• Asian-Australasian Journal of Animal Sciences
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• v.25 no.1
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• pp.66-74
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• 2012

The effects of exogenous fibrolytic enzymes (EFE; a mixture of two preparations from Trichoderma spp., with predominant xylanase and ${\beta}$-glucanase activities, respectively) on colonization and digestion of ground barley straw and alfalfa hay by Fibrobacter succinogenes S85 and Ruminococcus flavefaciens FD1 were studied in vitro. The two levels (28 and 280 ${\mu}g$/ml) of EFE tested and both bacteria were effective at digesting NDF of hay and straw. With both substrates, more NDF hydrolysis (p<0.01) was achieved with EFE alone at 280 than at 28 ${\mu}g$/ml. A synergistic effect (p<0.01) of F. succinogenes S85 and EFE on straw digestion was observed at 28 but not 280 ${\mu}g$/ml of EFE. Strain R. flavefaciens FD1 digested more (p<0.01) hay and straw with higher EFE than with lower or no EFE, but the effect was additive rather than synergistic. Included in the incubation medium, EFE showed potential to improve fibre digestion by cellulolytic ruminal bacteria. In a second batch culture experiment using mixed rumen microbes, DM disappearance (DMD), gas production and incorporation of $^{15}N$ into particle-associated microbial N ($^{15}N$-PAMN) were higher (p<0.001) with ammoniated (5% w/w; AS) than with native (S) ground barley straw. Application of EFE to the straws increased (p<0.001) DMD and gas production at 4 and 12 h, but not at 48 h of the incubation. EFE applied onto S increased (p<0.01) $^{15}N$-PAMN at 4 h only, but EFE on AS increased (p<0.001) $^{15}N$-PAMN at all time points. Prehydrolysis increased (p<0.01) DMD from both S and AS at 4 and 12 h, but reduced (p<0.01) $^{15}N$-PAMN in the early stage (4 h) of the incubation, as compared to non-prehydrolyzed samples. Application of EFE to barley straw increased rumen bacterial colonization of the substrate, but excessive hydrolytic action of EFE prior to incubation decreased it.

• Journal of The Korean Society of Grassland and Forage Science
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• v.38 no.2
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• pp.106-111
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• 2018

This study was conducted to investigate the relationship between changes of rumen microflora and bloat in Jersey cow. Jersey cows (control age: 42 months, control weight: 558kg; treatment age: 29 months, treatment weight 507kg) were fed on the basis of dairy feeding management at dairy science division in National Institute of Animal Science. The change of microbial population in rumen was analyzed by using next generation sequencing (NGS) technologies due to metabolic disease. The diversity of Ruminococcus bromii, Bifidobacterium pseudolongum, Bifidobacterium merycicum and Butyrivibrio fibrisolvens known as major starch fermenting bacteria was increased more than 36-fold in bloated Jersey, while cellulolytic bacteria community such as Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens was increased more than 12-fold in non-bloated Jersey. The proportion of bacteroidetes and firmicutes was 33.4% and 39.6% in non-bloated Jersey's rumen, while bacteroidetes and firmicutes were 24.9% and 55.1% in bloated Jersey's. In conclusion, the change of rumen microbial community, in particular the increase in starch fermenting bacteria, might have an effect to occur the bloat in Jersey cow.

• Asian-Australasian Journal of Animal Sciences
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• v.29 no.8
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• pp.1136-1144
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• 2016

Leucaena silage was supplemented with different levels of molasses and urea to study its nutritive value and in vitro rumen fermentation efficiency. The ensiling study was randomly assigned according to a $3{\times}3$ factorial arrangement in which the first factor was molasses (M) supplement at 0%, 1%, and 2% of crop dry matter (DM) and the second was urea (U) supplement as 0%, 0.5%, and 1% of the crop DM, respectively. After 28 days of ensiling, the silage samples were collected and analyzed for chemical composition. All the nine Leucaena silages were kept for study of rumen fermentation efficiency using in vitro gas production techniques. The present result shows that supplementation of U or M did not affect DM, organic matter, neutral detergent fiber, and acid detergent fiber content in the silage. However, increasing level of U supplementation increased crude protein content while M level did not show any effect. Moreover, the combination of U and M supplement decreased the content of mimosine concentration especially with M2U1 (molasses 2% and urea 1%) silage. The result of the in vitro study shows that gas production kinetics, cumulation gas at 96 h and in vitro true digestibility increased with the increasing level of U and M supplementation especially in the combination treatments. Supplementation of M and U resulted in increasing propionic acid and total volatile fatty acid whereas, acetic acid, butyric acid concentrations and methane production were not changed. In addition, increasing U level supplementation increased $NH_3$-N concentration. Result from real-time polymerase chain reaction revealed a significant effect on total bacteria, whereas F. succinogenes and R. flavefaciens population while R. albus was not affected by the M and U supplementation. Based on this study, it could be concluded that M and urea U supplementation could improve the nutritive value of Leucaena silage and enhance in vitro rumen fermentation efficiency. This study also suggested that the combination use of M and U supplementation level was at 2% and 1%, respectively.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.9
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• pp.1397-1401
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• 2007

An in situ experiment was conducted to find out whether Tween 80 improves rice straw digestion through increased adhesion of major fibrolytic bacteria. Rice straw was sprayed with various levels of Tween 80 non-ionic surfactant or SDS ionic surfactant 24 h before incubation in the rumen of Holstein steers. Dry matter (DM) disappearance and adhesion of F. succinogenes, R. flavefaciens and R. albus on rice straw after in situ incubation were measured by real-time PCR. Application of Tween 80 increased DM disappearance, which was more noticeable at an application level of 1% compared to lower application levels. Application of SDS resulted in an opposite response in DM disappearance with highest reduction in DM disappearance at 1% level. In a subsequent in situ experiment, higher Tween 80 was applied to rice straw in an attempt to find the optimum application level. Tween 80 at 2.5% gave better DM disappearance than 1% with a similar result at 5%. Therefore, an adhesion study was carried out using rice straw treated with 2.5% Tween 80. Our results indicated that Tween 80 reduced adhesion of all three major rumen fibrolytic bacteria to rice straw. Present data clearly show that improved DM disappearance by Tween 80 is not due to increased bacterial adhesion onto substrates.

• Korean Journal of Soil Science and Fertilizer
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• v.45 no.4
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• pp.515-523
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• 2012

The study investigated the biochemical methane potential (BMP) assay of cellulose supplementing with mixed methanogens and cellulolytic bacteria to improve anaerobic digestion for methane production. For the BMP assay, 7 different microbial supplementation groups were consisted of the cultures of mixed methanogens (M), Fibrobacter succinogenes (FS), Ruminococcus flavefaciensn (RF), R. albus (RA), RA+FS and M+RA+FS including control. The cultures were added in the batch reactors with the increasing dose levels of 1% (0.5 mL), 3% (1.5 mL) and 5% (2.5 mL). Incubation for the BMP assay was carried out for 40 days at $38^{\circ}C$ and anaerobic digestate obtained from an anaerobic digester with pig slurry as inoculum was used. In results, 5% FS increased total biogas and methane production up to 10.4~22.7% and 17.4~27.5%, respectively, compared to other groups (p<0.05). Total solid (TS) digestion efficiency showed a similar trend to the total biogas and methane productions. Generally the TS digestion efficiency of the FS group was higher than that of other groups showing at the highest value of 64.2% in the 5% FS group. Volatile solid (VS) digestion efficiencies of 68.4 and 71.0% in the 5% FS and the 5% RF were higher than other groups. After incubation, pH values in all treatment groups were over 6.4 indicating that methanogensis was not inhibited during the incubation. In conclusion, the results indicated that the hydrolysis stage for methane production in anaerobic batch reactors was the late-limiting stage compared with the methanogenesis stage, and especially, as the supplementation levels of F. succinogenes supplementation increased, the methane production was increased in the BMP assay compared with other microbial culture addition.

• Korean Journal of Soil Science and Fertilizer
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• v.45 no.6
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• pp.1049-1057
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• 2012

The study investigated the biochemical methane potential (BMP) assay of pig slurry supplemented with mixed methanogens and cellulolytic bacteria to improve anaerobic digestion for methane production. For the BMP assay, 7 different microbial supplementation groups consisted of the cultures of mixed methanogens (M), Fibrobacter succinogenes (FS), Ruminococcus flavefaciensn (RF), R. albus (RA), RA+FS, M+RA+FS, and control. The cultures were added in the batch reactors with the increasing dose levels of 1% (0.5 mL), 3% (1.5 mL) and 5% (2.5 mL). Incubation for the BMP assay was carried out for 60 days at $38^{\circ}C$ using anaerobic digestate obtained from an anaerobic digester with pig slurry as inoculum. In results, 5% RF and RA+FS increased total biogas up to 8.1 and 8.4%, respectively, compared with that of control (p<0.05). All 5% microbial culture supplements significantly increased methane production up to 12.1~17.9% compared with that of control (p<0.05). Total solid (TS) and volatile solid (VS) digestion efficiencies showed no relationship to the increased supplementation levels of microbial cultures. After incubation, pH values in all treatment groups ranged between 7.527 and 7.657 indicating that methanogensis was not inhibited during the incubation. In conclusion, the results indicated that both hydrolysis and methanogenesis stages for methane production in anaerobic batch reactors were influenced by the supplemented microorganisms due to the chemical characteristics of pig slurry, but only the 5% supplementation level of all microbial culture supplements used in the experiment affected methane production.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.10
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• pp.1325-1332
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• 2010

This study was conducted to evaluate the effects of the particle size (PS) of alfalfa hay (AH) and soybean hull (SH) on milk production of dairy cows and the population of major cellulolytic bacteria in the rumen. Eight lactating Holstein cows, averaging $590{\pm}33\;kg$ BW and $47{\pm}13$ days in milk (DIM), were assigned in a $4{\times}4$ Latin square design to a $2{\times}2$ factorial arrangement of treatments: alfalfa hay particle size (fine vs. coarse) combined with soy hull (zero or substituted as 50% of AH). The cows were fed diets formulated according to NRC (2001). Physically effective factor (pef) and physically effective fiber (peNDF) contents of diets increased by increasing AH particle size and inclusion of SH in the diets (p<0.01). Dry matter intake was not significantly affected by treatments but intake of peNDF was increased marginally by increasing the PS of AH (p = 0.08) and by SH inclusion (p<0.01) in the diets. Milk production was increased by feeding diets containing SH (p = 0.04), but it was not affected by the dietary PS. Milk fat content was increased by increasing AH particle size (p = 0.03) and decreased by SH substitution for a portion of AH (p<0.01). The numbers of total bacteria and cellulolytic species were not affected by PS of AH or by SH. F. succinogenes was the most abundant species in the rumen followed by R. albus and R. flavefaciens (p<0.01). This study showed that SH cannot replace the physically effective fiber in AH having either coarse or fine particle size. In diets containing SH, increasing of diet PS using coarse AH can maintain milk fat content similar to diets without SH. Particle size and peNDF content of diets did not affect the number of total or fibrolytic bacteria in the rumen.

• Asian-Australasian Journal of Animal Sciences
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• v.28 no.4
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• pp.530-537
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• 2015

The objective of this study was to evaluate the in vitro effects of flavonoid-rich plant extracts (PE) on ruminal fermentation characteristics and methane emission by studying their effectiveness for methanogenesis in the rumen. A fistulated Holstein cow was used as a donor of rumen fluid. The PE (Punica granatum, Betula schmidtii, Ginkgo biloba, Camellia japonica, and Cudrania tricuspidata) known to have high concentrations of flavonoid were added to an in vitro fermentation incubated with rumen fluid. Total gas production and microbial growth with all PE was higher than that of the control at 24 h incubation, while the methane emission was significantly lower (p<0.05) than that of the control. The decrease in methane accumulation relative to the control was 47.6%, 39.6%, 46.7%, 47.9%, and 48.8% for Punica, Betula, Ginkgo, Camellia, and Cudrania treatments, respectively. Ciliate populations were reduced by more than 60% in flavonoid-rich PE treatments. The Fibrobacter succinogenes diversity in all added flavonoid-rich PE was shown to increase, while the Ruminoccocus albus and R. flavefaciens populations in all PE decreased as compared with the control. In particular, the F. succinogenes community with the addition of Birch extract increased to a greater extent than that of others. In conclusion, the results of this study showed that flavonoid-rich PE decreased ruminal methane emission without adversely affecting ruminal fermentation characteristics in vitro in 24 h incubation time, suggesting that the flavonoid-rich PE have potential possibility as bio-active regulator for ruminants.

• 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.25 no.10
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• pp.1364-1373
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• 2012

Four crossbred (75% Holstein Friesian) lactating dairy cows, with an average live weight of $418{\pm}5$ kg and $36{\pm}10$ d in milk were randomly assigned according to a $2{\times}2$ factorial arrangement in a $4{\times}4$ Latin square design to evaluate the effects of cassava hay (CH) and rice bran oil (RBO) on feed intake, nutrient digestibility, ruminal fermentation, milk yield, and milk composition. Factor A was non-supplementation or supplementation with CH in the concentrate. Factor B was supplementation with RBO at 0% or 4% in the concentrate mixture. The four dietary treatments were (T1) control (Concentrate with non-CH plus 0% RBO; C), (T2) Concentrate with CH plus 0% RBO (CH), (T3) Concentrate with non-CH plus 4% RBO (RBO), and (T4) Concentrate with CH plus 4% RBO (CHRBO). The cows were offered concentrate, at a ratio of concentrate to milk production of 1:2, and urea-lime treated rice straw was fed ad libitum. Urea-lime treated rice straw involved 2.5 g urea and 2.5 g $Ca(OH)_2$ (purchased as hydrated lime) in 100 ml water, the relevant volume of solution was sprayed onto a 100 g air-dry (91% DM) straw, and then covering the stack with a plastic sheet for a minimum of 10 d before feeding directly to animals. The CH based concentrate resulted in significantly higher roughage intake and total DM intake expressed as a percentage of BW (p<0.05). Ruminal pH, $NH_3$-N, BUN and total VFA did not differ among treatments, while RBO supplementation increased propionate, but decreased acetate concentration (p<0.05). Furthermore, the population of total ruminal bacteria was significantly lower on the RBO diet (p<0.05). In contrast, the total ruminal bacteria and cellulolytic bacteria on the CH diet were higher than on the other treatments. Supplementation with CH increased (p<0.05) F. succinogens and R. flavefaciens populations, whereas the populations of B. fibrisolvens and M. elsdenii were increased on the RBO diet. In addition, supplementation with CH and RBO had no effect on milk production and composition in dairy cows, while fatty acid composition of milk was influenced by RBO supplementation, and resulted in significantly lower (p<0.05) concentrations of both short-chain and medium-chain FA, and increased (p<0.05) the proportion of long-chain FA in milk fat, as well as significantly increased cis-9, trans-11 CLA and total CLA. In conclusion, RBO or CH exhibited specific effects on DMI, rumen fermentation, microbial population, milk yield and composition in lactating dairy cows, which were not interactions between CH and RBO in the diets. Feeding lactating dairy cows with RBO could improve fatty acid in milk fat by increasing cis-9, trans-11 CLA.