纳米氧化锌对断奶仔猪肠道菌群平衡及黏膜免疫的影响

发布时间：2018-03-13 15:03

本文选题：纳米氧化锌　切入点：断奶仔猪　出处：《中国农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：高剂量氧化锌(2000 mg Zn/kg)被证实具有良好的抑制仔猪断奶腹泻的效果,但其会造成严重的环境锌污染。为降低使用量,减少污染,纳米氧化锌被开发出来并应用于仔猪饲料。纳米氧化锌相比于传统氧化锌具有较小的粒子结构与较大的比表面积,但是否能够在低剂量下(500 mg Zn/kg)有效地抑制仔猪腹泻,及其作用机理有待研究。本试验拟通过动物实验,比较低剂量纳米氧化锌和传统高剂量氧化锌抑制仔猪腹泻的效果差别。为研究纳米氧化锌对仔猪肠道菌群结构及黏膜屏障功能的影响,通过运用高通量测序技术,研究纳米氧化锌对断奶仔猪前肠和后肠菌群结构的影响,以及纳米氧化锌对肠道通透性、肠道形态、肠道微生物代谢产物、肠道紧密连接蛋白与肠道干细胞标志蛋白的调控规律和机制。本试验共分为两个部分:试验一:研究不同类型氧化锌对断奶仔猪腹泻率、生产性能和肠道屏障的影响。选用120只28日龄初始体重为6.5 ± 0.5 kg杜×长×大三元杂交断奶仔猪,随机分为4个处理组,每个处理6个重复,分别饲喂基础日粮、添加2000 mg Zn/kg普通氧化锌组、添加500 mg Zn/kg包被氧化锌组、添加500 mg Zn/kg纳米氧化锌组。饲养42 d,试验开始和结束时对仔猪称重,每天记录仔猪的生长性能和腹泻发生率。饲养试验结束后,进行仔猪肠道通透性试验,并屠宰取样,取回肠和结肠肠段组织,进行紧密连接蛋白和肠道干细胞标志蛋白检测。实验结果显示,与对照组相比,在不影响采食量的前提下,纳米氧化锌组仔猪的饲料转化率显著提高(P0.05),且纳米氧化锌与传统氧化锌和包被氧化锌的改善效果接近(P0.05)。添加纳米氧化锌在第1-3周和第4-6周的腹泻发生率相比对照组显著降低(P0.05),添加纳米氧化锌组与传统氧化锌和包被氧化锌相比没有差异。与对照组相比,纳米氧化锌处理组能够有效降低肠道通透性(P0.05),能够差异性的增加回肠和结肠绒毛高度,从而显著增加绒毛高度与隐窝深度的比值(P0.05)。蛋白免疫印迹结果显示,与对照组和传统氧化锌处理组相比,回肠紧密连接蛋白Claudin-1、Claudin-3和Occludin的表达量在包被氧化锌和纳米氧化锌组显著增加(P0.05),Claudin-5的表达量无显著性差异(P0.05);结肠中紧密连接蛋白Claudin-5的表达量在包被氧化锌和纳米氧化锌组显著高于对照组和传统氧化锌组(P0.05),Claudin-3和Occludin的表达量在传统氧化锌、包被氧化锌和纳米氧化锌处理组中均增加显著(P0.05)。回肠干细胞标志蛋白Lgr5和Bmil的表达量显著上升(P0.05),结肠中Bmil的表达量显著上升(P0.05),但Lgr5的表达量则无显著变化(P0.05)。试验二:研究纳米氧化锌对断奶仔猪肠道微生物区系的影响。在试验一的基础上,收集回肠和结肠内容物,运用Illumina二代测序技术,检测微生物菌群结构以及微生物代谢产物的浓度;取回肠和结肠肠段进行肠道短链脂肪酸受体表达量的检测。微生物测序结果表明,纳米氧化锌添加到日粮中,回肠微生物的多样性显著上升,其中回肠厚壁菌门的毛螺菌科、乳酸菌科、韦荣球菌科和红蝽菌科比例显著上升(P0.05)。变形菌门的肠杆菌科的比例明显降低(P0.05)。结肠菌群多样性虽然没有显著的变化,但其有益菌和致病菌的比例发生显著性变化,有益菌毛螺菌科、瘤胃菌科、韦荣球菌科和乳酸杆科比例提高,链球菌科、普雷沃氏菌科和理研菌科等比例下降(P0.05)。微生物代谢产物结果表明,饲喂纳米氧化锌后,回肠乙酸浓度显著下降,丁酸浓度显著上升(P0.05)。结肠中,乙酸、丙酸和丁酸浓度均显著上升(P0.05)。同时,脂肪酸受体检测结果显示,纳米氧化锌处理后,脂肪酸受体GPR41的表达量相比于对照组显著上升,与传统氧化锌和包被氧化锌处理组类似。综上所述,低剂量纳米氧化锌(500 mg Zn/kg)具有与高剂量传统氧化锌(2000 mg Zn/kg)类似,能够显著降低仔猪的腹泻发生率,提高生长性能,其作用机制可能是通过改善断奶仔猪肠道微生物菌群结构、提高肠道黏膜屏障功能和局部黏膜免疫来实现的。
[Abstract]:Zinc Oxide high dose (2000 mg Zn/kg) has been shown to have good inhibition of post weaning diarrhea effect, but it will cause serious environmental pollution. In order to reduce zinc consumption, reduce pollution, nano Zinc Oxide have been developed and applied to the piglet feed. Compared with Zinc Oxide nano particle structure a larger surface area than the traditional Zinc Oxide has a small, but whether it in a low dose (500 mg Zn/kg) effectively inhibited the diarrhea of piglets, and its mechanism remains to be studied. This paper through animal experiment, compared with low dose and high dose of traditional nano Zinc Oxide Zinc Oxide inhibit piglet diarrhea effect. In order to study the effect of nano Zinc Oxide on intestinal bacteria in piglets group structure and the mucosal barrier function, through the use of high-throughput sequencing technology, influence of nano Zinc Oxide on Weaned Piglets foregut and hindgut microflora, and nano Zinc Oxide Intestinal permeability, intestinal morphology and intestinal microbial metabolites, intestinal tight junction protein and intestinal stem cell marker protein regulation and mechanism. The test is divided into two parts: Test: a study of different types of Zinc Oxide on Weaned Piglets diarrhea rate, affect the production performance and intestinal barrier. A total of 120 28 day old initial the weight of 6.5 + 0.5 kg Duroc * Landrace * Yorkshire crossbred weaned piglets were randomly divided into 4 groups with 6 replicates of each treatment were fed basal diet supplemented with 2000 mg Zn/kg, Zinc Oxide group, adding 500 mg Zn/kg package by Zinc Oxide group, adding 500 mg nano Zn/kg Zinc Oxide group. After 42 d. And at the end of the piglets weighing the start of the test, the incidence rate of every record on growth performance and diarrhea. After the end of the feeding trial, of intestinal permeability in piglets, and slaughtered, retrieve the intestines and colon segment into the organization. For the tight junction protein and intestinal stem cell marker protein detection. The experimental results show that compared with the control group, without affecting the food intake, Zinc Oxide group nano piglet feed conversion rate increased significantly (P0.05), and Zinc Oxide and Zinc Oxide and traditional nano package is close to Zinc Oxide (P0.05) to improve the effect of adding nano. Zinc Oxide in the 1-3 week and the 4-6 week incidence of diarrhea was significantly decreased compared to the control group (P0.05), adding nano Zinc Oxide group and traditional Zinc Oxide and Zinc Oxide package showed no difference compared with control group, treatment group of nano Zinc Oxide can effectively reduce the intestinal permeability (P0.05), can increase the difference of the ileum and colon villi the height, and increase the ratio of villus height and crypt depth (P0.05). Western blot results showed that compared with the control group and the traditional Zinc Oxide group, the ileum is closely connected to the egg White Claudin-1, expression of Claudin-3 and Occludin by Zinc Oxide and Zinc Oxide group increased significantly in the nano package (P0.05), no significant difference in the expression of Claudin-5 (P0.05); the expression of tight junction protein Claudin-5 in the colon by Zinc Oxide and Zinc Oxide nano group significantly higher than that of the control group and the traditional group of Zinc Oxide in the package (P0.05) the expression of Claudin-3 and Occludin, in the traditional Zinc Oxide, Zinc Oxide and Zinc Oxide nano coated treatment group were increased significantly (P0.05). The ileum stem cell marker protein expression of Lgr5 and Bmil increased significantly (P0.05), significantly increased the expression of Bmil in colon (P0.05), but the expression of Lgr5 was not significant change (P0.05). Experiment two: the influence of nano Zinc Oxide on the intestinal microflora of weaned piglets. On the basis of Experiment 1, collecting ileum and colon contents, using the two generation Illumina sequencing technology, detection of microorganisms Microbial community structure and microbial metabolites concentration; retrieve the intestines and colon segment of intestinal short chain fatty acid receptor expression of microbial detection. The sequencing results showed that the nano Zinc Oxide was added to the diets, significantly increased the diversity of microorganisms in the ileum, ileum Firmicutes hair spirillaceae, lactic acid bacteria, Veillonella, increased significantly, and the proportion of red bug bacteria (P0.05). The proportion of Enterobacteriaceae Proteobacteria decreased significantly (P0.05). The colonic microflora diversity although no significant changes, but the proportion of bacteria and pathogens remarkable changes in bacteria, Mao spirillaceae, rumen bacteria families, Veillonella, and Lactobacillu, increase the proportion of streptococcaceae, and Poulet Was was a decline in the proportion of RIKEN bacteria (P0.05). The results showed that microbial metabolites, with nano Zinc Oxide after the ileum decreased the concentration of acetic acid, butyric acid concentration Increased significantly (P0.05). In the colon, acetic acid, propionic acid and butyric acid concentration increased significantly (P0.05). At the same time, fatty acid receptor assay showed that nano Zinc Oxide after the treatment, the expression of fatty acid receptor GPR41 compared with the control group increased significantly, and the traditional Zinc Oxide and Zinc Oxide group was similar. In conclusion, low the dose of nano Zinc Oxide (500 mg Zn/kg) with high doses of traditional Zinc Oxide (2000 mg Zn/kg), can significantly reduce the incidence of diarrhea, improve the growth performance and its mechanism may be through improving piglet intestinal microflora structure, improve intestinal mucosal barrier function and local mucosal immunity to achieve.

【学位授予单位】：中国农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S828.5

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