Research On Linseed Oil Regulating Synthesis And Metabolism Of Polyunsaturated Fatty Acids In Livers And Breast Muscles Of Ducks

A balanced diet of n-6/n-3PUFA is very important for human health and the prevention and control of obesity,but the fatty acid composition of duck meat under conventional feeding mode is not reasonable.Therefore,optimizing the fatty acid composition of duck meat is an important aspect to achieve a healthy diet.The study was to investigate the combined effects of linseed oil on growth performance,slaughter indexes,meat quality,plasma biochemical indexes and fatty acid profile in Runzhou White Crested ducks,in order to get duck products rich in n-3 PUFA.On the other hand,the effects of linseed oil on hepatic fat mechanism was investigated from the perspectives of liver metabolism and gene expression,and the molecular regulatory network of linseed oil regulating PUFA synthesis and metabolism was constructed combined with findings at a cellular level.All the results may provide reference for the study of lipid metabolism mechanism in ducks.The main results are as follows:In experiment one,totally 224 28-day-old Runzhou White Crested ducks(half male and half female)were divided into control group(basal diet+2%duck oil),low linseed oil group(basal diet+1.5%duck oil+0.5%linseed oil),middle linseed oil group(basal diet+1%duck oil+1%linseed oil)and high linseed oil group(basal diet+2%linseed oil),respectively.There were 4 replicates in each group and 14 ducks in each replicate(half male and half female).After 28 days of experiment,the comprehensive effects of linseed oil on production phenotype,slaughter performance,meat quality,plasma biochemical indices and liver morphology of ducks were determined.The results showed that adding linseed oil less than 2%had no significant effect(P>0.05)on production performance,slaughter performance and conventional meat quality indexes of ducks.The fatty acid content in breast muscle and leg muscle of middle linseed oil group was up to 2.02%and 4.72%,respectively.After linseed oil supplementation,the plasma triglyceride(TG)level was significantly decreased(P<0.05)to 0.63 mmol/L,and the total cholesterol(TC)and high density lipoprotein(HDL-C)levels were increased significantly(P<0.05).The fat content of liver was decreased as the proportion of linseed oil increased.In conclusion,adding 2%linseed oil in duck diet can improve plasma HDL-C level,promote TG transportation and utilization,and reduce fat deposition in liver without significant influence in performance.In experiment two,24 individuals from each group were randomly selected to collect liver and muscle samples,GC-MS method were used to detect the composition of fatty acids,a total of 12 male duck liver samples from the control group and high linseed oil group were selected for metabolites analysis effected by linseed oil through LC-MS method detection,in order to explore the changes of metabolites.The results showed that the proportion of n-6/n-3 PUFA in liver and breast muscle was significantly decreased(P<0.01)after linseed oil supplementation,from 13.98 to 4.38 in liver and from 32.94 to 8.54 in breast muscle.With the increase of linseed oil supplemental level,contents of n-3 PUFA,just as α-linolenic acid(ALA),eicosapentaenoic acid(EPA)and docosahexaenoic acid(DHA)in liver and breast muscle were significantly increased(P<0.001).There was no significant difference(P>0.05)in the total n-6 PUFA content,but the linoleic acid(LA)level was significantly increased.In addition,dihomo-gammalinolenic acid(DGLA)and arachidonic acid(ARA)levels were decreased in liver(P<0.001).Correlation analysis showed that gender had no significant effect on most indexes(P>0.05).While n-3 series PUFA in liver and breast muscle were significantly positively correlated(P<0.01).N-3 PUFA level,especially DHA level in liver,was significantly negatively correlated with body weight(P<0.01).Through the non-target metabolomics analysis,there were 91 different expressed metabolites,of which 49 were up-regulated and 42 were down-regulated.Among them,the levels of LA,ALA,EPA and DHA,Monolinolenin(MLN),1,2-dilinoleoylglycerol(DLG)and 1-stearoyl-2docosahexaenoyl-sn-glycero-3-phosphocholine(SDGPC)were all up-regulated.While the level of docosatetraenoic acid(DTA),2-arachylglycerol(2-AG)and 1-stearoyl-2arachidonoyl-sn-glycerol(SAG)were decreased.KEGG enrichment analysis showed that most of the different expressed metabolites were significantly enriched in linolenic acid metabolism pathway,polyunsaturated fatty acid synthesis pathway,ABC transporter and immune-related pathway.In conclusion,the fatty acid composition results were consistent with metabolome results.Linseed oil supplementation increased the levels of LA and n-3 PUFA(ALA,EPA and DHA)and downstream metabolites(MLN and DLG,etc.)in liver.Competition inhibited the levels of n-6 PUFA(DGLA,ARA and DTA)and downstream metabolites(2-Ag and SAG,etc.),leading to duck meat products rich in n3 PUFA.In experiment three,samples used for non-target metabolome analysis were further selected to investigate the effects of linseed oil on gene expression and lipid metabolism in liver of ducks by transcriptome,proteome and combined analysis.The results showed that 890 differentially expressed mRNAs were screened by transcriptome analysis,of which 547 were up-regulated and 343 were down-regulated.KEGG enrichment analysis showed that the differential mRNA was mainly enriched in pathways related by fatty acids,such as linolenic acid metabolism,arachidonic acid metabolism,PPAR signaling pathway,polyunsaturated fatty acid synthesis,steroid biosynthesis and so on.The results of proteomic analysis showed that 78 differential proteins were obtained,including 44 up-regulated proteins and 34 down-regulated proteins.KEGG enrichment analysis showed that the differential expressed proteins were mainly enriched in linolenic acid metabolism pathway,polyunsaturated fatty acid synthesis pathway,triglyceride and glycerol phospholipid metabolism pathway,TCA cycle and other pathways.Multi-omics KEGG pathway integration analysis showed that the differentially differentiated expressed substances were mainly enriched in unsaturated fatty acid synthesis pathway,ALA metabolic pathway,glycerophospholipid metabolic pathway.The expression pattern(metabolite-gene-metabolite)showed that PUFA-Fads2-PUFA,PA-Lpin2-DAG,Lpc-Lpcat3-PC,PL-Pla2g4-PUFA,LA-Cpt1b-LLC played an important role in liver metabolism induced by ALA.Combined gene quantitative and protein analysis showed that linseed oil had a greater effect on PUFA synthesis gene in liver than on breast muscle,and the expression and distribution of fatty acid desaturase and elongase of very long chain fatty acids genes in liver may play a decisive role in PUFA synthesis and metabolism in liver.In experiment four,the duck intramuscular adipocytes of were isolated and cultured from the 25E of Runzhou White Crested embryo ducks by collagenase digestion method.To construct the induction differentiation system of duck intramuscular adipocytes was constructed by the induction medium supplemented with 500μM IB MX,1 μM DEX,10 μg/mL insulin,1 μM rosiglitazone and 300 μM oleic acid.Results showed that with the increase of differentiation time,lipid droplets gradually increased and accumulated to form large lipid droplets,the lipid droplets increased significantly(P<0.01)by Oil Red O staining extraction assay and triglyceride assay,mRNA levels of Fasn,Cpt1b,Cpt2,Plin1,Plin2,Fabp1 and Fabp2 were significantly increased(P<0.01).To further investigate the effects of ALA on the induction and differentiation of duck intramuscular adipocytes and lipid metabolism,OA group(300 μM oleic acid),10%ALA group(270 μM oleic acid+30 μM linolenic acid),20%ALA group(240 μM oleic acid+60 μM linolenic acid),40%ALA group(180 μM oleic acid+120 μM linolenic acid)was arranged for intramuscular adipocytes study.The result showed that the activity of intramuscular adipocytes increased significantly(P<0.05)after ALA supplementation,while there was no significant difference(P>0.05)in the quantitative results of oil red O extraction and TG content.The expression levels of most lipid metabolism genes,especially Plinl and Cptlb genes,were significantly increased(P<0.01)after ALA supplementation.Along with the increase of ALA,fatty acid desaturase genes were firstly increased and then decreased,and except for Elovl2,the expression levels of other elongase of very long chain fatty acids genes were significantly decreased(P<0.05).In conclusion,ALA supplementation did not affect the induction and differentiation process of duck intramuscular adipocytes,but enhance the cell cell viability,and resulted in the activation of lipid metabolism and the change of PUFA synthesis pathway involved by Plinl,Cpt1b,Elovl2,Fads1,Fads2 genes.In conclusion,adding 2%linseed oil in the diet of Runzhou white crested ducks could promote the metabolism of plasma triglyceride,reduce the fat deposition in liver,increase the level of n-3 PUFA in liver and breast muscle,and obtain duck products rich in n-3 PUFA without affecting performance,slaughter performance and conventional meat quality indexes.In duck liver,PUFA synthesis and metabolism pathways were changed due to the increase of PUFA synthesis substrate ALA and the change of gene expression and distribution of Fads2,Elovl2,Lpin2,Lpcat3,Pla2g4,Cpt1b,Ptgs2 and Tbxasl.In addition,n-3 PUFA(ALA,EPA and DHA),LA and their downstream metabolites were up-regulated,while n-6 PUFA(DGLA,ARA and DTA)and their downstream metabolites were competitively down-regulated.In addition,the oxidation utilization of PUFA was promoted.PUFA in breast muscle not only come from the transport of fatty acid producing tissues such as liver,but also come from the production of breast muscle,among which Elovl2,Fads2 and other genes play an important role in this process.The results can provide reference for functional duck meat production and the mechanism of duck n-3 PUFA synthesis and metabolism.