Oh et al.
Journal of Animal Science and Technology (2017) 59:27
DOI 10.1186/s40781-017-0150-8
RESEARCH Open Access
Potency of cashew nut shell liquid in
rumen modulation under different dietary
conditions and indication of its surfactant
action against rumen bacteria
Seongjin Oh1,2, Yasuyuki Suzuki1,2, Shusuke Hayashi1,2, Yutaka Suzuki1,2, Satoshi Koike1,2 and Yasuo Kobayashi1,2*
Abstract
Background: Cashew nut shell liquid (CNSL) is an agricultural byproduct containing alkylphenols that has been
shown to favorably change the rumen fermentation pattern only under experimentally fixed feeding conditions.
Investigation of CNSL potency in rumen modulation under a variety of feeding regimens, and evidence leading to
the understanding of CNSL action are obviously necessary for further CNSL applications. The objective of this study
was to evaluate the potency of CNSL for rumen modulation under different dietary conditions, and to visually
demonstrate its surfactant action against selected rumen bacteria.
Methods: Batch culture studies were carried out using various diets with 5 different forage to concentrate (F:C)
ratios (9:1, 7:3, 5:5. 3:7 and 1:9). Strained rumen fluid was diluted with a buffer and incubated with each diet. Gas
and short chain fatty acid (SCFA) profiles were characterized after 18 h incubation at 39 °C. Monensin was also
evaluated as a reference additive under the same conditions. Four species of rumen bacteria were grown in pure
culture and exposed to CNSL to determine their morphological sensitivity to the surfactant action of CNSL.
Results: CNSL supplementation decreased total gas production in diets with 5:5 and 3:7 F:C ratios, whereas the F:C
ratio alone did not affect any gas production. Methane decrease by CNSL addition was more apparent in diets with
5:5, 3:7, and 1:9 F:C ratios. An interactive effect of CNSL and the F:C ratio was also observed for methane production.
CNSL supplementation enhanced propionate production, while total SCFA production was not affected. Monensin
decreased methane production but only in a diet with a 1:9 F:C ratio with increased propionate. Studies of pure
cultures indicated that CNSL damaged the cell surface of hydrogen- and formate-producing bacteria, but did not
change that of propionate-producing bacteria.
Conclusion: CNSL can selectively inhibit rumen bacteria through its surfactant action to lead fermentation toward
less methane and more propionate production. As CNSL is effective over a wider range of dietary conditions for such
modulation of rumen fermentation in comparison with monensin, this new additive candidate might be applied to
ruminant animals for various production purposes and at various stages.
Keywords: Cashew nut shell liquid, Forage to concentrate ratio, Rumen bacteria, Rumen modulation
* Correspondence: [email protected]
1
Graduate School of Agriculture, Hokkaido University, Sapporo, Hokkaido
060-8589, Japan
2
Laboratory of Animal Function and Nutrition, Graduate School of
Agriculture, Hokkaido University, Sapporo 060-8589, Japan
© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Oh et al. Journal of Animal Science and Technology (2017) 59:27 Page 2 of 7
Background fermentation, monensin was also employed in in vitro
Enteric methane is mainly produced in the gastrointestinal evaluation in the same manner.
tract of ruminant animals during their feed utilization,
and is closely related to loss of energy in feed stuff [1]. Methods
Many studies have been carried out using feed additives to Rumen fluid used
modulate rumen fermentation in order to improve feed Rumen content was taken from two ruminally cannu-
efficiency, and the quantity and quality of animal products lated Holstein dry cows at the experimental farm of
such as meat and milk [2–4]. Although ionophore anti- Hokkaido University, Sapporo, Japan. The donor cows
biotic additives, represented by monensin, have been used were fed twice daily (08:00 and 17:00) with a 50% con-
in many countries, the use of these additives has been centrate (Monster 18; Mercian, Tokyo, Japan) and 50%
gradually prohibited due to concerns regarding public orchardgrass hay diet, which contained 18.2% crude pro-
health [5, 6]. Natural additive sources (e. g. plant extracts, tein, 41.2% neutral detergent fiber and 2.16 Mcal
essential oils etc.) appear to be alternative additives for metabolizable energy/kg on a dry matter basis. The ob-
rumen modulation [7–9]. tained rumen contents from cows were equally mixed,
Cashew nut shell liquid (CNSL) is a byproduct of the placed into a bottle flushed with N2 gas, which was then
cashew nut industry and has been used as a material for transferred to the laboratory within 30 min. The rumen
products including paints, lacquers, coatings, and other content was then strained through 2 layers of surgical
products [10]. CNSL was recently evaluated as a poten- gauze for experimental use.
tial additive source for ruminants, because it contains
alkylphenols (anacardic acid, cardanol and cardol) that Batch culture
can favorably modify rumen microbiota and fermenta- Table 1 shows the chemical composition of the diet and the
tion [11]. Of these phenolics, anacardic acid is thought CNSL used. The experimental substrate was a mixture of
to have the highest antimicrobial activity [12, 13], and 0.2 g orchardgrass hay and concentrate, with five forage to
this notion was also supported by recent studies using concentrate (F:C) ratios (9:1, 7:3, 5:5. 7:3 and 1:9). These
anacardic acid and its source materials CNSL and materials were ground by using a 1 mm sieve-attached cut-
ginkgo fruit [11, 14]. Favorable shifts of microbiota ter mill. CNSL was obtained from Idemitsu Kosan Co. Ltd.
and fermentation were, however, confirmed in in vitro (Sodegaura, Chiba, Japan). Monensin (monensin sodium
evaluations only under fixed single dietary conditions. salt) was purchased from Sigma-Aldrich (Saint Louis, MO,
To employ this potent additive candidate in wider ap- USA). Both additives were evaluated for their potency for
plications, CNSL needs to be tested under various modulation of rumen fermentation by batch culture experi-
dietary conditions based on the purposes and stages ments using rumen fluid diluted (2×) with McDougall’s buf-
of animal production. fer [17] and the above substrates. Supplementation levels of
The mode of inhibition of bacteria by CNSL is consid- CNSL and monensin were 500 and 5 μg/mL, respectively.
ered to be the surfactant action of anacardic acid that It has been experimentally defined that these levels do not
physically breaks down the cell surface of bacteria. negatively influence total short chain fatty acid (SCFA)
Therefore, Gram-negative bacteria that possess an outer production [11, 18] and these levels were therefore recom-
membrane often exhibit tolerance to such a surfactant mended in the present study. Each additive was dissolved
[15, 16]. Although the inhibitory concentration of CNSL, in 99.5% ethanol, added to empty culture tubes and left
ginkgo fruit and their component phenolics against overnight to evaporate the ethanol. As a control, ethanol
rumen bacteria were determined in previous studies [11, was added and treated in the same manner. Diluted rumen
14], no direct observation of the cell morphology of fluid was added to each tube containing substrate with or
rumen bacteria that had been exposed to CNSL has without each additive, and the head space of the tubes was
been made. flushed with N2 gas, then sealed with a butyl rubber stopper
Evaluation of the above issues (the potency of and a plastic screw cap, and incubated at 39 °C for 18 h, as
CNSL depending on dietary conditions and the mode done by Watanabe et al. [11]. Four replicates were incu-
of action of CNSL) is important for proposing CNSL bated for each treatment. After incubation, the total gas of
application strategies in various feeding regimens for the head space was measured using a needle-attached pres-
ruminants based on an understanding of key CNSL sure gauge (Aϕ60B; GL Science, Tokyo, Japan) and was also
mechanisms. The objectives of the present study employed for gas composition analysis. The culture liquid
were, therefore, to evaluate the potency of CNSL was sampled and kept at −30 °C for SCFA analysis.
under various dietary conditions, and to determine its
surfactant action against selected rumen bacteria that Pure cultures
are responsible for rumen modulation. To evaluate The bacteria used were Ruminococcus flavefaciens C94,
the effectivity of CNSL for modulating rumen Butyrivibrio fibrisolvens D1, Megasphaera elsdenii LC1
Oh et al. Journal of Animal Science and Technology (2017) 59:27 Page 3 of 7
Table 1 Chemical composition of the experimental diet and Chemical analysis
the cashew nut shell liquid Gas and SCFA analyses were carried out essentially as
Content Hay Concentrate CNSL described by Oh et al. [14]. Head space gas was analyzed
g/kg using gas chromatography (GC-8A; Shimadzu, Kyoto,
Dry matter 971 969 983 Japan) with attached parallel columns, Porapak Q
(Waters, Milford, MA) and Molecular Sieve 13X
g/kg DM
(Restek, Bellefonte, PA), and a thermal conductivity de-
Crude protein 74 203 15
tector. Flame ionization detector-attached gas chroma-
Crude ash 63 61 12 tography (GC-14B; Shimadzu, Kyoto, Japan) was used
Ether extract 9 97 942 for SCFA analysis, using a fused silica capillary column
Neutral detergent fiber 743 345 – (ULBON HR-20 M, 0.53 mm i.d. × 30 m length, 3.0 μm
Acid detergent fiber 448 134 – film; Shinwa, Kyoto, Japan).
Water soluble carbohydrate 112 294 –
Statistical analysis
Nitrogen free extract – – 31
The data (n = 4) of rumen parameters obtained from
Ether extract fractions, % two batch culture studies (experiment 1: control vs.
Total alkylphenols – – 92.7 CNSL, and experiment 2: control vs. monensin) were in-
Total anacardic acid – – 62.3 dividually subjected to analysis of variance using the
monoenoic (15:1) – – 28.9 general linear model procedure of SPSS (version 16.0 J,
Tokyo, Japan). Tukey’s method was employed for mul-
dienoic (15:2) – – 8.8
tiple comparison across 5 different F:C ratios and 2
trienoic (15:3) – – 24.6
treatments (control vs. CNSL or monensin). Statistical
Total cardanol – – 8.9 significance was declared at P < 0.05.
monoenoic (15:1) – – 2.9
dienoic (15:2) – – 1.5 Results
trienoic (15:3) – – 4.5 Gas profiles
The effect of CNSL or monensin supplementation on
Total cardol – – 21.5
rumen gas production from diets with different F:C
monoenoic (15:1) – – 3.1
ratios is shown in Table 2. CNSL supplementation de-
dienoic (15:2) – – 4.3 creased total gas production from diets with 5:5 and
trienoic (15:3) – – 14.1 3:7 F:C ratios (P < 0.05), while the F:C ratio alone did
Othersa – – 7.3 not affect any gas production. CNSL strongly inhibited
a
unidentified fractions methane production, especially from diets with 5:5
(46%), 3:7 (46%), and 1:9 (51%) F:C ratios (P < 0.05). An
interactive effect between CNSL and diet (F:C ratio) was
and Selenomonas ruminantium.GA192, all of which are also observed for methane production (P < 0.01). No
a type strain of each species. These bacteria were anaer- specific accumulation of hydrogen was observed.
obically cultivated in rumen fluid containing medium Monensin supplementation did not alter total gas, CO2
[14]. When each bacterium was grown to the exponen- or hydrogen production. Methane was significantly de-
tial phase, ethanol (control) or CNSL was added creased by monensin only for a diet with a 1:9 F:C ratio,
(200 μg/mL final concentration) and incubation was showing 46% reduction (P < 0.05).
continued for 5 h. The culture was then analyzed using
scanning electron microscopy (SEM) as follows. Bacte- SCFA profiles
rial samples were washed with 20 mM K phosphate buf- The effect of CNSL or monensin supplementation on in
fer (pH 7.2), soaked in 2.5% glutaraldehyde in K vitro rumen SCFA profiles is shown in Table 3. The total
phosphate buffer and then fixed with 1% osmic acid in K concentration of SCFA was generally not affected by
phosphate buffer. The samples were then dehydrated CNSL, although an increase in SCFA by CNSL was ob-
using different ethanol concentrations of 50, 70, 90, and served for a diet with a 5:5 F:C ratio (P < 0.05).
99.5%. The next step was dehydration with iso- Proportions of acetate and butyrate were decreased by
amylacetate and a critical point drier (HCP-2, Hitachi, CNSL supplementation (P < 0.001), while that of propi-
Japan). The sample was then coated with gold-paradium onate was increased under all dietary conditions tested
by ion spatter and was observed using a high resolution (P < 0.001). The enhancement of propionate was more
scanning electron microscope (JSM-6301F, Japan pronounced for diets with 5:5, 3:7 and 1:9 F:C ratios. In
Electron, Tokyo, Japan). terms of the molar proportions of SCFA, additive effect,
Oh et al. Journal of Animal Science and Technology (2017) 59:27 Page 4 of 7
Table 2 Effect of cashew nut shell liquid and monensin supplementation under different dietary conditions on in vitro gas
production
Gases mL/ Forage to concentrate ratio P-value
culture
9:1 7:3 5:5 3:7 1:9
Control Treatment Control Treatment Control Treatment Control Treatment Control Treatment Additive Diet A×D
Cashew nut shell liquid
Total 4.00abc 3.58bc 4.84ab 4.27abc 4.87ab 3.32c 5.08a 3.56bc 4.81ab 3.69abc <0.001 0.173 0.213
gas
CO2 3.44ab 2.96ab 3.95ab 3.72ab 3.93ab 2.81b 4.08a 3.01ab 3.78ab 3.18ab <0.001 0.200 0.349
bc bc abc c ab c a c a c
CH4 0.54 0.60 0.88 0.54 (61) 0.93 0.50 (54) 0.98 0.53 (54) 1.02 0.50 (49) <0.001 0.103 0.008
(100) (111) (100) (100) (100) (100)
H2 0.01 0.02 0.01 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.744 0.466 0.428
Monensin
Total 4.62 4.44 5.75 4.80 5.47 4.82 5.17 4.68 5.31 4.39 0.009 0.442 0.742
gas
CO2 3.61 3.30 4.47 3.71 4.03 3.69 3.79 3.66 3.96 3.50 0.059 0.535 0.875
ab ab a ab a ab a ab a b
CH4 1.13 1.14 1.27 1.08 1.43 1.12 1.36 1.01 1.35 0.73 (54) <0.001 0.295 0.092
(100) (101) (100) (85) (100) (85) (100) (78) (100)
H2 0.01 0.01 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.001 0.642 0.949
Values in parenthesis are relative percentages of methane production in treatment to that in control
A × D indicates interaction between additive and diet effects
a-c
Means within a row with different superscripts significantly differ (P < 0.05)
Table 3 Effect of cashew nut shell liquid and monensin supplementation under different dietary conditions on in vitro short chain
fatty acid production
Parameters Forage to concentrate ratio P-value
9:1 7:3 5:5 3:7 1:9
Control Treatment Control Treatment Control Treatment Control Treatment Control Treatment Additive Diet A×D
mmol/dL
Cashew nut shell liquid
Total SCFA 9.57c 9.81bc 9.64bc 10.67ab 10.26bc 11.36a 10.71ab 10.71ab 10.67ab 10.49abc 0.004 <0.001 0.019
ab ab ab ab a a a bc a b
Acetate 6.44 6.41 6.40 6.70 6.72 6.83 6.82 6.35 6.74 6.12 0.078 0.048 0.003
Propionate 1.64e 2.11cde 1.71de 2.60b 1.90cde 3.32a 2.13bcd 3.30a 2.21bc 3.46a <0.001 <0.001 <0.001
n-Butyrate 1.13c 0.99de 1.20bc 1.07cd 1.30ab 0.94de 1.40a 0.84ef 1.36a 0.73f <0.001 0.011 <0.001
Monensin
Total SCFA 8.08b 7.96b 8.26b 8.29b 8.71ab 9.03ab 8.69ab 8.80ab 9.41a 8.73ab 0.650 <0.001 0.280
Acetate 5.20ab 5.00ab 5.15ab 4.92ab 5.24ab 5.16ab 5.07ab 4.81ab 5.38a 4.69b 0.002 0.416 0.228
cd d cd bcd bcd ab bc a ab a
Propionate 1.82 1.78 1.92 2.16 2.19 2.65 2.33 2.91 2.64 2.95 <0.001 <0.001 0.093
n-Butyrate 0.82d 0.90cd 0.95bcd 0.99abcd 1.03abc 1.04abc 1.09ab 0.92bcd 1.16a 0.93bcd 0.029 <0.001 0.001
molar %
Cashew nut shell liquid
Acetate 67.28a 65.35b 66.39ab 62.73c 65.48b 60.13d 63.72c 59.30de 63.22c 58.37e <0.001 <0.001 <0.001
Propionate 17.17f 21.46d 17.74f 24.34c 18.47ef 29.16b 19.90de 30.67b 20.72d 32.99a <0.001 <0.001 <0.001
b c ab c ab d a d ab e
n-Butyrate 11.80 10.03 12.43 10.05 12.63 8.31 13.08 7.87 12.72 6.90 <0.001 <0.001 <0.001
Monensin
Acetate 64.21a 62.53a 62.33ab 59.33cd 60.12bc 57.19d 58.34cd 54.58e 57.23d 53.78e <0.001 <0.001 0.280
Propionate 22.51de 22.27e 23.21de 26.00cde 25.02cde 29.29bc 26.67cd 33.05ab 28.11c 33.79a <0.001 <0.001 0.007
c abc abc abc abc abc a bc ab bc
n-Butyrate 10.19 11.27 11.52 11.87 11.87 11.49 12.51 10.47 12.37 10.57 0.034 0.119 0.001
A × D indicates interaction between additive and diet effects
a-f
Means within a row with different superscripts significantly differ (P < 0.05)
Oh et al. Journal of Animal Science and Technology (2017) 59:27 Page 5 of 7
diet effect and their interactive effect were significant exposure. The damaged cells of this bacterium had a
(P < 0.001). Regarding monensin addition, the total hairy surface in appearance and some cells were com-
SCFA level was not affected by monensin in comparison pletely broken and burst. Exposure of the bacterium
with control, whereas the proportion of acetate was Butyrivibrio fibrisolvens, which is a hydrogen and bu-
decreased in diets of all F:C ratios (P < 0.05), except for tyrate producer, to CNSL resulted in the formation of
the 9:1 ratio. An increase in the proportion of propion- bubble-like bumps on the cell surface. The length of
ate and a decrease in the proportion of butyrate were this bacterium also increased and cell division seemed
found with high concentrate diets (3:7 and 1:9 F:C to be inhibited. Thus, the surface structure of both of
ratios) (P < 0.05). A diet effect was therefore observed these bacterial species was apparently changed by ex-
for total SCFA, propionate and butyrate levels and pro- posure to CNSL. By contrast, there were no morpho-
portions of acetate and propionate (P < 0.001). logical changes in the cell surface or in cell size
following exposure of the two propionate-producing
Bacterial morphology bacterial species, Megasphaera elsdenii and Selenomo-
Figure 1 shows the morphology of 4 selected rumen nas ruminantium to CNSL.
bacterial species with or without exposure to CNSL,
as determined using SEM. The cell surface of Rumi-
nococcus flavefaciens, a hydrogen-producing fibrolytic Discussion
bacterium, suffered heavy damage following CNSL In the present study, responses of rumen fermentation
to CNSL under a variety of dietary treatments were in-
vestigated. As a reference additive, monensin was tested
in the same manner. The present batch culture studies
showed that both additives similarly changed gas and
SCFA production, which involved a decrease in me-
thane, acetate and butyrate production, and an increase
in propionate production. However, the extent of these
changes was greater for CNSL than for monensin
addition, at the recommended level for each additive,
and the effective range of diet that was effected was
broader in CNSL than in monensin addition (Tables 2 &
3). The present preliminary evaluation of the effect of
CNSL addition on the modulation of rumen fermenta-
tion supports the notion that not only can CNSL be
used in a fixed condition as indicated in a previous study
[11] but that it may also be used under a wide range of
dietary conditions. This finding is advantageous when
taking into consideration the potential use of CNSL
addition in the practice of ruminant feeding.
Although the reason why there is a broad CNSL effi-
cacy among diets is not apparent, the mode of action of
CNSL is simple (via surfactant action, see below) but
strong in comparison with other additives including
monensin. This activity could lead to clear microbial se-
lection followed by favorable fermentation changes in
terms of methane mitigation and propionate enhance-
ment. In addition, CNSL addition may prevent rumen
metabolic disorders such as lactic acidosis and feedlot
bloat when a high grain diet is fed, because CNSL in-
hibits the growth of the lactate-producing Streptococcus
bovis and decreases the viscosity of rumen fluid [11].
Applying these characteristics to practical use, CNSL
might be a useful additive for beef cattle receiving a high
Fig. 1 Morphological changes of rumen bacteria induced by cashew grain diet. However, whether the efficacy of CNSL is
nut shell liquid. Each bacterium was exposed to ethanol (control) or
long-lasting when continuously fed to animals for a long
cashew nut shell liquid (CNSL) for 5 h at log exponential growth phase
period of time [19], remains to be assessed.
Oh et al. Journal of Animal Science and Technology (2017) 59:27 Page 6 of 7
Modes of action of CNSL and monensin in rumen follows. Bacteria involved in propionate production can
modulation have been discussed in previous studies, in be selected more apparently by CNSL under the pres-
which CNSL was shown to have a surfactant activity ence of the substrate concentrate, while hydrogen- or
[11–14] and monensin was shown to have an ionophore formate-producing bacteria, sensitive to CNSL, can be
activity [18, 20]. However, no direct evidence of the sur- more suppressed with scanty of the substrate forage. As
face acting action of CNSL has been shown for bacteria, a result, shift of rumen microbiota became more demon-
especially for rumen bacteria. We observed the morph- strable, leading to more apparent fermentation changes
ology of representative rumen bacterial species exposed by CNSL. However, further evaluations in animal feeding
to CNSL using SEM. This analysis clearly proved that experiments are necessary for confirming the potency of
the cell surface of two species of rumen bacteria that CNSL as a rumen modulating agent.
produce hydrogen and formate was physically interfered
with upon CNSL exposure, while the cell surface of Conclusion
propionate-producing species did not show any change CNSL altered the in vitro rumen fermentation pattern
at all upon CNSL exposure (Fig. 1). These observations towards less methane and more propionate production
can be explained by the fact that the latter 2 species (S. under 5 different dietary conditions tested. CNSL sup-
ruminantium and M. elsedenii) possess an outer mem- plementation showed a greater extent of rumen modula-
brane that protects the cells from surfactant action, tion and showed efficacy over a wider coverage of diet
while the former 2 species (R. flavefaciens and B. fibrisol- composition in comparison with supplementation with
vens) lack such a membrane [21, 22], which means that the ionophore monensin. The surfactant action of CNSL
the surfactant CNSL can directly act against the bacterial against specific rumen bacteria was visually indicated to
cell surface to physically break it. According to be the main cause of rumen modulation. Feeding studies
Watanabe et al. [11], the minimum inhibitory concentra- are required to practically assess the potency of CNSL in
tions of CNSL (μg/ml) for the growth of these bacteria ruminants on various diets.
are 1.56 (R. flavefaciens), 3.13 (B. fibrisolvens), and >50
(M. elsedenii and S. ruminantium). These values appar- Abbreviations
CNSL: Cashew nut shell liquid; F:C ratio: Forage to concentrate ratio;
ently correspond to the physical sensitivity/strength of SCFA: Short chain fatty acid; SEM: Scanning electron microscopy
each bacterium against the surfactant action of CNSL as
shown by the present microscopic observations. This Acknowledgements
study provides the first and direct visual evidence for the Not applicable.
surfactant action of CNSL.
Funding
Theoretically, it is considered that other bacterial This work was supported by JSPS KAKENHI Grant Numbers 20,380,146 and
species are influenced by CNSL in the same manner, JP23380156. This funding agency is not involved in the present study
based on the presence and/or absence of an outer including collection, analysis and interpretation of data, and also in writing
manuscript.
membrane. This simple mode of action of CNSL
could successfully alter rumen microbiota, thereby Availability of data and materials
shifting metabolic hydrogen flow and leading to The datasets used and/or analyzed during this study are available from the
greater propionate production via fumarate and acryl- corresponding author on request.
ate pathways [23, 24]. Ruminococcus flavefaciens and
Authors’ contributions
Butyrivibrio fibrisolvens are known to be fiber de- SO and YK wrote the manuscript. YK designed the experiment. Yasuyuki S
graders and their end-products such as H2, formate and SH carried out analyses, while Yutaka S and SK contributed to the
and acetate, consistently contribute to methane pro- discussion. All authors read and approved this manuscript.
duction in the rumen [25, 26]. Additionally, other Ethics approval and consent to participate
rumen hydrogen and/or formate producers including All the study protocols conformed to the Guidelines for Animal Experiments,
Treponema bryantii were considered to be sensitive Hokkaido University (2007) and the Act on the Welfare and Management of
Animals (2005) with the approval of animal experimentation committee of
to CNSL and their abundance actually decreased both Hokkaido University.
when CNSL was supplemented to the continuous
culture RUSITEC [11] and also when CNSL was fed Consent for publication
to cattle [19]. These data also explain why rumen Not applicable.
methane production is depressed by CNSL.
Competing interests
As far as CNSL does not completely inhibit specific The authors declare that they have no competing interests.
group of bacteria in the rumen, the extent of fermenta-
tion change is essentially affected by type of substrate.
Publisher’s Note
Therefore, the reason for more pronounced effect of Springer Nature remains neutral with regard to jurisdictional claims in
CNSL under high grain diets could be explained as published maps and institutional affiliations.
Oh et al. Journal of Animal Science and Technology (2017) 59:27 Page 7 of 7
Received: 27 June 2017 Accepted: 24 October 2017 25. Latham MJ, Wolin MJ. Fermentation of cellulose by Ruminococcus
flavefaciens in the presence and absence of Methanobacterium ruminantium.
Appl Environ Microbiol. 1977;34:297–301.
26. Miller TL, Currenti E, Wolin MJ. Anaerobic biocenversion of cellulose by
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