组学技术研究亚急性瘤胃酸中毒对奶牛瘤胃微生物、代谢和上皮功能的影响

发布时间：2018-06-02 23:19

本文选题：奶牛 + SARA　；参考：《南京农业大学》2015年博士论文

【摘要】：瘤胃是反刍动物营养物质消化代谢的主要场所,其健康与否直接关系动物机体健康与生产性能的发挥。本论文拟系统研究高精料日粮对奶牛瘤胃健康和泌乳性能的影响,研究内容包括以下四方面。1.SARA对瘤胃细菌菌群结构与组成的影响试验选用4头装有瘤胃瘘管的泌乳奶牛,将其随机分为对照组(CON,40%精料)或SARA组(SARA,70%精料),采用2×2交叉试验设计。每试验期第12、17和21天晨饲后0 h采集瘤胃液样品,提取基因组DNA,进行焦磷酸测序。结果显示,SARA组奶牛瘤胃pH低于5.8的持续时间大于5小时,表明SARA模型成功构建。与对照组相比,SARA组奶牛瘤胃乳酸、丙酸、丁酸、戊酸、总VFA和LPS的浓度(P0.05)显著升高,乙丙比(P0.05)显著降低。SARA组奶牛瘤胃菌群丰度指数(Chao1和Ace)和多样性指数(Shannon)显著低于(P0.05)对照组。在门水平上,SARA组瘤胃变形菌门和拟杆菌门的相对丰度显著降低(P0.05),但厚壁菌门和放线菌门的相对丰度显著增加(P0.05)。属水平上,与对照组相比,SARA组奶牛瘤胃中普雷沃氏菌属、密螺旋菌属、厌氧支原体属、不动杆菌属、Papillibacter和unclassified Lentisphaerae,and unclassified bacteria的相对丰度显著降低(P0.05),而瘤胃球菌属、奇异菌属、双歧杆菌和unclassified Clostridiales的相对丰度显著升高(P0.05)。SARA组奶牛瘤胃内革兰氏阴性菌的比例显著下降,且瘤胃LPS浓度与拟杆菌门细菌拷贝数间存在显著负相关(r =-0.446,P= 0.029)。结果说明,高精料日粮诱发的SARA可导致瘤胃pH下降,VFA累积,LPS浓度升高;瘤胃细菌菌群结构紊乱,瘤胃细菌菌群的多样性降低。2.SARA对奶牛瘤胃代谢产物的影响瘤胃微生物菌群的改变与瘤胃代谢密切相关,本章拟通过研究SARA对奶牛代谢产物的影响,进一步解析SARA对瘤胃健康的影响。试验动物、试验设计与试验日粮同上。选用每试验期第12、17、21天晨饲前瘤胃液样品,进行GC-MS代谢组学检测。试验结果表明,瘤胃液样品中共检测到233种化合物,主要包括有机酸类、脂肪酸类、糖类、氨基酸类、嘌呤类、胺类、糖醇类等物质。统计显示,较对照组相比,SARA组黄嘌呤、次黄嘌呤、尿嘧啶等细菌降解产物含量显著提高(P0.05);LPS、生物胺、乙醇胺、戊二酸等有毒有害或促炎物质的含量显著提高(P0.05);丙氨酸、亮氨酸、甘氨酸、谷氨酸、亮氨酸等15种氨基酸的含量显著提高(P0.05)。对差异代谢物的通路富集分析结果显示,氨酰基tRNA生物合成,苯丙氨酸、酪氨酸和色氨酸生物合成,以及缬氨酸、亮氨酸和异亮氨酸生物合成三条通路发生显著富集(P0.05)。结果说明,高精料日粮诱发的SARA会导致瘤胃代谢紊乱,瘤胃内有毒或促炎物质含量升高,氨基酸合成增加,异常代谢产物的增加可能会影响奶牛瘤胃和机体健康。3.SARA对瘤胃上皮基因表达的影响与机制研究本试验旨在通过基因芯片技术研究SARA对瘤胃上皮基因表达的影响,并结合体外细胞培养手段揭示瘤胃上皮炎症相关基因对高精料日粮的响应模式及其可能机制。体内试验:试验动物、试验设计与试验日粮同上。于每期第21 d采集对照组和SARA组瘤胃上皮样品,进行基因芯片分析。结果显示,两组间共有245个基因存在差异。对差异基因进行通路富集分析和功能注释后,发现因子受体与其关联通路(cytokine-cytokine receptor pathway)显著富集(P0.05),通路中IL-1β、IL-2、IL-22、CCL19、CCL8、CX3CR1、CXCL6、INHBE、LEPR、PRL和TNFRSF9的表达量在SARA组奶牛瘤胃上皮中显著上调(P0.05),而通路中IL-6和IL15RA的表达量显著下调(P0.05),结果表明,瘤胃上皮可能已发生局部炎症反应。为验证基因芯片结果的准确性,使用qRT-PCR对IL-1β、IL-2和IL-6的mRNA表达量进行了测定。相关性分析显示,瘤胃上皮炎症因子与瘤胃环境因子(pH和LPS)存在关联(P0.05),为进一步验证这一结果,进行体外细胞培养试验。结果说明,瘤胃上皮细胞IL-1β、IL-2、IL-6和IL-8的mRNA表达量在LPS处理后显著上调(P0.05),而低pH值处理显著提高了瘤胃上皮细胞TNF-α的表达量,表明瘤胃环境因子pH和LPS可能在SARA引发瘤胃局部炎症反应过程中起着重要作用。4.SARA对奶牛泌乳性能及原奶细菌菌群的影响本章旨在研究SARA对奶牛泌乳性能及乳中细菌菌群结构和组成的影响。试验动物、试验设计与试验日粮同上。分别于每试验期第17、18和19天采集奶样,并于每试验期第12、17和21天饲喂后0 h和4 h采集尾静脉血,对所采集奶样中的细菌菌群结构和组成以及所采集血样中的相关指标进行分析。结果显示,对照组和SARA组奶牛的干物质采食量(DMI)、产奶量和乳成分无显著差异(P0.05)。较对照组相比,SARA组奶牛尾静脉血中白细胞、淋巴细胞数量和白蛋白的含量显著升高(P0.05),而总蛋白、球蛋白、胆固醇和低密度脂蛋白的含量显著降低(P0.05)。454测序结果显示,原奶中的优势菌群为放线菌门、后壁菌门、变形菌门和拟杆菌门。较对照组相比,SARA组奶牛原奶中乳房炎病原菌,包括Stenotrophomonasmaltophilia、Streptococcus parauberis、Brevundimonas diminuta 等细菌的相对丰度显著提高(P0.05);同时,SARA组奶牛原奶中嗜冷细菌,包括Pseudomonas、Brevundimonas、Sphingobacterium、Alcaligenes、肠杆菌属和乳杆菌属等的相对丰度也显著提高(P0.05)。然而,两组奶牛原奶中细菌菌群的丰度和多样性指数(Ace,Chao和Shannon)无显著差异。结果说明,本试验中,SARA虽未显著影响奶牛的泌乳性能,但可能影响奶牛乳腺健康,增加奶牛患乳房炎风险,并可能降低原奶和乳制品的感官质量,缩短液态奶的贮存期限。综上所述,本论文结合组学技术层层渐进、依次揭示了高精料饲喂诱发的SARA导致瘤胃细菌菌群结构改变,群落趋于简单化;代谢紊乱,有毒有害或促炎物质含量升高;瘤胃上皮基因表达发生改变,引发局部瘤胃炎症;原奶中细菌菌群中乳房炎致病菌含量增多,增加奶牛患乳房炎风险。研究结果有助于揭示瘤胃健康与宿主健康的内在关系,为奶牛的健康饲喂提供一定的理论依据。
[Abstract]:The rumen is the main place for nutrient digestion and metabolism of ruminants, and its health is directly related to the health and production performance of the animal body. This paper intends to systematically study the effects of high concentrate diet on the ruminal health and lactating performance of dairy cows. The contents of the study include the following four aspects of the structure and composition of the bacterial flora of the rumen bacteria in the following four aspects. 4 lactating cows with rumen fistula were randomly divided into control group (CON, 40% semen) or group SARA (SARA, 70% semen), and the 2 x 2 cross test was designed. The tumor gastric juice samples were collected at 0 h for 12,17 and 21 days in each test period, and genomic DNA was extracted and sequenced. The results showed that the pH in the rumen of the SARA group was less than 5.8. The duration of the SARA model was more than 5 hours. Compared with the control group, the rumen lactic acid, propionic acid, butyric acid, valerate, valerate, total VFA and LPS concentrations (P0.05) were significantly higher in the SARA group, and the ethylene propylene ratio (P0.05) significantly decreased the abundance index of the rumen group (Chao1 and Ace) and the diversity index (Shannon) of the.SARA group significantly lower than that of the (P0.05) control group At the gate level, the relative abundance of the rumen deformable bacteria gate and the bacteriobacteria in the SARA group decreased significantly (P0.05), but the relative abundance of the actinomycetes and the actinomycetes increased significantly (P0.05). Compared with the control group, the genus Poulet Was, the genus Acinetobacter, Acinetobacter, Papillibacter and UN in the rumen of the group SARA cows. The relative abundance of classified Lentisphaerae and and unclassified bacteria decreased significantly (P0.05), while the relative abundance of rumen, kiwi, Bifidobacterium and unclassified Clostridiales increased significantly (P0.05) in the rumen of the cow's rumen of the cow, and the LPS concentration in the rumen and the bacteriobacteria were copied. There was a significant negative correlation between the number of shellfish (R =-0.446, P= 0.029). The results showed that the SARA induced by high concentrate diet could lead to the decline of the rumen pH, the accumulation of VFA, the increase of LPS concentration, the disorder of the ruminal bacterial flora and the diversity of the rumen bacterial flora, which reduced the effects of.2.SARA on ruminal microbial flora and rumen metabolism. Closely related, this chapter intends to study the effects of SARA on the metabolic products of dairy cows and further analyze the effect of SARA on the health of the rumen. Experimental animals, experimental design and experimental diet are the same. The samples of the tumor and gastric juice before 12,17,21 day morning of each test period were selected for the GC-MS metabolomics test. The results showed that 233 of the samples of the tumor gastric juice detected a total of 233 Compounds, mainly including organic acids, fatty acids, carbohydrates, amino acids, purines, amines, amines, sugar alcohols, etc. statistics show that compared with the control group, the content of the biodegradation products, such as xanthine, hypoxanthine and uracil in the SARA group, is significantly increased (P0.05); LPS, biogenic amines, ethanolamine, glutaric acid and other toxic or probiotic substances The content of 15 amino acids, such as alanine, leucine, glycine, glutamic acid, leucine, and other 15 amino acids increased significantly (P0.05). The analysis of pathway enrichment for differential metabolites showed that amyl tRNA biosynthesis, phenylalanine, tyrosine and tryptophan biosynthesis, and the biosynthesis of valine, leucine and isoleucine by valine, valine and isoleucine. The pathway was significantly enriched (P0.05). The results showed that the SARA induced by high concentrate diet could lead to the disorder of rumen metabolism, the increase of toxic or proinflammatory substances in the rumen, the increase of amino acid synthesis, and the increase of abnormal metabolites may affect the ruminal gene expression of rumen by the rumen and the body's health.3.SARA. The aim of this study was to study the effect of SARA on the gene expression in the rumen epithelium by gene chip technology, and to reveal the response patterns and possible mechanisms of ruminal dermatitis related genes to high concentrate diet by means of cell culture methods in vitro. In vivo experiments: experimental animals, experimental design and experimental diets, at the same time. A control group and SARA were collected at twenty-first D per period. The results showed that there were 245 differences between the two groups. After the pathway enrichment analysis and functional annotation, we found that the factor receptor and its associated pathway (cytokine-cytokine receptor pathway) were significantly enriched (P0.05), IL-1 beta, IL-2, IL-22, CCL19, CCL8, CX3CR1, CXCL in the pathway. 6, the expression of INHBE, LEPR, PRL and TNFRSF9 was significantly up-regulated in the rumen epithelium of SARA dairy cows (P0.05), while the expression of IL-6 and IL15RA in the pathway was significantly down (P0.05). The results showed that the rumen epithelium may have local inflammatory response. To verify the accuracy of the gene chip results, qRT-PCR is used to express the IL-1 beta, IL-2, and the expressions. The correlation analysis showed that the rumen dermatitis factor was associated with the rumen environmental factors (pH and LPS) (P0.05). In order to further verify this result, the cell culture test was carried out in vitro. The results showed that the mRNA expression of the IL-1 beta, IL-2, IL-6 and IL-8 in the rumen epithelial cells was significantly up-regulated after LPS treatment (P0.05), while the low pH value was treated significantly. The expression of TNF- alpha in the rumen epithelial cells showed that the effects of the rumen environmental factors pH and LPS on the local inflammatory response of the rumen may play an important role in the effects of.4.SARA on lactating performance and the bacterial flora of the dairy cows. This chapter aims to study the effects of SARA on milk performance and the structure and composition of bacterial flora in dairy cows. Experimental animals, experimental design and experimental diet were the same. Milk samples were collected at 17,18 and 19 days of each test period respectively, and 0 h and 4 h were collected at the 12,17 and 21 days of each trial period. The structure and composition of bacterial flora in the milk samples and the related indexes in the collected blood samples were analyzed. The results showed that the control group and the SAR were the SAR. There was no significant difference in the dry matter intake (DMI), milk production and milk composition in the A group (P0.05). Compared with the control group, the white blood cells, the number of lymphocytes and the content of albumin in the tail vein blood of the SARA group increased significantly (P0.05), while the total protein, globulin, cholesterol and low density lipoprotein were significantly reduced (P0.05).454 sequencing results. The dominant bacteria in the original milk were actinomycetes, posterior wall, deformable bacteria and bacteriobacteria. Compared with the control group, the pathogens of mastitis in the milk of SARA dairy cows, including Stenotrophomonasmaltophilia, Streptococcus parauberis, Brevundimonas diminuta and other bacteria, were significantly increased (P0.05), and the original milk of SARA Group dairy cows was also milk. The relative abundance of Pseudomonas, Brevundimonas, Sphingobacterium, Alcaligenes, Enterobacteriaceae and lactobacilli also increased significantly (P0.05). However, there was no significant difference in the abundance and diversity index (Ace, Chao and Shannon) in the two groups of dairy cows. The results showed that in this experiment, SARA did not significantly affect milk. The lactating performance of cattle may affect the health of dairy cows, increase the risk of dairy cow's mastitis, reduce the sensory quality of milk and dairy products and shorten the storage period of liquid milk. In summary, this paper, combined with the technology layer gradually, reveals the structure change of the rumen bacterial flora induced by high concentrate feeding in SARA. The community tends to be simple; metabolic disorders, toxic or harmful or proinflammatory substances increase; the gene expression in the rumen epithelium changes, causes local tumor gastritis; the bacterial flora of the bacterial flora in the original milk increases the risk of mastitis in dairy cows. The results help to reveal the inherent relationship between the health of the rumen and the health of the host, and milk for milk. The health feeding of cattle provides a certain theoretical basis.
【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S858.23

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