The present study aimed to investigate the effect of age, breed, and sex of broilers, as well as a probiotic or phytobiotic product on mucosal morphology, bacterial metabolites, and immune traits in the ileum of broilers. A total of 2,880 one-day-old male and female broiler chicks from two breeds (Ross308® and Cobb500®) were randomly assigned to 72 pens. Broilers were offered a wheat-soybean diet without (CO), or with either a probiotic (PO; 2.4 × 109 CFU/kg of Bacillus subtilis DSM32324 and DSM32325 and B. amyloliquefaciens DSM25840) or a phytobiotic (PY; grape extract, 165 ppm procyanidin and 585 ppm polyphenols of the diet) product. The trial was conducted with a 3 × 2 × 2 factorial arrangement of diet, breed, and sex in a completely randomized design (6 replicate-pens per treatment). At day 7, 21, and 35, one chicken per pen was slaughtered for collecting ileal tissue to evaluate of histomorphology and mRNA expression, as well as ileal digesta to measure bacterial metabolites. Data were subjected to ANOVA (the main factors; age, diet, breed, and sex) and Four-Way ANOVA (interactions) using GLM procedure. Overall, the concentration of acetate and total short chain fatty acids reached the peak and lactate decreased to its lowest on day 21, but their concentrations at day 7 and 35 were similar (p > 0.05). Spermine, spermidine, and ammonia decreased after day 7, while putrescine and cadaverine increased after day 21 (p < 0.05). mRNA expression of cytokines, mucin 2 (MUC2) and claudin 5 (CLDN5) was similar; increased from day 7 to 21 and decreased afterward (p < 0.05). Villus height, crypt depth and villus surface area increased with age (p < 0.05). Acidic goblet cells (GC) number and density increased after day 21 (p < 0.05). Ross broilers showed higher D-lactate concentration and IFN-γ expression, while Cobb broilers had greater IL-4, IL-6 and TNF-α expression and higher total GC number (p < 0.05). Female displayed higher villus height and GC number and density (mixed and total GC) than male (p < 0.05). The effect of dietary treatment was not found on any investigated variables (p > 0.05). In conclusion, aging of broilers affected ileal histomorphology, cytokine expression, and barrier integrity, as well as bacterial activity. These observed impacts could be attributed to host-microbiota interaction and the direct effects of bacterial metabolites on intestinal cells and immune system.
