Effects Of Thymol And Carvacrol On Intestinal Epithelial Barrier And Immune Functions Of Broiler Chickens

Four experiments were conducted to study the effects of thymol and carvacrol-bearing essential oils on intestinal epithelial barrier and immune functions of broiler chickens.Exp.l was conducted to evaluate the modulatory effects of thymol and carvacrol bearing essential oils (EO) on lipopolysaccharide (LPS)-induced systemic inflammatory response in broiler chickens through a 2x2 factorial arrangement. According to our data, dietary EO supplementation had no influence on the growth performance or lymphoid organ weight of broiler chickens, but significantly decreased the production of MDA in liver (P< 0.05). At 3 h-post LPS injections, EO addition enhanced the mRNA expression of IL-1β and iNOS in the spleen, but inhibited TLR4 expression (P< 0.05). Whereas at 8 h-post injections, EO supplementation up-regulated the mRNA expression of IL-4 and down-regulated the mRNA expression of TNF-a and TLR4 (P< 0.05). These results suggest that dietary EO supplementation could improve the anti-oxidant ability of liver, and possess remarkable immune-modulatory effects in broiler chickens.Exp.2 was conducted to evaluate the modulatory effects of EO on coccidial vaccine challenge in broiler chickens through a 2×2 factorial arrangement. During the first week post challenge, BW gain and feed conversion efficiency was significantly decreased and jejunal morphology was damaged (P< 0.05) in challenged birds. Besides, coccidial challenge up-regulated the mRNA expression of inflammatory cytokines (IFN-y and IL-1β) and down-regulated the gene expression of occludin in jejunum (P< 0.05). Dietary EO supplementation decreased the oocyte numbers in the excreta, alleviated the bloody diarrhea and enhanced the mRNA expression of IL-4 in the jejunum (P< 0.05). However, EO did not improve the growth performance, jejunal histological parameters or mucosal disaccharides activity, indicating that EO supplementation could not completely alleviate the impaired intestinal functionality caused by coccidial challenge in broiler chickens.In Exp.3, the antibacterial activities of thymol and carvacrol were determined in vitro, and they both showed great antibacterial activity against pathogenic Escherichia coli, Clostridium perfringens, and Salmonella strains, and weak activity towards beneficial Lactobacillus strains. Besides, an additive effect was observed between thymol and carvacrol. Then a 2x4 factorial arrangement was conducted to evaluate the effects of EO supplementation (0,60,120 or 240 mg/kg) on broiler chickens challenged with C. perfringens. According to our data, dietary EO supplementation linearly alleviated the gut lesions and improved the ratio of villus height to crypt depth (P< 0.05), and the supplementation of 60 and 120 mg/kg EO increased the serum antibody titers against NDV and IBD in the challenged birds (P < 0.05). Regardless of challenge, the EO supplementation showed a tendency to linearly elevate the feed conversion efficiency (P< 0.10), linearly inhibited the mRNA expression of TLR2 and TNF-a in the ileum, and linearly decreased the counts of Escherichia in the ileum (P< 0.05). Also, the supplementation of 120 and 240 mg/kg EO increased the counts of Lactobacillus in the ceca (P< 0.05). These results suggest that dietary EO supplementation may alleviate C. perfringens induced gut damage through their modulation on intestinal bacteria and inhibition of TLR2-mediated inflammatory responses.In Exp.4, the antibacterial activities of thymol, carvacrol and lysozyme against C. perfringens were observed in vitro, and an additive effect was found between lysozyme and thymol/carvacrol. In the in vivo trial, broiler chickens fed 0,60,120 or 240 mg/kg EO, or the combination of EO and exogenous lysozyme were challenged with C. perfringens. Besides, a negative control group (non-EO or lysozyme supplementaed) without challenge was included. According to our data, the supplementation of lysozyme or 60/120 mg/kg EO alone remarkably decreased the mortality of birds during challenge, and decreased the translocated bacteria count in the liver on d 21 (P< 0.05). Also, the supplementation of EO or lysozyme alleviated gut lesions, improved intestinal histological parameters and enhanced the activity of mucosal disaccharides and serum lysozyme to a certain extent. Notably,120 mg/kg EO alone improved the mRNA expression of iNOS, occludin and claudinl in the small intestine (P< 0.05), increased the relative abundance of Firmicutes and Clostridiales whereas decreased the abundance of Proteobacteria and Cyanobacteria (P< 0.05). Besides,120 mg/kg EO alone changed the structure of microbial functional genes according to the metagenomics sequencing, and inhibited the relative abundance of several virulence factors of pathogenic bacteira (P< 0.05). Overall,120 mg/kg of the EO preparation was an optimum supplementation dose for protecting broiler chickens against C. perfringens challenge. The addition of lysozyme we used in the current study did not strengthen the benefical effets of EO.In conclusion, dietary supplementation of thymol and carvacrol-bearing EO could directly influence LPS-induced systemic inflammatory response in broiler.chickens through their immunomodulatory effects,_and alleviate C. perfringens-induced necrotic enteritis by affecting the intestinal microflora and improving the intestinal epithelial barrier function. However, EO supplementation could not completely alleviate the impaired intestinal functionality induced by coccidial challenge. Besides, the exogenous lysozyme we used was not capble of strengthening the benefical effects of EO on broiler chickens with necrotic enteritis.