Effects And Mechanisms Of Glucose Oxidase As Feed Additive On Growth Performance,Immune And Antioxidant Functions In White-feathered Broilers

The use of antibiotics in animal husbandry was widely adopted.However,due to the emergence of antibiotic resistance and other related issues,many countries have banned the use of antibiotics in animal feed.Therefore,the development of green and safe alternatives to antibiotics for feed has become a major requirement for the current animal husbandry industry.This thesis studies the effects and mechanisms of using glucose oxidase(GOD)as a potential alternative to growth-promoting antibiotics on the growth performance,immunity,and antioxidant capacity of broiler chickens.Additionally,the thesis laid the groundwork for the establishment of industry standards for the activity measurement of GOD.Experiment 1: We conducted a survey on the current application status of feed-grade GOD in the market and the establishment of relevant standards.We also conducted experiments to determine the key parameters for measuring the enzymatic activity of GOD.And we assessed the accuracy and precision of the standard method,leading to the development of a draft for the agricultural industry standard titled "Determination of Feed-grade Glucose Oxidase Activity." Currently,feed-grade GOD products in the Chinese market are mainly produced through liquid deep fermentation using specific strains of Penicillium notatum or Aspergillus niger.These products exist in liquid and solid forms.There are no nationally published standards for measuring the enzymatic activity of feed-grade GOD,with only one local standard and one organizational standard available.The standards for GOD enzymatic activity measurement in various enterprise are inconsistent.Based on the measurements of actual samples,the spectrophotometric method used in this study for determining GOD enzymatic activity is characterized by short detection time and small sample volume,meeting the requirements for accuracy and precision.Experiment 2: Effects of three different doses of GOD on the growth performance,indicators related to immune,antioxidant,as well as intestinal toxicity markers in the serum of broilers.And the comparison of the effects between the GOD supplementation and the Ant groups were conducted.A total of 420 oneday-old male Cobb 500 white feather broiler were randomly divided into five treatment groups,with six replicates in each group and 12 chickens in each replicate.The experiment lasted for 42 days.The treatment groups included a basal diet group(Ctr)and groups fed the basal diet supplemented with 50mg/kg chlortetracycline(Ant),300 U/kg,600 U/kg,and 1200 U/kg GOD(GOD300,GOD600,and GOD1200,respectively and the enzyme activity is determined by the industry standard method specified in Experiment 1).The results showed no significant differences in production performance among the five treatment groups(P > 0.05).After feeding the GOD-added diet,the levels of immunoglobulin A,transforming growth factor-β,superoxide dismutase,interleukin-10,and glutathione peroxidase activity in the serum of white-feathered broiler were significantly higher than those in the control group(P < 0.05),while the levels of alkaline phosphatase,malondialdehyde,and D-lactic acid were significantly lower(P< 0.05),and these changes were more pronounced in the GOD1200 group,with a trend similar to that of the antibiotic group.However,the changes in the serum levels of immunoglobulin A,glutathione peroxidase activity,and D-lactic acid in the antibiotic group were consistent with those in the control group at 42 days of age,with the former two being significantly lower than those in the GOD1200 group(P < 0.05),and the D-lactic acid significantly higher than that in the GOD1200 group(P < 0.05).In summary,adding a certain concentration of GOD to the feed can improve the functions of immune,antioxidant,and intestinal permeability,further promote healthy growth of white-feathered broiler.Experiment 3: The effects of GOD on intestinal barrier and microbial flora of white-feathered broiler(the experiment design is the same as Experiment 1).The effect of GOD on the results of immunohistochemistry and gene quantification of jejunal mucosa,as well as sequencing of the 16 sr RNA of chyme from the cecum of broiler,were evaluated.Results showed that although GOD did not significantly affect the jejunum morphology of the broiler(P > 0.05),it helped to reduce intestinal mucosal permeability and significantly increased the expression of tight junction protein ZO-1,Mucin-2(P < 0.05),and the concentration of secretory immunoglobulin A(P < 0.05)in the mucosa,which facilitated the formation of a stronger intestinal immune barrier and physical barrier in broilers.Although GOD did not significantly affect the diversity of microbial flora in the broiler cecum(P > 0.05),it caused significant differences in the composition of microbial genera between groups(P < 0.05).Colidextribacter,Oscillibacter,Flavonifractor,and Oscillospira,were identified as microbial biomarkers after the addition of GOD.Group comparisons revealed that the composition of microbial genera in the cecum of broilers was more complicated at 42 days of age than at 21 days of age.For the GOD group,especially when GOD1200 comparing with the antibiotic group,the abundance of the above-mentioned four biomarker genera,as well as beneficial genera such as Lactobacillus,UCG-005,and Roseburia,significantly increased(P < 0.05).By targeting quantitative analysis of short-chain fatty acids,which are important metabolic products of broiler cecal microbiota,it was found that most of them were significantly higher in the GOD1200 group.According to the functional prediction results of PICRUSt2,it is speculated that GOD can enhance the digestive and absorptive capacity of amino acids,lipids,energy metabolism,and proteins in the cecal microbiota of broilers.This improvement is beneficial for broilers to combat oxidative stress and play a role in protecting the intestine from exogenous contamination.To sum up,the addition of GOD can enhance the intestinal function of broilers from the physical mechanical,immune,and microbial barrier levels,and its effects on the intestine show more advantages than those of antibiotic additives.Experiment 4: Molecular mechanisms of GOD regulation on immune,antioxidant,and digestive absorption functions in broilers.Based on the results of Experiments 1 and 2,the jejunal mucosa of 42-day-old broilers in the GOD1200,Ctr,and Ant groups were selected as the research objects.Transcriptome sequencing results showed that adding GOD to the feed diet resulted in 1359 differentially expressed genes(DEGs)(P < 0.05)in the jejunal mucosa of broilers,including 929 upregulated genes and 430 downregulated genes,and of which 650 were significantly differentially expressed(P < 0.01).The top 10 significantly upregulated genes were mainly related to improving the intestinal barrier,reducing inflammation,promoting nutrient absorption,and antioxidant functions.Through GO analysis,it was found that a large number of DEGs were related to immune function,which were enriched in 17 immunerelated KEGG pathways.Among them,the antigen processing and presentation and the intestinal immune network for Ig A production were significantly enriched(P < 0.05).The DEGs related to digestive function were enriched in 9 KEGG pathways,of which 4 pathways related to protein,fat,carbohydrate digestion and absorption,and mineral absorption were significantly enriched(P < 0.05).Based on these results,key genes that may play an important role in regulating the immune and digestive absorption functions of broilers after GOD addition were screened,including PIGR,MEP1 B,SLC16A10,MEP1 A,CD36,and SLC2A5.The key genes affecting antioxidant function,SOD1,CAT,and NQO1,were also significantly upregulated after GOD1200 addition(P < 0.05).The mechanism of antibiotics on broilers may significantly differ from that of GOD.In comparison,the antioxidant-related genes GPx3,GPx7,and GPx8 were significantly upregulated(P < 0.05)among the antioxidant-related genes,and only one digestive function-related pathway significantly enriched(P < 0.05).However,it was found that both aureomycin and GOD have commonalities in their immunomodulatory mechanisms,as their DEGs were significantly enriched in 17 immune-related pathways,with both groups of DEGs up-regulated in the antigen processing and presentation pathway,and there were 8 common genes between the two groups in this pathway.In conclusion,the GOD-supplied diet can improve the health status of broiler chickens by regulating key genes and pathways associated with immune,antioxidant,and digestive functions in the jejunum,and the effects on immune regulation has similar part to that of antibiotic additives in broilers.In summary,the addition of GOD can improve the immunity and antioxidant function of white feather broiler,which is beneficial for the development of a stronger immune defense system.In addition,both the microbial functional prediction results and transcriptome sequencing results show that GOD also has a promotion effect on the digestion and absorption of nutrients.It exhibits a similar trend to aureomycin additives in terms of most functional regulation effects,and shows greater advantages in its impact on intestinal barrier function and microbial flora.This study systematically demonstrates that the use of GOD as a feed additive in the breeding process of broilers can serve as a substitute for growthpromoting antibiotics,providing data support and theoretical guarantee for further expansion of its application.