Effects of Vitamin D3 and 25(OH)D3 Supplementation on Growth Performance, Bone Parameters and Gut Microbiota of Broiler Chickens

Broiler chickens are commonly reared in closed housing systems with limited exposure to sunlight, thereby relying entirely on dietary sources of vitamin D. The hydroxylated metabolite 25-hydroxycholecalciferol [25(OH)D₃] has been proposed as a more potent form than native vitamin D₃ (cholecalciferol). This study evaluated the effects of dietary supplementation with vitamin D₃ alone or in combination with 25(OH)D₃ on growth performance, bone characteristics, and cecal microbiota in Ross 308 broilers. A total of 952 one-day-old male chicks were allocated to four treatments: a negative control (no vitamin D₃), a positive control (vitamin D₃ according to Ross 308 specifications), and a positive control supplemented with 25(OH)D₃ at 1394 or 2788 IU/kg, in a randomized design with 17 replicates per treatment and 14 birds per replicate. Over a 40-day feeding trial, diets containing vitamin D₃ (positive control) or supplemented with 25(OH)D₃ significantly improved final body weight, weight gain, average daily gain, and feed conversion ratio compared with the negative control (<i>p</i> < 0.01), with no significant differences among the positive control and 25(OH)D₃-supplemented groups, with a clear linear dose-dependent response. Although tibia ash and bone-breaking strength were not significantly affected, linear responses indicated a slight numerical trend toward improved skeletal mineralization with increasing 25(OH)D₃. Microbiota analysis indicated that 25(OH)D₃ affected cecal microbial ecology: low-dose inclusion showed reduced species richness and evenness, whereas high-dose inclusion restored richness to levels comparable to the positive control and enriched taxa associated with fiber fermentation and bile acid metabolism while reducing <i>Lactobacillus</i> dominance. In conclusion, supplementation with 25(OH)D₃ in addition to vitamin D₃ enhanced growth performance and selectively shaped the cecal microbiota of broilers, with suggestive benefits for bone mineralization. These findings highlight 25(OH)D₃ as a more potent source of vitamin D than cholecalciferol alone and support its practical use in modern broiler nutrition to improve efficiency, skeletal health, and microbial balance.