Non-pasteurized milk enhances early colonization and persistence of <i>Lactobacillus</i> species in dairy calves promoting a stable hindgut microbiome

Pasteurization is a standard method to minimize pathogen transmission from milk to calves, and its impact on calf morbidity and growth performance has been well-documented. However, its long-term effects on the development of the gut microbiota and the underlying mechanisms remain poorly understood. In this study, a longitudinal analysis of 15 newborn calves was conducted over their first month, employing high-throughput sequencing and microbial cultivation to explore the impact of continuous feeding with non-pasteurized vs pasteurized milk on the calf growth performance and hindgut microbiome. Fecal and milk samples were collected, and microbial communities were analyzed using 16S rRNA gene sequencing. Additionally, microbial isolates were cultivated and phylogenetically characterized. Non-pasteurized milk has no significant impact on calf body weight, but it preserves microbial resources and facilitates the transfer of beneficial microbes such as <italic>Limosilactobacillus reuteri</italic> (<italic>L. reuteri</italic>) from milk to the hindgut. Extensive cultivation of <italic>Lactobacillus</italic> from milk and feces identified 40 distinct clusters out of 218 isolates using Rep-PCR. Whole-genome sequencing of 40 <italic>L. reuteri</italic> strains confirmed the presence of shared isolates in milk and the hindgut of calves. These <italic>L. reuteri</italic> isolates exhibited a remarkable ability to metabolize diverse oligosaccharides, including FOS, GOS, and inulin, contributing to early carbohydrate metabolism in dairy calves. By demonstrating how non-pasteurized milk fosters early microbial colonization and promotes a functionally stable gut microbiota, this study provides critical insights into the microbial and functional dynamics of the calf GIT. These findings highlight the potential of raw milk's microbial resources for optimizing calf development.