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A total of 72 laying hens were used to investigate the effect of probiotic and lactose on body weight loss, tibia ash, antibody production against sheep red blood cell (SRBC), heterophile-to-lymphocyte (H/L) ratio and gut microbiota in a common moulting method for 14 d. Hens were randomly allocated to 6 experimental groups consisting of (i) full feed (FF), (ii) feed withdrawal (FW), (iii) FW with calcium (Ca), (iv) FW with Ca and offering 7 g/lit lactose in drinking water (CaL), (v) FW with Ca and offering 1 g/lit probiotic in drinking water (CaP), and (vi) FW with Ca and offering a mixture of lactose and probiotic in drinking water (CaLP). The results showed body weight loss in all FW groups were more than 25% that was significantly higher than FF group (p < 0.05). The relative organ weights of hens in FW groups were lower than FF group; especially, it was significant for liver and ovary (p < 0.05). No significant difference was observed between all groups for tibia ash. The highest H/L ratio was related to FW group and offering Ca and lactose numerically and probiotic alone significantly resulted in decrease of this ratio (p < 0.05). The results also showed no significant difference for antibody production against SRBC among experimental groups. The highest coliform bacteria numeration observed in FW group and lactose could significantly reduce this population (p < 0.05). Lactic acid bacteria (LAB) numeration demonstrated a significant difference among treatments, so that FF hens had higher LAB than others (p < 0.05). In addition, FW moulted hens had significantly lower LAB compared to other moulted hens (p < 0.05), except for Ca group. In conclusion, probiotic and lactose was effective in maintaining caecal microbiota balance and improving immunity in hens exposed to moulting.

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