| This study aims to investigate, under a condition of high concentrate feed, the relationship between the synthesis of milk proteins and the metabolism and utilization of their amino acid precursors as well as the molecular mechanism which resultis in this relationship. The research mainly includes the following three aspects:This study aims to investigate the rule and molecular mechanism of milk protein synthesis precursor of amino acid generation and utilization under the condition of high concentrate feed in our country, in order to expound influencing mechanism of milk protein synthesis. The main content of research includes the following three aspects:1 Effect of a high concentrate diet on the metabolic flow of milk proteins’ amino acid precursors and the milk protein content in lactating dairy cows fed a High-Concentrate Diet10 healthy lactating Holstein cows were selected in this experiment. The cows were randomly divided into 2 groups:forage-to-concentrate ratio 4:6 (low concentrate group) and 6:4 (high concentrate group). The experiment was performed as follow:1) Milk yield was recorded every day and milk composition was analyzed every week.2) The rumen pH value was detected; 3) Testing of the indexes in the plasma including biochemical indexes, free amino acids and LPS content. The results showed that,1) Milk yield was no changes during the early feeding. Begin with the fifth week; the milk yield was increased in the high concentrate diet group. From the fourteen week, the milk yield was reduced until the end of the experiment.2) At the beginning of the experiment, the milk protein and milk fat content is higher in the high concentrate group than that of low concentrate group. However, they were significantly decreased on the twenty week (P< 0.05). Lactose, total solid matter and dry matter of milk and milk urea nitrogen levels had no significant difference (P>0.05).3) With the fourth week, the rumen pH value was reduced in the high concentrate group and the duration of pH value below the 5.8 was more than 3 h. Thus, the cow’s rumen was in the state of SARA in the high concentrate group.4) Compared with the low concentrate diet, the contents of Asp, Ala, Tyr, Thr, Val, Trp and Ile in the jugular vein of dairy cows. The contents of LPS in plasma in high concentrate group was higher than that in low concentrate group, the levels of GOT, GPT and creatinine in high concentrate group were significantly increased (P<0.05). The results suggested that the milk yield, milk protein and milk fat was increased with the short term feeding high concentrate diet. Milk protein and milk fat was significantly decreased when the time of feeding was extended. The cow’s rumen fermentation is unusual and it is in a state of SARA, which resulted in increasing the plasma LPS content and decreasing the plasma total free amino acid content. These different changes might affect the milk quality, the rumen fermentation and health, but the related mechanism needs further research.2 Changes of Hepatic Amino Acids metabolic effect milk protein synthesis in lactating dairy cows fed a High-Concentrate DietThis section contains the following two series: Series 1:The changes of the distribution and redistribution of hepatic amino acids in lactating dairy cows with a High-Concentrate DietThe feeding and grouping of the lactating dairy cows were same as the first chapter. After 23 weeks. Hepatic vein and portal venous blood was collected by the liver vein intubation. Liver tissue sample was biopsy puncture to do the experiments as follow:1) Histological observation of morphological changes of liver lactating cows with high concentrate long-term feeding; 2) Determination the content of blood biochemical index; 3) Determination the content of amino acids in the plasma. The results was shown as follow,1) compared with low concentrate group, liver tissue morphological observation showed that the outline of central vein was fuzzy, hepatic cords is not clear, cytoplasm dyed deep, of those indicated the liver was in the faulted condition.2) The content of FAA in the portal vein (input) in the high concentrate group is higher than the content of hepatic vein (output), which indicated the amino acid depletion in the high concentrate diet was more than low concentrate group in the liver. Thereinto, the content of Ser, Gln, Gly and, Val, Ile, Lys and Arg is significantly higher than low concentrate group (P<0.05 & P<0.01).3) compared with low concentrate group, the content of total protein (TP), globulin (Glb) and glucose (Glu) in plasma were higher than in low concentrate group, and these indicators in input plasma was higher than the output plasma of liver, but the contents of albumin and urea has no obvious change. The creatinine content of input liver blood was significantly higher in the high concentrate group than that of low concentrate group (P<0.05), and it is more than the output of liver blood. The results suggested that high concentrate diet long-term feeding could damage the lactation cows liver tissue, increase the content of FAA, total protein, globulin and glucose, and enhance the synthesis of creatinine resulted in the amino acid depletion. The alteration of amino acid by distribution and redistribution was reduced the quantity demanded of milk protein synthesis in mammary. Thus, amino acid undersupply may be one of the reasons for milk protein decreased with high concentrate feeding.Series 2:The Comparative proteomic analysis of the hepatic metabolism of lactating dairy cows with a High-Concentrate DietFirst series research showed that the milk protein synthesis precursor of amino acid was depletion too much to undersupply result in the milk protein decreased under high concentration diet feeding during the lactation. In order to know the alteration of amino acid in the liver, we adopted the liver proteomics technology, through comparative analysis, further reveals the possible mechanism. Experiment was performed to separation liver proteomic by 2D-PAGE. The differences in protein obtained from the spectrum analysis and they were identified by MALDI-TOF-TOF MS. As a result,56 protein spots were differently expressed and the abundance is greater than 2.0, or less than 0.5 the differences of protein (P< 0.05), and 40 proteins were successfully identified. The identified proteins were involved in metabolic and cellular processes, including protein synthesis, protein folding and assembly, carbohydrate metabolism, amino acids, and nitrogen metabolism, etc. Screening and the identification of differentially expressed proteins was analyzed the protein interactions have centered on pyruvate carboxylase metabolism of protein interaction networks, including amino acids, and nitrogen metabolism, lipid metabolism and carbohydrate metabolism. Verification results showed that compared with low concentrate group, high concentrate feeding significantly raised aspartic acid transaminase (2.768±0.07 vs.1.001±0.04), guanidine acetic acid methyl transferase (3.289±0.11 vs.1.002±0.05) and glyceraldehyde-3-phosphate dehydrogenase (3.667±0.11 vs.1.027±0.17) gene mRNA expression levels (P<0.05). The results indicated that high concentrate feeding lactation cows of liver metabolism and defense mechanism has changed, the liver in sugar, fat, and amino acid catabolism was inhibited, creatine and protein (such as globulin and emergency) synthesis and antioxidant stress activation process was increased. Thus, these changes was increased the depletion of amino acid and resulted in milk protein depression.3 Changes of mammary Amino Acids uptake and utilization effect milk protein synthesis in lactating dairy cows fed a High-Concentrate Diet10 healthy lactating Holstein cows were selected in this experiment. The cows were randomly divided into 2 groups:forage-to-concentrate ratio 4:6 (low concentrate group) and 6:4 (high concentrate group). The experiment was performed as follow:1) Histological observation of morphological changes of mammary lactation cows with high concentrate long-term feeding; 2) Determination the content of amino acids in the mmary vein plasma and the somatic cell count in milk.3) Mammary tissue sample was biopsy puncture. The amino acid transport carrier of SLC7A8, SLC7A10, SLC16A10, SLC1A3, SLC7A7, SLC7A6 and SLC6A15 gene mRNA expression was detected by real-time PCR. The results showed:1) Compared with low concentrate group, breast tissue morphology observation showed that neutrophils was in the gland bubble, gland bubble structure are destroyed partly, gland bubble wall slightly swollen, of these indicated the mammary gland was damaged in the high concentrate group.2) Compared with low concentrate group, the total free amino acid and raw sugar amino acids were lower. Thereinto, the content of Asn, Gly, Tyr, Arg and Lys was significantly decreased (P< 0.05). The somatic cell counts in milk were increased and more than 250000 cell/mL.3) The abundances of the mRNA for amino acid transporter SLC16A10, SLC1A3 and SLC7A10 (P<0.05) were decreased, SLC6A15, SLC7A7 and SLC7A6 were increased (P<0.05) in the high concentrate group. These data indicate that the somatic cell counts in milk were increased and mammary gland bubble structure was damaged could induce the recessive mastitis in lactation cows with high concentrate diet feeding. Amino acid transport carrier of mammary gland gene mRNA expression level was decreased, subsequently, resulted in the milk protein synthesis of precursor amino acid transport depression, especial BCAA depressed. Amount of amino acids in the mmary vein is reduced, and that amino acid could use for synthesis of inflammatory factors, and decreased amino acid utilization, which affects the milk protein synthesis.Overviews of all, the high concentrate diet feeding were resulted in rumen metabolic abnormalities, hepatic metabolism and function changes. Of these alterations were affected the health and function of mammary gland, altered the milk protein precursor amino acid metabolism, influenced the milk protein synthesis. |
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