【作者】 李龙；

【导师】 赵辛；

【作者基本信息】 西北农林科技大学 ， 动物营养与饲料科学， 2015， 博士

【摘要】 在动物生产过程中不合理的使用抗生素饲料添加剂给食品安全和公共健康带来了巨大的威胁,因此人们迫切的寻找新的替代品以取代抗生素在动物生产中的作用。益生菌被认为是一种可能的抗生素替代品。在以往动物益生菌的筛选过程中,往往忽视益生菌菌株耐药性的检测,使其在实际应用中存在潜在风险,本研究利用西藏传统散养条件下的藏鸡胃肠道作为菌源来筛选出益生特性好、无获得性抗性的益生菌菌株,并进一步研究其对肉鸡生产性能和肠道屏障的影响。同时本试验还研究了西藏传统藏药红景天对高原条件下饲养肉鸡生长性能、健康和肠道发育的影响。为益生菌和红景天在家禽生产上的应用提供理论依据。取得的主要研究成果如下:试验一以健康藏鸡胃肠道为样本,利用MRS培养基分离乳酸菌并进行革兰氏染色和过氧化氢酶试验,共分离出乳酸菌113株。首先通过耐酸和耐胆盐试验筛选出16株抗逆性好的菌株,再通过抑菌试验、肠上皮细胞粘附试验、自凝集试验和耐热试验,对每一株菌株根据每一项测定指标指定的打分标准进行综合评分,其中LTC-55(唾液乳杆菌)和LTC-113(植物乳杆菌)菌株评分最高,分别为21和23分,并且这2株乳酸菌无溶血特性和抗生素耐药性,因此选择这2株乳酸菌菌株用于后续的活体试验。试验二研究筛选菌株对肉鸡生产性能、肠道消化酶活性和组织形态的影响。饲养试验采用单因素完全随机设计,360只1日龄AA肉鸡随机分为4个处理组:对照组(基础日粮)、抗生素处理组(金霉素,添加量为50mg/kg日粮)、LTC-55菌株处理组和LTC-113菌株处理组(2株乳酸菌的添加剂量均为109 CFU/kg日粮)。乳酸菌菌株制备成菌粉直接添加于饲料中。结果表明,日粮添加益生菌对0-21日龄肉鸡生产性能无显著影响(P>0.05)。而22-42日龄、0-42日龄,抗生素处理组、LTC-55处理组和LTC-113处理组的平均日增重和饲料转化率显著高于对照组(P<0.05),并且显著提高了42日龄肉鸡体重(P<0.05),且2株益生菌表现出与抗生素相似的促生长效果。28日龄时,益生菌LTC-55菌株处理组脂肪酶活性显著高于其它3个处理组(P<0.05)。在14、28和42日龄时,抗生素处理组和益生菌处理组均显著促进了十二指肠的肠道发育(P<0.05)。试验三研究益生菌对雏鸡沙门氏菌诱导的肠道屏障损伤的保护作用及其分子机理。80只1日龄健康雏鸡随机分为4个处理:阴性对照组、益生菌处理组(强饲109CFU植物乳酸杆菌LTC-113)、沙门氏菌处理组(强饲109CFU鼠伤寒沙门氏菌CVCC542)和益生菌沙门氏菌共处理组(强饲109CFU鼠伤寒沙门氏菌CVCC542和109CFU植物乳酸杆菌LTC-113)。结果显示沙门氏菌感染雏鸡8小时后,相比于阴性对照组雏鸡肠道通透性显著增加,盲肠ZO-1和CLDN-5基因m RNA表达量显著降低,CLDN-2基因m RNA表达量显著升高(P<0.05)。而益生菌与沙门氏菌共处理相比于沙门氏菌处理组显著降低了沙门氏菌在盲肠中的定植数量,并显著降低了内部器官肝脏和脾脏的沙门氏菌定植数量(P<0.05),消除了沙门氏菌感染引起的肠道通透性升高和紧密连接相关基因表达量的变化。相比于阴性对照组,益生菌LTC-113菌株处理提高了盲肠ZO-1基因m RNA表达量(P<0.05),但对雏鸡肠道通透性无显著影响。试验四从生产性能、健康和肠道发育3个角度研究大花红景天是否能够缓解西藏高原条件下肉鸡的缺氧应激。试验分为3个处理:对照组、低剂量红景天组(基础日粮+0.5%红景天)和高剂量红景天组(基础日粮+1.5%红景天)。试验结果表明,低剂量红景天组相比于对照组对肉鸡生产性能无显著影响,但高剂量红景天组显著降低了肉鸡14、28和42日龄时的平均日采食量,28和42日龄体重和肠道发育(P<0.05)。所有处理组腹水死亡率无显著差异(P>0.05),而低剂量红景天组相比于对照组和高剂量红景天组显著降低了肉鸡非腹水死亡率和总死亡率(P<0.05)。高剂量红景天显著提高了肉鸡28日龄时红细胞和血红蛋白水平(P<0.05)。综上所述,本研究从藏鸡胃肠道成功分离筛选到2株益生特性好,且安全的乳酸菌菌株,其能够通过改善肉鸡肠道消化酶活性和肠道发育,从而提高肉鸡的生产性能,并能通过稳定紧密连接基因的表达或减少沙门氏菌的定植保护雏鸡免受沙门氏菌感染造成的肠上皮屏障损伤。而日粮添加红景天可能通过缓解肉鸡的缺氧应激减少高原缺氧条件下肉鸡的死亡率。更多还原

【Abstract】 The overuse of antibiotics in animal production could pose a significant threat to food safety and public health. Thus, in order to reduce the antibiotic application, effective alternatives to antibiotics are urgently needed to maintain current animal production levels and health. Probiotics can be considered as a possible alternative to antibiotics. In the process of selecting animal probiotics, antibiotic resistance of probiotic strains is often neglected, so that the potential risks exist in its practical application. One objective of this study was to isolate, characterize and select lactic acid bacteria strains from the gastrointestinal tract of Tibet chickens which presumbably have lower antibiotic resistance, and further investigate its effect on growth performance and gut barrier of chicken. Our study also determined the effects of Rhodiola on production performance and health parameters of broilers raised at high altitude. The results from these studies would provide scientific evidence for the application of probiotics and Rhodiola in poultry production. The results for four experiments were shown as follows:In the first experiment, 113 lactic acid bacteria(LAB) strains were isolated from the gastrointestinal tract of Tibet chickens raised in Tibet. Lactic acid bacteria were isolated using a Man-Rogosa-Sharpe(MRS) medium and confirmed by Gram stain and catalase identification. Sixteen LAB strains showed high acid and bile tolerance. Each LAB strain was scored according to the results of acid, bile and heat tolerance, antimicrobial activity against selected enteric pathogens, adhesion to intestinal cells and aggregation. Among 16 LAB strains, LTC-55(Lactobacillus salivarius) and LTC-113(Lactobacillus plantarum) strains were selected for further in vivo studies because of their high score(21 and 23)..In the second experiment, 360 healthy Arbor Acres broilers were randomly allocated by body weithgt(BW) to 4 experimental treatments according to a completely randomized design. The control birds were fed the basal diet alone while birds on other treatments received the basal diet supplemented with 50mg/kg of aureomycin, 109cfu/kg LTC-55 or 109cfu/kg LTC-113. The two LAB strains were fermented and dry-freezed into powder before adding to the feed. The effect of dietary supplementation of different probiotic strains was investigated for production perfermance, intestinal morphological structure and digestive enzyme activities. There were no significant differences in average daily gain(ADG), average daily feed intake(ADFI) and feed conversion rate(FCR) of broilers during 1 to 21 d among different treatments(P>0.05). However, during 22 to 42 d and 1-42 d, ADG and FCR were improved in broilers fed diets supplemented with probiotic strains LTC-55 and LTC-113 compared with the control group(P<0.05). There was no difference between probiotics groups and the antibiotic group(P>0.05). The BW of 42-d-old broilers were significantly higher in the LTC-55 and LTC-113 groups compared with the control group(P<0.05), which was similar to the antibiotic group. In additon, broilers fed LTC-55 had higher lipase activities on days of 28 compared with other groups(P<0.05). The probiotic strains and antibiotic treatment improved the duodenum development on days of 14, 28 and 42(P<0.05).The third experiment was designed to investigate the effect of probiotics on Salmonella induced intestinal epithelial barrier disruption and its molecular mechanisms. Eighty 1-day-old healthy male Nick chicks were randomly allocated into 4 experimental treatments:(i) the negative control(no probiotics treatment and no Salmonella infection),(ii) the probiotics treated group(109 Lactobacillus plantarum LTC-113),(iii) the Salmonella-infected group(109 Salmonella Typhimurium CVCC542), and(iv) the probiotics-treated and Salmonella-infected group(109 Lactobacillus plantarum LTC-113 and 109 Salmonella Typhimurium CVCC542). Compared with the negative control group, Salmonella infection significantly increased the intestinal permeability, decreased m RNA expression level of ZO-1 and CLDN-5, and increased CLDN-2 m RNA level at 8 hours post infection(P<0.05). However, the changes due to Salmonella infection were eliminated by probiotics treatments. Meantime, probiotics treatments significantly decreased the Salmonella numbers in the liver, spleen and cecum(P<0.05). Treated with probiotics significantly increased the m RNA expression level of ZO-1 in comparison with negative control and Salmonella infection group(P<0.05), but have no effect on the intestinal permeability(P>0.05).The fourth experiment was designed to investigate whether Rhodiola crenulata(R. crenulata) could alleviate the negative effects of hypoxia on broiler chickens reared in Tibet Plateau. The effect of supplementing crushed roots of R. crenulata on production performance, health and intestinal morphology in commercial male broilers was investigated. Dietary treatments included CTL(basal diet), Low-R(basal diet + 0.5% R. crenulata) and High-R(basal diet + 1.5% R. crenulata). In comparison with broilers fed the control diet, Low-R had no effect on production performance(P>0.05) while High-R significantly decreased average daily feed intake at d14, 28 and 42, body weight at d28 and 42 and gut development(P<0.05). Ascites induced mortality did not differ among treatments(P>0.05). Nevertheless Low-R significantly reduced non-ascites induced mortality and total mortality compared with broilers fed CTL and High-R diets(P<0.05). Broilers fed the High-R diet had significantly increased blood red blood cell counts and hemoglobin levels at 28 d compared with other treatments(P<0.05).In conclusion, we successfully isolated and screened 2 LAB strains with good probiotic properties from Tibet chickens. Dietary supplementation with 2 lactic acid bacteria strains in broilers diet could improve intestinal digestion and absorption function; consequently enhance production performance, similar to the antibiotic treatment. In addition, 2 lactic acid bacteria strains could protect newly hatched chicks from Salmonella induced intestinal epithelial barrier disruption by stabilizing the expression of tight junction genes or decreasing the Salmonella colonization. Supplementation with Rhodiola could decrease mortality rate amd might reduce the effects of hypoxia on broilers reared at high altitude in Tibet.更多还原