Effects Of Roughage Quality On Milk Fatty Acid Composition And Its Mechanism In Dairy Cows

In this study,mammary gland samples of lactating Holstein cows fed different diets were collected for RNA-seq to investigate the expression of differentially expressed genes,and from the result of PPARG pathway and milk fatty acid percentage in different diet groups it was hypothized that variation in PPARG pathway was connected with milk fat synthesis to some extents.Thus,the function of PPARG in bovine mammary epithelial cells was verified by RNA interference and overexpression mediated by adenovirus,and saturated and unsaturated long chain fatty acids.Main results of the present study were as follows:1.This experiment studied differences of milk fatty acid percentage and transcriptional level of mammary gland of lactating cows fed diets differed in forage type and concentrate-roughage ratio.A randomized block design was adopted in this experiment.Thirty multiparous Chinese Holstein cows with similar body weight in their mid-lactation were randomly assigned to 3 treatment groups.Diets were: mixed forage treatment(MF)with concentrate-roughage ratio of 46:54;high concentrate corn stover treatment(CS1)with concentrate-roughage ratio of 65:35;low concentrate corn stover treatment(CS2)with concentrate-roughage ratio of 46:54.The experiment lasted for 12 weeks,including 3 weeks of preliminary period and 9 weeks of experimental period,with 3 weeks as one experimental stage.Milk samples were collected at each experimental stage and mammary glands were collected at the last day of the experiment.The results showed that,milk fat percentage was not altered between CS2 and MF(P > 0.05),while long chain fatty acids,mono-unsaturated fatty acids,and C18:3 in CS2 were higher than that in MF treatment,and de novo synthesized and saturated fatty acids were lower(P < 0.05);differentially expressed genes in PPARG pathway were higher in CS2 than MF,based on which it was speculated that PPARG alter fatty acid metabolism in bovine mammary epithelial cells to some extents.2.Adenovirus-mediated RNA interference was applied to mammary epithelial cells to explore the function of PPARG on milk fat synthesis.In this study gene and protein expression of PPARG was decreased by about 55%,suggesting successful inhibition of PPARG by adenovirous.When PPARG was down-regulated,expression of ACACA,FASN,SCD,ASCL1,AGPAT6,GPAM,and LPIN1 was reduced(P < 0.05),genes encoding transcription factors SREBF1 and PPARA was also suppressed(P < 0.05),whereas mRNA level of FAPB3 and DGAT1 was increased(P < 0.05).However,intracellular triacylglycerol accumulation was not affected(P > 0.05).3.Adenovirus overexpressing PPARG was constructed successfully that its gene and protein levels were sharply increased(P < 0.05).When PPARG was overexpressed,mRNA abundance of ACACA,FASN,SCD,ACSL1,AGPAT6,DGAT1,GPAM,LPIN1 and PPARA were enhanced significantly(P <0.05);however,level of LPL,CD36,and FABP3 was not altered(P > 0.05).In addition,intracellular triacylglycerol content was enhanced(P < 0.05).4.Fatty acids were natural ligands of PPARG.This experiment explored the influence of mixture of saturated or unsaturated long chain fatty acids on PPARG as well as other genes involved in milk fat synthesis.Exogenous concentrations of C16:0,C18:0,C18:1,C18:2,and C18:3 were selected based on condition that cell viability was not inhibited.Treatments were mixed saturated fatty acids(SFA)consisted of 50?M of C18:0 and 100?M of C16:0;and mixed unsaturated fatty acids(UFA)consisted of 75?M of C18:1,100?M of C18:2,and 3?M of C18:3.Results showed that both UFA and SFA inhibited expression of ACACA,FASN,SCD,FABP3,and SREBF1(P < 0.05);SFA down-regulated CD36,ACSL1,DGAT1,GPAM,PPARA(P < 0.05)while UFA had no effect on these genes.In addition,exogenous supplement of UFA or SFA increased intracellular triacylglycerol content as well as protein level of PPARG(P < 0.05).5.UFA or SFA supplied to PPARG inhibited bovine mammary epithelial cells could result in significant change in fatty acid metabolism.Both UFA and SFA inhibited ACACA,FASN,FABP3,and LPIN1 expression(P < 0.05);abundance of CD36 and ACSL1 was up-regulated(P < 0.05).UFA significantly increased mRNA expression of triacylglycerol synthesis related genes,PPARG and PPARA,and decreased SCD and SREBF1(P < 0.05).When PPARG expression was at a lower level,exogenous addition of long chain fatty acids did not influence triacylglycerol content and protein level of PPARG(P < 0.05).6.In bovine mammary epithelial cells where PPARG was overexpressed,both UFA and SFA reduced mRNA level of ACACA,FASN,and SREBF1(P < 0.01),and enhanced CD36,DGAT1,GPAM,and PPARG(P < 0.05).SFA promoted gene expression of SCD,LPL,ACSL1,FABP3,AGPAT6 and LPIN1(P < 0.01),whereas UFA did not show such effect on these genes.When PPARG expression was at a higher level,exogenous long chain fatty acids increased intracellular triacylglycerol content and protein expression of PPARG(P < 0.05).Overall,cows fed corn stover as the only roughage uptake more long chain fatty acids and inhibit fatty acid de novo synthesis in the mammary gland.In vitro studies with bovine mammary epithelial cells showed that transcription factor PPARG is a key regulator in milk fatty acid metabolism which is involved in regulating processes of fatty acid de novo synthesis,desaturation,and triacylglycerol accumulation.Long chain fatty acids could activate PPARG,and activated PPARG was involved in inhibition of fatty acid de novo synthesis genes and increase of intracellular triacylglycerol accumulation by exogenous long chain fatty acids.