• 한국초지조사료학회지
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• 제34권1호
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• pp.60-67
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• 2014

본 연구는 조사료의 반추위 발효가 진행됨에 따른 볏짚표면에 부착된 섬유소 분해 박테리아의 군집변화와 섬유소 소화율을 비교 관측하기 위하여 볏짚의 in situ 반추 발효를 실시하였다. 그리고 부착 박테리아의 군집 변화를 측정하기 위하여 RT-PCR 기법을 이용하여 F. succinogenes. R. albus와 R. flavefaciens의 군집을 모니터링 하였다. 본 연구를 수행하기 위하여 in situ 볏짚 발효를 0. 2, 4, 8, 12, 24시간 실시하였을 때 반추위내 볏짚의 in situ 분해는 발효 시간이 진행됨에 따라 가속화되어 발효 8~12시간 사이에 최고 분해 속도를 나타내었으나, F. succinogenes, R. flavefaciens과 R. albus는 모두 발효 0~1시간 사이에 볏짚 표면에 부착이 80% 이상 완료되어 이후 발효가 계속 진행되는 동안 일정 수준의 군락을 유지하는 것이 발견되었다. 그리고 반추위내 유입된 조사료의 표면에 초기 부착과정을 관찰하기 위하여 0, 5, 10, 30 및 60분 간격으로 볏짚의 in situ 샘플을 채취하여 조사하였을 때 F. succinogenes, R. flavefaciens 및 R. albus의 군락 모두 볏짚이 반추위 유입 후 5분 내에 상당량의 수가 부착함을 발견하였다. 또한 조사료의 반추위 발효 용이성에 따른 섬유소 분해 박테리아의 부착 정도를 관찰하기 위하여 0, 2, 4 및 8% NaOH를 처리한 볏짚을 12 및 24시간 in situ 배양 볏짚의 소화율과 부착 박테리아의 군집 변화를 관측하였을 때, 볏짚의 NaOH 처리 농도가 높아짐에 따라 in situ 소화율이 증가하였으며, 동시에 부착된 박테리아 군집의 증가 경향이 F. succinogenes, R. flavefaciens 및 R. albus의 3균주 모두 배양 12시간에 나타났으나 배양 24시간에서는 각기 다른 양상을 나타냈다. 따라서 본 연구결과는 반추위내 섬유소 발효과정에서 섬유소 분해 박테리아의 부착은 조사료의 반추위 유입 초기에 반드시 이루어지고, 발효 시간이 진행됨에 따라 조사료 표면에 안정된 군락을 형성하며, 섬유소 분해가 가속화된다는 사실을 보여 주었다.

• Asian-Australasian Journal of Animal Sciences
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• 제26권9호
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• pp.1276-1281
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• 2013

An in vitro experiment was conducted to evaluate the effect of methylcellulose on the attachment of major cellulolytic bacteria on rice straw and its digestibility. Rice straw was incubated with ruminal mixture with or without 0.1% methylcellulose (MC). The attachment of F. succinogenes, R. flavefaciens and R. albus populations on rice straw was measured using real-time PCR with specific primer sets. Methylcellulose at the level of 0.1% decreased the attachment of all three major cellulolytic bacteria. In particular, MC treatment reduced (p<0.05) attachment of F. succinogenes on rice straw after 10 min of incubation while a significant reduction (p<0.05) in attachment was not observed until 4 h incubation in the case of R. flavefaciens and R. albus. This result indicated F. succinogenes responded to MC more sensitively and earlier than R. flavefaciens and R. albus. Dry matter digestibility of rice straw was subsequently inhibited by 0.1% MC, and there was a significant difference between control and MC treatment (p<0.05). Incubated cultures containing MC had higher pH and lower gas production than controls. Current data clearly indicated that the attachment of F. succinogenes, R. flavefaciens and R. albus on rice straw was inhibited by MC, which apparently reduced rice straw digestion.

• 한국초지조사료학회지
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• 제37권1호
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• pp.35-43
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• 2017

본 연구는 반추동물이 볏짚을 소화하는 과정 중에 주요 섬유소 분해 박테리아의 분포 변화를 연구하여 반추위내 볏짚이용효율을 증진시키기 위한 진보적 연구 자료를 제공하고자 실시하였다. 본 연구를 수행하기 위하여 분쇄 볏짚을 기질로 사용한 In vitro 반추위 발효를 실시하였으며, 배양 0. 4, 8, 12, 48시간에 볏짚 건물 소화율 및 섬유소 분해 박테리아 균수 변화를 조사하였다. 섬유소 분해 박테리아 균수는 F. succinogenes. R. albus와 R. flavefaciens를 대상으로 Quantitative real-time PCR을 이용하여 16S rDNA의 copy No의 log값(Log copy No)을 측정하였다. 섬유소 분해 박테리아의 총 균수는 F. succinogenes, R. albus와 R. flavefaciens의 볏짚 표면 부착 및 배양액내 부유 박테리아의 총합으로 배양 시간에 따라 증식을 하여 24시간에서 최고 군집(29.0 Log copy No)을 형성하고, 이후 감소하였다. 이에 볏짚 소화율도 24시간까지 점점 큰 폭으로 증가하였고, 이후에는 점점 둔화되는 경향을 나타냈다. F. succinogenes, R. flavefaciens 및 R. albus는 배양 시작 즉시 볏짚 표면에 부착이 발생됨을 발견하였으며, 이때 이들의 부착 군집의 비율은 각각 34.5%, 84.4% 및 67.9%로 차지하였다. 그리고 이들 섬유소 분해 박테리아의 볏짚 부착 비율은 모든 균수가 공히 배양 4시간째부터 94.7% 이상 최고 수준에 이르렀고, 부착 균수는 균종에 따라 각각 배양 12시간 또는 24시간째에 최고 수준을 나타냈다. F. succinogenes, R. flavefaciens 및 R. albus의 최고 수준의 볏짚 표면 부착 속도는 배양 12시간에 시작되어 배양 24시간에 각각 10.33, 9.28 및 8.30 Log copy No/h/g DM에 도달하였다. 그리고 섬유소 분해 속도는 표면 부착 속도보다 좀 늦은 배양 24시간에 0.95% DM/h로 최고 정점에 도달하였다. 따라서 본 연구결과는 반추위 발효 진행 과정에서 섬유소 분해 박테리아 총균의 증식에 따라 조사료의 소화가 증가되며, 상당히 많은 섬유소 분해 박테리아들이 조사료 유입과 동시에 표면 부착이 발생하고, 이들 부착 섬유소 분해 박테리아의 활성화가 먼저 진행된 후에 섬유소 분해 활성화가 이루어져야 조사료 소화의 극대화를 가져올 수 있다는 사실을 제시하여 주었다.

• Asian-Australasian Journal of Animal Sciences
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• 제22권1호
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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.

• Journal of Animal Science and Technology
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• 제47권4호
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• pp.615-624
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• 2005

본 연구는 배양초기 pH 조건이 F. succino- genes의 섬유소 부착과 섬유소 소화에 미치는 영향을 보고자 실시하였다. 선정된 specific primer를 이용하여 F. succinogenes의 genomic DNA로부터 445bp의 16S rDNA 절편을 증폭하여 205bp의 internal control을 제작하였고, cPCR 결과로부터 박테리아 수를 계산할 수 있는 표준곡선의 회귀식($r^2$>0.99)을 얻을 수 있었다. In vitro 배양초기 pH에 따른 F. succ- inogenes의 cellulose 부착을 cPCR로 모니터링한 결과, 발효과정 전 기간동안 초기 pH가 6.8과 6.2일 때 cellulose 건물 g당 부착 균주의 수가 pH 5.6일 때 보다 높았으나, pH 6.8과 6.2 사이에서는 큰 차이는 없었다(p>0.05). Cellulose 분해는 배양시간이 진행됨에 따라 증가 되었으며, 분해 정도는 pH가 증가함에 따라 더 높았다. 배양초기 pH가 6.8, 6.2 그리고 5.8일 때 48시간동안 감소한 pH는 각각 0.24, 0.58 그리고 0.16 이었다. 가스 생산량은 발효 시간이 경과함에 따라 pH가 높을수록 더 많았다. 결론적으로 발효 초기 pH는 F. succinogenes에 의한 cellulose 소화, 가스 생산, pH 변화 및 cellulose 부착에 큰 영향을 주었으며, 특히, 낮은 pH(5.8)에서는 섬유소 소화 및 박테리아 부착을 현저한 감소 시켰다.

• Asian-Australasian Journal of Animal Sciences
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• 제20권5호
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• pp.802-810
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• 2007

Fibrobacter succinogenes, a Gram-negative, anaerobic ruminal bacterium is a major fibre digesting species in the rumen. It intensively degrades plant cell walls by an erosion type of mechanism, burrowing its way through the complex matrix of cellulose and hemicellulose with the release of digestible and undigested cell wall fragments. The enzymes involved in this process include a combination of glucanases, xylanases, arabinofuranosidase(s) and esterases. The genome of the bacterium has been sequenced and this has revealed in excess of 100 putative glycosyl hydrolase, pectate lyase and carbohydrate esterase genes, which is greater than the numbers reported present in other major cellulolytic organisms for which genomes have been sequenced. Modelling of the amino acid sequences of two glycanases, CedA and EGB, by reference to crystallized homologs has enabled prediction of the major features of their tertiary structures. Two dimensional gel electrophoresis in conjunction with mass spectroscopy has permitted the documentation of proteins over expressed in F. succinogenes grown on cellulose, and analysis of the cell surfaces of mutant strains unable to bind to cellulose has enabled the identification of candidate proteins with roles in adhesion to the plant cell wall substrate, the precursor to cellulose biodegradation.

• Asian-Australasian Journal of Animal Sciences
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• 제27권9호
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• pp.1285-1292
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• 2014

An in vitro experiment was conducted to evaluate the effects of Aspergillus oryzae culture (AOC) and 2-hydroxy-4-(methylthio)-butanoic acid (HMB) on rumen fermentation and microbial populations between different roughage sources. Two roughage sources (Chinese wild rye [CWR] vs corn silage [CS]) were assigned in a $2{\times}3$ factorial arrangement with HMB (0 or 15 mg) and AOC (0, 3, or 6 mg). Gas production (GP), microbial protein (MCP) and total volatile fatty acid (VFA) were increased in response to addition of HMB and AOC (p<0.01) for the two roughages. The HMB and AOC showed inconsistent effects on ammonia-N with different substrates. For CWR, neither HMB nor AOC had significant effect on molar proportion of individual VFA. For CS, acetate was increased (p = 0.02) and butyrate was decreased (p<0.01) by adding HMB and AOC. Increase of propionate was only occurred with AOC (p<0.01). Populations of protozoa ($p{\leq}0.03$) and fungi ($p{\leq}0.02$) of CWR were differently influenced by HMB and AOC. Percentages of F. succinogenes, R. albus, and R. flavefaciens (p<0.01) increased when AOC was added to CWR. For CS, HMB decreased the protozoa population (p = 0.01) and increased the populations of F. succinogenes and R. albus ($p{\leq}0.03$). Populations of fungi, F. succinogenes (p = 0.02) and R. flavefacien (p = 0.03) were increased by adding AOC. The HMB${\times}$AOC interactions were noted in MCP, fungi and R. flavefacien for CWR and GP, ammonia-N, MCP, total VFA, propionate, acetate/propionate (A/P) and R. albus for CS. It is inferred that addition of HMB and AOC could influence rumen fermentation of forages by increasing the number of rumen microbes.

• Asian-Australasian Journal of Animal Sciences
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• 제26권10호
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• pp.1459-1465
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• 2013

Two in vitro experiments were conducted to evaluate the effect of methylcellulose (MC) on i) bacterial detachment from rice straw as well as ii) inhibition of bacterial attachment and fiber digestibility. To evaluate the effect of MC on fibrolytic bacterial detachment (Exp 1), in vitro bacterial cultures with 0.1% (w/v) MC solution were compared with cultures without MC after 8 h incubation. The effect of MC on inhibition of bacterial attachment was determined by comparing with real-time PCR the populations of F. succinogenes, R. flavefaciens and R. albus established on rice straw pre-treated with 0.1% MC with those on untreated straw after incubation for 0, 6 and 12 h (Exp 2). The major fibrolytic bacterial attachment on rice straw showed significantly lower populations with either the addition of MC to the culture or pre-treated rice straw compared to controls (p<0.05). Also, the digestibility of rice straw with MC was significantly lower compared with control (p<0.05). The F. succinogenes population did not show detachment from rice straw, but showed an inhibition of attachment and proliferation on rice straw in accordance with a decrease of fiber digestion. The detachments of Ruminococcus species co-existed preventing the proliferations with subsequent reduction of fiber degradation by MC during the incubation. Their detachments were induced from stable colonization as well as the initial adhesion on rice straw by MC in in vitro ruminal fermentation. Furthermore, the detachment of R. albus was more sensitive to MC than was R. flavefaciens. These results showed the certain evidence that attachment of major fibrolytic bacteria had an effect on fiber digestion in the rumen, and each of fibrolytic bacteria, F. succinogenes, R. flavefaciens and R. albus had a specific mechanism of attachment and detachment to fiber.

• Asian-Australasian Journal of Animal Sciences
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• 제27권8호
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• pp.1088-1097
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• 2014

The study aimed to investigate the effects of gynosaponin on in vitro methanogenesis under different forage-concentrate ratios (F:C ratios). Experiment was conducted with two kinds of F:C ratios (F:C = 7:3 and F:C = 3:7) and gynosaponin addition (0 mg and 16 mg) in a $2{\times}2$ double factorial design. In the presence of gynosaponin, methane production and acetate concentration were significantly decreased, whereas concentration of propionate tended to be increased resulting in a significant reduction (p<0.05) of acetate:propionate ratio (A:P ratio), in high-forage substrate. Gynosaponin treatment increased (p<0.05) the butyrate concentration in both F:C ratios. Denaturing gradient gel electrophoresis (DGGE) analysis showed there was no apparent shift in the composition of total bacteria, protozoa and methanogens after treated by gynosaponin under both F:C ratios. The real-time polymerase chain reaction (PCR) analysis indicated that variable F:C ratios significantly affected the abundances of Fibrobacter succinogenes, Rumninococcus flavefaciens, total fungi and counts of protozoa (p<0.05), but did not affect the mcrA gene copies of methanogens and abundance of total bacteria. Counts of protozoa and abundance of F.succinogenes were decreased significantly (p<0.05), whereas mcrA gene copies of methanogens were decreased slightly (p<0.10) in high-forage substrate after treated by gynosaponin. However, gynosaponin treatment under high-concentrate level did not affect the methanogenesis, fermentation characteristics and tested microbes. Accordingly, overall results suggested that gynosaponin supplementation reduced the in vitro methanogenesis and improved rumen fermentation under highforage condition by changing the abundances of related rumen microbes.

• Asian-Australasian Journal of Animal Sciences
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• 제31권1호
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• pp.54-62
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• 2018

Objective: Due to the threat of global warming, the livestock industry is increasingly interested in exploring how feed additives may reduce anthropogenic greenhouse gas emissions, especially from ruminants. This study investigated the effect of Rhodophyta supplemented bovine diets on in vitro rumen fermentation and rumen microbial diversity. Methods: Cannulated Holstein cows were used as rumen fluid donors. Rumen fluid:buffer (1:2; 15 mL) solution was incubated for up to 72 h in six treatments: a control (timothy hay only), along with substrates containing 5% extracts from five Rhodophyta species (Grateloupia lanceolata [Okamura] Kawaguchi, Hypnea japonica Tanaka, Pterocladia capillacea [Gmelin] Bornet, Chondria crassicaulis Harvey, or Gelidium amansii [Lam.] Lamouroux). Results: Compared with control, Rhodophyta extracts increased cumulative gas production after 24 and 72 h (p = 0.0297 and p = 0.0047). The extracts reduced methane emission at 12 and 24 h (p<0.05). In particular, real-time polymerase chain reaction analysis indicated that at 24 h, ciliate-associated methanogens, Ruminococcus albus and Ruminococcus flavefaciens decreased at 24 h (p = 0.0002, p<0.0001, and p<0.0001), while Fibrobacter succinogenes (F. succinogenes) increased (p = 0.0004). Additionally, Rhodophyta extracts improved acetate concentration at 12 and 24 h (p = 0.0766 and p = 0.0132), as well as acetate/propionate (A/P) ratio at 6 and 12 h (p = 0.0106 and p = 0.0278). Conclusion: Rhodophyta extracts are a viable additive that can improve ruminant growth performance (higher total gas production, lower A/P ratio) and methane abatement (less ciliateassociated methanogens, Ruminococcus albus and Ruminococcus flavefaciens and more F. succinogenes.