Identification Of Active Components Of Walnut Green Husk Extract And Their Effects On Antioxidant Performance,Intestinal Morphology And Intestinal Flora Of Laying Hens In Late Laying Period

This experiment was conducted to identify the main active components and investigate the antimicrobial activities of different components of walnut green husk,and the effects of walnut green husk and its extracts on nutrient apparent availability,antioxidant performance,intestinal mucosal morphology and intestinal microflora of laying hens in late laying period,so as to provide a theoretical basis for the application of walnut husk extract in laying hens.The experiment was divided into two parts:Experiment one: The walnut green husk powder was extracted with 90% ethanol,and the solid residue was extracted with 20% ethanol.The 20% ethanol extract was separated by macroporous resin;90% ethanol extract was extracted with petroleum ether and ethyl acetate to obtain a petroleum ether extraction part and an ethyl acetate extraction part.The ethyl acetate extraction part was separated by silica gel column chromatography,and the samples were separated and purified further.The C and H spectra were determined by NMR.The molecular structure of the compounds was determined according to the spectral data and references.The inhibition zone of seven eluting components was determined with Escherichia coli as the test bacteria.The results showed that 26 compounds in walnut green husk extract were isolated and identified.The inhibition zone diameter of component D of 90% ethanol extract was the largest against on the tested bacteria(P<0.05),and it was not significantly different from the ampicillin inhibition zone diameter for the test bacteria(P>0.05).Experiment two: Walnut green husk and its extract were added into the late laying hens’ diet to explore the effects on the apparent utilization of nutrients,body antioxidant performance,intestinal mucosal morphology and intestinal flora in late laying hens.A total of 180 healthy 66 weeks old Hy-Line Variety Brown layers with body weight of 2.00 kg and laying rate of 85% were randomly divided into 3 groups,with 15 replicates in each group and 4 chickens in each replicate.The control group was fed with basal diet,and the walnut green husk group(group Ⅰ)was fed with the basic diet added with 1.0% walnut green husk powder,and the extract group(group Ⅱ)was fed with the basic diet added with 1.0% walnut green husk extract extracted with 25% ethanol.The pre-test period was 7 days and the trial period was 28 days.The pre-test period is 7 days,and the trial period is 28 days.In the last 7 days of the experiment,0.2 kg of excrement was collected every day,and 1.0 kg of basal diet was collected at the same time,and the apparent nutrient utilization rate was determined.At the end of the experiment,slaughter was carried out,liver tissue and blood samples were collected to determine the body’s antioxidant properties;intestinal tissue samples were collected to make intestinal tissue slices;the intestinal contents were collected to extract DNA for 16 S r DNA amplicon sequencing.The apparent availability of GE decreased significantly(P<0.05),while the apparent availability of Ca increased significantly(P<0.05),and the other nutrients availability were not significantly influenced by walnut green husk extract(P>0.05).Except for the ether extract and P,the apparent availability of most nutrients were significantly reduced by walnut green husk(P>0.05).Supplementation with walnut green husk and its extract had no influence on activity of glutathione reductase(GR),glutathione peroxidase(GSH-Px),total antioxidant capacity(T-AOC)in liver and serum,and concentration of malondialdehyde(MDA)in serum(P>0.05).The content of MDA in liver was significantly decreased by walnut green husk extract(P<0.05).The results showed that there was no significant effect on villus height,crypt depth and V/C value of duodenum,jejunum and ileum by addition of walnut green husk or its extract(P>0.05).There was no significant effect on duodenal microbial diversity(P>0.05).However,microbial diversity in ileum were significantly decreased by walnut green husk(P<0.05).Ileal Firmicutes increased by walnut green husk,and decreased by the extract.Duodenal and ileal Lactobacillus increased by walnut green husk.After adding the extract,ileal Lactobacillus decreased.It can be seen that adding 1% walnut green husk 25% ethanol extract in the late stage of laying can increase the apparent utilization of calcium in the body and inhibit liver lipid peroxidation.Direct addition of 1% walnut green husk will reduce the nutrient utilization of the diet,and cannot achieve the same effect as walnut green husk extract.It can be concluded that dietary addition of walnut green husk or its extract to laying hens does not change the intestinal structure and morphology.Walnut green husk significantly reduces the diversity and abundance of bacteria in the ileum.The abundance of Firmicutes and Lactobacillus in intestinal tract was increased by adding walnut green husk,and these was decreased by adding extract of walnut green husk.So,walnut green husk extract is a potential feed additive worthy of further development.