To study the efficiency of Bacillus spp. probiotics administered via different routes and doses, a 6-week grow-out trial was conducted using a total of 378 day-old mixed-sex ROSS308 broiler chickens in a completely randomized block design. Six experimental diets included probiotics added at two different inclusion rates into the feed (250 g/ton; PRO250, or 500 g/ton; PRO500), or in the drinking water (25 g/L; PRO-WS), or as a feed synbiotic (250 g probiotic + 250 g/ton prebiotic; SYN), compared to a negative (NC; without additives) and positive control (PC; with antibiotics) diets. The PRO-WS enhanced feed intake (p < 0.05) and tended to improve average daily gain and final body weight (p = 0.14). Broiler gut morphology in the duodenum including the villus height (p = 0.04), villus width (p = 0.05) and crypt depth (p = 0.02) were improved by PRO500. Firmicutes was the most abundant phylum, followed by Bacteroidetes. Streptococcaceae, Lachnoospiraceae, Peptostreptococcaceae, Ruminococcaceae, and Erysipe-lotrichaceae were the top five most abundant families. Antibiotic inclusion in PC reduced microbial beta-diversity and increased similarity compared to probiotic inclusion (p = 0.05). Probiotic inclusion reduced the relative abundance of Bacteroides fragilis, which is a commonly isolated pathogen and is considered as a marker for antimicrobial resistance. Overall, probiotic supplementation via feed or water may potentially improve the production performance of the broiler chickens, and water-soluble probiotics are potentially more effective. Probiotics, especially when added to water, suggest a promising feed additive to support gut microbial maturation and diversity, and may reduce resistant bacteria in broiler chickens. However, it is suggested that the best route for the administration of probiotics be further examined under commercial conditions to find the most effective and practical application method that yields the most consistent results.
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