Garlic and Its Bioactive Compounds: Implications for Methane Emissions and Ruminant Nutrition

Simple Summary Methane (CH4) produced by ruminants contributes as a source of anthropogenic greenhouse gases (GHG). Plant-derived bioactive compounds have been investigated for their potential to reduce CH4 emissions from ruminant livestock. Garlic contains bioactive organosulphur compounds, which have been reported to be effective in reducing CH4 emissions, but they have demonstrated inconsistent effects in reducing CH4 production in the rumen. This might be because different types of garlic-based supplements vary in their concentrations of bioactive compounds. Therefore, further investigation is needed, such as the mode of action and persistence of the bioactive compound, to determine whether these compounds can be used successfully to inhibit rumen methanogenesis. The present review discusses garlic and its potential contribution to reducing CH4 production by ruminant animals and discusses how differences in the diet and the concentration of bioactive compounds in garlic might contribute to inconsistent CH4 mitigation potential of garlic. Abstract Methane (CH4) emission from enteric fermentation of ruminant livestock is a source of greenhouse gases (GHG) and has become a significant concern for global warming. Enteric methane emission is also associated with poor feed efficiency. Therefore, research has focused on identifying dietary mitigation strategies to decrease CH4 emissions from ruminants. In recent years, plant-derived bioactive compounds have been investigated for their potential to reduce CH4 emissions from ruminant livestock. The organosulphur compounds of garlic have been observed to decrease CH4 emission and increase propionate concentration in anaerobic fermentations (in vitro) and in the rumen (in vivo). However, the mode of action of CH4 reduction is not completely clear, and the response in vivo is inconsistent. It might be affected by variations in the concentration and effect of individual substances in garlic. The composition of the diet that is being fed to the animal may also contribute to these differences. This review provides a summary of the effect of garlic and its bioactive compounds on CH4 emissions by ruminants. Additionally, this review aims to provide insight into garlic and its bioactive compounds in terms of enteric CH4 mitigation efficacy, consistency in afficacy, possible mode of action, and safety deriving data from both in vivo and in vitro studies.

. Global livestock emissions from supply chains, production activities, and products (adapted from [1]). This figure is excluded from the CC BY licence under which this article is published. Figure 1. Global livestock emissions from supply chains, production activities, and products (adapted from [1]). This figure is excluded from the CC BY licence under which this article is published. biotics, plant-derived bioactive compounds, and essential oils [27,[29][30][31][32]. Ionophores such as monensin have also been reported to inhibit rumen methanogenesis [33,34]. However, since the European Union (EU) banned antibiotics as feed additives in 2006 due to concerns about antimicrobial resistance in food supply chains [35], interest in using plant-based feed additives (essential oils, plant extracts, and plant-derived bioactive compounds) to reduce enteric CH 4 emissions has increased [36].
Dietary manipulation is an attractive and effective way to mitigate ruminant-derived CH 4 emissions due to the direct influence of feed on rumen fermentation patterns which can lead to decreased CH 4 production. Garlic contains a number of active metabolites that could impact rumen fermentation, decreasing CH 4 synthesis by rumen microbes and increasing propionate production in the rumen [37][38][39]. A detailed review of the literature around the potential use of garlic to decrease CH 4 emissions is presented in Section 3 of this review.

The Rumen Microbiome and Metabolic Pathways of CH 4 Synthesis in the Rumen
Ruminants have a unique digestive system comprised of four chambers: the reticulum, rumen, omasum, and abomasum [40,41]. The most significant among the four chambers (approx. 80% of the total volume) is the rumen, which contains a diverse and dynamic population of microorganisms that allow ruminants to break down plant material containing cellulose and hemicellulose via anaerobic fermentation [40,42]. Bacteria and protozoa account for the most significant fraction of microbial biomass (50-70%), followed by fungi (8-20%) [43,44]. These microorganisms make up a complex microbial ecosystem in the rumen, living in a symbiotic relationship with the ruminant hosts, which assists with the efficient conversion of plant biomass (rich in structural polysaccharides) into a major energy substrate i.e., VFA for the ruminant host [43,45]. For large herbivores such as dairy cows and beef cattle, this energy resource makes up 70% of the dietary energy [43].
To date, very few methanogenic species have been isolated from the rumen; Holotrich ciliate protozoa are highly active in the rumen and produce H 2 that methanogens use to produce CH 4 . The interactions between bacteria and protozoa are essential and could play a critical role in the CH 4 production pathways [44,49]. The removal of protozoa from the rumen is associated with decreased CH 4 emission [44,50].
In the symbiotic relationship between the ruminant and the rumen microbial ecosystem, ruminants maintain the rumen in an anaerobic state with a stable temperature of around 39 • C and a pH ideal for microbial growth [51][52][53]. Production of CH 4 in ruminants starts with different ruminal microorganisms, bacteria, protozoa, and fungi when they hydrolyse and ferment complex feed components such as proteins and polysaccharides into simple products, including amino acids, sugars, and alcohols [54].
The products are further fermented to VFA, H 2, and CO 2 by both the primary fermenters and other microbes that cannot hydrolyse complex polymers by themselves [55]. It enables the high conversion efficiency of cellulose and hemicellulose, and CH 4 represents a by-product of this process produced by certain microbes (methanogens) [56]. It is estimated that a cow produces 250-500 g/d CH 4 [57]. The gaseous waste products of enteric fermentation, CO 2 , and CH 4, are mainly removed from the rumen by eructation [52] . Methane synthesis in the reticulorumen is an evolutionary adaptation that enables the rumen ecosystem to dispose of excess H 2 , which may otherwise accumulate and inhibit carbohydrate fermentation and fibre degradation [58]. Disposal of excess H 2 produced by direct inhibition of CH 4 production results in increased concentrations of other H 2 sinks, such as propionate and butyrate [59]. Methanogens are at the bottom of this trophic chain and use the end products of fermentation as substrates ( Figure 2). which methanogens can utilise H2 as an electron donor to reduce CO2 to CH4. Newly rec-ognised methanogens use a range of methyl donor compounds and CO2 for CH4 production, suggesting that other pathways may be identified [61]. The draft genome of Candidatus Methanomethylophilus Mx1201, a methanogen isolated from the human gut belonging to the rumen cluster C, more recently categorised into the order Methanomassiliicoccales [64], contains genes for methylotrophic methanogenesis from methanol and tri-, di-, and monomethylamine [65]. In artificial systems, such as biogas production facilities, acetate is recognised as an important substrate for methanogens, which is referred to as acetoclastic methanogenesis [66]. A comprehensive understanding of the functionality of methanogens and their CH4-producing pathways may provide insights into effective CH4 abatement strategies. Biochemical pathways for CH 4 synthesis (adapted from [24]). This figure is excluded from the CC BY licence under which this article is published.
Methanogens are anaerobic microorganisms that have three coenzymes that have not been observed in any other microorganisms, which allow them to produce CH 4 from methyl coenzyme M [60]. It has been estimated that there are between 360-1000 species of methanogens; however, until this point, only six genera have been identified, and eight species have been cultured [53,61]. The predominant genus in the rumen is Methanobrevibacter, and from this genus, the most predominant species are ruminantium, smithii, and mobile [60]. Most methanogens grow at pH 6-8, although some species can survive in a wider range from 3-9.2 [49,62].
Three types of methanogenic pathways are involved in CH 4 synthesis, namely hydrogenotrophic (reduction of CO 2 coupled to the oxidation of H 2 ), methylotrophic (conversion of methyl-group-containing compounds), and acetoclastic [63]. The hydrogenotrophic pathway is generally recognised as the main pathway to remove H 2 , through which methanogens can utilise H 2 as an electron donor to reduce CO 2 to CH 4 . Newly recognised methanogens use a range of methyl donor compounds and CO 2 for CH 4 production, suggesting that other pathways may be identified [61]. The draft genome of Candidatus Methanomethylophilus Mx1201, a methanogen isolated from the human gut belonging to the rumen cluster C, more recently categorised into the order Methanomassiliicoccales [64], contains genes for methylotrophic methanogenesis from methanol and tri-, di-, and monomethylamine [65]. In artificial systems, such as biogas production facilities, acetate is recognised as an important substrate for methanogens, which is referred to as acetoclastic methanogenesis [66]. A comprehensive understanding of the functionality of Animals 2022, 12, 2998 6 of 33 methanogens and their CH 4 -producing pathways may provide insights into effective CH 4 abatement strategies.

Targeted Manipulation of Ruminant Metabolic Pathways to Reduce CH 4 Synthesis
Methane production in the rumen can represent a loss of up to 12% digestible energy [57] . Decreasing enteric CH 4 emissions by ruminants without compromising animal production is desirable as a strategy both to decrease global warming effects and to improve feed conversion efficiency [16,67]. The type of feed and the presence of electron acceptors other than CO 2 in the rumen will significantly influence the presence and activity of H 2 producers and users [54,57]. This is because pathways other than methanogenesis can also consume H 2 and thus potentially compete with and decrease methanogenesis in the rumen [54].
Dietary manipulation may rechannel the H 2 produced during ruminal fermentation from CH 4 production to propionate synthesis in the rumen [68,69]. However, the rumen ecosystem is very complex, and the ability of this system to efficiently convert complex carbohydrates to VFA is partly due to the effective removal of H 2 by reducing CO 2 to produce CH 4 . Thus, inhibition of methanogenesis is often short-lived, as the system's ecology is such that it often returns to the initial level of CH 4 production through various adaptive mechanisms [58]. Issues surrounding chemical residues, toxicity, and high cost, can also limit the utilisation of this strategy in animal production [70].
Another potential pathway is a targeted effect on certain microbial populations [31,71]. Plant-derived bioactive compounds are volatile components and aromatic lipophilic compounds which contain chemical constituents and functional groups such as terpenoids, phenolics, and phenols, which have potent antimicrobial activities. [32,[72][73][74][75]. Methanogenesis decreases with the application of plant-derived bioactive compounds, primarily by reducing protozoa. Methanogenesis decreases by disrupting cell membranes due to the lipophilic nature of plant-derived bioactive compounds, decreasing protozoa and methanogens [71,76]. Therefore, the inclusion of plant-derived bioactive compounds in ruminant diets is a potential strategy to mitigate rumen CH 4 synthesis [77].
A targeted approach to reducing CH 4 emissions by dietary manipulation will therefore: (i) need to have a long-term effect by overcoming any adaptation to dietary changes, (ii) should not have a detrimental effect on the digestion of other dietary nutrients, which may occur if the rumen microbiome is altered in any way, (iii) should not have negative impact on animal health, and (iv) should not make animal-origin food products unsafe for human consumption.

The Need to Exploit Plant-Derived Bioactive Compounds
In livestock production, the use of antibiotics as growth promotors in animal feed is highly objectionable due to their residual effects and the risk of antimicrobial resistance development [78]. Garlic (Allium sativum) has been applied pharmaceutically since ancient times in nearly every known civilisation, has been widely used as a foodstuff in the world, and is "generally recognized as safe" (GRAS) as a food flavouring agent by the U.S. FDA, making them ideal candidates to use as feed additives in livestock production [79]. However, plant-derived bioactive compounds also exhibit antimicrobial activity and, therefore, can affect the rumen microbial ecosystem directly [36,[80][81][82].
Antimicrobial properties of organosulphur compounds in garlic have shown a bactericidal effect [83][84][85][86], and hence, garlic extract and some of their compounds have been extensively investigated as a potential way to modify the rumen microbiome. Garlic is a plant that can greatly alter microbial ecosystems within the gastro-intestinal tract (GIT) of cattle [87]. Table 1 shows previously reported antimicrobial activities from garlic and its compounds (antifungal, antiprotozoal, antibacterial). The complex composition of garlic also involves a paradoxical outcome in the GIT microbiome [88]; at the same time, garlic is rich in indigestible polysaccharides, such as fructans, which act as a prebiotic for specific GIT microbiota [89].
In recent years, plant-derived bioactive compounds (e.g., organosulphur, saponins, and tannins) with diverse biological activities have been investigated for their potential as alternatives to growth-promoting antibiotics in ruminant production [72,90,91] and their potential mechanism of action as rumen modulators and inhibitors of CH 4 production in the rumen [91,92]. To date, garlic supplementation in ruminant diets has shown a variable CH 4 reduction in both in vitro and in vivo studies [87,93,94]; these are summarised in Table 2.

Effect of Garlic on CH 4 Emissions: In Vitro Assessments
Based on batch culture and dual flow continuous culture studies, the supplementation of garlic oil (300 mg/L) and allicin (a sulphur-containing bioactive compound in garlic; 300 mg/L) decreased CH 4 yield (mL/g dry matter (DM)) by 73.6 and 19.5%, respectively, compared with control basal diets consisting of 50:50 forage:concentrate ratio, over 24 h [37]. The inclusion of garlic extracts at 1% of the total volume of rumen fluid containing 0.3 g of timothy grass decreased CH 4 yield (mL/g DM) by 20% compared to control after 24 h incubation [95]. Garlic powder supplementation at 16 mg/200 mg of substrate resulted in reducing CH 4 yield (mL/g DM) by 21% with basal diets comprising 60:40 forage:concentrate ratio over 72 h using swamp buffalo rumen fluid in batch cultures [29]. The supplementation of a combination of garlic oil at 0.25 g/L, nitrate at 5 mM, and saponin at 0.6 g/L reduced CH 4 yield (mL/g DM) by 65% at day two and by 40% at day eighteen compared with the control basal diet consisting of 50:50 forage:concentrate ratio in batch cultures [48].
The effects of a combination of garlic powder and bitter orange (Citrus aurantium) extract (Mootral) using a semi-continuous in vitro fermentation (Rumen Simulation Technique, RUSITEC) demonstrated that the treatment effectively decreased CH 4 yield by 96% (mL/g DM) by altering the archaeal community without exhibiting any negative effects on fermentation [96]. The study showed that a mixture of garlic and citrus extracts effectively decreased CH 4 production in all feeding regimens without adversely affecting nutrient digestibility. Furthermore, a mixture of garlic and citrus extracts supplementation improved rumen fermentation by increasing the production of total VFA.
The supplementation of whole garlic bulb decreased CH 4 yield (mL/g DM) by 55% at 0.5 mL/30 mL in batch culture using rumen liquor of buffalo as inoculum without affecting the protozoa population [97]. The inclusion of garlic at the rate of 135 mg/g of substrate resulted in more than 20% inhibition in CH 4 yield (mL/g DM), with no effect on gas production and a slight increase (2%) in in vitro DM degradability [98]; although such an inclusion rate is rather unrealistic for application at the commercial level. The effect of the inclusion of garlic oil on CH 4 and VFA production based on in vitro is also influenced by diet and dose-dependent factors [99].
Some studies on ruminants have shown that garlic extracts improved nutrient use efficiency by decreasing energy loss as CH 4 or ammonia nitrogen in continuous rumen culture [39,100,101]. Almost complete inhibition of methanogenesis has been demonstrated using garlic oil distillate without affecting feed organic matter degradation in experiments using RUSITEC [102]. These studies have consistently shown the potential of garlic supplementation in reducing CH 4 production [48,103], while the effect on short-chain fatty acids (SCFA) production is more variable. Previous studies also observed an increase in total SCFA concentrations with moderate garlic oil concentrations [37]. Additionally, most studies reported an increase in the molar proportion of butyrate, often accompanied by a decrease in acetate proportion, whereas the effects on other SCFA and digestibility can vary [37,48,103].
Variations in the concentration and effect of individual substances in garlic extract and the type of diet can contribute to these differences [37,104]. Since different garlic varieties can vary substantially in different concentrations of compounds that affect CH 4 emissions, the efficacy of garlic in reducing CH 4 emissions may also depend on the variety [29,105]. However, the role of garlic and its bioactive compounds in enteric CH 4 mitigation still remains unclear due to limited data on the mode of action related to CH 4 mitigation potential .

Effect of Garlic on CH 4 Emissions: In Vivo Assessments
Based on an in vivo study, the supplementation of a feed additive based on citrus and garlic extracts (Mootral) at 15 g/d in steers' diets caused a decrease of 23% in CH 4 yield after 12 weeks [106]. Steers (n = 20) receiving the Mootral treatment had lower CH 4 production than the steers receiving the control treatment over time with no effect on DMI, average daily gain, and feed conversion efficiency. Dietary supplementation of allicin at 2 g/d for 42 d decreased CH 4 yield (mL/g DM) by 6% compared to a control diet in sheep [107]. The inclusion of garlic extract directly affects rumen archaea, which are the microorganisms primarily responsible for CH 4 synthesis in the rumen [37]. This hypothesis is supported by further in vivo research that reported the effect of garlic oil on the diversity of methanogenic archaea in the rumen of sheep [108]. The supplementation of garlic oil at different doses (20 g-35 g/kg DM/day) resulted in CH 4 reduction (mmol/L of VFA) at 21.96 [109]. A decrease in CH 4 production scaled to digested NDF intake when diallyl disulphide (DAD) was supplemented at 4 g/d in sheep [110]. The supplementation of 7% coconut oil and 100 g/d of garlic powder in buffalo diet improved the rumen ecology by increasing amylolytic and proteolytic bacteria while the protozoal population decreased by 68-75% and the CH 4 yield (g/kg DMI) decreased by 9% without changing nutrient digestibility [111]. Other studies demonstrated no long-lasting effects on CH 4 production when anti-methanogenic treatments (essential garlic oil and linseed oil at 3 µL/kg BW and 1.6 mL/kg BW, respectively) were given to neonatal lambs [112]. However, early-life intervention induced modifications in the composition of the rumen bacterial community of lambs that persisted after the intervention ceased with little or no effect on archaeal and protozoal communities [112].
Feeding garlic bulbs at the rate of 1% of DMI resulted in 11% inhibition in CH 4 yield (g/kg DMI) in sheep (fed a diet with a 50:50 concentrate-to-roughage ratio), along with an increase in nutrient digestibility. Methane was decreased up to 31% when supplemented with garlic powder at the rate of 2% of DMI without affecting the digestibility of nutrients and milk composition compared to the control group in lactating murrah buffaloes [113]. The supplementation of freeze-dried garlic leaves (FDGL) at 2.5 g/kg DM/day of sheep diet resulted in a reduction of CH 4 yield (g/kg DMI) by 9.7% [114].
Bioactive compounds derived from plants also have antimicrobial activity and, therefore, can affect the rumen microbial ecosystem. Although it might be argued that similar to the concept of developin antimicrobial resistance, there is a risk of microbes developing resistance to garlic bioactive compounds after long exposure periods. The antimicrobial properties of organosulphur compounds from garlic include a bactericidal effect. Garlic extract and some of its compounds have been studied extensively as potential means to modify the rumen microbiome. Reports on the effect of garlic on CH 4 emissions, both in vitro and in vivo, are inconsistent between studies and applications in terms of efficient livestock production and limited ability to maintain its effects over longer periods of time. This may be due to the effect of garlic supplementation on rumen fermentation depending on the type and dosage of garlic components which vary in bioactive components, substrate composition, and composition of microbial population in the inoculum.

Bioactive Compounds in Garlic That Decrease CH 4 Emissions and the Potential Effect on Biochemical Pathways
Garlic contains the organosulphur compounds allicin (C 6 H 10 S 2 O), alliin (C 6 H 11 NO 3 S), diallyl sulphide (C 6 H 10 S), diallyl disulphide (C 6 H 10 S 2 ), and allyl mercaptan (C 3 H 6 S) [137][138][139][140] (Figure 3). These compounds are widely known for their unique therapeutic properties and health benefits, as they act as antioxidants to scavenge free radicals [141]. Garlicderived organosulphur compounds demonstrate different biochemical pathways that may provoke multiple inhibitions [142]. One potential pathway for the direct inhibition of methanogenesis by garlic is via the inhibition of CH 4 -producing microorganisms such as archaea [142]. Archaea possess unique glycerol-containing membrane lipids linked to long-chain isoprenoid alcohols, which are essential for cell membrane stability. The synthesis of isoprenoid units in methanogenic archaea is catalysed by the enzyme hydroxyl methyl glutaryl coenzyme A (HMG-CoA) reductase. Garlic oil is a potent inhibitor of HMG-CoA reductase Gebhardt and Beck [142]; as a result, the synthesis of isoprenoid units is inhibited, the membrane becomes unstable, and cells die. The effect of garlic bioactive compounds in ruminants has been reported in Table 3.

Bioactive Compounds in Garlic That Decrease CH4 Emissions and the Potential Effect on Biochemical Pathways
Garlic contains the organosulphur compounds allicin (C6H10S2O), alliin (C6H11NO diallyl sulphide (C6H10S), diallyl disulphide (C6H10S2), and allyl mercaptan (C3H6S) [1 140] (Figure 3). These compounds are widely known for their unique therapeutic pro ties and health benefits, as they act as antioxidants to scavenge free radicals [141]. Ga derived organosulphur compounds demonstrate different biochemical pathways may provoke multiple inhibitions [142]. One potential pathway for the direct inhibitio methanogenesis by garlic is via the inhibition of CH4-producing microorganisms suc archaea [142]. Archaea possess unique glycerol-containing membrane lipids linked long-chain isoprenoid alcohols, which are essential for cell membrane stability. The s thesis of isoprenoid units in methanogenic archaea is catalysed by the enzyme hydro methyl glutaryl coenzyme A (HMG-CoA) reductase. Garlic oil is a potent inhibito HMG-CoA reductase Gebhardt and Beck [142]; as a result, the synthesis of isopren units is inhibited, the membrane becomes unstable, and cells die. The effect of garlic active compounds in ruminants has been reported in Table 3. Diallyl sulphide (DAS) has shown small effects on rumen microbial fermenta [37]. It has been suggested in various studies that the antimicrobial potency of allyl phides in garlic oil increases with each additional S atom [143,144]. This could exp why supplementation of DAD (which contains two S atoms) resulted in more poten fects compared with diallyl sulphide (DAS) (containing one S atom). Supplementatio DAD at 80 μL/L/day and propyl propane thiosulphinate (PTS) at 200 μL/L/day stron inhibited CH4 yield (g/kg DMI) by 62% and 96%, respectively, in batch cultures after incubation of the ruminal fluid of goats [131]. Diallyl sulphide (DAS) has shown small effects on rumen microbial fermentation [37]. It has been suggested in various studies that the antimicrobial potency of allyl sulphides in garlic oil increases with each additional S atom [143,144]. This could explain why supplementation of DAD (which contains two S atoms) resulted in more potent effects compared with diallyl sulphide (DAS) (containing one S atom). Supplementation of DAD at 80 µL/L/day and propyl propane thiosulphinate (PTS) at 200 µL/L/day strongly inhibited Animals 2022, 12, 2998 14 of 33 CH 4 yield (g/kg DMI) by 62% and 96%, respectively, in batch cultures after 24 h incubation of the ruminal fluid of goats [131].
Supplementation of allicin at 2 g/head/day effectively enhanced OM, N, NDF, and ADF digestibility and decreased daily CH 4 yield (g/kg DMI) in ewes, probably by decreasing the population of ruminal protozoa and methanogens [107]. Supplementary allicin can also decrease the ruminal concentration of ammonia by 14% but can increase the total VFA produced by up to 14.3% [100,107,110]. Significant increases in the populations of F. succinogenes, R. flavefaciens, and B. fibrisolvens in ewes supplemented with allicin have also been observed [135]. It is well established that CH 4 production has been positively correlated with more acetate production and negatively correlated with increased propionate production [145] because propionate synthesis is a main pathway for H 2 consumption, representing a competitive and alternative pathway to methanogenesis [70,146]. Allicin has been found to alter rumen VFA production so that less acetate and more propionate and butyrate are produced, and this may be due to an abundance of the Prevotellaceae and Veillonellaceae families [112]. Prevotellaceae is one of the predominant families in rumen fluid, and it is well known to produce propionate by utilising H 2 produced during carbohydrate fermentation [147].
Dietary garlic constituents are transformed into various metabolites in a biological system. Busquet, Calsamiglia, Ferret, Carro and Kamel [37] observed that allyl mercaptan is a common metabolite of allium-derived compounds as obtained after incubation of allicin and other allyl sulphides in fresh blood at 37 • C or gastric fluids [137]. Diallyl disulphide and allyl mercaptan resulted in a less potent effect than garlic oil in increasing in vitro rumen fermentation and decreasing CH 4 production, suggesting a possible synergistic effect between the different compounds present in the garlic oil [37]. In the specific case of garlic oil, the CH 4 mitigating effect may be directly attributed to the toxicity of organosulphur compounds, such as diallyl sulphide and allicin, to the methanogens [148].
Garlic extracts have demonstrated effectively decreased CH 4 production and improved rumen fermentation by increasing the production of total VFA at 200 g/kg of the feed [130]. Supplementation with garlic extracts has been associated with a lower abundance of the family Methanobacteriaceae, the major CH 4 producer in the rumen [96]. This was connected to the toxicity of the organosulphur compounds of garlic, such as diallyl sulphide and allicin, in inhibiting certain sulphydryl-containing enzymes essential for the metabolic activities of methanogenic archaea [48]. This interaction has been demonstrated by the loss of activity of some thiol-containing enzymes (e.g., papain and alcohol dehydrogenases) and by the reaction between different organosulphur compounds and cysteine to form other substances by a thiol-disulphide exchange reaction [143].
The constituents of dietary garlic are converted into various metabolites in biological systems, which can cause synergistic effects between different compounds in garlic. It can therefore cause different forms of garlic to have different bioactive components. This compound can potentially impact CH 4 reduction, which is directly related to the toxicity of organosulphur compounds to methanogens.

Chemical Composition of Garlic
Garlic contains volatile oils and protein, comprising 1-3.6 g/kg and 160-170 g/kg, respectively [137]. In addition, it is a rich source of sulphur, potassium, phosphorus, magnesium, sodium, and calcium [119]. The sulphur content in garlic varies from 5 to 37 g/kg of DM [119]. Garlic products can be classified into garlic essential oils, garlic oil macerate, garlic powder, and garlic extract [153].

Effects of Garlic on Rumen Fermentation
Garlic powder and garlic oil exhibit activities on modifying rumen fermentation parameters, improving nutrient digestibility, decreasing rumen protozoa numbers, and decreasing CH 4 emissions, and the effect of garlic extracts on the rumen microbiome have been comprehensively investigated [149,151]. The latest findings on the effect of garlic on ruminant animal productivity are summarised for both in vitro (Table 4) and in vivo determinations ( Table 5).
Supplementation of garlic oil at 0.8 g/d did not greatly affect ruminal fermentation parameters (total VFA concentration and individual VFA molar proportions) but increased ammonia and microbial crude protein [152]. In addition, garlic oil altered rumen fatty acid profile by increasing the concentration of certain fatty acids e.g., t11-18:1 (TVA) and c9, t11-CLA. This appeared to be achieved as a consequence of inhibition of the final step of biohydrogenation, which can lead to the accumulation of TVA in the rumen [152]. Garlic powder supplementation at 80 g/d in steers could enhance ruminal propionate production and reduce the acetate/propionate (C 2 :C 3 ) ratio by 10%, decreasing protozoa population while increasing N retention and absorption in ruminants [91]. Similarly, Ahmed, Yano, Fujimori, Kand, Hanada, Nishida and Fukuma [130] showed similar finding in in vitro studies; the supplementation of garlic and citrus extract at 20% of the substrate could improve the production of total VFA and propionate and reduce C 2 :C 3 ratio by 27%.
The effect of garlic oil and other organosulphur compounds (diallyl disulphide and allyl mercaptan) on rumen microbial fermentation in batch culture have been reported as resulting in lower molar proportions of acetate and higher proportions of propionate and butyrate upon supplementation of diallyl disulphide (DAD) (30 and 300 mg L −1 culture fluid) and allyl mercaptan (300 mg L −1 culture fluid) [37]. Moreover, there was a decrease in CH 4 yield (mL/g DM) of 73.6, 68.5, and 19.5% upon administration of garlic oil, DAD, and allyl mercaptan at 300 mg/L, respectively, which may help to improve the efficiency of energy use in rumen fermentation [37]. The effects of cinnamaldehyde and garlic oil have been investigated on rumen fermentation in a dual-flow continuous culture [154]. They reported that the inclusion of garlic oil at 312 mg/L increased the small peptide plus amino acid N concentration and the proportion of propionate and butyrate and decreased the proportion of acetate and branch-chained VFA, which indicate that garlic oil affected the fermentation profile and can be used as modulators of rumen microbial fermentation [37]. However, in the experiment of Kamel, Greathead, Tejido, Ranilla and Carro [39], three levels of DAD (0.5, 5, and 10 mg/L) were investigated, but none of the treatments had a suppressing effect on CH 4 production. Furthermore, DAD supplementation at 56 and 200 mg/kg DM levels failed to decrease CH 4 production in vivo [150]. Other studies reported that DAD supplementation in sheep diet only tended to decrease CH 4 yield relative to OM digested and that its potential to reduce CH 4 production in sheep was low; despite that, it improved digestibility and energy use efficiency by promoting the growth of anaerobic rumen fungi which might increase fibre digestion [110].    ↑ NH 3 -N by nitrate at days 10 and 18 ↓ CH 4 ; Feed digestion by the combinations (binary and ternary) of garlic oil with the other inhibitors at days 10 and 18; NH 3 -N by saponin, alone or in combinations, and garlic oil alone at day 2; Total VFA by garlic oil alone or garlic oil-saponin combination; Methanogens [48] Batch culture Concentrate and wheat straw at a 50:50 ratio Garlic powder 2-6% of DMI ↓ CH 4 ; C 3 ; C 5 [113]     Reports of garlic's effect on rumen fermentation are inconsistent between studies. This might be the effect of various factors, such as the dose administered, the composition of the substrate, and the composition of the microbial population in the inoculum [99]. Garlic oil and garlic powder tested at high doses showed the highest impact in reducing CH 4 emission. However, the dose level needs to be considered on how much it can be fed at the farm level.

Effects of Garlic on Rumen Microbiota
Garlic has been found to modify the microbial population profile in continuous culture experiments, reducing specifically the Provotella spp. (mainly P.ruminantium and P. briyantii) while other microbial populations remain unaffected [92,155]. Provotella spp. is mainly responsible for protein degradation and amino acid deamination, suggesting that garlic oil may also affect protein metabolism in which dehydrogenase activity is required to suppress deamination when using CH 4 inhibitors [156].
Endo and ectosymbiotic methanogens of protozoa can contribute around 25% of CH 4 emission from sheep rumen fluid, but the effect of garlic by-products on protozoa numbers was highly variable between different studies [49,143]. The effect of garlic powder supplementation at 4 mg/200 mg DM in vitro fermentation systems has shown a decrease in protozoa population by 60% [29]. Supplementing a basal diet with raw garlic or garlic oil at 500 mg/kg DM decreased the number of rumen protozoa in sheep by 35% [105]. Most studies that investigated the effect of garlic components on the population of methanogens were carried out in vitro. The inclusion of garlic oil at 100 and 250 mg/L decreased methanogenic bacterial activity by 68.5 and 69%, respectively (Chaves, He, Yang, Hristov, McAllister and Benchaar [103]). Supplementation of garlic oil at 1 g/L effectively reduced the in vitro abundance of F. succinogenes, R. flavefaciens, and R. albus without affecting total bacteria and could reduce the abundance of archaea and protozoa population by 16.5 and 8%, respectively (Patra and Yu [32]). In addition, the increase in the population of those three cellulolytic bacteria (F. succinogenes, R. flavefaciens, and R. albus) could be more probably explained by the reduced populations of the protozoa that engulf bacteria [32].
Observations of the reduction of methanogens coincide with those of in vitro results. In addition, the decreased population of protozoa could also be responsible for the reduction in methanogens, as the total methanogen population declined in absolute number as well as in proportion to the total bacterial population in the absence of protozoa [157]. Garlic powder supplementation at 80 g/d did not affect the amylolytic or cellulolytic bacteria population but decreased the protozoa population by 41% (Wanapat, Khejornsart, Pakdee and Wanapat [151]). Supplementation of plant extracts (mixture of garlic and citrus extract) at 10% and 20% of the substrate reduced Methanobacteriaceae, which is the major CH 4 producer in the rumen, by 94.07 and 92.70, respectively (Ahmed, Yano, Fujimori, Kand, Hanada, Nishida and Fukuma [130]). Furthermore, 20% PE effectively increased the abundance of H 2 -consuming groups such as Prevotellaceae and Veillonellaceae and reduced some H 2 -producing bacteria.
Garlic showed positive effects on rumen fermentation, improving nutrient digestibility and altering the rumen microbiome by decreasing the number of protozoa and decreasing CH 4 emissions. However, the effects are inconsistent between studies. In addition, future research should aim to understand the mode of action of garlic and its bioactive compounds in regard to enteric CH 4 mitigation.

Conclusions and Future Perspectives
Significant amounts of research have been conducted to identify strategies to reduce entric CH 4 emissions, as this is a major contributor to global warming. Understanding rumen function and dynamics have been found to be important in determining dietary strategies to mitigate CH 4 production in the rumen. Interactions between bacteria and protozoa are crucial and play a critical role in ruminal CH 4 production pathways. The main target of dietary manipulation is either via direct inhibition of methanogens, or by altering metabolic pathways leading to the reduction of substrates for methanogenesis. Garlic and its bioactive compounds, such as allicin (C 6 H 10 S 2 O), diallyl sulphide (C 6 H 10 S), diallyl disulphide (C 6 H 10 S 2 ), and allyl mercaptan (C 3 H 6 S), have demonstrated inconsistent effects in decreasing CH 4 production during rumen fermentation. This may be due to various reasons: firstly, different types of garlic contain different amounts of bioactive compounds. Secondly, the composition of the basal diet can affect the action of garlic-origin bioactive compounds by modulating rumen metabolism. However, generally increasing the dietary dose of garlic and/or its bioactive compounds results in a decrease in CH 4 production. Further research is needed to understand how organosulphur compounds in garlic influence methanogens and their metabolic pathways, providing insight into effective CH 4 mitigation strategies. Generally, there will not be a single "silver bullet" for agricultural GHG emissions. Rather, this approach will have a shorter-term impact but could be combined with other dietary strategies to prevent adverse effects on rumen digestibility and fermentation. There are real opportunities for the feed industry to develop garlic-based feed additives to reduce CH 4 emission from ruminant production. Given the far-reaching consequences of rumen fermentation on ruminant nutrition, food production, and the environment, it is not surprising that many studies have been undertaken to understand microbial populations in the rumen and ultimately manipulate them to maximise productivity while reducing the environmental impact of ruminant production.

Funding:
We would like to thank the Indonesia Endowment Fund for Education (LPDP) from the Ministry of Finance, the Republic of Indonesia for supporting this study via a scholarship to N.F.S. The APC was funded by the University of Reading.