Uptake Efficiency Of Leucine In Dairy Cows And Its Effect On Milk Protein Synthesis

In this paper, studies were focused on the effect of different dietary pattern on leucine absorption in mammary gland of lactating dairy cows and leucine influences on milk protein synthesis in dairy cows.The objective of this study was to illustrate mechanism of the milk protein synthesis to provide a basis for manipulating of milk protein.The purpose of experiment1was to detect the extraction efficiency of amino acid in different pattern of diet. One was consisted of cornstalk which was used by free-range farms, the other was a complete feed applied by large-scale farms. The blood in external pudendal artery and vein was collected continuously. The result indicated that the different diet supplied different amount of amino acids, but it had no effect on the extraction efficiency of amino acid.The aim of experiment2was to provide polyclonal antibody against αs-casein for detecting the expression of αs-casein in mammary epithelial cells cultured in vitro. New Zealand White Rabbits were uesd to produce rabbit anti-bovine as-casein antibody which provides powerful material for the detection of αs-casein by Western-blot.The objective of experiment3was to further explore the function of Leucine to the expression of casein. Mammary epithelial cells cultured in vitro were used in this study. The result of identification indicated that the expression of CSN3was the most stable. Epithelial cells were supplemented with different concentration of Leucine. The result indicated that CSN3gene was highly expressed when the concentration of Leucine was118.1mg/L to236.2mg/L.The experiment4was conducted to find the mechanism of Leucine in manipulating milk protein. In order to remove the influence of the interaction between amino acids, DMEM/F12medium was diluted100-fold to weak the role of other amino acid and keep health of the cells. Many studies have shown that mTOR signal pathway may work on mediating protein synthesis. Cells were treated with optimum concentration of Leucine which was got above and with LY294002to inhibite mTOR to explore its mechainism. The expression of CSN1S1and synthesis of αs-casein and κ-casein were detected to confirm the effection of Leucine to casein besides CSN3. The results found that expression level of CSN3, CSN1S1gene, κ-casein and as-casein were increased by leucine (P<0.05) significantly. However, inhibitor LY294002 weakened its roles (P<0.01) definitely. Meanwhile, Adding Leucine and LY294002had tendency to increased the expression of genes related to mTOR pathway. but both the effection of them were not significant. Proliferation of the cells supplemented with leucine was not significantly changed, but it was decreased by inhibitor LY294002observably (P<0.05).