Dietary supplementation with a multi-strain Bacillus subtilis probiotic improves digestive efficiency, growth performance, and meat quality in densely stocked broilers

• Multi-strain B. subtilis improved growth efficiency under high stocking density. • Optimal performance achieved at 0.5% probiotic inclusion level. • Probiotic enhanced OM, NDF, and GE digestibility in broilers. • Moderate doses improved haemato-immune status and reduced stress. • Excessive dosage caused gut enlargement and impaired growth. • Meat quality improved at intermediate probiotic inclusion levels. Bacillus subtilis has attracted attention as a robust probiotic candidate to replace antibiotic growth promoters ( AGPs ) in poultry, owing to its spore-forming resilience and capacity to modulate gut function. However, inclusion levels and the efficacy of multi-strain B. subtilis mixtures remain underexplored, particularly at high stocking densities, when birds are more susceptible to physiological and environmental stressors. This study evaluated the effects of incremental levels of a multi-strain B. subtilis probiotic mixture on growth performance, nutrient digestibility, physiological responses, carcass traits, and meat quality of broiler chickens reared under intensive conditions. A total of 900 one-day-old Ross 308 broiler chicks were assigned to six dietary treatments in a completely randomized design, with 36 pens (13 birds/0.75 m²) serving as experimental units. Diets comprised a basal diet without additives ( PRB0 ), a positive control containing zinc bacitracin ( POSC ), and four diets supplemented with 0.3, 0.4, 0.5, or 0.6% of the B. subtilis probiotic mixture ( PRB3 – PRB6 ). Diets were fed from the starter to the finisher phases. Probiotic supplementation influenced growth responses across the graded inclusion levels. Birds receiving the 0.5% inclusion level consistently exhibited the most favourable body weight gain and feed conversion efficiency compared with the other treatments. Probiotic supplementation enhanced the digestibility of organic matter, neutral detergent fibre, and gross energy, whereas crude protein digestibility exhibited a quadratic response. Haemato-biochemical profiles suggested improved physiological status in birds receiving mid-level probiotic inclusions (PRB3 – PRB5). Carcass yield and major meat quality traits were unaffected by dietary treatments, while minor effects on gut morphological indices were observed. Collectively, these findings suggest that a 0.5% inclusion level may be a promising probiotic supplementation strategy for broiler production under high stocking density.