Longitudinal assembly of the hindgut microbiota reveals fermentative maturation during early life in dairy calves

Introduction The pre-weaning period is a critical developmental phase during which the calf gut microbiota undergoes ecological succession, shaping its taxonomic composition and functional potential. Because the rumen is not fully developed at birth, the hindgut initially represents the primary site of microbial fermentation. Understanding microbial colonization and fermentative maturation during the transition from milk to solid feed is essential to elucidate host–microbiota interactions and their implications for calf health and performance. Methods Holstein dairy calves ( n = 27) were monitored from birth to 70 days, across weaning. Fecal microbiota composition, a proxy for the hindgut microbial ecosystem, was characterized by 16S rRNA gene sequencing. Fecal volatile fatty acid (VFA) profiling was used to investigate fermentative activity. Random Forest models were applied to identify microbial configurations associated with performance-related traits. Results Longitudinal changes in fecal microbiota followed a multistage process of ecological succession comprising three broad developmental stages: neonatal, intermediate, and post-weaning. Fecal VFA profiles showed marked temporal changes from the first days of life. Spearman correlation analyses revealed timepoint-specific associations between bacterial genera and fecal VFA molar proportions. Random Forest models identified microbial configurations significantly associated with starter intake. Higher intake was characterized by earlier enrichment of taxa associated with fermentative metabolism. Later microbial shifts were consistent with more advanced rumen maturation in high-intake calves. Discussion Multistage succession, changes in fecal VFA profiles, and the evolving pattern of microbiota–VFA associations alongside dietary transition were consistent with progressive maturation of the hindgut fermentative community. Random Forest findings suggest that starter intake trajectories are associated with distinct patterns of hindgut ecosystem maturation. Overall, the results support the hypothesis that fecal microbial development, progressive fermentative maturation, and nutritional adaptation are coordinated processes throughout the study period, suggesting that fecal microbiota reflects the progressive fermentative and physiological maturation of the calf.