Modulation of gut microbiota in rainbow trout by dietary protein-to-energy ratio and calcium carbonate through chyme and nutrient digestibility changes

• The link between diet, digestion and gut microbiota in rainbow trout was investigated. • Microbiota differ among gut regions linked to chyme composition and digestion. • High dietary protein-to-energy ratio reduces inter-individual microbial variability. • Diet alters the gut microbiota associated with changes in magnesium digestibility. • Results support feed strategies to improve nutrient use in aquaculture species. Modulating feed buffering capacity via diet formulation is of increasing interest in livestock due to its ability to regulate gastrointestinal conditions and nutrient utilization. This study hypothesizes that dietary macronutrient composition and calcium carbonate ( CaCO 3 ) modulate fish gut microbiota, potentially improving the effects of feed buffering capacity. Thus, it explores the relationship between microbiota modulation and chyme changes induced by different protein-to-energy ( P:E ) ratios (16.1 mg/kJ vs . 26.4 mg/kJ) and CaCO 3 supplementation in diets of rainbow trout ( Oncorhynchus mykiss , 284 ± 2.5 g). This multifactorial experiment also evaluated the effect of gut region (proximal vs . distal) and of postprandial time (3 h vs . 7 h). After a 6-week feeding trial, none of the tested factors showed differences in estimated bacterial richness (Chao1 index, P > 0.05), while the proximal intestine exhibited higher bacterial diversity (Simpson index) than the distal gut ( P = 0.007). In addition, inter-individual microbial variability was significantly different between intestinal regions, qualitatively (Jaccard distance; F = 7.98, R 2 = 0.05, P = 0.001) and quantitatively (Bray-Curtis distance; F = 96.19, R 2 = 0.40, P = 0.001), which was correlated with differences in chyme pH, relative water and mineral fluxes, and crude protein apparent digestibility ( P < 0.05). Jaccard distances also revealed different inter-individual microbial variability in fish fed the high P:E diet without CaCO 3 addition ( HP:E-LC ) with respect to fish fed rest of the diets ( F = 2.08, R 2 = 0.01, P = 0.007), associated with magnesium digestibility ( Pseudo-F = 1.47, P = 0.009). Furthermore, fish fed the high P:E diets showed a reduction in the relative abundance of the phylum Bacteroidota in both intestinal regions ( P < 0.05), and of the genus Phaeodactylibacter in the proximal intestine ( P = 0.002), which may reflect differences in the content of carbohydrates and lipids used as substrates by these taxa. Meanwhile, CaCO 3 supplementation reduced the abundance of the phylum Desulfobacterota in the proximal intestine ( P = 0.04), potentially due to its sensitivity to high pH values. Moreover, the genus Geobacillus was only present in the proximal intestine of fish fed the HP:E-LC diet and was positively correlated with magnesium digestibility ( r s = 0.61, P = 0.01), supporting improved nutrient utilization. In conclusion, both P:E ratio and CaCO 3 supplementation modulate fish gut microbiota in correlation with changes in digestion kinetics induced by the diet macronutrient composition and feed buffering capacity respectively, while maintaining the typical commensal microbiota of rainbow trout.