AHDB

Feed additives in ruminant nutrition

Can feed additives reduce methane and improve performance in

ruminants?

Poppy Frater
Beef and Sheep Scientist

June 2014
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Summary

There is a growing range of feed additives, aimed for use in ruminant diets, that offer
potential to improve rumen fermentation efficiency while reducing methane output. This
review aims to determine how effectively those additives on the market and under
development are meeting these claims.

Methane production is the dominant mechanism for hydrogen disposal in the rumen, a
necessary activity for healthy rumen functioning. Potential opportunities to reduce methane
production involve: inhibiting the methaneogenic archea and/or the accompanying protozoa,
supplying an alternative hydrogen disposal mechanism, encouraging other natural hydrogen
disposal routes and slowing fermentation to improve fermentation efficiency.

A recent review completed by Herefordshire University for the European Food Safety
Authority and a Food and Agriculture Organisation report on technical options for methane
mitigation form the basis of this report. From these, the most promising feed additives were
identified and effectiveness discussed.

Plant extracts, in various forms, hold significant potential for reducing methane, including:
fatty acids, dietary lipids, essential oils and condensed tannins. Polyunsaturated fatty acids
and essential oils are thought to have a similar mode of action to monensin (an ionophore
currently prohibited for use in the EU); they favour propionate producing bacteria in the
rumen, thereby encouraging another hydrogen sink.

Dietary lipids, or their constituent fatty acids alone, reduce methane emissions but at high
intakes they can reduce digestibility and dry matter intake.

Plant saponins are thought to reduce rumen protozoa numbers which can affect the methane
producing bacteria and slow down protein turnover in the rumen, increasing transport of
microbial nitrogen to the duodenum.

Garlic oil is the essential oil with most supporting evidence for methane reduction, but effects
on performance are variable.

A promising group of chemical additives are electron receptors. Nitrate is an electron

receptor that acts as an alternative hydrogen sink to methane, but if used in high protein diets,
excess ammonia production is likely. Nitrate toxicity is a risk to ruminants, but this can be
overcome with gradual introduction of nitrate to the diet.
Overall, most additives require further long term studies in the live ruminant to determine
how effective they are in commercial systems. To be sustainable and taken up by the
industry, the feed additive would need to be effective over long periods of time, non-toxic for
animals, the environmental and consumers and cheap enough for standard use in animal
feeds.
Contents
1. Introduction ..................................................................................................................................... 6

2. Feed additives to reduce methane production in ruminants .......................................................... 10

2.1 EU regulation ........................................................................................................................ 10

2.2 European Food Safety Authority report ................................................................................ 10

2.1.1. Additive development ......................................................................................................... 11

2.3 Food and Agriculture Organization review........................................................................... 14

2.3 The most promising feed additives for reducing methane ......................................................... 16

2.3.1 Fatty acids and plant oils ...................................................................................................... 16

2.3.4 Essential oils (Armoracia rusticana (horseradish) extract, Mentha microphylla, Carvacrol
(oregano))...................................................................................................................................... 21

2.3.5 Plant extracts: Saponins ....................................................................................................... 25

2.3.6 Electron receptors ................................................................................................................ 26

3. Persistence..................................................................................................................................... 32

4. Conclusion .................................................................................................................................... 33

5. References ..................................................................................................................................... 34
 Feed additives in ruminant nutrition

1. Introduction
Feed additives are products used in animal nutrition to improve the quality of feed and the
quality of food from animal origin, or to improve the animals’ performance and health. They
are categorised as follows:

 Technological additives (e.g. preservatives, antioxidants, emulsifiers, stabilising

agents, acidity regulators, silage additives)
 Sensory additives (e.g. flavours, colorants)
 Nutritional additives (e.g. vitamins, minerals, amino acids, trace elements)
 Zootechnical additives (e.g. digestibility enhancers, gut flora stabilizers)
 Coccidiostats and histomonostats (additives used in poultry diets for health
reasons)

In recent years, zootechnical additives1 (the focus of this review from here on) have shown
vast growth in the research field and, consequently, in the feed market. Their suggested
mode of action varies, but in general, they aim to manipulate the rumen fermentation
environment to achieve greater efficiencies.

This range of feed additives has potential to deliver the following improvements in ruminant
nutrition:

1. Increase feed conversion efficiency (FCE) and productivity

2. Stabilise rumen pH to reduce acidosis risk
3. Increase dry matter intake (DMI)
4. Reduce methanogenesis
5. Enhance rumen development
6. Reduce pathogen load and shedding
7. Improve meat quality
8. Enhance rumen stability during dietary transitions
9. Buffer against dietary health risks (e.g. mycotoxins)

1
For the remainder of this review, zootechnical feed additives will be referred to solely as feed additives
This review aims to:

 Inform producers/advisors of the most promising feed additives for ruminants in

terms of methane reduction
 Provide information on the other effects, risks and practicalities of the feed additives
 Where the feed additive is not commercially available, describe those most promising
for future development

1.1 The potential of feed additives

Methane (CH4) is a by-product of carbohydrate breakdown in the reticulo-rumen. Methane
production is energy inefficient, wasting 2-15% of digested energy (McCrabb and Hunter,
1999, Johnson and Ward, 1996, Blaxter and Clapperton, 1965).

Dietary manipulation could reduce methane production while improving performance. A

simplified diagram taken from a Food and Agriculture Organisation report (Alexander N.
Hristov et al., 2013) demonstrates carbohydrate breakdown in Figure 1.

Figure 1: Carbohydrate metabolism in the rumen, taken from Alexander N. Hristov et al.
(2013)

Reaction stoichiometry is the term to describe the relationship between substances as they
interact in chemical reactions. It is used to estimate the quantity of products formed during a
reaction. If we can understand how carbohydrates break down (the reaction stoichiometry)
and have an understanding of the effect of a specific feed additive on the process, we can
predict how that additive will affect the various product quantities.

In general, the reaction stoichiometry for carbohydrate breakdown has been described by Van
Soest (1994) as follows:

Glucose + ammonia microbes + methane + carbon dioxide + volatile fatty acids*

*butyrate, formate, lactate, propionate, acetate

Note: methanogenic archea are anaerobic, therefore this only occurs under anaerobic conditions

Therefore, the main products of carbohydrate breakdown in the rumen are volatile fatty acids
(VFA), methane and carbon dioxide (CO2). Alcohols and lactate are also formed.

The basic problem in anaerobic metabolism is the storage of oxygen (i.e. as carbon dioxide)
and disposal of hydrogen (i.e. as methane) (Van Soest (1994)). Methane works as a hydrogen
sink to remove hydrogen from the rumen for healthy rumen functioning.

Methane is commonly referred to as “2H” sink because pairs of protons and electrons are
donated and accepted in metabolic reactions. If we can replace this process with an
alternative, there is potential to reduce methane emissions and improve energy-use efficiency,
but in-doing so caution is required not to replace the methane with another pollutant. Ideally,
the hydrogen will be redirected to a useful form of nutrition for the animal.

Although methane can be produced from VFA and alternative sinks for hydrogen do exist in
other environments (acetate, for example), these processes appear to be of little significance
in the rumen (Russell and Wallace, 1997). As shown in Figure 1, acetate and butyrate
produce four and two hydrogen molecules respectively and propionate is a 2H sink.
Therefore propionate is the VFA to encourage if the objective is to reduce methane
production as it decreases the overall amount of 2H available to reduce carbon dioxide to
methane.
There is potential to manipulate carbohydrate breakdown to reduce methane production and
potentially improve performance in the following ways:
  Encourage propionate production, thereby providing an alternative H sink (problem
with this approach is that propionate has a lower affinity for hydrogen than methane)
 Adding an alternative hydrogen sink into the diet , e.g. nitrate (this has health risks
and may increase ammonia output unless utilised by microbes)
 Encourage the production of acetate and butyrate, as these utilise the hydrogen before
it can be directed to produce methane (less potential here as acetate and butyrate still
produce hydrogen)
 Inhibit or eliminate the methanogenic microorganisms, thereby forcing the use of
other hydrogen sink pathways (problematic if these organisms have other functions in
the rumen)
 Slow down fermentation to improve nitrogen and energy-use efficiency

To be sustainable and taken up by the industry, the feed additive would need to be effective
over long periods of time, non-toxic for animals, the environment and consumers and cheap
enough for standard use in animal feeds.

1.2 The relationship between methane and dry matter intake

To determine how effective an additive is at reducing methane, it is important that the studies
report the effect using the same units, per land area or per unit of product, for example. In
addition, we should also look out for a reduction in methane that is simply due to a reduction
in feed intake and/or a reduction in performance.

Dry matter intake is an important determinant of methane output. A meta-analysis showed

on average, for every kilogram of dry matter intake approximately 30 litres of methane is
produced (Mills et al., 2008). Therefore, when studies report a reduction in methane and a
reduction in dry matter intake, the additive may be reducing digestibility or palatability of the
feed.
 2. Feed additives to reduce methane production in ruminants

2.1 EU regulation
According to EU Regulation 1831/2003, only additives that have been authorised by the
European Food Safety Authority (EFSA) can be placed on the market in the EU.

Authorised additives can be put on the market solely for the specific use described for
authorisation. Companies or researchers wishing to get an additive authorised must submit
an application and a dossier to EFSA which must:

 Enable assessment of additives based on the current state of knowledge

 Demonstrate compliance with the fundamental principles for authorisation
 Have a minimum of three long term in vivo studies showing significant effects for the
relevant target species/category. These should be carried out at least two different
locations, one of which should be in the EU. The studies should include the lowest
dose proposed by the applicant to enable recommendations on dose to be made.
 Demonstrate the safety of the product ((EFSA), 2012).

Probiotics (fungi, bacteria, enzymes) are the only feed additives categorised under
zootechnical additives for beef and sheep production and therefore, technically, should be the
only feed additives in that category on the market in the UK. However, some additives, with
the claim of offering a dietary nutritional component, may appear on the catalogue of feed
materials European Commission (2013).

2.2 European Food Safety Authority report

A recent review completed by Herefordshire University for the European Food Safety
Authority (EFSA) conducted a meta-analysis of feed additives aimed at mitigation of
environmental impacts of livestock (Lewis et al., 2013).

Many meta-analyses on feed additives report substantial variation in additive effects on

animal performance and other measured variables due to differences in the methodology
employed across the different experiments (Eugène et al., 2008, Sales, 2011, Desnoyers et
al., 2009).

This makes it difficult to determine the efficacy of a substance, as it is hard to disentangle the
effect of the additive from any interaction with other factors. Additionally, the variation in
different diets would likely influence the results (Desnoyers et al., 2009). The review
covered 246 substances in ten major groups, as illustrated in Table 1.

Table 1: The substance groups covered by the EFSA report (Lewis et al., 2013)

Substance group Number of substances Number of studies

Organic acids & their salts 24 47
Fatty acids 12 15
Amino acids 8 11
Plant & animal oils, fats 16 33
Plant extracts
General 6 6
Saponins 7 34
Tannins 27 28
Essential oils & spices 42 48
Antibiotic drugs
General 11 13
Ionophores 9 47
Bacteria, enzymes & yeasts 29 62
Mineral salts and complexes 11 9
Miscellaneous substances
Chemical substances 21 31
Natural substances 23 29
TOTAL 246 -

2.1.1. Additive development

Unfortunately, many of the additives, particularly those that had the greatest effect (Table 2)
were only tested in vitro, and not in vivo. In vitro research gives insight into the potential of
the additive with different substrates, but it does not account for the capacity for rumen
microbes to adapt to the additives and degrade them (Benchaar and Greathead, 2011).
Additionally, dose rate in vitro can be significantly higher than when fed to the ruminant,
therefore a response can be induced that may not be practically feasible in a live animal
(Benchaar and Greathead, 2011).
Table 2: Summary of the top ten feed additives for methane reduction taken from the EFSA
report (Lewis et al., 2013)

Substance group Substance Methane reduction (%) No. of Species

and range in brackets studies
Essential oils Mentha -96 (-92 to -100) 1 Sheep
microphylla
Plant extracts: Rheum -75 (No range) 1 Sheep
tannins officinale
(root)
Fatty acids Linolenic -71 (-46 to -99) 3 Sheep (1) and
acid cattle (2)
Miscellaneous 9, 10- -70 (-58 to -91) 2 Cattle (1) and
chemical substances anthraquinon goats (1)
e
Miscellaneous 2- -61 (-25 to -97) 1 Cattle
chemical substances iodopropane
Fatty acids Lauric acid -59 (-11 to -99) 4 Cattle
Plant and animal oil, Palm kernel -58 (-32 to -85) 2 Cattle
fats oil
Miscellaneous Pyromellitic -57 (-7 to -100) 3 Cattle
chemical substances diimide
Essential oils Carvacrol -56 (-13 to -98) 1 Sheep
Plant extracts: Cashew nut -55 (-36 to -70) 2 Cattle
general shell liquid
n.b. these were all in vitro studies

Looking only at the substances with five or more studies and some in vivo experimentation,
we can ascertain which substances have the greatest evidence to reduce methane production
(Table 3). Tables 2 and 3 were used to compile a list of the most promising feed additives on
which to base the rest of the review (Table 4).
Table 3: Summary of substances with five or more studies including some in vivo
experiments from the EFSA report (Lewis et al., 2013)

Substance Substance Average Number of Species Total

group methane change in vivo studies
in vivo studies
Fatty Acids Myristic acid -36.3 2 Sheep 5
Plant & animal Coconut oil -23.8 5 Cattle (4) 10
oils, fats and Sheep
(1)
Plant & animal Canola oil -16.0 3 Cattle 6
oils, fats
Essential oils & Allium -12.0 1 Sheep 9
Spices arenarium oil
Organic acids Fumaric acid -11.5 2 Cattle and 5
and their salts Sheep
Antibacterial Monensin -11.3 10 Cattle (9) 21
drugs: sodium and Sheep
Ionophores (1)
Plant extracts: Tea saponins -10.1 7 Cattle (3) 7
Saponins and Sheep
(4)
Plant extracts: Quillaja -4.3 4 Cattle (3) 9
Saponins saponaria and Sheep
extract (1)
Plant extracts: Yucca -2.4 5 Cattle (4) 10
Saponins Schidigera and Sheep
extract (1)
Organic acids Fumaric acid, 0.0 1 Cattle 12
and their salts sodium salt
 2.3 Food and Agriculture Organization review
A recent report, Mitigation of Greenhouse gas emissions in livestock production; a review of
technical options for non-CO2 emissions, of the Food and Agriculture Organization of the
United Nations (Alexander N. Hristov et al., 2013) discussed the major feed additive groups
with potential to reduce methane as follows:

Inhibitors (i.e. targeted chemical compounds that inhibit rumen archea, e.g
bromochloromethane)

Verdict: Bromochloromethane is promising but cannot be used because it is an ozone

depleting substance, there was no sufficient data for other substances in this category

Electron receptors, e.g. nitrate

Verdict: Nitrate may be promising in low protein diets that may benefit from non-
protein nitrogen supplementation. An adaptation period is crucial and must beware of
pollution swapping (i.e. ammonia). Long term effects unknown.

Ionophores

Verdict: Likely to provide a moderate CH4 mitigating effect, prohibited in Europe.

Plant bioactive compounds (includes tannins, saponins, essential oils)

Verdict: hydrolysable and condensed tannins show promise but intake and animal
production may be compromised. Tea saponins require more long term studies.

Most essential oils or their active ingredients do not reduce methane. Studies that
demonstrated a reduction were over short timescales, therefore longer studies are
required.

Dietary lipids (also termed plant and animal oils)

Verdict: Effective at reducing methane but uptake will depend on cost-effectiveness

and potential negative effects on feed intake and productivity. Distillers grains were
also categorised here, but results have been variable, some increases in methane
observed due to increased fibre intake.
Exogenous enzymes

Verdict: May increase feed efficiency but inconsistent results.

Direct fed microbials

Verdict: Insufficient evidence, although yeast appears to stabilise pH and promote

rumen function in high concentrate diets.

Using the results of the two reviews, the most promising additives are summarised in Table 4.

Table 4: Most promising feed additives of the EFSA and FAO reports, as categorised by the
reports

Substance group Substances

Fatty Acids Myristic acid Linolenic acid, Lauric acid
(table 2)
Plant & animal oils, fats/dietary lipids Coconut oil, Canola oil, Palm Kernel oil
Essential oils and spices Allium arenarium oil, Mentha microphylla,
Carvacrol
Plant extracts: Saponins Tea saponins, Quillaja saponaria extract,
Yucca Schidigera extract
Electron receptors Nitrate, sulphate

Taking into account the findings of the EFSA and FAO reports, the next section will provide
further details of the mode of action of these additives, describe the practicality of feeding
them, health or environmental risks and effects on other pollutants.
2.3 The most promising feed additives for reducing methane

2.3.1 Fatty acids and plant oils

Table 5: Characteristics of the most promising fatty acids

Fatty acid Characteristics Main sources

Myristic acid Saturated, Coconut oil,
Medium chain Palm kernel oil
Lauric acid Saturated, Coconut oil
Medium chain
Linolenic acid Polyunsaturated, Linseed
Long chain
Linoleic acid Unsaturated, long Soybean,
chain, omega 6 sunflower
Oleic acid Monounsaturated, Rapeseed meal,
long chain Canola oil

Medium-chain saturated fatty acids have strong antimicrobial properties (Hristov et al.,
2004), particularly affecting methanogenic archea (Machmuller et al., 2003). This inhibition
should reduce methane production. Conversely, it has been suggested that long chain fatty
acids do not have this mode of action (Mosoni et al., 2008).

Polyunsaturated fatty acids have toxic effects on cellulolytic bacteria and protozoa (Doreau
and Ferlay, 1995). They act against lactate producers (Kubo et al., 1993, Nagaraja et al.,
1997) thereby favouring propionate producers.

Coconut oil contains lauric and myristic acid. Both fatty acids have substantial potential to
reduce methane production (Tables 2 and 3) but there is a large range in effects observed and
lauric acid is only explored in vitro in the EFSA report. There is some indication that the
effect of the two fatty acids on methane production is additive (Machmüller and Kreuzer,
1999, Soliva et al., 2004).

Linolenic acid and linoleic acid also reduce methane by an average of 71% and 51%,
respectively, in vitro (Lewis et al., 2013) but further work is required in vivo.
Diet effect

Diet composition will affect the success of the additive, but results are not definitive.

Machmuller et al. (2003) investigated the interaction of myristic acid supplementation with
diet type (concentrate: forage ratios: 1 : 1·5 and 1 : 0·5) and dietary calcium (Ca) content (4.2
and 9g/kg DM) in sheep. Myristic acid suppressed methane in both diets, although more in
the concentrate diet than the forage diet and the highest dose of Ca reduced this effect.

When averaged across both diets, myristic acid increased DMI, as a result, methane
emissions reported in kg/megajoule (MJ) of gross energy intake and g/kg organic matter
digested were reduced for both diet types.

Lovett et al. (2003) showed no interaction between diet (forage to concentrate ratios: 65: 35,
40: 60 and 10: 90) and coconut oil supplementation in finishing beef heifers. Although
supplementing with coconut oil reduced dry matter intake, this was outweighed with greater
reductions in methane, therefore methane produced per kg DMI, liveweight gain, carcase
gain and as percentage of gross energy intake was reduced.

Martin et al. (2009) explored linseed supplementation in dairy cows on a hay- and maize
silage-based diet. In the four treatments (0, 5, 10 and 15% linseed in ration), the cows on the
maize silage diet produced less methane than those on the hay-based diet. The methane
reduction effect from linseed supplementation was greater on the hay-based diet than the
maize diet, perhaps this is due to a greater potential to improve rumen fermentation efficiency
in the hay based diet. 15% extruded linseed with a maize diet had the lowest total methane
emissions (l/d). The authors did not present results on dry matter intake, however, they found
that supplementation did not alter milk yield, suggesting extruded linseed (providing an
expected oil supplementation of 6% of the diet) is a viable methane mitigation option.

Feeding

The best way to feed dietary lipids of interest is unclear. Wholeseeds tend to be cheaper than
the refined oils (Martin et al., 2010), but sunflower seeds have been reported to reduce NDF
digestibility in heifers (Beauchemin et al., 2007) and average daily gain may be enhanced
with the oil form compared to whole seed/bean form (refined soy oil, crossbred beef bulls
(Jordan et al., 2006a)).
In terms of methane reduction, the oil form was better at reducing methane when feeding soy
(young bulls, Jordan et al. (2006a)) and linseed (lactating dairy cows, Martin et al. (2008))
than the whole form, but sunflower seed has been reported to reduce methane more than
sunflower oil (Aberdeen angus heifers, Beauchemin et al. (2007)). For many seeds, the hard
seed coating is difficult to digest by enzymes, therefore crushing, bruising, extrusion or
expansion is required to release the oil (Raes et al., 2004). These papers suggest the oil form
would be the most attractive option in terms of performance and methane reduction as long as
the cost of the ingredient is outweighed by improved performance.

Unintended consequences

Only five studies additionally looked at ammonia emissions in the EFSA report (three in vivo
and two in vitro), illustrating that lauric acid and myristic acid have potential reduce
ammonia emissions too (Lewis et al., 2013).

The fatty acids above are described as slight to strong skin and eye irritants and lauric acid is
toxic to aquatic life (Lewis et al., 2013).

Performance effects
Many studies report a reduction in methane with various types of fat supplementation to
ruminant diets but this is often accompanied by a reduction in dry matter intake and
digestibility (Table 6). When this is reported on a fat-corrected milk basis, increased feed
efficiency is observed in dairy production (Eugene et al., 2008, Rabiee et al., 2012).

Table 6 is adapted from a recent review of the benefits of supplementary fats in ruminant
rations (Rasmussed and Harrison, 2011). From this table, we can see that, apart from one
study, all the plant oils in the studies had potential to decrease methane. However,
digestibility and intake were reduced in some studies, particularly with high dose rates
therefore the negative effect on production is likely to increase methane output per unit of
liveweight gain.
Table 6: In vivo studies analysing the effects of plant oils on methane and other parameters (adapted from (Rasmussed and Harrison, 2011))

Animal n Durati Ration (forage/concentrate dry Fat source (% of Methane NH3 DMI Digestibility Reference
on matter basis) DM) (%)
Charolais/Limousin 4 93d 50/50 Coconut oil -18 NS NS (Jordan et al.,
heifers 1 (10%) 2006c)
250g/day
Copra meal* -14.9 NS decrease
(10%)
250 g/day
coconut oil
Charolais/Limousin 1 35d 50/50 Coconut oil -20.5 NS NS (Jordan et al.,
heifers 6 (14%) 2006b)
250g/day
Coconut oil -39 decreas decrease
(42%) e
375g/day
Charolais/Limousin 3 103 d 10/90 Soya oil (10%) -40 NS (Jordan et al.,
bulls 6 2006a)
Soybean (12%) -25.3 decreas
e
Lactating cows 5 12 wk 70/30 Cotton seed -23 NS decreas (Grainger et al.,
(48%) e 2010)
Lactating cows 1 4X 45/55 TMR Sunflower seed -10 increa decreas decrease (Beauchemin et
6 28d (3.3%) se e al., 2009)
Linseed oil -18 NS decreas decrease
(3.3%) e
Rape seed (3.3%) -16 NS NS NS
Huzhou sheep 3 60 d 60/40 Soya oil (3%) -14 decre Decrease (Mao et al., 2010)
2 ase
Swiss White Hill 1 3x 60/40 Coconut oil (6%) -26 NS (Machmüller et al.,
sheep 2 21d 2000)
Sunflower seed -27 Decrease
(6%)
Linseed oil (6%) -10 Decrease
Table 6 continued: In vivo studies analysing the effects of plant oils on methane and other parameters (adapted from (Rasmussed and Harrison, 2011))
Animal n Durati Ration (forage/concentrate) Fat source (% of Methane NH3 DMI Digestibil Reference
on DM) (%) ity
Holstein bulls 1 21 d 75/25 Sunflower oil -22 decrease (McGinn et al., 2004)
6 (5%)
400g/d
Lactating Holstein 3 319d 50/50 TMR Cotton seed (4%) NS increa (Johnson et al., 2002)
cows 6 se
Cotton seed NS increa
(5.6%) se
Rape seed (4%) NS increa
se
Rape seed (5.6%) NS increa
se
Lactating Holstein 1 18 d 60/40 TMR Myristic acid -36 NS decrease (Odongo et al., 2007b)
cows 2 (5%)
Lactating Holstein 8 4 wk 65/35 Linseed oil -64 decre decrease (Martin et al., 2008)
cows (5.7%) ase
Extruded linseed -38 decre decrease
(5.7%) ase
Crude linseed -12 NS decrease
(5.7%)
Huzhou Sheep 8 21 d 60/40 Coconut oil (7%) -38 (Yuan et al., 2007)

*copra meal based concentrate with 250g of Coconut/day from copra meal
DMI = dry matter
intake
TMR = total mixed
ration
NS = no statistically significant effect
2.3.4 Essential oils
Essential oils cover a wide variety of products extracted from plants using steam distillation
with water or aqueous alcohol.

Mode of action

Similar to ionophores and polyunsaturated fatty acids, essential oils are thought to select
against gram positive bacteria, favouring propionate producing bacteria, thereby encouraging
the alternative hydrogen sink to methane – propionate.

Response

Within the category 'Essential oils', 42 substances were covered by the EFSA review and 31
were found to offer potential for reducing ruminal methane and ammonia, in many cases
reduction in these pollutants occurred simultaneously (Lewis et al., 2013). The most
promising essential oil with over 5 studies, that included in vivo work, was Allium arenarium
oil (garlic oil) which reduced methane by 12 % in vivo and an average of 36% reduction was
reported in vitro (Lewis et al., 2013).

Garlic oil is unique in that some active compounds present in the oil are not present in the
whole plant; they are formed during the steam treatment process (Pentz and Siegers, 1996).
It is active against a wide range of gram positive and gram negative bacteria, fungi, parasites
and viruses – this is likely due to interaction with the sulfhydryl groups (-SH) of other active
compounds (Reuter et al., 1996). The activity of the oil is more powerful than the individual
compounds alone – suggesting synergism between constituent compounds (Busquet et al.,
2005b).

Performance and practicality

Garlic and other essential oils could affect the palatability of the feed. Garlic oil and diallyl
disulphide (garlic oil constituent) additions to mature ewe diets did increase feed refusal
initially (Klevenhusen et al., 2011). With time, the sheep adjusted to diallyl disulphide
additions and feed intake resumed to the level of the control (no additive). This adjustment
did not occur with the garlic oil treatment.
Robertson et al. (2006) looked at improving the palatability of straw by adding different food
flavourings and found that applying a low rate of garlic flavouring (0.05 g/kg (0.005%)) to
straw improved acceptability to sheep. However, higher rates of garlic inclusion (5, 10, 15,
20, and 25% DM) reduced diet palatability (Nolte and Provenza, 1992a, Nolte and Provenza,
1992b).

Performance effects of added garlic to ruminant diets are variable, as illustrated in Table 7.

Diet effect

There are few studies investigating the interaction between diet and garlic supplementation.
One in vitro study suggests garlic oil additives are more effective with moderate concentrate
diets (50 : 50 alfalfa hay : concentrate ) than high concentrates (15 : 85 barley straw :
concentrate) at reducing the acetate:propionate ratios (Mateos et al., 2013). In both diets the
garlic oil reduced methane production significantly.

Risks

High rates of any plant extracts may be toxic, therefore dose will need careful consideration.
No adverse effects on the target species or human health (as typical usage doses) have been
identified for these substances.

The main barrier to the use of garlic in dairy diets it the risk of milk taint (Babcock, 1938),
but there is no evidence to suggest it will impact meat quality. In fact, Strickland et al.
(2011) found that lamb reared on diets containing garlic additives was accepted by taste
panellists more positively than those reared on garlic-free diets.
Table 6: Studies reporting the effect of garlic supplementation in ruminants

Form Dose Diet Animals Response Author

ADG Dry FCE (DM Methane Other

matter intake:kg
intake growth)
Garlic 200mg/kg Barley based 10 ewe lambs/treatment NS NS NS NA Meat quality (Chaves et al.,
oil dietary measurements: NS, 2008)
DM Carcase
characteristics: NS,
except liver weight
(+20%)
VFAs, pH and
ammonia: NS
Garlic 250 mg/kg Milk, forage and 12 pre-ruminant Holstein +18 +8% NS NA Faecal score (Ghosh et al.,
extract* BW/day/c concentrate (~1:6 cross calves, weaned just decreased an average 2011)
alf conc:forage ratio) after birth, 5 days old of one score (four
point scoring scale),
NS change in feed
cost/kg gain
Garlic 250 mg/kg Milk, forage and 38 pre-ruminant Holstein +44% +12% -44% NA Faecal score (Ghosh et al.,
extract* BW/day/c concentrate (~1:6 cross calves, weaned just decreased an average 2010)
alf conc:forage ratio) after birth, 5 days old of 0.8 score (four
point scoring scale),
30% reduction in feed
cost/kg gain
Table 6 continued: Studies reporting the effect of garlic supplementation in ruminants

Form Dose Diet Animals Response Author

ADG Dry FCE Methane Other

matter (DM
intake intake:kg
growth)
Raw 53 g/kg Concentrate based Brown swiss x Limousin NS NS NS NS (Staerfl et al.,
Garlic dietary DM crossbred bulls 2012)
** (1.5% DMI)
Raw Diet Pelleted ration 40 Merino wether lambs -18% NS NS NA Worm count: NS (Strickland et al.,
Garlic inclusion: aged 6 months 2009)
0.9%
1.8% -22% NS NS NA

3.6% -41% NS +62% NA

Garlic 4g/day Lucerne hay and Four ruminally fistulated NA NA NA NA No effect on organic (Vakili et al.,
oil*** concentrate at Baloochi lambs matter digestibility 2011)
45:55ratio or fibre digestibility
*prepared using electric mixer and filtered through muslin cloth, **acacia tannin, maca and lupines also compared, ***Tumeric powder and monensin also compared
ADG = average daily gain, BW = body weight, DM = dry matter, DMI = dry matter intake, FCE = feed conversion efficiency, NA = not applicable to study, NS = no statistically significant
effect, VFA = volatile fatty acids.
2.3.5 Plant extracts: Saponins
Although there has been a lot of research interest in tannins and other plant extracts, saponins
were the only plant extract- based additive that had sufficient evidence described in the EFSA
review to reduce methane emissions.

Saponins are sugar and non-carbohydrate complexes that characteristically foam when
shaken in water. They are steroid or triterpene glycoside compounds found in a variety of
plants.

Commercial additives are derived from Yucca shidigera or Quillaya saponaria and Sapindus
sp (temperate and tropical plant species).

As Table 3 shows, in vivo effects of saponin addition in the studies for the EFSA report show
mean methane reductions of 2.4%, 4.3% and 10.1% for Yucca Schidigera extract, Quillaja
saponaria and Tea saponins, respectively.

Mode of action

In vitro and in vivo experiments show how saponins can be used to remove or reduce
protozoa numbers in the rumen (described in a review by Wina et al. (2005)). Protozoa cause
protein turnover by predating on bacteria. Theoretically their removal should lead to an
increase in the number of bacteria in the rumen, this slows the protein turnover leading to an
increase in bacterial N flow to the duodenum. Therefore, the removal of protozoa may
increase nitrogen utilisation leading to improvements in performance output and reductions in
ammonia produced. This theory has been supported by in vitro work of Makkar and Becker
(1996), whereby quillaja saponins (0.4-1.2 mg/mL) on a hay substrate increased efficiency of
microbial protein synthesis.

In addition, some methanogens are closely associated with protozoa, therefore the reduction
of protozoa may also reduce methanogen activity.

Diet effect

A review of studies by Wina et al. (2005) indicated that improvements in liveweight gain are
most likely to be seen with saponin addition to high roughage diets, although this was not
clear cut, some moderate to high roughage diets still showed no growth enhancing effect
(Hussain and Cheeke, 1995). Also, sex can interplay with the response: a higher growth
response was reported in male lambs compared to females lambs with 40 mg quillaja
saponins/kg in low roughage diets (Hussain and Cheeke, 1995).

Performance and practicality

The main issue with feeding saponins to ruminants is evidence that the rumen microbe adapt
and protozoa return to pre-supplementation levels (Newbold et al., 1997), intermittent
additions may mitigate this effect (Thalib et al., 1996). There is also evidence that they can
be degraded in the rumen (Wang et al., 2000). Further long term experimentation will need
to fully explore this effect and develop ways to overcome it.

Saponins have been described to reduce fibre digestibility but not total tract digestibility,
however acid detergent fibre (ADF) digestibility of a legume-grass diet was reduced and
neutral detergent fibre digestibility of a grass diet were reduced with addition of S.Saponaria
fruit (Abreu et al., 2004), further emphasising the important diet-additive interactions.

Risks

Some saponin containing plants are toxic which can lead to photosensitisation and subsequent
liver and kidney degeneration, and gut problems. However it is unlikely these would be used
in commercial additives.

2.3.6 Electron receptors

Electron receptors such as nitrate and sulphate were not considered in the EFSA review, yet
are prominent in other recent reviews on methane mitigation (Alexander N. Hristov et al.,
2013, Hristov et al., 2013). They offer an alternative a hydrogen sink to carbon dioxide,
thereby reducing methane emissions and potentially improving digestion efficiency
(Ungerfeld and Kohn, 2006). Nitrate (NO3−) likely holds the greatest potential for this
because it has a greater affinity for hydrogen (H2) than carbon dioxide therefore – instead of
methane – nitrite and ammonia are produced. Ammonia generated would supply fermentable
nitrogen for animals receiving protein deficient diets where ammonia is limiting protein
synthesis for microbial growth (Dijkstra et al., 1998). Additionally, the reaction to produce
nitrite is more energetically efficient than methanogenesis (Ungerfeld and Kohn, 2006).
Risks

Sudden introduction of nitrate into the diet may induce methaemoglobinemia, a state of
general anoxia; in mild cases this will depress animal behaviour but in severe cases can be
fatal (Ozmen et al., 2005). Methaemoglobinemia is caused by nitrite in the blood. The
accumulation of nitrite in the rumen is readily absorbed across the rumen wall and converts
blood haemoglobin (Hb) from the ferrous (Fe2+) to the ferric (Fe3+) form- methaemoglobin
(MetHb)- which is incapable of transporting oxygen (Morris et al., 1958). Gradual
introduction of nitrate into the diet can allow the rumen microbes to adapt and increase their
ability to reduce nitrite (Alaboudi and Jones, 1985). In animals unadapted to nitrate in their
diet, the capacity of the microbes to reduce nitrate to nitrite, exceeds the capacity for nitrite
reduction (Lewis, 1951). MetHb formation coincides with reduced oxygen intake, carbon
dioxide production and metabolic rate (Takahashi et al., 1998).

A comprehensive review suggests that sheep can tolerate up to 4% nitrate in high quality
concentrate or forage based diets without showing clinical signs of methamoglobinemia
(Leng, 2008).
Table 7: Studies reporting nitrate supplementation in ruminant diets

Paper Additive(s) Form Administration Dose Animals Diet Adaptation Methanoglobin Methane
period >2% of change (%
(days) haemoglobin? difference
in output
(L/day)
(Li et Nitrate Calcium Dietary pellet 3% of DM 10 weaned Limited info. 7 No -34
al., nitrate ewe lambs (P=0.06)
2012) (1.9% (Poll dorset
nitrate) sire x
Dohne
ewe)
(van Nitrate Calcinit: Added to 8.8% of DM 20 lactating TMR-Corn silage 28 Yes, max. -16
Zijderv 5Ca(NO3) concentrates holstein- based ration observed:4.2%
eld et 2NO3·10H fresian (66:34, of Hb
al., 2O; 75% cows 33.2 forage:concentrate)
2011) NO3 in ±6.0kg Isoenergetic and
DM milk/d isonitrogenous
104±58 d
in milk at
start
(van Nitrate and 5Ca(NO3) Added to 2.6% of DM 20 male Maize silage basal 28 Yes-max. Nitrate -
Zijderv Sulphate 2·NH4NO3 concentrates cross bred ration (90%) plus observed: 7% of 32%
eld et ·10H2O;75 lambs concentrates based Hb for high Sulphate -
al., % NO3 in lambs on soybean mean nitrate inclusion 16%,
2010) DM and additives diet Both -47%
(highly (10%).
soluble Urea substitutes
and nitrate in sulphate
available experiment
nitrate)
MgSO4
Table 7 continued: Studies reporting nitrate supplementation in ruminant diets

Paper Additive(s) Form Administr Dose Animals Diet Adaptation Methanoglobin Methane
ation period >2% of change (%
(days) haemoglobin? difference
in output
(L/day)
(Nolan Nitrate Potassium Sprinkled 4% of DM 8 merino Chaffed oaten hay 18 No -23
et al., nitrate (KNO3) on hay wethers 1kg/day
2010) rumen-
cannulated
sheep

(Guo et i) Nitrate-N Sodium nitrate in vitro 12.6% DM 3 60% roughage NA Not measured -74
al., ii) urea N (NaNO3) cannulated (corn stover cubes
2009) iii) tryptone-N (a Simmental and alfalfa hay
pancreatic digest x Luxi pellets) and 40%
of casein and a steers mixed concentrate
source of amino (ground corn,
acids and soybean mean and
peptides) soy hulls)
Table 7 continued: Studies reporting nitrate supplementation in ruminant diets

Paper Additive(s) Form Administr Dose Animals Diet Adaptation Methanoglobin Methane
ation period >2% of change (%
(days) haemoglobin? difference
in output
(L/day)
(Sar et Nitrate with or 30%(w/v) Via fistula 1.3g 4 Chopped timothy 7 Yes-peaked at -50
al., without: aqueous solution 30 mins NaNO3/kg0. ruminally- hay, alfalfa hay 18.4% in No diff.
2004) Beta 1-4 galacto- of NaNO3/kg0.75 after 75
of body fistulated cube, and nitrate-only with GIS
oligosaccharides morning wgt wethers concentrate treatment or nisin
or Nisin GOS-oligomate meal (40:40:20 on DM
55 GOS basis)
20g/day Fed at maintenance
Nisin-Sigma energy level
Nisin
3mg/kg0.75
of BW
(Alabo Nitrate Initially low Added to 2.5g 4 Dorset- 44% cereal grain, 63 No NA
udi and dose of nitrate ration KNO3/kg Columbia 50% hay
Jones, (NO3-) alone body crossbred
1985) (0.05% of BW) wgt/day ewes
then potassium reached in
nitrate (KNO3-) 0.5g
increments
Unintended consequences

Nitrate may divert hydrogen away from other uses such as propionogenesis- therefore the
VFA profile often shifts away from propionate and butyrate in favour of acetate (Farra and
Satter, 1971, Allison and Reddy, 1984, Alaboudi and Jones, 1985). This could potentially
reduce the efficiency of the rumen as acetate production is less energy efficient than
propionate and butyrate production.

Additionally, it can lead to an increase in ammonia production, which can be a good source
of nitrogen for microbes but in high protein diets it is likely to be excreted, thereby producing
another pollutant.

Effect longevity

Although overall, potassium nitrate addition did reduce methane emissions in the study by
Nolan et al. (2010) (further details in table 10) after the immediate reduction observed post
feeding, there was a gradual rise in methane over the two hours between feeding periods.
The authors suggest a ‘slow release’ form of nitrate might enhance methane mitigation and,
in addition, reduce nitrate and nitrite absorption from the rumen and thereby reduce the
potential for nitrite toxicity.

More in vivo studies are needed to fully understand the impact of nitrate supplementation on
whole-farm GHG emissions (animal, manure storage and manure-amended soil), animal
production and animal health. The long-term effects of these compounds have
not been established.
 3. Persistence

Of the studies considered in the EFSA review, very few studied the long term effects of
feeding additives. Where the mode of action depends on shifting the microbial population of
the rumen there is always potential for adaption of the microbes to take place over time and
the response to diminish, this issue was highlighted in a review by McAllister and Newbold
(2008). Where the mode of action is related to providing exogenous alternative hydrogen
sinks then there may be less potential for adaptation over time to occur.

Sauer et al. (1998) reported that ruminal microflora adapted over time to the use of
monensin, a view supported by Guan et al. (2006) who found that monensin reduced methane
production by around a third but this benefit was short-lived and methane production
gradually returned to its pre-supplementation level over a 16 week period. However, Odongo
et al. (2007a) studied the effects of monensin on methane production in steers and identified a
modest yet long term (6-months) reduction in methane of around 7%.

In a study with dairy cows on pasture, Woodward (2006) examined the effect of vegetable
and fish oils on milk production and CH4 emission in short- (14 days) and long-term (12
weeks) experiments. Lipids significantly decreased CH4 production in the short-term study,
but this effect was not observed after 11 weeks of feeding lipids in the long-term study.

Promising results in vitro demonstrating inhibition of methane production is not always seen
in vivo. Several processes in the live animal, such as: adaptation of the microbes to the
additive or degradation of the additive, toxicity for the host animal, negative effects on
overall digestion and productive performance can temper the results achieved in vivo
(VanNevel and Demeyer, 1996).
 4. Conclusion

Several strategies have been explored to mitigate methane production using feed additives in
ruminants. Recent reviews suggest that plant extracts (dietary lipids, fatty acids, essential oils
and saponins) and some chemical substances (electron receptors) hold the most potential.

Condensed tannins and electron receptors (e.g., nitrate) have known toxicity risks to animals,
therefore dose and adaptation periods are necessary. All substances require more in vivo
studies over long time periods to determine whether the effect will be sustained.

Their successful implementation will depend on the cost-effectiveness and practicality in

commercial farm systems. Solutions must be evaluated in combination with other aspects of
livestock production.
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