Effects Of Seabuckthorn Leaves And Flavonoids Of Seabuckthorn Leaves On Production Performance, Blood Indexes And Lipid Metabolism Of Altay Sheep

Object:This study was conducted to investigate the optimal parameters of ultrasonic extraction of flavonoids from seabuckthorn leaves,the effects of seabuckthorn leaves and flavonoids of seabuckthorn leaves on Altay sheep performance,slaughter performance,serum index and immune index and lipid metabolism,and to reveal the regulation mechanism of seabuckthorn leaves and flavonoids of seabuckthorn leaves on lipid metabolism at organ,tissue,cell,molecular and metabolic levels of Altay sheep.Methods:The single factor experiment and orthogonal design experiment were used to determine the optimum conditions for extracting flavonoids from seabuckthorn leaves.taking ethanol volume fraction,solid-liquid ratio,ultrasonic time and ultrasonic power as test factors,and taking the extraction rate of flavonoids from seabuckthorn leaves as index.Using single factor randomized trial design,105 male Altay lambs with similar health,age and weight were randomly divided into control group?Con group?,3 seabuckthorn leaves test groups?SJY-1 group,SJY-2 group and SJY-3 group?and three flavone of seabuckthorn leaves test groups?SLF-1 group,SLF-2 group and SLF-3 group?.Each group had three replicates,with 5repeats between replicates.The control group was fed with basic diet.The SJY-1 group,SJY-2group and SJY-3 group were fed with 2.5%,5.0%and 7.5%seabuckthorn leaves in the basic diet to replace the same amount of wheat straw.The SLF-1 group,SLF-2 group and SLF-3 group were fed with 0.10%,0.25%and 0.50%flavonoids of seabuckthorn leaves.The experimental period was 63 days,including 7 days preliminary trial period and 56 days in trial.The feeding standard was formulated according to Chinese sheep feeding standard?NY-T 816-2004?.The sheep house was disinfected before the experiment.The experimental sheep were fed in repetitive groups twice a day at 8:00 and 19:00,respectively.Each day,they were fed with a little food left.drinking water freely,The methods were 8:00 and 19:00 per day respectively,with a slight surplus of food.The test sheep drank water freely every day.The experimental sheep were vaccinated and insect repellent and stomach-strengthening in the preliminary trial period.Before morning feeding on the 56th day of the trial period,6 sheep from each group were randomly selected to collect blood for 5 ml,and centrifuge at the scene.The serum was collected and packed into 3 parts,take it back to the laboratory with liquid nitrogen,store it in the refrigerator at-80?refrigerator,and one was measure the serum biochemical indexes by automatic biochemical analyzer,and one was detection of Immunocytokines by Immune Kit,and one was analyzed metabolomics determination by gas chromatography-mass spectrometry.NIST and KEGG databases were used to identify differentially metabolized metabolites and analyze their metabolic pathways.Before morning feeding on the 56th day of the trial period,6 sheep from each group were randomly selected,and about 50ml of rumen chyme was extracted by self-made in vivo rumen sampler.Rumen chyme was collected and its pH value was determined rapidly.After treatment,The chyme samples were brought back to the laboratory with liquid nitrogen and frozen at-70?refrigerator for the determination of NH₃-N and VFA.On the second day after the end of the trial period,3 sheep were slaughtered in each group.After slaughter,tail fat,abdominal fat,subcutaneous fat,biceps femoris muscle and intermuscular fat were quickly taken with sterilized living sampling forceps.Three repeated samples were taken from each part of each sheep.Each sample was taken about 2g,placed in a Eppendorf tube,immediately put into a liquid nitrogen tank,and brought back to the laboratory.The samples were stored in a refrigerator at-70 C for testing.Results:1)The single factor results of extracting flavonoids from seabuckthorn leaves were as follows:when the volume fraction of ethanol was 75%,the extraction rate increased significantly?P<0.05?;when the ratio of solid-liquid was 1:35?g/mL?,the extraction rate reached the highest?P<0.05?;when the extraction time was 45 minutes,the extraction rate increased significantly;and when the ultrasonic power was 90%,the extraction rate reached the highest.The results of orthogonal experiment were as follows:the optimal combination of four factors was A2B2C3D3,i.e.,75%ethanol volume fraction,solid-liquid ratio of 1:35,ultrasonic time of 48minutes and ultrasonic power of 95%.The factors affecting the extraction of flavonoids from seabuckthorn leaves by range analysis were as follows:ultrasonic time?A?>ultrasonic power?D?>ethanol volume fraction?C?>solid-liquid ratio?B?.2)Compared with the Con group,the seabuckthorn leaves groups and significantly increased FBW and ADG?P<0.05?,the SLF-2 and SLF-3 groups significantly increased ADG,the SJY-2,SJY-3 and SLF-2 groups ignificantly increased ADFI?P<0.05?,and the feed conversion rate of the experimental group increased.The net meat percentage was significantly increased?P<0.05?and the fat percentage and abdominal fat percentage were decreased?P<0.05?by adding medium to high proportion of seabuckthorn leaves and flavonoids of seabuckthorn leaves.3)With the addition of seabuckthorn leaves and flavonoids of seabuckthorn leaves,acetic acid,propionic acid,butyric acid and VFA in the rumen of Altay sheep increased significantly?P<0.05?.The SJY-3 group significantly decreased the pH value?P<0.05?.There was no significant difference in acetic acid/propionic acid and acetic acid/VFA between groups?P>0.05?.The concentration of NH₃-N in rumen was significantly decreased in the middle and high proportion of flavonoids in seabuckthorn leaves and seabuckthorn leaves?P<0.05?.4)Compared with Con group,in the abdominal fat,FAS gene expression was extremely significantly decreased in SJY-1 group,SJY-2 group and SJY-3 group?P<0.01?,FAS gene expression was significantly decreased in SLF-1 group,SLF-2 group and SLF-3 group?P<0.05?,HSL gene expression was extremely significantly increased in SJY-3group?P<0.01?,HSL gene expression was significantly increased in SJY-1 group,SLF-2 group and SLF-3 group?P<0.05?,and Leptin gene was significantly decreased in SLF-2 group and SLF-3 group?P<0.05?.In tail fat,FAS gene expression was extremely significantly decreased in SJY-2 and SJY-3 groups?P<0.01?,and significantly decreased in SJY-1 group?P<0.05?;HSL gene expression was extremely significantly increased in SJY-2 and SJY-3 groups?P<0.01?;HSL and Leptin gene expression were significantly increased in SLF-2 and SLF-3 groups?P<0.05?.In subcutaneous fat,FAS gene expression in SJY-3 group,SLF-1 group,SLF-2 group and SLF-3 group decreased significantly?P<0.05?.In intramuscular fat,FAS gene expression was significantly increased in SJY-3 group and SLF-3 group?P<0.05?;HSL gene expression was extremely significantly decreased in SJY-3 group?P<0.01?;HSL gene expression was significantly decreased in SJY-2 group,SLF-2 group and SLF-3 group?P<0.05?.In muscle,FAS gene expression was extremely significantly increased in SJY-3 group?P<0.01?,and significantly increased in SLF-2 and SLF-3 groups?P<0.05?;HSL gene expression was significantly decreased in SLF-2 and SLF-3 groups?P<0.05?.There was no significant difference in other indicators among groups?P>0.05?.5)The serum total protein in SLF-1 group increased extremely significantly?P<0.01?,while that in SJY-2 group and SLF-2 group increased significantly?P<0.05?.There was significant increase in albumin in SLF-1 and SLF-2 groups?P<0.05?.Globulin in SLF-1 group increased extremely significantly?P<0.01?,while globulin in SJY-2 group and SLF-2 group increased significantly?P<0.05?.Serum urea nitrogen in SJY-3group and SLF-3 group decreased extremely significantly?P<0.01?,while serum urea nitrogen in SJY-2 group decreased significantly?P<0.05?.Serum cholesterol and high density lipoprotein cholesterol increased significantly in SJY-2 and SLF-2 groups?P<0.05?.Serum low density lipoprotein cholesterol increased extremely significantly in SJY-2 group and SLF-1 group?P<0.01?,and increased significantly in SJY-1 group,SJY-3 group and SLF-2 group?P<0.05?.Alkaline phosphatase decreased significantly in SJY-2 group,SJY-3 group and SLF-3 group?P<0.05?;other indicators were not significant among groups?P>0.05?.Serum CD4 concentration in SJY-3 group and SLF-3 group increased significantly?P<0.05?.Serum IL-1 concentration in SJY-2 group,SLF-2 group and SLF-3 group increased significantly?P<0.05?.Serum IFN-gamma concentration in SJY-1 group and SLF-2 group increased significantly?P<0.05?.6)By added seabuckthorn leave,the contents of three amino acids and glycerol,glucose,galactose,fumaric acid,linoleic acid and octadecanoic acid in serum of Altay sheep were significantly increased?P<0.05?,and the contents of beta-hydroxybutyric acid,palmitic acid,oleic acid,oleic amide,stearamide and cholesterol were significantly decreased?P<0.05?.Seabuckthorn leaves are involved in 12 metabolic pathways.Flavonoids of seabuckthorn leaves could significantly increased five amino acids,four organic acids and three carbohydrates in serum of Altay sheep?P<0.05?,and significantly decreased beta-hydroxybutyric acid,palmitic acid,oleic amide and cholesterol?P<0.05?.Flavonoids of seabuckthorn leaves are involved in 20 metabolic pathways.Conclusion:Under the conditions of this experiment,?1?Establish and optimize the ultrasonic extraction technology of flavonoids from seabuckthorn leaves.The optimum parameters are 75%ethanol volume fraction,1:35?g/mL?solid-liquid ratio,95%ultrasonic power and 48 min extraction time.?2?Feeding experiments showed that flavonoids of seabuckthorn leaves and seabuckthorn leaves could significantly increase the content of VFA and each components in rumen chyme of Altay sheep,reduce the concentration of NH₃-N,promote rumen metabolism,and microbial protein synthesis,thereby increasing daily gain,feed intake and net meat rate,reducing carcass fat rate and abdominal fat rate of Altay sheep.?3?By analyzing the serum biochemical indexes of Altay sheep,it was found that seabuckthorn leaves and flavonoids of seabuckthorn leaves could increase serum total protein,reduce serum urea nitrogen,promote the growth of Aletay sheep;increase HDL-C promote the transportation and metabolism of cholesterol,reduce fat;reduce the content of alkaline phosphatase,protect normal physiological functions of liver and kidney.By measuring the cellular immune factors,it was found that seabuckthorn leaves and flavonoids of seabuckthorn leaves could regulate a single immune factor,which might improve its immune performance.4)Metabonomics analysis showed that flavonoids of seabuckthorn leaves and seabuckthorn leaves regulated serum glycine,valine,palmitic acid,oleic amide,cholesterol,etc,affecting the amino acid and lipid metabolism pathways.The changes of these substances result in lipid synthesis and decomposition.It has been confirmed that flavonoids of seabuckthorn leaves and seabuckthorn leaves could change the expression level of lipid metabolism related gene.