Alleviating Effect Of Bacillus Licheniformis On The Heat Stress That Laying Hens Suffer And The Underlying Mechanisms

This thesis included four parts. Firstly, biological and probiotic properties of the selected Bacillus licheniformis were evaluated in vitro. And then, production experiment was carried out to assay the effect of different doses of Bacillus licheniformis on the egg production, egg quality, and serum indices of heat-stressed laying hens. So, the optimal dosage was determined. At last, the influence of the Bacillus licheniformis on intestinal barrier function and microflora of heat-stressed hens was evaluated to explore the underlying mechanisms for the alleviating effects on the heat stress suffered by hens.Exp. 1 Biological and probiotic properties of the Bacillus licheniformisThe aim of this study was to investigate the growth characteristics, tolerance to acid and bile salt, and adhesive capability of the Bacillus licheniformis. The results showed that growth of the Bacillus licheniformis came into logarithmic phase from 12 h and it lasted to about 22 h. pH of the fermentation broth declined at earlier stage, but increased later. The dissolved oxygen concentration decreased gradually. After being subjected to pH 2 for 3 h, loss of activity was 24%. For pH 3 and 4 for 3 h, the loss was less than 10%. 0.3% bile salt decreased survival of the Bacillus licheniformis to 89.1% after working for 3 h. Fermentation products of the Bacillus licheniformis could significantly inhibit the growth of Escherichia coli, Salmonella pullorum, and Staphylococcus aureus on agar plate and their adhesion to mucin in vitro.Exp. 2 Effects of the Bacillus licheniformis on egg production, egg quality, and serum hormone levels of heat-stressed laying hensHeat stress depressed daily egg production (2～9 d), average egg production and feed intake of hens, lowered egg shell strength, shell thickness, albumen height, and Haugh unit, lowered serum FSH, LH, and P levels, boosted ACTH and CORT levels, elevated serum IL-1, TNF-α, and endotoxin levels of laying hens. 1000 mg/kg dietary Bacillus licheniformis enhanced daily egg production (2, 5, 6, and 9 d), average egg production and feed intake of heat-treated hens, increased the egg shell strength, decreased serum IL-1, TNF-α, and endotoxin levels, bring down serum ACTH and CORT levels, and raise FSH and P levels of heat-stressed hens (P < 0.05).Exp. 3 Effects of the Bacillus licheniformis on intestinal barrier function of heat-stressed laying hensHeat damaged structure of intestinal villi in hens, decreased villus height and V:C of ileum and cecum, enhanced crypt depth of cecum, decreased the number of IELs and area positive for sIgA, and increased mast cell number in ileum and cecum. Shorts stress increased the number of goblet cells in the cecum, while long stress decreased it. Heat depressed the mRNA expressing of occluding and claudin-1 in ileum and cecum, and EGF in cecum (P < 0.05). 1000 mg/kg dietary Bacillus licheniformis could improve structure of intestinal villi, enhance villus height of ileum and cecum and V:C of cecum, decreased crypt depth of cecum, increased the number of IELs and area positive for sIgA, and decreased mast cell number in ileum and cecum (P < 0.05). Also, it stabilized the distribution of goblet cells. 1000 mg/kg dietary Bacillus licheniformis could enhance the mRNA expressing of claudin-1 in ileum and cecum, and EGF in cecum (P < 0.05).Exp. 4 Effects of the Bacillus licheniformis on intestinal microflora of heat-stressed laying hensDGGE band patterns of PCR products for intestinal microbial V3 region showed similarities within groups were low. Heat stress reduces the number of DGGE bands and Shannon index for ileum and cecum, reduced the cecal concentrations of Lactobacillus and Bifidobacterium, and increased E. coli concentrations of ileum and cecum (P <0.05). Diet supplemented with 1000 mg/kg Bacillus licheniformis increased the number of DGGE bands and Shannon index for cecum, and increased the concentration of Lactobacillus in cecum of layers under heat stress (P <0.05). Five common or specific bands could be affiliated to Lactobacillus reuteri, Enterococcus sp., Escherichia coli, Clostridium perfringens, and Lactobacillus johnsonii, respectively.