【作者】 朱岩丽；

【导师】 李福昌；

【作者基本信息】 山东农业大学 ， 基础兽医学， 2013， 博士

【摘要】 家兔消化道疾病是制约家兔养殖业发展的一个重要因素，由于抗生素的大量使用会造成细菌耐药性和肉制品安全等问题，而通过日粮调控来预防消化道腹泻被普遍认可。根据家兔的生理特点，日粮纤维在家兔饲料组分中占重要地位，饲粮淀粉是主要能量来源，保证家兔健康而又有充分的经济回报是广大养殖户所追求的。因此，日粮中合理的纤维/淀粉在家兔生产中具有重要意义。本研究选用平均体重相近的断奶新西兰肉兔200只，根据日粮NDF/淀粉为2.3，1.9，1.4，1.0，随机分成4组，每组50只，正式期40天（42-82日龄）。围绕试兔生产指标、盲肠发酵、肠道黏膜发育、特异性及非特异性免疫、肠道微生物多样性及丰度等方面评价了日粮纤维/淀粉对试兔机体内环境的影响，并利用蛋白质组学研究初步探讨了健康与腹泻组试兔肠道蛋白表达差异，综上所述得出以下系列结果：1.调控NDF/淀粉对试兔生产、盲肠发酵、肠道黏膜发育的影响各处理组试兔82日龄时，每组选10只屠宰，测定各项相关指标。结果表明：饲粮NDF/淀粉对试兔平均日增重、料重比影响显著（P<0.05），随着NDF/淀粉的降低，平均日增重（ADG）先升高后降低，料重比以日粮Ⅰ组最高(P<0.05)，为4.51:1；饲粮纤维水平对家兔腹泻指数影响极显著（P<0.01），Ⅰ、Ⅱ组腹泻指数最低，在0.026-0.03之间，而第Ⅳ组最高，为3.27。随着NDF/淀粉降低，胃相对重量以日粮Ⅱ组高于与Ⅲ、Ⅳ组，差异显著（p<0.05），但与日粮Ⅰ组差异不显著（P>0.05）。小肠相对重量、胃重、小肠重和胃内容物重各组间差异显著（P<0.05）；四组日粮处理对小肠长度、小肠相对长度、盲肠重、盲肠相对重差异不显著（P>0.05）。盲肠氨氮浓度有随着NDF/淀粉的下降而升高的趋势（P>0.05），NDF/淀粉仅对丙酸的含量有显著影响(P<0.05），对pH、乙酸含量和丁酸含量及乙酸/（丙酸+丁酸）的影响均不显著（P>0.05）；试兔十二指肠肠绒毛的高度随着NDF/淀粉的降低而先升高后降低，在处理组Ⅱ达到最大值，851μm,影响极显著（P<0.01），综上所述，我们认为日粮NDF和淀粉配比在1.9能获得家兔较好的生产性能和胃肠道良好发育。2.调控NDF/淀粉对试兔非特异性免疫的影响利用ELISA、流式细胞计数、免疫组化等方法监测试兔分别在52、62、72和82日龄时的非特异性免疫指标变化，结果显示，日龄、日粮和日粮×日龄均对试兔圆小囊和蚓突中M细胞数量影响差异显著（P<0.001），饲喂日粮I（高纤维/低淀粉）的试兔在52-62日龄时，M细胞的数量最高（P<0.001），并且低纤维/高淀粉日粮在家兔52日龄时，M细胞数量最低（P<0.001），试兔62日龄时，除日粮I组外，其他三个日粮各组间M细胞数量差异不显著(P>0.05)。试兔72-82日龄，各日粮处理组间M细胞数量差异均不显著(P>0.05)，在光学显微镜下观察M细胞形态变化发现，62-82日龄日粮Ⅰ组比其它日粮组的M细胞尺寸明显增大；日粮×日龄主要对回肠和十二指肠内SIgA水平的影响有有交互作用，在52和62日龄时，日粮Ⅱ组中试兔回肠内SIgA水平显著最低（P<0.05），其它三组试兔SIgA水平在52日龄时无显著差异（P>0.05），62日龄时日粮Ⅰ组最高。72-82日龄各组差异不显著，而对十二指肠SIgA水平的影响主要集中在72日龄，以日粮Ⅳ组最低（P<0.05）。日粮调控对外周血的IgG水平的影响仅限于82日龄(P=0.05)，而对CD4+/CD8+的影响不显著(P>0.05)。结果表明NDF/淀粉升高可以改善局部肠道黏膜免疫，但仅限于试验早期，对局部黏膜免疫的影响（52-62日龄）早于外周免疫（82日龄）。结合临床腹泻指数认为日粮NDF/淀粉为1.9时可以维护家兔肠道健康。3.调控NDF/淀粉对试兔肠道菌群发育的影响分别提取试兔52、62、72和82日龄时各处理组盲肠内细菌总基因组DNA，通过高通量测序技术，获得肠道菌群丰度及多样性等生物学信息，并依据采样时间对不同日粮处理组进行比较。调控NDF/淀粉可以从细菌门的水平改变试兔早期盲肠内不同门类菌群的相对丰度，并改变种属水平的盲肠菌群的多样性及相对丰度。研究得出表明高纤维日粮（日粮Ⅰ）在早期增加了Bacteroidetes门，降低Firmicutes门丰度，而高淀粉日粮（日粮Ⅳ）组Firmicutes菌门丰度在整体试验期较稳定。在种属水平，其中高纤维日粮（日粮Ⅰ、Ⅱ组）盲肠内发现有Escherichia和Bifidobacteria；同时也在日粮Ⅰ、Ⅲ组发现致病性细菌Desulfovibrionales。Ruminococcus内被认为含有大量分解纤维素细菌种类，但是日粮因素对其数量影响不大。主成份分析、聚类分析以及Venn图分析表明82和72日龄时菌群丰度基本趋于一致，共有菌群随日龄增加。DGGE图谱聚类分析结果表明试兔个体间菌群差异较大，但随着年龄的增长，各日粮组菌群逐渐聚到一个分支上。综合上述结果，认为日粮DNF/淀粉在早期（52-62日龄）可以改变肠道菌群的丰度和多样性，当NDF/淀粉为1.9时肠道菌群分布较合理。4.非病原性腹泻兔与健康兔间蛋白质组学研究利用二维电泳技术和蛋白质质谱分析技术找出健康与腹泻试兔间的表达差异蛋白，结果发现得出两组共有蛋白数14个，而腹泻组特有的蛋白质点55个；在两组共有蛋白中健康组有13个蛋白点下调，1个蛋白点上调。选出GRP94和а-Smoothmuscleactin两个蛋白做免疫印迹试验印证了蛋白表达结果。结果表明在腹泻状态下а-Smoothmuscleactin蛋白表达上调，GRP94蛋白在腹泻状态下检出，而健康组没有该蛋白表达。更多还原

【Abstract】 Enteric diseases frequently occur in rabbit restricted the farming development. Due to the extensive use of antibiotics in breeding can lead to bacterial resistance and food safety issues, and consumers as well as breeders require alternative breeding strategies including diet that improve the health of animals without using in-feed antibiotics.Two hundred weaner rabbits were randomly assigned to4groups(Ⅰ、Ⅱ、 Ⅲ、Ⅳ) with50replicates in each group, in which they were fed each diet with NDF/starch2.3,1.9,1.4,1.0, respectively. Around the trial rabbit production performance, cecal fermentation, gastrointestinal tract development, intestinal membrane shape, non-specific immunity, intestinal microbial diversity and abundance etc, which were determined to evaluate the effect of dietary fiber/starch on rabbit body environment. Proteomics was to explore intestinal protein expression differences between the health and diarrhea group trial rabbits. The results are summarized as follows:1. The effect of differnt NDF/starch on rabbit production, cecal fermentation, gastrointestinal developmentTen82-day old trial rabbit per treatment group were slaughtered to determined the relevant datas. Dietary NDF/starch had significantly influences on average daily gain (ADG) and feed/gain (F/G)(P<0.05),ADG and F/G deteriorated with decreasing NDF and increasing starch, F/R was the highest(4.51:1) in Diet I (P<0.05); Diarrhea index were affected by dietary NDF/starch (P<0.01), the diarrhea index was the lowest (0.026-0.03) in Diet Ⅰ and Ⅱ and largest in Group Ⅳ(3.27). The stomach weight (g/kg SW), small intestine weight (g/kg SIW) was influenced significantly among all treatment groups (P<0.05), The stomach weight (SW/LW, g/kg), small intestine weight (SIW/LW,g/kg) in Diet Ⅱ all were largest compared to Diet Ⅲ>Ⅳ treatment groups(P<0.05), and no difference with Diet Ⅰ. The NH3-N concentration increased with the descended of dietary NDF/starch, but every treatment had no significant influence (P>0.05); SIW/LW and SW was significant difference among four dietary treatments (P<0.05), while SIL SIL/LW, CW, CW/LW was not significant. Acetic acid was affected significantly by dietary NDF. Dietary NDF/starch had significant influenced illus height of the duodenum (p<0.05) and was the largest in Group Ⅱ (851μm). In conclusion, the appropriate fiber/starch in the diet of weaner to82-day-old growing rabbit is1.9.2. The effect of different NDF/starch on rabbits’ Non-specific immuneUsing ELISA, flow cytometry, immunohistochemistry method, the following parameters were assessed:the number of membranous epithelial (M) cells in the appendix, total SIgA titers in the gut, total IgG levels in the serum and the CD4+/CD8+T cell ratio in the peripheral blood; as these measures were taken to determine the effect of diet with age, samples were obtained at52,62,72and82days of age. Monitoring the trail rabbits’non-specific immune changes at52,62,72,82days of age, respectively. The effect of Diet×age on trail rabbits’M cells number had significantly different (P<0.001). The number of M cells was the highest in Diet I at52and62days of age (P<0.00\), while was the lowest in Diet Ⅳ at52days (P<0.001). However, at72and82days of age, there was no difference (P>0.05) in M cell number among the four diets treatments. Using immunohistochemistry, the high fiber/low starch diet (Diet I) resulted in an increase in M cell size and number when compared with the other diets. Diet×age effected on SIgA levels in the ileum and duodenum at52and62days (P<0.05), which was no difference at72-82days of age. The SIgA level change of duodenal mainly concentrated in the72days of age and the diet IV group had the lowest (P<0.05). Serum IgG titers were only affected (P=0.05) at82days of age. The ratio of CD4+/CD8+T cells in the peripheral blood was not affected. The study revealed that increased levels of fiber might improve mucosal functionality, but only during the early stage of growth of rabbits. The effect of dietary fiber/starch levels on intestinal immunity was greater than their effect on humoral immunity in the peripheral blood. Combined with clinical diarrhea index,dietary NDF/starch1.9rabbits can maintain intestinal health.3. The effect of different NDF/starch on trail rabbit intestinal flora abundance and diversityIn this work, we used a pyrosequencing-based analysis of16S rRNA genes to compare the cecal microbiota of rabbits fed diets containing different dietary fiber/starch ratio to determine the how the overall microbiome structure varies according to age (from52to82days of age) and diet. The different categories of bacterial phyla and species relative abundance in rabbits’cecum can be changed in early period through regulating NDF/starch. Research results show that high-fiber diets (Diet I) increased the abundance of Bacteroidetes phyla and decreased the abundance of Firmicutes during early peirod, While the abuandance of Firmicutes phyla bacteria from high-starch diet (diet IV) group was relatively stable in trial period. In the species level, including high-fiber diet (Diet Ⅰ,Ⅱ) were found Escherichia and Bifidobacteria in rabbits’ cecum,While found Desulfovibrionales for pathogenic bacteria from Diet Ⅰ, Ⅲ group.the cellulose-decomposing bacteria belonging to the genus Ruminococcus which are not affected by the composition of the diet. A weighted UniFrac analysis revealed that abundance of the gut microbiome was similar at72and82days old. A hierarchical clustering heatmap analysis based on the bacterial community profiles at the family level revealed that the major samples at72and82days of age grouped together. By Venn diagram, all samples have more OTUs in common at72and82days of age. DGGE profiles cluster analysis results show that the individual flora difference among trial rabbit was found. However, flora from all diets treatments gradually gathered a branch with aging. Conclusions:Our data suggest that the bacterial community in the rabbit cecum is able to change and adapt gradually to reach a new equilibrium in response to a nutritional disturbance. More reasonable distribution of intestinal flora was established when the NDF/starch1.9. 4. Intestinal proteomics research between Non-pathogenic diarrhea rabbit and health rabbitUnder the application of two-dimensional gel electrophoresis and mass ctrometry experiments,we finally obtained organization different proteins of14shared proteins of health, including1up-regulated protein and13proteins down in health rabbits. With the application of two-dimensional LC-MS analysis, we could obtain55specific protein of diarrhea group. In order to confirm the accuracy of our experiment, we selected two proteins (GRP94, a-Smooth muscle actin) among health and diarrhea rabbits’ tissues by Dimensional gel electrophoresis and mass spectrometry experiments respectively and verified the accuracy of two-dimensional gel electrophoresis by immunity signature experiment. We verified the accuracy of Two-dimensional LC-MS experiment by immunity signature.更多还原