【作者】 高爱武；

【导师】 侯先志；

【作者基本信息】 内蒙古农业大学 ， 动物营养与饲料科学， 2008， 博士

【摘要】 本论文通过体内分别驱除厌氧真菌和原虫的方法,系统研究了高纤维日粮条件下(精粗比为20:80)绵羊瘤胃微生物在纤维物质降解中的作用及其相互关系,同时检测它们对瘤胃发酵参数、瘤胃纤维降解酶类和绵羊瘤胃食糜流通速率的影响,旨在为最大可能发挥瘤胃微生物的纤维降解活性,合理调控瘤胃微生态系统,提高反刍动物瘤胃降解纤维物质的能力提供科学依据。同时,本研究对real-time PCR方法与传统厌氧滚管方法定量瘤胃微生物的效果进行了比较,以寻求更为合理准确定量瘤胃微生物的技术手段。试验分两部分,为分别驱除厌氧真菌和原虫对绵羊瘤胃微生物种群及纤维物质降解的影响。每期试验选用6只体况良好、体重35Kg左右,年龄1.5岁左右,安装有永久性瘤胃瘘管和十二指肠瘘管的蒙古绵羊。试验动物随机分为两组,分别为对照组(Control, C)和处理组,每组3只绵羊。试验1处理组驱除真菌(Elimination of fungi, EF)后进行采样。待采样结束后,向该组试验羊瘤胃内重新引入厌氧真菌(Introduction of fungi, IF),直到真菌总数恢复到驱除前的水平,再进行采样。试验2处理组驱除原虫(Defaunation, DF)后进行采样。待采样结束后,向该组试验羊瘤胃内重新引入原虫(Refaunation, RF),直到原虫总数恢复到驱除前的水平,再进行采样。试验结果如下:1驱除及重新引入厌氧真菌对绵羊瘤胃微生物种群及纤维物质降解的影响驱除厌氧真菌显著降低了绵羊瘤胃DM、NDF和ADF的降解率(p<0.05),但没有影响瘤胃液相食糜流通速率和消化道固相食糜流通速率。瘤胃细菌总数、纤维分解菌和原虫数量在驱真菌后显著增加(p<0.05),三种主要纤维分解细菌中,黄色瘤胃球菌和产琥珀酸丝状杆菌数量明显增加(p<0.05),白色瘤胃球菌虽也呈现增加趋势,但变化不显著。驱除厌氧真菌导致绵羊瘤胃羧甲基纤维素酶活性显著降低,滤纸酶活、木聚糖酶活和果胶酶活也出现不同程度的下降,但并不明显。同时也导致瘤胃总挥发性脂肪酸浓度和乙酸浓度显著降低(p<0.05)。瘤胃pH值呈现升高趋势,而NH3-N浓度呈现降低趋势,但均不显著。2驱除及重新引入厌氧真菌对绵羊瘤胃微生物种群及纤维物质降解的影响驱除原虫导致绵羊瘤胃DM、NDF和ADF降解率显著降低(p<0.05),瘤胃细菌总数显著增加(p<0.05),但对瘤胃内可培养的纤维分解细菌数量没有影响。滚管培养显示瘤胃真菌数量不受驱除原虫的影响,而real-time PCR检测的真菌量在驱原虫后显著下降(p<0.05),二种方法的结果并不一致。白色瘤胃球菌和黄色瘤胃球菌的数量在驱原虫后显著增加(p<0.05),产琥珀酸丝状杆菌的数量却显著下降(p<0.05)。驱除原虫显著降低了绵羊瘤胃CMCase活性、滤纸酶活性和果胶酶活性(p<0.05),但没有影响木聚糖酶的活性,对瘤胃液相食糜流通速率和消化道固相食糜流通速率也无影响。驱除原虫对绵羊瘤胃内pH值和总挥发性脂肪酸浓度没有影响,但使丙酸的摩尔比例显著增加,丁酸的摩尔比例显著降低(p<0.05)。NH3-N浓度在驱除原虫后也显著降低(p<0.05)。3 Real-time PCR方法与传统厌氧滚管方法定量瘤胃微生物的效果比较Real-time PCR对瘤胃总细菌和总真菌的检测结果显示,细菌总数的量比传统厌氧培养方法高10倍左右,而真菌的数量更是高出培养方法1000倍以上,且灵敏性和准确性方面也优于传统滚管培养方法。对三种主要纤维分解细菌的定量结果显示,real-time PCR方法可以在体内对单一的瘤胃纤维分解微生物进行定量研究,结果较为准确。本研究的结果表明,瘤胃厌氧真菌和原虫在瘤胃纤维物质降解中发挥着重要作用,瘤胃微生物在生长过程中或存在相互的促进,或存在一定程度的抑制作用,与微生物的种类有关。瘤胃厌氧真菌或原虫的缺失会导致瘤胃纤维降解纤维素酶活性的显著下降和瘤胃微生态环境的变化,不利于纤维的降解。Real-time PCR是一种更加准确、便捷的定量瘤胃微生物的方法。更多还原

【Abstract】 The aim of cureent research was to study effects of ruminal microorganisms and their interactions on fiber digestion, rumen fermentation parameters, fibrolytic enzyme activity and digesta passage rate under high forage diets (forage to concentrate ratio was 80: 20) by eliminating anaerobic fungi (EF) and defaunation (DF) respectively in sheep for pursuing the reasonable suggestion to improve the utilization of fiber in rumen. Ruminal microorganism count was conducted by real-time PCR and traditional roll tube method respectively to hunt for precise quantification approach of rumen microbes.Two trials were carried out and each trial included 6 healthy, 35 Kg body weight and 1.5 aged Mongolia sheep randomly assigned into two groups (control and treatment). In trial I treatment group, the anaerobic fungi were eliminated. After the sampling, the anaerobic fungi were introduced (IF) into the rumen of sheep. The same way was used in trial 2, in which samples were collected two times after defaunation (DF) and refaunation (RF).After EF, the digestibility of dry matter (DM), neutral detergent fiber (NDF) and acid detergent fiber (ADF) were significantly decreased (p<0.05), but the digesta passage rate was not changed. Total quantities of bacterial, cellulolytic bacterial and protozoa were significantly increased (p<0.05). Among three main cellulolytic bacteria species (R.albus, R.flavefaciens and F.succinogenes), the quantities of R.flavefaciens and F.succinogenes were significantly increased (p<0.05). The EF resulted in a decrease (p<0.05) of activity of CMCase, total VFA and acetate, whereas the activity of FPA, xylanase and pectinase were not changed. The ruminal pH value increased and the concentration of NH3-N decreased after EF, but was not statisticaly significant.The DF resulted in a significant decrease (p<0.05) in digestibility of DM, NDF and ADF. After DE, the total ruminal bacterial quantities were significantly increased (p<0.05), whereas the cellulolytic bacterial counts were not affected by DF. The quantities of ruminal anaerobic fungi were not changed by traditional roll tube method, but quanitites of rumianl fungus were significantly decreased (p<0.05) by using real-time PCR detection, which indicated that two methods were not consistent. Quantities of R.albus and R.flavefaciens significantly increased but f F.succinogenes significantly decreased (P<0.05). The DF significantly decreased the activity of CMCase, FPA and pectinase, but had little effect on the xylanase. The ruminal pH value and total VFA were not affected by DF, while the concentration of propionate was significantly increased and concentration of butyrate and NH3-N was significantly decreased (p<0.05).The results of real-time PCR detection of ruminal microorganisms showed that the quantities of total rumianl bacteria and fungi were 10 and 1000 times higher than that of traditional roll tube respectively. The sensitivity and accuracy of real-time PCR were also superior to the traditional method. The results of three main cellulolytic bacteria quantities by real-time PCR suggested that it could be a useful tool in the quantitative research of single ruminal microorganism.The results of current study indicated removal of anaerobic fungi or protozoa would lead to a decrease of fiber degradation and cellulolytic enzyme activity, and it also brought about a change of ruminal microbial ecosystem. Real-time PCR was a more accurate and convenient method for quantitative study of ruminal microorganisms.更多还原