Effects Of Allium Mongolicum Regel Essential Oil On Lipid Metabolism And Components Of Volatile Flavor Compounds And Its Relevant Mechanism In Mutton Sheep

This experiment studied the effects of AMO on sheep of meat and fat metabolism and volatile flavor compounds,and explored its mechanism from the aspects of rumen bacterial composition and the gene expression of transcription factors and enzymes related to fat metabolism.In vitro test:three healthy,three-month-old Duhan hybrid sheep with a weight of（37.1±0.5）kg were selected as rumen fluid donor sheep.Different levels of AMO were added to the culture substrate for 7 treatment groups:0 mg（CON）,0.02 mg（T1）,0.04 mg（T2）,0.06 mg（T3）,0.08 mg（T4）,0.1 mg（T5）,0.12 mg（T6）,3 replicates per treatment group.The optimum amount of AMO was selected,providing reference data for in vivo experiments.In vivo test:This test adopt a single factor completely random design to select thirty F1 generations of 6-month-old Duhan hybrid sheep with good body condition and similar weight（34.5 kg±2.5 kg）,and they were randomly divided into two groups with three replicates in each group and repeat five sheep each.The control group（CON）was fed a basal diet,and the test group（AMO）added 40 mg/kg AMO to the basal diet.The test period was 75 days,including 15 days for the pre-test period and 60 days for the positive test period.During the experiment,jugular vein blood was collected from all sheep every15 days.After the experiment,six sheep in each group were slaughtered to collect their rumen fluid,longissimus dorsi,subcutaneous fat and omental fat.In vitro test:divided the cells into blank control group（complete medium supplemented with 0.1%DMSO）and different concentrations of AMO（0.1,1,10,50,100μg/m L）into each well,and set 6 wells for each concentration,6 wells were also set for the blank control.1.The main active ingredient of AMO.AMO has determined 210 active ingredients.The main active ingredients are methallyl trisulfide（21.3%）,dimethyl trisulfide（10.77%）,diallyl trisulfide（7.65%）,2-methylhexane（6.09%）,Allyl methyl disulfide（5.42%）,3-methyl hexane（5.21%）,diallyl disulfide（4.19%）,dimethyl tetrasulfide（3.63%）,allyl methyl Disulfide（1.73%）,heptane（1.62%）,n-hexane（1.34%）,C₅H₁₂S₄（1.14%）,etc.,these components account for 70.09%of the total.The remaining active ingredients accounted for 29.91%.2.The effects of AMO on rumen fermentation of mutton sheep in vitro.After 24 hours,the p H value of the fermentation broth of the T2,T3,T4,and T6 groups were lower than that of the CON（P<0.05）.The NH₃-N concentration of T1,T2,T3 group were higher than that of the CON（P<0.05）,and the NH₃-N concentration of T4 group was lower than that of the CON（P<0.05）.The concentration of MCP in the T4 group was higher than that in the CON（P<0.05）.At 24 h,the concentrations of butyric acid and valeric acid in the T4group were higher than those in the CON（P<0.05）.The concentration of TVFA in each test group of AMO were higher than that of the CON（P<0.05）.At 24 h,the substrate degradation rate of T4 and T5 groups were higher than that of the CON（P<0.05）.0.08mg/2 g substrate AMO increased the p H,NH₃-N,MCP concentration,and butyric acid,valeric acid and TVFA content in the fermentation broth（P<0.05）,so this concentration is the optimum addition amount in in vitro.3.The influence of AMO on serum biochemical indexes of mutton sheep.On the 60day,the content of non-esterified fatty acids（NEFA）in AMO serum was higher than CON（P<0.05）.At 45 d and 60 d,the growth hormone（GH）content in AMO serum was higher than CON（P<0.05）.After adding AMO to diet,the p H value and NH₃-N concentration in the rumen were reduced（P<0.05）,and the concentration of acetic acid,propionic acid,and TVFA were increased（P<0.05）.Adding AMO to the mutton sheep diet increased cellulase activity,α-amylase activity and pepsin activity in rumen fluid（P<0.05）.Compared with the CON,the composition of SFA,C16:0 and C18:0 in the longissimus dorsi muscle of the AMO group decreased（P<0.05）,and n6/n3 decreased（P<0.05）.The composition of C18:1cis-9,C18:2cis-9,tan-11,CLA,C18:3n6,C18:3n3,n-3PUFA,and n-6PUFA increased（P<0.05）.Compared with the CON,the composition of SFA,C8:0,C16:0,and C18:0 in the subcutaneous fat of the AMO group was decreased（P<0.05）.The composition of MUFA,C18:1cis-9,PUFA,C18:2cis-9,tan-11,CLA,C20:2,C20:5n3,n-3PUFA,n-6/n-3 increased（P<0.05）.Compared with the CON,the C12:0 and C18:1cis-9 composition of omental fat in the AMO group decreased（P<0.05）.The composition of C14:0,C14:1,PUFA,C18:3n3,and C20:4n6 increased（P<0.05）.In the longissimus dorsi muscle,the total amount of hydrocarbon compounds,alcohol compounds,ketone compounds,and ester compounds in the AMO group increased（P<0.05）,while the total content of aldehyde compounds decreased（P<0.05）.1,6,10-Trimethylhexadecane,1,6-Dimethyl-4-（1-methylethyl）-naphthalene,1-octene-3ol,2-methyl-2-ring The content of oxyethyl-cyclobutanone,hexanal,2-ethylhexyl isobutyl phthalate,and ethyl acetate increased（P<0.05）,while the content of heptaldehyde decreased（P<0.05）.In subcutaneous fat,compared with the CON,the total amount of ester compounds increased（P<0.05）,while the total amount of aldehydes,acids and ether compounds decreased（P<0.05）.ComparedwiththeCONtheAMOgroup cis2-methyl-cyclopentanol,2,6,6-Trimethyl Cyclohexanone,3,3-diphenyl-acetophenone,hexanal,phenylacetaldehyde,The content of 2-methyl-1-butanol acetate increased（P<0.05）.Compared with the CON,the contents of nonanal,cinnamaldehyde,pterin-6-carboxylic acid and n-butyl ether were reduced（P<0.05）.4.The influence of AMO on the composition of rumen microbe in mutton sheep.In this experiment,a total of eleven bacterial phyla were identified in the rumen.Among them,Bacteroides and Firmicutes were the two most dominant phyla.At the phylum level,AMO decreased the abundance of Bacteroides and Spirobacteria（P<0.05）,and increased the abundances of Firmicutes,Actinomycetes and Verrucomicrobia（P<0.05）.A total of eleven species were identified in this experiment with an abundance greater than 1%,of which the main dominant species was Prevotella.Adding AMO increased the abundance of Prevotella and Prevotellaceae＿UCG-003（P<0.05）.Adding AMO reduced the abundance of Succiniclasticum,norank＿f＿＿F082,Christensenellaceae＿R-7＿group and norank＿f＿Muri baculaceae（P<0.05）.5.The effect of AMO on fatty acid and lipid metabolism.Compared with the CON,the m RNA expression of FAS,ACACA,SCD,FABP,SREBP1c,and Pref-1 in the AMO group were decreased（P<0.05）,while the m RNA expression of LPL,HSL,PRKAA2,and PPARγwere increased（P<0.05）.compared with the CON,the AMO group increased（P<0.05）the gene expression of SCD,LPL,PPARγand SREBP1c.Compared with the CON,the AMO group reduced the gene expression of DGAT1,PRKAA2,and Pref-1（P<0.05）.Compared with the CON,the AMO group increased the gene expression of FAS,ACACA,LPL,PPARγ,and SREBP1c（P<0.05）.Compared with the CON,the AMO group reduced the gene expression of HSL,CPT1β,PRKAA2 and FABP（P<0.05）.6.The effect of AMO on the differentiation of 3T3-L1 preadipocytes.Compared with normally differentiated pre-adipocytes,after 50μg/m L and 100μg/m L AMO treatment of pre-adipocytes,the TG concentration in adipocytes decreased significantly by 21.95%and65.85%（P<0.05）.Compared with normally differentiated preadipocytes,3T3-L1preadipocytes treated with 100μg/m L AMO reduced PPARγ,C/EBPα,C/EBPβ,SREBP1c,PGC-1,a P2 at different stages（P<0.05）.The gene expression of C/EBPδincreased the gene expression of C/EBPδ.Compared with 3T3-L1 preadipocytes without AMO treatment,the phosphorylation level of p38 in 3T3-L1 preadipocytes treated with 10,50 and 100μg/m L were increased（P<0.05）.Compared with 3T3-L1 preadipocytes not treated with AMO,the phosphorylation level of ERK of 3T3-L1 preadipocytes treated with 50 and 100μg/m L was increased（P<0.05）.After 50μg/m L and 100μg/m L AMO were treated with mature 3T3-L1 adipocytes at48 h and 72 h,the intracellular glycerol content increased significantly（P<0.05）.When 10μg/m L,50μg/m L and 100μg/m L AMO treated mature 3T3-L1 adipocytes,the intracellular triglyceride content was reduced（P<0.05）.Compared with mature adipocytes,the 3T3-L1 mature adipocytes treated with 50μg/m L and 100μg/m L AMO increased（P<0.05）the gene expression of ATGL and HSL after 72 hours of treatment.In summary,AMO regulated the composition and content of fatty acids and volatile flavors in the longissimus dorsi muscle and subcutaneous fat by affecting the structure of the rumen bacterial flora and the expression of lipid metabolism-related genes.