【摘要】：細(xì)胞凋亡是維持內(nèi)環(huán)境穩(wěn)定的重要機(jī)制,它對機(jī)體免疫系統(tǒng)的影響很大,凋亡功能過度旺盛或不足,均能引起自身免疫疾病[1]。已發(fā)現(xiàn)的細(xì)胞凋亡的誘導(dǎo)因素有很多,通常的研究主要集中在外界環(huán)境或人為添加物質(zhì)對其的影響,對于奶牛自身細(xì)胞凋亡的規(guī)律報道較少,而對于泌乳階段和產(chǎn)奶水平對細(xì)胞凋亡的影響尚未見報道。本試驗的目是研究不同產(chǎn)奶水平和泌乳階段的奶牛外周血淋巴細(xì)胞凋亡的差異情況。在以往的研究中發(fā)現(xiàn),夏季高溫條件下奶牛的淋巴細(xì)胞凋亡率會因熱應(yīng)激的影響而大幅升高。本試驗通過飼喂不同能量水平、不同能量來源和添加過瘤胃賴氨酸和蛋氨酸的9種日糧,比較各日糧配比下奶牛淋巴細(xì)胞凋亡率的差異,以期找到降低淋巴細(xì)胞凋亡的營養(yǎng)調(diào)控措施。本研究進(jìn)行了以下幾個方面的試驗:試驗一:根據(jù)產(chǎn)奶量和泌乳階段,選擇6組奶牛,每組8頭,共計48頭。分別為泌乳盛期高產(chǎn)牛組、泌乳盛期低產(chǎn)牛組、泌乳中期高產(chǎn)牛組、泌乳中期低產(chǎn)牛組、泌乳后期高產(chǎn)牛組、泌乳后期低產(chǎn)牛組。對各組試驗牛進(jìn)行尾靜脈采血,采用流式細(xì)胞儀檢測各組的外周血淋巴細(xì)胞凋亡率。結(jié)果表明:在泌乳盛期高產(chǎn)奶牛的淋巴細(xì)胞凋亡率極顯著高于低產(chǎn)奶牛(P0.01);泌乳中期高產(chǎn)組奶牛的淋巴細(xì)胞凋亡率極顯著高于低產(chǎn)奶牛(P0.01);泌乳后期兩個組差異不顯著。將各泌乳期的高產(chǎn)、低產(chǎn)奶牛進(jìn)行綜合對比發(fā)現(xiàn),高產(chǎn)奶牛的淋巴細(xì)胞凋亡率與低產(chǎn)奶牛差異顯著(P0.05);泌乳末期的淋巴細(xì)胞凋亡率顯著高于泌乳盛期(P0.05),且隨著泌乳時間的延長,淋巴細(xì)胞凋亡率逐漸上升。試驗二:選擇72頭奶牛,根據(jù)體重、產(chǎn)奶量、胎次以及泌乳階段相近的原則,先將奶牛配對處理后再隨機(jī)分為9組,每組8頭。采用L9(34)正交試驗設(shè)計,以日糧DM中能量水平、碳水化合物類型和瘤胃保護(hù)性氨基酸(RPAA)為試驗因子,共設(shè)計9種日糧對奶牛進(jìn)行飼喂,30天后尾靜脈采血,測定奶牛外周血淋巴細(xì)胞凋亡率。結(jié)果表明:能量水平能夠顯著(P0.05)的影響淋巴細(xì)胞的凋亡,隨著能量梯度的增加,淋巴細(xì)胞的凋亡率逐漸降低;不同能量來源對各組影響差異不顯著;從過瘤胃氨基酸添加水平上來看,分別添加10g/d.Head RPMet和30g/d.Head RPLys在降低淋巴細(xì)胞凋亡率上效果最好。試驗三:試驗設(shè)計同試驗二,在采血后測定淋巴細(xì)胞凋亡相關(guān)基因的表達(dá)。結(jié)果表明:隨著能量水平的提高,Bcl-2基因的表達(dá)量升高,P53基因的表達(dá)降低;添加過瘤胃蛋氨酸和賴氨酸能夠影響B(tài)cl-2基因的表達(dá),添加量分別為10g和30g時,Bcl-2的表達(dá)量最高;隨著過瘤胃氨基酸量的添加量的增加,Bax的表達(dá)量逐漸升高,P53的表達(dá)逐漸降低。本研究發(fā)現(xiàn),不同泌乳階段高低產(chǎn)奶牛的淋巴細(xì)胞凋亡率不同;能量水平和補(bǔ)飼過瘤胃氨基酸能夠影響淋巴細(xì)胞凋亡率,在一定范圍內(nèi)凋亡率與能量水平和補(bǔ)飼劑量密切相關(guān)。
[Abstract]:Apoptosis is an important mechanism to maintain the stability of the internal environment. It has a great influence on the immune system. The function of apoptosis is exuberant or insufficient, which can cause autoimmune diseases [1]. Many factors have been found to induce apoptosis. Most of the studies focus on the effects of external environment or artificial addition of substances on apoptosis, but there are few reports on the regularity of apoptosis in dairy cattle. However, the effect of lactation stage and milk production level on cell apoptosis has not been reported. The purpose of this experiment was to study the difference of apoptosis in peripheral blood lymphocytes of dairy cows at different milk production levels and lactation stages. In previous studies, it was found that the apoptosis rate of lymphocytes in dairy cows increased significantly under high temperature in summer due to the effect of heat stress. In this study, nine diets were fed with different energy levels, different energy sources and supplementation of rumen lysine and methionine. In order to find out the nutritional control measures to reduce lymphocyte apoptosis. The experiment was carried out in the following aspects: experiment 1: according to the milk yield and lactation stage, 6 groups of cows were selected, 8 cows in each group, a total of 48 cows. The cows were divided into three groups: high yield cattle group, low productivity cattle group, middle lactation high yield cattle group, mid lactation low productivity cattle group, late lactation high yield cattle group and late lactation low productivity cattle group. The blood samples were collected from the tail vein and the apoptosis rate of peripheral blood lymphocytes was detected by flow cytometry. The results showed that the apoptosis rate of lymphocytes in high-yielding cows was significantly higher than that in low-yield cows (P0.01), and the apoptosis rate of lymphocytes in high-yielding cows was significantly higher than that in low-yield cows (P0.01), but there was no significant difference between the two groups in the late lactation period. The results showed that the apoptosis rate of lymphocytes in high-yielding dairy cows was significantly higher than that in low-yielding cows (P0.05), and the apoptosis rate of lymphocytes in late lactation stage was significantly higher than that in peak lactation period (P0.05), and with the prolongation of lactation time, the apoptosis rate of lymphocytes was significantly higher than that of lactation period (P0.05). The apoptosis rate of lymphocytes increased gradually. Experiment 2: 72 cows were selected. According to the principle of similar body weight, milk yield, birth order and lactation stage, the cows were divided into 9 groups at random, 8 in each group. Using L9 (34) orthogonal design, nine diets were designed to collect blood from tail vein of dairy cattle after 30 days of feeding with energy level, carbohydrate type and rumen protective amino acid (RPAA) as experimental factors. The apoptosis rate of peripheral blood lymphocytes in dairy cattle was measured. The results showed that the energy level could significantly affect the apoptosis of lymphocytes (P0.05), and with the increase of energy gradient, the apoptosis rate of lymphocytes decreased gradually, but different energy sources had no significant effect on the apoptosis of lymphocytes. From the level of rumen amino acid supplementation, 10g/d.Head RPMet and 30g/d.Head RPLys had the best effect on reducing the apoptosis rate of lymphocytes. Experiment 3: the expression of apoptosis-related genes in lymphocytes was determined after blood collection. The results showed that: with the increase of energy level, the expression of Bcl-2 gene increased and the expression of p53 gene decreased, and the expression of Bcl-2 gene was affected by the addition of rumen methionine and lysine, and the expression of Bcl 2 was the highest when the dosage was 10 g and 30 g, respectively. With the increase of the amount of amino acid in rumen, the expression of Bax increased gradually and the expression of p53 decreased gradually. The results showed that the apoptosis rate of lymphocytes was different in dairy cows at different lactation stages, and the energy level and rumen amino acid supplementation could affect the apoptosis rate of lymphocytes, and the apoptosis rate was closely related to the energy level and supplementary feeding dose in a certain range.
【學(xué)位授予單位】：河南農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S823

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