Evaluation of the effects of dietary particle fractions on fermentation profile and concentration of microbiota in the rumen of dairy cows fed grass silage-based diets

The study evaluated the effects of three different theoretical particle lengths (TPL) of grass silage on the distribution of particle fractions of the diet and the resulting effects on fermentation profile and concentrations of protozoa and mixed bacterial mass in the rumen of three lactating Holstein cows fed total mixed rations (45% grass silage, 5% grass hay and 50% concentrate) ad libitum. Decreasing TPL of grass silage (long, medium, short) reduced particles retained on the 19-mm sieve of the Penn State Particle Separator, while particle fractions from 8 mm to 19 mm and smaller than 8 mm were increased. Different TPL did not affect pH and the concentration of volatile fatty acids in the rumen. However, lowering the TPL from long to medium increased significantly the bicarbonate concentration, acetate proportion and protozoal number in the rumen, whereas the proportion of bacterial protein in ruminal digesta and its amino acid concentration were significantly increased by the short TPL. For the current feeding conditions, it can be concluded that increasing the fraction of particles between 8 and 19 mm and probably even the fraction below 8 mm by decreasing TPL of grass silage do not adversely affect rumen conditions and can be beneficial in terms of optimising concentration and activity of ruminal microbiota in high-yielding dairy cows.     

Evaluation of the effects of dietary particle fractions on fermentation profile and concentration of microbiota in the rumen of dairy cows fed grass silage-based diets

The study evaluated the effects of three different theoretical particle lengths (TPL) of grass silage on the distribution of particle fractions of the diet and the resulting effects on fermentation profile and concentrations of protozoa and mixed bacterial mass in the rumen of three lactating Holstein cows fed total mixed rations (45% grass silage, 5% grass hay and 50% concentrate) ad libitum. Decreasing TPL of grass silage (long, medium, short) reduced particles retained on the 19-mm sieve of the Penn State Particle Separator, while particle fractions from 8 mm to 19 mm and smaller than 8 mm were increased. Different TPL did not affect pH and the concentration of volatile fatty acids in the rumen. However, lowering the TPL from long to medium increased significantly the bicarbonate concentration, acetate proportion and protozoal number in the rumen, whereas the proportion of bacterial protein in ruminal digesta and its amino acid concentration were significantly increased by the short TPL. For the current feeding conditions, it can be concluded that increasing the fraction of particles between 8 and 19 mm and probably even the fraction below 8 mm by decreasing TPL of grass silage do not adversely affect rumen conditions and can be beneficial in terms of optimising concentration and activity of ruminal microbiota in high-yielding dairy cows.     

Effects of faba bean,blue lupin and rapeseed meal supplementation on nitrogen digestion and utilization of dairy cows fed grass silage-based diets

There is increasing interest in using locally produced protein supplements in dairy cow feeding. The objective of this experiment was to compare rapeseed meal (RSM), faba beans (FBs) and blue lupin seeds (BL) at isonitrogenous amounts as supplements of grass silage and cereal based diets. A control diet (CON) without protein supplement was included in the experiment. Four lactating Nordic Red cows were used in a 4 × 4 Latin Square design with four 21 d periods. The milk production increased with protein supplementation but when expressed as energy corrected milk, the response disappeared due to substantially higher milk fat concentration with CON compared to protein supplemented diets. Milk protein output increased by 8.5, 4.4 and 2.7% when RSM, FB and BL were compared to CON. The main changes in rumen fermentation were the higher propionate and lower butyrate proportion of total rumen volatile fatty acids when the protein supplemented diets were compared to CON. Protein supplementation also clearly increased the ruminal ammonia N concentration. Protein supplementation improved diet organic matter and NDF digestibility but efficiency of microbial protein synthesis per kg organic matter truly digested was not affected. Flow of microbial N was greater when FB compared to BL was fed. All protein supplements decreased the efficiency of nitrogen use in milk production. The marginal efficiency (amount of additional feed protein captured in milk protein) was 0.110, 0.062 and 0.045 for RSM, FB and BL, respectively. The current study supports the evidence that RSM is a good protein supplement for dairy cows, and this effect was at least partly mediated by the lower rumen degradability of RSM protein compared to FB and BL. The relatively small production responses to protein supplementation with simultaneous decrease in nitrogen use efficiency in milk production suggest that economic and environmental consequences of protein feeding need to be carefully considered.     

黄海日照海域部分可培养细菌的分离、初步鉴定及产琼胶酶细菌的筛选

海洋细菌在海洋的物质与能量循环中起着非常重要的作用。为了适应复杂多变的海洋环境,海洋细菌在物种和基因方面表现出极高的丰富度。[目的] 对中国黄海日照海域部分可培养细菌进行分离、初步鉴定,同时筛选产琼胶酶菌株。[方法] 对从日照海域河流入海口和潮间带沙样和海水中分离到的73株细菌进行了16S rRNA基因序列测定,并进行序列同源性分析。同时检测了这些菌株对琼脂的降解能力。[结果] 结果显示,分离到的73株细菌属于4个门、13个科、34个属。18株细菌可能是新分类单元,其中16株为潜在新种,2株为可能的新属。73株细菌中,5株菌具有降解琼脂的能力,最高的琼胶酶活可达到2.17±0.04 U/mL,其中4株嗜琼胶属的细菌降解琼脂糖的产物均为新琼四糖。[结论] 本研究丰富了人们对黄海海域可培养细菌多样性的理解,为新物种和新酶的研究提供了基础,并为琼胶酶的提取提供了高产菌株。     

Enumeration of Bacteroides succinogenes in the rumen of sheep fed maize-straw diets

Abstract Estimates of Bacteroides succinogenes in the rumen of two sheep fed a maize-straw diet exceeded 10⁸ per g of rumen ingesta and were even higher when the straw was supplemented with pelleted maize grain. A number of B. succinogenes strains were characterized in some detail and very few were found to ferment starch. Cells of freshly isolated strains lost viability more rapidly when grown on agar medium than did cells of strains which had been subjected to more transfers.     

Comparative growth rates of various rumen bacteria in clarified rumen fluid from cows and sheep fed different diets.

Pure cultures of strains of different species of rumen bacteria were grown in filter-sterilized rumen fluid supplemented with glucose, bicarbonate, and reducing agent (cysteine and sulfide). Growth rates were determined in a series of experiments. Strains of species most abundant in the rumen grew more rapidly than strains of less abundant bacteria. Ammonia, amino acids, and peptides increased growth rates to some extent, but the greatest stimulatory effect for less abundant bacteria was provided by other factors, present in yeast extract. Factors released from lysates of mixed rumen microbes stimulated growth, but their rate of release was slow. It was concluded that, besides energy and nitrogen sources, growth factors of an as-yet-undetermined nature probably play an important role in determining the predominance of different bacterial species in the rumen.     

Integration of the effects of animal and dietary factors on total dry matter intake of dairy cows fed silage-based diets

An empirical regression model for the prediction of total dry matter intake (DMI) of dairy cows was developed and compared with four published intake models. The model was constructed to include both animal and dietary factors, which are known to affect DMI. For model development, a data set based on individual cow data from 10 change-over and four continuous milk production studies was collected (n = 1554). Relevant animal (live weight (LW), days in milk (DIM), parity and breed) and dietary (total and concentrate DMI, concentrate composition, forage digestibility and fermentation quality) data were collected. The model factors were limited to those that are available before the diets are fed to animals, that is, standardized energy corrected milk (sECM) yield, LW, DIM and diet quality (total diet DMI index (TDMI index)). As observed ECM yield is a function of both the production potential of the cow and diet quality, ECM yield standardized for DIM, TDMI index and metabolizable protein concentration was used in modelling. In the individual data set, correlation coefficients between sECM and TDMI index or DIM were much weaker (0.16 and 0.03) than corresponding coefficients with observed ECM (0.65 and 0.46), respectively. The model was constructed with a mixed model regression analysis using cow within trial as a random factor. The following mixed model was estimated for DMI prediction: DMI (kg DM/day) = -2.9 (±0.56)+0.258 (±0.011) × sECM (kg/day) + 0.0148 (±0.0009) × LW (kg) -0.0175 (±0.001) × DIM -5.85 (±0.41) × exp (-0.03 × DIM) + 0.09 (±0.002) × TDMI index. The mixed DMI model was evaluated with a treatment mean data set (207 studies, 992 diets), and the following relationship was found: Observed DMI (kg DM/day) = -0.10 (±0.33) + 1.004 (±0.019) × Predicted DMI (kg DM/day) with an adjusted residual mean square error of 0.362 kg/day. Evaluation of the residuals did not result in a significant mean bias or linear slope bias, and random error accounted for proportionally >0.99 of the error. In conclusion, the DMI model developed is considered robust because of low mean prediction error, accurate and precise validation, and numerically small differences in the parameter values of model variables when estimated with mixed or simple regression models. The Cornell Net Carbohydrate and Protein System was the most accurate of the four other published DMI models evaluated using individual or treatment mean data, but in most cases mean and linear slope biases were relatively high, and, interestingly, there were large differences in both mean and linear slope biases between the two data sets.     

Comparative growth rates of various rumen bacteria in clarified rumen fluid from cows and sheep fed different diets.

Pure cultures of strains of different species of rumen bacteria were grown in filter-sterilized rumen fluid supplemented with glucose, bicarbonate, and reducing agent (cysteine and sulfide). Growth rates were determined in a series of experiments. Strains of species most abundant in the rumen grew more rapidly than strains of less abundant bacteria. Ammonia, amino acids, and peptides increased growth rates to some extent, but the greatest stimulatory effect for less abundant bacteria was provided by other factors, present in yeast extract. Factors released from lysates of mixed rumen microbes stimulated growth, but their rate of release was slow. It was concluded that, besides energy and nitrogen sources, growth factors of an as-yet-undetermined nature probably play an important role in determining the predominance of different bacterial species in the rumen.     

Enumeration and characterization of cellulolytic bacteria from refuse of a landfill

Enumeration and phenotypic characterization of aerobic cellulolytic bacteria were performed on fresh, 1 year old and 5 years old refuse samples of a French landfill site. Numbers of cellulolytic bacteria ranged from 1.1x10(6) to 2.3x10(8) c.f.u. (g dry wt.)(-1) and were lower in 5 years old refuse samples. A numerical analysis of phenotypic data based on 80 biochemical tests and performed on 321 Gram-positive isolates from refuse, revealed a high phenotypic diversity of cellulolytic bacteria which were distributed into 21 clusters. Based on the phenotypic analysis and the sequencing of 16S rDNA of five representative strains of major clusters, the predominant cellulolytic groups could be assigned to the family of Bacillaceae and to the genera Cellulomonas, Microbacterium and Lactobacillus. Furthermore, chemical parameters such as pH, carbohydrates and volatile solid contents influenced the composition of the cellulolytic bacterial groups which were reduced essentially to the family of Bacillaceae in the oldest refuse samples.     

Relationship between rumen methanogens and methane production in dairy cows fed diets supplemented with a feed enzyme additive

Aims: To investigate the relationship between ruminal methanogen community and host enteric methane (CH₄) production in lactating dairy cows fed diets supplemented with an exogenous fibrolytic enzyme additive. Methods and Results: Ecology of ruminal methanogens from dairy cows fed with or without exogenous fibrolytic enzymes was examined using PCR–denaturing gradient gel electrophoresis (PCR–DGGE) analyses and quantitative real‐time PCR (qRT‐PCR). The density of methanogens was not affected by the enzyme additive or sampling times, and no relationship was observed between the total methanogen population and CH₄ yield (as g per head per day or g kg^?1 DMI). The PCR–DGGE profiles consisted of 26 distinctive bands, with two bands similar to Methanogenic archaeon CH1270 negatively correlated, and one band similar to Methanobrevibacter gottschalkii strain HO positively correlated, with CH₄ yield. Three bands similar to Methanogenic archaeon CH1270 or Methanobrevibacter smithii ATCC 35061 appeared after enzyme was added. Conclusions: Supplementing a dairy cow diet with an exogenous fibrolytic enzyme additive increased CH₄ yield and altered the composition of the rumen methanogen community, but not the overall density of methanogens. Significance and Impact of the Study: This is the first study to identify the correlation between methanogen ecology and host CH₄ yield from lactating dairy cows.     

Magnesium requirement of some of the principal rumen cellulolytic bacteria

Information available on the role of Mg for growth and cellulose degradation by rumen bacteria is both limited and inconsistent. In this study, the Mg requirements for two strains each of the cellulolytic rumen species Fibrobacter succinogenes (A3c and S85), Ruminococcus albus (7 and 8) and Ruminococcus flavefaciens (B34b and C94) were investigated. Maximum growth, rate of growth and lag time were all measured using a complete factorial design, 2(3)×6; factors were: strains (2), within species (3) and Mg concentrations (6). R. flavefaciens was the only species that did not grow when Mg was singly deleted from the media, and both strains exhibited a linear growth response to increasing Mg concentrations (P<0.001). The requirement for R. flavefaciens B34b was estimated as 0.54 mM; whereas the requirement for R. flavefaciens C94 was >0.82 as there was no plateau in growth. Although not an absolute requirement for growth, strains of the two other species of cellulolytic bacteria all responded to increasing Mg concentrations. For F. succinogenes S85, R. albus 7 and R. albus 8, their requirement estimated from maximum growth was 0.56, 0.52 and 0.51, respectively. A requirement for F. succinogenes A3c could not be calculated because there was no solution for contrasts. Whether R. flavefaciens had a Mg requirement for cellulose degradation was determined in NH₃-free cellulose media, using a 2×4 factorial design, 2 strains and 4 treatments. Both strains of R. flavefaciens were found to have an absolute Mg requirement for cellulose degradation. Based on reported concentrations of Mg in the rumen, 1.0 to 10.1 mM, it seems unlikely that an in vivo deficiency of this element would occur.     

Effect of monensin feeding and withdrawal on populations of individual bacterial species in the rumen of lactating dairy cows fed high-starch rations

Population dynamics of ammonia-oxidizing bacteria (AOB) in a full-scale aerated submerged biofilm reactor for micropolluted raw water pretreatment was investigated using molecular techniques for a period of 1 year. The ammonia monooxygenase (amoA) gene fragments were amplified from DNA and RNA extracts of biofilm samples. Denaturing gradient gel electrophoresis (DGGE) profile based on the amoA messenger RNA approach exhibited a more variable pattern of temporal dynamics of AOB communities than the DNA-derived approach during the study. Phylogenetic analysis of excised DGGE bands revealed three AOB groups affiliated with the Nitrosomonas oligotropha lineage, Nitrosomonas communis lineage, and an unknown Nitrosomonas group. The population size of betaproteobacterial AOB, quantified with 16S ribosomal RNA gene real-time polymerase chain reaction assay, ranged from 6.63 × 10⁵ to 2.67 × 10⁹ cells per gram of dry biofilm and corresponded to 0.23–1.8% of the total bacterial fraction. Quantitative results of amoA gene of the three specific AOB groups revealed changes in competitive dominance between AOB of the N. oligotropha lineage and N. communis lineage. Water temperature is shown to have major influence on AOB population size in the reactor by the statistic analysis, and a positive correlation between AOB cell numbers and ammonia removal efficiency is suggested (r = 0.628, P < 0.05).     

Development and use of competitive PCR assays for the rumen cellulolytic bacteria: Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens

Competitive PCR assays were developed for the enumeration of the rumen cellulolytic bacterial species: Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens. The assays, targeting species-specific regions of 16S rDNA, were evaluated using DNA from pure culture and rumen digesta spiked with the relevant cellulolytic species. Minimum detection levels for F. succinogenes, R. albus and R. flavefaciens were 1-10 cells in pure culture and 10(3-4) cells per ml in mixed culture. The assays were reproducible and 11-13% inter- and intra-assay variations were observed. Enumeration of the cellulolytic species in the rumen and alimentary tract of sheep found F. succinogenes dominant (10(7) per ml of rumen digesta) compared to the Ruminococcus spp. (10(4-6) per ml). The population size of the three species did not change after the proportion of dietary alfalfa hay was increased. All three species were detected in the rumen, omasum, caecum, colon and rectum. Numbers of the cellulolytic species at these sites varied within and between animals.     

薇甘菊根际可培养固氮菌和氨化细菌的分离鉴定与促生作用

【目的】固氮菌和氨化细菌是氮循环产生生物有效氮的关键起始环节,直接影响了外来入侵植物的生长速度和扩散进程。然而,关于典型入侵植物薇甘菊根际可培养固氮菌和氨化细菌的研究尚未见报道,这在很大程度上制约了我们对薇甘菊根际高效的氮素转化机制的深刻理解。【方法】采用传统平板涂布培养法对野外采集的薇甘菊根际土壤中的可培养固氮菌和氨化细菌进行了分离鉴定,并进行了接种验证实验。【结果】结果表明,入侵植物薇甘菊根际土壤中的固氮菌和氨化细菌的菌群密度显著高于两个本地伴生植物(火炭母和鸡屎藤),其固氮效率及有机氮矿化效率也优于2个本地种;系统发育分析表明：薇甘菊根际的固氮菌菌株归类于5个属,分别为伯克霍尔德氏菌属(Burkholderia)、肠杆菌属(Enterobacter)、植物杆菌属(Phytobacter)、新肠杆菌属(Kosakonia)和根瘤菌属(Rhizobium);氨化细菌归类于7个属,分别为沙雷氏菌属(Serratia)、不动杆菌属(Acinetobacter)、假单胞菌属(Pseudomonas)、博德特氏菌属(Bordetella)、寡养单胞菌属(Stenotrophomonas)、苍...     

不同精粗比底物下瘤胃真菌和纤维降解细菌共培养发酵特性及菌群变化

采用体外厌氧共培养技术,研究了瘤胃真菌和纤维降解细菌在不同精粗比(A组为全粗料,B组3∶7,C组5∶5,D组7∶3,E组为全精料)底物下菌群变化及其共培养发酵特性。结果表明:与0h相比,发酵至24h时B组和C组的厌氧真菌数量有较大幅度的上升,A组和D组则有所下降,E组未检测到真菌生长;纤维降解细菌随精粗比的增加呈上升趋势。发酵至48h时,各组均未检测到真菌生长;从A组到C组细菌数量呈上升趋势,此后急剧下降。DGGE结果表明,A、B和C组(精粗比低于5∶5)的DGGE图谱相似,有11条共有条带,但是当精粗比上升到7:3时,条带数目显著下降。随精料比例的增加,整个发酵期共培养系统中pH值显著下降(P<0.05)。整个发酵期间,共培养系统发酵产生的VFA主要为乙酸,丙酸和丁酸的量较少,乙酸与丙酸比值从A组到C组呈下降趋势,此后呈上升趋势。随精料比例的上升,发酵48h时总挥发性脂肪酸浓度从A组到C组呈上升趋势,此后呈下降趋势。发酵48h的羧甲基纤维素酶活和木聚糖酶活均以A组最高,而α-淀粉酶活从A组到D组逐渐增大,而E组最低,仅为B、C、D组的1/4~1/3。     

Identification of non-catalytic conserved regions in xylanases encoded by the xynB and xynD genes of the cellulolytic rumen anaerobe Ruminococcus flavefaciens

xynB is one of at least four genes from the cellulolytic rumen anaerobe Ruminococcus flavefaciens 17 that encode xylanase activity. The xynB gene is predicted to encode a 781-amino acid product starting with a signal peptide, followed by an amino-terminal xylanase domain which is identical at 89% and 78% of residues, respectively, to the amino-terminal xylanase domains of the bifunctional XynD and XynA enzymes from the same organism. Two separate regions within the carboxy-terminal 537 amino acids of XynB also show close similarities with domain B of XynD. These regions show no significant homology with cellulose- or xylan-binding domains from other species, or with any other sequences, and their functions are unknown. In addition a 30 to 32-residue threonine-rich region is present in both XynD and XynB. Codon usage shows a consistent pattern of bias in the three xylanase genes from R. flavefaciens that have been sequenced.     

Feed intake and milk production in dairy cows fed different grass and legume species: a meta-analysis

The aim of this meta-analysis was to compare feed intake, milk production, milk composition and organic matter (OM) digestibility in dairy cows fed different grass and legume species. Data from the literature was collected and different data sets were made to compare families (grasses v. legumes, Data set 1), different legume species and grass family (Data set 2), and different grass and legume species (Data set 3+4). The first three data sets included diets where single species or family were fed as the sole forage, whereas the approach in the last data set differed by taking the proportion of single species in the forage part into account allowing diets consisting of both grasses and legumes to be included. The grass species included were perennial ryegrass, annual ryegrass, orchardgrass, timothy, meadow fescue, tall fescue and festulolium, and the legume species included were white clover, red clover, lucerne and birdsfoot trefoil. Overall, dry matter intake (DMI) and milk production were 1.3 and 1.6 kg/day higher, respectively, whereas milk protein and milk fat concentration were 0.5 and 1.4 g/kg lower, respectively, for legume-based diets compared with grass-based diets. When comparing individual legume species with grasses, only red clover resulted in a lower milk protein concentration than grasses. Cows fed white clover and birdsfoot trefoil yielded more milk than cows fed red clover and lucerne, probably caused by a higher OM digestibility of white clover and activity of condensed tannins in birdsfoot trefoil. None of the included grass species differed in DMI, milk production, milk composition or OM digestibility, indicating that different grass species have the same value for milk production, if OM digestibility is comparable. However, the comparison of different grass species relied on few observations, indicating that knowledge regarding feed intake and milk production potential of different grass species is scarce in the literature. In conclusion, different species within family similar in OM digestibility resulted in comparable DMI and milk production, but legumes increased both DMI and milk yield compared with grasses.     

In situ identification of carboxymethyl cellulose-digesting bacteria in the rumen of cattle fed alfalfa or triticale

A method was developed and used to arrest and stain reducing sugars (glucose) produced by bacteria with cell-surface-associated carboxymethyl cellulase (CMCase) and endoglucanase activities (CMC bacteria) in the rumen of cows fed alfalfa or triticale. Precipitation of silver oxide on the surface of individual cells was observed using cellulolytic bacterial pure cultures with known CMCase activity and rumen mixed cultures. The CMC bacteria in the liquid and solid fractions of the rumen digesta were identified using fluorescence in situ hybridization (FISH) with currently available and newly designed oligonucleotide probes. The CMC bacteria contributed between 8.2% and 10.1% to the total bacterial cell numbers. Most of the CMC bacteria (75.2-78.5%) could be identified by FISH probing. The known cellulolytic populations Ruminococcus flavefaciens, R.?albus, and Fibrobacter succinogenes constituted 44.5-53.1% of the total. Other CMC bacteria identified hybridized with the probe Clo549 (11.2-23.0%) targeting members of an uncharacterized genus in Clostridia, the probe Inc852 (8.9-10.7%) targeting members of the family Incertae Sedis III and unclassified Clostridiales, and the probe But1243 (相似文献