Probiotic application to hatching egg surface supports microbiota development and acquisition in broiler embryos and hatchlings

Abstract

In modern poultry production, hatchlings primarily acquire their initial gut microbiota from the hatchery environment. This, in turn, results in delayed gut microbiota acquisition and a less diverse microbiota. However, the gut microbiota in the hatchling and the neonate is crucial for the normal development of the immune system and healthy gut function. Hence, promoting healthy microbiota development and acquisition in hatchlings is critical. To this end, we determined the potential for probiotic spray application to hatching eggs to support microbiota acquisition in the hatchling. A total of 100 hatching eggs (Ross 308) were either sprayed with phosphate-buffered saline (PBS; Control) or probiotics [∼9 log CFU/egg of Lacticaseibacillus rhamnosus NRRL B-442 (LR) or Lacticaseibacillus paracasei DUP 13076 (LP)] during incubation. Six eggs were sacrificed for sample collection at each sampling point. Eggshells were washed with sterile PBS buffer and collected at embryonic day (D) 0, 7, 14, 18, and 20. Chorioallantoic membrane (CAM) was collected at D7, 14, 18, and 20, and intestine at D14, 18, and 20. At hatch, chicks were euthanized, and the cecal and ileal samples were collected for microbiota characterization. Results indicate that spray application of LP and LR significantly modulated the microbiota associated with the eggshell, CAM, embryonic intestine, and hatchling gut. Further predictive analysis revealed CAM to be a significant source for the microbiota associated with the hatchling gut. Also, application of LP and LR led to significant enrichment in Lactobacillus populations and potential probiotic taxa, including Enterococcus, in the hatchling gut. Moreover, functional profile analysis revealed that microbial communities associated with the hatchling gut microbiota in the probiotic groups were enriched for nutrient and energy metabolism, which could not only support embryo development but also post-hatch growth. In conclusion, in ovo spray application of probiotics to the egg's surface can be a potential approach to support microbiota acquisition in hatchlings.

Keywords: Broiler embryo; Hatchling; In ovo probiotic application; Microbiota acquisition.

Fig. 1

Changes in the eggshell associated microbial communities between different groups through incubation. (A) Principal coordinate analysis (PCoA) of the bacterial communities of different groups at different time points. (B) Stacked bar charts showing the relative abundance of bacterial community (mean value) at the family level. Bacterial families with relative abundance < 1 % are grouped as others.

Fig. 2

Changes in chorioallantoic membrane (CAM) associated microbial communities between different groups through the incubation. (A) Principal coordinate analysis (PCoA) of the bacterial communities of different groups at different time points. (B) Stacked bar charts showing the relative abundance of bacterial community (mean value) at the family level. Bacteria families with relative abundance < 1 % are grouped as others. (C) Differentially enriched taxonomic profiles of CAM in descending order at (C1) day 14, (C2) 18, and (C3) 20 (p ≤ 0.05, LDA score>3.5, one against one strategy). (D) Differentially enriched functional profiles of CAM in descending order at (D1) day 14, (D2) 18, and (D3) 20 (p ≤ 0.05, LDA score>3.5, one against one strategy).

Fig. 3

Changes in microbial communities of hatchling gut (cecum and ileum) between different groups. (A) Principal coordinate analysis (PCoA) of the bacterial communities of different groups. (B) Stacked bar charts showing the relative abundance of bacterial community (mean value) at the family level. Bacteria families with relative abundance < 1 % are grouped as others. (C) Differentially enriched taxonomic profiles of (C1) cecum and (C2) ileum in descending order (p ≤ 0.05, LDA score>3.5, one against one strategy). (D) Differentially enriched functional profiles of (D1) cecum and (D2) ileum in descending order (p ≤ 0.05, LDA score >3.5, one against one strategy).

Fig. 4

Top biomarkers characterized in (A) chorioallantoic membrane (CAM), (B) cecum, and (C) ileum at the family level using random forest model (Top 10 biomarkers in CAM and ileum, and all characterized biomarkers in cecum). Biomarkers are ranked by descending order of importance.

Fig. 5

Relative source proportions contributing to the hatchling gut microbiota using source tracking. The height of the flow between bars demonstrates the average proportions of predicted sources contributing to the cecum and ileum microbiota in each group.