| Environmental stress is one of the main factors that endanger livestock and poultry production,which often has adverse effects on growth,product quality,production performance and immune performance of livestock and poultry,and then causes huge economic losses.Chinese medicine feed additives have multiple effects,which can solve the above problems caused by environmental changes to a certain extent.As a precious plant for both medicine and food,Eucommia ulmoides Oliver is rich in a variety of active components,which has high medicinal health value and has a good application prospect in the production of feed additives.Eucommia ulmoides Oliver is mainly used by taking bark,and leaves are generally discarded and rich in resources.The separation,preparation and functional elaboration of effective components in Eucommia ulmoides Oliver leaves are the basic premise of research and development and application of Eucommia ulmoides Oliver functional feed additives.In this paper,the active components of Eucommia ulmoides Oliver leaves(Eucommia ulmoides extracts)were isolated and prepared and the stress protective effects of chicks were studied.The main findings are as follows:1.Extract process of Eucommia ulmoides extracts.The ratio of solid to liquid,extraction time and extraction times were optimized by single factor experiment;the optimum extraction process parameters were obtained by orthogonal test: the ratio of solid to liquid was 1:32,the extraction time was 2h,and the extraction time was 5times.The extraction rate of Eucommia ulmoides extracts is 46.03%,and the content of chlorogenic acid is 21.06mg/g.Optimize and establish the process as follows:Drying Eucommia ulmoides leaves at 80-90℃→Pulverize and sieving with 80 mesh sieve→Add 32 times volume distilled water and soak for 2h→Water bath at 80-90℃for 2.5h → 10000 rpm,Centrifuge for 5min → Supernatant was dried at 60 ℃ →Pulverize and sieving with 80 mesh sieve→Eucommia ulmoides extracts.2.Components of Eucommia ulmoides extracts.LC-MS was used to analyze the biological active components of Eucommia ulmoides extracts.21 kinds of chemical components can be detected in Eucommia ulmoides extracts,including 6 iridoids,5flavonoids,4 phenylpropanoids,2 lignans and 4 other components,including higher content of antioxidant substances such as Asperulosidic acid(25.87mg/g),chlorogenic acid(16.92mg/g),Kaempferol(10.03mg/g)and aucubin(7.87mg/g).3.Protective effect of Eucommia ulmoides extracts on heat stress chicks.Chicks were alternately and repeatedly treated with heat stress at "35-37℃＿6h→23℃＿18h" for 15 days.Chicks in the experimental group were fed with 0.8% Eucommia ulmoides extracts.The feed intake,growth and development,pathological changes,antioxidant and immune substances were analyzed.Compared with the control group,the weight and daily gain of chicks in Eucommia ulmoides extracts group increased by 10.03%(P<0.05)and 18.17%(P<0.05),respectively,and the feed-weight ratio decreased by 13.04%(P<0.05);The indexes of heart,spleen,bursa of fabricius,duodenum and pancreas in the experimental group were higher,and the development was not significantly affected;The structure of heart,duodenum and gizzard of chickens in heat stress experimental treatment group is relatively complete and there are few inflammatory cells,while the tissues and organs of chickens in control group are seriously damaged in the same period.;Serum T-AOC,SOD,GSH-PX increased by 9.54%(P>0.05),7.93%(P<0.05),7.49%(P<0.05),MDA decreased by 29.99%(P<0.05);Serum Ig A,Ig G,Ig M increased by 10.95%(P<0.05),8.43%(P<0.05),3.51%(P<0.05).The experimental results showed that Eucommia ulmoides extracts has a good protective effect on chickens under high temperature stress,which can improve the influence of high temperature on the growth and development of chickens,reduce the damage of tissues and organs caused by environmental stress,improve the antioxidant level and enhance the immune regulation function of chickens.4.Protective effect of Eucommia ulmoides extracts on cold stress chicks.Chicks were treated with alternating cold stress at "10-14℃＿6h→23℃＿18h" for 15 days,and the chicks in the experimental group were fed with 0.8% Eucommia ulmoides extracts.The analysis of feed intake,growth and development,pathological changes,antioxidant and immune substance content showed that the weight and average daily gain of chicks in the Eucommia ulmoides extracts group increased by 15.88%(P<0.05)and 27.25%(P<0.05)compared with those in the control group at 15 days,while the feed-weight ratio decreased by 32.81%(P<0.05)at 7 days;The indexes of liver,bursa of fabricius,duodenum and pancreas in experimental group were higher than those in control group;Under the condition of low temperature stress,the structure of heart,duodenum and muscle stomach of chicks is relatively complete,arranged regularly and there are few inflammatory cells,while the tissues and organs of control chicks are seriously damaged;Serum T-AOC,SOD and GSH-PX increased by 13.59%(P<0.05),6.49%(P<0.05)and 3.24%(P>0.05)respectively,while MDA decreased by 21.85%(P<0.05).Serum Ig A,Ig G and Ig M increased by 3.23%(P<0.05),13.70%(P<0.05)and 2.83%(P<0.05),respectively.Eucommia ulmoides extracts can obviously improve and reduce the effect of low temperature stress on chicks.5.Molecular regulation mechanism of Eucommia ulmoides extracts on stress protection of chicks.The c DNA library of spleen of chicks under heat stress and cold stress for 0d,11 d and 15 d was constructed and the transcriptome was sequenced.GO and KEGG enrichment analysis showed that the differential genes of Eucommia ulmoides extracts group annotated that the number of immune and defense-related pathways was 25 and 7 respectively,mostly compared with the control group,under heat stress;There are 16,12 and 3 pathways related to immunity,defense and growth in Eucommia ulmoides extracts under cold stress.Co-expression analysis of transcription factors showed that genes related to immunity,growth and development of chicks in Eucommia ulmoides extracts group were co-expressed with transcription factors related to growth and development and immune regulation,such as Homeobox,CUT,TF-b ZIP,Frok＿head,PAX,ZBTB and zf-MIZ under heat and cold stress.The results of RNA-seq showed that feeding Eucommia ulmoides extracts can enhance the molecular regulation function of chicks by initiating gene transcription of immune-related,growth and development,defense and so on,and achieve stress protection and regulation of chicksThe above research shows that Eucommia ulmoides extracts has obvious protective effect on stressed chicks treated by high temperature and low temperature environment,and can reduce the influence of adverse environment on the growth and development of chicks.Its protective effect can be realized by starting the transcription of related regulatory pathway genes,increasing the content of antioxidant substances in serum and enhancing the immune function of the body.The research results laid a foundation for the development and utilization of Eucommia ulmoides leaves feed additives. |
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