Abstract 1. This study was carried out to investigate the effects of 1, 25-dihydroxycholecalciferol (1, 25 (OH)2 D3) and a hydroalcoholic extract of Withania somnifera (WS) root on performance, mineral retention, bone mineralisation, bone mechanical and bone histological characteristics of broiler chicks. 2. A 2 × 3 × 2 factorial experiment consisted of a positive control diet with adequate Ca and a negative control diet (Ca concentration reduced by 30%), three concentrations of WS (0, 75 and 150 mg/kg diet), and two concentrations of 1, 25 (OH)2 D3 (0 and 0.5 μg/kg diet). 3. A total of 600 male one-d-old Ross 308 broiler chicks were randomly distributed into 60 floor pens, with 10 birds each. Each treatment was replicated 5 times (50 birds). Diets were given ad libitum from one to 42d of age. On d 21 and 42, one bird per replicate was killed and tibiae were removed. 4. Dietary treatments did not affect feed intake and feed conversion. The maximum body weight gain (2475 g) was noted in birds fed on a diet adequate in Ca and supplemented with 75 mg/kg WS. 5. The Ca and P retentions were significantly higher in birds that were given a diet with 30% less Ca. Supplementation of 150 mg/kg WS significantly improved Ca retention in birds receiving a negative control compared to those given a positive control diet (83.0% vs. 66.3%). Ca retention was significantly improved with the addition of 0.5 μg/kg 1, 25 (OH)2 D3 to the diet containing 75 mg/kg WS, regardless of dietary Ca concentration (79.5 vs. 73.3 and 77.9 vs. 68.9). 6. On d 21, birds that received WS had significantly higher tibia Ca compared to those of controls. No significant effects on tibia Ca were noted at 42 d. Birds given a negative control diet supplemented with 75 mg/kg WS and 0.5 μg/kg 1, 25 (OH)2 D3 displayed a similar tibia Ca compared to those given only 150 mg/kg WS. Dietary supplementation of 1, 25 (OH)2 D3 significantly increased tibia Ca and tibial mineralised zone width in birds at 42 d of age. 7. Tibia shear force and stiffness were significantly increased by supplementation of WS. Administration of 1, 25 (OH)2 D3 resulted in a significantly lower width of the proliferative zone at 21 d of age. Reduction of the Ca supply significantly reduced the mineralised zone and cortical thickness at this age. 8. The present study showed that WS supplementation improved Ca retention, bone calcification and mechanical properties with no adverse effects on performance. Also, synergistic effects of WS and 1, 25 (OH)2 D3 were observed on Ca retention and bone calcification.

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