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

Objective: To assess longitudinal stability of targeted quantitative PCR (qPCR)-based fecal microbial and metabolomic markers in healthy puppies from 8 weeks to 1 year of age. Methods: This prospective longitudinal cohort study was conducted at the Clinic for Small Animal Internal Medicine, Vetsuisse Faculty, University of Zurich, Switzerland, from August 2023 to January 2025. Clinically healthy client-owned puppies were sampled at 8 to 12 weeks, 16 weeks, 6 months, and 1 year. Eligible dogs had no gastrointestinal signs, no previous systemic antibiotics, and negative parasitology. Primary outcomes were the qPCR-based dysbiosis index, selected qPCR-targeted bacterial taxa, fecal bile acids, long-chain fatty acids, and sterols, quantified by mass spectrometry. Mixed-effects models assessed time point associations. Results: 23 dogs contributed 62 fecal samples. Dysbiosis index values and most targeted bacterial taxa remained within reference intervals, with no significant longitudinal changes. Bacteroides spp abundance was lower at 8 to12 weeks than at 6 months (difference, -0.73 logDNA copies/g feces; 95% CI, -1.40 to -0.06; Tukey-adjusted P = .028). Secondary unconjugated bile acids dominated at all ages. The concentration of 12-oxo-lithocholic acid was lower at 16 weeks than at 1 year (ratio, 0.33; 95% CI, 0.14 to 0.79; Tukey-adjusted P = .007) but not after multiple-testing adjustment. Long-chain fatty acid and sterol profiles showed no age-related changes. Conclusions: The qPCR-based dysbiosis index and targeted microbial and metabolomic markers were largely stable from 8 weeks to 1 year, supporting early functional maturation of measured markers. Clinical Relevance: These findings establish age-specific baseline information for fecal bacterial and metabolite markers, supporting interpretation in young dogs with gastrointestinal disease.