| The key problem of poultry industry is to reduce fat deposition and improve meat quality.The key metabolic intermediate or its structural analogues,as a chemical signal,ccould alter the direction of nutrient metabolism.Using key metabolic intermediate or its structural analogues to regulate the nutrients metabolism pathway not only could improve the animal growth performance,but also ensure the quality and safty of animal products.As a structural analogue of citrate,the potential function of(-)-hydroxycitric acid may be to regulate the conversion of glucose metabolism and lipid metabolism,while there is no any report about the effect of(-)-hydroxycitric acid on the glucose metabolism in birds.This study was aimed to investigate the changes of serum metabolites and hepatic proteins at different developmental stages in chicken embryo.And to explore the effects of(-)-hydroxycitrate[(-)-HCA]on metabolic changes and mechanism in chicken embryos.The research mainly includes the following five aspects:1 Based Serum Metabolomics Analysis Reveals Simultaneous Interconnecting Changes during Chicken Embryonic DevelopmentDue to native hyperglycemia and resistance to exogenous insulin,chickens had been used in the studies of metabolism and obesity.But no detail information is available on comprehensive changes of serum metabolites at the different stage in chicken embryos development.Thus,180 fertilized eggs(65±0.2 g)were selected to determine the changes of major functional serum metabolites at incubation day 14(E14),19(E19)and hatching day 1(H1)using liquid chromatography-mass spectrometry quadrapole time of flight system(LC/MS-QTOF)method.Fertilized eggs were placed into an electric forced-draft incubator at 37±0.5? and 65%relative humidity,with rocking at an angle of 90°at 30min intervals.Principal component analysis(PCA),partial least squares-discriminate analysis(PLS-DA)and variable important for the projection(VIP)were applied to identify differential metabolites.Metabolite Set Enrichment Analysis(MESA)was applied for the analysis of the associated pathways of these differential metabolites during chicken embryos development.Results showed that 5 center metabolites were significantly changed from E14 to E19 and 7 metabolites were significantly altered from E19 to HI in chicken embryos development.Protein synthesis was promoted by increasing the concentrations of L-glutamine and threonine,and gonadal development was promoted through increasing estrone content from 14-day old to 19-day old in chicken development.These results indicated that serum glutamine,threonine and estrone contents may be considered as the candidate indicators for the assessment of embryonic development.2-oxoglutaric acid mainly contributed to improving the citric cycle and it plays an important role in improving the growth at the later development of chicken embryos;the decreasing of L-glutamine,L-isoleucine and L-leucine contents from 19-day old to hatching day in chicken embryos development implied their possible functions as the feed additive at early post-hatch period of broiler chickens in order to satisfy the growth and development.2 Based on proteomics analysis reveals the changes of hepatic proteins during chicken embryonic development180 fertilized eggs(65±0.2g)were selected to clarify the change pattern of proteins at E145 E19 and H1.Fertilized eggs were placed into an electric forced-draft incubator at 37±0.5??and 65%relative humidity,with rocking at an angle of 900 at 30min intervals.Proteomic approach based isobaric tags for relative and absolute quantitation(iTRAQ)was employed to identify the changes of proteins expression levels in liver.Results showed that 26 center proteins with differential expression were identified at E19 compared with E14.During this stage,fatty acid degradation(upregulated ACAA2,CPT1A and ACOX1),protein folding rate and disulfide bond formation efficiency(upregulated PDIs,CALR3,LMAN1 and UBQLN1),and gluconeogenesis(upregulated ACSSI,AKRIAI,ALDH3A2,ALDH7A1 and FBP2)were enhanced.15 center proteins with differential expression were identified at HI compared with E19.During this stage,glycolysis(downregulated AKR1A1,LDHA and LDHB;upregulated PGM2)and purine metabolism(upregulated NT5C)were decreased or inhibited,while ribosome(upregulated RPL18A,RPL19,RPLP1,RPS2 and RPS3A)was enhanced.These results indicated that glycolysis was not the main way to produce energy during the later developmental stage.Importantly,we found that ACAA2,CPT1A and ACOX1 might be key factors to control fatty deposition in chicken embryo development3 Effects of(-)-hydroxyeitrate on lipids droplets accumulation in chicken embryosTo evaluate the safety of(-)-HCA and its effect on lipids droplets accumulation during chicken embryonic development,240 fertilized eggs were divided into six groups and injected with(-)-HCA at concentration of 0,0.1,0.5,1.0,10.0 and 50mg/kg(n=40).(-)-HCA was dissolved in 0.25%DMSO solution.Fertilized eggs were placed into an electric forced-draft incubator at 37±0.5,C and 65%relative humidity,with rocking at an angle of 900 at 30min intervals.At 19 days of incubation,serum and tissues samples were collected for further analysis.Results showed that no significant differences were observed on embryo weight,absolute liver weight,absolute spleen weight,absolute heart weight and absolute kidney weight,while embryo weight with a downward trend;There were no differences were observed on albumin,globulin,TP,Cr,P,AST,ALT and LDH in serum,while BUN content were significantly decreased in 0.1-50 mg/kg(-)-HCA treated group(P<0.05);The number of red blood cells and white blood cells had no change in each group;HE results showed that no pathological changes were observed;PAS and Oil Red O staining results showed that number of glycogen granules were significantly increased and number of lipid droplets were decreased in 1-50 mg/kg(-)-HCA treated groups(P<0.01);Serum and hepatic lipid parameters results showed that serum and hepatic TG contents were significantly decreased in 1-50mg/kg(-)-HCA treated groups(P<0.05),hepatic NEFA and glycogen contents,activity of HL and LPL were significantly increased in 1-50 mg/kg(-)-HCA treated groups(P<0.05),LDL-C content was significantly decreased in 0.5-50 mg/kg(-)-HCA treated groups(P<0.05),glucose content was significantly increased in 0.5-1 mg/kg(-)-HCA treated groups(P<0.05),ADP content was significantly increased in 0.5-50 mg/kg(-)-HCA treated groups(P<0.01),contents of TC,HDL-c,T3,INS and GLU were no changes in each group;ACLY,ME1 and SREBP-1c mRNA expression levels were significantly decreased in 0.5-10 mg/kg(-)-HCA treated groups(P<0.05),FAS mRNA expression levels were significantly decreased in 1-10 mg/kg(-)-HCA treated groups(P<0.05),PPAR? mRNA expression levels were significantly increased in 0.5-50 mg/kg(-)-HCA treated groups(P<0.05).These results indicated that injected with 0.1-50mg/kg(-)-HCA could be considered safe in chicken embryos.In addition,(-)-HCA treatment inhibited triglyceride synthesis via decreasing lipogenesis-related genes(ACLY,ME19 SREBP-1c and FAS)mRNA expression levels and accelerated lipolysis through increased PPARa mRNA expression levels.4 Serum differential metabolites and key metabolic pathways analysis regulated by(-)-hydroxy citrate in embryonic broilers360 fertilized eggs were divided into two groups and injected with(-)-HCA at concentration of 1 mg/kg.(-)-HCA was dissolved in 0.25%DMSO solution.Fertilized eggs were placed into an electric forced-draft incubator at 37±0.5? and 65%relative humidity,with rocking at an angle of 90°at 30min intervals.The liver tissue samples and serum samples were collected at E14,E19 and HI.LC/MS-QTOF method was applied to determine the changes of major functional serum metabolites.PCA,PLS-DA and VIP were applied to identify differential metabolites.MESA was applied for the analysis of the associated pathways of these differential metabolites during chicken embryos development as treated with(-)-HCA.Results showed that differential metabolites were identified at E19 and HI were belong to four categories:sugars,lipids,amino acids and other categories.PCA results showed that samples were significantly separate in control group and(-)-HCA treated group.At E19,(-)-HCA could promote protein synthesis by increasing the contents of glutamine,threonine,arginine,alanine,and glutamate;accelerate citrate cycle through increasing the contents of malic acid,citric acid,fumarate and isocitric acid;accelerate gluconeogenesis by increasing the contents of glucose and fructose and decreasing the content of lactose;and it could promote lipolysis through decreasing PGE2 content and increasing decanoyl-CoA content.At H1,(-)-HCA promotes protein synthesis by increasing glutamine,threonine,alanine,leucine,and glutamate levels;accelerate citrate cycle through increasing the contents of malic acid,citric acid,fumarate and isocitric acid;it could inhibit or reduce the progress of glycolysis by reducing the content of 3-phospho-D-glycerophosphate and lactic acid.The results suggested that(-)-HCA could promote protein synthesis,glycogen synthesis,and fat oxidation through the redistribution of metabolites in the later embryo developmental stage,ultimately inhibiting or reducing fat deposition.And(-)-HCA could promote the growth and development of embryos and improves their ability to cope with environmental changes.5 The screening and identification of key effector proteins related to lipid metabolism regulated by(-)-hydroxycitrate in embryonic broilers360 fertilized eggs were divided into two groups and injected with(-)-HCA at concentration of 1 mg/kg.(-)-HCA was dissolved in 0.25%DMSO solution.Fertilized eggs were placed into an electric forced-draft incubator at 37±0.5? and 65%relative humidity,with rocking at an angle of 90°at 30min intervals.The liver tissue samples and serum samples were collected at E19 and HI.Proteomic approach based iTRAQ was employed to identify the changes of proteins expression levels in liver.Results showed that 8 center proteins with differential expression were identified at E19 between control group and(·)-HCA treated group.At E19,fatty acid degradation were enhanced(upregulated ACSL1,ACSL5,CPT1 and ACOX2),gluconeogenesis was enhanced and glycolysis was inhibit or reduce(upregulated FBP2,ALDH3A2 and GPI,downregulated ALDOC);At HI,fatty acid degradation were enhanced(upregulated CTP2,ACAT1,ALDH7A1,ECHSI,ECI2 and ACAA2,downregulated ALDH9A1),luconeogenesis was enhanced and glycolysis was inhibit or reduce(upregulated ACSSI,downregulated PFKL and LDHB),citrate cycle was enhanced(upregulated IDH1 and ACO1),these results were consistent with metabolomics results. |
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