Longitudinal long-read microbiome profiling in a canine model reveals how age, diet, and birth mode shape gut community dynamics.

The gut microbiome undergoes dynamic age-related changes shaped by diet and maternal factors. Here, we present a species-level, long-read 16S rRNA survey of the developing gut microbiome in a translational canine model, profiling 89 purebred Hungarian Pumis across early-life and reproductive stages. We collected 456 fecal samples longitudinally: 60 puppies followed from birth to 81 weeks, their mothers sampled during pregnancy and lactation, and adult controls from six kennels. We recorded detailed dietary metadata and reproductive status throughout the study. Age was the strongest determinant of alpha diversity, with a rapid increase during weaning and stabilization by 6 months of age. Beta diversity analyses revealed structured compositional transitions from early developmental phases to adulthood, including a shift toward more uniform, adult-like communities. Within-kennel variation was modest, consistent with shared environmental exposures. Mixed-effects models showed robust associations between specific taxa and age, diet, and kennel, while SparCC-inferred co-occurrence networks indicated increasing ecological complexity with age. We also demonstrated that the delivery mode-vaginal versus cesarean-impacted early-life microbiome composition: