Targeted quantitative polymerase chain reaction-based bacterial and fecal metabolite markers show early stability in healthy puppies after 8 weeks of age.

Fecal microbiome and metabolite markers are largely stable from 8 weeks onward, enabling early diagnostic interpretation in puppies.

This prospective longitudinal cohort study evaluated the stability of targeted quantitative PCR (qPCR)-based fecal microbial and metabolomic markers in healthy puppies from 8 weeks to 1 year of age. Twenty-three client-owned puppies with no gastrointestinal signs, no prior systemic antibiotic exposure, and negative parasitology contributed 62 fecal samples collected at four time points: 8–12 weeks, 16 weeks, 6 months, and 1 year. Primary outcomes included the qPCR-based dysbiosis index (DI), selected bacterial taxa abundances, fecal bile acids, long-chain fatty acids (LCFAs), and sterols quantified via mass spectrometry. Mixed-effects models were used to assess longitudinal associations. The DI and most targeted bacterial taxa remained within established reference intervals throughout the study with no significant longitudinal changes. A notable exception was Bacteroides spp., which showed lower abundance at 8–12 weeks compared to 6 months. Secondary unconjugated bile acids predominated at all ages, consistent with an early functionally mature microbiome. The concentration of 12-oxo-lithocholic acid was lower at 16 weeks versus 1 year, though this did not survive multiple-testing correction. LCFA and sterol profiles showed no age-related changes. These findings indicate that qPCR-based microbial and metabolomic markers achieve early functional stability by 8 weeks of age in healthy puppies. The study provides age-specific baseline reference data for fecal bacterial and metabolite markers, which is clinically valuable for interpreting diagnostic results in young dogs presenting with gastrointestinal disease.

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