Effects And Mechanism Of Chitosan On Growth, Intestinal Barrier And Immunity In Piglets

The objectives of present study were:to grasp systematically the effects of diets added with chitosan on growth,diarrhea,intestinal barrier,immunity and oxidation resistance in weanling piglets, to reveal the mechanism of chitosan on growth and intestinal barrier,and to explore the feasibility of chitosan applied to feed additive replacing antibiotic,to feel furtherly a suitable adding proportion,to provide theoretical basis for exploiting green feed additive.Experiment 1 Effects of Chitosan on Growth Performance and IGF-I in PigletsIn order to study the effects of chitosan on growth,diarrhea and their mechanism on growth performance in piglets,fifty weanling piglets with 21 days old were challenged with Enterotoxigenic Escherichia coli during preliminary trial period and randomly assigned to five treatment groups fed a maize-soybean meal diet containing either no addition,50 mg/kg chlortetracycline,200,300 or 400 mg/kg chitosan for 21 d. The results showed as follows:1) The chitosan’s D.D was 91.25%; 2) Compared with the control group,every experimental group could improve daily feed intake (P>0.05), decrease the ratio of feed to gain(P<0.05) and diarrhea, average daily gain increased respectively 15.8%,9.2%,16.8% and 21.1%(P>0.05); 3)The contents of IGF-I in serum were improved in 200(P>0.05)、300(P<0.05)and 400(P<0.05) mg/kg chitosan groups, IGF-I mRNA expression in jejunal(P<0.05)and ileal(P>0.05)mucosa was increased in 300 mg/kg chitosan group,but these effects were not significant in the chlortetracycline group(P>0.05). These results indicate:Diet supplemented with 200,300 or 400 mg/kg chitosan can improve feed conversion rate,promote growth performance,decrease diarrhea in weanling piglets, furthermore, the effect and the concentration have positive correlation,and further confirms that the growth-promoting effect is correlated with regulating IGF-I growth axis.Experiment 2 Effects of Chitosan on Intestinal Microbiota in PigletsIn order to know the effects of chitosan on intestional microbiota, and to reveal the mechanism of chitosan on intestinal barrier,microbial flora in jejunum and caecum was analysised by 16S rDNA PCR-DGGE technology, then the amounts of main microorganisms in intestine were detected by Real-time PCR in this experiment. The result of DDGE showed:In jejunum, microbial diversity was lower in chitosan group and chlortetracycline group than control group.Advantage bands lay in Lactobacillus, but the relative amounts of Aureus, E.coli and Salmonella were decreased significantly in chitosan group.The total bacterial counts were decreased significantly in chlortetracycline group, moreover, there was little difference in same treatment; In caecum, the effect of either chitosan or chlortetracycline was weak,moreover, intestinal bacteria similarity was decreased in the same treatment,there were great differences in different treatment,but chitosan still had inhibition role on Aureus. The result of Real-time PCR showed:Compared with the control group,the amounts of Lactobacillus, Bifidobacterium, E.coli and Streptococcus were decreased in chlortetracycline group; The amounts of intestinal Lactobacillus and Bifidobacterium were increased, the amounts of E.coli and Streptococcus were decreased in chitosan group, moreover,the effect was better in jejunum than duodenal and ileum. The result indicated:Chitosan can effectively promote the quantity of beneficial bacterias and inhibit the reproduction of harmful germs, but chlortetracycline is harmful to all kinds of bacteria.Experiment 3 Effects of Chitosan on Intestinal Mucosal Mechanical Barrier Function in PigletsIn order to explore the effect of chitosan on intestinal mechanical barrier, and to reveal the mechanism of chitosan on intestinal barrier, DAO activity and D-lactate level in plasma, ET-1 and NO contents in serum were detected by ELISA, the villus height and width, crypt depth,and the radio of villous height and crypt in jejunum and ileum were detected under light microscop, tight junction protein Occludin,ZO-1 and Claudin-1 expressions in jejunal mucosa were detected by immunohistochemical method,furthermore, the expression of Occludin protein was detected by western blotting,Occludin,ZO-1 and Claudin-l mRNA expression in jejunal mucosa were detected by Real-time PCR.The result showed:1) DAO activity was decreased in 200(P>0.05),300(P<0.05) and 400(P<0.05)mg/kg chitosan groups, D-lactate and ET-1 level was decreased (P<0.05)in three chitosan groups,but DAO activity and D-lactate level were increased(P>0.05) in chlortetracycline group.NO level was not significant different in all experimental group(P>0.05); 2) The villus height(P<0.05) and radio of villous height and crypt(P<0.01 in ileum and P<0.05 in jejunum) was increased, the villus width was decreased(P<0.05), the crypt depth was reduced(P<0.01 in 300 mg/kg chitosan group, P<0.05 in chlortetracycline group); 3) Occludin(P<0.01), ZO-1(P<0.05), Claudin-1(.P>0.05) protein and gene expressions were increased in 300 mg/kg chitosan group, but were not significant different in chlortetracycline group(.P>0.05). The results indicated:Chitosan can improve intestinal mucosal mechanical barrier function in piglet by preventing intestinal permeability from increasing, and by inducing Occludin and ZO-1 expression which improve intestinal closely connectivity.Experiment 4 Effects of Chitosan on Intestinal Mucosa Immunologic Barrier Function in PigletsIn order to explore the effect and mechanism of chitosan on intestinal mucosal immunologic barrier,the quantities of intestine mucosal intraepithelial lymphocytes and mast cells were counted by HE staining method,and goblet cells were counted by PAS staining method,TLR4,sIgA and Calprotectin protein expression in intestinal mucosa were detected by immunohistochemical method, IL-1β, IL-6, TNF-a and TLR4 mRNA relative expression in jejunal mucosa were detected by Real-time PCR in this experiment. The results showed:Compared with the control group,l)Intestine mucosal intraepithelial goblet cells (in jejunum P<0.05, in ileumP>0.05)and lymphocytes cells(P<0.05 in chitosan group, P>0.05 in chlortetracycline group)were increased, the quantity and degranulation of mast cells were decreased (P<0.05)in chitosan group and chlortetracycline group; 2) Jejunal mucosa TLR4 and calprotectin protein expression were decreased in chitosan group and chlortetracycline group(P<0.05).Jejunal mucosa slgA protein expression was increased in chitosan group(P<0.05),but had no significant changes in chlortetracycline group (P>0.05); 3) Jejunal mucosa TLR4 mRNA expression was decreased(.P<0.05), IL-6 mRNA expression was increased (P<0.05 in chitosan group, P>0.05 in chlortetracycline group), TNF-a mRNA expression had no significant changes in chitosan and chlortetracycline group(P>0.05), but IL-1βmRNA expression were increased in chitosan group(P>0.05) and decreased in chlortetracycline group(P>0.05). The results indicated:Chitosan can decrease inflammatory, affect directly intestinal mucosal immune molecular and improve intestinal mucosal immunologic barrier function,furthermore,the effect is more significant in jejunum.Chlortetracycline also can decrease intestinal inflammatory.Experiment 5 Effects of Chitosan on immunity and Anti-oxidation in PigletsIn order to analysis on the immunity and oxidation resistance of chitosan in piglets, and to explore its mechanism on growth, IgM and IgG levels in serum were detected using immune projective turbidimetry, IL-1β、IL-6 and TNF-αin serum contents were detected by ELISA, antioxidant indexes in serum were detected by uv spectrophotometry in this experiment. The results showed:1) IgG and IgM levels were increased in 200,300 or 400 mg/kg chitosan groups, which was increased with the concentration of chitosan,but were decreased in chlortetracycline groups(P>0.05). IL-1β, TNF-a and IL-6 contents in serum were no significant effect(P>0.05)in chitosan groups and chlortetracycline group.2) The activities of SOD and GSH-Px(F<0.05 in 300,400 mg/kg chitosan groups and chlortetracycline group,P>0.05 in 200 mg/kg chitosan group) and the content of T-AOC(P<0.05 in 300 and 400 mg/kg chitosan, P>0.05 in 200 mg/kg chitosan and chlortetracycline groups) were increased, MDA level was decreased(P<0.05),but the activities of POD and NOS were no significant effect in all experimental group. The results indicated:Chitosan and chlortetracycline can enhance the ability of oxidation resistance in piglets, moreover, chitosan can improve body humoral immunity, the effect and concentration also have positive correlation.General conclusionsTo sum up, the study draw main conclusions that the effect and mechanism of chitosan act on piglets as below:1)Chitosan can promote the growth performance and humoral immunity of early weanling piglet, furthermore, the effect and concentration have positive correlation, which relate with what chitosan can regulate 1GF-1 to induce growth axis, relief oxidative stress, and improve intestinal barrier function; 2)Chitosan can maintain intestinal structure,decrease intestinal permeability,improve intestinal mucosa close connectivity, promote the growth of beneficial bacterias and inhibit the reproduction of harmful germs, affect directly mucosal immune molecular,decrease intestinal inflammatory response and improve intestinal mucosa immunity,so as to protect the intestinal barrier of weanling piglets challenged with enterotoxigenic Escherichia coli from injuring, to promote the digest and ingest of the nutrient,and to reduce enterogenous infection; 3) Chitosan has the potential of replacing chlortetracycline as a new kind of feed additive for pig.