Probiotic spray on incubating chicken eggs accelerates the establishment of a healthy jejunal microbiota

Establishing beneficial microbiota in chickens earlier in life is advantageous for optimum production and health. This study was conducted to investigate the colonization of probiotics in the intestines of specific pathogen free chicken embryos using a coarse spray of probiotics on incubating eggs to accelerate the establishment of a healthy jejunal microbiota in neonatal chickens. Enterococcus faecalis were sprayed on incubating eggs on 12, 15 and/or 17 days of embryonation (DOE) to explore the optimum day(s) needed for colonization. Colonization was confirmed by direct culture and whole genome sequencing of intestinal contents of embryos. Colonization was best observed when probiotics were sprayed on two separate days - 15 and 17 DOE. Further, effects of coarse spray of E. faecalis, Ligilactobacillus salivarius, Lactobacillus plantarum, Bifidobacterium pullorum subsp. gallinarum, and Pediococcus acidilactici on incubating eggs individually or as a mixture in jejunal microbiota were assessed at 2, 10, 20, and 30 days post hatch (DPH) using 16S rRNA amplicon sequencing. Coarse spray application of L. salivarius, L. plantarum and the mixture of probiotics lead to significantly faster convergence trajectories towards a mature reference profile [represented by 30 DPH with no probiotics delivery (negative control)], compared to groups that received probiotics by the oral route. Beta diversity analysis showed the largest separation between groups at 2 DPH. Over 10, 20, and 30 DPH, all communities gradually converged, with the group that received the sprayed probiotic mixture retaining a unique profile for the longest period. The group that received the sprayed probiotic mixture consistently outperformed the negative control group and the group given oral probiotics on 2 DPH at both species and genus levels in terms of richness. This confirms the beneficial effect of a probiotic mixture spray compared to natural colonization or short-term oral application. In conclusion, spray application of probiotics as a coarse spray is an industry feasible technique and noninvasive method for introducing probiotics to chicken embryos leading to a rapid establishment of healthy jejunal microbial composition.