Disentangling host identity and storage time effects on gut microbiota composition in captive migratory birds using absolute and relative quantification.

Understanding how gut microbiota support migratory birds is essential; yet, fecal sample freshness is often a challenge, particularly for rare species that cannot be captured directly. Here, we collected fecal samples from multiple captive migratory bird species at Nanchang Zoo and grouped them by post-defecation time (0, 1, 2, and 4 h). Using both relative and absolute quantification, we assessed the effects of host identity, short-term storage, and their interaction on gut microbiota composition. Species identity and quantification method significantly shaped microbiota profiles. Absolute quantification revealed Firmicutes (763,405.73 copies/μL) and Proteobacteria (340,231.03 copies/μL) as dominant in Grey-crowned Cranes, whereas relative quantification indicated Firmicutes (96.74%) predominated in Swan Geese and Proteobacteria (30.30%) in Black-necked Cranes. Red-crowned Cranes showed higher species richness than Black Swans and Swan Geese, with a significantly greater Shannon index than the latter. PCoA demonstrated clear interspecific differences, especially between crane and waterfowl lineages. Storage time had no significant effects on alpha and beta diversity across 6 species, except for reduced richness in Swan Geese at 2 and 4 h. While overall community structure was stable, a few conditionally rare taxa displayed time-sensitive shifts shortly after defecation. Our findings highlight that both host identity and quantification approach are critical determinants of avian gut microbiota profiles and emphasize that fecal sample freshness mainly affects rare taxa. This study provides methodological insights for optimizing fecal sampling protocols in field-based microbiome research on migratory birds.