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Feed additives as a strategic approach to reduce enteric
methane production in cattle: modes of action,
effectiveness and safety
M. Honan
A
, X. Feng
A
, J.M. Tricarico
B
and E. Kebreab
A,C
A
Department of Animal Science, University of California, Davis, 2111 Meyer Hall, One Shields Avenue,
Davis, CA, 95618, USA.
B
Innovation Center for US Dairy, 10255 West Higgins Road, Suite 900, Rosemont, IL 60018, USA.
C
Corresponding author. Email: ekebreab@ucdavis.edu
Abstract. Increasing consumer concern in greenhouse-gas (GHG) contributions from cattle is pushing the livestock
industry to continue to improve their sustainability goals. As populations increase, particularly in low-income
countries, the demand for animal-sourced foods will place further pressure to reduce emission intensity. Enteric
methane (CH
4
) production contributes to most of the GHG from livestock; therefore, it is key to mitigating such
emissions. Feed additives have primarily been used to increase animal productivity, but advances in understanding the
rumen has resulted in their development to mitigate CH
4
emissions. The present study reviewed some of the main feed
additives with a potential to reduce enteric CH
4
emissions, focusing on in vivo studies. Feed additives work by either
inhibiting methanogenesis or modifying the rumen environment, such that CH
4
production (g/day) is reduced. Feed
additives that inhibit methanogenesis or compete with substrate for methanogens include 3-nitroxypropanol (3NOP),
nitrates, and halogenated compounds containing organisms such as macroalgae. Although 3NOP and macroalgae affect
methyl–coenzyme M reductase enzyme that is necessary in CH
4
biosynthesis, the former is more specificto
methanogens. In contrast, nitrates reduce CH
4
emissions by competing with methanogens for hydrogen. However,
nitrite could accumulate in blood and be toxic to ruminants. Rumen modifiers do not act directly on methanogens but
rather on the conditions that promote methanogenesis. These feed additives include lipids, plant secondary compounds
and essential oils. The efficacy of lipids has been studied extensively, and although supplementation with medium-
chain and polyunsaturated fatty acids has shown substantial reduction in enteric CH
4
production, the results have been
variable. Similarly, secondary plant compounds and essential oils have shown inconsistent results, ranging from
substantial reduction to modest increase in enteric CH
4
emissions. Due to continued interest in this area, research is
expected to accelerate in developing feed additives that can provide options in mitigating enteric CH
4
emissions.
Keywords: greenhouse gases, methanogens, rumen function, ruminants.
Received 22 May 2020, accepted 23 November 2020, published online 2 February 2021
Introduction
The livestock sector is crucial for food and nutrition security
globally, with a projected increase of 80% in consumer
demand by 2050 for beef (Nadathur et al.2017).
Approximately 83% of global milk is produced from cattle
(Visioli and Strata 2014) and, by the end of the decade, milk
output is anticipated to have grown by 33% and 9% in
developing and developed countries respectively (OECD/
FAO 2018). Globally, beef is the third-most consumed
meat, contributing 320 million tons of product to world
food supply, representing 79% of total sourced meat (Opio
et al.2013; Ritchie and Roser 2019). Nutritional benefits from
ruminants are pronounced as they have the ability to convert
fibre-dense forages that are indigestible to humans into high-
quality bioavailable nutrient sources. In fact, 86% of the feed
consumed by livestock worldwide is not considered edible for
human consumption (Mottet et al.2017). At the same time,
ruminants occupy more land than do any other livestock
species and their enteric methane (CH
4
) emissions
contribute to total anthropogenic greenhouse gases (GHG;
Knapp et al.2014). Enteric CH
4
is under increased scrutiny
due to its heightened potency compared with carbon dioxide
(CO
2
) in the atmosphere, and the 39% it contributes to the
sector’s total emissions (Gerber et al.2013;IPCC2013).
Heightened attention on climate change by scientists,
governments and consumers is challenging the livestock
industry to reduce GHG emissions. Arguments for consumers
to shift towards plant-based diets have gained traction;
however, constructing diets on the basis of the level of
GHG emissions will not necessarily have a positive
CSIRO PUBLISHING
Animal Production Science Review
https://doi.org/10.1071/AN20295
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SPECIAL ISSUE
correlation with nutritional provision (Payne et al.2016).
Dietary manipulation has been studied over the past few
decades as a strategy to reduce enteric CH
4
emissions and
could be assimilated into management practices, notably
through feed additives (Cottle et al.2011). Feed additives
are used in livestock diets to improve feed-use efficiency,
quality of animal-source foods, and animal performance and
health. These additives include vitamins, amino acids, fatty
acids, minerals, pharmaceutical compounds, fungal products
and steroidal compounds. Recent advances in understanding
methanogenesis have led to the development and discovery
of feed additives that can reduce CH
4
emissions to varying
degrees. The present review aims to provide a concise
summary of feed additives currently available, or in
development, with some potential to reduce CH
4
emissions
from ruminants. The secondary objective of the review is to
summarise information on mode of action, efficacy, safety and
readiness for adoption of anti-methanogenic feed additives.
Although the focus is on feed additives tested in vivo,
some in vitro studies are also discussed if there is paucity
of in vivo trials for an additive or to help explain mode of
action.
Rumen methanogenesis
Methane production can be substantial in ruminants,
representing up to 12% of gross energy intake that could
potentially be utilised for physiological processes, but,
instead, is released into the atmosphere through eructation
(Beauchemin et al.2009a). However, CH
4
synthesis
represents a significant metabolic sink for reducing
equivalents (hydrogen, H
2
) that would otherwise accumulate
in the rumen and create an unfavourable environment for
fermentative digestion processes (Morgavi et al.2010).
Hydrogen itself does not accumulate due to methanogen
activity, instead, methanogens participate in interspecies H
2
transfer, and dispose of the reducing equivalents from other
metabolic processes (Bergman 1990; McAllister et al.1996).
Hydrogen synthesis is a self-limiting process that relies on
separate and distinct reducing equivalent consumption
pathways so as to continue production. Cellulose-degrading
activity in both bacteria and fungi increases in the presence of
methanogens, which contributes to the principle of rumen
syntrophic relationships (Bauchop and Mountfort 1981;
Sasaki et al.2012).
Rumen methanogenesis is performed strictly by archaea
(Hook et al.2010). A methanogenesis pathway is presented in
a simplified diagram (Fig. 1), which includes the convergence
of pathways known to occur in a Methanosarcina spp. Lambie
et al.(2015) categorised methanogens on the basis of
their metabolic pathways, as follows: hydrogenotrophic,
acetoclastic and methylotrophic that can yield CH
4
in the
rumen from Methanosarcina spp. Methanogens reduce CO
2
with H
2
(hydrogenotrophic), source a methyl group from
acetate (acetoclastic), or a methyl group from compounds
such as methanol, methylthiol, dimethylamine, and mono-,
di-, tri- methylamine (methylotroph). Formate contributes
to methanogenesis as an electron donor within the
Hydrogenotrophic
Carbon Dioxide (CO2)
Formyl-MF
Formyl-H4SPt
Methenyl-H4SPt
Methyl-H4SPt
Methyl-SCoM
Methane (CH4)
Methylene-H4(S)Pt
Upon inhibition of
methanogenesis the
fate of metabolic
hydrogen is redirected
VFA
Pathways
Introduced
electron
acceptors
Eructated
as gas
Acetate
Acetyl-Pi
Acetyl-CoA
Methylthiol
Methanol
Mono + Di + Tri
methylamine
M
e
t
h
y
l
o
t
r
o
p
h
i
c
A
c
e
t
o
c
l
a
s
t
i
c
A
l
t
e
r
n
a
t
i
v
e
H
2
s
i
n
k
s
Fig. 1. Simplified methanogenesis pathway from Methanosarcina barkeri CM
1
, adapted from Lambie et al.(2015). The three pathways depicted include
hydrogenotrophic (carbon dioxide utiliser), acetoclastic (acetate utiliser) and methylotroph (methyl-group utiliser), which all have the potential to donate a
methyl group and form methane.
BAnimal Production Science M. Honan et al.
hydrogenotrophic pathway, representing ~16–18% of CH
4
in
batch- and continuous-culture experiments (Seedorf et al.
2014; Ungerfeld 2015; Hungate et al.1970). Coenzyme M
requires a methyl group for the reduction to CH
4
, which is
provided through each of these pathways. Methane mitigation
could be achieved by directly targeting methanogens or
modifying the rumen environment to shift the metabolic
pathways away from methanogenesis or reduce substrates
for the archaea.
Rumen inhibitors
Feed additives classified as CH
4
inhibitors directly act on the
methanogenesis pathway (Fig. 1) in a way that can disrupt the
process and reduce CH
4
production (g/day). Methanogens
prevent H
2
accumulation in the rumen, which otherwise may
lead to adverse effects on fibre degradability and animal
performance (Ellis et al.2008). Given the importance of
efficient fibre digestion, the use of CH
4
inhibitors must
balance between reducing CH
4
production and avoiding
negative impacts on animal performance and welfare.
Inhibition of methanogenesis requires a redirection of
reducing equivalents, H
2
in this case, to alternative sinks,
instead of CO
2
, unless the inhibitor’s mode of action is a
highly competitive electron acceptor. Malik et al.(2015)
argued that H
2
clearance through pathways such as
reductive acetogenesis and propionogenesis also has the
advantage of energy conservation into end products such as
meat and milk. Several of these alternative sinks will be
reviewed herein and may also be implemented as
independent feed additives or with an inhibitor. Studies
have shown a decrease in CH
4
emissions paired with an
increase in H
2
emissions without the addition of an
alternative sink (e.g. Roque et al.2019b), indicating that
elevated H
2
concentration in the rumen may not necessarily
result in decreased fermentation, and hence, productivity.
3-nitroxypropanol (3NOP, marketed as Bovaer in the
European Union)
Methyl–coenzyme M reductase (MCR) is the enzyme that
catalyses the final step of the methanogenesis pathway from
intermediate methyl–CoM to CH
4
as illustrated in Fig. 1.Asa
nickel enzyme, MCR can catalyse this step only when its Ni
ion is in the +1-oxidation state and can be inactivated due to
the existing redox potential (Duin et al.2016). The position of
3NOP binding to an active site of MCR places the reducing
nitrate group in close proximity to Ni(I), a distance in which
electrons could be transferred. Although 3NOP inhibits
methanogenesis and reduces methanogen growth, it does
not negatively affect other microbial groups in the rumen
(Duin et al.2016).
More than 15 studies have been conducted using 3NOP,
showing a marked reduction of enteric CH
4
emissions with a
range of effectiveness. 3NOP added to ruminant diets in
small quantities has been shown to persistently reduce
enteric CH
4
emissions by inhibiting an important step in the
methanogenesis metabolic pathway, without apparent negative
side effects (Hristov et al.2015). Figure 2shows a forest
plot illustrating the effect sizes as a mean difference between
the control and treatment-group mean CH
4
production. For
example, Vyas et al.(2016) reported that with 0.2 g 3NOP/kg
dry-matter (DM) supplementation, CH
4
production in
backgrounding and finishing beef cattle reduced 37.6% and
84.3% compared with the control group, whereas Vyas et al.
(2018), using the same amount of supplementation in
Additives Counts Min Max
Seaweed
Fatty acids
3NOP
Oregano
Tannins
Nitrate
Agolin
Monensin
Biochar
Cinnamon
Garlic
Saponins 5
2
5
6
50
3
59
15
13
36
10
5 −269
−211
−240
−298
−188
−100
−42
−92
−9 −103.6 [−112.4, −94.8]
−84.5 [−90.0, −79.0]
−66.4 [−68.9, −63.9]
−48.0 [−53.3, −42.7]
−46.1 [−49.9, −42.3]
−32.8 [−34.1, −31.5]
−27.7 [−31.9, −23.5]
−15.6 [−16.9, −14.3]
−10.0 [−12.0, −8.0]
−10.0 [−12.7, −7.3]
−3.6 [−7.0, −0.2]
−3.3 [−6.2, −0.4]
−14
−1−21
−18
−8
−18
−120.0 −100.0 −80.0 −60.0 −40.0 −20.0 0.0
14
10
23
1
31
27
5
1
9
MD 95%Cl
Mean difference of methane production (g/d)
Fig. 2. Forest plot of mean difference (MD) of methane production for different feed additives, counts of studies, minimum and maximum of MD. Only
studies conducted in vivo were included in the analysis.
Reducing enteric methane through feed additives Animal Production Science C
backgrounding phase (0.2 g/kg DM) of beef cattle, found a
54.1% reduction in CH
4
production. These authors reduced
the level of supplementation of 3NOP to 0.125 g/kg DM
during the finishing phase and reported 53.8% reduction in
CH
4
production. There was also an improvement in gain-to-
feed ratio during treatment, with a 7% drop in DM intake
(DMI). Similarly, Martinez-Fernandez et al.(2018) reported a
decrease in CH
4
production of 38% and daily weight gain of
0.571 kg/day compared with the control in steers
supplemented with 0.30 g 3NOP/kg DM. Hristov et al.
(2015) demonstrated that CH
4
production in lactating cows
was reduced by 30% by feeding 3NOP at 0.04–0.08 g/kg DM
without affecting feed intake and milk production. Lopes et al.
(2016) reported a 31% decrease in CH
4
production in lactating
dairy cattle fed diets supplemented with 0.06 g/kg DM. In a
meta-analysis of the anti-methanogenic effects of 3NOP,
Dijkstra et al.(2018) reported that enteric CH
4
production
was reduced 39% in dairy and 22% in beef cattle at a mean
dose of 0.123 g/kg DM. Additive dose and the neutral
detergent fibre (NDF) content of diet had a significant
impact on the effectiveness of 3NOP in reducing enteric
CH
4
emissions. Furthermore, an increase in 3NOP dose of
0.010 g/kg DM from the mean dose further reduced CH
4
production by 2.56 0.55%. Similarly, Jayanegara et al.
(2018) reported that the methanogenic archaea population
was reduced through 3NOP supplementation and the
magnitude of reduction was positively correlated with
3NOP dose in small and large ruminants. Addition of
3NOP is also associated with shifting H
2
production in the
rumen and results in an increase in molar proportion for
propionate and decreases acetate production (Haisan et al.
2014;Kimet al.2019;Lopeset al.2016).
There are no known adverse effects of supplementing
3NOP on the animal or the subsequent product. The feed
additive 3NOP continues to be studied and, after approval by
regulatory bodies, it is expected to be on the market in the near
future.
Halogens
Plant species that accumulate halogenic compounds in their
tissues have been investigated for their potential to reduce
enteric CH
4
emissions. Halogens are elements that hold a
large, negative electron affinity and seek to combine with
other compounds to reach stability through satisfaction of the
valence shell in the rumen environment (Gribble 2004).
Bromoform and chloroform are halogens that have been
found to interfere directly with the methanogenesis pathway
by serving as competitive inhibitors (or analogues) of the
MCR, preventing the final catalysis step (Goel et al.2009).
The mode of action is through reacting with reduced vitamin
B12 and inhibiting the cobamide-dependent methyl-transferase
step of methanogenesis (Wood et al.1968; Chalupa 1977). The
B12-dependent methyl-transferases also play an important role in
one carbon metabolism in acetogenic bacteria (Banerjee and
Ragsdale 2003), and, therefore, halogenated compounds may
have an effect on reductive acetogenesis.
At supplementation level of 1.50–1.59 g/kg DM (2.6 g/100 kg
liveweight; mean liveweight = 288 kg) of chloroform–
cyclodextrin, steers have demonstrated a 30–35% reduction
in enteric CH
4
production, with no detectable differences in
rumen fermentability (Martinez-Fernandez et al.2016). Steers
dosed daily with 0.267 g/kg DM of chloroform were shown to
decrease 94–95% of CH
4
production within 4–5 days of
treatment. However, CH
4
production has been shown to
slowly recover to 62% of the pre-treatment levels by Day
42 of treatment (Knight et al.2011). The macroalgae species
Asparagopsis taxiformis and A. armata have been evaluated
for their mitigation potential both in vitro and in vivo (Roque
et al.2019a,2019b). Asparagopsis spp. contain relatively
high concentrations of bromoform and other halogenated
compounds such as bromochloromethane (Paul et al.2006;
Machado et al.2016). An in vitro trial analysing effectiveness
across seaweed species found A. taxiformis to be the most
effective species among 20 freshwater and marine macroalgae
in reducing CH
4
output (98.9%), but also reduced total gas
production (62%), likely indicating inhibition of digestion
(Machado et al.2016). Increasing the dose to 5% in vitro,
Roque et al.(2019a) reported a 95% reduction in the level of
CH
4
production. Three papers have been published so far,
reporting the effect of Asparagopsis spp. in sheep, dairy and
beef cattle in vivo.Liet al.(2018) supplemented A. taxiformis
at 67.5 g/kg DM (30 g/kg of organic matter, OM) in sheep diets
and reported a reduction of up to 80% in enteric CH
4
production. However, rumen volatile fatty acid (VFA)
concentrations in the 0%, 0.5%, 1.0%, 2.0% and 3.0%
macroalgae inclusion groups declined from 92.0, to 86.5,
74.9, 69.1 and 65.4 mM respectively. Reductions in VFA
concentrations are not desirable as they provide energy to
the ruminant. In lactating dairy cattle, Roque et al.(2019b)
observed up to 67.2% reduction in CH
4
intensity (g/kg milk
produced) using A. armata at an inclusion rate of 18.3 g/kg
DM (10 g/kg of OM). In Brangus beef cattle, Kinley et al.
(2020) reported a reduction of enteric CH
4
production of up to
98% by supplementing a feedlot diet with A. taxiformis at 3.26
g/kg DM (2 g/kg of OM). In addition, there was an
improvement of 42% in average daily gain with a
supplementation level of 1.63 g/kg DM (1.0 g/kg of OM)
and it went up to 53% at an inclusion rate of 3.26 g/kg DM (2.0
g/kg of OM). The study by Kinley et al.(2020) reported a
greater effectiveness at a lower dose than did that of Roque
et al.(2019b), which was likely due to the large differences in
the bromoform concentration in A. taxiformis and A. armata,
while also acknowledging the inclusion of monensin in the
Kinley et al.(2020) experimental diets. The bromoform
concentration in Roque et al.(2019b) study was 1.32 mg/g
compared with 6.55 mg/g in the Kinley et al.(2020)
study.
Sourcing naturally occurring halogens circumvents the
need to use synthetic halogens. Historically, these synthetics
have had detrimental effects on the environment (Gribble
2004). Kinley et al.(2020) and Roque et al.(2019b) tested
for residual bromoform content in meat (or edible offal) and
milk respectively. In both cases, concentrations of bromoform
were either undetectable or not significantly different from
the control, suggesting no safety issues arising from the
active ingredient. At present, A. taxiformis is not produced
commercially; so, accessibility is an issue. The use of
DAnimal Production Science M. Honan et al.
macroalgae also needs to be approved by regulatory agencies
before widespread use by producers.
Nitrate
Adding nitrate to ruminant diets can be an effective CH
4
mitigation strategy because nitrate competes with
methanogens for H
2
in the rumen. Nitrate (NO
3
–
) is reduced
to nitrite (NO
2
;NO
3
+H
2
!NO
2
+H
2
O) and further to
ammonia (NH
4+
;NO
2
+3H
2
+2H
+
!NH
4+
+2H
2
O) by rumen
microbes. However, small quantities of nitrous oxide may also be
produced (Latham et al.2016). This pathway is highly
competitive with methanogens for H
2
utilisation in the
rumen due to greater changes in Gibbs energy than with
methanogenesis (CO
2
+4H
2
!CH
4
+2H
2
O) pathway
(Villar et al.2020). The result is a redirection of H
+
flow
from CO
2
to nitrate reduction, thereby reducing the generation
of CH
4
(Olijhoek et al.2016).
About 24 in vivo studies showed that the efficacy of nitrate
additives varied widely, ranging from +1.25% to 29.8%, and
may be affected by several factors. A meta-analysis conducted
by Feng et al.(2020) investigated the potential explanatory
variables for anti-methanogenic effects of in vivo nitrate
supplementation in cattle. These included DMI, roughage
proportion, NDF content, crude protein (CP) content,
bodyweight, nitrate dose, cattle type, and CH
4
measurement
methods. The authors reported that nitrate significantly
reduced CH
4
emissions in a dose–response manner and the
mitigating effect increased with the level of nitrate inclusion.
Methane production reduced 14.6% in cattle supplemented
with nitrate at 17.7 g/kg DM (Feng et al.2020). Hulshof et al.
(2012) reported that nitrate supplementation increased
ammonia-nitrogen concentrations in the rumen by 34%,
decreased propionate concentrations by 16%, but did not
affect the total VFA concentrations. Persistency of nitrate
was tested by van Zijderveld et al.(2011a), by including 21
g/kg DM during four successive 24-day periods and a
consistent 16% reduction in daily CH
4
production (g/day)
and yield (g CH
4
/kg DMI) was demonstrated. An additive
effect of nitrate and linseed oil was reported by Guyader et al.
(2015a) in multiparous, non-lactating dairy cattle. These
authors reported that adding 4% linseed oil to 3% calcium
nitrate further reduced CH
4
production from 22.8% (nitrate
only) to 33.0%.
Concerns about the toxicity of the intermediate product of
nitrate, namely nitrite, to ruminants necessitate management,
as animal poisoning may occur via methaemoglobinemia
(Latham et al.2016). Nitrite is toxic in blood because it
converts haemoglobin to methaemoglobin, which is
incapable of carrying oxygen. Blood methaemoglobin
concentrations in ruminants increase with a greater nitrate
consumption and could cause nitrate poisoning (Lee and
Beauchemin 2014). Apparent nitrate-poisoning symptoms
such as depressed feed intake, slow or no weight gain,
reproduction failure, respiratory distress, coma and death
have been reported in previous studies with methaemoglobin
concentrations of 30–40% of total haemoglobin (Bruning-Fann
and Kaneene 1993). Lee and Beauchemin (2014) discussed
several critical factors related to nitrate toxicity, including the
dietary nitrate concentrations, nitrate consumption rate,
incomplete reduction of nitrate and nitrite to ammonia, and
rumen outflow rates. Toxic effects of nitrite on the populations
of main cellulolytic bacteria, which may be caused by the
negative effects of nitrate/nitrite on cellulolytic and xylanolytic
activity, have also been observed (Iwamoto et al.2002; Asanuma
et al.2015; Granja-Salcedo et al.2019). However, the risk of
nitrate toxicity can be reduced by gradual acclimation of
ruminants to dietary nitrate or utilisation of encapsulated
nitrate (Lee and Beauchemin 2014). Currently, nitrate
inclusion may not be advisable in commercial operations due
to its potential toxicity. However, a denitrifying probiotic,
Paenibacillus fortis, that can enhance nitrite detoxification in
nitrate treatedruminants, has been identified (Lathamet al.2019).
If successful, nitrate and the probiotic might be a practical
mitigation strategy to reduce CH
4
production from ruminants.
Rumen modifiers
The rumen environment can be modified with feed additives to
limit the growth of methanogens and to suppress CH
4
production, without targeting the specific methanogenesis
pathway. The factors influencing CH
4
production include
those involved in H
2
and carbohydrate metabolism
(Morgavi et al.2010). Understanding rumen metabolic
processes that affect CH
4
formation is still advancing;
however, feed additives were used to modify the rumen
environment to reduce CH
4
production without
compromising animal health or productivity. This section
discusses feed additives that can potentially reduce CH
4
production by modifying the rumen environment.
Dietary lipids
Dietary lipids modify the rumen environment in several
ways, including (1) toxic characteristics on methanogens
and protozoa, (2) hydrogenation of unsaturated fatty acids
(alternative H
2
sink) and (3) shifts to propionic production,
leading to reduction of enteric CH
4
production (Johnson and
Johnson 1995; Beauchemin et al.2008,2009b). Efficacy of
lipids to reduce CH
4
emissions are dependent on the form and
level of supplementation, as well as the source and fatty acid
profile (Beauchemin et al.2008; Eugène et al.2008). Several
meta-analyses were conducted to estimate the impact of
dietary lipids on CH
4
production (e.g. Beauchemin et al.
2008;Eugèneet al.2008; Martin et al.2010). For example,
Beauchemin et al.(2008) evaluated 17 studies in sheep, beef
and dairy cattle and reported a 5.6% reduction in CH
4
production for every 1% additional inclusion of
supplemental fat. In dairy cattle, Eugène et al.(2008)
reported a decrease of 9% through lipid-supplementation
(average 6.4%) compared with control diets (average 2.5%),
mostly as a consequence of reduced DMI. Similarly, Patra
(2013) reported 3.77% decline in CH
4
emissions for each
percentage inclusion of lipid in dairy cattle diets. Prediction
inconsistencies by the inclusion of supplemental lipid are
likely to be due to differences in lipid source and diet
composition. In a review, Rasmussen and Harrison (2011)
reported that the most effective fatty acid profiles that reduce
CH
4
production were medium-chain (8–16 carbon chains;
Reducing enteric methane through feed additives Animal Production Science E
MCFA) and polyunsaturated (PUFA) fatty acids. However,
reductions in DMI due to high levels of dietary lipids are
well characterised and ration formulation programs often are
set not to exceed 6–7% of total DMI (NRC 2001).
Medium-chain fatty acids
These include lauric, myristic, capric and caprylic acids
(Hollmann et al.2012). In vitro studies have reported coconut
oil, which contains 75% of MCFA, to reduce CH
4
production
by 43–85% (Dong et al.1997;Machmülleret al.1998).
Application of coconut oil in in vivo trials also showed
similar patterns in CH
4
reduction (Hollmann et al.2012).
Ruminants fed diets containing 13, 27 and 33 g coconut oil/
kg DM had 3%, 37% and 45% reduction in CH
4
output
compared with the control respectively. DMI, solids-
corrected milk yield, and milk fat yield (no difference
between the two greatest levels of inclusion on milk fat
yield) decreased linearly with an increase in coconut oil
application. Inclusion of myristic acid at a rate of 50.0 g/kg
DM in dairy cattle diets reduced CH
4
production by 36%, but
also reduced milk fat by 2.4%, with a tendency to reduce DMI
(Odongo et al.2007). Lauric acid had no negating effects on
methanogenesis in dairy cattle when they received it at 10.0 g/
kg DM (Hristov et al.2009). Within the same trial, the
treatment group receiving 21.6 g/kg DM of coconut oil
reduced their CH
4
production by 61% compared with the
control.
Polyunsaturated fatty acids
Polyunsaturated fatty acids have also been shown to reduce
CH
4
production. For example, Bayat et al.(2015)found
that enteric CH
4
production reduced by 29.5% with
supplementation of 60 g/kg DM of camelina oil, but other
parameters such as milk yield and milk components were
compromised. In contrast, Duthie et al.(2018) did not find
significant differences in enteric CH
4
production in steers fed
increasing amounts of dietary lipid sourced from maize
distillers dark grains, which increased diet ether extract
from 24 to 37 g/kg DM for 17 weeks. Supplementation of
diets with cottonseed oil has been shown to decrease enteric
CH
4
production by ~42% (Nogueira et al.2020). These
authors suggested that bio-hydrogenation of lipids served as
an alternative H
2
sink, and with each percentage point of lipid
added to the diet, CH
4
production was reduced by 8%. Further
characterisation and understanding of the impact and longevity
of dietary lipid inclusion on methanogenesis would be
valuable in selecting plant sources and estimating their
impact. Dietary lipid additives (both MCFA and PUFA)
show substantial decreases in CH
4
production with a wider
range of effectiveness compared with other feed additives
(Fig. 2).
Probiotics
Microorganisms included in diets are often referred to as
probiotics, cultures, or direct-fed microbials. Introducing
microorganisms to a digestive microbiome is practiced on
farms to influence the rumen flora for improved digestion.
Results of feeding fungi, yeast or bacteria to reduce CH
4
production have not been consistent in studies conducted
in vitro or in vivo. Application of live yeast cultures (various
strains of Saccharomyces cerevisiae) have not been shown to
significantly change CH
4
production, rumen fermentation or
apparent total tract nutrient digestibility in dairy cattle (Bayat
et al.2015). Additionally, inclusion of either a dead or live
form of S. cerevisiae has little to no impact on nutrient
digestibility or rumen fermentation patterns in beef heifers
(Vyas et al.2014). A meta-analysis by Darabighane et al.
(2019) using data from 1990–2016 observed no significant
reduction in CH
4
production through the use of probiotics.
Introducing propionate-producing bacteria has been
evaluated as a possible solution because propionate
production consumes H
2
as a reducing equivalent and,
thereby, competes with methanogenesis (Ungerfeld 2013).
This has not been effective with all strains of bacteria but
Propionibacterium thoenii T159 reduced CH
4
production
by 20% and increased VFA production by 21% in a study
that screened 31 different strains within in vitro models (Chen
et al.2020). However, in lactating primiparous cows,
P. freudenreichii 53-W was shown to increase CH
4
production by 27% (Jeyanathan et al.2019). The mechanisms
of reduction in CH
4
production (if any) are still unknown and
could be either directly by microbes or indirectly through
metabolites that affect the rumen microbiome (Doyle et al.
2019). Jeyanathan et al.(2019) found no effect on CH
4
output
when feeding Lactobacillus pentosus D31, and L. bulgaricus
D1 in vivo. Currently, there is no concrete evidence that
probiotics are an effective method of CH
4
mitigation.
Acetogenesis, or reductive acetogenesis, is another H
2
-
utilising metabolic pathway in which acetogens utilise CO
2
and H
2
as substrates to produce acetate. While more
prevalent in other mammalian guts, acetogens cohabit with
methanogens in the rumen, but are either lacking a substantial
population density, preferred environment conditions, or the
competitiveness to be the favourable pathway of H
2
‘disposal’
(Joblin 1999). Redirection of H
2
into the acetogenesis pathway
to yield acetate would allow the recapture of energy compared
with the loss due to methanogenesis. Enhancing this pathway
in the rumen may be approached by sourcing acetogens from
other ecosystems and transplanting them into the rumen
(Gagen et al.2014) or uncovering a method to enhance the
existing rumen acetogen population if they can outcompete
native methanogens.
Biochar
Organic matter that has undergone pyrolysis, commonly
known as biochar, has a wide range of impacts on livestock
systems due to its unique characteristics. Biochar has been
utilised for generations as a remedy for digestive disorders
and is sourced by the livestock industry to address issues
surrounding animal husbandry, metabolism and waste
management (Kalus et al.2019; Schmidt et al.2019).
Abatement of CH
4
production through the application of
biochar has been shown in soil (Yu et al.2013)and
compost (Sonoki et al.2013). Considering that there is
already an existing market for biochar as a beneficial feed
additive, in vivo evidence for GHG mitigation will be
FAnimal Production Science M. Honan et al.
significant (Schmidt et al.2019). Possible mechanisms have
been elucidated through a study that observed that application
of biochar to paddy soils stimulated methanotrophic
proteobacteria and reduced CH
4
, despite methanogens also
being stimulated (Feng et al.2012). Additionally, biochar may
provide a habitat for methanogens or possibly absorb gases
when consumed due to its porous nature, but the mechanisms
of action for CH
4
mitigation in cattle are not well understood
(Terry et al.2019;Manet al.2020).
Rice husks sourced for biochar and fed at an inclusion rate
of6g/kgDMreducedCH
4
production by 22%, increased
liveweight gain by 25%, and had no impact on DMI over a
98-day period (Leng et al.2012). Biochar supplemented at
8g/kgDMreducedCH
4
production by 9.5% in growing steers
and 18.4% in finishing steers (Winders et al.2019). Contrary
to these findings, inclusion levels of ‘pine-enhanced biochar’
at 5, 10 and 20 g/kg DM in the diets of Angus ·Hereford
heifers did not reduce CH
4
emissions (Terry et al.2019).
However, it altered the microbiota, notably selecting against
Fibrobacter species, which is one of the dominating phyla of
the rumen responsible for cellulose degradation (Béra-Maillet
et al.2004). The wide variation in effectiveness precludes
biochar as proven feed additive to reduce CH
4
production at
present. More research, particularly in vivo, is required to
understand the conditions under which biochar can mitigate
CH
4
production.
Ionophores
Ionophores, such as monensin, alter rumen microbial populations
to improve digestive efficiency by depriving methanogens of
substrates that are typically provided by Gram-positive bacterial
and ciliate protozoal populations(Russell and Strobel 1989;Hook
et al.2010). This fermentation shift favours the production of
propionateover acetate, which reduces theamount of H
2
available
for methanogens.
A meta-analysis by Appuhamy et al.(2013) quantitatively
determined the impact of monensin in cattle. In beef cattle
supplemented with monensin at an average monensin dose of
0.032 g/kg DM, CH
4
production was reduced by 19 g/day,
which was further reduced as the NDF content of the diet
increased. In dairy cattle, CH
4
production was reduced by 6 g/
day at the same average dose and was further reduced as the
dietarylipidcontentincreased.Appuhamyet al.(2013)concluded
that although there were reductions in CH
4
production through
supplementation with monensin, the effect was transient, lasting
~6 weeks. In contrast, Benchaar (2020) reported no suppression
effect of monensin on CH
4
output when it was administered to
dairy cattle (0.024 g/kg DM), but there was an increase in the
proportion of a biohydrogenation intermediate, thus altering
rumen metabolism patterns.
The antimicrobial nature of ionophores has caused a
concern to human health (Guan et al.2006;Hooket al.
2010). Long-term use of ionophores is limited due to a low
efficacy, transient nature and safety concerns.
Plant secondary compounds
Plant secondary compounds are primarily synthesised in
response to their environmental conditions and not for specific
physiological function (Morrissey 2009). Some plant secondary
compounds that may possess antimethanogenic properties are
variable in composition due to environmental condition in
which they are grown. Seasonal variation, pollution, diseases,
pests, storage, injuries and pollination activity influence
secondary-compound production and composition (Figueiredo
et al.2008). These compounds are not commonly extracted or
isolated before feeding to ruminants because of time and cost
considerations, which may contribute to their concentrations
being inconsistent. These obstacles present a challenge in
determining or predicting efficacy.
Tannins
Tannins are soluble, phenolic compounds that accumulate
within plant tissues likely due to ongoing metabolic processes
and contribute to the plant defence system (Swanson 2003).
The CH
4
mitigation mechanisms of tannins are not well
understood but may be due to a combination of factors,
including a reduction in fibre digestibility (decrease in H
2
production) or a direct inhibition of methanogens (Tavendale
et al.2005).
Jayanegara et al.(2012) conducted a meta-analysis
describing the relationship between rumen CH
4
formation
and the level of dietary tannin (hydrolysed or condensed)
inclusion between in vivo and in vitro models. These
authors reported that low levels of inclusions of tannins in
animal experiments often yielded inconsistent results on CH
4
production, but that variability seemed to diminish at higher
doses, leading to setting the threshold for detecting treatment
differences in animals to be >20 g/kg DM of tanniferous
inhibitors. Furthermore, reduction in CH
4
production was
often followed by a suppression in OM and fibre
digestibility. Methane measurements from goats fed Kobe
lespedeza, a forage containing condensed tannins at 151,
101 and 49.9 g/kg of DM led to a 54%, 52% and 32%
reduction compared with the control group respectively
(Animut et al.2008). Supplementing beef cattle diets with
tannic acid at a 26 g/kg DM inclusion rate, CH
4
production
decreased 33.6%, but the digestibility of DM and CP, and the
concentration of VFA were negatively affected (Yang et al.
2017). Investigating different tannin-containing hays, Stewart
et al.(2019) found small burnet (Sanguisorba minor)fedto
Angus cows and heifers to reduce CH
4
production in
comparison to a diet containing alfalfa hay (209 vs 289 g
CH
4
/day respectively). However, CP and DM digestibility was
affected negatively.
Grape marc or pomace contains high concentrations of
condensed tannins and it is a readily available biowaste
from the viticulture industry. Moate et al.(2014) fed dried
pelleted (274 g/kg DM) or ensiled grape marc (269 g/kg DM)
to dairy cattle and found that the dried form was the most
effective in reducing CH
4
. The authors reported that the CH
4
production in dairy cattle fed the control, dried and ensiled
grape marc was 470, 375 and 389 g CH
4
/day. More recently,
Caetano et al.(2019) fed ensiled grape marc at a rate of
31.2 g/kg DM, which equates to ~3–4 kg/day of ensiled grape
marc (estimated on the basis of reported DMI). Treatment
inclusion in the study of Caetano et al.(2019) study led to a
Reducing enteric methane through feed additives Animal Production Science G
14% reduction in CH
4
production; however, it ultimately
decreased the energy availability of the diet due to the
greater contents of lignin and acid detergent fibre in the
treatment diet. Cattle have exhibited intoxication
sensitivities to tannins, particularly if diets do not meet
nutrient requirements for growth or milk production.
However, such issues can be avoided through appropriate
dosages and adaptation periods paired with properly
formulated diets (Doce et al.2013).
Flavonoids
Flavonoids are not known to have extensive CH
4
reduction
potential, but anti-microbial properties of the compounds have
been reviewed (Patra and Saxena 2010). Several in vitro trials
(Oskoueian et al.2013;Kimet al.2015) have been conducted
to gain a better understanding of antimicrobial characteristics
and its relation to methanogenesis, but studies utilising in vivo
models are scarce. Kim et al.(2015) studied the mitigation
potential of four plants containing flavonoids in vitro,byusing
rumen fluid sourced from a single cow. In all treatments, CH
4
production was reduced by 39–48%; however, results such as
this have not yet been translated into animal models.
Flavonoids derived from mulberry leaves (~1.3 g/kg DM)
did not influence methanogenesis to a detectable level in
sheep, but they increased digestibility (Chen et al.2015).
Rutin trihydrate, a flavonoid, was given to dairy cattle at a
dose of 100 mg/kg bodyweight, which led to an elevated
plasma glucose, b-hydroxybutyrate and albumin, but did not
suppress CH
4
production (Stoldt et al.2016).
Saponins
Saponins have been studied for their capacity to alter rumen
fermentation by reducing protozoal communities, thus
lowering H
2
availability and the production of CH
4
(Hess
et al.2003). Saponins are commonly found in low quantities in
legume plants such as kidney beans, soya beans, chickpeas and
green peas (Shi et al.2004). Holtshausen et al.(2009)
conducted a two-part study on saponins derived from Yucca
schidigera and Quillaja saponaria and their effect on CH
4
production in vitro and in vivo. Inclusions of 15, 30, or 45 g/kg
DM of Y. schidigera and Q. saponaria decreased CH
4
production ranging from 6 to 26% in vitro. However,
in vivo study in dairy cattle using whole-plant Y. schidigera
and Q. saponaria powders at 10 g/kg of DM did not show
an impact on rumen fermentation. Cross-bred cattle
supplemented with soapnut, a saponin-containing plant, did
not have significant reductions in CH
4
production
(Poornachandra et al.2019). Tea saponins offered to ewes
led to a decrease in CH
4
production if scaled to metabolic
weight; otherwise, no differences were observed in absolute
values (Liu et al.2019). The same supplement was offered
to steers at 2.44 and 3.85 g/kg DM, but no impact on gas
output was observed (Ramírez-Restrepo et al.2016). Lack of
results in reducing CH
4
production may be linked to low
concentrations of saponins within additives. However, in
some circumstances, high concentrations of saponins have
been linked to bloat through foaming properties, but no
strong conclusions have been drawn (Lindahl et al.1954;
Sen et al.1998). Low-level inclusions may have antiprotozoal
and mild antibacterial characteristics and can be incorporated
into livestock diets through a variety of plant options.
Essential oils
Essential oils (EO) are naturally occurring chemical
compounds extracted from plants and used in fragrances
and cosmetics and, to a lesser extent, pharmaceutical
products for humans and animals. Volatile in nature, the
EO contribute to the phenotypic expression of the plant
including colour and scent (Edris 2007; Benchaar et al.
2008). Consumption of EO has been observed to affect
rumen microbial communities and fermentation patterns in
a varying manner, depending on the EO source (Benchaar and
Greathead 2011). Many EO hold a high affinity for lipid and
bacterial membranes, leading to disruption, but the broad
antimicrobial effect is likely to be due to a combination of
mechanisms (Helander et al.1998). EO are non-specificin
nature; therefore, there is a concern for their inclusion in diets
because they may affect favourable microbe populations,
leading to a decrease in feed efficiency. Numerous plants
such as cinnamon, lemongrass, ginger, garlic, juniper
berries, eucalyptus, thyme, citrus, oregano, mint, rosemary
and coriander have been screened in vitro (Becnhaar et al.
2008; Nanon et al.2015). However, only few have been
studied in vivo. Some studies include the whole plant
(Olijhoek et al.2019) into a diet, while other extract the
EO before inclusion in a more concentrated treatment
(Lejonklev et al.2016), which introduces another level of
variability.
Oregano contains EO compounds carvacrol and thymol that
may stimulate general antimicrobial properties in the rumen
(Kolling et al.2018). Only two in vivo studies (Tekippe et al.
2011;Hristovet al.2013) have shown reduction of CH
4
production of up to 40% in dairy cattle. Hristov et al.
(2013) did not observe any adverse effects of
supplementation (8.7, 18.9 and 28.2 g Origanum vulgare
leaves/kg DM) on feed efficiency, rumen pH or VFA
concentrations. In contrast, several other studies have
shown no significant impact of supplementing oregano on
CH
4
production. For example, lactating dairy cattle
supplemented with oregano oil and carvacrol at 0.05 g/kg
DM did not express any anti-methanogenic properties
(Benchaar 2020). Kolling et al.(2018) reported a reduction
in CH
4
yield (in g/kg digestible DMI), but no reductions
surrounding other CH
4
emission parameters such as
protozoal count, by using 0.56 g oregano extract/kg DM in
lactating dairy cattle. Olijhoek et al.(2019) reported no
significant reduction in dairy cows supplemented with either
18–53 g oregano plant/kg DM from Origanum vulgare ssp.
vulgare containing 0.12% EO of oregano DM, or 7–21 g
oregano DM/kg of DM from Origanum vulgare ssp. hirtum
containing 4.21% EO of oregano DM. The authors speculated
that the differences in reported effectiveness could be related
to the duration of measurement (1–8 h post intake in those that
reported reductions vs >24 h in studies with no effect). The
observation by Hristov et al.(2013) who reported a linear
decline in effectiveness after feed intake lends support to
HAnimal Production Science M. Honan et al.
measurement duration contributing to differences in reported
effectiveness.
Garlic (Allium sativum) contains organosulfur compounds,
specifically diallyl disulfide, as its main EO component.
Organosulfur compounds are suspected of having a toxic
effect on the enzyme system of the methanogenic archaea,
inhibiting their activity, while also suppressing protozoal
populations (Busquet et al.2005a; Soliva et al.2011).
Soliva et al.(2011)reporteda91%reductioninCH
4
with
300 mg/L garlic oil in vitro,associatedwithanincreasein
bacterial counts and reduction in protozoa. Similarly, Busquet
et al.(2005a) observed a 73.6% reduction in CH
4
production
in vitro by using similar concentrations of garlic oil. However,
most in vivo cattle studies have not found an impact of garlic
oil on CH
4
production. For example, van Zijderveld et al.
(2011b) used diallyl disulfide at 0.056 g/kg DM in dairy cattle
and observed no reduction in CH
4
production. Staerflet al.
(2012) using dried garlic bulbs (treatment standardised for
15 g allicin/kg DM) in feedlot cattle reported no significant
effect on CH
4
production measured at 5, 9 and 11 months of
age. Similarly, Meale et al.(2014) reported no detectable
differences in enteric CH
4
or CO
2
production in animals
supplemented with garlic oil (15 g allicin/kg DM). Sheep
models have reported similar results of no detectable
difference in enteric CH
4
production (Patra et al.2011;
Klevenhusen et al.2011); however, goat models supplemented
with L propyl–propane–thiosulfinate, another organosulfur
compound found in garlic, suppressed CH
4
production by
roughly 33% (Martinez-Fernandez et al.2013). Nevertheless,
in their subsequentexperiment, Martinez-Fernandez et al.(2014),
usingthe same compound in goatsin vivo,did not findasignificant
reduction in enteric CH
4
production.
Lemongrass (Cymbopogon spp.) has been assessed in vitro
for potential antimicrobial effects due to citral, an aldehyde
sourced from the EO fraction contributing to aromatic
characteristic of the plant (Pawar et al.2014;Jochet al.
2016; Singh et al.2018). While CH
4
was not measured,
Wanapat et al.(2008) detected an improvement in
microbial protein supply, DM digestibility and microbial
populations when Brahman-native beef cattle consumed
18.5 g lemongrass powder/kg DM. In lactating Barki goats,
4 g/kg DM elicited a slight increase in protozoal counts and
CH
4
production (Khattab et al.2017).
Supplementing cinnamaldehyde and cinnamon oil
(containing 78% cinnamaldehyde) to dairy cattle diet at
inclusion rates ranging from 0.003 to 0.16 g/kg did not reduce
CH
4
production (Benchaar 2016). Methanogen numbers
decreased in a study adding 0.5 g/kg DM of cinnamon oil, but
the study did not measure gases directly, so any CH
4
reduction
was speculative (Khorrami et al.2015). Eugenol, an active
EO component of cinnamon, was added to diets at 0.025, 0.050
or 0.075 g/kg DM, but no treatment group demonstrated a
difference in enteric CH
4
compared with the control (Benchaar
et al.2015). Shifts away from acetate production and towards
propionogenesis have been observed in artificial conditions
when cinnamon-sourced additives were introduced (Busquet
et al.2005b). Inclusion of EO in livestock diets has not
rendered any safety concerns for animal husbandry or
consumption of subsequent products.
Essential oil blends
Taking advantage of the unique composition among plants,
some studies have used an EO ‘blend’or ‘complex’containing
extracts from multiple plants. The antimicrobial nature of a
variety of the EO may imply a capacity to modify rumen
fermentation. EO blends have demonstrated a greater feed
efficiency and a higher production of energy-corrected milk in
dairy cattle through modification of rumen fermentation
(Elcoso et al.2019;Silvaet al.2020). Blends have become
commercially available, typically containing at least two
different EO. For example, Agolin Ruminant (Agolin,
Bière, Switzerland; AR) contains a blend of eugenol,
geranyl acetate and coriander EO. Agolin Ruminant is an
antimicrobial EO product and has shown 20% reduction in
CH
4
intensity in dairy cattle (Hart et al.2019). Klop et al.
(2017) alternated AR (0.17 g/kg DM) with lauric acid (0.65 g/
kg DM) for 2-week periods over 10 weeks, but CH
4
production
was not altered. Elcoso et al.(2019) estimated 15% lower CH
4
production in lactating dairy cattle consuming AR. Castro-
Montoya et al.(2015) fed 0.0128 and 0.0240 g AR/kg DM to
dairy and beef cattle respectively, but detected only tendencies
towards CH
4
reduction in both groups, with no significant
differences occurring.
Mootral
is synthesised from natural products including
garlic- and flavonoid-containing citrus extract and has
demonstrated anti-methanogenic properties (Eger et al.
2018;Roqueet al.2019c;Vranckenet al.2019). The garlic
component in Mootral
targets methanogenic archaea
populations and protozoal communities in the rumen and
has led to nearly complete inhibition of CH
4
production
in vitro at a dosage of 2 g experimental mixture/day,
without compromising bacterial population (Eger et al.
2018). The experimental mix contained 1.5% (w/w) allicin
and 45% (w/w) polyphenolics (Eger et al.2018). A 23.2%
decrease in CH
4
yield (26.8% expressed in CH
4
production)
was observed in Angus ·Hereford crosses after 12 weeks of
treatment by supplementing Mootral
at 1.58 g/kg DM (Roque
et al.2019c). Adverse effects on DMI, ADG and feed
efficiency were not detected over the 12-week trial.
Lactating cattle offered Mootral incorporated in pellets at a
rate of 0.640 g/kg DM for Holstein-Friesian and 1.21 g/kg DM
for Jersey herd experienced suppression of CH
4
of 20.7% and
38.3% respectively (Vrancken et al.2019). Additionally,
3–5% increase in milk yield across breeds was observed
with increased feed efficiency in the Jersey cattle. Further
research is required to determine the effective dose and
magnitude of reduction from ruminants supplemented with
Mootral
.
Conclusions
Several feed additives provide a promising option that could
increase the sustainability of animal-sourced foods by
substantially reducing enteric CH
4
emissions. Rumen
inhibitors have shown potential of up to 98% reduction in
enteric CH
4
production, although they differ in accessibility
and risk to animal welfare. Although none of the inhibitors
are currently on the market, on the basis of the volume of
available literature, 3NOP may be offered to producers in the
Reducing enteric methane through feed additives Animal Production Science I
near future, with nitrate and microalgae to follow after further
research. Rumen modifiers including EO, tannins, saponins,
biochar and lipids can be sourced globally but vary in
composition and are not always effective. Consistency is a
factor to consider with plant-based feed additives, but it can
be addressed, as demonstrated, in commercial applications such
as Mootral
and Agolin Ruminant. Direct-fed microbials or
probiotics have not demonstrated strong evidence to be
considered a rumen modifier to suppress CH
4
production. Due
to increased interest in this area, research is expected to
accelerate in production of feed additives that reduce enteric
CH
4
production.
Conflicts of interest
The authors declare no conflicts of interest.
Acknowledgements
We are grateful to the California Air Resources Board for supporting the
study under Project #17RD018.
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