| Feed probiotics are safe,have no residues,and have probiotic properties such as promoting the growth of livestock and poultry,immunity and anti-infection.They are ideal products in the era of "anti-infection",which meet the development trend and industrial value-added demand,and have great research and development potential.In order to test the application potential of compound probiotics in poultry production,a feeding experiment was carried out with broilers as experimental animals.First of all,in order to prepare a compound probiotic preparation which meets the production standards and has an ideal bacterial quantity,the strains in this study included three strains of lactic acid bacteria selected,one strain of Bacillus subtilis(B.subtilis)(BYS2),preserved and one strain of yeast.(CICC 1355)The strains were fermented in the pilot plant to prepare compound probiotics.These strains were fermented in the pilot workshop to make composite probiotics.Then,in order to test the probiotic effect of animals fed with compound probiotics,this study conducted a feeding experiment for broilers to evaluate the changes of innate immunity and acquired immunity,intestinal flora,growth performance and other changes of broilers fed with compound probiotics.In addition,in order to evaluate and confirm whether the immune resistance of animals fed with probiotics is enhanced,this study analyzed the survival rate of broilers after the challenge of pathogenic Escherichia coli(E.coli)(CVCC 1512),and the expression of immune-related genes one day and three days after the challenge.This study is mainly divided into the following three parts:1.Screening of lactic acid bacteria and preparation of compound probioticsIn order to screen lactic acid bacteria with better probiotic performance in vitro,this study evaluated the ability of 10 strains of lactic acid bacteria to tolerate acid and bile salt and to inhibit intestinal pathogenic E.coli.The results showed that Lactobacillus plantarum(21809),Enterococcus faecalis(24203)and Lactobacillus acidophilus(6091)not only had good acid resistance,but also had a survival rate of more than 84% after being cultured for 6 hours under0.3% bile salt conditions.Moreover,these three strains had a strong ability to inhibit E.coli,and the diameter of inhibition zone was 17 mm～18 mm.Therefore,in this study,the above three strains of lactic acid bacteria,one strain of B.subtilis and one strain of yeast purchased were fermented through the standard fermentation process in the pilot plant,and the compound probiotics was prepared.Among them,three strains of lactic acid bacteria were made into compound preparations of lactic acid bacteria,and five strains were made into compound preparations of probiotics(used in group B and group C respectively in animal experiments).2.To test the effect of compound probiotics on the innate and acquired immunity of broilersIn order to test the effect of feeding compound probiotics on the immune performance of broilers.In this study,the serum immunoglobulin expression of broilers at 28 and 42 days of age was detected by ELISA.The results showed that Ig A,Ig G and Ig M of broilers in the compound probiotics group(group B and group C)were higher than those of the control group(group A)at 28 and 42 days of age.And the Ig of group C was significantly higher than that of of antibiotic group(group D,colistin sulfate)at 42 days of age(P<0.05).The results indicated that feeding compound probiotics could improve the humoral immunity level of broilers,and the effect of probiotic compound group was better.Then,in order to test the effect of feeding compound probiotics on the acquired immunity of broilers,the antibody titers of broilers after immunization were detected by hemagglutination inhibition test.The results showed that the antibody titers of H9 subtype avian influenza(AI)and Newcastle disease(ND)of broilers in groups B and C were higher than those in groups A and D(P>0.05).The results indicated that feeding compound probiotics to animals could improve the vaccine titer and antibody level.Finally,in this study,the immune-related genes in the spleen and small intestine of 28-day-old broilers were detected by fluorescence quantitative PCR,the results showed that compared with the control group(group A),the genes of TLR2,TLR4 receptors and downstream cytokines such as IL-1β,IL-8,IL-10,AVBD2 and AVBD6 were more up-regulated in the compound probiotics group(group B and group C),which indicated that feeding compound probiotics could regulate innate immunity,make the body enter the immune defense state,and prevent the pathogen infection.At the same time,at the age of 28 days,the indexes of spleen,thymus and bursa of fabricius of broilers in the compound probiotics group(groups B and C)increased,while the changes in the antibiotic group(group D)were not as significant as those in the compound probiotics group.In a word,compound probiotics can promote the development of immune organs of broilers,and can regulate the innate and acquired immune responses of broilers,which is conducive to the resistance of broilers to pathogen infection.3.To test the effect of compound probiotics on the caecal flora,immune resistance and growth performance of broilersIn order to test the effects of compound probiotics on intestinal flora,immune resistance and growth performance of broilers,after feeding for 28 and 42 days,the changes of the caecum flora of broilers were detected by 16 S r RNA high-throughput sequencing.The results showed that compared with the control group(group A),after feeding the compound probiotics,the relative abundance of Firmicutes and Faecalibacterium in the cecum of broilers increased(P<0.05),while the relative abundance of Bacteroidetes and other in broilers decreased(P<0.05)at 28 days of age.In addition,the diversity and species abundance of intestinal flora in the compound probiotics group increased,while the species abundance in the antibiotic group(D group)decreased.At the same time,compared with the control group,after 28 days of broilers feeding with compound probiotics,the villi height and the ratio of villi height to crypt depth of broilers increased(P<0.05).The results showed that the compound probiotics could regulate the intestinal flora of broilers and promote the development of small intestinal villi,which was beneficial to the healthy growth of broilers.At the same time,this study examined the survival rate and immune-related gene expression of 28-day-old broilers infected with enteropathogenic E.coli.The results showed that compared with the control group,the survival rate of the experimental groups were increased by 30%,35% and 50%,respectively,indicating that the compound probiotics could improve the anti-infection ability of broilers.In addition,after 3days of infection with E.coli,the spleen,small intestinal receptors(TLR2,TLR4)and downstream interleukins(IL-1β,IL-8,IL-10),defensins(Av BD2,Av BD6)and other of broilers in the compound probiotics group were generally up-regulated,which indicated that the compound probiotics could regulate the innate immunity of broilers and promote the body to resist diseases.In addition,this study tested the growth performance related indexes of broilers for 28 days and 42 days.The results showed that compared with the control group(group A),the average body weight and average daily gain of broilers in the experimental group increased significantly(P<0.05),and the ratio of feed to gain of the probiotics group decreased significantly(P<0.05).The results showed that feeding compound probiotics could increase the weight gain,reduce the feed-to-weight ratio and improve the growth performance of broilers.To sum up,compound probiotics,especially the compound of different types of probiotics,can improve the growth performance of broilers such as weight gain,and improve the composition and morphology of the intestinal flora by regulating innate immunity and acquired immunity,enhance the immunity and disease resistance of broilers.This study provides data support and theoretical reference for the application of compound probiotics in production. |
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