Grass supplementation to a pellet-based diet fails to enrich gut microbiomes with wild-like functions in captive-bred hares

Reintroducing captive-bred animals into the wild often faces limited success, with the underlying causes frequently unclear. One emerging hypothesis is that maladapted gut microbiota may play a significant role in these challenges. To investigate this possibility, we employed genome-resolved metagenomics to analyze the taxonomic and functional differences in the gut microbiota of 45 wild and captive European hares (<i>Lepus europaeus</i>), as well as to assess the impact of fresh grass supplementation to a pellet-based diet aimed at pre-adapting captive hares to wild conditions. Our analyses recovered 860 metagenome-assembled genomes, with 87% of them representing novel species. We found significant taxonomic and functional differences between the gut microbiota of wild and captive hares, notably the absence of Spirochaetota in captive animals and differences in amino acid and sugar degradation capacities. While grass supplementation induced some minor changes in the gut microbiota, it did not lead to statistically significant shifts toward a more wild-like microbial community. The increased capacity for degrading amino acids and specific sugars observed in wild hares suggests that, instead of bulk grass, dietary interventions tailored to their specific dietary preferences might be necessary for pre-adapting hare gut microbiota to wild conditions.IMPORTANCEThis study sheds light on the role of gut microbiota in the success of reintroducing captive-bred animals into the wild. By comparing the collection of 860 near-complete genomes of wild and captive European hares, we identified significant taxonomic and functional differences, including the absence of key microbial groups in captive hares. Grass supplementation to a pellet-based diet yielded limited success in restoring a microbiota similar to that of wild counterparts, highlighting the need for more tailored approaches to mimic natural diets. With 87% of recovered microbial genomes representing novel species, this research also enriches our understanding of microbial diversity in wildlife. These findings emphasize that maladapted gut microbiota may hinder the survival and adaptation of reintroduced animals, suggesting that microbiome-targeted strategies could improve conservation efforts and the success of animal rewilding programs.