Establishment Of A Rapid Method For Determination Of Intramuscular Fat And Identification Of Key Candidate Genes For Differential Deposition Of Intramuscular Fat And Abdominal Fat In Chickens

Chicken is a nutritious and healthy food that is widely recognized by consumers for its high protein and low fat with high edible and nutritional value.Animal fat deposition has an important impact on carcass meat production and consumer eating quality,with intramuscular fat(IMF)content favoring a variety of meat properties,while abdominal fat(AF)is often treated as waste in chicken farming.However,the heterogeneity of lipid composition between IMF and AF and the different regulatory mechanisms are not known.In addition,traditional methods for chicken meat testing are time-consuming,tedious operation steps,and destructive to the samples,which cannot meet the requirements of rapid,and real-time development of modern meat industry,and realizing real-time monitoring and rapid testing of chicken meat quality is essential for the development of meat industry.Therefore,in this study,a rapid method for the determination of IMF content in chickens was first established based on low-field nuclear magnetic resonance(LF-NMR);then the overall lipid profiles of chicken IMF and AF were detected by non-targeted lipidomics,and the main differential lipids leading to heterogeneous deposition of IMF and AF were further identified using principal component analysis(PCA),indicating differences in lipid profiles and metabolism between the two lipid pools.And then key differentially expressed genes(DEGs)significantly associated with major lipid molecules were mined by transcriptomics combined with WGCNA analysis,and regulatory networks of key DEGs were constructed.Finally,the potential differential lipid regulatory functions of these key genes were validated by in vivo and ex vivo experiments.The main results were as follows:Part 1.Establishment of a rapid LF-NMR method for the determination of IMF content in chickenIn this study,the distribution curve of transverse chilling time(T2)of chicken meat was firstly constructed by CPMG sequence,and then the optimal conditions for adding aqueous Mn Cl2 solution were explored.The results showed that 1.5 m L of aqueous 20% Mn Cl2 solution added at 50℃,mixed for 5 min and left for more than30 min could separate the water and fat signal peaks and obtain stable fat peaks.A calibration curve with R2=0.9998(with chicken oil mass as the horizontal coordinate and fat peak area as the vertical coordinate)was obtained using chicken oil as the standard sample for fat content determination.In addition,the repeatability of the method ranged from 1.05% to 1.26%,and the stability and reliability of the fat peak area of the samples could be guaranteed under these detection conditions.A significant correlation(P < 0.05)existed between the LF-NMR method and the conventional method(Soxhlet extraction)with an R2 of 0.9984.This study showed that LF-NMR can be used for the rapid,determination of fat content in different parts in chicken,and it is reproducible with high consistency.Part 2.Differences in lipid composition of intramuscular fat and abdominal fat deposition in chickenIn this study,we conducted a non-targeted lipidomic analysis of IMF in pectoralis tissue and AF in abdominal fat tissue.733 lipids from 29 subclasses were identified in IMF and AF,including 423 differential lipid molecules(DLMs),which consisted mainly of glycerolipids(GLs),glycerophospholipids(GPs),sterolipids(SPs),and sphingolipids(STs);among them,307 DLMs were upregulated and 116 DLMs were downregulated in pectoralis IMF,which showed clear changes in lipid composition.The DLMs upregulated in the IMF were mainly glycerophospholipids(GPs),including most phosphatidylcholine(PC,PC(P)and PC(O)),phosphatidylethanolamine(PE,PE(P)and PE(O)),phosphatidylglycerol(PG)and phosphatidylinositol(PI),whereas DLMs upregulated in AF were mainly glycerolipids(GLs),including most triacylglycerols(TG)and diacylglycerols(DG).In addition,the relative contents of TG and DG were significantly higher in AF than in IMF(P < 0.01),whereas the relative contents of PC,PC(P),PE,PG,SM,PI,PC(O),and PE(P)were lower than in IMF(P < 0.01).The results of this study further refined the differences in individual phospholipid molecules or individual triglyceride molecules between IMF and AF.Part 3.Identification of major lipids and differential lipid characteristics of intramuscular fat and abdominal fat in chickenIn this study,30 major lipids including 13 glycerolipid molecules common to IMF and AF,12 IMF-specific glycerophospholipid molecules and 5 AF-specific glycerolipid molecules were identified by PCA analysis,which can be used as potential lipid biomarkers to distinguish IMF from AF.TG,PC and PC(P)are the major lipid compound subclasses of chicken IMF,12 of PC and PC(P)major lipid molecules contributed uniquely and primarily to IMF deposition,and five major lipid molecules of DG contributed uniquely and primarily to AF deposition.In addition,the distribution of fatty acid locations in IMF and AF was significantly different,with polyunsaturated fatty acids(PUFA)from the major differential lipids of pectoralis IMF preferentially distributed at the sn-2 position of PC and PC(P),with no significant selection for the location of TG molecules.It is suggested that the unique biochemical characteristics and metabolic mechanisms of adipose tissue from different parts may be responsible for the differences in lipid composition in breast and abdominal fat in chicken.Part 4.Transcriptome co-expression analysis of WGCNA identifies key genes for differential deposition of intramuscular fat and abdominal fat in chickensIn this study,11 modules were identified as significantly positively associated with PC and PC(P)lipid molecules in pectoralis(P ≤ 0.05);In abdominal adipose tissue,8 modules were found to be significantly associated with AFW,AFP,TG lipid molecules and DG lipid molecules(P ≤ 0.05).Transcriptomics combined with WGCNA analysis identified five key gene sets that interacted and linked in different ways to form a network regulating lipid deposition in IMF and AF.Key DEGs in breast regulate IMF deposition mainly through pathways involved in cellular response to cytokine stimulation,response to hormones,lipid metabolism,protein phosphorylation,kinase activity and regulation of lysosomes;The key DEGs in abdominal fat regulate AF deposition mainly through pathways involved in regulation of insulin-like growth factor(IGF)transport and uptake,cholesterol metabolism,isoprene-like metabolic processes,glycerolipid metabolic processes and regulation of systemic processes to regulate AF deposition.It was shown that the regulatory pathways or processes involved in key genes affecting the differential deposition of IMF and AF were significantly altered.Part 5.Validation of expression of key candidate genes for intramuscular fat and abdominal fat deposition in chickensIn this study,the expression characteristics of TMEM164 and ZNF488 genes were observed in vivo and in vitro.The TMEM164 gene expression was higher in breast,leg muscle and kidney and at lower levels in spleen,duodenum and liver;The ZNF488 gene was most highly expressed in abdominal fat and at lower levels in pectoralis.Furthermore,q PCR results of TMEM164 and ZNF488 in high and low IMF in pectoralis and high and low AFP in abdominal fat tissue were consistent with transcriptome sequencing data,confirming the reliability of the key gene set obtained from WGCNA analysis.The expression of TMEM164 was progressively upregulated in the intramuscular preadipocyte induction differentiation model and significantly downregulated in the late of abdominal preadipocyte induction differentiation model.The expression of ZNF488 gradually increased in the abdominal preadipocyte differentiation model,but did not change significantly in the intramuscular preadipocyte differentiation model,implying that TMEM164 may be mainly involved in the positive regulation of intramuscular fat deposition and has some influence on the negative regulation of abdominal fat deposition.Whereas ZNF488 has a potential unique positive regulation function on abdominal fat deposition.In conclusion,this study established a method for rapid determination of IMF content,and the lipidomic and transcriptomic results indicated that there are differences in the lipid profiles and metabolism of IMF and AF,meanwhile also identified key regulatory genes for IMF deposition in pectoralis and AF deposition in abdominal fat.Finally,the molecular mechanisms underlying the differential deposition of IMF and AF were elucidated.