• 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.

• Journal of Life Science
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• v.28 no.12
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• pp.1477-1488
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• 2018

This study investigated the muscles, intestines, and gonads of Sulculus diversicolor supertexta to examine the diversity of microbial communities within examples collected from the Jeju Coast. Using different media, initial pure isolation in MA, 1% BHIA, and 1% TSA indicated that the muscles, intestines, and gonads supported more communities, respectively. In analysis of relative similarity with 16s rRNA sequencing, 190 pure colonies were isolated, and further analysis with NBLAST identified 71 species, 39 genera, 25 families, and five phyla. Homogeny with the reference strain was 91-100%. Microbial communities in S. supertexta consisted of gamma and alpha Proteobacteria (48%), Actinobacteria (32.5%), Firmicutes (16.9%), Deinococcus-Thermus (1.3%), and Bacteroides (1.3%). In all tissue, Psychrobacter cibarius in Moraxellaceae was dominant. Alteromonadaceae, Enterobacteriaceae, Pasturellaceae, Moraxellaceae, Rhodobacteraceae, Geminicoccaceae, Dietziaceae, Intrasporangiaceae, Microbacteriaceae, Micrococcaceae, Micromonosporaceae, Streptomycetaceae, Aerococcaceae, Bacillaceae, Paenibacillaceae, Planococcaceae, and Staphylcoccaceae were commonly isolated across all tissues, and Flavobacteriaceae, Corynebacteriaceae, Yesiniaceae, Vibrionaceae, Hahellaceae, Pseudomonadaceae were also identified from the intestines. In microbial monitoring of four harmful bacteria, Streptomyces albus (96%) showed antibacterial activity against all four strains, and Agrococcus baldri (99%) and Psychrobacter nivimaris (99%) presented against E. Coli and E. aerogens. In addition, some strains with low homogeny were isolated and further experiments are therefore required, for example to refine the antimicrobial substances including new strain investigations. These additional experiments would aim to establish generic resources for the microbial communities in S. Supertexta and provide basic data for applied microbiological research.

• Asian-Australasian Journal of Animal Sciences
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• v.12 no.3
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• pp.358-365
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• 1999

The effects of dry roasting whole lupin seeds (lupinus albus, WLS) at 110, 130 or $150{^{\circ}C}$ for 15, 30 or 45 minutes on the in sacco rumen degradation characteristics, optimal heating conditions of time and temperature and in vitro enzyme digestibility were determined. Ruminant degradation characteristics (RDC) of crude protein (CP) of WLS were determined by in sacco technique in dairy cows. Measure ROC were soluble (S), undegradable (U), potentially degradable (D) fractions, lag time (TO) and rate of degradation (Kd) of insoluble but degradable fraction. Based on measured ROC, percentage bypass CP (%BCP) and bypass CP (BCP in g/kg, DM) were calculated. Degradability of CP was significantly reduced by dry roasting (p<0.001). The interaction of dry roasting temperature and time had significant effects on D (p<0.05), Kd (p<0.01), U (p<0.01), %BCP (p<0.001) and BCP (p<0.001) but not on S (p=0.923>0.05). With increasing time and temperature, S, D, Kd and U varied from 31.8%, 67.4%, 10.3%/h and 0.8% in the raw WLS (RWLS) to 27.1 %, 35.8%, 3.6%/h, 38.4% in $150{^{\circ}C}/45\;min$, respectively. All these effects resulted in increasing %BCP from 25.9 in RWLS to 61.0% in the $150{^{\circ}C}/45\;min$. Therefore BCP increased form 111.2 to 261.2 g/kg DM, respectively. Both %BCP and BCP at $150{^{\circ}C}/45\;min$ increased nearly 2.5 times over the RWLS. The effects of dry roasting on %BCP and BCP seemed to be linear up to the highest value tested. Although ROC had been altered by dry roasting, the In vitro perpsin-cellulase digestibility was generally unchanged. It was concluded that dry roasting was effective in shifting CP degradation from rumen to the lower gastrointestinal tract to potential reduce unnecessary N loss in the rumen. It might be of great value in successfully synchronizing the rhythms of release of nitrogen and energy in the rumen, thus achieving a more efficient fermentation of diets with high proportions of lignocellulosic resources. To determine the optimal dry roasting conditions, the digestibility of each treatment in the cows will be measured in the next trial using mobile bags technique.

• Journal of Microbiology and Biotechnology
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• v.5 no.2
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• pp.74-79
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• 1995

Sequence analysis of a DNA region encompassing the site of hybridization to actl, the gene for type II minimal polyketide synthase (PKS) for actinorhodin biosynthesis, from Streptomyces ablus revealed three more complete open reading frames additional to the already found two genes, plausibly encoding ${\beta}-ketoacyl$ synthase/acyl transferase (KS/AT) and chain length determining factor (ClF). The open reading frames (ORFs) were named salA, salD, and salE, from the upstream. In the homology analysis of the deduced amino acid sequences, SalA resembles the Streptomyces glaucescens Tcml, decaketide cyclase, SalD resembles acyl carrier protein in type II PKS, and SalE resembles the Actlll ketoreductase, The whole 4.4 kb of DNA sequence obeys the same conservation pattern as other type II PKSs. Therefore, we suggest that the 4.4 kb DNA from Streptomyces albus encompasses genes encoding enzymes for polyketide biogenesis in the organism and its organization is type II. The exsitence of SaIA, an analogue of the aromatic cyclase, revealed a relatedness of the 4.4 kb DNA with the aromatic PKS.

• Asian-Australasian Journal of Animal Sciences
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• v.24 no.8
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• pp.1086-1091
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• 2011

This experiment was designed to investigate the effect of lerak extract on the dynamic of rumen microbes in the in vitro fermentation of diet with different ratios of forage and concentrate. In vitro fermentation was conducted according to the method of Tilley and Terry (1963). The design of experiment was a factorial block design with 2 factors. The first factor was the ratio of forage and concentrate (90:10, 80:20, and 70:30 w/w) and the second factor was the level of lerak extract (0, 0.6, and 0.8 mg/ml). Total volatile fatty acid (VFA) concentration, proportional VFA and NH3 concentration were measured at 4 h incubation. Protozoal numbers in the buffered rumen fluid after 4 and 24 h of incubation were counted under a microscope. Bacterial DNAs of buffered rumen fluid were isolated from incubated samples after 24 h of incubation using a QiaAmp kit. Total bacteria, Fibrobacter succinogenes, Ruminococcus albus, and Prevotella ruminicola were quantified using real time polymerase chain reaction (PCR). Lerak extract markedly reduced protozoal numbers in buffered rumen fluid of all diets after 24 h of incubation. Total bacteria did not change with lerak extract addition. While no difference in F. succinogenes was found, there was a slight increase in R. albus number and a significant enhancement in P. ruminicola number by increasing the level of lerak extract in all diets. Propionate concentration significantly increased in the presence of lerak extract at level 0.8 mg/ml. It was concluded that the addition of lerak extract could modify rumen fermentation and had positive effects on rumen microbes.

• Asian-Australasian Journal of Animal Sciences
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• v.25 no.3
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• pp.325-334
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• 2012

The objective of this study was to determine the roughage to concentrate (R:C) ratio with rain tree pod meal (RPM) supplementation on in vitro fermentation using gas production technique. The experiment design was a 6${\times}$4 factorial arrangement in a CRD. Factor A was 6 levels of R:C ratio (100:0, 80:20, 60:40, 40:60, 20:80 and 0:100) and factor B was 4 levels of RPM (0, 4, 8 and 12 mg). It was found that gas kinetic, extent rate (c) was linearly increased (p<0.01) with an increasing level of concentrate while cumulative gas production (96 h) was higher in R:C of 40:60. In addition, interaction of R:C ratio and RPM level affected $NH_3-N$ and IVDMD and were highest in R:C of 0:100 with 0, 4 mg of RPM and 40:60 with 8 mg of RPM, respectively. Moreover, interaction of R:C ratio and RPM level significantly increased total volatile fatty acids and propionate concentration whereas lower acetate, acetate to propionate ratios and $CH_4$ production in R:C of 20:80 with 8 mg of RPM. Moreover, the two factors, R:C ratio and RPM level influenced the protozoal population and the percentage of methanogens in the total bacteria population. In addition, the use of real-time PCR found that a high level of concentrate in the diet remarkably decreased three cellulolytic bacteria numbers (F. succinogenes, R. flavefaciens and R. albus). Based on this study, it is suggested that the ratio of R:C at 40:60 and RPM level at 12 mg could improve ruminal fluid fermentation in terms of reducing fermentation losses, thus improving VFA profiles and ruminal ecology.

• Asian-Australasian Journal of Animal Sciences
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• v.25 no.6
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• pp.806-811
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• 2012

This study was conducted to evaluate effects of plant extracts on methanogenesis and rumen microbial diversity in in vitro. Plant extracts (Artemisia princeps var. Orientalis; Wormwood, Allium sativum for. Pekinense; Garlic, Allium cepa; Onion, Zingiber officinale; Ginger, Citrus unshiu; Mandarin orange, Lonicera japonica; Honeysuckle) were obtained from the Plant Extract Bank at Korea Research Institute of Bioscience and Biotechnology. The rumen fluid was collected before morning feeding from a fistulated Holstein cow fed timothy and commercial concentrate (TDN; 73.5%, crude protein; 19%, crude fat; 3%, crude fiber; 12%, crude ash; 10%, Ca; 0.8%, P; 1.2%) in the ratio of 3 to 2. The 30 ml of mixture, comprising McDougall buffer and rumen liquor in the ratio of 4 to 1, was dispensed anaerobically into serum bottles containing 0.3 g of timothy substrate and plant extracts (1% of total volume, respectively) filled with $O_2$-free $N_2$ gas and capped with a rubber stopper. The serum bottles were held in a shaking incubator at $39^{\circ}C$ for 24 h. Total gas production in all plant extracts was higher (p<0.05) than that of the control, and total gas production of ginger extract was highest (p<0.05). The methane emission was highest (p<0.05) at control, but lowest (p<0.05) at garlic extract which was reduced to about 20% of methane emission (40.2 vs 32.5 ml/g DM). Other plant extracts also resulted in a decrease in methane emissions (wormwood; 8%, onion; 16%, ginger; 16.7%, mandarin orange; 12%, honeysuckle; 12.2%). Total VFAs concentration and pH were not influenced by the addition of plant extracts. Acetate to propionate ratios from garlic and ginger extracts addition samples were lower (p<0.05, 3.36 and 3.38 vs 3.53) than that of the control. Real-time PCR indicted that the ciliate-associated methanogen population in all added plant extracts decreased more than that of the control, while the fibrolytic bacteria population increased. In particular, the F. succinogens community in added wormwood, garlic, mandarin orange and honeysuckle extracts increased more than that of the others. The addition of onion extract increased R. albus diversity, while other extracts did not influence the R. albus community. The R. flavefaciens population in added wormwood and garlic extracts decreased, while other extracts increased its abundance compared to the control. In conclusion, the results indicated that the plant extracts used in the experiment could be promising feed additives to decrease methane gas emission from ruminant animals while improving ruminal fermentation.

• Asian-Australasian Journal of Animal Sciences
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• v.13 no.10
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• pp.1377-1387
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• 2000

Two laboratory techniques: (1) an in vitro method with two procedures for measuring protein degradabilities and (2) an in vitro method with three procedures for measuring protein solubility, were investigated to determine which laboratory techniques could most accurately predict the quantity of rumen protein degradation kinetics of legume seeds after dry roasting under various conditions, in terms of (1) rumen protein disappearance ($D_j$, where j=0, 2, 4, 8, 12, 24 and 48 h incubation), (2) rumen protein effective degradability (EDCP), (3) the parameters describing rumen degradation characteristics (the soluble fraction: S, the potentially degradable fraction: D, undegradable fraction: U, lag time: T0 and the degradation rate: Kd) and (4) rumen bypass protein (BCP), which were determined by the method accepted internationally at present, in sacco nylon bag technique using the standardized Dutch method. Feeds evaluated were the raw and dry roasted whole faba (Vicia faba) beans (WFB) and whole lupin (Lupinus albus) seeds (WLS), each was dry roasted under various conditions (at 110, 130 or $150^{\circ}C$ for 15, 30 or 45 min). In vitro protein degradability ($D_1$_Auf and $D_{24}$_Auf) were determined using the modified Aufr re method by enzymatic hydrolysis for 1 h and 24 h using a protease extracted from Streptomyces griseus in a borate-phosphate buffer. In vitro protein solubility ($bf_1$_S, $bf_2$_S, $bf_3$_S) was measured in a borate-phosphate buffer with three different procedures. Results from laboratory techniques (in vitro) were correlated and linearly regressed with in sacco results. Of the three procedures of in vitro protein solubility evaluated, none of them could predict in sacco results with good precision. The highest Pearson correlation coefficient ($R^2$) was less than 0.50. Of two procedures of in vitro protein degradability studied, the $D_1$_Auf values were closely correlated with in sacco parameters: Kd, EDCP and %BCP with high R' values: 0.82, 0.85 and 0.85, respectively, and closely correlated with in sacco $D_j$ at 2, 4, 8 and 12 h rumen incubation with high $R^2$ values: 0.83, 0.91, 0.93 and 0.83, respectively. The $D_{24}$_Auf values could not predict in sacco results. The highest $R^2$ value was less then 0.40. These results indicated that in vitro protein solubility measured in borate-phosphate failed to identify differences in the rate and extent of protein degradation of legume seeds after dry roasting under various conditions and thus should not be used to predict rumen degradation, particularly for heat processed feedstuffs. But in vitro protein degradability using the modified Aufr re method by enzymatic hydrolysis for 1 h or possibly an intermediate time (>1 h and <24 h) is a promising laboratory procedure to detect effectiveness of dry roasting legume seeds on rumen protein degradation characteristics and could be used as a simple laboratory method to predict the rate and extent of protein degradation in the rumen in sacco with high accuracy. The equations to predict EDCP, Kd and BCP of dry roasted legume seeds (WLS and WFB) under various conditions are as follow: For both: EDCP (%)=-1.37+1.06*$D_1$_Auf ($R^2=0.85$, p<0.01). For both: Kd (%/h)=-21.81+0.49*$D_1$_Auf ($R^2=0.82$, p<0.01). For both: %BCP=103.37-1.07*$D_1$_Auf ($R^2=0.85$, p<0.01).

• Asian-Australasian Journal of Animal Sciences
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• v.26 no.11
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• pp.1583-1591
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• 2013

The objective of this study was to investigate microbial population in the rumen of dairy steers as influenced by supplementing with dietary condensed tannins and saponins and different roughage to concentrate ratios. Four, rumen fistulated dairy steers (Bos indicus) were used in a $2{\times}2$ factorial arrangement in a $4{\times}4$ Latin square design. The main factors were two roughage to concentrate ratios (R:C, 60:40 and 40:60) and two supplementations of rain tree pod meal (RPM) (0 and 60 g/kg of total DM intake). Chopped 30 g/kg urea treated rice straw was used as a roughage source. All animals received feed according to respective R:C ratios at 25 g/kg body weight. The RPM contained crude tannins and saponins at 84 and 143 g/kg of DM, respectively. It was found that ruminal pH decreased while ruminal temperature increased by a higher concentrate ratio (R:C 40:60) (p<0.05). In contrast, total bacterial, Ruminococus albus and viable proteolytic bacteria were not affected by dietary supplementation. Numbers of fungi, cellulolytic bacteria, Fibrobactor succinogenes and Ruminococus flavefaciens were higher while amylolytic bacteria was lower when steers were fed at 400 g/kg of concentrate. The population of Fibrobactor succinogenes, was found to be higher with RPM supplementation. In addition, the use of real-time PCR technique indicated that the population of protozoa and methanogens were decreased (p<0.05) with supplementation of RPM and with an increasing concentrate ratio. Supplementation of RPM and feeding different concentrate ratios resulted in changing the rumen microbes especially, when the animals were fed at 600 g/kg of concentrate and supplemented with RPM which significantly reduced the protozoa and methanogens population.

• Journal of Animal Environmental Science
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• v.9 no.1
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• pp.27-34
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• 2003

This experiment was carried out to investigate the effect of dietary supplementation of fermented microbial complex(Eco-Farm$^{(R)}$) on performance of finishing pigs and indoor air quality in finishing building. A total 135 crossbred [(Landrace ${\times}$ Yorkshire) ${\times}$ Duroc] pigs were randomly arranged into nine groups and assigned to three treatments. Pigs were fed a basal diet supplemented with 0, 0.5 and 1% level of fermented microbial complex(Eco-Farm$^{(R)}$) until the market weight for 40 days of the experimental period. Average daily feed intake and feed conversion ratio were significantly improved (p<0.05) with dietary supplementation of 0.5% fermented microbial complex (Eco-Farm$^{(R)}$): however, average daily gain was not affected by dietary supplementation of fermented microbial complex(Eco-Farm$^{(R)}$). Indoor ammonia and hydrogen sulfide concentrations in the finishing building were significantly(p<0.05) decreased by dietary supplementation of fermented microbial complex(Eco-Farm$^{(R)}$) compared with those of control, however, indoor carbon dioxide concentration was not affected by dietary supplementation of fermented microbial complex(Eco-Farm$^{(R)}$). In conclusion, the results obtained from this experiment suggest that the dietary supplementation of fermented microbial complex(Eco-Farm$^{(R)}$) for finishing pigs improved performance and indoor air quality in the finishing building.hing building.