| This study aims to explore the rule of milk fat synthesis precuesor of fatty acid generation and metabolism under the condition of high concentrate feeding in our country,in order to expound influencing mechanism of milk fat synthesis.The main content of research includes the following aspects:1 Effects of high-concentrate diet on milk quality and body health in lactating goatsThe purpose of this research is to study the effects of high-concentrate diet on milk quality and body health of lactating goats(Capra hircus).8 healthy mid-lactation goats were randomly divided into 2 groups,using 2×2 Latin square design with 2 replications.The goats were fed with forage with different concentrate ratios,4:6(the control group)and 6:4(the high-concentrate group),for 8 weeks each Latin square cycle.Milk samples were taken to measure milk yield everyday.milk composition and rumen pH were measured every 2 weeks,and the contents of MPO,AKP and NAGase in skim milk were measured at the same time.At the end of each cycle(8 weeks),blood was taken from jugular vein,simultaneously liver and breast samples were collected by living donor.The contents of LPS,lactic acid and histamine in blood were determination by enzymatic colorimetric method;the related biochemical indicators in peripheral blood were detected by automatic blood analyzer or enzyme colorimetric method;morphological changes were observed by HE staining of liver and breast tissue.Results:Comparing the two groups,the daily milk yield in high-concentrate group was higer than that of the control group,but the rate of milk fat and milk protein decreased significantly(P<0.05);milk somatic cell counts continued to rise with the extension of feeding time(P<0.05).The rumen fluid PH value of high-concentrate group always lower than the control group,and SARA occurred after feeding 2 weeks.The LPS content in peripheral blood had an extremely significant increase after feeding 8 weeks(4.67±0.30 vs 9.25±1.02).At the same time,GOT activity and NEFA content in blood were significantly increased(48.00±2.30 vs 64.67±1.45,575.89±30.49 vs 812.23±51.51);albumin content decreased significantly(26.13±1.18 vs 24.23±1.33);glucose and LDL-C were significantly increased in high-concentrate(3.02±0.35 vs 3.31±0.13,0.51±0.12 vs 0.69±0.08).The concentrations of NAGase,AKP and MPO in skim milk showed significant(P<0.05)or extremely significant(P<0.01)increase.The results of HE staining showed that the liver tissue appeared to have a fatty degeneration and inflammatory cell infiltration;while mammary gland lumen showed a large number of inflammatory cells and the structure was not integrated.Combined with the above tests,this research indicated that there was no significant changes in milk yield and milk quality in the short term feeding of high concentrate diet on lactating goats,but the long term feeding could lead to the decreased milk fat and milk protein.High-concentrate diet caused the rumen pH decreased and LPS increased,the mammary gland and liver were in the state of chronic or latent inflammation.The changes of milk precursors in blood may be one of the reasons to cause decreased milk quality.2 Changes of mik fat precuror distribution and redistribution as well as its mechanism in the liverThe purpose of this research is to study the relationship between different concentrate to forage diet feeding and changes of hepatic lipid metabolism,then investigate the mechanism of milk fat depression caused by high-concentrate diet.6 healthy mid-lactation goats with hepatic vascular fistula,were randomly divided into 2 groups,experiment design according to the fourth chapter.At the end of each cycle(8 weeks),blood was collected through hepatic portal vein and hepatic vein,liver samples were collected by living donor liver biopsy.The contents of lipid metabolism related indexes and TG,NEFA,total cholesterol and glycogen in liver were determined;The differential expression proteins in liver was analyzed by two-dimensional electrophoresis(2-DE)and MAIDI-TOF/TOF;The gene and protein expression of hepatic glucolipid metabolism related enzymes were determined.Results:1)The contents of TG and NEFA in the portal vein(input)in the high-concentrate group were higher than that in hepatic vein(output),and the differences were extremely significant(P<0.01)and significant(P<0.05),which indicated the consumption of milk fat precursors were more in the high-concentrate group.The contents of glucose,total cholesterol and BHBA in input blood showed lower than that in output blood,but the differences were not significant(P>0.05).The content of TG in liver tissue was significantly higher than that in control group(0.83±0.02 vs 2.27±0.51),NEFA content decreased significantly(76.21±6.76 vs 55.43±3.81).2)The results of proteomics showed,52 protein spots of more than 2-fold difference in both dietary modes were revealed,and of which 51 were identified.The identified proteins mainly involved in lipid metabolism,glucose metabolism,protein metabolism and stress response.The results of protein interaction analysis showed ApoA-I,AC02 and HSPA1B were in the core.3)Compared with the control group,high-concentrate diets feeding significantly increased the mRNA expression levels of FAS(1.00±0.16 vs 3.09±0.45),SCD-1(1.00±0.18 vs 3.53±0.33),DGAT2(1.00±0.22 vs 1.55±0.03),HMGCS2(1.00±0.22 vs 2.20±0.23)and ACSL1(1.00±0.06 vs 1.46±0.10),significantly decreased the mRNA expression levels of PEPCK(1.00±0.10 vs 0.58±0.02).The phosphorylation level of ACC1 and the protein expression level of ACSL1 were significantly higher than those in control group(23.00±3.13 vs 53.76±5.62,1.39±0.09 vs 1.80±0.07).The results showed that long term feeding of high concentrate diet caused the amount of NEFA input the liver higher than that output the liver,and the supply of milk fat precursors was reduced.This may be one of the reasons for milk fat depression under the feeding of high concentrate diet.The synthesis of TG,glucose and ketone body in liver were increased,gluconeogenesis and lipid catabolism were blocked,anti-oxidative stress process was activated,these factors leaded to the excessively consumption of NEFA and other precursors in liver.3 The endocrine mechanism and signaling pathway by which a high concentrate diet causes redistribution of mik fat precuror in lactating goatsWe have found that long-time feeding of high concentrate diet on lactating goats can cause the redistribution of milk fat precursors and change of glucolipid metabolism in liver.In order to investigate the endocrine mechanism of milk fat depression caused by milk fat redistribution,this experiment was focussed on the changes of blood hormone and possible signal pathways.Experiment animals and design were accorded to the fifth chapter.In the end of each circle,blood was collected through jugular vein and liver samples by living donor biopsy.Hormone contents in peripheral blood were detected.Protein and mRNA expression levels of factors involved in PI3K-Akt pathway were determined by real-time PCR and Western Blot.The results showed that 1)Insulin levels in peripheral blood were significantly increased(P<0.05)in high concentrate group,IGF-I decreased significantly(P<0.05).2)In high concentrate group,SREBP-1c and LXR mRNA expression levels were significantly increased(1.00±0.10 vs 1.96±0.14,1.00±0.07 vs 1.45±0.46),PPARa and PPAR?mRNA expression levels were significantly decreased(1.00±0.29 vs 0.70±0.10,1.00±0.23 vs 0.58±0.11).3)IR,PI3K and Akt mRNA expression levels were significantly increased in high concentrate group(1.00±0.13 vs 1.42±0.18,1.00±0.07 vs 1.45±0.16,1.00±0.06 vs 1.67±0.04),PI3K protein level and S6K phosphorylation level were significantly higher than those in control group(0.81±0.03 vs 1.26±0.05,25.09±2.98 vs 37.21±3.88).The results suggested that long-time feeding of high concentrate diet on lactating goats caused the secretion of insulin increased and then insulin-PI3K-Akt pathway was activated.The up-regulated SREBP-lc(key transcription factors of lipid synthesis)promoted lipid synthesis in liver.And the synthesis of milk fat was reduced because of the excessive consumption of milk fat precursors. |
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