Investigation of the Effect of TiO₂ as a Dietary Marker on Broiler Intestinal Fermentation: Combination of Ex Vivo Simulation and In Vivo Approach

The impact of dietary inert digestibility markers on gut microbiota and intestinal fermentation remains poorly understood. This study investigated the effects of titanium dioxide (TiO₂) supplementation at 4 kg/ton feed, representing a typical dose used in animal nutrition studies, on fermentation dynamics and microbial composition in broiler chickens using combined ex vivo and in vivo approaches. Ex vivo fermentations were conducted using ileal and caecal microbiota and substrates collected from 32-day-old broiler chickens, with direct TiO₂ supplementation, with gas production and volatile fatty acid (VFA) profiles as main measurements. In parallel, 392 broiler chickens were fed diets with or without TiO₂ for 32 days, and ileal and caecal digesta were analysed for fermentation end-products and microbial composition using shotgun metagenomic sequencing. A second ex vivo experiment was performed using microbiota adapted to dietary TiO₂. In the first ex vivo model, TiO₂ reduced gas production and acetic acid concentration in the ileum (p < 0.05), whereas in the caecum it increased gas production, total eubacterial counts, and branched-chain fatty acids (BCFA) (p < 0.05). In vivo, TiO₂ did not affect growth performance or organ development but significantly increased isobutyric acid and total BCFA concentrations in the caecum (p < 0.05). Metagenomic analysis revealed increased caecal alpha diversity (Shannon index) and enrichment of taxa associated with amino acid metabolism, including Massilicoli timonensis, Blautia merdavium, Rubneribacter badeniensis, and Mediterraneibacter caccavium. The second ex vivo experiment showed similar trends, with increased gas and BCFA production. Collectively, these findings indicate that TiO₂ can modulate intestinal fermentation and microbial composition in a segment-specific manner, suggesting that dietary markers may not be biologically inert.