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.     

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。     

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。     

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,每天采集一个时间点,共采集5次。实时荧光定量PCR(探针法)检测9种瘤胃菌群的数量,并用16S rRNA高通量测序技术分析菌群多样性。结果表明:试验组溶纤维丁酸弧菌、黄色瘤胃球菌、产琥珀酸丝状杆菌、埃氏巨球型菌、牛链球菌和栖瘤胃普雷沃氏菌数量均高于对照组(P<0.05),反刍兽甲烷短杆菌数量低于对照组(P<0.05),嗜淀粉瘤胃杆菌和白色瘤胃球菌在两组间差异不显著;试验组中菌群多样性低于对照组(P<0.05),拟杆菌门、纤维杆菌门、密螺旋体门含量升高,厚壁菌门含量降低(P>0.05);理研菌科_RC9菌属含量升高,克里斯滕森菌属_R-7含量降低(P<0.05)。综上,酵母发酵饲料能够影响瘤胃菌群数量,改善瘤胃菌群结构,降低瘤胃菌群的多样性。     

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.     

Detection and identification of rumen bacteria constituting a fibrolytic consortium dominated by Fibrobacter succinogenes

A fibrolytic consortium, dominated by the rumen cellulolytic bacterium Fibrobacter succinogenes, was artificially constructed on hay stems to detect and identify rumen bacteria that can potentially interact with F. succinogenes . Consortium-bacterial members were determined by DGGE and sequencing analysis targeted bacterial 16S rDNA. An artificial consortium was formed in a 2-step incubation of hay stems; the first step with group 1, 2 or 3 F. succinogenes strains, the second step with rumen fluid. After consortium formation, morphologically different bacteria were observed in association with F. succinogenes . DGGE exhibited more than 30 bands, the pattern of which depended on the F. succinogenes group. Sequencing suggested that Butyrivibrio fibrisolvens, Pseudobutyrivibrio ruminis , Clostridium sp., F. succinogenes group 2, Prevotella ruminicola and unclassified Bacteroides were prominent in the group 1 consortium and that Treponema bryantii , B. fibrisolvens , Acinetobacter sp, and Wolinella succinogenes were prominent in the group 2 consortium. However, in the group 3 consortium, F. succinogenes -like bacteria were microscopically undetectable, whereas cellulolytic Ruminococcus albus and F. succinogenes group 1 were prominent, suggesting that the group 3 cannot be a core member of this consortium. This study is the first attempt to identify bacterial members of a fibrolytic consortium dominated by a specific bacterium.     

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浓度与微生物菌群密度间的互作关系。结果表明:藏绵羊冷季瘤胃中乙酸与丙酸浓度及比值(A/P)显著高于暖季(P<0.05),其他SCFA浓度在暖、冷季间差异不显著(P>0.05);各种SCFA所占比例分析发现,乙酸比例在冷季显著高于暖季(P<0.05),丁酸比例在暖季显著高于冷季(P<0.05),异丁酸比例在暖、冷季间差异不显著(P>0.05)。随着季节的变化,藏绵羊瘤胃微生物中黄色瘤胃球菌(Ruminococcus flavefaciens)、白色瘤胃球菌(Ruminococcus albus)、产琥珀酸丝状杆菌(Fibrobacter succinogenes)、溶纤维丁酸弧菌(Butyrivibrio fibrisolvens)、嗜淀粉瘤胃杆菌(Ruminobacter amylophilus)与产甲烷菌(Methanogenic bacteria)的相对密度在暖季显著高于冷季(P<0.05)。相关性分析表明,乙酸浓度与黄色瘤胃球菌相对密度,丙酸浓度与白色瘤胃球菌相对密度呈极显著正相关(P<0.01),乙酸浓度与白色瘤胃球菌、嗜淀粉瘤胃杆菌、溶纤维丁酸弧菌、产甲烷菌相对密度,丙酸浓度与产琥珀酸丝状杆菌相对密度呈显著负相关(P<0.05),丁酸浓度与6种微生物菌种相对密度无显著相关性(P>0.05)。由此可见,藏绵羊瘤胃微生物菌群密度与SCFA浓度间的互作存在明显差异,这种互作机制可能在藏绵羊应对暖、冷季的适应性方面发挥着重要作用,研究结果将为藏绵羊产业发展及种质资源保护利用提供参考。     

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。     

16S rRNA定量分析技术对绵羊瘤胃固相纤维降解菌的研究

本试验选择3只安装永久性瘤胃瘘管、体重25￣30kg的羯羊,使用特异性16SrRNA目标探针进行斑点杂交研究绵羊瘤胃固相纤维降解菌的动态变化规律。结果表明:3种纤维分解菌的RNA占总菌数RNA的15.76%;在各时间点产琥珀酸拟杆菌的相对含量均高于黄化瘤胃球菌和白色瘤胃球菌相对含量。     

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)。     

Effects of isobutyrate supplementation on ruminal microflora,rumen enzyme activities and methane emissions in Simmental steers

The objective of this study was to evaluate the effects of isobutyrate supplementation on rumen microflora, enzyme activities and methane emissions in Simmental steers consuming a corn stover‐based diet. Eight ruminally cannulated Simmental steers were used in a replicated 4 × 4 Latin square experiment. The treatments were control (without isobutyrate), low isobutyrate (LIB), moderate isobutyrate (MIB) and high isobutyrate (HIB) with 8.4, 16.8 and 25.2 g isobutyrate per steer per day respectively. Isobutyrate was hand‐mixed into the concentrate portion. Diet consisted of 60% corn stover and 40% concentrate [dry matter (DM) basis]. Dry matter intake (averaged 9 kg/day) was restricted to a maximum of 90% of ad libitum intake. Population of total bacteria, cellulolytic bacteria and anaerobic fungi were linearly increased, whereas that of protozoa and total methanogens was linearly reduced with increasing isobutyrate supplementation. Real‐time PCR quantification of population of Ruminococcus albus, Ruminococcus flavefaciens, Butyrivibrio fibrisolvens and Fibrobacter succinogenes was linearly increased with increasing isobutyrate supplementation. Activities of carboxymethyl cellulase, xylanase and β‐glucosidase were linearly increased, whereas that of protease was linearly reduced. Methane production was linearly decreased with increasing isobutyrate supplementation. Effective degradabilities of cellulose and hemicellulose of corn stover were linearly increased, whereas that of crude protein in diet was linearly decreased with increasing isobutyrate supplementation. The present results indicate that isobutyrate supplemented improved microflora, rumen enzyme activities and methane emissions in steers. It was suggested that the isobutyrate stimulated the digestive micro‐organisms or enzymes in a dose‐dependent manner. In the experimental conditions of this trial, the optimum isobutyrate dose was approximately 16.8 g isobutyrate per steer per day.     

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;     

Degradation of wheat straw and alkaline hydrogen peroxide-treated wheat straw by Ruminococcus albus 8 and Ruminococcus flavefaciens FD-1

Degradation of wheat straw (WS) and alkaline hydrogen peroxide (AHP)-treated wheat straw (AHPWS) by Ruminococcus albus 8 and Ruminococcus flavefaciens FD-1 was determined by measuring the growth (OD600) of each bacterium and determining DM disappearance (DMD) of the substrate. Complex medium and defined medium with or without the addition of phenylpropanoic acid (PPA) and phenylacetic acid (PAA) were used. Tubes were incubated at 39 degrees C for 8 d. Both OD600 and DMD indicated that AHPWS was degraded to a much greater extent by either bacterium (R. flavefaciens FD-1, 60.8 +/- 1.8% and R. albus 8, 42.3 +/- 3.5%) vs untreated WS (R. flavefaciens FD-1, 16.5 +/- 1.8% and R. albus 8, 8.6 +/- 6%) in the complex medium. Most degradation occurred between d 1 and 4. With the complex medium, addition of PPA and PAA did not stimulate degradation by either bacterium. When the defined medium was used, the addition of PPA and PAA enhanced (P less than .05) degradation of AHPWS (39.6 +/- 2.6%) vs AHPWS with no added PPA and PAA (24.9 +/- 7.6%) by R. albus 8. There was no synergistic effect on degradation when the two species were co-cultured with either WS or AHPWS as the substrate. No effect of PPA and PAA on disappearance of AHPWS was observed for R. flavefaciens FD-1 or when the two bacteria were grown together. Dry matter disappearance analysis showed that R. flavefaciens FD-1 degraded AHPWS more rapidly (6.1 mg/d) than R. albus 8 did (4.2 mg/d) in complex medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

海子水牛瘤胃微生物的宏基因组学分析

为系统探讨海子水牛瘤胃内的微生物组成及木质纤维素降解酶系,本试验利用基于高通量测序的宏基因组学技术对海子水牛(2.5岁左右,平均体重为493 kg)瘤胃液样本进行组学分析。结果表明,共获得了77 283 638条reads,并拼接出744 712个scaffold。经prodigal分析后,共预测出827 044个基因。通过基因注释发现海子水牛瘤胃中含有多种木质纤维素降解微生物,如生黄瘤胃球菌(Ruminococcus flavefaciens)、白色瘤胃球菌(Ruminococcus albus)、产琥珀酸丝状杆菌(Fibrobacter succinogenes)及栖瘤胃普雷沃氏菌(Prevotella ruminicola)。此外,还发现有38 011个基因编码蛋白质具有木质纤维素降解酶活性,其中糖苷水解酶(GH)基因数量最多(17 877个),糖基转移酶(GT)(8 637个)、碳水化合物结合模块(CBM)(4 693个)和碳水化合物酯酶(CE)基因(4 214个)数量次之,多糖裂合酶(PL)(1 296个)和辅助氧化还原酶(AA)基因(934个)数量较少。在GH基因中,归属于GH2、GH43、GH97、GH3家族的基因较多,且编码蛋白质具有寡糖降解酶活性的基因数量最多。此外还发现了少量的纤维小体组分蛋白基因。结合其他物种肠胃宏基因组中GH基因比对分析,发现海子水牛瘤胃中的纤维素酶、半纤维素酶和分支降解酶比例与奶牛瘤胃较为接近。由此可见,海子水牛瘤胃内含有丰富的木质纤维素降解微生物及酶系,这将为筛选具有工业应用潜力的酶基因奠定理论基础。     

日粮粗蛋白质水平对育肥湖羊瘤胃微生物组成和发酵参数的影响

为研究日粮粗蛋白质水平(crude protein,CP)对育肥湖羊瘤胃微生物、pH及VFA含量的影响,选取54只4月龄[(32.25±2.50)kg]湖羊公羔,采用完全随机分组设计,将试验羊分为3个处理组,每个处理组18个重复,每个重复1只羊,3个处理组日粮CP水平分别为11.5%、14.0%、16.5%,试验期共70 d,包括7 d预试期,63 d正试期。试验期结束后,每组选择10只试验羊进行屠宰,测定屠宰性能并采集瘤胃内容物,用于瘤胃微生物DNA提取。结果表明:1)不同CP处理组对宰前活重、胴体重、屠宰率、瘤胃总重、瘤胃组织重及尾脂重无显著影响(P>0.05)。2)11.5%CP组瘤胃液氨态氮显著低于14.0%CP及16.5%CP组(P<0.001),但14.0%CP组与16.5%CP组无显著差异(P>0.05)。瘤胃液pH随日粮蛋白水平的增加有增加的趋势(P<0.1)。3)总挥发性脂肪酸(total volatile fatty acids,TVFA)浓度不受日粮中CP水平的影响(P>0.05),其中乙酸、丙酸、异丁酸、丁酸、戊酸比例均不受CP水平的影响(P>0.05),但异戊酸的比例随日粮中CP水平的增加有增加趋势(P=0.098)。4)在不同CP处理组中,瘤胃内溶纤维丁酸弧菌含量最高,但各处理组间无显著差异(P>0.05),其中白色瘤胃球菌、产琥珀酸丝状杆菌、普雷沃氏菌的含量均不受CP水平的影响(P>0.05),而黄色瘤胃球菌11.5%CP组显著低于14.0%CP组(P<0.05),16.5%CP组与11.5%CP组及14.0%CP组无显著差异。综上所述,日粮不同CP水平不影响试验羊屠宰性能,但能够影响瘤胃液pH及氨态氮含量,随CP水平的增加,异戊酸的比例有增加的趋势,14.0%CP组黄色瘤胃球菌数最多。     

发酵玉米蛋白粉对奶牛瘤胃体外发酵特性及微生物菌群的影响

本试验旨在研究全混合日粮（TMR）中添加发酵玉米蛋白粉（fermented corn gluten meal,FCGM）对奶牛瘤胃体外发酵特性及微生物菌群的影响。选用3头体重（600±25）kg,安装永久性瘤胃瘘管的荷斯坦奶牛作为瘤胃液供体,发酵底物为TMR,分为对照组和3个试验组,各组分别在发酵液中添加0、0.3、0.6、0.9 g/L FCGM（干物质基础）,每个处理3个重复。记录体外发酵12、24、36和48 h产气量,测定体外发酵12、24和48 h发酵液pH、体外干物质消失率（IVDMD）、纤维素酶活性、氨态氮（NH₃-N）、挥发性脂肪酸（VFA）和菌体蛋白浓度,并测定体外发酵24 h发酵液中瘤胃微生物菌群相对丰度。结果显示：①添加不同水平FCGM组的体外产气量（除12 h外）、慢速产气部分、潜在产气部分和有效产气速率均显著或极显著高于对照组（P < 0.05;P < 0.01）;②与对照组相比,添加不同水平FCGM处理组的发酵液pH显著或极显著低于对照组,纤维素酶活性、菌体蛋白、挥发性脂肪酸、氨态氮含量和体外干物质消失率均显著或极显著升高（P < 0.05;P < 0.01）,且0.9 g/L FCGM组达到最高。③添加0.6和0.9 g/L FCGM组发酵液中白色瘤胃球菌、黄色瘤胃球菌、产琥珀酸丝状杆菌、牛链球菌、普雷沃氏菌、溶纤维丁酸弧菌、嗜淀粉瘤胃杆菌、真菌和原虫相对丰度均显著高于对照组（P < 0.05）,且0.9 g/L FCGM组达到最高,而产甲烷菌相对丰度显著低于对照组（P < 0.05）,且0.9 g/L FCGM组达到最低。综上所述,TMR中添加FCGM可提高体外发酵产气量,增加发酵液内纤维素酶活性、VFA、NH3-N及菌体蛋白含量,提高瘤胃内某些纤维降解菌、蛋白降解菌、淀粉降解菌、真菌和原虫相对丰度,降低产甲烷菌相对丰度,调节瘤胃微生物菌群结构,改善瘤胃发酵,其中以添加0.9 g/L FCGM为宜。     

Dynamics of methanogenesis,ruminal fermentation and fiber digestibility in ruminants following elimination of protozoa: a meta-analysis

Background: Ruminal microbes are vital to the conversion of lignocellulose-rich plant materials into nutrients for ruminants.Although protozoa play a key role in linking ruminal microbial networks,the contribution of protozoa to rumen fermentation remains controversial; therefore,this meta-analysis was conducted to quantitatively summarize the temporal dynamics of methanogenesis,ruminal volatile fatty acid(VFA) profiles and dietary fiber digestibility in ruminants following the elimination of protozoa(also termed defaunation).A total of 49 studies from 22 publications were evaluated.Results: The results revealed that defaunation reduced methane production and shifted ruminal VFA profiles to consist of more propionate and less acetate and butyrate,but with a reduced total VFA concentration and decreased dietary fiber digestibility.However,these effects were diminished linearly,at different rates,with time during the first few weeks after defaunation,and eventually reached relative stability.The acetate to propionate ratio and methane production were increased at 7 and 11 wk after defaunation,respectively.Conclusions: Elimination of protozoa initially shifted the rumen fermentation toward the production of more propionate and less methane,but eventually toward the production of less propionate and more methane over time.     

茶皂素对奶牛瘤胃发酵及瘤胃微生物区系的影响

本试验旨在研究茶皂素对奶牛瘤胃发酵及瘤胃微生物区系的影响。试验选用12头健康状况良好,体重相近的荷斯坦奶牛,随机分为4组,均饲喂基础饲粮,各组分别灌服0(对照)、20、30、40 g/d茶皂素,茶皂素与水混匀灌服,进行预试期14 d,正试期35 d的饲养试验。正试期期间每隔7 d,在晨饲前1 h用口腔采样器采集瘤胃液测定瘤胃发酵指标,用实时定量PCR(qRT-PCR)法测定瘤胃微生物含量。结果表明:1)与对照组相比,茶皂素显著降低了瘤胃液p H(30、40 g/d组)、氨态氮的浓度(20、30、40 g/d组)(P0.05),但均未超过正常范围值;茶皂素显著提高了微生物蛋白(30、40 g/d组)、丙酸(20、30、40 g/d组)和丁酸浓度(20、30、40 g/d组)(P0.05),30 g/d组的微生物蛋白浓度提高了20.20%;但茶皂素对总挥发性脂肪酸和乙酸浓度的影响不显著(P0.05)。2)与对照组相比,茶皂素各组的瘤胃液原虫、溶纤维丁酸弧菌含量均显著降低(P0.05),甲烷菌、白色瘤胃球菌、产琥珀酸丝状杆菌、黄色瘤胃球菌、真菌含量没有显著变化(P0.05)。综上所述,补饲茶皂素改善了奶牛瘤胃发酵模式,并显著影响了奶牛瘤胃微生物区系,30 g/d的剂量对奶牛较为适宜。     

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。     

饲料来源对育肥湖羊生产性能、养分消化及瘤胃微生物组成的影响

为评价日粮粗饲料来源对育肥湖羊生产性能、养分消化及瘤胃微生物组成的影响。选择60日龄健康湖羊公羔(22.9±1.2) kg 120只,根据体重相近原则随机分成4组,每组5个重复,每个重复6只羊,分别以玉米秸秆(CS)、玉米芯(CC)、葵花籽壳(SH)和油菜秸秆(RS)作为粗饲料来源,添加比例均为20%,4组精料配比一致,预试期7 d,正试期70 d。在饲喂的49~55 d,每组选择5只羊采用全收粪法进行消化代谢试验,测定干物质(DM)、有机物质(OM)、中性洗涤纤维(NDF)、酸性洗涤纤维(ADF)和氮(N)的表观消化率。试验结束后,每组选择15只羊进行屠宰,采集瘤胃内容物,提取瘤胃微生物DNA,对瘤胃细菌进行绝对定量。结果显示:1)CS组湖羊干物质采食量显著高于其他3组(P<0.05),且末体重和平均日增重显著高于RS组(P<0.05),其他组间差异不显著(P>0.05)。2)CS和CC组湖羊DM和ADF消化率显著高于RS组(P<0.05),且其OM和NDF消化率显著高于SH组和RS组(P<0.05);SH组湖羊N消化率显著高于RS组,其ADF消化率与其他3组湖羊差异均不显著(P>0.05)。3)CS组和CC组湖羊瘤胃中白色瘤胃球菌和普雷沃氏菌数量均显著高于RS组(P<0.05),而SH组湖羊瘤胃中两种细菌的数量与其他3组湖羊均无显著差异(P>0.05),CS组和CC组湖羊瘤胃中黄色瘤胃球菌和产琥珀酸丝状杆菌数量均显著高于SH组和RS组(P<0.05),4组湖羊瘤胃中反刍兽新月单胞菌数量差异不显著(P>0.05)。结果表明:相较于葵花籽壳组日粮和油菜秸秆组日粮,玉米秸秆组和玉米芯组日粮不仅可以确保育肥湖羊的生产性能,并且有利于纤维分解菌的生存,可促进纤维素在瘤胃中的降解。