**Background:** The overuse of antibiotics in livestock production has led to bacterial resistance and environmental contamination, prompting a search for effective alternatives. Clostridium butyricum, a butyric acid-producing probiotic, has shown potential to improve growth, immunity, and intestinal health in poultry. This study aimed to evaluate C. butyricum as an antibiotic substitute in broiler chickens.
**Methods:** A total of 330 one-day-old Ross 308 broilers were randomly assigned to five treatment groups with six replicates each (11 birds per replicate). The groups were: CON (basal diet), AM (basal diet + 150 mg/kg aureomycin), CBL (basal diet + 2 × 10^8 CFU/kg C. butyricum), CBM (basal diet + 4 × 10^8 CFU/kg), and CBH (basal diet + 8 × 10^8 CFU/kg). The experiment lasted 39 days with a two-phase feeding program (starter days 0–21, finisher days 22–39). Growth performance was measured at days 21 and 39. Blood samples were collected for serum biochemical and immunoglobulin analysis. Jejunal segments were collected for histological examination of villus height and crypt depth. Immune organ weights (thymus, spleen, bursa of Fabricius) were recorded and expressed as a proportion of body weight.
**Key Results:** Growth performance: Final BW was significantly higher in the CBM group (1733.02 g) compared to CON (1620.17 g, p < 0.05), with linear (p < 0.01) and quadratic (p < 0.05) dose responses. ADG from days 22–39 was highest in CBM (65.78 g/day) versus CON (59.61 g/day, p < 0.05), with linear (p = 0.014) and quadratic (p = 0.044) effects. Overall ADG (days 1–39) showed linear improvement (p < 0.01). No significant differences were observed in ADFI or FCR across groups.
INTESTINAL MORPHOLOGY
On day 21, C. butyricum supplementation quadratically decreased jejunal crypt depth (p < 0.01) and increased V/C ratio (linear p = 0.01, quadratic p < 0.01). On day 39, villus height was linearly increased (p < 0.001), with CBH showing the highest value (1689.48 μm) versus CON (1070.10 μm, p < 0.001). V/C ratio was linearly (p < 0.01) and quadratically (p < 0.001) increased, with all C. butyricum groups higher than AM (p < 0.001).
SERUM BIOCHEMISTRY
On day 21, serum TG was lower in CBL (0.30 mmol/L) versus AM (0.39 mmol/L, p < 0.05). Serum UREA was lower in CBL (0.41 mmol/L) and CBH (0.43 mmol/L) versus AM (0.59 mmol/L, p < 0.05). On day 39, serum TC was higher in CBM (4.24 mmol/L) versus AM (2.95 mmol/L, p < 0.01). Serum ALB was higher in all C. butyricum groups versus AM (p < 0.01).
IMMUNITY
Thymus index was linearly increased by C. butyricum at both day 21 (p < 0.01) and day 39 (p < 0.01). On day 39, CBH had the highest thymus index (3.10 g/kg) versus CON (2.17 g/kg, p < 0.05). Bursa of Fabricius index showed a quadratic response at day 39 (p < 0.05). Serum IgG on day 21 was higher in CBL (7.27 g/L) and CBH (7.65 g/L) versus AM (5.91 g/L, p < 0.05). On day 39, IgA was lower in AM (0.86 g/L) versus CON (1.25 g/L, p < 0.05), and IgG was higher in CBM (8.34 g/L) versus AM (6.42 g/L, p < 0.05).
**Clinical Implications:** This study demonstrates that dietary supplementation with C. butyricum, particularly at 4 × 10^8 CFU/kg, can effectively replace antibiotics as a growth promoter in broiler production. The probiotic improved growth performance, enhanced intestinal morphology (longer villi, shallower crypts, higher V/C ratio), stimulated immune organ development, and maintained favorable serum biochemical profiles. Notably, the antibiotic group (aureomycin) showed no significant growth-promoting effects and had lower immunoglobulin levels compared to probiotic groups, supporting the potential of C. butyricum as a safe, non-residue-producing alternative. These findings have practical implications for the poultry industry seeking to reduce antibiotic use while maintaining productivity and bird health.