Effects Of Coniugated Linoleic Acid (CLA)-Induced Milk Fat Denression On Milk Fat Globule Size And Its Mechanism

Milk fat is present in milk in the shape of spherical lipid droplets,called milk fat globules(MFGs).MFGs are derived from lipid droplets(LDs)in mammary epithelial cells.Mammary cells form MFGs by generating LDs,encapsulating phospholipids and releasing them into milk.As the basic traits of milk fat itself,the size of MFGs reflects the differences in mammary gland function,animal species,genetics and nutrition.Early studies in our laboratory found that conjugated linoleic acid(CLA)can cause milk fat depression,but its mechanism is not clear.This experiment explained the regulation mechanism of MFGs’ size by feeding CLA to Holstein cows,from the relationship of fatty fat acid,phospholipid,genes and size of MFGs,which provided the basis for the molecular mechanism of CLA-induced MFD phenomenon from multiple angles.This study was conducted in two aspects:1.In order to study the effect on the size of fat globules,16 Holstein cows with similar body condition during mid-lactation were randomly divided into two groups,each with 8 heads,and the experiment lasted for 14 days.The control group(CON group)was fed with basal diet for 14 days,and the experimental group(CLA group)was fed with basal diet plus 400 g/d CLA for 7 days,and then changed to only basal diet for 7 days.During the experiment,two groups of milk were collected every day to analyze the changes of milk production performance,milk composition,MFGs size and the distribution ratio.The number of MFGs was counted,and the MFGs of different particle sizes were measured by the gravity separation.The results showed that CLA treatment had no significant effect on milk production performance,milk protein and lactose of dairy cows(P>0.05),but significantly decreased the content of milk fat(P<0.0l),indicating that CLA induced milk fat depression in dairy cows.As the test time continues,the milk fat globules size D[3,2],D[4.3],Dv(10),Dv(50)and the number of MFGs all showed a trend of first decreasing and then increasing.The specific surface area showed a trend of first increasing and then decreasing(P<0.01);At the same time,CLA treatment also caused the proportion of small-diameter fat globules to increase first and then decrease,and the proportion of large-diameter fat globules first decreased and then increased(P<0.01).After gravity separation,Size of MFGs in each layer had varying degrees reduction after CLA treatment(P<0.05).2.In order to explore the regulation mechanism of milk fat globule size,14 lactating cows were randomly divided into two groups,each with7 heads,and the experiment lasted for 8 days.The control group(CON group)was fed basal diet,and the experimental group(CLA group)was fed basal diet+400 g/d CLA.Milk and blood were collected on the 8th day of feeding,the composition and content of fatty acids and phospholipids in milk were analyzed,the relative expression of fat synthesis genes in milk fat was detected by fluorescence quantitative PCR,and the difference of metabolites in milk and plasma was detected by metabonomics.It was found that CLA treatment reduced the content of short-chain and medium-chain fatty acids(C6:0,C8:0,C10:0,C16:0),while increased the content of long-chain fatty acids(C18:0,C18:1 and C18:2)(P<0.05).Lipidomic analysis found that concentration of PC compounds(including PC(16:0/16:0),PC(16:0/18:0),etc.)and PE compounds(including PE(16:0/18:1),PE(18:0/18:1),etc.)and LPE compounds(including LPE(18:1)and LPE(18:2))decrease in milk,while the concentration of PI compounds(including PI(18:0/18:1),PI(18:1/18:1),etc.)and PS compounds(PS(18:1/18:2))increase(P<0.05 or P<0.01).Gene expression analysis found that CLA treatment significantly reduced the relative expression of fat synthesis regulation factors SREBP1 and PPARγ gene in milk(P<0.01),and the relative expression of key fat synthesis enzyme genes FASN,SCD and LPL were also down-regulated to varying degrees(P<0.05).Analysis of milk metabolomics found CLA treatment reduced the levels of decanoic acid and lysophosphatidylcholine in milk,increased the leves of lipid metabolites such as linoleic acid and oleic acid;decreased the levels of amino acid metabolites such as serine and glycine,increased the levels of aspartic acid and phenylalanine;decreased the levels of 12-hydroxystearic acid and 2-aminoethane sulfonic acid,and increased the levels of organic acid metabolites such as DL-2-aminocrylic acid.Plasma metabolomics analysis found CLA treatment decreased the level of lipid metabolites in plasma,such as methyl stearate and palmitaldehyde,increased the level of cholesterol and stearamide;decreased the level of amino acid metabolites such as isoleucine and histidine,increased the level of glycine and cysteine metabolism(P<0.05).All the above results showed that CLA caused milk fat depression and also caused the changes of size and proportion of milk fat globules,and the change of the content of fatty acids,phospholipids and gene in milk played an important role in regulating the MFGs size,and could cause changes in the content of milk and plasma metabolites.