Maternal separation (MS) is a widely used model of early-life stress, yet the biological pathways transmitting maternal stress signals to offspring remain incompletely understood. Breast milk represents a primary biological interface between mother and infant, and stress-induced changes in milk composition may influence offspring development through the gut microbiota-brain axis. Here, we investigated whether MS alters the breast milk metabolome and whether such changes are associated with offspring gut microbiota composition, serum metabolite profiles, and serotonin-related gene expression in the prefrontal cortex. Using an integrated multi-omics approach in mice, we found that MS dams had elevated myristic acid and ethanolamine levels in breast milk. MS offspring exhibited reduced A. muciniphila abundance in the cecal microbiota, elevated myristic acid and decreased inosine in cecal contents, decreased inosine in serum, and reduced Tph2 and Htr1b expression in the prefrontal cortex. Oral myristic acid supplementation in neonatal pups reduced A. muciniphila abundance, and inosine supplementation selectively increased Htr1a expression. Spearman correlation analyses using paired individual data revealed a negative association between cecal myristic acid and cecal inosine levels, and a positive correlation between cecal inosine and prefrontal Tph2 expression, supporting quantitative links among these components. Collectively, these findings identify coordinated associations among maternal stress-induced milk fatty acid changes, offspring gut microbiota, microbial metabolites, and serotonin-related gene expression, providing an exploratory framework for understanding milk-mediated effects on the developing gut-brain axis.
