Effects Of Combination Of Protease And Probiotic On Growth Performance And Intestinal Health Of Broilers

In order to further alleviate the negative impact of the ban on antibodies on the farming industry,and to develop the use of more environmentally friendly and efficient green feed additives,this study was conducted to compare the effects of proteases and probiotics(Clostridium butyricum,Enterococcus faecalis and Bacillus coagulans)added to broiler diets and to investigate their mechanisms of action,to provide a theoretical basis for the selection of bacterial enzyme combinations with good resistance replacement effects in broiler production.The trial was conducted with 648 healthy 1-day-old Arbor Acres Plus broilers of similar initial weight,completely randomised to six treatment groups,each with six replicates of 18 chickens each.The dietary treatments were: 1)Basic rations(control,CON group);The basal diet was corn-soybean meal type.2)Base diet plus 500 mg/kg of calcium oxytetracycline(antibiotic group,AN group).3)Base diet with 100 mg/kg of protease(protease group,PRO group).4)Base diet with 100 mg/kg of protease and 100 mg/kg of Clostridium butyricum(Clostridium butyricum group,PRO+CB group).5)Base diet plus 100 mg/kg of protease and 100 mg/kg of Enterococcus faecalis(Enterococcus faecalis group,PRO+EF group).6)Base diet with 100 mg/kg of protease and 150 mg/kg of Bacillus coagulans(Bacillus coagulans group,PRO+BC group).The trial lasted 42 days.Experiment 1: Investigating the effect of protease and probiotic addition on the growth performance and digestive function of broilers.The results indicate that : 1)Compared to the control group,the Enterococcus faecalis and Bacillus coagulans groups significantly increased body weight at 42 days of age(BW)and average daily gain(ADG)from 22 to 42 days of age and from 1 to 42 days of age in broilers(P < 0.05);The experimental groups significantly reduced the feed to gain ratio(F/G)from 22 to 42 days of age(P <0.05),the Enterococcus faecalis group also significantly reduced the feed to weight ratio from 1 to 42 days of age(P <0.05).Compared to the antibiotic group,Average daily feed intake(ADFI)from 22 to 42 days of age and from 1 to 42 days of age in the Enterococcus faecalis and Bacillus coagulans groups,Enterococcus faecalis and Bacillus coagulans groups was significantly increased(P < 0.05).2)Compared to the control group,significantly lower jejunal p H in the PRO+EF group(P < 0.05),Compared to the antibiotic group,Significant increase in jejunal p H in the PRO,PRO+CB and PRO+BC groups(P < 0.05).3)Compared to control and antibiotic groups,Intraduodenal trypsin activity was enhanced in the PRO and PRO+CB groups,but the difference was not significant(P >0.05).4)Compared to the control group,Relative m RNA expression of solute carrier family 6 member 14 and solute carrier family 7 member 2 of the duodenal amino acid transport carrier was significantly increased in all experimental groups(P < 0.05);The relative m RNA expression of ileal SLC7A9 in AN,PRO,PRO+CB and PRO+EF groups and ileal SLC6A14 in AN,PRO+CB and PRO+EF groups was significantly higher(P < 0.05).Compared to control and antibiotic groups,The relative m RNA expression of ileal SLC7A2 and SLC15A1 in the PRO,PRO+CB and PRO+EF groups and ileal SLC7A2 in the PRO+BC group was significantly higher(P < 0.05).Experiment 2: Investigating the effect of combined protease and probiotic addition on gene expression of intestinal physical and mucus barriers and intestinal mucosal inflammatory immune factors in broilers.The results suggest that: 1)Compared to the control group,Both the protease addition group and the bacterial enzyme combination addition group significantly increased the duodenal villus height and significantly decreased the ileal crypt depth in broilers(P <0.05);The Enterococcus faecalis group significantly increased the duodenal and ileal villus height to crypt depth ratio(V/C)(P <0.05),The Bacillus coagulans group also significantly increased the ileal villi crypt ratio(P < 0.05).Compared to the antibiotic group,The Enterococcus faecalis group significantly reduced the depth of the duodenal crypts and significantly increased the V/C of the duodenum(P < 0.05).2)Compared to the control group,Clostridium butyricum and Enterococcus faecalis groups significantly reduced diamine oxidase(DAO)activity in broiler serum(P < 0.05).Compared to the antibiotic group,Protease,Clostridium butyricum,Enterococcus faecalis and Bacillus coagulans groups significantly reduced serum DAO activity in broilers(P < 0.05).3)Comparison with the control group,Duodenal mucin 2(MUC-2)gene expression was significantly higher in all test groupsand gene expression of duodenal trefoil factor TEF3 was significantly increased in the protease,Enterococcus faecalis and Bacillus coagulans groups(P < 0.05).Significantly higher jejunal closure protein 1(Claudin-1)gene expression in the antibiotic,protease,Clostridium butyricum and Bacillus coagulans groups,The expression of jejunal TEF3 gene was also significantly up-regulated in the protease(P<0.05),Clostridium butyricum and Enterococcus faecalis groups(P<0.05).Gene expression of ileal occludin(Occludin),Mucin-2,Claudin-1 was significantly up-regulated in the Enterococcus faecalis group(P<0.05);Protease group and Bacillus coagulans group significantly up-regulated Claudin1 gene expression in broiler ileum(P<0.05).4)Comparison with the control group,Relative m RNA expression of duodenal tumour necrosis factor alpha(TNFα)and interleukin 2(IL2)was significantly increased in each test group(P<0.05).Relative m RNA expression of jejunal TNFα,IL2 and IL2 in the ileum was significantly higher in the group with the addition of each bacterial enzyme combination(P<0.05).In addition,the levels of intestinal pro-inflammatory factors in the added group of each bacterial enzyme combination were comparable to those in the antibiotic group.Experiment 3: Investigating the effect of protease and probiotic addition on the cecum microbiota of broiler chickens.The results indicate that: Comparison with the control group,The Chao1 index,Faith_pd index and Shannon index of the Protease,Enterococcus faecalis and Bacillus coagulans groups were all increased to some extent;The Enterococcus faecalis group increases the relative abundance of Bacteroidetes and Bacteroides.The antibiotic,protease,Clostridium butyricum and Bacillus coagulans groups increased the relative abundance of Firmicutes.Antibiotic group and Clostridium butyricum group increased the relative abundance of Phascolarctobacterium.Compared to the control and antibiotic groups,The relative abundance of beneficial bacteria such as Bacteroidetes and Bacteroides.was increased in the combined Bacillus enzyme addition group.In conclusion,the combination of protease and probiotics can improve the intestinal morphology and structure of broilers,increase the abundance of beneficial bacteria,and promote intestinal health of broilers.Adding protease in combination with Enterococcus faecalis and Bacillus coagulans could improve the growth performance and enhance the intestinal physical barrier function of broilers.Protease combined with Clostridium butyricum and Enterococcus faecalis can enhance intestinal mucus barrier function,relieve intestinal inflammation,improve immunity,and promote the expression of amino acid transporters in small intestine.The combination of protease and Enterococcus faecalis can also increase intestinal alpha diversity index,improve intestinal p H,and promote intestinal health of broilers.Under the conditions of this experiment,considering the actual addition effect,adding protease and Enterococcus faecalis on the diet of broilers has the best effect,reaching the level similar to that of antibiotic group,which is of great significance in the current anti-antibiotic culture environment.