3种瘤胃纤维分解菌实时定量PCR方法的建立

为探索以减毒胞内侵袭菌介导的黏膜免疫对宿主动物胃肠道微生态的影响,本试验以白色瘤胃球菌、黄化瘤胃球菌和产琥珀酸丝状杆菌3种主要瘤胃纤维分解菌16S rRNA分别设计引物,以山羊瘤胃液提取细菌总DNA,分别扩增3种纤维分解菌目的DNA片段,并连接至pMD-18 T Vector上,经PCR和测序鉴定后,以不同稀释度的重组质粒为模板进行荧光定量PCR反应。结果显示,扩增得到的3种瘤胃纤维分解菌目的片段与已知菌种相应片段的同源性大于99%;以不同稀释度重组pMD-18 T为模板建立的荧光定量PCR扩增曲线差异明显,绘制标准曲线的相关系数均接近1,熔解曲线均呈单一峰值。因此,本试验成功建立了3种瘤胃主要纤维分解菌的实时定量PCR方法,为减毒胞内侵袭菌介导的黏膜免疫研究奠定了基础。瘤胃纤维分解菌;Real-time PCR;标准曲线;     

瘤胃总甲烷菌实时荧光定量PCR方法的构建

试验构建了瘤胃总甲烷菌实时荧光绝对定量PCR的标准品及标准曲线用于总甲烷菌的定量测定,并提取瘤胃微生物总DNA, 以甲烷菌特异性引物进行PCR扩增, 回收纯化PCR产物,与pBS-T载体连接并转化到大肠杆菌,再用氨苄西林培养基筛选阳性重组质粒, 提取含目的片段质粒DNA, 通过PCR及测序鉴定重组质粒.根据OD值确定浓度, 将梯度稀释的质粒作为模板, 进行荧光定量PCR反应并作出标准曲线.结果表明: 所构建的标准曲线具有很高的相关性(R2=0.992), 并获得了高扩增效率产物.     

瘤胃总甲烷菌实时荧光定量PCR方法的构建

试验构建了瘤胃总甲烷菌实时荧光绝对定量PCR的标准品及标准曲线用于总甲烷菌的定量测定,并提取瘤胃微生物总DNA,以甲烷菌特异性引物进行PCR扩增,回收纯化PCR产物,与pBS-T载体连接并转化到大肠杆菌,再用氨苄西林培养基筛选阳性重组质粒,提取含目的片段质粒DNA,通过PCR及测序鉴定重组质粒.根据OD值确定浓度,将梯度稀释的质粒作为模板,进行荧光定量PCR反应并作出标准曲线.结果表明:所构建的标准曲线具有很高的相关性(R2=0.992),并获得了高扩增效率产物.     

两种山羊瘤胃纤维分解菌16S rDNA序列的体外扩增和鉴定

从山羊瘤胃内容物中提取瘤胃细菌总DNA,以瘤胃中两种主要纤维分解菌为目的菌种,分别依据其16S rDNA序列设计引物,利用PCR技术对其16S rDNA 进行体外扩增,对扩增产物测序后进行同源性分析.结果表明:以提取的瘤胃细菌总DNA为模板,采用PCR技术可成功地从山羊瘤胃内容物中扩增出黄色瘤胃球菌和产琥珀酸丝状杆菌16S rDNA的特异性片段;测序后与GenBank中的序列进行比对,表明黄色瘤胃球菌与原序列(AF104841)的同源性达到99.4%,产琥珀酸丝状杆菌与原序列(AJ505937)的同源性达到94.6%.     

运用Real-time PCR方法研究日粮添加豆油与胡麻油对肉牛瘤胃纤维分解菌数量的影响

本研究分别以产琥珀酸丝状杆菌(Fibrobacter succinogenes)、黄色瘤胃球菌(Ruminococcus flavefaciens)、白色瘤胃球菌(Ruminobacter albus)和溶纤维丁酸弧菌(Butyrivibrio fibrisolvens)16S rDNA序列设计引物,运用Real-time PCR技术研究日粮中添加豆油与胡麻油对肉牛上述4种瘤胃纤维分解菌数量的影响.结果表明,与对照组(CK)相比,添加豆油组(LOC1)和胡麻油(LOC2)组,产琥珀酸丝状杆菌(Fibrobacter succinogenes)、黄色瘤胃球菌(Ruminococcus flavefaciens)、白色瘤胃球菌(Ruminobacter albus)和溶纤维丁酸弧菌(Butyrivibrio fibrisol-vens)数量显著减少(P<0.05),分别降低了78%和31%、30%和36%、27%和23%、6%和13%.通过该方法的结果表明日粮中添加4%的豆油和胡麻油显著减少了瘤胃中纤维分解菌,对产琥珀酸丝状杆菌的影响明显.而且采用Real-time PCR方法对瘤胃纤维分解菌进行定量,可以快速有效反映出在日粮改变的情况下菌的数量变化趋势,相对于传统计数方法更直观、快捷与准确.     

内蒙古白绒山羊瘤胃中内纤毛虫及三种纤维分解菌的定量研究

试验采用荧光定量PCR技术,在不同精粗比日粮条件下对内蒙古白绒山羊瘤胃内容物中内纤毛虫和3种主要纤维分解菌进行定量研究。结果表明:日粮中粗料比例增加,内纤毛虫(Ento-dinium)的数量降低,组间差异不显著(P>0.05);日粮中粗料比例增加,产琥珀酸丝状杆菌(Fibrobacter succinogenes)、黄色瘤胃球菌(Ruminococcus flavefaciens)和白色瘤胃球菌(Ruminococcus albus)的数量均增加,组间差异不显著(P>0.05)。     

瘤胃纤维分解菌对钙离子需要量的研究

产琥珀酸丝状杆菌、黄色瘤胃球菌和白色瘤胃球菌3种主要的瘤胃优势纤维分解菌表现出对Ca2+的需要。本试验评价了Ca2+在纤维分解菌生长和纤维素降解中的作用。采用最大生长量或生长率、降解度和延滞时间为评价标准确定Ca2+的需要量。除了产琥珀酸丝状杆菌A3c,其它菌群在无Ca2+的培养基中重复接种都不能生长。随着纤维二糖培养基中Ca2+浓度的增加,产琥珀酸丝状杆菌A3c的生长速率加快,滞后时间减慢,而产琥珀酸丝状杆菌S85的最大生长量和生长率都有所增加。瘤胃球菌在纤维二糖培养基中以上检测指标都没有变化。在以纤维素作为唯一底物的培养基中,两种产琥珀酸丝状杆菌都表现出对Ca2+的绝对需要。菌株A3c的需要量为0.36～0.42 mg/kg,菌株S85的需要量大于0.64 mg/kg。当Ca2+浓度增加时,所有瘤胃球菌菌株对纤维素的降解率都增加,然而,瘤胃球菌在无Ca2+存在的培养基中与产琥珀酸丝状杆菌添加Ca2+对纤维素的降解率相似。虽然瘤胃球菌组成中可能需要Ca2+以降解纤维素,但本研究中没有得到证明。尽管Ca2+在黄色瘤胃球菌降解纤维素中作用还不知道,但有可能与纤维素酶的分泌和活化有关。根据对瘤胃中Ca2+浓度的报道,体内这种菌可能不会出现Ca2+缺乏。     

鸡内参基因β-actin和GAPDH实时定量PCR重组质粒标准品的构建

本研究从SPF鸡皮肤中提取总RNA,并反转录为cDNA;以该cDNA为模板,通过聚合酶链式反应(PCR)分别扩增出看家基因β-actin和GAPDH,将PCR产物连入pGEM-T easy载体,转化DH5α菌,经蓝白斑筛选和质粒酶切鉴定和DNA含量测定,得到定量的β-actin和GAPDH基因融合的重组质粒(pGEM T-actin和pGEM T-GAPDH),即实时定量PCR内参标准品.构建成功的标准品经10倍梯度稀释,作为模板,获得扩增效率良好及可信度高的标准曲线.构建成功的标准品可大量制备,可用于鸡的靶基因定量PCR所需的内参标准品.     

羊草茎在奶牛瘤胃中降解特性及其对食糜纤维分解菌数量的影响

本试验旨在研究羊草茎降解特性和紧密吸附于茎的3种主要纤维分解菌的动态变化。选用羊草茎为试验材料,将其纵切6份后装入尼龙袋投入瘤胃中,分别在6,12,24,48和72 h取出,利用扫描电子显微镜观察超微结构变化;取粉碎后羊草茎进行尼龙袋试验,分别在0.5,2,6、12,24,48和72 h取出,测定不同时间点中性洗涤纤维(neutral detergent fiber,NDF)降解率和吸附在茎中3种主要纤维分解菌的数量变化。结果表明,薄壁组织和韧皮部可被瘤胃微生物降解,维管束会伴随薄壁组织的降解而发生脱落。羊草茎和食糜不同时间点纤维分解菌数量均为产琥珀酸丝状杆菌>白色瘤胃球菌>黄色瘤胃球菌,茎中产琥珀酸丝状杆菌和黄色瘤胃球菌数量在24 h达峰值,分别为10⁹和10⁵ copy/g羊草茎,白色瘤胃球菌在12 h达峰值,为10⁸ copy/g羊草茎。瘤胃食糜中3种纤维分解菌的数量在24 h内基本处在一个恒定的水平,而羊草茎NDF降解率在72 h内逐渐提高,羊草NDF降解率与瘤胃食糜中3种纤维分解菌数量不同步,这可能与纤维分解菌分泌的酶活力存在滞后有关。     

氨化秸秆与苜蓿组合对其营养成分及瘤胃微生物区系的影响

本试验通过体外产气法探讨不同比例快速氨化玉米秸秆(ACS)替代苜蓿(AM)对其营养成分及瘤胃微生物区系的影响,为提高粗饲料利用率和降低饲养成本提供科学依据。结果表明:苜蓿干草和氨化玉米秸秆以100∶0、80∶20、60∶40、50∶50、40∶60、20∶80和0∶100比例进行组合后各组干物质含量相差较小,粗蛋白质、粗脂肪和粗灰分的含量随氨化秸秆添加比例的提高而不断降低,酸性洗涤纤维和中性洗涤纤维的含量随着氨化秸秆添加比例的提高而不断增加;不同比例组合对瘤胃发酵液中产琥珀酸丝状杆菌、黄色瘤胃球菌、白色瘤胃球菌、真菌和原虫相对于总菌的数量产生了不同程度的影响。随着发酵时间的延长,黄色瘤胃球菌和原虫的相对数量显著降低(P 0.05),而产琥珀酸丝状杆菌、白色瘤胃球菌和真菌的相对数量则显著上升(P 0.05),溶纤维丁酸弧菌的相对数量无明显变化。在发酵48 h时,AM∶ACS(20∶80)组的黄色瘤胃球菌和产琥珀酸丝状杆菌的相对数量显著高于其他各组(P 0.05),分别为0.036%和0.26%。20%的苜蓿干草和80%的氨化秸秆组合可有效提高其营养成分的互补,增强瘤胃内主要纤维分解菌的活性,有助于真菌的定植,促进瘤胃微生物的发酵。     

Quantitative comparisons of select cultured and uncultured microbial populations in the rumen of cattle fed different diets

ABSTRACT: BACKGROUND: The number and diversity of uncultured ruminal bacterial and archaeal species revealed by 16S rRNA gene (rrs) sequences greatly exceeds that of cultured bacteria and archaea. However, the significance of uncultured microbes remains undetermined. The objective of this study was to assess the numeric importance of select uncultured bacteria and cultured bacteria and the impact of diets and microenvironments within cow rumen in a comparative manner. RESULTS: Liquid and adherent fractions were obtained from the rumen of Jersey cattle fed hay alone and Holstein cattle fed hay plus grain. The populations of cultured and uncultured bacteria present in each fraction were quantified using specific real-time PCR assays. The population of total bacteria was similar between fractions or diets, while total archaea was numerically higher in the hay-fed Jersey cattle than in the hay-grain-fed Holstein cattle. The population of the genus Prevotella was about one log smaller than that of total bacteria. The populations of Fibrobacter succinogenes, Ruminococcus flavefaciens, the genus Butyrivibrio, and R. albus was at least one log smaller than that of genus Prevotella. Four of the six uncultured bacteria quantified were as abundant as F. succinogenes, R. flavefaciens and the genus Butyrivibrio. In addition, the populations of several uncultured bacteria were significantly higher in the adherent fractions than in the liquid fractions. These uncultured bacteria may be associated with fiber degradation. CONCLUSIONS: Some uncultured bacteria are as abundant as those of major cultured bacteria in the rumen. Uncultured bacteria may have important contribution to ruminal fermentation. Population dynamic studies of uncultured bacteria in a comparative manner can help reveal their ecological features and importance to rumen functions.     

Long-term defaunation increases the abundance of cellulolytic ruminococci and methanogens but does not affect the bacterial and methanogen diversity in the rumen of sheep

Protozoa are commensal eukaryotes in the rumen of herbivores. Protozoa are large producers of hydrogen, which is utilized by methanogenic archaea to produce methane, a greenhouse gas. The removal of protozoa from the rumen (defaunation) decreases methanogenesis, but also negatively affects fiber digestion, which is the main function of the rumen. The aim of this study was to examine the effect of long-term defaunation on the structure of the microbiota and particularly methanogenic archaea and fibrolytic bacteria to better understand the microbial mechanisms responsible for the decrease in methanogenesis and fibrolysis. The trial was conducted in 5 adult sheep subjected successively to long-term defaunation (2 yr), refaunation (12 wk), and short-term defaunation (10 wk). Methanogens were enumerated by quantitative PCR targeting the rrs (16S ribosomal RNA subunit) and mcrA (methyl coenzyme-M reductase) genes. The rrs gene was used to quantify the 3 major culturable rumen cellulolytic bacterial species (i.e., Fibrobacter succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens) and total bacteria. Bacterial and methanogen diversity was also examined by PCR-DGGE (PCR-denaturing gradient gel electrophoresis) analysis targeting the rrs and mcrA genes, respectively. Total rumen bacterial density estimated as rrs copies per gram of DM of rumen content increased in response to long- and short-term defaunation (+1 log, P < 0.001), but without noticeable shifts in diversity. Defaunation increased the rrs copies per gram of DM of rumen content of R. albus and R. flavefaciens (+2 log, P < 0 0.001), but did not affect that of F. succinogenes. Despite a 20% reduction in methane emission in the 2 defaunated periods, the mcrA and rrs copies of methanogens per gram of DM of rumen content increased (+1 log, P < 0.001) in the absence of protozoa, whereas the diversity of the dominant methanogenic community was not modified. This study shows no major difference between long- and short-term defaunation in abundance and diversity of bacteria and archaea. It also provides evidence that monitoring the abundance and diversity of methanogens is not sufficient to comprehend the microbial mechanisms leading to a reduction in methane emissions by ruminants. This study also reports for the first time in sheep a selective effect of defaunation on the abundance of cellulolytic bacterial species.     

A comparison of enzymatic and molecular approaches to characterize the cellulolytic microbial ecosystems of the rumen and the cecum

We used RNA probes and enzyme activities to compare the cellulolytic microbial ecosystems of the rumen and the cecum. Four rumen- and cecum-cannulated wethers were fed a diet of barley plus hay (60:40). Digesta samples were collected 1 h before feeding and 3, 6, and 9 h after feeding for measurements on microbial populations, and 1 h before feeding and 3 and 6 h after feeding for digestion measurements, pH, and VFA. Polysaccharidase and glycosidase specific activities of solid-adherent microorganisms were measured respectively by the amount of reducing sugars released from xylan or avicel or p-nitrophenol from the p-nitrophenol derivatives of xylose and glucose. The distribution and amounts of the three main cellulolytic bacterial species (Fibrobacter succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens) were determined by dot-blot hybridization using specific 16SrRNA-targeting probes. Enzyme activities were higher in the rumen than in the cecum and before feeding than at 3 h after feeding. The sum of the three cellulolytic bacterial species represented, on average, 4.5% of the total bacterial RNA in the two compartments and did not vary with sampling time. The cellulolytic bacterial community structure was different in the two compartments, with F. succinogenes as the main species in the rumen and R. flavefaciens in the cecum. The lower cellulolytic activity in the cecum than in the rumen could not be ascribed to any difference in the structure of the cellulolytic bacterial community between these two compartments, and other hypotheses related to digestion are proposed.     

酵母发酵饲料对瘤胃菌群数量及多样性的影响

本试验研究日粮中添加酵母发酵饲料对蒙古绵羊瘤胃菌群数量及多样性的影响.选用10只安装永久性瘤胃瘘管且体重约40 kg的14月龄蒙古羯羊,分为对照组和试验组,每组各5只.对照组饲喂基础日粮,试验组饲喂基础日粮+酵母发酵饲料(13.37％).预试期15 d,正试期5 d.于正试期晨饲后0、3、6、9、12 h依次采集瘤胃液...     

藏绵羊暖、冷季瘤胃微生物菌群密度与其短链脂肪酸浓度互作分析

针对藏绵羊暖、冷季营养供给严重不均衡的生产实际,本试验旨在解析藏绵羊应对冷季营养严重匮乏的适应机制.试验选择1周岁(±1月龄)健康的放牧藏绵羊母羊12只,采用气相色谱仪和荧光定量PCR仪分别测定藏绵羊暖(7月)、冷(12月)季瘤胃短链脂肪酸(SCFA)浓度和微生物菌群密度,分析藏绵羊暖、冷季瘤胃SCFA浓度与微生物菌群...     

Effect of incremental levels of fish oil supplementation on specific bacterial populations in bovine ruminal fluid

The objective of this study was to determine the effects of incremental replacement of dietary linoleic acid by >20-carbon polyunsaturated fatty acids (PUFA) on changes in population of ruminal micro-organisms associated with fibre digestion and biohydrogenation using real-time PCR of bacterial 16S rRNA sequences. Four beef steers with ruminal cannulas were randomly assigned to control (CK, 65:35 forage to concentrate), CK with 3% sunflower oil plus 1% fish oil (S3F1), 2.5% sunflower oil plus 1.5% fish oil (S2.5F1.5) or 2% sunflower oil plus 2% fish oil (S2F2) in a 4 × 4 Latin square design with 21-day periods. Ruminal fluid was collected on day 15 of each period. Compared with CK, oil addition led to lower ruminal acetate and butyrate but greater propionate concentration. DNA copy number of Anaerovibrio lipolytica in ruminal fluid was greater with oil (average 5.38 vs. 3.62 × 10(5) DNA copy number), particularly with S2F2 relative to CK. Fibrobacter succinogenes and Butyrivibrio fibrisolvens DNA copy number decreased by 74% (1.06 vs. 4.01 × 10(5)) and 39% (5.16 vs. 8.42 × 10(7)) in response to S2F2 compared with CK. DNA copy numbers of Ruminococcus flavefaciens and Ruminococcus albus were not affected by incremental fish oil. Results suggest that greater availability of PUFA with >20 carbons (i.e. eicosapentaenoic acid and docosahexaenoic acid) promoted changes in bacterial populations that are relevant for fibre digestion and biohydrogenation.