Research On The Identification Method And Mechanism Of Meat From Pasture-fed And Concentrate-fed Sheep/Goats Based On Multi-omics

Feeding method is crucial factor in influencing mutton quality.Relevant studies have shown that different feeding methods can make differences in fat deposition and flavor substance content in mutton.With the improvement of living standards,people will prefer high-quality mutton from pasture-fed in the purchasing process.In order to increase sales and make profits,some illegal traders will sell the mutton from concentrate-fed as high-quality mutton from pasture-fed.This behavior not only infringes the rights and interests of consumers,but also impacts the mutton from pasture-fed industry and hinders its development.Therefore,in order to achieve high price for good quality and promote the development of high quality industry,it is necessary to develop an effective identification method for mutton with different feeding methods.At present,the research hotspots of mutton authenticity identification mainly focus on the identification of origin and adulteration between varieties,while there are few reports on the identification of mutton authenticity between two feeding methods.In this paper,metabolomics and lipidomics methods are used to analyze small molecular metabolites in mutton with different feeding methods,screening differential metabolites as markers,to establish the stable and reliable prediction model,subsequently,with the help of the transcriptome study,discusses the regulatory mechanism of metabolites involved in upstream,realize the mutual authentication between omics,so as to provide basis for improving the quality of mutton.The main contents and results are as follows:A pretreatment method for polar and non-polar metabolites in mutton and a non-targeted metabonomics and lipidomics detection method using UHPLC-QTOF-MS were established.85 samples of mutton from pasture-fed and 53 samples of mutton from concentrate-fed were tested.Multivariate statistical analysis method was used to characterize the metabolic profile of pasture-fed and concentrate-fed mutton.Difference metabolites were screened to establish discriminant models,and the models were verified with actual samples.The results show that the PCA scores plots showed the separation of mutton from pasture-grazing and concentrated-fed groups into distinctive clusters based on their metabolite profiles in non-targeted metabolomics and lipidomics.Subsequently,based on the supervised OPLS-DA model,with P<0.05,FC>1.2 or<0.83 and VIP>1 were used as thresholds to screen difference markers.15 biomarkers were initially qualitatively identified in the positive ion mode of non-targeted metabolomics.Among them,,the standard substances are used to accurately qualitate12 differences.Under the negative ion mode,10 biomarkers were initially qualitatively identified.20biomarkers were initially qualitatively identified in the positive ion mode of non-targeted lipidomics.Under the negative ion mode,5 biomarkers were initially qualitatively identified.The structures of the selected lipid markers were analyzed according to the mass spectrometry fragmentation rules reported in the literature.The discriminant models were established based on the difference markers obtained in the positive ion mode,and 59 actual samples were used to verify the models.The correct discrimination rates of non-targeted metabolomics and lipidomics were 89.83%and 98.3%,respectively.These compounds play important roles in the flavor,taste,water retention,color,buffering and antioxidant properties of meat,and affect the nutritional quality of mutton.On the basis of non-targeted omics,an LC-MRM-MS targeted metabolomics analysis method was established for 15 polar metabolites and 20 non-polar lipid metabolites.85 samples of mutton from pasture-fed and 53 samples of mutton from concentrate-fed were tested.In addition,the 12 polar metabolites were relatively quantified using solvent standards,and the models with strong predictive ability were further screened to construct models,and the actual samples were predicted.The results show that these differences detected by the targeted method can distinguish mutton from pasture-fed and mutton from concentrate-fed in unsupervised analysis.Through ROC analysis,the metabolites with strong predictive ability were selected,11 markers were screened by targeted metabolomics,and 6markers were screened by targeted lipidomics.The actual samples were distinguished,and the correct identification rates were 89.83%and 100%,respectively,showing good discriminant ability.In order to better reflect the whole situation of gene transcription to downstream metabolism in mutton of different feeding methods,realize the complementation of data,and more fully illustrate the complex regulation mechanism of multiple metabolic pathways involved in the different metabolites of mutton of different feeding methods.High-throughput RNA-seq technology was used to perform transcriptome sequencing in mutton of different feeding methods,and differentially expressed genes were screened.Functional annotation and metabolic pathway enrichment analysis were performed,and then conjoint analysis with metabolomics was performed.The results showed that a total of 162differentially expressed genes were screened between pasture-fed and concentrate-fed mutton samples using p-adjusted<0.01 and Log₂(FC)>2.32 or<-2.32 as screening conditions,among which 50 genes were up-regulated and 112 genes were down-regulated.The KEGG for these genes was significantly enriched in AMPK signaling pathway,PI3K-Akt signaling pathway and other pathways,while 33pathways were preliminarily predicted by differential metabolites.Through joint analysis,some key pathways involved in differential genes and metabolites were identified.These differential metabolites and genes have complex regulatory relationships,which are the important reasons for the differences in nutritional quality of mutton under different rearing methods.