| Due to the widespread use and abuse of antibiotic products in the breeding industry,the adverse consequences of drug-resistant bacteria and drug residues have forced people to find new methods and products to replace the use of antibiotics.The lactic acid bacteria as the most studied and most widely used probiotics,have become an ideal antibiotic substitute in the breeding industry to promote better and faster development of animal husbandry.This study intends to isolate,identify and screen the dominant lactic acid bacteria with good growth performance,strong tolerance and strong ability to inhibit the growth of pathogenic bacteria from the intestines of healthy Bian chicken,and conduct related safety tests on them,and finally use them together with antibiotics in laying hen breeding,research and compare the effects of them on the weight,immune function,antioxidant capacity and antibody level of laying hens,providing a theoretical basis for the application of lactic acid bacteria as antibiotic substitutes in poultry production,and for the development of probiotics feed additives provide reference.Test 1 Isolation,identification and screening of Bian chicken-derived lactic acid bacteria: Lactic acid bacteria with good growth characteristics,strong tolerance and excellent antibacterial properties were isolated,identified and screened from the intestines of healthy Bian chicken.The contents and mucosa in the duodenum,jejunum,ileum and cecum of healthy laying hens were used as samples,lactic acid bacteria with MRS selective medium were isolated,and count the number of lactic acid bacteria in each intestinal tract,and identified by gram staining,strain biochemical test and 16 S rDNA sequence analysis.Lactic acid bacteria were mainly distributed in the cecum,with less jejunum and ileum,and the duodenum the least.Through gram staining,strain biochemical test and 16 S rDNA sequence analysis,five strains of lactic acid bacteria were successfully isolated and identified,strains I(Lactobacillus crispatus)and L(Lactobacillus johnsonii),G(Lactobacillus salivarius),Y(Lactobacillus plantarum)and F(Lactobacillus acidophilus).And then through the growth curve determination,vitro acid tolerance,bile salt tolerance test and bacteriostasis test(Elidinella coli,Salmonella and Staphylococcus aureus)for the screening of dominant lactic acid bacteria.The growth curve showed that the G,Y and F strains entered the logarithmic growth phase earlier than the I and L strains,and the growth rate was faster;the vitro acid tolerance test showed that G,Y and F strains were less affected by lower p H than I and L strains;the bile salt tolerance test showed that strain G,Y and F were less affected by high concentrations of bile salts than strain I and L;the gastric juice tolerance test showed that G,Y and F strains had better tolerance to artificial gastric juice;the intestinal juice tolerance test showed that G,Y and F strains had stronger ability to tolerate artificial intestinal juice;the bacteriostatic test showed that the diameter of inhibition zone of G,Y and F strains were larger than I and L strain.After the above steps screened,three dominant strains G,Y and F were obtained.Test 2 Safety evaluation of three dominant lactic acid bacteria: Relevant safety tests were carried out on the selected three dominant strains to ensure their safety so that they could be put into practical applications.The safety tests mainly included hemolysis test,antibiotic susceptibility test,inter-strain antagonism test and mouse test.The hemolysis test showed that none of the three strains caused hemolysis and had no effect on red blood cells;the antibiotic susceptibility test showed that three strains were sensitive to most antibiotics;the antagonistic test between strains showed that the three strains had no antagonism to each other,and the joint growth effect was good.Gavage test of mixed bacteria liquid of three dominant strains in mice,the results showed that all mice were normal,weight increased,there was no significant difference between the test group and the control group(P>0.05);the blood routine indexes of the test group and the control group were in the normal range,and there was no significant difference(P>0.05);the biochemical test of the test group results of alanine aminotransferase,aspartate aminotransferase,blood urea nitrogen and creatinine in the control group and the control group were all within the normal range,and there was no significant difference(P>0.05);the bacterial translocation test showed that no bacterial translocation occurred;the sections of mouse liver and kidney microscopic examination showed that the liver and kidney of the control group and the experimental group were not abnormal.The results showed that the G,Y and F three strains of lactic acid bacteria and their mixed bacterial liquid were safe and could be used in actual production.Test 3 Preliminary study on the Bian chicken-source mixed lactic acid bacteria instead of antibiotics for feeding laying hens: The three dominant bacteria strains were mixed to form a mixed bacterial liquid,and antibiotics were added to the diet of laying hens to study the effects of lactic acid bacteria and antibiotics on the weight,immune function,antioxidant capacity and the influence of antibody levels of laying hens.The feeding experiment adopted a single factor and completely random design,the test divided into control group,antibiotic group,high-dose group,medium dose group and low-dose group.The results showed that,the weight of the antibiotic group,low-dose group,middle-dose group and high-dose group were significantly higher than the control group(P<0.05),and there was no significant difference between the middle-dose group and the antibiotic group(P>0.05);the Ig A,Ig G and Ig M of the antibiotic group,low-dose group,middle-dose group and high-dose group were significantly higher than those in the control group(P<0.05),and the Ig A,Ig G and Ig M in the middle-dose group and antibiotic group were no significant difference(P>0.05);the spleen index,thymus index and bursa index of the antibiotic group,low-dose group,middle-dose group and the high-dose group were significantly higher than those of the control group(P<0.05),the spleen index,thymus index and bursa index of the middle-dose group and the antibiotic group were not significantly different(P>0.05);the blood MDA content of the control group was significantly higher than the antibiotic group,middle dose group and high dose group(P<0.05),the blood SOD content of the antibiotic group,middle dose group and high dose group were significantly higher than the low dose group and control group(P<0.05),the content of GSH-Px and T-AOC of the antibiotic group,low-dose group,middle-dose group and high-dose group were significantly higher than the control group(P<0.05).The antibody levels(Newcastle disease virus antibody titer)of the test group and the control group both increased to varying degrees,and the test group was significantly higher than the control group(P<0.05),and there was no significant difference between the medium dose group and the antibiotic group(P>0.05).The results of the test showed that mixed lactic acid bacteria can promote the growth performance,immune function,anti-oxidant capacity and antibody level of laying hens,and have similar effects to antibiotics,and the best addition amount of the lactic acid bacteria mixed bacteria liquid was 109 CFU/m L(medium dose group). |
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