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。     

实时荧光定量PCR对瘤胃纤维分解菌定量方法的构建

试验构建了白色瘤胃球菌、黄色瘤胃球菌及产琥珀酸丝状杆菌等3种瘤胃纤维分解菌实时荧光绝对定量PCR的标准品及标准曲线,以用于纤维分解菌的定量测定。提取瘤胃微生物总DNA,以各菌特异性引物进行PCR扩增,回收纯化PCR产物,与pMD18-T Vector连接并转化到大肠杆菌。用氨苄青霉素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反应做出标准曲线。结果表明:所构建的3条标准曲线具有很高的相关性(R2>0.999),并获得了高扩增效率产物(白色瘤胃球菌为101%、黄色瘤胃球菌为98.0%、产琥珀酸丝状杆菌为97.7%),说明成功构建了瘤胃纤维分解菌实时绝对定量PCR的标准品和标准曲线,为定量研究瘤胃纤维分解菌奠定基础。     

银杏叶提取物对山羊瘤胃纤维分解菌的影响

以3只安装永久性瘤胃瘘管的山羊为试验动物,以淀粉、纤维素和酪蛋白为底物,银杏叶提取物在瘤胃培养液底物的浓度设为0(对照组)、0.30%、0.60%、0.90%、1.20%5个水平,利用相对定量PCR法分析银杏叶提取物对山羊瘤胃内3种主要纤维分解菌的影响,结果表明,银杏叶提取物能够极显著提高产琥珀酸拟杆菌的数量(P<0.01),显著提高黄色瘤胃球菌和白色瘤胃球菌的数量(P<0.05)。     

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。     

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;     

不同长链脂肪酸组合对体外瘤胃细菌发酵和群体结构的影响

本试验旨在研究不同长链脂肪酸组合对体外培养瘤胃细菌发酵和群体结构的影响。以3头瘤胃瘘管奶牛提供瘤胃液,对照(A)组底物含5%脂肪酸钙,试验组培养底物中硬脂酸、油酸、亚油酸和亚麻酸的含量分别为1.5%、1.0%、0.5%和1.5%(B组),1.5%、1.0%、1.5%和1.0%(C组),1.0%、1.5%、1.5%和0.5%(D组)以及1.5%、0.5%、0.5%和1.0%(E组)。在培养后0、3、6、12、18、24 h采集培养液,测定p H、氨氮浓度和瘤胃细菌含量。结果表明:1)培养液p H在组间的差异不显著(P0.05);C组的培养液氨氮浓度显著高于B、D组(P0.05)。2)除白色瘤胃球菌,其他菌属含量在组间存在显著差异(P0.05)。其中琥珀酸拟杆菌、生黄瘤胃球菌、蛋白溶解梭菌和嗜淀粉瘤胃杆菌含量在B组较高;C组溶纤维丁酸弧菌、埃氏巨球菌、降解淀粉瘤胃球菌以及瘤胃总细菌含量显著高于其他各组(P0.05)。培养液埃氏巨球菌含量最高,为优势菌。综合得出,脂肪酸组合对瘤胃总细菌和大部分细菌种属含量有显著影响,这与发酵模式有关。     

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。     

饲粮中添加酵母培养物对舍饲牦牛瘤胃发酵参数及微生物区系的影响

本试验旨在研究饲粮中添加酵母培养物(YC)对舍饲牦牛瘤胃发酵参数及微生物区系的影响。试验选取4岁、初始体重为(180.31±29.73)kg的健康麦洼公牦牛36头,随机分为4组,每组9个重复,每个重复1头牦牛,分别饲喂YC添加水平为0(Ⅰ组)、0.5%(Ⅱ组)、1.0%(Ⅲ组)和1.5%(Ⅳ组)的全混合日粮。预试期10 d,正试期80 d。结果表明:1)随着饲粮YC添加水平的升高,瘤胃微生物蛋白(MCP)含量显著升高(P<0.05),pH有呈线性降低的趋势(P=0.058),氨态氮(NH3-N)含量有呈二次曲线变化的趋势(P=0.055),乙酸比例及乙酸/丙酸呈显著线性降低(P<0.05),丙酸和丁酸比例呈显著线性增加(P<0.05),总挥发性脂肪酸(TVFA)含量无显著变化(P>0.05)。2)舍饲牦牛瘤胃微生物多样性指数除Chao指数外,其余各指数组间无显著差异(P>0.05)。3)门水平上,4组的优势菌门均为拟杆菌门、厚壁菌门和变形菌门。随着饲粮YC添加水平的升高,疣微菌门的相对丰度呈显著线性升高(P<0.05),Saccharibacteria的相对丰度呈显著线性降低(P<0.05),互养菌门的相对丰度呈显著二次曲线变化(P<0.05),变形菌门和纤维杆菌门的相对丰度有呈二次曲线变化的趋势(P=0.065,P=0.064)。属水平上,普雷沃菌属、拟杆菌目BS11和理研菌科RC9为优势菌属,随着饲粮YC添加水平的升高,克里斯滕森菌科R7的相对丰度呈显著线性降低(P<0.05),与pH和NH3-N含量呈显著正相关(r>0.500,P<0.05),奎因氏菌属的相对丰度呈显著线性降低(P<0.05),与TVFA含量呈显著正相关(r=0.460,P<0.05),瘤胃球菌科UCG-001和瘤胃球菌科UCG-014的相对丰度呈显著线性降低(P<0.05),与pH呈显著正相关(r>0.500,P<0.05),瘤胃球菌科UCG-011和瘤胃球菌属2的相对丰度呈显著二次曲线变化(P<0.05)。综上所述,在舍饲牦牛饲粮中添加YC显著影响了瘤胃中微生物的丰富度,影响门和属水平微生物的组成,进而对瘤胃发酵参数产生影响。     

饲粮中燕麦干草含量对绵羊瘤胃液pH及微生物区系的影响

本试验旨在研究饲粮燕麦干草含量对绵羊瘤胃液p H及微生物区系的影响。选取9只体况和体重[(70.32±2.14)kg]相近、装有永久性瘘管的德国美利奴与蒙古羊杂种公羊,采用3×3拉丁方设计,随机分为3组,每组3只,各组分别采用全株玉米青贮、全株玉米青贮+燕麦干草(1∶1)(混合组)、燕麦干草为粗饲料。饲粮精粗比34.50∶65.50。进行3期饲养试验,每期20 d,15 d预试期,5 d采样期。采集饲喂前(0 h)和饲喂后1、3、5和7 h的瘤胃液,测定p H,采用实时定量PCR方法测定微生物相对含量。结果表明:1)全株玉米青贮组的瘤胃液p H在1、5 h均显著低于燕麦干草组(P0.05),在3 h极显著低于混合组(P0.01);2)混合组和燕麦干草组瘤胃液真菌的相对含量在0 h均极显著高于全株玉米青贮组(P0.01),燕麦干草组在5 h真菌相对含量显著高于全株玉米青贮组(P0.05);3)混合组原虫的相对含量在1、5 h显著低于全株玉米青贮组(P0.05);4)饲喂后5 h,混合组和燕麦干草组的纤维分解菌相对含量均较高,其中燕麦干草组黄色瘤胃球菌相对含量显著高于全株玉米青贮组(P0.05),白色瘤胃球菌和产琥珀酸丝状杆菌的相对含量极显著高于全株玉米青贮组(P0.01)。综上所述,在精粗比为34.50∶65.50的饲粮中采用全株玉米青贮+燕麦干草(1∶1)的粗饲料,有利于维持绵羊瘤胃内环境的稳态及瘤胃微生物的生长,白色瘤胃球菌和产琥珀酸丝状杆菌为优势菌。     

白色瘤胃球菌DNA提取的优化试验研究与分析

白色瘤胃球菌实为一种瘤胃内纤维分解菌,其利用所分泌的纤维素酶,来降解纤维素.本试验利用白色瘤胃球菌这一特性,首先基于DNA提取对其加以优化,为深入研究奠定基础.     

Degradation of cellulose and forage fiber fractions by ruminal cellulolytic bacteria alone and in coculture with phenolic monomer-degrading bacteria.

We hypothesized that bacterial species capable of metabolizing phenolic monomers may act as catalysts for forage fiber breakdown by increasing microbial access to cell wall polysaccharides. Ruminal cellulolytic bacteria alone and in combination with phenolic-degrading bacteria were examined for differences in their ability to degrade fiber fractions of alfalfa or bromegrass. Electron micrographs of Fibrobacter succinogenes S85 cultured in combination with the ruminal phenolic-degrading organisms Eubacterium oxidoreducens G41 and Syntrophococcus sucromutans S195 indicated that bromegrass was degraded more extensively by the triculture than by the monoculture. The sequential detergent system was used to quantify the digestibility of fiber components from alfalfa and bromegrass. F. succinogenes incubated with the two phenolic-degrading organisms did not degrade more cell wall material than did F. succinogenes alone. However, with two other ruminal cellulolytic organisms, Clostridium longisporum B6405 and Ruminococcus albus B6403, greater (P less than .05, P less than .10, respectively) amounts of hemicellulose were degraded (72 h in vitro fermentation) from whole-plant alfalfa when E. oxidoreducens and S. sucromutants were combined with the cellulolytic species than when their monocultures were tested. Similar increases were not observed using a NDF preparation of alfalfa as the substrate. Based on these in vitro experiments, it does not seem that E. oxidoreducens and S. sucromutans play an important role in improving forage fiber degradation by cellulolytic ruminal bacteria.     

Sulfur influences on rumen microorganisms in vitro and in sheep and calves

When continuously cultured ruminal microbes were given orchardgrass hay and sufficient sulfuric acid or hydrochloric acid to maintain a pH of 5.5, fermentation and numbers of protozoa were reduced compared with cultures whose pH was controlled with phosphoric acid. Likewise, when sulfur-deficient, purified diets were supplied to cultures, less methane (mmol X liter-1 X d-1), 3.2 vs 32.6, was produced and fewer cellulolytic bacteria (log10/ml), 5.8 vs 7.2 were present than when cultures were given the same diet supplemented with .3% elemental sulfur. The rumen of sheep fed the .04% sulfur diet had reduced digesta weights (1.69 vs 3.2 kg) compared with sheep fed the diet with .34% sulfur at the same intake. There also was reduced methanogenesis 12.3 vs 25.8 mmol X liter-1 X d-1) and reduced numbers of cellulolytic bacteria (7.4 vs 8.4 log10/ml) in sulfur-deficient sheep in comparison to sulfur-supplemented sheep. In growing calves, the same types of bacteria predominated in the rumen, but more facultative anaerobic bacteria were isolated from calves fed .04% sulfur than from calves fed diets with .34 to 1.72% sulfur. None of the dietary levels of sulfur appeared toxic. Regardless of treatment, volatile fatty acids were more predominant than lactic acid as end-products of fermentation of ruminal microbes in fermenters, sheep and calves. The greater methanogenesis and the greater cellulolytic bacterial numbers of sulfur-supplemented sheep compared with sulfur-deficient in vitro cultures, is interpreted to be the result of recycling of sulfur to the rumen in sheep where it is efficiently scavengered by ruminal bacteria.     

日粮补充异丁酸对犊牛生长性能、瘤胃发酵和纤维分解菌菌群的影响

为探索异丁酸对犊牛生长性能、瘤胃发酵、纤维分解菌菌群和酶活性的影响,试验选用15日龄荷斯坦犊牛36头,随机分成4组,对照组、低水平异丁酸组(LIB)、中水平异丁酸组(MIB)和高水平异丁酸组(HIB)分别饲喂异丁酸0、3、6和9g·d^-1,预饲15d,分别于60日龄(断奶)和90日龄测定体重和采集瘤胃液进行分析。结果表明,MIB组和HIB组犊牛干物质采食量、日增重和经济效益显著高于对照组(P<0.05)。90日龄时HIB组犊牛瘤胃pH值显著低于对照组和LIB组(P<0.05)。MIB和HIB组瘤胃总挥发性脂肪酸和乙酸浓度显著高于对照组和LIB组(P<0.05);MIB和HIB组犊牛60日龄时瘤胃乙酸/丙酸显著高于对照组(P<0.05),90日龄时显著高于对照组和LIB组(P<0.05)。60和90日龄时MIB和HIB组瘤胃异丁酸均显著高于对照组(P<0.05)。60日龄时MIB和HIB组木聚糖酶和α-淀粉酶显著高于对照组(P<0.05),MIB和HIB组纤维二糖酶、果胶酶、溶纤维丁酸弧菌、黄色瘤胃球菌和产琥珀酸丝状杆菌显著高于对照组和LIB组(P<0.05);90日龄时MIB和HIB组各种瘤胃酶活力和纤维分解菌均显著高于对照组和LIB组(P<0.05)。结果显示,日粮补充异丁酸促进了犊牛瘤胃纤维素分解菌的生长,提高了纤维素分解酶的活性,进而促进了犊牛瘤胃发酵和生长发育。由于MIB与HIB之间各指标差异不显著,兼顾经济效益,在本试验条件下异丁酸的最佳添加量为6.0g·d^-1。     

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.     

Oral administration of Lactobacillus plantarum and Bacillus subtilis on rumen fermentation and the bacterial community in calves

The objective of this study was to assess the effect of dietary probiotics on rumen fermentation and the bacterial community in dairy calves. Twelve Holstein calves were randomly allocated to three treatments: a basal diet, the basal diet supplemented with Lactobacillus plantarum GF103 (LB) or basal diet supplemented with a mixture of Lactobacillus plantarum GF103 and Bacillus subtilis B27 (LBS). A milk replacer was fed to calves from 8 days of age. A starter and alfalfa hay was offered ad libitum from 21 and 28 days of age, respectively, and the orts were weighted daily. The ruminal fluid was sampled at 56 and 83 days of age to determine the rumen fermentation characteristics. The bacterial community was analyzed by denaturing gradient gel electrophoresis (DGGE) and the number of certain bacteria was quantified by real‐time polymerase chain reaction. The ratio of total dry matter intake to average body wieght was higher in the control (P < 0.05). The DGGE fingerprint of the 16S ribosomal RNA gene was affected by the blended probiotics at 83 days of age. The number of Ruminococcus albus was lower in the LB and LBS treatment (P < 0.05). Oral administration of the probiotics affected the rumen bacterial community and the numbers of cellulolytic bacteria decreased.     

Variation of bacterial communities and expression of Toll-like receptor genes in the rumen of steers differing in susceptibility to subacute ruminal acidosis

In order to determine differences in the ruminal bacterial community and host Toll-like receptor (TLR) gene expression of beef cattle with different susceptibility to acidosis, rumen papillae and content were collected from acidosis-susceptible (AS, n=3) and acidosis-resistant (AR, n=3) steers. The ruminal bacterial community was characterized using PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and quantitative real time PCR (qRT-PCR) analysis. Global R analysis of bacterial profile similarity revealed that bacterial diversity was significantly different between AR and AS groups for both rumen content (P=0.001) and epithelial (P=0.002) communities. The copy number of total bacterial 16S rRNA genes in content of AS steers was 10-fold higher than that of AR steers, and the copy number of total 16S rRNA genes of epimural bacteria in AR steers was positively correlated with ruminal pH (r=0.59, P=0.04), and negatively correlated with total VFA concentration (r=-0.59, P=0.05). The expressions of host TLR2 and 4 genes were significantly higher in AR steers compared to those in AS steers. These findings enhance our understanding about the ruminal microbial ecology and host gene expression changes that may be useful in the prevention of ruminal acidosis.     

Occurrence of 2-aminoethylphosphonic acid in feeds, ruminal bacteria and duodenal digesta from defaunated sheep

A quantitative method of analysis for 2-aminoethylphosphonic acid (AEP) was developed using reverse-phase HPLC. The detection limit for AEP was 15 nM, and the detector response (peak area) was linear from AEP levels up to 100 microM (R = .99). Mean recovery of AEP added to strained ruminal fluid from faunated sheep was 98.2%. When AEP was added to a fermentation mixture at a concentration of 22.6 micrograms/ml, 78% disappeared during a 24-h incubation. 2-Aminoethylphosphonic acid was readily detected in preparations of mixed ruminal ciliate protozoa as well as in mixed and pure strains of ruminal bacteria, feedstuffs, and ruminal fluid and duodenal digesta from defaunated sheep. The occurrence of AEP in feed and bacterial hydrolysates was confirmed by organic phosphorus analyses. The concentration of AEP in mixed ruminal protozoa was three times greater than its concentration in mixed ruminal bacteria (4,304 vs 1,383 micrograms/g DM, respectively). The AEP values for pure ruminal bacterial cultures ranged from 733 micrograms/g DM in Bacteroides succinogenes B21a to 1,166 micrograms/g DM in Butyrivibrio fibrisolvens H17c. Ruminal fluid and duodenal digesta from defaunated sheep contained AEP concentrations of 30 micrograms/ml and 90 micrograms/g DM, respectively. The concentration of AEP in feedstuffs ranged from 25 micrograms/g DM in wheat straw to 263 micrograms/g DM in oats. Because AEP occurrence is not limited to ruminal ciliate protozoa, it is of little value as a marker for protozoal presence in or passage out of the rumen.     

Feasibility of using total purines as a marker for ruminal bacteria

A procedure for measuring total purine content of mixed ruminal bacteria was adapted for use in the determination of purines in pure cultures of ruminal bacteria. Recovery of adenine and guanine, alone or in mixture, was quite variable. The problem was traced to solubility of the silver salt of adenine in the acid wash solution. When the precipitating solution was used as the wash, recovery of the purines was over 97%. Recovery of a 1:1 mixture of adenine and guanine added to yeast RNA was 100.6+/-3.2%. Purine, protein, and bacterial concentrations were determined for 10 pure cultures of ruminal bacteria: Butyrivibrio fibrisolvens, D16f, H10b, and H17c; Fibrobacter succinogenes B21a; Lachnospira multiparus D25e; Lactobacillus lactis ARD26e; Prevotella ruminicola H15a; Ruminococcus albus 7; Ruminococcus flavefaciens B34b; and Streptococcus bovis ARD5d. The CV for the most-probable-number (MPN) assay (bacterial concentrations), purine analysis, and protein analysis were 55.86, 5.25 and 6.52%, respectively. Considerable variation was found among bacterial species and strains when purine and protein concentrations were compared as the amount per individual cell. More consistent values were obtained when these components were expressed on a dry matter basis. Purine:protein ratios for the 10 pure cultures ranged from .023 to .1299, with a mean value of .0883. For samples of mixed bacteria separated from ruminal fluid, this ratio was found to average .0306, which is approximately one-third of the value for the pure cultures. The value determined for the mixed bacterial sample is similar to previously reported values. Based on the ratio obtained with the pure cultures, the microbial protein flow out of the rumen has probably been overestimated in most previous reports. Limited studies suggest that the samples of mixed ruminal bacteria used as a standard are probably contaminated with feed particles containing protein, which results in lower purine:protein ratios.     

Synergistic fibrolysis in the rumen by cellulolytic Ruminococcus flavefaciens and non-cellulolytic Selenomonas ruminantium: Evidence in defined cultures

To investigate the rumen bacterial interaction between cellulolytic Ruminococcus flavefaciens and non‐cellulolytic Selenomonas ruminantium, fiber digestibility and fermentation products were determined in defined cultures consisting of these two species. Avicel, orchardgrass hay, rice straw and alfalfa hay were used as substrates for 72 h incubation to monitor digestibility, volatile fatty acids, succinate, lactate and bacterial number. In monoculture, R. flavefaciens digested the fiber sources at 21–32%, while S. ruminantium strains did not. When R. flavefaciens was cocultured with one of three different strains (GA192, S137 and S150) of S. ruminantium, fiber digestion exceeded the value recorded by R. flavefaciens alone. In particular, cocultures with S. ruminantium S137 showed significantly higher digestibility for all the fiber sources than R. flavefaciens alone (P < 0.05). Propionate production and growth of S. ruminantium was notable in all cocultures but not in monocultures. Succinate was accumulated in monoculture of R. flavefaciens, while the accumulation was not observed in cocultures. These results indicate that R. flavefaciens provides fiber hydrolysis products to S. ruminantium as growth substrates. In addition, S. ruminantium could activate R. flavefaciens by rapidly consuming the products. Such cross‐feeding between cellulolytic and non‐cellulolytic bacteria could enhance fiber digestion, although the extent of the enhancement may depend on strain combinations.