Abstract
Early microbial colonization is critical for poultry gastrointestinal development. This study investigated the effects of in ovo injection of Lactobacillus reuteri strain 21 (LR21) into the amniotic cavity at embryonic day 18 on intestinal colonization, development, and growth performance in broiler chickens. As indicated by FITC labeling, LR21 effectively colonized the embryonic intestine, accumulating in the proventriculus and gizzard within 24 h and migrating to the ileum and rectum by 48 h. Meanwhile, LR21 injection significantly increased daily body weight gain and daily feed intake from days 1 to 7 (P < 0.05), while had no effect on hatchability or birth weight. It significantly reduced abdominal yolk sac weight (P < 0.05) and tended to increase small intestine weight (0.05 < P < 0.1). Duodenal villus height and villus height to crypt depth ratio increased significantly in newborns (P < 0.05), with persistent effects at day 7. LR21 promoted duodenal crypt stem cell proliferation by upregulating Wnt/β-catenin pathway genes (Wnt3a, LRP5, c-Myc) and the stem cell marker Lgr5 (P < 0.05). By day 7, LR21 also suppressed Notch signaling components (Notch2, Dll1, Dll4), concomitant with increased goblet cell density and Muc2 expression (P < 0.05). Non-targeted metabolomics indicated enrichment of tryptophan metabolism, with elevated levels of indole-3-carboxaldehyde (I3A). In vitro, I3A treatment (100 μM) stimulated enteroid expansion and stem cell proliferation via Wnt/β-catenin activation, while also inhibiting Notch signaling and boosting Muc2 expression (P < 0.05). Taken together, our findings indicate that in ovo administration of LR21 enhances early-growth performance and intestinal development in broilers by enabling probiotic pioneer colonization, which raises I3A levels and thereby activates Wnt/β-catenin while suppressing Notch signal pathway.