产奶水平和泌乳阶段对奶牛淋巴细胞凋亡的影响及热应激下细胞凋亡的营养调控

发布时间：2018-08-12 08:50

【摘要】：细胞凋亡是维持内环境稳定的重要机制,它对机体免疫系统的影响很大,凋亡功能过度旺盛或不足,均能引起自身免疫疾病[1]。已发现的细胞凋亡的诱导因素有很多,通常的研究主要集中在外界环境或人为添加物质对其的影响,对于奶牛自身细胞凋亡的规律报道较少,而对于泌乳阶段和产奶水平对细胞凋亡的影响尚未见报道。本试验的目是研究不同产奶水平和泌乳阶段的奶牛外周血淋巴细胞凋亡的差异情况。在以往的研究中发现,夏季高温条件下奶牛的淋巴细胞凋亡率会因热应激的影响而大幅升高。本试验通过饲喂不同能量水平、不同能量来源和添加过瘤胃赖氨酸和蛋氨酸的9种日粮,比较各日粮配比下奶牛淋巴细胞凋亡率的差异,以期找到降低淋巴细胞凋亡的营养调控措施。本研究进行了以下几个方面的试验:试验一:根据产奶量和泌乳阶段,选择6组奶牛,每组8头,共计48头。分别为泌乳盛期高产牛组、泌乳盛期低产牛组、泌乳中期高产牛组、泌乳中期低产牛组、泌乳后期高产牛组、泌乳后期低产牛组。对各组试验牛进行尾静脉采血,采用流式细胞仪检测各组的外周血淋巴细胞凋亡率。结果表明:在泌乳盛期高产奶牛的淋巴细胞凋亡率极显著高于低产奶牛(P0.01);泌乳中期高产组奶牛的淋巴细胞凋亡率极显著高于低产奶牛(P0.01);泌乳后期两个组差异不显著。将各泌乳期的高产、低产奶牛进行综合对比发现,高产奶牛的淋巴细胞凋亡率与低产奶牛差异显著(P0.05);泌乳末期的淋巴细胞凋亡率显著高于泌乳盛期(P0.05),且随着泌乳时间的延长,淋巴细胞凋亡率逐渐上升。试验二:选择72头奶牛,根据体重、产奶量、胎次以及泌乳阶段相近的原则,先将奶牛配对处理后再随机分为9组,每组8头。采用L9(34)正交试验设计,以日粮DM中能量水平、碳水化合物类型和瘤胃保护性氨基酸(RPAA)为试验因子,共设计9种日粮对奶牛进行饲喂,30天后尾静脉采血,测定奶牛外周血淋巴细胞凋亡率。结果表明:能量水平能够显著(P0.05)的影响淋巴细胞的凋亡,随着能量梯度的增加,淋巴细胞的凋亡率逐渐降低;不同能量来源对各组影响差异不显著;从过瘤胃氨基酸添加水平上来看,分别添加10g/d.Head RPMet和30g/d.Head RPLys在降低淋巴细胞凋亡率上效果最好。试验三:试验设计同试验二,在采血后测定淋巴细胞凋亡相关基因的表达。结果表明:随着能量水平的提高,Bcl-2基因的表达量升高,P53基因的表达降低;添加过瘤胃蛋氨酸和赖氨酸能够影响Bcl-2基因的表达,添加量分别为10g和30g时,Bcl-2的表达量最高;随着过瘤胃氨基酸量的添加量的增加,Bax的表达量逐渐升高,P53的表达逐渐降低。本研究发现,不同泌乳阶段高低产奶牛的淋巴细胞凋亡率不同;能量水平和补饲过瘤胃氨基酸能够影响淋巴细胞凋亡率,在一定范围内凋亡率与能量水平和补饲剂量密切相关。
[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.
【学位授予单位】：河南农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S823

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