【摘要】：由于缺乏優(yōu)質(zhì)牧草,常給泌乳奶牛飼喂高精料日糧以達(dá)到高產(chǎn)奶的目的,然而,長(zhǎng)期過度飼喂高精料會(huì)引起奶牛瘤胃異常發(fā)酵,從而影響奶牛健康,最終降低其泌乳性能。肝臟生理機(jī)能是機(jī)體重要的保護(hù)屏障,乳成分前體物也會(huì)經(jīng)肝臟代謝而進(jìn)行營(yíng)養(yǎng)的分配與重分配,從而影響免疫與生產(chǎn)性能。本文以泌乳山羊?yàn)閯?dòng)物模型,研究長(zhǎng)期采食高精料對(duì)瘤胃、血液生理指標(biāo)及乳品質(zhì)的影響,并采用2D-MALDITOF/TOF、western blot和real-time PCR技術(shù)研究肝臟蛋白質(zhì)譜的變化,以揭示高精料日糧對(duì)泌乳山羊肝臟免疫與代謝的影響及機(jī)制;并通過體外原代細(xì)胞培養(yǎng)實(shí)驗(yàn),探索內(nèi)源性脂多糖LPS和瘤胃代謝產(chǎn)物對(duì)肝細(xì)胞代謝的影響。1高精料對(duì)奶山羊生產(chǎn)性能的影響選擇12只泌乳中期關(guān)中奶山羊,隨機(jī)分成高精料組(精粗比為65:35)和低精料組(精粗比為35:65),試驗(yàn)期12周。每周記錄奶產(chǎn)量,檢測(cè)乳品質(zhì)指標(biāo)。試驗(yàn)結(jié)束時(shí),采集瘤胃液、血液和肝臟組織等樣品。pH計(jì)檢測(cè)瘤胃液pH值;氣象色譜法檢測(cè)揮發(fā)性脂肪酸含量;顯色基質(zhì)法檢測(cè)瘤胃液LPS濃度;全自動(dòng)血液生化分析儀檢測(cè)血漿中白蛋白、膽固醇、血糖等生理生化指標(biāo)。結(jié)果表明:高精料日糧顯著降低奶山羊瘤胃液pH(P0.01);升高瘤胃液中丙酸(P0.01)、丁酸(P0.01)、異丁酸(P0.05)、戊酸(P0.01)、異戊酸(P0.01)以及總揮發(fā)性脂肪酸VFA(P0.05)的濃度;降低乙酸/丙酸的比值(P0.01),顯著增加瘤胃液中LPS((P0.01)濃度。與低精料日糧相比,高精料日糧顯著增加山羊乳產(chǎn)量(P0.01)、增加乳糖(P0.01)、乳脂(P0.01)、乳蛋白(P0.01)的含量;增加血液白蛋白(P= 0.07)和低密度脂蛋白膽固醇(P0.05)。2高精料對(duì)奶山羊肝臟蛋白表達(dá)譜及脂代謝的影響與低精料組相比,高精料組山羊肝體比顯著升高(P0.05);肝臟CRP mRNA(P0.05)、SAA mRNA(P0.05)、CD14 mRNA(P0.05)表達(dá)顯著升高,但 TNF-a mRNA表達(dá)有下降的趨勢(shì)(0.05P0.1)。用2-DE/MALDI-TOF技術(shù)鑒定肝臟蛋白表達(dá)譜,共得到20個(gè)蛋白點(diǎn),這些蛋白參與機(jī)體氧化應(yīng)激、線粒體功能以及葉酸代謝等的調(diào)節(jié)。采用ELLSA方法檢測(cè)結(jié)果表明,與低精料組山羊相比,高精料組山羊肝臟總抗氧化能力顯著降低(P0.01)。Real-time PCR檢測(cè)結(jié)果表明,線粒體基因COX3 mRNA表達(dá)有上升趨勢(shì)(0.05P0.1),ATP8 mRNA表達(dá)顯著上調(diào)(P0.05)。與低精料組山羊比較,高精料組山羊肝臟膽固醇含量顯著降低(P0.01)。Real-time PCR 結(jié)果表明,ABCA1(P0.01)、ABCG1(P0.01)、LXRa(P0.01)、LXRβ(P0.05)和SREBP2(P0.01)mRNA表達(dá)水平顯著上升;肝臟ACAA2(P0.05)、ACSS1(0.05P0.1)、FASN(P0.05)、PPARa(P0.05)、SREBP1c(P0.05)、DGAT1(P0.05)和DGAT2(P0.05)mRNA表達(dá)水平均顯著上調(diào)。3 丁酸鈉、LPS對(duì)山羊肝細(xì)胞糖脂代謝的影響選擇2.42kg、45日齡波雜小山羊,處死后分離肝細(xì)胞,貼壁培養(yǎng)4h后,在生長(zhǎng)培養(yǎng)基中培養(yǎng)72 h,無(wú)血清培養(yǎng)基饑餓細(xì)胞24 h。用5 mmol/L 丁酸納、100 ng/mL LPS以及5 mmol/L 丁酸納和100 ng/mL LPS共同處理肝細(xì)胞24 h,收集細(xì)胞上清及細(xì)胞。檢測(cè)細(xì)胞活力,上清液中葡萄糖含量;檢測(cè)細(xì)胞中糖脂代謝相關(guān)基因表達(dá)與蛋白含量的差異。結(jié)果顯示,丁酸鈉顯著升高肝細(xì)胞活性(P0.05);丁酸鈉處理顯著降低上清液中葡萄糖含量(P0.01)。丁酸鈉和LPS均顯著下調(diào)G6Pase mRNA表達(dá)(P0.05),丁酸鈉下調(diào) PCK2(P0.05)、PKLR(P0.05)、COX1(P0.05)、SCD(P0.01)mRNA 轉(zhuǎn)錄。LPS 上調(diào) ND4(0.05P0.1)和 PFKL(0.05P0.1)mRNA 表達(dá)。蛋白免疫印跡結(jié)果表明,丁酸納處理增加GPR41蛋白表達(dá)(P0.1);丁酸納處理引起細(xì)胞AMPK蛋白表達(dá)升高(0.05P0.1);LPS顯著增加肝細(xì)胞AMPK(P0.05)和 p-AMPK(0.05P0.1)的蛋白含量。以上研究結(jié)果表明:長(zhǎng)期飼喂高精料的奶山羊在增加乳產(chǎn)量的同時(shí),瘤胃發(fā)生酸中毒,肝臟抗氧化能力降低,線粒體氧化磷酸化增強(qiáng),脂肪酸氧化增強(qiáng);丁酸納和LPS處理原代細(xì)胞,丁酸鈉通過AMPK通路抑制糖異生,LPS通過AMPK通路激活線粒體氧化磷酸化。
[Abstract]:Due to the lack of high quality pasture, high concentrate diet is often fed to lactating cows for the purpose of high milk production. However, long term overfeeding of high concentrate can cause abnormal fermentation in the rumen of dairy cows, which will affect the health of dairy cows and ultimately reduce their lactation performance. In this paper, lactating goats were used as animal models to study the effects of long term diet high concentrate on rumen, blood physiological indexes and milk quality, and the changes of liver protein mass spectrometry were studied by 2D-MALDITOF/TOF, Western blot and real-time PCR techniques to reveal high essence. Effects and mechanisms of diet on liver immunity and metabolism in lactating goats, the effects of endogenous lipopolysaccharide LPS and rumen metabolites on hepatocyte metabolism were investigated by primary culture in vitro. The effects of.1 high concentrate on milk goat production performance were selected in 12 mid-term dairy goats, which were randomly divided into high concentrate group (65:). 35) and low sperm group (35:65), the test period was 12 weeks. Milk production was recorded every week and milk quality was detected. At the end of the experiment, the pH value of tumor gastric juice was detected by.PH, the content of volatile fatty acids was detected by the method of meteorologic chromatography, the concentration of LPS in the gastric juice was detected by the chromogenic matrix method, and the automatic blood biochemical analysis was carried out. The results showed that high concentrate diet significantly reduced pH (P0.01) of milk goats gastric juice, increased propionic acid (P0.01), butyric acid (P0.01), isobutyric acid (P0.05), valerate (P0.01), isovaleric acid (P0.01) and total volatile fatty acid VFA (P0.05) in the tumor gastric juice, and reduced the ratio of acetic acid / propionic acid. Value (P0.01) significantly increased the concentration of LPS ((P0.01) in gastric juice. Compared with low concentrate diet, high concentrate diet increased goat milk yield (P0.01), increased lactose (P0.01), milk fat (P0.01), milk protein (P0.01) content, increased serum albumin (P= 0.07) and low density lipoprotein cholesterol (P0.05).2 high concentrate on the liver protein expression profiles of dairy goats The effect of lipid metabolism on the liver body ratio of the high concentrate group was significantly higher than that in the low concentrate group (P0.05), and the expression of CRP mRNA (P0.05), SAA mRNA (P0.05) and CD14 mRNA (P0.05) in the liver increased significantly, but the expression of TNF-a mRNA was decreased. The protein expression profiles of the liver were identified with 20 protein points and these proteins were obtained. The results of ELLSA method showed that the total antioxidant capacity of the liver in the high concentrate group was significantly lower than that of the low sperm group (P0.01).Real-time PCR detection results showed that the expression of COX3 mRNA in the mitochondria was rising (0.05P0.1) and ATP8 mRNA table. The cholesterol content in the liver of the high concentrate group was significantly lower than that of the low sperm group (P0.05). The results of the liver cholesterol in the high concentrate group were significantly lower (P0.01).Real-time PCR results showed that ABCA1 (P0.01), ABCG1 (P0.01), LXRa (P0.01), LXR beta (P0.05) and PCR were significantly increased. (P0.05), the expression level of DGAT1 (P0.05) and DGAT2 (P0.05) mRNA significantly up-regulated the sodium butyrate and LPS on the glycolipid metabolism of goat liver cells, selected 2.42kg, 45 day old goat, separated from the liver cells after death, and after adherent culture 4h, the 72 h was cultured in the growth medium, and the serum-free medium starvation cells 24 h. was 5 sodium butyric acid, 100 LPS and 5 mmol/L butyrate and 100 ng/mL LPS were used to treat 24 h of liver cells, collect cell supernatant and cell. Detect cell vitality, glucose content in supernatant, detect the difference of gene expression and protein content in cells. The results show that sodium butyrate significantly increases the activity of liver cells (P0.05); sodium butyrate treatment is significantly reduced. The content of glucose (P0.01) in the supernatant (P0.01). Sodium butyrate and LPS decreased the expression of G6Pase mRNA (P0.05), sodium butyrate downregulated PCK2 (P0.05), PKLR (P0.05), COX1 (P0.05), and the egg white immunoblotting showed that butyrate treatment increased the expression of protein. The increased expression of AMPK protein (0.05P0.1) was caused by acid treatment; LPS significantly increased the protein content of AMPK (P0.05) and p-AMPK (0.05P0.1) in the liver cells. The above results showed that the milk goats fed with high sperm for a long time increased the milk yield, and the rumen was acidosis, the liver antioxidant capacity was reduced, the mitochondrial oxidative phosphorylation was enhanced, and the lipid was increased. The oxidation of fatty acid was enhanced. Sodium butyrate and LPS treated primary cells. Sodium butyrate inhibited gluconeogenesis through AMPK pathway. LPS activated mitochondrial oxidative phosphorylation through AMPK pathway.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S827.5

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