Preparation Of Antibiotic Substitute And Its Effect On Growth Performance And Intestinal Microflora Of Broilers

In order to develop safe,efficient,green and residue-free functional feed additives for replacing antibiotics to ensure the safety of animal foods,glucose oxidase(GOD),Lactobacillus acidophilus and Curcumin were selected in this research.The best compatible compound additive was developed through antibacterial experiments in vitro,and the effect of compound additives on the growth performance,intestinal microflora of broilers challenged by Escherichia coli was deeply explored.The experiments were divided into three parts,and the main results were as follows:The first focused on the preparation of antibiotic substitute.:The first part was the screening of in vitro inhibitory concentration of GOD,Lactobacillus acidophilus and Curcumin and the screening of the best combination of the three inhibitory concentrations.(1)Through the antibacterial experiment in vitro,it was found that GOD had a strong antibacterial effect on Escherichia coli proliferation when its concentration was between 0.005%and 0.200%in the presence of 2%glucose,the greater the GOD concentration,the stronger the antibacterial effect(P<0.05);Lactobacillus acidophilus inhibited Escherichia coli proliferation with an optimal viable counts of 1 × 105～1×107 CFU/mL;Curcumin inhibited Escherichia coli proliferation at the optimal concentrations of 0.020%～0.060%,the higher Curcumin concentration,the stronger antibacterial effect(P<0.05).(2)Through the antibacterial experiment in vitro for the combination of GOD,Lactobacillus acidophilus and Curcumin,it was indicated that the combination of 0.005%GOD,0.020%Curcumin and 1 X 105 CFU/mL Lactobacillus acidophilus was effective to inhibit Escherichia coli proliferaton,which was better than a single factor(P<0.05),indicating that the combination had the synergistic effect on inhibiting Escherichia coli proliferation,which was equivalent to 150.0 mg/kg chlortetracycline hydrochloride.In addition,the combination of 1 X 105 CFU/mL Lactobacillus acidophilus and 0.005%GOD was was equivalent to 75.0 mg/kg chlortetracycline hydrochloride for inhibiting the proliferation of Escherichia coli.(3)The pH values of GOD,Lactobacillus acidophilus and Curcumin or their combination in Escherichia coli co-culture solution decreased with longer incubating time(P<0.05).(4)0.005%GOD and 0.020%Curcumin had no obvious inhibitory effect on Lactobacillus acidophilus proliferation(P>0.05);therefore,the combination of 0.005%GOD,0.020%Curcumin and 1 × 105 CFU/mL Lactobacillus acidophilus was prepared and made as the feed additives for the following experiment in vivo.The second part was to study the effect of feed additives on the growth performance and intestinal microflora of broilers after replacing the antibiotics.(1)Three hundred and fifty healthy 1-day-old AA broilers were selected and randomly divided into 7 groups,and each group was designed with 5 replicates,and each repicate with 10 chickens.The whole feeding management was divided into two stages:the early stage(1～21 d)and the later stage(22～42 d).Group A:the basal diet;group B 75.000 mg/kg chlortetracycline hydrochloride;groups C,D and E:0.03%,0.06%and 0.09%compound additives;group F:0.03%compound additive+0.50%glucose;group G:0.50%glucose.The results showed that diarrhea rate in group F in the early feeding period was significantly lower than that of groups A and B(P<0.05);the final weight and ADG in group F in the late feeding period were significantly higher than those of groups A and B(P<0.05);diarrhea rate,daily feed intake/daily gain(F/G)were significantly lower than that in groups A and B(P<0.05);the CP.EE,Ca and TP metabolic rates in group F were significantly higher than those in groups A and B(P<0.05);the carcass weight,full eviscerated weight and half eviscerated weight in group F were significantly higher than those in groups A and B(P<0.05).(2)According to the analysis of intestinal tissue morphology,the jejunal villi height of broilers in groups C and F was significantly increased compared with groups A,B and G(P<0.05):the crypt depth of the jejunum tissue in group F was significantly lower than that in groups A,B,C and G(P<0.05);the ratio of the villus height to the crypt depth in the jejunum tissue in groups C.F and G groups was significantly higher than that in groups A and B(P<0.05),in which it was the highest in group F,indicating that the compound additives could improve the intestinal tissue morphology of broilers.(3)The microflora analysis of broiler jejunum contents by 16S rDNA high-throughput sequencing showed that group F had significantly improved Proteobacteria,Bacteroidetes,and Cyanobacteria,compared with group A.The abundance of Bacteroideles and Cyanobacteria in groups F and B was significantly different(P<0.05);group F had significantly improved the abundance of Novosphingohium,Aquabacterium,and Azospirillum than group A(P<0.05);group F significantly reduced the abundance of Lactobacillus salivarius compared with group A(P<0.05),group F significantly increased the abundance of Ruminococcus than groups A and B(P<0.05).It was showed that the compound feed additive could increase the Alpha diversity of intestinal microbes in broilers for maintaining the intestinal microecological balance of broilers.The third part focused on the effect of feed additives on growth performance and gut microbiota of broilers challenged by Escherichia coli.(1)When the broiler was raised for 42 d in the last experiment,all the broilers were weighed after 12 h fast.The broilers with the medium body weight were selected from the basal diet group,the Chlortetracycline group,0.03%additive group,0.03%additive+0.5%glucose group,and 0.5%glucose group,respectively.6 male and 6 female were selected in each group,in which they were given with their previous diets individually.6 broilers in each group(half male and half female)were put in the saline group,and the other 6 broilers(half male and half female)were put in the Escherichia coli group.Each broiler was raised in each cage.The experimental design were groups A,B,C,D and E for the saline group,and groups F,G,H,I and J were for Escherichia coli.Groups A and F were fed with basal diet;groups B and G were fed with the basal diet containg 75.000 mg/kg Chlortetracycline;groups C and H were fed with the basal diet containing 0.03%compound additives;groups D and I were fed with the basal diet containing 0.03%compound additive and 0.5%glucose;groups E and J were fed with the basal diet containg 0.5%glucose.(2)Starting at 12:00,each broiler in the saline group was given with 1.00 mL sterile normal saline by intraperitoneal injection,and each broiler in the Escherichia coli group was given with 1 mL of Escherichia coli(The viable counts were 2.3 X 108 CFU/mL)by intraperitoneal injection.The experimental period was 14 d.The results showed that the diarrhea rate of broilers was significantly reduced in groups C and D,compared with groups B;which was also decrased in groups H and I,compared with groups A,B,and J(P<0.05),indicating that the compound feed additive was better than Chlortetracycline to reduce diarrhea rates of broilers challenged by Escherichia coli.(3)Through 16S rDNA high-throughput sequencing analysis of microbiota in rectum contents of broilers,it was found that group H significantly increased the abundance of Lactobacillus at genus level,compared with groups A,B,C,F and G(P<0.05);groups C and H significantly reduced the abundance of Acinetobacter,compared with group A(P<0.05);group F significantly increased the abundance of Bacteroides(P<0.05),compared with groups A,B,C,G and H;group F significantly increased the abundance of Shigella,compared with groups A,B,C and H(P<0.05);group C significantly increased the abundances of Aquabacterium and Oscillospira,compared with groups A,F,G and H(P<0.05);group F significantly increased the abundance of Oscillospira,compared with group A(P<0.05).It was found that the compound additive in group C could increase the abundance of beneficial bacteria,compared with groups A and B;group H had the same function,compared with groups F and G.It can be concluded that the compound feed additive is very helpful to maintain the balance of broiler intestinal microflora and promote gut health and production performance of broilers.