The γ-ray Irradiation Treatment Of Entermorpha Polysaccharide And The Effects On The Performance,Antioxidant Capacity,and Intestinal Barrier Functions In Juvenile Broilers

Intestinal diseases of broilers cause great losses to the broiler breeding industry.Maintaining intestinal health of broilers plays an important role in maximizing the growth performance of broilers.Enteromorpha polysaccharide（EP）,which could improve growth performance,enhance immunity and protect intestinal health,was the main active ingredient of Enteromorpha.Its biological activity is closely related to its molecular structure.And irradiation can destroy the glycosidic bonds of polysaccharides,reduce the molecular weight of polysaccharides,expose the functional groups of polysaccharides,and thus improve the antioxidant effect of polysaccharides.Therefore,this experiment was conducted to investigate the effects ofγ-irradiated Enteromorpha polysaccharide on the performance,antioxidant capacity and intestinal barrier functions of broilers on the basis of exploring the effects ofγ-ray irradiation on the physical and chemical properties of Enteromorpha polysaccharide,such as molecular weight（molecular structure）and antioxidant activity in vitro.This study is divided into two parts.Experiment 1:Effects ofγ-ray irradiation on physicochemical properties and antioxidant activity of Enteromorpha polysaccharide in vitro.The aim of this study was to investigate the effects of different doses ofγ-ray irradiation on the molecular weight,physical and chemical properties and antioxidant activity of Entermorpha polysaccharide in vitro.Four kinds of EP were obtained by using⁶⁰Coγray as irradiation source with the irradiation intensity was 2×10⁶Ci,the irradiation dose was 25,50,100,150 kGy,respectively and the dose rate was 4 Gy/s.The molecular weight and physicochemical properties of polysaccharide before and after modification were analyzed with unirradiated EP as control,and the irradiated polysaccharide with high antioxidant activity were screened out.The results were as follows:1)Compared with the control group,the average molecular weight（Mn）and average molecular weight（Mw）of irradiated samples decreased with the increase of irradiation dose（P<0.05）,the polydispersity slightly decreased to about 1.25,and remained at a relatively constant level under different doses;2)The EP samples treated with different irradiation doses had similar FT-IR spectra,indicating thatγradiation had no significant effect on EP functional group status;3)Scanning electron microscopy showed that EP in the control group was a complete sphere of different sizes.After irradiation,the fragment size of the sample was smaller than that of the untreated sample,and the number of small fragments increased with the increase of the irradiation dose.4)At 25,50,100 and 150kGy,the solubility of EP is 99.10%,99.50%,99.82%and 99.90%,respectively,and the initial solubility of EP is 98.70%while at 0 kGy.5)After radiation treatment,the scavenging ability of EP on DPPH radical and O₂^·-radical was significantly enhanced,and the highest scavenging ability was obtained at 150 kGy dose;when the irradiation dose was 150 kGy,EP’s scavenging ability of hydroxyl radical（HO^·）was significantly enhanced.In conclusion,EP irradiated at 150 kGy has the best antioxidant biological function.Therefore,EP irradiated at 150 kGy（IEP）is selected for the next animal experiment.Experiment 2:Effects of native andγ-irradiated Enteromorpha polysaccharide on the performance,antioxidant capacity and intestinal barrier functions of broilers.A total of 300 healthy one-day-old AA broilers with similar body weight were randomly divided into 3 treatment groups with 10 replicates per group and 10 broilers per replicate.The three treatment groups were control group,Entermorpha polysaccharide（EP）group andγ-irradiated Entermorpha polysaccharide（IEP）group.Broilers in the control group were fed a basal diet,and those in the EP group were fed the basal diet supplemented with 400 mg/kg EP,and those in the IEP group were fed the basal diet supplemented with400 mg/kg IEP,the trail lasts 21 days.The test results are as follows:1)There were no significant differences in growth performance parameters among the three groups,but body weight in the 21 d（P=0.053）and average daily gain（P=0.054）in the IEP group had an increasing trend compared with control group and EP group;2)Both the EP and IEP had higher serum T-AOC（P<0.05）and SOD（P<0.05）activities than those in the control group,additionally,the IEP tended to increase serum CAT activity（P=0.056）compared with the control group.IEP-administrated birds showed higher ileal CAT mRNA level（P<0.01）when compared with that in the EP group;the ileal greater relative mRNA level of GPx2（P<0.05）,HMOX1（P<0.05）,Nrf2（P<0.05）and Keap1（P<0.05）were presented in the EP group when compared with those in the control group;3)The IEP group had highest ileal villus height（VH）（P<0.01）and villus height to crypt depth（V/C）ratio（P<0.01）among the three groups;the ileal gene expression of Mucin-2（P<0.01）and Occludin（P<0.05）in the EP group was higher than that in control group;the ileal gene expression of and Occludin（P<0.01）in the IEP group was higher than that in control group;4)In the ileum,compared with the control group,the IEP supplementation significantly inhibited the key inflammatory-related factor NF-κB（P<0.01）,HSP70（P<0.01）,HSP90（P<0.01）,IL-1β（P<0.05）,IL-6（P<0.01）,TNF-α（P<0.05）,and IFN-γ（P<0.05）and the EP supplementation showed significant decrease in the mRNA of HSP70（P<0.01）,HSP90（P<0.01）,and IL-1β（P<0.05）;5)In the cecal microflora,increased levels of Chao1（P<0.05）,ACE（P<0.05）,and Shanonn indices（P<0.05）in the IEP treatment were observed as compared to the control group.Simpson（P<0.05）index was significantly decreased by EP or IEP treatment compared to the control group;forβ-diversity,Venn diagram showed 417 shared OTU in the three groups,and 18,7,and 31 unique OTU in Con,EP,and IEP group,respectively.The principal coordinate analysis（PCA）presented closely clustered bacterial community among samples in the three groups.At genus level,the IEP supplement showed greater abundances of Barnesiella（P<0.05）,Negativibacillus（P<0.05）and Rikenella（P<0.05）when compared with those in the EP group.Additionally,the EP supplement significantly increased the relative abundances of Alistipes（P<0.05）when compared to the control group;6)No significant differences were observed in the cecal acetate,propionate,butyrate and velarate among three groups（P>0.1）.In conclusion,γ-irradiation did not affect the antioxidant capacity of EP,and IEP improved the ileal morphology of broilers,kept the ileum at a low level of inflammation,and promoted the cecal microecological balance,which was helpful to further understand the effect of irradiation on enhancing the function of EP,and provided a basis for the production and application of irradiated polysaccharide additives.