The swine IsoLoop model of the gut host-microbiota interface enables intra-animal treatment comparisons to advance 3R principles.

Understanding gut-host microbiota interactions requires models that replicate human physiology while providing region-specific resolution, translational precision, and minimal animal use. To this end, we developed the IsoLoop model, a swine gut loop platform enabling intra-animal, multi-treatment comparisons. Microbiota-depleted ileal loops were surgically created in pigs, maintaining neurovascular integrity while isolating them from the anastomosed digestive tract. In Experiment 1, loops were inoculated with human fecal microbiota (HFM) or HFM combined with Peptacetobacter hiranonis. In Experiment 2, they were inoculated with Clostridioides difficile. Host-microbiota interactions were compared with respective controls in each experiment. The IsoLoop model reduced animal use by 75% compared to conventional whole-animal designs. Following antibiotic-induced depletion, loops re-established microbial diversity by day 5, despite reduced richness and loss of taxa, including Lactobacillus. HFM transplantation in microbiota-depleted loops induced robust transcriptomic recovery, enriched Akkermansia and Bifidobacterium, and restored specific metabolic pathways, although taxonomic and metabolic restoration remained incomplete and divergent. P. hiranonis promoted normal ileum-like metagenomic functional convergence, activated epithelial repair pathways, and increased specific secondary bile acids. C. difficile challenge recapitulated early infection pathology in IsoLoops. The IsoLoop model offers an ethical and precise platform for investigating host-microbiota crosstalk, localized enteric pathologies, and therapeutic interventions.