Essential Oils and Opportunities to Mitigate Enteric Methane Emissions

8/9/2019 Essential Oils and Opportunities to Mitigate Enteric Methane Emissions

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Animal Feed Science and Technology 166–167 (2011) 338–355

Contents lists available at ScienceDirect

Animal Feed Science and Technology

journal homepage: www.elsevier.com/locate/anifeedsci

Essential oils and opportunities to mitigate enteric methane emissionsfrom ruminants

Chaouki Benchaar a,∗, Henry Greathead b

a Agriculture and Agri-Food Canada, Dairy and Swine Research and Development Centre, P.O. Box 90, STN-Lennoxville, Sherbrooke, Quebec, Canada J1M 1Z3b University of Leeds, Faculty of Biological Sciences, Leeds, United Kingdom

a r t i c l e i n f o

Keywords:

Plant extract

Essential oil

Enteric methane emission

Mitigation

a b s t r a c t

The well documented antimicrobial activity of essential oils has prompted interest in

whether these bioactive compounds can be used to selectively inhibit rumen methano-

genesis. A number of studies have recently evaluated the ability of essential oils to reduce

enteric CH4 production. Most studies conducted have been in vitro and short term. Essen-

tial oils derived from thyme, oregano, cinnamon, garlic, horse radish, rhubarb and frangula

have decreased CH4 production in vitro in a dose dependent manner. However, inhibi-

tion of CH4 production occurred at high doses (i.e., >300mg/L of culture fluid) and was, in

many cases, associated with a decrease in total volatile fatty acid concentrations and feed

digestion. Some essential oils, such as garlic, cinnamon, rhubarb and frangula, may exert

a direct effect on methanogens. Evidence for in vivo antimicrobial activity of essential oils

has been equivocal to date, probably because of the capacity of rumen microbes to adapt

and degrade these secondary metabolites. Further, many of the concentrations of essential

oils that have favourably affected rumen fermentation in vitro are too high for in vivo useas they would likely have deleterious effects on efficiency of rumen fermentation, palata-

bility and possibly cause toxicity. Based on available results, it appears that some essential

oils (e.g., garlic and its derivatives and cinnamon) reduce CH4 production in vitro. However,

there isa need for in vivo investigation to determine whether these compounds can be used

successfully to inhibit rumen methanogenesis. The challenge remains to identify essential

oils that selectively inhibit rumen methanogenesis at practical feeding rates, with lasting

effects and without depressing feed digestion and animal productivity.

This article is part of the special issue entitled: Greenhouse Gases in Animal Agriculture –

Finding a Balance between Food and Emissions, Guest Edited by T.A. McAllister, Section Guest

Editors; K.A. Beauchemin, X. Hao, S.McGinnandEditor for Animal Feed Science and Technology,

P.H. Robinson.

Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved.

1. Introduction

There is interest in reducing CH4 emissionsfrom domestic ruminants. Methane is a potentgreenhouse gas(GHG) andit has

a globalwarming potential25 times that of CO2. According toan FAO report(Steinfeldet al., 2006), livestock account for ∼37%

Abbreviations: ADF, aciddetergent fibre; ATP, adenosine triphosphate;CP, crudeprotein; DM, dry matter; GHG,greenhouse gas;NDF, neutral detergent

fibre; VFA, volatile fatty acid; DAS, diallyl sulphide; DADS, diallyl disulphide; DATS, diallyl trisulphide.∗ Corresponding author. Tel.: +1 819 780 7117; fax: +1 819 564 5507.

E-mail address: [email protected](C. Benchaar).

0377-8401/$ – see front matter. Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved.doi:10.1016/j.anifeedsci.2011.04.024

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C. Benchaar, H. Greathead / Animal Feed Science and Technology 166–167 (2011) 338–355 339

24.3%

4.8%3.1%

62.1%

0

10

20

30

40

50

60

70

Thymus

heymalis L.

Thymus zygis

sub. Gracilis

Carvacrol

Thymol

24.3%

4.8%3.1%

62.1%

0

10

20

30

40

50

60

70

Thymus

heymalis L.

Thymus zygis

sub. Gracilis

Carvacrol

Thymol

C o m

p o n e n t ( % )

Fig. 1. Concentrations (%) of carvacrol and thmyol in essential oils extracted from two species of thyme (adapted from Martínez et al., 2006).

of global anthropogenic CH4 emissions with most of it from enteric fermentation in ruminants, which is produced as a result

ofmicrobialfermentationoffeedsintherumenand,toalesserextent,inthehindgut.EntericCH4 isalossofproductiveenergy

typically between 2 and 12% of gross energy intake in ruminants depending on level of feed intake and diet composition

( Johnson and Johnson, 1995; Boadi et al., 2004). Therefore, reducing enteric CH4 emissions from ruminants is beneficialfrom a nutritional (i.e., improved feed efficiency and animal productivity) and environmental (i.e., reduced contribution

of the agricultural sector to total GHG emissions) perspective. Accordingly, several dietary strategies have been suggested

to mitigate enteric CH4 emissions from ruminants (McAllister et al., 1996; Boadi et al., 2004; Beauchemin et al., 2009).

Ionophores such as monensin have been extensively investigated for their ability to reduce CH4 production in ruminants

and their effectiveness has been demonstrated, although their inhibitory effects do not always persist (Beauchemin et al.,

2008, 2009).

In North America, ionophores are used in dairy and beef cattle diets to improve efficiency of milk and meat production.

However, use of ionophores in livestock production is not permitted in Europe after the ban on growth promoters in January

2006 (OJEU, 2003). Although this ban is currently limited to the EU, there is increased pressure from the public in other

parts of the world, including North America, to ban or restrict use of antimicrobials in food animal production for other

than therapeutic purposes. For example, a recent report of the Pew Commission on Industrial Farm Animal Production

in the United States (PCIFAP, 2008) recommended restricting use of antimicrobials in food animal production in order to

reduce the risk of antimicrobial resistance to antibiotics used to treat infections in humans. Consequently, research has beenvery intensive, particularly in Europe, to develop alternatives to antibiotic growth promoters in livestock production. Plant

secondary metabolites, due to their well documented antimicrobial activity, are viewed as potential alternatives.

Plants produce an array of diverse secondary metabolites which, when extracted and concentrated, may exert antimicro-

bial activities against a wide variety of microorganisms including bacteria, fungi and viruses (Chao et al., 2000; Greathead,

2003; Burt, 2004). Severalstudies, most of them invitro, have been publishedon effects of essentialoils andtheir components

on rumen microbial fermentation with a focus on N metabolism and volatile fatty acid (VFA) concentrations (Calsamiglia

et al., 2007; Benchaar et al., 2008b). However, thepotential of essentialoils andtheir constituentsto selectively inhibit rumen

methanogenesis has only recently been evaluated. Over the last 5 years, there has been an increased body of knowledge in

this area. This review aims to provide a critical evaluation of the potential of plant derived essential oils to inhibit rumen

methanogenesis. A number of studies have recently been published on the capacity of herbs, spices and plants to reduce

rumen CH4 production (Bodas et al., 2008; Garcìa-González et al., 2008a,b; Patra et al., 2010) but without identifying the

secondary metabolites responsible. For the purpose of our review, only data from studies that have specifically investigated

impacts of essential oils on methanogenesis are discussed.

2. Essential oils

2.1. Definition

Essential oils are complex mixtures of volatile lipophilic secondary metabolites. Traditionally extracted from plants by

boiling water and steam distillation, methods also include solvent extraction, supercritical CO2 extraction, and expression

extraction (i.e., a method used to extract essential oils from plants). They are plant specific and are responsible for a plant’s

characteristic flavour and fragrance. There can be a great deal of variation in essential oilyield andcomposition among plants

of the same species, and within different parts of the same plant (Cosentino et al., 1999; Burt, 2004). For instance, Martínez

et al. (2006) reported that the composition of the essential oil extracted from Thymus varied depending on the species. The

volatile fraction of the essentialoils extracted from Thymus zygisssp.Gracilliscontained higher concentrations of thymol, and

a lower concentration of carvacrol, than oil extracted from Thymus hymmalis Lange (Fig. 1). Delaquis et al. (2002) reported


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340 C. Benchaar, H. Greathead / Animal Feed Science and Technology 166–167 (2011) 338–355

Table 1

Chemical composition (% of total oil) of Oreganum vulgare ssp. hirtuma essential oil samples from different regions of Greece (adapted from Vokou et al.,

1993).

Athos Peninsula (650m) Kriti Island (550 m) Mount T aygetos ( 400 m) Evoia I sland ( 260 m)

Thymol 46.7 0.8 30.0 90.2

Carvacrol 11.9 74.2 51.0 2.5

p-Cymene 12.0 9.1 7.6 3.8

-Terpinene 16.0 4.1 5.2 0.6

-Thujene 0.8 1.0 0.5-Pinene 0.6 1.0 0.2Camphene 0.1 0.1

1-Octen-3-ol 0.4 0.2 0.7 0.7

3-Octanol 0.1 0.1 0.3 0.1

Myrcene 2.2 2.2 0.9

-Phellandrene 0.1 0.1 0.1-Terpinene 2.5 1.2 0.9-Phellandrene 0.3Limonene 0.2

-Phellandrene + limonene 0.3 0.1trans-Sabinene hydrate 0.7 0.6 0.7 0.4

Terpinolene 0.2

cis-Sabinene hydrate 0.4 0.2 0.2 0.1

Borneol 0.2 0.2 0.2 0.1

Naphthalene 0.4 0.2

Terpinen-4-ol 1.3 0.6-Terpineol 0.1Methylthymol 3.5

-Caryophyllene 1.0 0.8 0.2Farnesene 0.1 2.2 0.4

Caryophyllene oxide 1.3

a Plants were sampled at the flowering stage, air-dried and then distilled.

that the essential oil obtained from seeds of coriander (CoriandrumsativumL.) had a different composition from the essential

oil of cilantro, which was obtained from immature leaves of the same plant. Only terpenes were detected in coriander oil

while cilantro oil contained an additional heterogeneous mix of components that included alcohols, aldehydes, alkanes and

terpenes. Linalool was the most abundant compound in both oils, although the concentration was higher in coriander than

in cilantro (70.0 versus 25.6%). The composition of essential oils from a particular plant species can differ among harvesting

seasons and geographical locations.Vokou et al.(1993) observedthatconcentrations of themajoressential oils (i.e., carvacrol,

thymol, -terpinene and p-cymene) from Oregano (Origanum vulgare ssp. hirtum) varied among the geographical areas inGreece from which the plant was harvested (Table 1).

While it was once believed that secondary metabolites, and thus essential oils, had no function in the plant, it is now

generally accepted that they provide the plant with protection from abiotic and biotic stressors, as well as being attractants

to organisms that pollinate and disperse seeds (Wink and Schimmer, 1999). An alternative hypothesis is that they are a

metabolite reservoir for times when primary metabolites are in surplus (Gottlieb, 1990). While this hypothesis can account

for the variability in secondary metabolite composition among plants within species, it does not explain stress induced

changes in concentrations in plants (McNaughton et al., 1985; Marriott, 2000).

2.2. Chemistry and metabolic pathway of synthesis

Essential oils are typically composed of terpene and phenylpropene secondary metabolites. Generally, compounds from

one class dominate. For example clove (Syzygium aromaticum) essential oil is composed mainly of phenylpropenes (Dewick,

2002), whereas oregano (Oreganum vulgare) is composed mainly of terpenes (Vokou et al., 1993).

The isoprenes (C5) isopentenyl diphosphate and dimethylallyl diphosphate are the basic building blocks of terpenes

(Fig. 2). In plants, they are synthesised using the citric acid cycle intermediate acetyl-CoA (mevalonate pathway), and the

glycolytic intermediates glyceraldehydes 3-phosphate and pyruvate (deoxyxylulose pathway) as precursors (Fig. 3; Dewick,

2002). Condensation of isopentenyl diphosphate and dimethylallyl diphosphate produce the monoterpene (C10) geranyl

diphosphate, from which linalyl diphosphate and neryl diphosphate are formed by isomerisation reactions. From these three

monoterpenes, a range of linear and cyclic monoterpenes are formed (e.g., limonene, thymol, carvacrol, linalool, carvone,

geranyl acetate). The larger and less volatile sesquiterpenes (C15) are formed by addition of isopentenyl diphosphate to

geranyl diphosphate, which yields farnesyl diphosphate, the precursor of other sesquiterpenes including zingerberine and

artemesinin.

The phenylpropenes are synthesised via the shikimate pathway (Fig. 3), which is used by plants and microorganisms

(Sangwan et al., 2001) to synthesise aromatic amino acids phenylalanine and tyrosine. Deamination of phenylalanine yields

cinnamic acid, the basic phenylpropyl building unit (i.e., 6C aromatic ring with a 3C chain attached; Fig. 2) which, upon

hydroxylation, yields 4-coumaric acid. This is thought to be the most common pathway by which 4-coumaric acid is synthe-


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Fig. 2. The basic building units of the terpenes and phenylpropenes, the C5 isoprene unit (a) and the C6C3 phenylpropyl unit (b), respectively.

Fig. 3. An overview of the pathways responsible for synthesising terpenes and phenylpropenes, the principle metabolites found in plant essential oils

(adapted from Benchaar et al., 2009).


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sised (Dewick, 2002), but it may also be formed from deamination of tyrosine. From 4-coumaric acid, via a series of reactions,

the phenylpropenes are synthesised (e.g., cinnamaldehyde, eugenol, anethole, myristicin and safrole; Fig. 3).

While terpenes andphenylpropenes dominate the composition of essentialoils, they are notthe only types of compounds

in essential oils. For example, garlic essential oil contains a range of organo-sulphur compounds (e.g., diallyl sulphide (DAS),

diallyl disulphide (DADS), diallyl trisulphide (DATS), vinyl-dithiins and adjoenes) which originate from -glutamyl cysteinestorage dipeptides (Corzo-Martínez et al., 2007). These dipeptides are hydrolysed and oxidised into the cysteine sulphoxides;

alliin, isoalliin and methiin ( Jones et al., 2004). Alliin is the major cysteine sulphoxide in garlic. Upon processing (i.e., crushing

and cutting) the cysteine sulphoxides are rapidly cleaved by the enzyme alliinase to yield thiosulphinates (e.g., allicin),

pyruvate and ammonia (Amagase et al., 2001). The thiosulphinates are unstable and rapidly decompose to sulphides, which

are abundant in garlic essential oil.

2.3. Mechanisms of action (antimicrobial properties)

Many of the component secondary metabolites in essential oils, and thus the essential oils themselves, exhibit antimi-

crobial activity. The sensitivity of microbes to essential oils varies, and it is this property which is of interest to ruminant

nutritionists, as it lends itself to applications for changes in rumen fermentation via selection for or against specific groups

of microorganisms.

What determines the antimicrobial activity of a secondary metabolite is unclear, but the presence of oxygen and S in

the chemical structure would appear to be important. Hydrocarbons (e.g., p-cymene, -terpinene and R(+)-limonene) havevariable antimicrobial activity (Oh et al., 1967; Cosentino et al., 1999; Dorman and Deans, 2000; Cristani et al., 2007).

However, secondary metabolites containing oxygen, such as phenols, and S such as in sulphides (Ross et al., 2001; Corzo-

Martínez et al., 2007) tend to exhibit strong antimicrobial activity. The hydroxyl group is believed to be instrumental in

disrupting normal ion transport across the cytoplasmic membrane (Ultee et al., 2002) and in inactivating microbial enzymes

(Burt, 2004).

Hydrophobicity appears to be crucialfor antimicrobial activity. It means essential oils will preferentially partitionfrom an

aqueous phase into the lipid bilayer of the cytoplasmic membrane where they accumulate. It is from within the lipid bilayer

that essential oils are believed to orchestrate one or more of their antimicrobial effects by altering membrane permeability

and so disrupting ion transport processes and interacting with membrane proteins, and/or other cytoplasmic components.

This essential oil mediated response is achieved either from within the cytoplasmic membrane or by diffusion into the

cytoplasm. As essential oils are mixtures of many secondary metabolites, it is likely that there are a number of mechanisms

of action (Fig. 4).

The antimicrobial activity of thymol and carvacrol, phenolic monoterpenes, found in high concentrations in oregano

essential oil, is well documented (Helander et al., 1998; Ultee et al., 1999, 2000a,b). They, and oregano essential oil, have

both been shown to increase fluidity and permeability of the cytoplasmic membrane leading to loss of cell contents and cell

lysis (Trombetta et al., 2005; Di Pasqua et al., 2007; Paparella et al., 2008; Xu et al., 2008). Possibly in response to increased

fluidity of the cytoplasmic membrane causedby thymol and carvacrol,an increased ratioof saturated:unsaturated membrane

fatty acids was reported in bacteria exposed to these phenolic monoterpenes (Di Pasqua et al., 2006, 2007).

Both thymol and carvacrol have been reported to dissipate H+ and K+ ion gradients, and thus proton motive force,

leading to depletion of intracellular ATP concentration as a result of either inhibiting ATP synthesis or increasing rates of

ATP hydrolysis (Lambert et al., 2001). A proton transfer mechanism has been proposed by Ultee et al. (2002) in which the

hydroxyl group of these phenolic monoterpenes acts as a trans-membrane proton carrier (Fig. 4), a mechanism of action not

dissimilar to ionophore antibiotics (Bergen and Bates, 1984).

The antimicrobial mechanism of action of the phenylpropene eugenol, a major component of clove essential oil, has been

suggested to be via inhibition of glycolytic enzyme activity resulting in an inability of the microbe to utilise intracellular

glucose (Gill and Holley, 2004). Membrane effects have also been reported, the ratio of saturated to unsaturated fatty acids

fatty is increased in the cytoplasmic membranes of eugenol treated bacteria (Di Pasqua et al., 2006), and there is leakage

of K from eugenol treated bacteria (Walsh et al., 2003). Gill and Holley (2004) discount these effects as responsible for

the bacteriocidal effects of eugenol, as they were unable to detect changes in extracellular ATP concentrations in bacteria

exposed to eugenol.

The antimicrobial activity of the phenylpropene cinnamaldehyde, a major component of cinnamon oil, is suggested to

arise from disruption of the cytoplasmic membrane (Ouwehand et al., 2010). This results in depletion of cellular ATP and

impaired glucose import (Gill and Holley, 2004) and inactivation of bacterial enzymes (Burt, 2004; Gill and Holley, 2004).

Helander et al. (1998) were unable to detect any evidence of membrane associated damage in cinnamaldehyde treated Gram

negative bacteria.

Antimicrobial activity of garlic essential oil seems to be associated with allicin and diallyl sulphides. In the case of

the later, antimicrobial activity increases with the number of sulphur atoms so, whereas DAS has very little activity, the

polyallylsulphides (DATS) have a great deal (Münchberg et al., 2007). The S -allyl moiety (S–CH2–CH CH2) of allicin has been

shown to interact with proteins and amino acids containing sulfhydryl (–SH) groups (Fujisawa et al., 2009), which is likely

to disrupt cell function. While the same mechanism of action is frequently associated with the diallyl sulphides, a number

of other mechanisms have been proposed, which are described by Münchberg et al. (2007).


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Fig. 4. Schematicoverviewof some of thesitesand mechanisms of antimicrobial activity of essentialoilsin thebacterial cell (adapted from Benchaaret al.,

2009).

The antimicrobial activity of essential oils tends to be selective against Gram positive bacteria. For example, there is

evidence to suggest that Gram positive bacteria may be more sensitive to oregano essential oil and its constituent phenols

than Gram negative bacteria (Cosentino et al., 1999; Lambert et al., 2001). Smith-Palmer et al. (1998), Trombetta et al. (2005)

and Fujisawa et al. (2009) have reported similar observations for other essential oils. The outer membrane of Gram negative

bacteria is highly charged and is thought to act as a barrier to essential oils. It has been suggested that essential oils activeagainst Gram negative bacteria contain secondary metabolites which are small enough to pass through porin proteins in

the outer membrane and so able to access the cytoplasmic membrane (Nikaido, 1994; Dorman and Deans, 2000). In the

case of allicin, protein in the outer membrane of Gram negative bacteria has been suggested to bind to the S -allyl moiety,

deactivating it before it reaches the cytoplasmic membrane and cytoplasm (Fujisawa et al., 2009).

3. Effects of essential oils onCH4 production

3.1. Thymol

Thymol, a phenolic monoterpene [(5-methyl-2-(1-methylethyl)phenol)] is one of the major compounds of thyme (Thy-

mus vulgaris) and organo (Origanum vulgaris) essential oils. Thymol has been shown to have a broad spectrum of activity

against a variety of Gram positive and Gram negative bacteria (Dorman and Deans, 2000; Lambert et al., 2001; Walsh et al.,

2003). Several studies have investigated effects of thymol or essential oils with high thymol content on rumen fermentation


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(Castillejos et al., 2006, 2008; Martínez et al., 2006; Benchaar et al., 2007a), but only a few have specifically determined its

effects on rumen methanogenesis (Table 2).

Evans and Martin (2000) examined effects of increasing concentrations of thymol (50, 100, 200, and 400 mg/L of culture

fluid) on in vitro fermentation as 24h batch culture incubations of d-glucose by mixed rumen bacteria (Table 2). Methane

concentration was not affected when thymol was supplied at 50, 100, and 200 mg/L of culture fluid. However, at 400 mg/L,

thymol increased the pH of the medium, drastically decreased (−94%) CH4 concentration along with acetate and propionate

concentrations (−44 and −78%, respectively). A higher pH and a reduction in VFA concentrations are an indication of an

overall inhibition of rumen microbial fermentation, and these changes would not be nutritionally beneficial to the host

animal if the same effects were expressed in vivo.

Macheboeuf et al. (2008) evaluated in batch cultures (16 h incubation), effects of thyme (T. vulgaris; 470 g/kg thymol,

200 g/kg terpinene and 200 g/kg p-cymene) on rumen fermentation. A minimum of 300 mg/L of thymol provided as is, or

via thyme oil, was required to inhibit CH4 production with a concomitant decrease in total VFA production, acetate and

propionate production (Table 2). More recently, Benchaar and Chiquette (unpublished) observed a 32% reduction in CH4concentration when thymol was supplied in vitro in a 24 h batch culture incubation at 300mg/L of culture fluid (Table 2),

which occurred with decreased concentrations of total VFA, acetate and propionate.

Interestingly, in the study by Macheboeuf et al. (2008), the extent of reduction in CH4 production was more pronounced

with thyme oil (providing 300mg/L of thymol) than when thymol was provided alone in incubations at the same con-

centration (−62 versus −32%, respectively), which suggests that other constituents in thyme essential oil contributed to

its antimicrobial activity. The thyme essential oil used in the study by Macheboeuf et al. (2008) also contained 200 g/kg

p-cymene and 200 g/kg terpinene. The monoterpenes p-cymene [1-methyl-4-(1-methylethyl)-benzene] and -terpinene[1-methyl-4-(1-methylethyl)-1,4-cyclohexadiene] are the precursors of thymol [5-methyl-2-(1-methylethyl)phenol] and

carvacrol [2-methyl-5-(1-methylethyl)-phenol] in ThymusandOriganumspecies (Cosentinoet al., 1999; Jerkovic et al., 2001;

Ultee et al., 2002). Compared to the phenolic monoterpenes, p-cymene and-terpinene appear to have limited antimicro-bial activity, particular alone (Cosentino et al., 1999; Dorman and Deans, 2000; Ultee et al., 2000a), possible due to a lack

of a hydroxyl group. However in one study these two non-phenolic monoterpenes exhibited antibacterial activity against

the Gram positive bacterium Staphylococcus aureus and the Gram negative bacterium Escherichia coli (Cristani et al., 2007).

The antimicrobial activity of p-cymene was also demonstrated against rumen bacteria. In a 6 h in vitro batch culture study,

Chaves et al. (2008b) observed that p-cymene at 20mg/L reduced methanogenic activity of rumen bacteria (mol of CH4/gbacterial N/min), and CH4 concentration in the fermentation gases, without altering the VFA profile or VFA concentration

(Table 2), perhaps suggesting that this compound acts via by directly inhibiting methanogens.

In addition to thymol and p-cymene, essential oil extracted from Thymus also contains carvacrol (Cosentino et al., 1999;

Martínez et al., 2006). Synergistic effects have occurred against Bacillus cereus in vitrowhen p-cymene was combined with

the phenolic carvacrol (Ultee et al., 2000b). Ultee et al. (2002) suggested that the antimicrobial activity of p-cymene is due to

its accumulation in the plasma membrane, causing the membrane to expand and allow leakage of ions. Cristani et al. (2007)

suggested that non-phenolic monoterpenes, such as p-cymene and -terpinene, are able to diffuse through the plasmamembrane into the bacterial cytoplasm. This supports evidence indicating that they potentate antimicrobial effects of the

phenolic monoterpenes by facilitating their transport across the plasma membrane (Ultee et al., 2002).

3.2. Carvacrol

Carvacrol [2-methyl-5-(1-methylethyl)-phenol] is a phenolic compound mainly found in the essential oil fraction of

Origanum and Thymus. As for thymol, the high antimicrobial activity of carvacrol has been attributed to the presence of a

hydroxyl group in the phenolic structure (Dorman and Deans, 2000; Ultee et al., 2002; Burt, 2004).

Macheboeuf et al. (2008) observed a linear decrease in CH4 production when carvacrol was supplied at 225 (−13%), 300

(−32%), 450 (−85%) and 750 (−98%) mg/L in batch culture (Table 2). In the same study, CH4 production was not affected

by addition of O. vulgare oil (890g/kg carvacrol, and 50g/kg thymol) at concentrations of 150 and 300 mg/L, while at 450

and 750 mg/L CH4

was markedly inhibited (−63 and −97%, respectively). At 450 mg/L of thymol plus carvacrol, the essential

oil of O. vulgare was less inhibitory than its main component carvacrol (−63 versus −85%), suggesting that minor compo-

nents in Origanum essential oil acted antagonistically with carvacrol. Benchaar and Chiquette (unpublished) confirmed the

antimethanogenic effect of carvacrol when it was used at 225, 250, 300, and 350 mg/L in vitro (Table 2).

In the studies of Macheboeuf et al. (2008) and Benchaar and Chiquette (unpublished), inhibition of methanogenesis with

oregano and its main component carvacrol occurred concomitantly with a reduction in acetate, propionate and total VFA

concentrations. In the rumen, Gram positive bacteria are generally acetate and butyrate producing bacteria, while Gram

negative bacteria are generally propionate producing (Stewart, 1991). Therefore, results suggest that rumen Gram positive

and Gram negative bacteria were sensitive to oregano oil and carvacrol, which agrees with findings of Sivropoulou et al.

(1996) and Lambert et al. (2001) who demonstrated that both of these essential oils inhibited several strains of Gram positive

and Gram negative pathogenic bacteria.

Macheboeuf et al. (2008) evaluated the thymol chemotype of O. vulgare (210g/kg carvacrol, and 350 g/kg thymol) for its

effects on rumen fermentation and CH4 production (Table 2). At 134 mg/L (providing 75mg/L of carvacrol and thymol) it

did not exhibit antimicrobial activity, and had no effect on CH4

, total VFA, acetate or propionate production. At 268 mg/L,

providing 150 mg/L of carvacrol and thymol, CH4 production was unchanged although production of VFA and propionate


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Table 2

Effects of essentials oils and essential oil components on rumen fermentation characteristics.

Essential

oil/essential

component

Test system Dose Substrate VFA Acetate Propionate Methane Reference

Anethol Batch (6 h) 20 mg/L Soluble starch = = = = Chaves et al.

(2008b)

Cinnamon leaf

(760g/kgeugenol)

Batch (6 h) 250 mg/L Soluble starch = = – −

Garlic (15g/kg

allicin)

Batch (6 h) 100 mg/L Soluble starch = = − −

Garlic (15g/kg

allicin)

Batch (6 h) 250 mg/L Soluble starch = = − −

Juniperberry

(350 g/kg

-pinene)

Batch (6 h) 20 mg/L Soluble starch = = = −

p-Cymene Batch (6 h) 20 mg/L Soluble starch = = = −

Thymol Batch (24 h) 100 mg/L F:C (50:50) = = = = Benchaar and

Chiquette

(unpublished)

Batch (24 h) 130 mg/L F:C (50:50) = = = =

Batch (24 h) 160 mg/L F:C (50:50) = = = =

Batch (24 h) 200 mg/L F:C (50:50) =−

= =Batch (24 h) 220 mg/L F:C (50:50) = − = =

Batch (24 h) 240 mg/L F:C (50:50) − − − =

Batch (24 h) 260 mg/L F:C (50:50) − − − =

Batch (24 h) 280 mg/L F:C (50:50) − − − −

Batch (24 h) 300 mg/L F:C (50:50) − − − −

Carvacrol Batch (24 h) 150 mg/L F:C (50:50) = = = = Benchaar and

Chiquette

(unpublished)

Batch (24 h) 175 mg/L F:C (50:50) = − = =

Batch (24 h) 185 mg/L F:C (50:50) = − = −

Batch (24 h) 200 mg/L F:C (50:50) − − = =

Batch (24 h) 225 mg/L F:C (50:50) − − = −

Batch (24 h) 250 mg/L F:C (50:50) − − − −

Batch (24 h) 300 mg/L F:C (50:50) − − − −

Batch (24 h) 350 mg/L F:C (50:50) − − − −

Eugenol Batch (24 h) 300 mg/L F:C (50:50) = = = = Benchaar and

Chiquette

(unpublished)

Batch (24 h) 400 mg/L F:C (50:50) = = = −

Batch (24 h) 500 mg/L F:C (50:50) = = = −

Batch (24 h) 600 mg/L F:C (50:50) − − = −

Batch (24 h) 700 mg/L F:C (50:50) − − − −

Batch (24 h) 800 mg/L F:C (50:50) − − − −

Batch (24 h) 900 mg/L F:C (50:50) − − − −

Anethum

graveolensa

(400g/kg

carvone;

320g/kg

limonene)

Batch (16 h) 2.5 mM F:C (25:75) − = = = Macheboeuf

et al. (2008)

Batch (16 h) 5.0 mM F:C (25:75) − = − =

Batch (16 h) 10.0 mM F:C (25:75) − − − −

Batch (16 h) 25.0 mM F:C (25:75) − − − −

Cinnamomun

verumb (790g/kg

cinnamaldehyde)

Batch (16 h) 1.0 mM F:C (25:75) = = = = Macheboeuf

et al. (2008)

Batch (16 h) 3.0 mM F:C (25:75) − = − −

Batch (16 h) 5.0 mM F:C (25:75) − − − −

Batch (16 h) 10.0 mM F:C (25:75) − − − −

Cinnamaldehyde Batch (16 h) 1.0 mM F:C (25:75) = = = = Macheboeuf

et al. (2008)

Batch (16 h) 2.0 mM F:C (25:75) = = = −

Batch (16 h) 3.0 mM F:C (25:75) − = − −

Batch (16 h) 5.0 mM F:C (25:75) − − − −


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Table 2 (Continued)

Essential

oil/essential

component


Carvacrol Batch (16 h) 1.5 mM F:C (25:75) − − − − Macheboeuf

et al. (2008)

Batch (16 h) 2.0 mM F:C (25:75) − − − −

Batch (16h) 3.0mM F:C (25:75) − − − −

Batch (16h) 5.0mM F:C (25:75) − − − −

Origanum vulgarec

(890g/kg

carvacrol;

50 g/kg thymol)

Batch (16 h) 1.0 mM F:C (25:75) = = − = Macheboeuf

et al. (2008)

Batch (16 h) 2.0 mM F:C (25:75) − = − =

Batch (16 h) 3.0 mM F:C (25:75) − − − −

Batch (16 h) 5.0 mM F:C (25:75) − − − −

Thymol chemotype

of Origanum

vulgarec

(350g/kg

thymol; 210g/kg

carvacrol)


et al. (2008)

Batch (16h) 1.0mM F:C (25:75) − = − =

Batch (16 h) 2.0 mM F:C (25:75) − − − −

Batch (16 h) 3.0 mM F:C (25:75) − − − −

Thymol Batch (16 h) 1.0 mM F:C (25:75) = = − = Macheboeuf

et al. (2008)

Batch (16 h) 2.0 mM F:C (25:75) − − − −

Batch (16h) 3.0mM F:C (25:75) − − − −

Batch (16 h) 6.0 mM F:C (25:75) − − − −

Thymus vulgarisc

(470g/kg

thymol; 200g/kg

terpinene;

200g/kg

p-cymene)


et al. (2008)

Batch (16 h) 1.0 mM F:C (25:75) − = − =

Batch (16 h) 2.0 mM F:C (25:75)− − − −

Batch (16h) 3.0mM F:C (25:75) − − − −

Garlic (7g/kg

allicin)

Batch (17 h) 300 mg/L F:C (50:50) − − + − Busquet et al.

(2005b)

Diallyl disulfide Batch (17 h) 300 mg/L F:C (50:50) − − + −

Ally mercaptan Batch (17 h) 300 mg/L F:C (50:50) − − = −

Thymol Batch (24 h) 50 mg/L Glucose NR = = = Evans and

Martin (2000)

Batch (24 h) 100 mg/L Glucose NR = = =

Batch (24 h) 200 mg/L Glucose NR = = =

Batch (24 h) 400 mg/L Glucose NR − − −

Allicin Batch (24 h) 0.5 mg/L F:C (50:50) = = = = Kamel et al.

(2008)

Batch (24 h) 5.0 mg/L F:C (50:50) = = = =

Batch (24 h) 10.0 mg/L F:C (50:50) = = = =

Diallyl disulfide Batch (24 h) 0.5 mg/L F:C (50:50) = = = = Kamel et al.

(2008)

Batch (24 h) 5.0 mg/L F:C (50:50) = = = =

Batch (24 h) 10.0 mg/L F:C (50:50) = + = =

-Cyclodextrin–horseradish oil

complex

Batch (6 h) 170 mg/L Corn starch + − + − Mohammed

et al. (2004)

850 mg/L Corn starch + − + −

1700 mg/L Corn starch + − + −

-CD-peppermint Batch (6 h) 83 mg/L Cellobiose/glucose (1:1) = = = = Tatsuoka et al.(2008)

Batch (6 h) 167 mg/L Cellobiose/glucose (1:1) = = = =


10/18


Table 2 (Continued)

Essential

oil/essential

component



-CD-peppermint Batch (6 h) 83 mg/L Cellobiose/glucose (1:1) = = = = Tatsuoka et al.(2008)



Peppermint

(Mentha piperita)

Batch (24 h) 0.33 mg/L F:C (50:50) = = = − Agarwal et al.

(2009)

Batch (24 h) 1.0 mg/L F:C (50:50) − + − −

Batch (24 h) 2.0 mg/L F:C (50:50) − + − −

Crina® Ruminants

(thymol,

eugenol, vanillin

limonene)

In vivo

(beef cattle)

1000mg/d

(137mg/kg DMI)

TMR (F:C = 75:25) = = = = Beauchemin

and McGinn,

2006

-Cyclodextrin–horseradish

oil complex

In vivo (steers) 80 g /d

(20g/kg DMI)

F:C (60:40) = − + – Mohammed

et al. (2004)

VFA, volatile fatty acids; F:C, forage to concentrate ratio; NR, not reported; +, increase; −, decrease; =, no change; DMI, dry matter intake; -CD, -cyclodextrin; and -CD, -cyclodextrin.

a Doses were calculated considering carvone as the main active molecule in Anethum graveolens essential oil (Macheboeuf et al., 2008).b Doses were calculated considering cinnamaldehyde as the main active molecule in Cinnamomunverum essential oil (Macheboeuf et al., 2008).c Doses were calculated consideringthe sumof carvacrol, and thymol as main components inOriganum vulgare, thymol chemotype of Origanum vulgare

and Thymus vulgaris essential oils (Macheboeuf et al., 2008).

decreased (−18 and −50%, respectively). When the essential oil was supplied to provide 300 mg/L of thymol and carvacrol,

CH4 production decreased (−60%) along with production of VFA (−67%), acetate (−54%) and propionate (−73%). At the

highest concentration of 450 mg/L of active components, fermentation was almost completely inhibited as evidenced by

decreases in CH4 (−95%), total VFA (−93%), acetate (−91%) and propionate (−99%).

Results from Macheboeuf et al. (2008) showed that at 300mg/L of active components, the antimethanogenic effect of the

essential oil from thymol chemotype of O. vulgarewas higher (−60%) than its main components carvacrol andthymol (−32%)

were included individually at the same concentration in vitro. This suggests that the effects of these two molecules on rumen

microbial fermentation is additive, which was supported when it was shown that the minimum inhibitory concentration

of oregano essential oil and its two main constituents, thymol and carvacrol, against S. aureus and Pseudomonas aeruginosawas lower as a mixture compared to either compound alone (Lambert et al., 2001).

3.3. Eugenol

Eugenol (4-allyl-2-methoxyphenol) is a phenolic monoterpene present in high quantities in clove bud (S. aromaticum)

and cinnamon leaf (Cinnamomum cassia) essential oils. Eugenol has been shown to have antimicrobial activity against Gram

positive and Gram negative bacteria (Dorman and Deans, 2000; Walsh et al., 2003).

Patra et al. (2010) investigated boiling water, methanol and ethanol extracts of clove bud for their effects on CH4 pro-

duction in vitro using 24h batch cultures. Methane production (ml/kg of DM) was not affected by clove water extract while

addition of methanol and ethanol extracts of this spice (at 2.5 ml/g of substrate) inhibited CH4 (ml/g DM) production by 35

and 83%, respectively, but digestibility of feed was also reduced. However, this inhibitory effect cannot be attributed only to

the essential oil fraction of the plant, because S. aromaticum also contains high concentration of tannins (Patra et al., 2010),

a factor that may explain the higher effectiveness of solvent versus aqueous extractions.

Chaves et al.(2008b) investigated effects of cinnamon leaf oil(containing 760g/kg eugenol) on fermentationend products

and methanogenesis in vitro (Table 2). When supplied at 250mg/L, cinnamon leaf oil reduced CH4 production without

adversely affecting total VFA production, although it did alter the relative proportions of VFA produced. It did not affect the

molar proportion of acetate, but it decreased propionate and increased butyrate. Interestingly, in the study by Chaves et al.

(2008b), monensin did not reduce CH4 production to the same extent as cinnamon leaf essential oil (i.e., −57 versus −70%,

respectively). Results from Chaves et al. (2008b) suggest that cinnamon oil did not exert its antimicrobial effects through

the same mechanism as monensin, but rather that inhibition of CH4 production by this essential oil was due to the direct

inhibition of rumen methanogens.

Benchaar and Chiquette (unpublished) studied effects of eugenol on rumen microbial fermentation and CH4 production

using in vitro batch culture incubations over 24h (Table 2). At 300 mg/L, this compound did not exert any effect on VFA or

CH4 concentrations. Lack of change in total VFA concentration would be viewed as desirable if it was accompanied by a

decline in CH4

production. Indeed, at 400 and 500 mg/L, eugenol decreased CH4

production by −30 and −35%, respectively,

without altering VFA concentrations Benchaar and Chiquette (unpublished), thereby corroborating results of Chaves et al.


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(2008b) that, at doses of 400–500 mg/L, the inhibitory effect of eugenol appears to target methanogens rather than other

rumen bacteria. However, when eugenol was supplied at higher doses of 600, 700, 800 and 900 mg/L, the reduction in CH4production was accompanied by a strong reduction in total VFA production (up to −88%) and feed digestion was inhibited

(Benchaar and Chiquette, unpublished). Taken together, results from studies of Chaves et al. (2008b) and Benchaar and

Chiquette (unpublished) indicate that the range between beneficial and detrimental effects of eugenol on rumen microbial

ecosystem is narrow, which limits potential use of this essential oil compound in practical feeding situations to mitigate

enteric CH4 emissions from ruminants.

3.4. Cinnamaldehyde

Cinnamaldehyde (3-fenil-2-propenal phenol), a non-phenolic phenylpropene, is a major component of cinnamon bark (C.

cassia) essential oil. Both Gram positive and Gram negative bacteria have been reported to be sensitive to cinnamon oil and

cinnamaldehyde (Ouattara et al., 1997; Helander et al., 1998; Smith-Palmer et al., 1998). Cinnamaldehyde has been shown

to exhibit antimicrobial activity similar to that of the phenolicsthymol and carvacrol (Helander et al., 1998). However, unlike

thymol and carvacrol, it does not have a hydroxyl or acid group to act as a proton carrier to disrupt the outer membrane or

deplete the intracellular ATP pool (Helander et al., 1998). Alternatively, the antimicrobial properties of cinnamaldehyde are

thought to arise through its carbonyl group binding and inactivating microbial enzymes (Burt, 2004).

To our knowledge, only one study has investigated effects of cinnamon bark oil and its main component cinnamaldehyde

on rumen methanogenesis. Macheboeuf et al. (2008) showed in vitro (Table 2) that supplying 132mg/L cinnamaldehyde

had no effect on CH4, total VFA, acetate or propionate production. At 264 mg/L, cinnamaldehyde slightly decreased CH4production, by 13%, without altering VFA, acetate or propionate production, suggesting that, at this dose, it inhibited rumen

methanogenesis by acting directly against rumen methanogens. At 396mg/L, cinnamaldehyde decreased production of CH4by19%,aswellasproductionofVFA(−13%),acetate(−7%)and propionate (−22%). At 661mg/L, it almostcompletely inhibited

CH4 production (−94%) and dramatically reduced VFA (−60%), acetate (−55%) and propionate (−92%) concentrations. Such

changes indicate that, at high doses, the antimicrobial activity of cinnamaldehyde is sufficient to almost completely inhibit

microbial metabolism.

When cinnamon bark essential oil (Cinnamomum verum, containing 790g/kg cinnamaldehyde) was evaluated under the

same experimental conditions in the same study, the minimum concentration required to inhibit rumen methanogenesis

was 500 mg/L (providing 396 mg/L of cinnamaldehyde), which reduced CH4 production by 26% (Macheboeuf et al., 2008).

This decrease was more than the 19% when cinnamaldehyde was included alone at the same concentration in vitro. These

results disagree with those of Busquet et al. (2006), who found that cinnamaldehyde inhibited in vitro CH4 production to a

higher extent than cinnamon oil containing 590 g/kg cinnamaldehyde. Macheboeuf et al. (2008) suggested that, although

cinnamaldehyde is the main active component of cinnamon bark oil, other components present in low concentrations may

interact with cinnamaldehyde. Indeed the essential oil fraction of cinnamon oil bark has been reported to also contain

up to 80g/kg eugenol (Davidson and Naidu, 2000). Synergism between cinnamaldehyde and eugenol has been reported

(Burt, 2004), which may explain the more pronounced antimethanogenic activity of cinnamon bark essential oil versus

cinnamaldehyde.

3.5. Garlic oil and its constituents

Intact whole garlic contains several sulphur containing compounds with the major being -glutamyl-S -allyl-l-cysteinesand S -allyl-l-cysteine sulphoxides (e.g., alliin). These compounds are converted into thiosulphinates, such as allicin, through

enzymatic reactions when raw garlic is cut or crushed (Fenwick and Hanley, 1985; Amagase et al., 2001; Amagase, 2006).

During oilextraction by steam distillation, allicin degrades to form a variety of fatsoluble organosulphur compounds, includ-

ing diallyl trisulphide, diallyl disulphide, and diallyl sulphide (Block, 1985; Lawson, 1996). These S containing compounds

have activity against a wide range of Gram positive and Gram negative bacteria, and their effects against pathogenic bac-

teria are well documented (Reuter et al., 1996; Ross et al., 2001). However, the potential of garlic oil and its derivatives to

selectively inhibit rumen methanogenesis has only recently been explored.

Busquet et al. (2005b) were the first to report effects of garlic essential oil and two of its compounds (i.e., diallyl disulphide

and allyl mercaptan) on CH4 production (Table 2). When added at 300mg/L in 17h in vitro batch culture fermentations,

allyl mercaptan decreased CH4 production by 19.5%, and VFA concentration, without altering digestibility. At the same

concentration, garlic and diallyl disulphide reduced CH4 production by −74 and −69%, respectively, but DM digestibility

and VFA concentration were also depressed. That diallyl disulphide reduced CH4 production to the same extent as the oil

fraction of garlic may indicate that this S containing compound is responsible for most of the antimethanogenic activity

of garlic oil. Effects of garlic essential oil on rumen methanogenesis was accompanied by a decrease in the proportion of

acetate, whereas propionate and butyrate increased. High butyrate concentrations as a result of inclusion of garlic oil to in

vitro incubations may indicate that it alters ruminal fermentation differently than monensin which tends to decrease the

acetate to propionate ratio and butyrate concentration (Calsamiglia et al., 2007).

The inhibitory effect of garlic essential oil on in vitro CH4 production was confirmed by Chaves et al. (2008b), who

reported that at 100 and 250 mg/L, decreased CH4

production by 69 and 72%, respectively, with no adverse effect on VFA

concentration (Table 2). Interestingly, in Busquet et al. (2005b) and Chaves et al. (2008b) the inhibitory effect of monensin on


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CH4 production was less pronounced than either garlic essential oils or its active component, diallyl disulphide. Collectively,

these results suggest that, unlike monensin, which specifically inhibits rumen Gram positive bacteria, the antimethanogenic

effect of garlic and its main components may be the result of direct inhibition of rumen methanogens.

Busquetet al.(2005a,b) hypothesized that theinhibitory effectof garlicoil andits sulphur containing compoundscould be

mediated via inhibition of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase or HMGR),

an enzyme that has an important role in synthesis of isoprenoid ethers, the main component of archaeal cell membranes.

Several studies have shown that organosulphur compounds depress activity of HMG-CoA, the rate limiting enzyme in

cholesterol biosynthesis (Gebhardt, 1993; Gebhardt and Beck, 1996).

Contradictory results have been reported on effects of allicin on rumen methanogenesis. Busquet et al. (2005b) reported

that allicin (3, 30, 300 and 3000mg/L) had no effects on the concentrations of VFA, acetate or propionate, suggesting that

rumen methanogenesis was not inhibited, although CH4 was not directly measured. Using a rumen simulation technique,

Hart et al. (2006) observed that addition of a commercial source of allicin (Neem Biotech Ltd., Cardiff, UK) at concentrations

of 2 and 20mg/L did not affect production of VFA, acetate or propionate. However, at 20mg/L, CH4 production was almost

totally inhibited (i.e., −94%). In the same study, analysis by real time PCR suggested a direct effect of allicin on decreasing

methanogen DNA (i.e., methanogen numbers), with no effect on total bacterial DNA. Therefore, it seems that allicin can have

a selective effect on microbial populations in the rumen (Hart et al., 2008). However, these observations were not confirmed

by Kamel et al. (2008), who reported that CH4 production was unaffected by addition of allicin at 0.5, 5 and 10mg/L in 24h

in vitro incubations, which was consistent with its lack of effect on VFA concentration and molar proportions of acetate and

propionate (Table 2). Given the extent to which CH4 production was reduced (i.e., −94%) in the study of Hart et al. (2006),

one may expect that allicin should have also resulted in inhibition of rumen methanogenesis in Kamel et al. (2008), even if

the dosage was lower (10 versus 20 mg/L).

It is possible that rumen microbes are able to adapt to and degrade some S containing components of garlic oil (Hart et al.,

2008). Indeed, Ohene-Adjei et al. (2008) observed that feeding lambs with garlic essential oil at 200 mg/kg DM intake did not

affect total copy number of archaeal 16S rRNA, but increased diversity of methanogenic archaea includingMethanosphaera

stadtmanae, Methanobrevibacter smithii, and some uncultured groups. The increased diversity of methanogens may be a

reflection of the ability of ruminal microbes to adapt to these compounds. Using an in vitro batch culture system over 24h,

Busquet et al. (2005b) observed that, at high concentrations of 300mg/L, diallyl disulphide exhibited antimicrobial activities

by decreasing VFA concentration and acetate molar proportion, but increased the molar proportion of propionate, which

should result in lower CH4 production. However, such effects were not observed by Busquet et al. (2005b) when the same

compound was evaluated at the same concentration in a continuous culture system of 8 d incubation, suggesting possible

adaptation and/or degradation of diallyl disulphide by rumen microbes. The adaptation of rumen microbes to essential oils

may result from shifts in microbial populations and/or changes in individual microbial species (Benchaar et al., 2008b).

3.6. Horseradish essential oil

Extensive research has been conducted in Asia, particularly Japan, to evaluate the potential of the essential oil of

horseradish to manipulate rumen microbial fermentation and decrease ruminal CH4 production. Horseradish ( Armoracia

rusticana) is a perennial plant of the Brassicaceae family. The main component of horseradish essential oil is allyl isothio-

cyanate, a non-phenolic sulphur-containing compound that has been shown to have antimicrobial activity (Tajkarimi et al.,

2010).

Mohammed et al. (2004) examined effects of increasing levels (170, 850, 1700mg/L) of -cyclodextrin encapsulatedhorseradish essential oil on rumen microbial fermentation in 6 h batch cultures (Table 2), and observed that CH4 produc-

tion decreased linearly with increasing essential oil levels. These changes were accompanied by a shift in VFA proportions

towards more propionate and less acetate, with accumulation of H2 and an increase in VFA concentration. The higher VFA

concentration is somewhat surprising as it is not consistent with the antimicrobial activity of horseradish essential oil. The

increased H2 concentration is an indication of a possible direct effect of horseradish essential oil on methanogens. If such

accumulation of H2 occurs in vivo, fermentation activity would be depressed and feed digestion and animal performance

adversely affected (Beauchemin et al., 2009).

In the same study, Mohammed et al. (2004) observed that at a high inclusion rate of 20g/kg dietary DM, encapsulated

horseradish essential oil decreased CH4 production by 19% in steers without affecting diet digestibility, but DM intake

was depressed by 10% (Table 2). Supplementation with horseradish oil increased propionate molar proportion with a

decline in acetate and the numbers of methanogens, but with no effect on total numbers of protozoa, total viable bacteria,

sulphate-reducing bacteria, cellulolytic bacteria or acetogens. However, horseradish essential oil decreased the number of

methanogens in vivo confirming that the antimethanogenic activity of horseradish essential oil is mainly modulated via the

direct inhibition of methanogens.

3.7. Peppermint oil

Essential oils from Mentha species and several of their components (menthol, menthone, p-cymene, limonene, linalol,

-pinene,

-pinene and 1,8-cineole) have been thought to possess antimicrobial activities against Gram positive and Gram

negative bacteria (Marotti et al., 1994; Imai et al., 2001).


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Conflicting results have been reported on effects of peppermint oilon rumen microbialfermentation andrumenmethano-

genesis. Tatsuoka et al. (2008) observed no antimethanogenic activity of cyclodextrin ( or )-peppermint oil complex (83,167 and 250 mg/L of culture fluid) on in vitrobatch culture fermentation, which was consistent with the lack of effect of this

essential oil on total VFA concentration and molar proportions of acetate and propionate (Table 2).

Agarwal et al. (2009) evaluated effects of peppermint essential oil (0.33, 1.0 and 2.0 ml/L of culture fluid) on rumen

methanogenesis using a 24h in vitro batch culture system (Table 2). Methane production (ml/g digestible DM) decreased

linearly with increasing levels of peppermint oil, but diet digestibility was also depressed at all levels while total VFA

concentration was depressed only at the two highest inclusion levels. The reduction in CH4

production was accompanied by

a decrease in numbers and activity of protozoa (i.e., holotrichs and spirotrichs). Similar results were observed by Ando et al.

(2003) who reported a decrease in the total numbers of protozoa, includingEntodinum, Isotrica andDiplodium in rumen fluid

from steers supplemented with 200g/d (54g/kg DM intake) of sun-dried peppermint. About 25% of rumen methanogens are

associated with protozoa (Newbold et al., 1995) and, therefore, a part of the antimethanogenic effect of peppermint essential

oil may be due to an antiprotozoal activity.

In the study by Agarwal et al. (2009), the number of methanogens increased at the lowest level (0.33 ml/L) of peppermint

oil supplementation, which contradicts the reduction in CH4 production. However at higher doses of 1.0 and 2.0 ml/L,

peppermint oil reduced numbers of methanogens, which agrees with the reduced CH4 production. The pattern of VFA

production was not typical of that when CH4 production is inhibited (i.e., a lower acetate to propionate ratio). In fact, at

the lowest level of peppermint oil, the acetate to propionate ratio remained unaffected while, at higher concentrations, it

increased. This is an indication that excess H2 resulting from inhibition of methanogen activity and/or numbers was not

directed towards alternative H2 sinks such as propionate. Results of Agarwal et al. (2009) suggest that the antimethanogenic

activity of peppermintoil is mainlydue to itsdirect effecton activity and/ornumbers of methanogens andprotozoa. However,

care must be taken to ensure that achieving CH4 suppression using peppermint oil is not through a general reduction in

fermentation activity, as observed by Agarwal et al. (2009).

3.8. Rhubarb and frangula essential oils

Garcìa-González et al. (2008b) conducted a study to screen 158 plants, herbs and spices for their potential to reduce CH4production invitro. Of the plant sources examined, in addition to garlic bulbs ( Allium sativum), rhizomes and roots of rhubarb

(Rheumofficinale) and bark of frangula(Frangulaalnus) had antimethanogenic properties. When rhubarb and frangula plants

were included in in vitro incubations at 135 mg/g DM or 1400mg/L culturefluid, CH4 production as mmol/g DM decreased by

75 and 45% compared to control. This change was accompanied by a lower acetate to propionate ratio (−40 and −29%), and

reduction in total VFA production (−15 and −13%) and DM digestibility (−4 and −5%), for rhubarb and frangula, respectively.

In a subsequent dose response 24h in vitro study (Garcìa-González et al., 2008a), both plants caused similar changes in

rumen fermentation, resulting in a linear decrease in CH4

production and in the acetate to propionate ratio when added at

500, 950, 1400 and 1850mg plant DM/L culture fluid. The production of butyrate also increased at all dosages. At higher

doses (i.e., 1400 and 1850 mg/L), rhubarb and frangula adversely affected substrate degradation as reflected by reduced total

VFA production, a response which did not occur at lower dosages of 500 and 950 mg/L.

Interestingly, in the same study, effects of rhubarb and frangula on CH4 and acetate to propionate ratio were similar

to those with monensin at 5M. However, while monensin decreased butyrate production, the plant additives increasedbutyrate production (Garcìa-González et al., 2008a). Another reported difference between the plant additives and monensin

wasthe effectof treatmenton H2 recovery estimated stoichiometrically. Indeed, H2 recovery decreasedat alldosesof rhubarb

andfrangulawhileit wasnot affected by monensin. Basedon theseobservations, Garcìa-González et al. (2008a) hypothesized

that theactive compoundsof rhubarb andfrangulaplants reduceCH4 productionprimarilybe directly inhibiting methanogen

activity and/or numbers.

It is noteworthy that Garcìa-González et al. (2008a) did not identify the secondary metabolites responsible for the

antimethanogenic activity of rhubarb and frangula plants. Although other secondary metabolites such as tannins, oxalic

acid and alkaloids are in rhubarb and frangula plants, anthraquinone derivatives are the major secondary compounds and

may account for most of the antimethanogenic activity of these plants. Garcìa-López et al. (1996) reported that, in vitro,

9,10-anthraquinone decreased CH4 production and the acetate to propionate ratio, but resulted in accumulation of H2, sug-

gesting a direct inhibitory effect of this major component of rhubarb and frangula essential oils on methanogen activity

and/or numbers.

3.9. Mixtures of essential oils and/or essential oil compounds

Additive, antagonistic and synergistic effects have occurred between components of essential oils (Burt, 2004), suggesting

that combinations of essential oils of different composition, or specific combinations of essential oil secondary metabolites,

may result in additive and/or synergetic effects which may enhance efficiency of rumen microbial fermentation. As a con-

sequence, a number of commercial products combining different essential oils and/or essential oil compounds are currently

available on the market, but limited research is available on the potential of commercial products to specifically inhibit

CH4

production in ruminants (Benchaar et al., 2008b, 2009). Evaluating the effects of a commercial mixture of essential oils

(Crina® Ruminants; Akzo Nobel Surface Chemistry Ltd., St. Albans, Hertfordshire, UK) consisting of thymol, eugenol, vanillin


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and limonene, McIntosh et al. (2003) observed in vitro that inhibition of growth of the methanogenM. smithii occurred only

after 1000mg/L of the product was included. In an in vivo study, Beauchemin and McGinn (2006) observed no change in

CH4 production, although feed digestibility decreased by 7% when beef cattle were supplemented 1 g/d of this same product

(Table 2). Assuming a rumen volume of 30 L, the maximum ruminal concentration of essential oils (i.e., achieved immediately

after feeding) in the study by Beauchemin and McGinn (2006) would be ∼33mg/L, which is 33 times lower than that used

in the in vitro study of McIntosh et al. (2003). Comparison between these studies suggests that caution should be taken in

extrapolation of in vitro data to in vivo conditions. Indeed, many of the concentrations of essential oils that have elicited

favourable fermentation responses in vitro are far too high for in vivo application (Beauchemin et al., 2009).

4. Challenge and future directions

The antimicrobial activity of essential oils against a variety of microorganisms has been demonstrated in several studies.

Essential oils and their constituents have been shown to inhibit growth of pathogenic bacteria such as E. coli O157:H7,

Salmonella spp. and S. aureus. The well documented antimicrobial activity of essential oils has prompted researchers to

examine their potential to modify rumen microbial populations in order to enhance efficiency of rumen fermentation and

improve nutrient utilisation. The resurgence of interest in using essential oils in ruminant nutrition and production has

increased, particularly in Europe after the ban on the use of growth promoting antibiotics, including ionophores, in livestock

production.

In recent years, the number of research papers published on effects of essential oils and their components on rumen

microbial fermentation has increased (see reviews by Calsamiglia et al., 2007; Benchaar et al., 2008b, 2009). Most of this

research is short term andhas used invitro systems. As thesecompounds are relatively novel supplements in animal nutrition

(Wallace, 2004), their effects on rumen microbial fermentation and overall nutritional effects are unknown. Moreover, the

range of essentialoils available is extensive (>3000; Vande Braak andLeijten, 1999) and so researchers have relied heavily on

in vitromodels of batch and continuous culture techniques to investigate effects of essential oils and their main constituents

on rumen fermentation and, consequently, attempt to predict in vivo effects.

Because of the importance of enteric CH4 production in ruminant nutrition and production (i.e., loss of energy and contri-

bution to GHG emissions), a number of studies have investigated the potential of essential oils and essential oil components

to selectively inhibit rumen methanogenesis. Based on results to date (see Table 2), it appears that phenolic compounds

(i.e., thymol, eugenol, carvacrol) or essential oils with high concentrations of these phenolics, cinnamon oil and its main

component cinnamaldehyde, garlic essential oil and its derivatives, in particular diallyl disulphide, and other essential oils

(i.e., horseradish, rhubarb, frangula essential oils) may be effective, at least in vitro, in decreasing CH4 production. However,

use of essential oils in ruminant nutrition to reduce enteric CH4 emissions could be limited for several reasons:

(1) From published in vitro studies, it appears that high concentrations of essential oils (i.e., >300mg/L of culture fluid) are

required to inhibit rumen methanogenesis. These levels are far too high to be achieved in vivo and are impractical in

terms of feeding due to potentially deleterious effects on efficiency of rumen fermentation and palatability, possible

toxicity issues (Benchaar et al., 2008b, 2009), and high cost.

(2) Because of the high dosage required to reduce CH4 production, and because of the wide spectrum of activity of essential

oils, beneficial effects of CH4 inhibition are often counterbalanced by an overall inhibition of total VFA production and

feed digestion which, of course, is not desirable due to negative consequences on animal productivity.

(3) Microbialpopulationsexhibit a remarkable capacity to adapt to and/ordegrade a wide variety of plant secondarymetabo-

lites such as saponins and tannins (Newbold et al., 1997; Makkar et al., 1995; Makkar, 2003) and the same appears to be

true for essential oils, particularly at ‘low’ dosage rates in vitro. Indeed, rumen microbes adapted to essential oils when

they were administered at ‘low’ doses (Cardozo et al., 2004: 0.22 mg/L; Busquet et al., 2005c: 2.2 mg/L), but at higher

doses (Busquet et al., 2005a: 300mg/L; Fraser et al., 2007: 500 mg/L) effects of essential oils appears to be sustained

over time (e.g., 9 d of continuous culture). However these levels are very high and impractical for feeding to animals due

to potentially negative effects on efficiency of fermentation in the rumen. McIntosh et al. (2003) showed that bacterial

species such as P. ruminicola and P. bryantiiadapted and were able to grow at higher concentrations of essential oils. The

diversity of methanogens has been shown to increase when animals were fed essential oils (Ohene-Adjei et al., 2008),

which is further evidence to support the ability of rumen microbes to adapt to essential oils. This adaptive response may

explain why, to date, responses in vitro (i.e., short term exposure) are not as marked as in vivo (i.e., long term exposure).

In fact, a number of in vivo studies reported no effects on feed intake, total VFA concentrations and VFA pattern (i.e.,

acetate to propionate ratio) when essential oils and/or their main components as single compounds and/or mixtures

were fed to dairy cows (Benchaar et al., 2006b, 2007b, 2008a; Yang et al., 2007; Spanghero et al., 2009; Santos et al.,

2010), beef cattle (Beauchemin and McGinn, 2006; Benchaar et al., 2006a; Yang et al., 2010a,b,c), and sheep (Newbold

et al., 2004; Chaves et al., 2008a,c; Malecky et al., 2009). Although enteric CH4 emissions were not quantified in these

studies, the lack of effects of essentials oils on feed intake and rumen fermentation characteristics suggest that under

these conditions, no changes in rumen methanogenesis occurred. The adaptation of rumen microbes to essential oils

represents a major challenge for use of these compounds to mitigate enteric CH4 emissions from ruminants.

(4) Despite the increased interest in using essential oils to manipulate rumen fermentation, little research has been com-

pleted to determine the fate of essential oils and their compounds in the gastro-intestinal tract of animals, especially


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ruminants. There are indications that some essential oils, such as terpenes, can be degraded within the gastro-intestinal

tract. Cluff et al. (1982) reported that 80% of the monoterpenoids contained in sagebrush ( Artemisia tridentata L.) dis-

appeared from the rumen of wild mule deer. White et al. (1982) observed a similar extent of disappearance (i.e., 77%)

of sagebrush monoterpenoids from the stomach of pigmy rabbits. More recently, Malecky et al. (2009) reported high

rates of disappearance of the terpenes linalool, p-cymene and- and -pinene from the rumen of dairy goats. Differentmechanisms have been proposed to explain the disappearance of essential oil terpenes from the digestive tract. These

include, bioconversion by the rumen microflora (Welch and Pederson, 1981; Schlichtherle-Cerny et al., 2004), transfer to

the gas phase of the rumen due to volatility of terpenes resulting in their loss during eructation (Cluff et al., 1982; White

et al., 1982) andabsorption across theruminal wall into theblood system andexcretionin theurine (Michiels et al., 2008;

Malecky et al., 2009). The degradation and/or conversion of terpenes within the gastrointestinal tract may explain the

discrepancy in results of in vitro and in vivo studies (Benchaar et al., 2008b, 2009). However, given the diversity of essen-

tial oils in terms of chemical structure, it is possible that differences exist in rates at which microbial populations degrade

these secondary metabolites. For example, Broudiscou et al. (2007) investigated the extent of disappearance of the 10

monoterpenes limonene,-myrcene, -ocimene, -pinene, sabinene,-terpinene and thymol, and the sesquiterpenescamphene, -caryophyllene and -copaene when these compounds were added at a high dose of 2000 mg/L in a 24hin vitro batch culture fermentation. Results revealed that thymol, camphene, -caryophyllene and (−)-limonene werepoorly degraded, whereas -copaene, myrcene, -ocimene, -pinene and sabinene were highly degraded. However,although supplied in high concentrations, most terpenes investigated did not inhibit microbial activity. Michiels et al.

(2008) reported that thymol, carvacrol, eugenol and cinnamaldehyde were mainly, and almost completely, absorbed

from the stomach and the proximal small intestine in piglets after oral administration. Substantial degradation of thy-

mol, carvacrol, cinnamaldehyde and eugenol has occurred in the cecum (Michiels et al., 2008), and degradation and

absorption may also occur in the rumen. Based on these considerations, further research is necessary to determine the

extent to which essential oils can be degraded and/or metabolized in the digestive tract of ruminants.

(5) Little information is available on transfer of plant secondary metabolites such as essential oils into animal products (e.g.,

meat and milk) and their possible toxicity to humans. There is evidence that essential oils can be absorbed from different

parts of the digestive tract of animals and so the potential of residues in animal products cannot be excluded. Indeed

studies of Viallon et al. (2000) and Tornambé et al. (2006) have shown transfer of terpenes in forages to milk of grazing

cows, and that these essential oils can modify the organoleptic properties of dairy products.

Plant extracts are perceived as safe because they are produced naturally. However essential oils evolved to protect

plants against herbivores, pathogens and abiotic stressors (Greathead, 2003). Therefore they can be toxic to animals

if consumed at high doses. Little is know on toxicity mechanisms, and toxic doses, for animals and there is a need to

research the tolerance of animals to toxicity of plant derived essential oils.

Although several essential oils are considered as safe (GRAS) by the United States FDA, some research has shown that

these compounds can be toxic. For example, a number of essential oil components (e.g., carvacrol, cinnamaldehyde,

eugenol, thymol) have been registered by the European Commission for use as flavourings in foodstuffs. However essen-

tial oil compounds such as estragole and methyl eugenol were deleted from the list in 2001 due to genotoxic properties

(Burt, 2004). The S containing compounds in garlic and onion have been shown to be responsible for haemotoxic effects

in beef cattle (Rae, 1999) and horses (Pearson et al., 2005). The cytotoxicity of organo-sulphur compounds from garlic

has been demonstrated as cell damage (Amagase, 2006). Use of essential oils as feed additives in livestock production

must also be safe for the feed manufacturing personnel and farm workers. Indeed these compounds have been reported

to be potentially irritating and cause allergic dermatitis (Burt, 2004), suggesting that caution should be taken by users

in handling of such feed additives.

(6) It has been claimed that, unlike conventional antibiotics, it is not possible for microorganisms to develop resistance

to antimicrobial plant extracts such as essential oils. The reasons include that essential oils are complex mixtures of

chemical compounds, many of which possess antimicrobial activity with different modes of action making it impossible

for microorganisms to establish resistance mechanisms (Briskin, 2000) and that the mode of action of plant extracts

against microorganisms is structural rather than at the level of DNA (Mellor, 2000). These claims can be challenged

because microbial resistance is not limited to a single mode of action and neither is it dependent on the mode of action

being at the level of DNA.Theoretically, allthat is required for the creation of resistantbacteria is for bacteria to randomly

acquire a genotype that confers a survival advantage in the presence of the compound(s) with antimicrobial properties

of interest, be it an antibiotic or an essential oil. Creation of resistant bacterial populations is facilitated by exchange

of genes, in particular those responsible for resistance, which are carried on plasmids, between bacteria of the same

and related species by a process of conjugation or bacterial mating. As yet, there has been little research to investigate

bacterial resistance to herbs and spices with antibacterial properties. However, Brul and Coote (1999) reviewed this

topic and Nelson (2000) reported that S. aureus developed resistance to tea tree (Melaleuca alternifolia) essential oil.

More research is warranted to determine the capacity of rumen bacteria to develop resistance to essential oils.

5. Conclusions

Based on the considerations described in this review, it appears that the potential of essential oils to selectively inhibit

rumen methanogenesis has been mostly demonstrated in vitrowhen these secondary metabolites were used at high dosage


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levels. In vivo application of essential oil based feed additives may be limited by adaptation of rumen microbes to these

compounds and the capacity of the rumen microflora to degrade and/or metabolize these secondary metabolites. Tox-

icity to animals, users and consumers, palatability and effects on organoleptic quality of animal products require further

research to ensure that the compounds can be safely used in livestock production to enhance animalproductivityand reduce

environmental impacts of animal production.

Conflict of interest statement

None.

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Essential Oils and Opportunities to Mitigate Enteric Methane Emissions