Probiotic efficacy and mechanism of a pigeon derived Ligilactobacillus salivarius strain in promoting growth and intestinal development of pigeons

Abstract

Background: With the gradual rise of antibiotic-free farming practices, the exploration of novel, green, and low-pollution alternatives to antibiotics has become one of the key research focus in the field of agricultural science. In the development of antibiotic alternatives, probiotics, particularly host-associated probiotics, have been found to play a significant role in enhancing the production performance of livestock and poultry. However, research on and application of probiotics specifically for meat pigeons remain relatively underdeveloped.

Objective: To assess and investigate the probiotic efficacy and mechanisms during homologous lactic acid bacteria (LAB) transplant to host-pigeons, LAB strains with good probiotic properties were isolated from the intestinal contents of 28-day-old Mimas pigeons. And then measured the production indexes, intestinal flora, and intestinal transcriptomics of the hosts after instillation of LAB strains.

Methods: A total of 360 at 1-day-old pigeons were randomly divided into four groups and gavaged 0.4 mL Ligilactobacillus salivarius S10 with concentration of 0, 10⁸, 10⁹, and 10¹⁰ CFU/mL, designated as the control group (CG), the low concentration group (LG), the medium concentration group (MG), and the high concentration group (HG), respectively.

Results: The findings revealed that an optimal concentration of 10⁹ CFU/mL L. salivarius S10, a dominant strain isolated and screened, enhanced the growth performance and intestinal development of young pigeons. 16S rRNA gene sequencing analysis demonstrated a significant increase in the abundance of Lactobacillus, Pantoea_A and Enterococcus_H and a significant reduction in the abundance of Clostridium_T in the pigeon ileum (p < 0.05) under selected concentration treatment. Transcriptomic profiling of the ileum revealed 1828 differentially expressed genes (DEGs) between CG and MG. Notably, DEGs involved in the MAPK signaling pathway, such as RAF1, PDGFRB, and ELK4, were significantly correlated with differential ileal bacteria, suggesting that modulation of intestinal flora can influence the expression of genes related to cell proliferation and differentiation in the ileum, which is potentially important in promoting the growth and development of pigeons.

Conclusion: Ligilactobacillus salivarius S10 possesses the potential to be used as a probiotic for pigeons, which can influence the expression of gut development-related DEGs by regulating the intestinal flora, and further improve the growth performance of pigeons. This research provides a scientific foundation for developing pigeon-specific probiotics and promotes healthy farming practices for meat pigeons. Furthermore, it opens new avenues for improving the economic efficiency of pigeon farming.

Keywords: Ligilactobacillus salivarius; intestinal flora; intestinal transcriptomics; pigeon; production performance.

Figure 1

Isolation, purification and identification of LAB strains. (A) MRS agar medium containing 2% CaCO₃ at the dilution of 10⁻⁴. (B) Depict the colony morphology of the isolated strains after three times of purification. (C) Depict the Gram staining of the isolated strains after three times of purification. (D) S1–S20 represents the results of 16S rRNA gene amplification for isolated strains. (E) Phylogenetic tree of isolated strains.

Figure 2

Growth and acid-producing capacity of isolated strains. (A) Growth curve. (B) Acid-producing ability.

Figure 3

Ileum morphology by HE staining (14.0 ×). (A) Ileum morphology of CG. (B) Ileum morphology of MG. Scale bar: 200 μm.

Figure 4

The diversity of the ileal microbiota. (A) Venn diagram. (B) Rarefaction curve. (C) Alpha diversity analysis. (D) PCoA based on Weighted UniFrac algorithm. (E) Sample distances based on Weighted UniFrac algorithm. Sample sizes for each group are as follows: CG (n = 6), MG (n = 6).

Figure 5

Differences in the ileal microbiota composition and associations of differential bacteria with production performance and intestinal development. (A) Bacterial community compositions at phylum level. (B) Bacterial community compositions at genus level. (C) LEfSe analysis. (D) Heatmap of the Spearman’s correlation analysis between differential ileal bacteria and production indexes of pigeons. The red and blue color represents a positive and negative correlation, respectively. *Indicates a difference at p < 0.05, **Indicates a difference at p < 0.01. (E) Heatmap of the Spearman’s correlation analysis between differential ileal bacteria and ileal morphological indexes of pigeons. The red and blue color represents a positive and negative correlation, respectively. *Indicates a difference at p < 0.05, **indicates a difference at p < 0.01. Sample sizes for each group are as follows: CG (n = 6), MG (n = 6).

Figure 6

Ileal transcriptomics. (A) PCOA diagram between two groups of samples. (B) Volcano plot of DEGs between two groups. (C) Cluster heatmap. (D) Dot plot of the GO enrichment analysis. (E) Histogram of KEGG enrichment analysis. (F) Heatmap of the Spearman’s correlation analysis between differential ileal bacteria and DEGs in the MAPK signaling pathway. The red and blue color represents a positive and negative correlation, respectively. *Indicates a difference at p < 0.05, **Indicates a difference at p < 0.01. (G) FPKM values of DEGs randomly selected from outside the MAPK signaling pathway. *Indicates a difference at p < 0.05, **indicates a difference at p < 0.01, ***indicates a difference at p < 0.001, ****indicates a difference at p < 0.0001, same below. (H) Relative mRNA expression levels of DEGs randomly selected from outside the MAPK signaling pathway. (I) FPKM values of DEGs randomly selected from the MAPK signaling pathway. (J) Relative mRNA expression levels of DEGs randomly selected from the MAPK signaling pathway. Sample sizes for each group are as follows: CG (n = 3), MG (n = 3).