Effect Of Lps On The Milk Fat And Protein Synthesis In Dairy Cows

The objectives of this thesis were to evaluate the effect of LPS on milk protein and milk fat synthesis in dairy cows using ex vivo tests and vitro tests. In ex vivo tests, the main objective was to study the effect of LPS on fatty acids, amino acids and physicochemical index. In vitro tests, the main objective was to study the mechanism of LPS on milk fat and milk protein synthesis.Experiment 1, The objective of this study was designed to evaluate plasma fatty acids profile, lipid indices, milk fat composition, the content of synthesis precursors of milk fat and milk fatty acids profile in response to LPS challenge following intra-external pudic arterial. Six multiparous, lactating Holstein cows received a single, small dose of LPS (Escherichia coli O111:B4) in 10 mL of sterile saline (0.9% NaCl) solution (0.01μg/kg BW) or sterile saline solution alone (control treatment) in one of a two-week randomized crossover study. The results showed that plasma fatty acids profile was not significantly affected (P>0.05), but tended to increase the content of T-FA, SFA, SLFA and CLFA. Compared to the control, LPS challenge increased mammary gland uptake for the length of carbon chain more than sixteen in various degrees (P>0.05) excepted for C20 (P <0.01). The challenge increased the plasma content of CHOL (P<0.05), HDLD (P>0.05) and LDLD (P>0.05). Compared to the control, LPS significantly decreased milk fat percent (P>0.05) and milk fat yield (P>0.05). The challenge also reduced the content of NEFA (P<0.01) for synthesis precursor of milk fat, the content of BHA (P>0.05) first decreased then increased as the time. Saturated fatty acids (P>0.05) and short-chain fatty acids (P>0.05) decreased following LPS challenge, but increased unsaturated fatty acids and middle-long chain fatty acids (P>0.05), and the de-saturated indices was affected. It is concluded that LPS mobilized lipid metabolism for the inflammation and LPS is one of the important factors affecting milk fat synthesis.Experiment 2, The objective of this study was designed to evaluate plasma amino acids profile and blood physicochemical indices in response to LPS challenge following intra-external pudic arterial. The results showed that LPS significantly increased milk protein percentage (P<0.05) and tended to increase casein percent (P>0.05), but decreased milk protein yield (P>0.05) and casein yield (P>0.05). The content of EAA and BCAA first increased the highest point at 6 h after infusion and then decreased (P>0.05), the result of NEAA was converse (P>0.05).There was no siginificant effect on amino acids of plasma (P>0.05) compared to the control, but tended to increase the content of his and met and decreased the content of ser, glu, gly, arg, thr, ala, pro, cys, val, lys, iie, leu and phe. The content of GH, BUN, TP and TBIL were decreased and the content of INS and GLU were increased as LPS challenge (P>0.05). LPS increased the activity of ALT (P<0.01), AST (P<0.01), ALP (P<0.01) and LD (P>0.05). These results indicated that LPS mobilized amino acids to synthetize acute phase protein so disturbed milk protein synthesis.Experiment 3, The objective of this study was to determine the effect of LPS on plasma protein and milk proteins using two-dimensional electrophoresis gel (2-DE) coupled with MALDI-TOF-TOF spectrometry. Plasma proteins at 0, 6, 12 and 24 h after LPS infusion were separated by two-dimensional electrophoresis. After visualization proteins with Coomassie Brilliant Blue G-250, protein spots were detected by PDQuest 8.0.1 software and differential proteins were identified by MALDI-TOF-TOF spectrometry. The result showed that milk SCS started to increase after infusing LPS and peaked at 6 h, and then gradually decreased to the level before infusing LPS at 24 h. In plasma, eight protein spots were identified to be four proteins including vitamin D-binding protein precursor, serpin A3-6, alpha-1 antitrypsin and serpin A3-1 precursor. These proteins were up-regulated at 6, 12 and 24 h in cows after infusing LPS, but there was not significantly different among 6 to 24 h. In skimed milk, three protein spots were identified to be two proteins includingβ-lactoglobulin andα- elongation factor. There was no effect on whey protein. These results indicated that vitamin D-binding protein precursor, serpin A3-6, alpha-1 antitrypsin and serpin A3-1 precursor may play an important role in response to LPS challenge, and further study was needed.Experiment 4, The objective of this study was to investigated the expression of fatty acid metabolic-related gene in mammary epithelial cells of dairy cow when incubated with 0 ng.mL-1 LPS, 0.1 ng.mL-1 LPS and 10 ng.mL-1 LPS for 24 h with RT-qRCR. LPS did not significantly affect fatty acid-binding protein FABP3 and FABP4 mRNA expression (P>0.05), but caused a marked increase in transporter CD36 mRNA expression (P<0.01). Treated with 0.1 ng.mL-1 LPS increased the fatty acid synthesis enzyme FASN, ACACA, ACSS2 and fatty acid metabolism gene PPARG, PPARGC1A mRNA expression (P>0.05) compared to the control, On the other hand, differential regulation of fatty acid synthesis enzyme FASN, ACACA, ACSS2 and fatty acid metabolism gene PPARG, PPARGC1A mRNA expression were observed in 10 ng.mL-1 LPS group. It was conclude that LPS disturb fatty acid metabolism, low dose (0.1 ng.mL-1) LPS mobilize fatty acid metabolism, but high dose (10 ng.mL-1) inhibit fatty acid synthesis, which needs to study further in translation leve1.Experimemt 5, The objective of this study was to investigated the expression of protein metabolic-related gene in mammary epithelial cells of dairy cow when incubated with 0 ng.mL-1 LPS, 0.1 ng.mL-1 LPS and 10 ng.mL-1 LPS for 24 h with RT-qRCR. The results showed that LPS increased S6K1and 4EBP1 mRNA expression (P>0.05) and in a dose effect. Compared to the control, LPS did not significantly affect mTOR expression (P>0.05). STAT5 mRNA expression (P<0.05) and JAK2 mRNA expression (P>0.05) were increased. It was conclude that LPS affect protein metabolism through mTOR signaling pathway and JAK2/STAT5 passage.In summary, On the one hand, LPS mobilized lipid metabolism for the inflammation and decreased SCFA synthesis, but increased the uptake of MCFA and LCFA in mammary gland. Overall, LPS decreased milk fat percent and milk fat yield when LPS level was 0.01μg/kg BW. The mechanism was LPS affect fatty acids uptake, activation and transcription gene. LPS was one of the important factors affecting milk fat synthesis. On the other hand, LPS mobilized amino acids to synthetize acute phase protein so as to disturb amino acids composition for milk protein synthesis. The mechanism was LPS affected mTOR signaling pathway and JAK2/STAT5 passage.