• Asian-Australasian Journal of Animal Sciences
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• v.22 no.1
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• pp.131-138
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• 2009

Among rumen microbes, bacteria play important roles in the biological degradation of plant fiber due to their large biomass and high activity. To maximize the utilization of fiber components such as cellulose and hemicellulose by ruminant animals, the ecology and functions of rumen bacteria should be understood in detail. Recent genome sequencing analyses of representative fibrolytic bacterial species revealed that the number and variety of enzymes for plant fiber digestion clearly differ between Fibrobacter succinogenes and Ruminococcus flavefaciens. Therefore, the mechanism of plant fiber digestion is also thought to differ between these two species. Ecology of individual fibrolytic bacterial species has been investigated using pure cultures and electron microscopy. Recent advances in molecular biology techniques complement the disadvantages of conventional techniques and allow accurate evaluation of the ecology of specific bacteria in mixed culture, even in situ and in vivo. Molecular monitoring of fibrolytic bacterial species in the rumen indicated the predominance of F. succinogenes. Nutritive interactions between fibrolytic and non-fibrolytic bacteria are important in maintaining and promoting fibrolytic activity, mainly in terms of crossfeeding of metabolites. Recent 16S rDNA-based analyses suggest that presently recognized fibrolytic species such as F. succinogenes and two Ruminococcus species with fibrolytic activity may represent only a small proportion of the total fibrolytic population and that uncultured bacteria may be responsible for fiber digestion in the rumen. Therefore, characterization of these unidentified bacteria is important to fully understand the physiology and ecology of fiber digestion. To achieve this, a combination of conventional and modern techniques could be useful.

• Asian-Australasian Journal of Animal Sciences
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• v.16 no.1
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• pp.44-49
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• 2003

Effects of supplementation with ruminal epithelial cells on fiber-degrading activity and cell growth of Ruminococcus albus (R. albus, strain 7) was tested using a basal substrate of rice straw and formulated concentrate. Cultures of R. albus alone and R. albus with rumen protozoa were grown at $39^{\circ}C$ for 48 h with an 8.4% crude protein (CP) substrate, 33% of the CP supplemented with either ruminal epithelial cells or defatted soybean meal. The ruminal epithelial cells had lower amounts of rumen soluble and degradable protein fractions as compared to defatted soybean meal, as determined by an enzymatic method, and the same was found with amino acid composition of protein hydrolysates. Ruminal epithelial cells were directly utilized by the R. albus, and resulted in greater growth of cell-wall free bacteria compared to defatted soybean meal. The effect of epithelial cells on bacterial growth was enhanced by the presence of rumen protozoa. In consistency with cultures of R. albus and R. albus with rumen protozoa, fermentative parameters such as dry matter degradability and total volatile fatty acid did not differ between supplementation with ruminal epithelial cells or defatted soybean meal.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.2
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• pp.199-202
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• 2004

Cellulolytic bacterial strains have been isolated from the faeces of wild (blackbuck, Antilope cervicapra; nilgai, Baselophus tragocamelus chinkara, Gazella gazella spotted deer, Axis axis and hog deer, Cervus porcinus) and rumen liquor of domestic (sheep, Ovis aries) ruminants. Five best cellulose degrading bacterial isolates (Ruminococcus sp.) were used as microbial feed additive along with buffalo rumen liquor as inoculum to study their effect on digestibility of feed and enzyme production in in vitro conditions. The bacterial isolate from chinkara (CHI-2) showed the highest per cent apparent dry matter (DM) digestibility ($35.40{\pm}0.60$), true dry matter digestibility ($40.80{\pm}0.69$) and NDF ($26.38{\pm}0.83$) digestibility (p<0.05) compared to control ($32.73{\pm}0.56$, $36.64{\pm}0.71$ and $21.16{\pm}0.89$, respectively) and other isolates at 24 h of incubation with lignocellulosic feeds (wheat straw and wheat bran, 80:20). The same isolate also exhibited the highest activities of fibre degrading enzymes like carboxymethylcellulase, xylanase, ${\beta}$-glucosidase and acetyl esterase. The bacterial isolate from chinkara (Gazella gazella) appears to have a potential to be used as feed additive in the diet of ruminants for improving utilization of nutrients from lignocellulosic feeds.

• Asian-Australasian Journal of Animal Sciences
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• v.5 no.1
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• pp.159-164
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• 1992

The present study was done to examine whether inoculated and established bacteria in the digestive tract of germ-free (GF) chickens affect growth performance, energy availability, nitrogen utilization and neutral detergent fibre (NDF) digestibility of the host bird fed a high-fibre diet. Gnotobiotic (GB) chicks were made from GF birds by co-inoculating with Ruminococcus albus, and Staphylococcus warneri, only the latter of which was established in the chicken gut. No difference was detected among conventional (CV), GF and GB birds in body weight gain, food intake or food efficiency from 7 to 21 d of age. The amount of nitrogen retained was larger in CV than in GF and GB chicks. DE and ME values of the diet and NDF digestibility were higher in CV birds than in GF and GB counterparts. It was concluded, therefore, that the established bacterium S. warneri did not give any beneficial effects on the host bird as judged by growth performance, energy availability, nitrogen utilization, and NDF digestibility.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.6
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• pp.880-884
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• 2001

Rumen microbiology research has undergone several evolutionary steps: the isolation and nutritional characterization of readily cultivated microbes; followed by the cloning and sequence analysis of individual genes relevant to key digestive processes; through to the use of small subunit ribosomal RNA (SSU rRNA) sequences for a cultivation-independent examination of microbial diversity. Our knowledge of rumen microbiology has expanded as a result, but the translation of this information into productive alterations of ruminal function has been rather limited. For instance, the cloning and characterization of cellulase genes in Escherichia coli has yielded some valuable information about this complex enzyme system in ruminal bacteria. SSU rRNA analyses have also confirmed that a considerable amount of the microbial diversity in the rumen is not represented in existing culture collections. However, we still have little idea of whether the key, and potentially rate-limiting, gene products and (or) microbial interactions have been identified. Technologies allowing high throughput nucleotide and protein sequence analysis have led to the emergence of two new fields of investigation, genomics and proteomics. Both disciplines can be further subdivided into functional and comparative lines of investigation. The massive accumulation of microbial DNA and protein sequence data, including complete genome sequences, is revolutionizing the way we examine microbial physiology and diversity. We describe here some examples of our use of genomics- and proteomics-based methods, to analyze the cellulase system of Ruminococcus flavefaciens FD-1 and explore the genome of Ruminococcus albus 8. At Illinois, we are using bacterial artificial chromosome (BAC) vectors to create libraries containing large (>75 kbases), contiguous segments of DNA from R. flavefaciens FD-1. Considering that every bacterium is not a candidate for whole genome sequencing, BAC libraries offer an attractive, alternative method to perform physical and functional analyses of a bacterium's genome. Our first plan is to use these BAC clones to determine whether or not cellulases and accessory genes in R. flavefaciens exist in clusters of orthologous genes (COGs). Proteomics is also being used to complement the BAC library/DNA sequencing approach. Proteins differentially expressed in response to carbon source are being identified by 2-D SDS-PAGE, followed by in-gel-digests and peptide mass mapping by MALDI-TOF Mass Spectrometry, as well as peptide sequencing by Edman degradation. At Ohio State, we have used a combination of functional proteomics, mutational analysis and differential display RT-PCR to obtain evidence suggesting that in addition to a cellulosome-like mechanism, R. albus 8 possesses other mechanisms for adhesion to plant surfaces. Genome walking on either side of these differentially expressed transcripts has also resulted in two interesting observations: i) a relatively large number of genes with no matches in the current databases and; ii) the identification of genes with a high level of sequence identity to those identified, until now, in the archaebacteria. Genomics and proteomics will also accelerate our understanding of microbial interactions, and allow a greater degree of in situ analyses in the future. The challenge is to utilize genomics and proteomics to improve our fundamental understanding of microbial physiology, diversity and ecology, and overcome constraints to ruminal function.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.2
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• pp.200-207
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• 2007

In vitro rumen incubation studies were conducted to determine effects of initial pH on bacterial attachment and fiber digestion. Ruminal fluid pH was adjusted to 5.7, 6.2 and 6.7, and three major fibrolytic bacteria attached to rice straw in the mixed culture were quantified with real-time PCR. The numbers of attached and unattached Fibrobacter succinogenes, Ruminococcus flavefaciens and Ruminocococcus albus were lower (p<0.05) at initial pH of 5.7 without significant difference between those at higher initial pH. Lowering incubation media pH to 5.7 also increased bacterial numbers detached from substrate regardless of bacterial species. Dry matter digestibility, gas accumulation and total VFA production were pH-dependent. Unlike bacterial attachment, maintaining an initial pH of 6.7 increased digestion over initial pH of 6.2. After 48 h in vitro rumen fermentation, average increases in DM digestion, gas accumulation, and total VFA production at initial pH of 6.2 and 6.7 were 2.8 and 4.4, 2.0 and 3.0, and 1.2 and 1.6 times those at initial pH of 5.7, respectively. The lag time to reach above 2% DM digestibility at low initial pH was taken more times (8 h) than at high and middle initial pH (4 h). Current data clearly indicate that ruminal pH is one of the important determinants of fiber digestion, which is modulated via the effect on bacterial attachment to fiber substrates.

• Asian-Australasian Journal of Animal Sciences
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• v.28 no.10
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• pp.1433-1441
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• 2015

This study examined changes of rumen fermentation, ruminal bacteria biodiversity and abundance caused by nitrate addition with Ion Torrent sequencing and real-time polymerase chain reaction. Three rumen-fistulated steers were fed diets supplemented with 0%, 1%, and 2% nitrate (dry matter %) in succession. Nitrate supplementation linearly increased total volatile fatty acids and acetate concentration obviously (p = 0.02; p = 0.02; p<0.01), butyrate and isovalerate concentration numerically (p = 0.07). The alpha (p>0.05) and beta biodiversityof ruminal bacteria were not affected by nitrate. Nitrate increased typical efficient cellulolytic bacteria species (Ruminococcus flavefaciens, Ruminococcus ablus, and Fibrobacter succinogenes) (p<0.01; p = 0.06; p = 0.02). Ruminobactr, Sphaerochaeta, CF231, and BF311 genus were increased by 1% nitrate. Campylobacter fetus, Selenomonas ruminantium, and Mannheimia succiniciproducens were core nitrate reducing bacteria in steers and their abundance increased linearly along with nitrate addition level (p<0.01; p = 0.02; p = 0.04). Potential nitrate reducers in the rumen, Campylobacter genus and Cyanobacteria phyla were significantly increased by nitrate (p<0.01; p = 0.01).To the best of our knowledge, this was the first detailed view of changes in ruminal microbiota by nitrate. This finding would provide useful information on nitrate utilization and nitrate reducer exploration in the rumen.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.1
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• pp.100-110
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• 2017

Objective: The gastrointestinal tract of sheep contain complex microbial communities that influence numerous aspects of the sheep's health and development. The objective of this study was to analyze the composition and diversity of the microbiota in the gastrointestinal tract sections (rumen, reticulum, omasum, abomasum, duodenum, jejunum, ileum, cecum, colon, and rectum) of sheep. Methods: This analysis was performed by 454 pyrosequencing using the V3-V6 region of the 16S rRNA genes. Samples were collected from five healthy, small tailed Han sheep aged 10 months, obtained at market. The bacterial composition of sheep gastrointestinal microbiota was investigated at the phylum, class, order, family, genus, and species levels. Results: The dominant bacterial phyla in the entire gastrointestinal sections were Firmicutes, Bacteroidetes, and Proteobacteria. In the stomach, the three most dominant genera in the sheep were Prevotella, unclassified Lachnospiraceae, and Butyrivibrio. In the small intestine, the three most dominant genera in the sheep were Escherichia, unclassified Lachnospiraceae, and Ruminococcus. In the large intestine, the three most dominant genera in the sheep were Ruminococcus, unclassified Ruminococcaceae, and Prevotella. R. flavefaciens, B. fibrisolvens, and S. ruminantium were three most dominant species in the sheep gastrointestinal tract. Principal Coordinates Analysis showed that the microbial communities from each gastrointestinal section could be separated into three groups according to similarity of community composition: stomach (rumen, reticulum, omasum, and abomasum), small intestine (duodenum, jejunum, and ileum), and large intestine (cecum, colon, and rectum). Conclusion: This is the first study to characterize the entire gastrointestinal microbiota in sheep by use of 16S rRNA gene amplicon pyrosequencing, expanding our knowledge of the gastrointestinal bacterial community of sheep.

• Asian-Australasian Journal of Animal Sciences
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• v.7 no.3
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• pp.389-396
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• 1994

Effects of chemical treatments on in sacco and in vitro digestibility of barley straw by rumen fluid and pure cultures of cellulolytic bacteria were studied to evaluate the pretreatment and to improve the poor quality feed. Chemicals were applied by dissolving them in water equivalent to 40% of the weight of the straw (dry matter basis). Pretreatment with 5% NaOH yielded the largest increase in sacco digestion followed by pretreatment with 2% $(NH_4)_2SO_3$, 2.6% $NH_4OH$, 1.6% $NaHSO_3$ and untreated straw (control). In sacco dry matter digestibility of straw treated with NaOH and $(NH_4)_2SO_3$ continued to increase as the concentration of chemical increased (1 to 7.5%), as it was the in vitro dry matter loss by leaching. Treatment of barley straw with 5% NaOH enhanced significantly (p < 0.01) in vitro digestibility by rumen fluid, Fibrobacter suceinogenes and Ruminococcus albus though the fermentation products by cellulolytic bacteria were low, whereas the treatment with 5% $(NH_4)_2SO_3$ inhibited in vitro digestibility by F. succinogenes and R. albus together with lower fermentation products. Dry matter loss by leaching and bacterial digestion from barley straw treated with NaOH and $(NH_4)_2SO_3$ suggested the effect of pretreatment with these chemicals were based on leaching, and the cellulolytic bacteria had little to do with digestion.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.6
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• pp.818-823
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• 2008

The interaction of fibre degrading microbes and methanogens was studied using two forages, lucerne (Medicago sativa) hay and maize (Zea mays) hay, as substrate and 2-bromoethanesulphonic acid (BES) as an additive in an in vitro gas production test. Gas and methane production (ml/g dry matter) were significantly higher (p<0.05) on lucerne as compared to maize hay. Inclusion of BES in the incubation medium significantly suppressed methane emission irrespective of substrate. The population density of total bacteria, fungi, Ruminococcus flavefaciens and Fibrobacter succinogenes was higher, whereas that of methanogens was lower with maize hay as compared to lucerne as substrate. BES suppressed methanogen population by 7 fold on lucerene and by 8.5 fold on maize at 24 h incubation as estimated by real time-PCR. This suppression was accompanied by almost complete (>98% of control) inhibition of methanogenesis. The proportion of acetate decreased, whereas that of propionate increased significantly by inclusion of BES, resulting in narrowing of acetate to propionate ratio. In vitro true digestibility (IVTD) of lucerne was significantly higher as compared to maize but BES inclusion did not affect IVTD.