In ovo galactooligosaccharides and sodium butyrate induce segment-specific changes in gut microbiota of broiler chickens

Early-life in ovo interventions are increasingly explored as tools to modulate poultry gut ecosystems and intestinal function. However, high-throughput evidence describing ileal and cecal microbiota responses following embryonic exposure remains limited. This study evaluated whether a single in ovo administration of a prebiotic (galactooligosaccharides) or a short-chain fatty acid donor (sodium butyrate) resulted in differences in gut microbial communities of broiler chickens at day 42 of age. On embryonic day 12, 1,068 fertile Ross 308 eggs were assigned to four treatments: non-injected control, saline-injected control, galactooligosaccharides (3.5 mg/0.2 mL), or sodium butyrate (0.3%/0.2 mL). Birds were reared to day 42, and ileal and cecal digesta were collected from 10 birds per group. Full-length 16S ribosomal RNA gene sequencing was used to characterize microbial composition and diversity. Galactooligosaccharides increased richness and diversity while maintaining dominance of Lactobacillus and promoting Romboutsia and unclassified Peptostreptococcaceae. Sodium butyrate induced a stronger restructuring of the microbiota, accompanied by reduced Lactobacillus abundance and enrichment of multiple Clostridia-associated genera together with representatives of Bacteroidota; community structure differed among treatments. In the cecum, galactooligosaccharides increased diversity and promoted expansion of taxa affiliated with Lachnospiraceae and Ruminococcaceae, including Faecalibacterium, Blautia, Barnesiella , and Sporobacter . In contrast, sodium butyrate reduced microbial diversity while enriching several Clostridia-associated taxa. Discriminant analysis identified distinct marker taxa in the ileum and cecum. In the ileum, sodium butyrate showed a broader range of discriminant taxa, whereas in the cecum most discriminant markers belonged to the class Clostridia. These findings demonstrate that galactooligosaccharides and sodium butyrate differentially modulate microbial communities in the ileum and cecum, highlighting the distinct effects of prebiotic and postbiotic in ovo interventions on gut microbiota composition.