Supplementation of the probiotic Lactobacillus rhamnosus GG to pre-weaning calves decreases diarrhea incidence by modulating gut bacteria and associated metabolites

Abstract

The aim of the present study was to investigate whether Lactobacillus rhamnosus GG (LGG) could be utilized as an effective strategy to promote growth performance of calves and to further study its impact on decreasing diarrhea. A total of 18 healthy Holstein calves (age 8 ± 3 days, weight 42.48 ± 1.04 kg) were raised in a centralized manner to minimize differences and then divided into two groups: one group received LGG supplementation (1 × 10¹⁰ CFU per calf per day), while the other served as the control (CON). Each group consisted of 9 replicates, with 1 calf per replicate. The experimental period spanned 6 weeks. The experimental results demonstrated an increase of the average daily growth (P = 0.094) and a highly significant reduction in diarrhea (P = 0.001) for the LGG group compared with the CON group. The supplementation of LGG resulted in an increased abundance of endogenous beneficial bacteria in the gut including Lysinbacillus (P = 0.012) and Rikenellaceae_RC9_gut_group (P = 0.041) while reducing levels of pathogenic bacteria in the Escherichia-Shigella (P = 0.066). These particular groups could serve as biomarker bacteria for calf diarrhea and gut health. The alteration of metabolite production and metabolic pathways were also closely associated with the variation of bacterial composition in the gut microbiome of calves, of which included enrichment of biosynthesis of unsaturated fatty acids, α-linolenic acid and steroid biosynthesis. Overall, LGG administration had a positive impact on improving the intestinal homeostasis, alleviating diarrhea, and enhancing growth performance by modulating gut bacteria and metabolites in pre-weaning calves. Thus, LGG could be a potential alternative to prophylactic antibiotic treatment that would lower associated costs and provide new insights into preventing and treating calf diarrhea.

Keywords: Calf; Diarrhea; Gut bacteria; Lactobacillus rahamnosus GG; Metabolomics.

Fig. 1

Community abundance and diversity of feces sample from calves. Alpha diversity was evaluated based on the (A) Chao 1 and (B) Simpson indices using OTU abundance. (C, D) Bacterial similarity between CON and LGG groups using PCoA scatter plots as indicated. CON = control group (n = 6); LGG = fed the Lactobacillus rhamnosus GG group (n = 6); W = week; OTU = operational taxonomic unit; PCoA = principal component analysis.

Fig. 2

Gut bacteria composition spectrums in calves. Taxonomic distributions of calf fecal bacteria at the (A) phylum and (B) genus levels (top 10). (C, D and E) Statistical analysis using the Wilcoxon rank-sum test of genus-level bacteria that significantly differed between CON and LGG group calves. (F) LDA score histograms were used to identify bacterial genera that differed significantly between LGG and CON groups calves in the sixth week (LDA score >2). (G) Microflora distribution of feces bacteria from phylum to genus levels in sixth week in LGG and CON groups calves as indicated using LefSe. CON = control group (n = 6); LGG = fed the Lactobacillus rhamnosus GG group (n = 6); W = week; LDA = linear discriminant analysis; LefSe = linear discriminant analysis effect size.

Fig. 3

Gut bacteria interaction networks. (A) CON and LGG groups calves gut bacteria co-occurrence network at week 6. Relationships between the orders (B) Solibacillus, (C) Lysinibacillus, (D) Rikenellaceae_RC9_gut_group, and (E) Ruminococcaceae_UCG_005 with Escherichia-Shigella in the gut. CON = control group (n = 6); LGG = fed the Lactobacillus rhamnosus GG group (n = 6); W = week.

Fig. 4

Effects of LGG on feces metabolic profiles of calves. (A) PLS-DA of feces metabolic from calves at week 6. (B) Differential metabolites from calf feces at week 6. The VIP >1 and P < 0.05 were the standard screening criteria for significant differences in metabolites. (C) Feces metabolites of calves in CON and LGG groups at week 6 (top 20). (D) KEGG pathway enrichment analysis of metabolites at week 6 (top 20 pathways). The horizontal axis indicates the proportion of metabolites in each pathway relative to the total number of significantly differential metabolites. The height of the bars represents the number of differential metabolites enriched in each pathway. CON = control group (n = 6); LGG = fed the Lactobacillus rhamnosus GG group; W = week; VIP = variable importance for the projection; PLS-DA = partial least squares discriminant analysis; KEGG = Kyoto Encyclopedia of Genes and Genomes.

Fig. 5

Relationships between (A) VFA and bacteria abundance and (B) Gut bacteria and metabolites. R values were displayed in different colors. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001. The legend on the right is the color interval of different R values. VFA = volatile fatty acid.