In vitro indications for favourable non-additive effects on ruminal methane mitigation between high-phenolic and high-quality forages.
ABSTRACT Feeding plants containing elevated levels of polyphenols may reduce ruminal CH4 emissions, but at the expense of nutrient utilisation. There might, however, be non-additive effects when combining high-phenolic plants with well-digestible, high-nutrient feeds. To test whether non-additive effects exist, the leaves of Carica papaya (high in dietary quality, low in polyphenols), Clidemia hirta (high in hydrolysable tannins), Swietenia mahagoni (high in condensed tannins) and Eugenia aquea (high in non-tannin phenolics) were tested alone and in all possible mixtures (n 15 treatments). An amount of 200 mg DM of samples was incubated in vitro (24 h; 39oC) with buffered rumen fluid using the Hohenheim gas test apparatus. After the incubation, total gas production, CH4 concentration and fermentation profiles were determined. The levels of absolute CH4, and CH4:SCFA and CH4:total gas ratios were lower (P < 0·05) when incubating a combination of C. papaya and any high-phenolic plants (C. hirta, S. mahagoni and E. aquea) than when incubating C. papaya alone. Additionally, mixtures resulted in non-additive effects for all CH4-related parameters of the order of 2-15 % deviation from the expected value (P < 0·01). This means that, by combining these plants, CH4 in relation to the fermentative capacity was lower than that predicted when assuming the linearity of the effects. Similar non-additive effects of combining C. papaya with the other plants were found for NH3 concentrations but not for SCFA concentrations. In conclusion, using mixtures of high-quality plants and high-phenolic plants could be one approach to CH4 mitigation; however, this awaits in vivo confirmation.
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ABSTRACT: BACKGROUND: Mixtures of plant species provide biochemical diversity to pastures which may enhance productivity while decreasing reliance on herbicides and insecticides. All plants contain secondary metabolites (PSMs) that interact in plant communities in a variety of ways. For instance, tannins are a group of highly reactive chemical compounds with the potential to interact with other PSM such as alkaloids and saponins, neutralizing their negative effects. Our objective was to determine whether tannins modify the foraging behavior of sheep grazing on varieties of alfalfa, birdsfoot trefoil and tall fescue with high and low concentrations of saponins, tannins and alkaloids, respectively.RESULTS: Lambs that received intraruminal infusions of tannins increased their consumption of the high-saponin variety of alfalfa and the high-alkaloid variety of tall fescue relative to lambs not infused with tannins (controls). Lambs infused with tannins and then offered choices among the three high-PSM varieties of the forages also manifested higher consumption of the high-alkaloid variety of tall fescue than control lambs. In contrast, lambs infused with tannins reduced their consumption of the high-tannin variety of birdsfoot trefoil. Thus lambs modified their foraging behavior as a function of the presence/absence of tannins in their rumens.CONCLUSIONS: These results indicate that ruminants are able to discriminate the specific post-ingestive effects of forage varieties with high concentrations of PSM, and that PSM complementarities are likely to increase the efficiency of use of diverse forages with different biochemistries. Copyright © 2009 Society of Chemical IndustryJournal of the Science of Food and Agriculture 11/2009; 89(15):2668 - 2677. · 1.76 Impact Factor
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ABSTRACT: The aim of the current study was to investigate the associative effects of a cornstalk-based diet supplemented with alfalfa (Medicago sativa) hay on intake, digestibility, nitrogen (N) metabolisation, rumen environment and hematological parameters in Xiaoweihan sheep. We also investigated the optimal range of alfalfa hay to achieve positive associative effects and avoid negative effects. Xiaoweihan sheep (n=5; fitted with rumen T-cannula) were fed five cornstalk-based diets in a 5×5 Latin square design. Diets contained 0, 50, 150, 300, 450 g alfalfa, and were supplemented with 100 g concentrate, respectively. Our results suggested that supplementation of 300 g alfalfa hay reduced (PLivestock Science - LIVEST SCI. 01/2008; 113(1):87-97.
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ABSTRACT: Whether foraging on pastures or rangelands, herbivores encounter plant species that differ in their concentrations of nutrients. They also all contain various secondary compounds that at too high doses can be toxic, but at the appropriate dose many of these toxins may have medicinal benefits. The quantity of forage an animal consumes depends on the other forages it selects because nutrients and toxins interact. Food intake also depends on an individual's morphology and physiology, and marked variation is common, even among closely related animals, in needs for nutrients and abilities to cope with toxins. Thus, individuals can better meet their needs when offered a variety of foods that differ in nutrients and toxins than when constrained to a single food. Nonetheless, we have focused on a few species, often grown in monoculture, and we have reduced concentrations of secondary compounds with little appreciation for their roles in protecting plants against herbivores, pathogens, and competitors. In nature, where diversity of plants is the rule and not the exception, eating a variety of foods is how animals cope with, and may benefit from, secondary compounds. The potential benefits of creating mixtures of plant species whose nutrient and secondary compound profiles complement one another are obvious, though much remains to be learned about how to reconstruct agro-ecosystems with plants that complement and enhance one another structurally, functionally, and biochemically.Behavioral Education for Human, Animal, Vegetation, and Ecosystem Management (BEHAVE). 01/2007;
In vitro indications for favourable non-additive effects on ruminal methane
mitigation between high-phenolic and high-quality forages
Anuraga Jayanegara1,2, Svenja Marquardt1, Elizabeth Wina3, Michael Kreuzer1and Florian Leiber1*
1ETH Zurich, Institute of Agricultural Sciences, Universitaetstrasse 2, 8092 Zurich, Switzerland
2Department of Nutrition and Feed Technology, Faculty of Animal Science, Bogor Agricultural University, Bogor 16680,
3Indonesian Research Institute for Animal Production, PO Box 221, Bogor 16002, Indonesia
(Submitted 2 November 2011 – Final revision received 19 March 2012 – Accepted 2 April 2012 – First published online 28 May 2012)
Feeding plants containing elevated levels of polyphenols may reduce ruminal CH4emissions, but at the expense of nutrient utilisation.
There might, however, be non-additive effects when combining high-phenolic plants with well-digestible, high-nutrient feeds. To test
whether non-additive effects exist, the leaves of Carica papaya (high in dietary quality, low in polyphenols), Clidemia hirta (high in
hydrolysable tannins), Swietenia mahagoni (high in condensed tannins) and Eugenia aquea (high in non-tannin phenolics) were
tested alone and in all possible mixtures (n 15 treatments). An amount of 200mg DM of samples was incubated in vitro (24h; 39oC)
with buffered rumen fluid using the Hohenheim gas test apparatus. After the incubation, total gas production, CH4concentration and fer-
mentation profiles were determined. The levels of absolute CH4, and CH4:SCFA and CH4:total gas ratios were lower (P,0·05) when incu-
bating a combination of C. papaya and any high-phenolic plants (C. hirta, S. mahagoni and E. aquea) than when incubating C. papaya
alone. Additionally, mixtures resulted in non-additive effects for all CH4-related parameters of the order of 2–15% deviation from the
expected value (P,0·01). This means that, by combining these plants, CH4in relation to the fermentative capacity was lower than that
predicted when assuming the linearity of the effects. Similar non-additive effects of combining C. papaya with the other plants were
found for NH3concentrations but not for SCFA concentrations. In conclusion, using mixtures of high-quality plants and high-phenolic
plants could be one approach to CH4mitigation; however, this awaits in vivo confirmation.
Key words: Ruminants: Methanogenesis: Phenolic compounds: Forage
Various investigations are currently under way to identify and
test means for mitigating CH4emissions that originate from
ruminants due to the activity of methanogenic archaea
during feed fermentation(1). As a product of fermentative
digestion, CH4emission levels depend considerably on the
quantity and composition of feeds consumed(2). The potential
of mitigating CH4emissions by the extracts of phenolic com-
pounds, which are synthesised in the intermediary metabolism
of plants, has been demonstrated experimentally(3–5). Also,
the direct inclusion of plants containing phenolics in ruminant
diets reduced CH4 emissions compared with control diets,
both in vitro(6)and in vivo(7,8).
A major drawback in implementing diets with doses of phe-
nolics aimed at reducing CH4emissions is often a decline in
the digestibility of the feed and therewith the productivity of
the animals(9), even at dosages where toxic side effects are
excluded. As a consequence, there are often no or only
small declines in CH4per unit of digested feed and, therefore,
food produced. Analysing a larger dataset by principal
components analysis illustrated that plants with high forage
quality are arranged opposite to those with a high CH4-
mitigating potential(10). This indicates that achieving both
goalssimultaneously is difficult.
et al.(8,11), where low-quality tropical Brachiaria hay was
combined with highly tanniferous shrub forage, have shown
that any reduction in CH4was associated with a correspond-
ingly lower utilisation of dietary energy. This may be different
when high-phenolic plants are combined with high-quality
feeds. So far, studies specifically designed to measure the addi-
tivity or non-additivity of the effects (i.e. non-linear effects,
elsewhere also defined as associative effects(12)when investi-
gating combinations of plants) in the context of ruminal CH4
emissions are scarce.
In the present study, we hypothesised that combining
plants characterised by different phenolic profiles with one
have general favourable
*Corresponding author: F. Leiber, fax þ41 44 632 1128, email email@example.com
Abbreviations: CT, condensed tannins; HT, hydrolysable tannins; NTP, non-tannin phenolics; SCFA, short-chain fatty acids.
British Journal of Nutrition (2013), 109, 615–622
q The Authors 2012
British Journal of Nutrition
non-additive effects in terms of lower CH4emission relative to
the productivity of ruminal fermentation in vitro. We specifi-
cally looked at in vitro CH4emissions per unit of SCFA or
total gas as indicators of fermentation productivity. For this
purpose, the leaves of four tropical plants were selected as
model forages. Carica papaya was chosen as forage of high
quality. Clidemia hirta, Swietenia mahagoni and Eugenia
aquea represented forages containing appreciable amounts
of total phenolics but a different phenolic profile. These
plants were selected on the basis of a previous screening
experiment(10). With this kind of experimental design, it is
possible to demonstrate in vitro the non-additivity of the
properties of differing forages, which in the best case can be
used as a first step towards developing forage-based diets
with lowered methanogenic
impaired ruminal fermentation efficiency. However, with this
approach, it is not possible to distinguish whether the result-
ing effects are dose–response effects of any single compound
or interaction effects of different compounds. Although in vitro
evidence has a limited applicability for the situation in vivo, it
provides additional information about the occurrence of such
complementary effects of feeds with differing profiles in plant
secondary compounds on nutrition processes in ruminants,
which have already been described in vivo in another con-
text(13). The present study aimed to indicate areas on which
future in vivo experiments could focus.
Materials and methods
Experimental design and plant material
All plant samples were incubated both individually and in all
possible mixtures (Table 1). The mixtures consisted of two,
three or four plants represented in equal proportions. This
resulted in a total of fifteen treatments. The leaves of
C. papaya were characterised by high crude protein contents,
low contents of fibre and lignin as well as favourably high
in vitro organic matter digestibility(10). This differed clearly
from the properties of the three high-phenolic plants selected
that were rich in phenolics (7- to 9-fold levels of total pheno-
lics compared with C. papaya). Concerning the phenolic pro-
file, C. hirta is rich in hydrolysable tannins (HT), S. mahagoni
contains particularly high levels of condensed tannins (CT) as
well as appreciable levels of HT and non-tannin phenolics
(NTP), and E. aquea is especially rich in NTP and lignin.
The selected plants were considered to be suitable models
for the purpose of comparing plants with quite similar con-
tents but different categories of phenolic compounds. Even
though not very common globally and not used as the sole
feed, the leaves of C. papaya, C. hirta, S. mahagoni and
E. aquea are used either as ruminant feeds in rural areas
(C. papaya and C. hirta) or as traditional veterinary medicinal
plants in the areas around Bogor on the Java island of
Indonesia(10). Since the present study aimed at the basic
question of non-additivity when combining single forages
instead of formulating complex diets, no standardisation was
applied for other nutrients such as crude protein or neutral-
detergent fibre, although this might have interfered with the
effects of phenolic compounds.
The leaves of the experimental plants were collected in
November 2008 from the area of the Indonesian Research
Institute for Animal Production, Ciawi, Bogor, located at an
elevation of 350m above sea level. About 3kg fresh matter
of each plant species was sampled. Each sample consisted
of leaves from several individual plants from the same species.
The samples were immediately air-dried in a greenhouse for
2d, followed by oven-drying overnight at 508C. After drying,
the samples were ground to pass a 1mm sieve, and then sub-
jected to chemical analysis and in vitro incubation. As the
same batches of the four feeds as those tested in an earlier
study were used in the present study, no new compositional
analyses were performed and the analytical procedures have
been described in detail in Jayanegara et al.(10). Briefly, for
C. papaya, C. hirta, S. mahagoni and E. aquea, the following
concentrations (g/kg DM) were analysed: crude protein, 386,
129, 112 and 199; neutral-detergent fibre, 155, 232, 281 and
479; total extractable phenolics, 25, 216, 207 and 169; total tan-
nins, 8, 212, 138 and 67; CT, 0, 10, 86 and 40; HT, 8, 202, 52
and 27; NTP, 17, 4, 69 and 102(10).
In vitro procedure and analyses
An amount of 200mg DM of individual plants or mixtures was
incubated with 10ml of rumen fluid and 20ml of buffer sol-
ution using the Hohenheim gas test apparatus(14)with modi-
fied syringes(15). The latter have two outlets, in which the
first outlet is designed for filling and emptying the liquid
phase and the second allows sampling from the gas phase.
Incubation was carried out for 24h at 398C in four subsequent
runs, comprising two treatment replicates per run (n 8). This
was complemented for each run by three syringes without
feed, with standard hay and concentrate. Both standard hay
and concentrate were obtained from the Institute of Animal
Nutrition, University of Hohenheim, Stuttgart, Germany. Data
on the expected amounts of total gas produced from incu-
bation of the standards were used in comparison with those
actually measured, in order to monitor whether incubation
Table 1. Treatment formulation and amounts (mg DM) of
plants incubated in vitro
P, Carica papaya; C, Clidemia hirta; S, Swietenia mahagoni;
E, Eugenia aquea.
A. Jayanegara et al.616
British Journal of Nutrition
went in a normal way and to adjust total gas(14). The donor of
rumen fluid was a rumen-cannulated lactating Brown Swiss
cow; the fluid was taken before the morning feeding. In
order to prevent any previous adaptive processes to the phe-
nolics, the cow received hay made from a ryegrass–white-
clover ley with ad libitum access and 0·5kg/d of dairy cow
concentrate (UFA 149, UFA AG). The cow was cared for
according to the Swiss guidelines for animal welfare. After col-
lection, rumen fluid was strained through four layers of gauze
(1mm pore size, Type 17; MedPro Novamed AG) in order to
filter out any feed particles.
The volume of fermentation gas produced during 24h of
incubation was read from the calibrated scale on each glass
syringe. The liquid phase in each syringe was decanted.
Subsequently, 0·15ml of fermentation gas were withdrawn
with a Hamilton syringe (Hamilton AG) through a gas-tight
septum covering the outlet. This gas sample was injected
into a gas chromatograph (Model 5890 Series II; Hewlett Pack-
ard) for measuring CH4and H2concentrations. NH3and pH of
the incubation liquid were determined with a potentiometer
(Model 632; Metrohm) equipped with the corresponding elec-
trodes. Total NH3was calculated from NH3concentration and
the volume of the incubation liquid. SCFA were analysed using
HPLC (LaChrom, L-7000 series; Hitachi Limited) equipped
with an UV–VIS detector, read at 210nm(10). Total viable
bacterial and protozoal numbers were counted by direct
microscopic counting using
(Blau Brand) with depths of 0·02 and 0·1mm, respectively.
For bacterial counting, samples were treated with Hayem sol-
ution (HgCl2, 2·5mg/ml; Na2SO4, 25mg/ml; NaCl, 5·0mg/ml).
Viability of bacteria was accounted for by counting only
moving individuals. Before protozoal counting, samples
were treated with 1:10 diluted formalin (40/100, w/v in
water). Only intact protozoa and no fragments were counted.
Calculations and statistical analysis
Following Menke & Steingass(14), gas production from the
blank was subtracted from all samples incubated to obtain
the net gas production. Subsequently, gas production from
the hay standard (44·43ml gas/200mg DM; 24h incubation)
was divided by the measured net value of the hay standard
to provide the correction factor FH. Similarly, gas production
from the concentrate standard (65·18ml/200mg DM; 24h
incubation) was divided by the measured net gas production
of the concentrate standard to provide FC. The average
value of FH and FC was used for the adjustment. Data on
CH4 and H2 concentrations were transformed into volume
(ml, ml) data by multiplying the measured concentrations
with the measured total gas volume. To get a direct meaning-
ful relationship of CH4to SCFA, molar amounts were calcu-
lated by assuming a density of 0·67kg/m3for CH4 gas at
1·013 bar and 168C(16).
The data generated were subjected to a mixed model of
ANOVA. Incubation runs, serving as a block in the ANOVA
model, were considered as random effects, while plant treat-
ments were included as fixed effects. If fixed effects were
significant at P,0·05, multiple comparisons among means
were made using Tukey’s post hoc test. Before ANOVA, bac-
terial and protozoal counts were transformed into their logar-
ithmic units. All statistical analyses were performed using SPSS
statistical software version 17.0(17).
The main purpose of the experiment was to reveal the pre-
sence of non-additive effects. Non-additive effects were
defined as the deviations of the observed values (obtained
by measurements) from the expected values (calculated as
arithmetic means of the values obtained by the respective indi-
vidual plant incubations). These differences were analysed for
all plant mixtures using a paired t test and presented as
(observed value 2 expected value)/expected value £ 100%
following Niderkorn et al.(12). All expected ratio values were
calculated by the actually measured values for each individual
incubation. This means that the expected values were not cal-
culated from the already averaged ratio values, as shown in
Tables 2 and 3. For the ratio of CH4:total gas (ml/l), the
n)/((total gas plant1þ total gas planti2n)/n), where n is the
number of involved plant species. The same principle was
applied for calculating the expected value for the ratio of
Effects of plants and combinations of plants on in vitro
The total SCFA amount was superior when C. papaya was
incubated compared with the other three plants (P,0·05),
where E. aquea was inferior to C. hirta and S. mahagoni
(Table 2). The acetate:propionate ratio was shifted towards
acetate when E. aquea was incubated compared with the
other three forages (P,0·05). Combining the different forages
resulted in values for SCFA production and proportions of
acetate and propionate being always intermediate between
those obtained from individual incubations. For the molar pro-
portions of butyrate, valerate and their iso forms, the effects
were less clear.
Total gas produced from incubation with C. papaya was
twice that with C. hirta and S. mahagoni (P,0·05), and the
latter two plants produced twice as much total gas compared
with E. aquea (P,0·05; Table 3). The volumes of CH4and H2
(which on average amounted to only about 0·1% of that
of CH4) varied, when total gas varied. Nevertheless, the gas
composition differed among the treatments. Incubating
S. mahagoni led to the lowest CH4:total gas ratio among the
individual plants (P,0·05), while incubating C. papaya pro-
duced twice the level. The lowest H2:total gas proportion
was found when E. aquea (40ml/l) was incubated and the
highest with C. papaya (110ml/l; P,0·05; data not shown).
The amounts of CH4in relation to the amounts of SCFA after
incubation differed (P,0·05) between each of the forage
species, and were ranked in the order of C. papaya .
C. hirta . S. mahagoni . E. aquea. The concentration of
NH3in the incubation liquid was highest when C. papaya
was incubated alone, whereas the ratio of NH3-N:dietary N
was highest with E. aquea incubation and differed (P,0·05)
Non-additive effects in rumen methane mitigation 617
British Journal of Nutrition
from the values measured with the other plants. There were
some differences in the pH of the incubation liquid, but not
(P.0·05) in the logarithmic counts of viable bacteria and pro-
tozoa across all experimental treatments (data not shown).
In general, combining either C. hirta, S. mahagoni or
E. aquea with C. papaya in binary mixtures resulted in
lower total gas production compared with the incubation of
C. papaya alone (P,0·05). CH4-related variables (CH4,
CH4:SCFA and CH4:total gas) were also reduced by mixing
these plants (P,0·05) compared with the incubation of
C. papaya alone. The mixing of C. hirta and/or E. aquea
with S. mahagoni resulted in a lower (P,0·05) CH4:total gas
ratio than all mixtures including C. papaya. Furthermore, all
plant mixtures caused a decrease in the ratio of CH4:SCFA
compared with pure C. papaya incubation (P,0·05). The
lowest ratios were found with the mixtures containing no
C. papaya; however, this resulted in a very low level of
SCFA production.The mixtures
increased ruminal NH3 concentrations compared with the
mixtures without this plant.
Non-additive effects of using plant mixtures
No non-additive effects on total SCFA concentration were
observed when combining any plant species (Table 4).
Binary mixtures including C. papaya did not result (P.0·05)
in non-additive effects on total gas production. However,
when C. papaya was combined with two or all three high-
production were observed. Combining C. papaya with any
Table 2. Effect of plant species or species combinations on in vitro incubation liquid SCFA profiles (observed values, n 8)
Molar proportion of total SCFA
Treatments* Total SCFA (mmol/l)C2
C2, acetate; C3, propionate; C4, butyrate; C5, valerate; C2:C3, acetate:propionate ratio.
a-iMean values within a column with unlike superscript letters were significantly different (P,0·05).
*C, Clidemia hirta; E, Eugenia aquea; P, Carica papaya; S, Swietenia mahagoni.
Table 3. Effect of plant species or species combinations on in vitro rumen fermentation measurements (observed values, n 8)
a-jMean values within a column with unlike superscript letters were significantly different (P,0·05).
*C, Clidemia hirta; E, Eugenia aquea; P, Carica papaya; S, Swietenia mahagoni.
A. Jayanegara et al.618
British Journal of Nutrition
of the other plants (C. hirta, S. mahagoni and E. aquea) led to
non-additive effects (at least at P,0·01) in terms of CH4emis-
sion, either when expressed as the absolute CH4amount or as
the CH4:SCFA and CH4:total gas ratios. Non-additive effects,
apparent as negative deviations from the values predicted
when assuming additive responses to the plant combinations,
were observed for mixtures of two, three or four plants, and
ranged from 5 to 15%, 2 to 15% and 7 to 10% for CH4,
CH4:SCFA and CH4:total gas, respectively. In contrast, no
non-additive effects (P.0·05) on CH4emissions were found
when combining the high-phenolic plants only, i.e. C. hirta,
S. mahagoni and E. aquea, in 2- or 3-fold mixtures. For the
molar amount of CH4produced per molar amount of SCFA
synthesised, all multiple combinations comprising C. papaya
resulted in non-additive effects (at least at P,0·05). For the
binary combinations, such non-additive effects were found
only for C. papaya together with S. mahagoni and for
C. hirta together with E. aquea (P,0·05). With regard to
NH3-related variables, non-additive effects (negative devi-
ation; P,0·01) of all mixtures of C. papaya combined with
the other plants were observed for NH3and NH3-N:dietary N.
The magnitude of the effects was considerable with deviations
of mostly more than 220%. No such effect was found
with combinations of the high-phenolic plants, except for
the mixture of S. mahagoni and E. aquea.
In natural environments, ruminants select diets from various
forage resources such as grasses, forbs, shrubs and tree
leaves. These plants may largely vary in their nutritional com-
position such as energy, protein, vitamins and minerals, and in
contents of plant secondary metabolites. Under such con-
ditions, interactions between different kinds of forages and
chemical constituents may occur, which might influence
intake behaviour, digestion, well-being and performance(13,18).
Niderkorn & Baumont(19)described that mixing two or more
different forages can even result in a different response for
various parameters (higher or smaller) from that expected if
just considering the average of the effects of the individual
plants. Yet few studies have specifically investigated the
non-additive effects of dietary ingredients, or plants character-
ised by specific compounds, on rumen fermentation and
digestibility in vitro(20,21)and in vivo(22,23). The results
reported so far are quite variable; some mixtures showed
non-additive effects (either favourable or unfavourable) and
others were simply additive.
There is still particularly little information available on the
non-additive effects of mixed feeds on CH4emissions(24,25).
Even less literature is available for mixtures involving plants
differing in phenolic profiles(12). This is of a particularly
high interest as feeds rich in total phenolics cannot be fed
alone and it depends on the nature of the relationship
between compounds whether their basic anti-methanogenic
potential(26,27)is enforced, unchanged or decreased by the
combination with plants of high forage quality. In this context,
a favourable non-additive effect would mean that the combi-
nation of a high-phenol ‘plant X’ with a high-quality ‘plant
Y’ would reduce the anti-methanogenic potential coming
from plant X to a proportionately lesser extent than the
feeding value of plant Y. This concept is simplistic in a way
because it cannot answer the question whether any non-
additive effects found are based on a non-linear dose–
response relationship with a single compound or whether
they emerge from an interaction of various plant compounds.
However, even though non-linearity cannot be traced back to
the level of compounds involved (which may also include sec-
ondary compounds other than phenolics), this perspective
might indicate the potential of complementarity on the level
of forages, which is the relevant level in practical livestock
feeding, particularly in smallholder farms of developing tropi-
Non-additive effects of plant mixtures on ruminal
methane formation in relation to the level of ruminal
Phenolics have been shown to reduce the population of
methanogenic archaea in the rumen(4)and, therefore, to miti-
gate CH4emissions. In addition, phenolics interact with other
chemical plant constituents such as protein and carbohydrates
(both fibre and non-fibre carbohydrates) via hydrogen
Table 4. Non-additivity of the effects of the plant mixtures (difference in observed values to expected values, in percentage of the expected
values†) on in vitro rumen fermentation parameters (n 8)
Treatments‡Total SCFA Total gasCH4
CH4:SCFA CH4:total gas pHNH3
*P,0·05, **P,0·01, ***P,0·001.
†Mean values of the individual plants present in the mixtures incubated individually.
‡C, Clidemia hirta; E, Eugenia aquea; P, Carica papaya; S, Swietenia mahagoni.
Non-additive effects in rumen methane mitigation 619
British Journal of Nutrition
bonds(28). Inhibition of carbohydrate digestion leads to a lower
formation of H2, which is a substrate for methanogenesis(29). If
this is the major way to mitigate CH4emissions, there is no
advantage in implementing this supplementation strategy
into practical feeding as productivity of the animals is concomi-
tantly hampered. The addition of high-phenolic plants to high-
quality forages might result in an even larger depression in
feed utilisation as nutrients of an inherently higher digestibility
might be transformed into indigestible compounds. This was
confirmed in the present study by the adverse non-additive
effects found with some mixtures in total gas production. How-
ever, such unfavourable non-additive effects mainly occurred
for mixtures containing S. mahagoni, which is rich in CT con-
tent (numerically also for the mixture with C. papaya). Apart
from the inhibitory effects of CT on the growth and activity
of rumen micro-organisms and the enzymes secreted(30),
a stable complex between CT and other chemical constitu-
ents(11,31)might explain non-additive effects which decrease
fermentative activity in combinations including S. mahagoni.
The SCFA amounts did not show this effect even in combi-
nations including S. mahagoni. This indicates that the hypoth-
esis that combinations with high-phenolic forages cause
non-additive decreases in ruminal productivity is neither fully
supported nor disproved by the present results. However,
the NTP in E. aquea are likely to possess no or a smaller bind-
ing capability. Also, complexes with HT are degradable under
ruminal conditions(30), which is relevant for C. hirta being rich
in HT. However, it should be noted that C. hirta, when fed at
a high proportion (0·5 parts of the total ration; air-dry basis),
may lead to hepato- and nephrotoxicity and be associated
with gastroenteritis in goats as HT may be absorbed(32).
Concerning CH4production relative to total gas or SCFA,
substantial non-additive effects were found when incubating
C. papaya together with the high-phenolic plants or their mix-
tures (not significant for some binary combinations concern-
ing SCFA). Even though there could be a certain bias in total
gas, as some of the gas could be CO2 released from the
buffer, both variables pointed to the same direction and thus
indicate that methanogenesis was generally more decreased
than fermentation productivity when adding the high-
phenolic plants to C. papaya. It appears that, in combination
with a high-quality plant, the phenolic compounds inhibit
CH4production more than expected but not at the cost of a
more than proportionate impairment of ruminal productivity.
In comparison with a high-phenolic plant, a high-quality
plant containing low levels of phenolics might not be affected
as much in the forage value, whereas the phenolics are still
able to exert their direct anti-methanogenic property. This
means that the stoichiometry of CH4 formation might be
affected. If CH4formation decreases more than the amounts
of either total gas or SCFA, both indicating fermentative pro-
ductivity, there has to be an alternative sink for the emerging
H2. The H2concentrations in the present study were lower by
a factor of 1000 compared with CH4, which is in accordance
with other studies and makes it generally difficult to explain
alterations in the concentration of CH4with those in H2(33).
The concentration of H2was never increased in treatments
with negative non-additive effects on CH4 traits. For the
combination with the strongest non-additive effects on CH4
(C. papaya, S. mahagoni and E. aquea), the amount of gaseous
H2also underwent a negative non-additive effect. Thus, there
seemed to have been generally less H2present in the incu-
bation unit. Lower ruminal H2concentrations can be a conse-
However, this was not the case with the treatments discussed
here. A shortcoming of the present study is that the concen-
trations of nutrients serving as fermentation substrates alter
too much between the different forage combinations and thus
do not allow for clear stoichiometric comparisons. Further-
more, non-phenolic plant secondary compounds, such as
papain in the papaya leaves, that have not been measured
could have interfered. Although the reasons for this disparity
of non-additive effects remain unclear, the results of the present
study might offer opportunities to develop diets that are conco-
mitantly effective for production and CH4mitigation.
According to Niderkorn et al.(12)and Robinson et al.(21)
non-additive effects of feeds on total gas production in vitro
may even be clearly more pronounced after a shorter incu-
bation time of 3·5 or 8h, when compared with 24h. A lack
of significant non-additive effects on CH4at 24h of incubation
was also observed by Goel et al.(34). In that study, combining
different levels of the leaves from Carduus pycnocephalus,
a plant containing phenolics in unknown concentration,
with hay or concentrate did not lead to differences between
the observed and expected values for in vitro CH4emissions.
The time effect, however, was not measured in the present
study, and all effects found were present after 24h.
Almost no non-additive effects were found for mixtures of
the high-phenolic plants except with regard to CH4:total gas
and CH4:SCFA. Hypothetically, non-additive effects for mix-
tures of plants containing high concentrations of different phe-
nols could be significant in two extreme cases: either the
effects of different phenolics are mutually strengthening their
activity, which would result in less CH4than expected from
incubating individual feeds, or they would counterbalance
each other, which would result in the opposite. The response
pattern in non-additive effects on CH4found in the present
study suggests that such effects are stronger when combining
individual plants that are distinctly different in their general
CH4 production potential than when combining forages
containing similarly high levels of potentially CH4-inhibiting
constituents. The latter plants already produced low CH4emis-
sions (2·5, 2·1 and 1·3ml CH4/200mg DM with C. hirta,
S. mahagoni and E. aquea, respectively, when incubated
alone. This favourably compares with the levels of 2·7, 1·6
and 1·0ml CH4/200mg DM described earlier in Jayanegara
et al.(10)). In both experiments, this was far below the level
found with C. papaya (8·1 and 7·4ml CH4/200mg DM,
respectively). Even though the high-phenolic plants differed
in their phenolic profiles, CH4production potential seems to
be especially determined by total phenolic contents rather
than by the specific phenolic fraction (NTP, CT or HT)(10)or
other potentially effective compounds.
Generally, the incubations resulted in SCFA concentrations
and a pH that are comparable with other experiments incubat-
ing high-phenolic feeds(10,35). The resulting pH was high, but
A. Jayanegara et al.620
British Journal of Nutrition
according to Van Kessel & Russell(36), this should not have
impaired methanogenesis. However, since the pH in the
rumen is expected to be clearly lower in vivo, this is one
factor which makes it necessary to confirm the results also
in in vivo experiments.
Non-additive effects of plant mixtures on ruminal
The non-additive effects of the plant mixtures on NH3vari-
ables followed the pattern found with the CH4-related vari-
ables, i.e. they were significant and negative for the mixtures
containing C. papaya, and even to a higher magnitude than
the CH4variables. Again, the largely contrasting NH3-gene-
rating properties between C. papaya (27mmol/l) and the
plants characterised by high total phenolics (ranging from
8 to 11mmol/l) might have opened room for generating
non-additive effects. Mixing forages with high levels of rumin-
ally degradable protein with those elevated in phenolics could
therefore be particularly useful to prevent excessive degra-
dation of protein into NH3as non-additive effects enhance
this process. The different groups of phenolics have different
protein-binding capacities. While NTP, by definition, do not
bind proteins, this is different with both HT and CT, and the
bonds formed with CT are particularly resistant(30). However,
in the present study, only limited non-additive effects on
NH3 production were found when incubating the plants
with different types of phenolics alone when compared with
incubations of the mixtures with C. papaya.
The general presence of the non-additive effects of plant
combinations with different phenolic profiles might also be
reflected by the fact that combinations of shrub species that
contained different classes of plant secondary metabolites
enhanced intake by ruminants(37–39). This could be explained
by the attempt to achieve a better nutrient balance, to find the
optimum medicinal benefit and to minimise the harmful
effects of each of the toxins(13,37,38,40).
Combining plants containing phenolics with the high-quality
leaves of C. papaya reduced ruminal CH4emissions more than
predicted from the arithmetic means. This was independent of
the respective phenolic profile. These non-additive effects of
plant combinations were also apparent in the ratio of
CH4:SCFA. Provided the confirmation of such effects in vivo,
this could mean that such mixtures of high-quality and high-
phenolic forages could help to mitigate CH4without corre-
spondingly extensively reducing ruminal nutrient utilisation.
The mixtures would also prevent excessive degradation of pro-
teininto NH3.Whetheror notnon-additiveeffectsofcombining
such plants can be recovered in vivo and on which compound
interactions they mechanistically rely merits further studies.
A. J. is grateful to the Indonesian Department of National Edu-
cation for providing a Directorate General of Higher Education
(DIKTI) scholarship. All authors are grateful to Carmen Kunz,
Muna Mergani and Irene Zbinden for their assistance during
the experimental period. The responsibilities of the authors
were as follows: A. J. performed the experiment and statistical
analysis, and wrote the manuscript; S. M. revised the manu-
script; E. W. collected the experimental plants and advised
on the writing of the manuscript; M. K. supervised the project
and revised the manuscript; F. L. was the project leader,
designed the experiment and revised the manuscript. The
authors declare that there is no conflict of interest.
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