresponding groups (# P < 0.05, ## P < 0.01).

Figure 3. Serum oxidative stress level of broilers. (A) MDA content, (B) SOD activity, (C) GSH activity, (D) H₂O₂ content. * indicates significant difference compared to the corresponding control (*P < 0.05, **P < 0.01). # indicates statistically significant difference between corresponding groups (# P < 0.05, ## P < 0.01).

Figure 4. Effects of probiotics on TJ-related mRNA expression levels of genes in jejunum of broilers. (A) ZO-1 gene expression levels, (B) Occludin gene expression levels, (C) Claudin1 gene expression levels. * indicates significant difference compared to the corresponding control (*P < 0.05, **P < 0.01). # indicates statistically significant difference between corresponding groups (# P < 0.05, ## P < 0.01).

Figure 5. Welch’s t-test analysis for the significant changes of differential microbiota at phylum and genus levels in the caecum contents of broilers. (A) The Venn diagram of OTUs; (B) Microbial composition in the caecum at the phylum level; (C) Microbial composition in the caecum at the genus level; (D) Differences in the phylum level of caecum microorganisms; (E) Differences in the genus level of caecum microorganisms.

Figure 6. α-diversity and β-diversity analysis on the caecum microbiota. (A) Alpha diversity indices, including Sob index, Shannon index, Simpson index, Chao1 index, ACE index and Good’s coverage; (B) Rank-abundance distribution curve; (C) PCoA plot; (D) Analysis of similarities. The ACE and Chao1 indexes represent the community richness of the microbiota, the Shannon and Simpson indexes represent the community diversity of the microbiota. The rank-abundance curve is used to display species abundance and evenness.

Figure 7. Predicted microbial function of caecum microbiota in broilers. Statistics were conducted by Welch’ s t-test, with P-value < 0.05 indicating a significant difference in microbial function at Level 2 of KEGG pathways.