Interactive effects of lysine restriction, vitamin fortification, and insoluble fiber on intestinal morphometry in broilers fed reduced-crude-protein diets

This 2 × 2 × 2 + 1 factorial trial examined digestible lysine:CP (6.2 vs. 5.7%), vitamins (standard vs. enhanced), and fiber (0 vs. 0.5% Arbocel®) effects on jejunal morphometry in 756 male Ross 308 broilers (d10-40) fed 12% reduced-CP diets vs. control. Protein and lysine reduction had limited effects on overall intestinal dimensions, though jejunal length declined (<i>P</i> = 0.041). Insoluble fiber significantly enlarged cecal surface area (<i>P</i> = 0.023, +15.2%). Vitamin × fiber and lysine × fiber interactions significantly influenced intestinal segment weights and lengths (<i>P</i> < 0.05). CP reduction markedly decreased epithelial length (-12.3%), villus height (-10.5%), villus surface area (VSA) (-18.7%), and wall thickness (<i>P</i> ≤ 0.001), while lysine restriction alone had minimal impact except on crypt number (<i>P</i> = 0.002). Vitamin fortification enhanced epithelial length (+8.4%), villus height (+9.1%), and enterocyte count (+11.2%), in HV2F0 birds. Synergistic fiber × vitamin and fiber × lysine interactions significantly modulated epithelial structure (<i>P</i> < 0.05). AI-based ImageJ analysis outperformed Cell^ A, detecting 5.2% shorter villi and 10.9% deeper crypts, confirming its superiority in microstructural assessment. CP and lysine reductions altered goblet cell number and crypt dimensions, whereas fiber increased goblet cell area and ratios (+14.6%). Vitamin effects on goblet metrics were modest but significant (<i>P</i> = 0.032). In conclusion, while CP and lysine reduction perturbed jejunal microstructure, synergistic supplementation with vitamins and insoluble fiber effectively supported mucosal integrity, highlighting their compensatory role in maintaining gut health under protein-reduced conditions.