Effect Of Hormone Supplementation On Milk Fat And Milk Protein Synthesis In Two And Three-Dimensional Cultured Bovine Mammary Epithelial Cells

The aim of this study was to determine the effect of supplementation of prolactin(PRL),insulin(INS),growth hormone(GH)and the ratios between them on milk fat and protein synthesis in bovine mammary epithelial cells that cultured under two-dimensional(2D)and three-dimensional(3D)model.The suitable supply mode of hormone for improving milk components synthesis and the molecular mechanism of hormone regulation milk fat and protein synthesis were clarified through genes expression of transcriptional regulation factors and genes associated with fat and protein synthesis.Result of this thesis provided theory basis for direction of milk fat synthesis.This study contained four trials.Each trail was composed of two part of content.In part one,effect of different concentration of PRL(0,100,300,500,1000 g/mL),INS(0,25,100,400,800 ng/mL),GH(0,10,30,100 ng/mL)and different ratio of these three hormones[0.(20:1:2).(7.3:1:1.8)]on triglyceride content(TAG),as well as mRNA expression of genes involved in milk fat and protein synthesis in 2D cultured BMECs were investigated.In part two the optimum concentration of PRL(0,100,200 ng/mL),INS(0,5,10 ng/mL),GH(0,10,30 ng/mL)and mixed supplementation(0,10:1:3,20:1:2,2:1:1)were used to comparative study the effect on synthesis of milk fat and protein both in 2D and 3D cultured BMECs.The results of trial one showed that compared with control group,100 ng/mL PRL significantly upregulated gene expressions of mTOR,S6K1,4EBP1,CSN1S1 and PRLR(P<0.05).The content of TAG in BMECs and gene expressions of ACC,DGAT2,FABP3 and PRLR were sig,nificantly higher in 300 ng/mL PRL group compared with those in control group(P<0.05).100?300 ng/ml PRL are optimal dosage in 2D model,considering its simulative effects on milk fat and protein synthesis,but high dosage of PRL suppress the milk fat and protein synthesis in 2D cultured model.The TAG content and gene expressions in 3D were significantly higher than that of 2D.The content of TAG in BMECs and gene expressions of ACC,FASN,SCD,DGAT and SREBP1,PPARG,4EBP1,CSN2,CSAN3,PRLR were significantly higher in 100 ng/mL prolactin group compared with those in control group(P<0.05).Compared with control group.200 ng/mL PRL significantly upregulated gene expressions of mTOR.AKT and CSN1S1(P<0.05).PRL significantly increased the gene expressions of FABP3(P<0.05).100 ng/ml PRL is optimal dosage in 3D model,considering its simulative effects on milk fat and protein synthesis.The results of trial two showed that the content of TAG in BMECs were significantly higher in 25 ng/ml and 100 ng/mL INS group compared with those in control group(P<0.05).25 ng/ml INS significantly upregulated gene expressions of ACC,SREBP1(P<0.05).100 ng/mL INS significantly upregulated gene expressions of 4EBP1,whereas S6K1,ACC,FASA,SCD.DGAT,SREBP1,FABP3,STAT5,AKT,CSN3 and INSR were significantly decreased(P<0.05).0?25 ng/ml INS are optimal dosage in 2D model,considering its simulative effects on milk fat and protein synthesis,but 100?800 ng/ml INS suppress the milk fat and protein synthesis in 2D cultured model.The TAG content and gene expressions in 3D were significantly higher than that of 2D.5 ng/ml INS group significantly upregulated gene expressions of ACC,PPARG,AKT,S6K1,CSN1S1.10 ng/ml INS group significantly upregulated gene expressions of FASN,DGAT2,FABP3,4EBP1,STAT5,INSR and TAG content(P<0.05).10 ng/ml INS is optimal dosage in 3D model,considering its simulative effects on milk fat and protein synthesis.The results of trial three showed that 10 ng/mL GH significantly upregulated gene expressions of FASN,DGAT2,STAT5,PARG,CSN1S1 and GHR(P<0.05).30 ng/mL GH significantly upregulated gene expressions of SREBP1 and TAG content(P<0.05).10?30 ng/ml GH are optimal dosage,considering its simulative effects on milk fat and protein synthesis,but 100?300 ng/ml GH suppress the milk fat and protein synthesis in 2D cultured model.The TAG content and gene expressions in 3D were significantly higher than that of 2D.10 ng/ml GH group significantly upregulated gene expressions of ACC,SCD,DGAT2,CSN2,CSN3,but downregulated the gene expression of S6K1(P<0.05).30 ng/ml GH group significantly upregulated gene expressions of FASN,SREBP1,PPARG,FABP3.AKT,mTOR.AEBP1,S6K1,STAT5,CSN1S1,GHR,IGFR and TAG content(P<0.05).30ng/ml INS is optimal dosage in 3D model,considering its simulative effects on milk fat and protein synthesis.The results of trial four showed that the gene expression of DGAT2,FABP3,SREBP1,CSN1S1,STAT5,mTOR,AKT,4EBP1,S6K1,GHR,IGFR and TAG content were siognificantly upregulated in treatment group ? than control group and other treatment groups(P<0.05).Treatment group ? is optimal ratio,considering its simulative effects on milk fat and protein synthesis in 2D cultured model.The TAG content and gene expressions in 3D were significantly higher than that of 2D(P<0.05).The gene expressions of ACC,FASN,DGAT2.FABP3,CSN1S1,CSN2,CSN3 and AKT were significantly upregulated in treatment group ?than those of treatment group ?,? and ?,and the gene expressions of SCD,PPARG,mTOR,4EBP1,S6K1 and STAT5 were significantly than control group(P<0.05).The genes expression of PRLR,IASR,GHR and IGFR were significantly higher in group ?Hand ? than in groups? and ?(P<0.05).Treatment group ? significantly upregulated gene expressions of SCD,SREBP1(P<0.05).Treatment group ? significantly upregulated gene expressions of 4EBP1,STAT5 and IGFR(P<0.05).Treatment group ?(10:1:3)is optimal ratio,considering its simulative effects on milk fat and protein synthesis in 3D cultured model(P<0.05)?...