Nutritional Interventions to Reduce Methane Emissions in Ruminants

Abstract

Methane is the single largest source of anthropogenic greenhouse gases produced in ruminants. As global warming is a main concern, the interest in mitigation strategies for ruminant derived methane has strongly increased over the last years. Methane is a natural by-product of anaerobic microbial (bacteria, archaea, protozoa, and fungi) fermentation of carbohydrates and, to a lesser extent, amino acids in the rumen. This gaseous compound is the most prominent hydrogen sink product synthesized in the rumen. It is formed by the archaea, the so-called methanogens, which utilize excessive ruminal hydrogen. Different nutritional strategies to reduce methane production in ruminants have been investigated such as dietary manipulations, plant extracts, lipids and lipid by-products, plant secondary metabolites, flavonoids, phenolic acid, statins, prebiotics, probiotics, etc. With the range of technical options suggested above, it is possible to develop best nutritional strategies to reduce the ill effects of livestock on global warming. These nutritional strategies seem to be the most developed means in mitigating methane from enteric fermentation in ruminants and some are ready to be applied in the field at the moment.

1. Introduction

Methane is the single largest source of anthropogenic greenhouse gases (GHG) produced in agricultural systems, especially in ruminant husbandry. It is estimated that 18% of the annual GHG emissions come from different types of livestock and that 37% of methane (CH₄) comes from fermentation processes in ruminants. As global warming is a main concern, the interest in mitigation strategies for ruminant derived methane is strongly increased over the last years. Enteric methane (~87%) is produced in rumen, the remaining 13% being released from fermentation in the large intestine [1]. Methane is a natural by-product of microbial fermentation of carbohydrates and, to a lesser extent, amino acids in the rumen. In rumen, the diverse and dense microbial populations consisting of protozoa, fungi and bacteria act on feed particles to degrade plant polysaccharides and produce volatile fatty acids (VFAs; mainly acetate, propionate and butyrate) and gases (CO₂ and H₂) as main end products. Methanogens use the excess of H₂ from NADH (reduced form of nicotinamide adenine dinucleotide) and CO₂ as the principal substrates to produce CH₄. About 82% of the CH₄ formed comes from H₂ reduction of CO₂, while about 18% is derived from formate. However, two genera of methanogens: the Methanosarcina and Methanosaeta can convert acetate to CO₂ and CH₄ (acetoclastic methanogenesis) [2]. Since methane contains energy, its emission during rumen fermentation is considered to be a loss of feed energy that is equivalent to 2–12% of dietary gross energy of animal feed.

2. Greenhouse gas effect

Since last few decades, the increased emission of GHG in the atmosphere has drawn worldwide attention due to global warming and stratospheric ozone depletion. The absorption and emission of infrared radiation by these atmospheric gases warm earth’s surface and lower atmosphere. It ultimately leads to increased air, land and ocean temperatures and which in turn can increase annual precipitation in high rainfall regions and decrease precipitation in regions of low rainfall [3]. Global warming is the increase in average temperature of the earth’s near-surface air and ocean since the mid-twentieth century and its projected continuation. In the twentieth century, average atmospheric temperature near the surface of the earth rose by 0.6 ± 0.2°C from 14°C. It is estimated that global temperature would increase by 1.4–5.8°C between 1990 and 2100. The Intergovernmental Panel on Climate Change (IPCC) concludes that most of the temperature increases since the mid-twentieth century is ‘very likely’ due to the increase in anthropogenic GHG concentrations.

The IPCC included six gases as GHG viz. CO₂, CH₄, nitrous oxide (N₂O), hydroflurocarbons, perflurocarbons and sulfur hexafluoride (SF₆). The first three gases in the atmosphere are produced as a result of agricultural and livestock activities. While CO₂ represents 73.5% of the total GHG, CH₄, N₂O and others represent 16.8%, 8.7% and 0.7% respectively. Since 1950, atmospheric CO₂ has increased 28%, while CH₄ has increased 70%. Methane, over the first 20 years after release, has 80-times more warming potential as a GHG than CO₂ [4]. Methane is also considered a highly potent GHG because of its ability to trap infrared radiation 20 times more effectively than CO₂ [5]. The warming potential of CO₂, CH₄ and N₂O is 1, 23 and 298, respectively. However, the life span of CH₄ in the atmosphere is 12 years while those of CO₂ and N₂O is 100 and 120 years respectively.

3. Livestock role in climate change

India possesses about one fifth of the world’s total livestock population, which is being held responsible for the large contribution to the GHG emission. The livestock industry contributes ~18% of global GHG emissions. It accounts for 35% of CH₄, 9% of CO₂ and 65% of human-related N₂O emissions [6]. Enteric fermentation [7] and storage of slurry [6] are the main sources of anthropogenic CH₄ emissions. The output of methane emitted from ruminants accounts for one fifth of that in atmosphere. Methane emissions from ruminant livestock (cattle, buffalo, sheep and goat) were estimated at ~2.2 billion tonnes of CO₂ equivalent, accounting for ~80% of agricultural CH₄ and 37% of the total anthropogenic CH₄ emissions [8].

4. Rumen fermentation and methanogenesis

Digestion of feed in the rumen is the result of anaerobic fermentation involving various groups of microbes (bacteria, archaea, protozoa, and fungi). Methane is formed by the archaea, the so-called methanogens, which utilize excessive ruminal H₂. The activity of H₂-utilizing methanogenic archaea in rumen reduces the end product inhibition of H₂, thereby allowing more rapid fermentation of feed. Methane keeps the partial pressure of H₂ in the rumen contents very low, promoting the regeneration of reduced pyridine nucleotides by H₂ gas formation through hydrogenase activity instead of formation of lactate and ethanol by alcohol- or lactate-dehydrogenases. Even a small amount of H₂ in rumen can limit the oxidation of sugar, VFAs conversion and hydrogenase activity, if alternative pathways for disposal are absent [9].

The major factors influencing CH₄ emissions from ruminants are: (a) level of feed intake, (b) type of carbohydrates fed and (c) alteration of the ruminal microflora. When CH₄ reduction is attempted, it is therefore necessary to consider alternative hydrogen sinks to methanogenesis. Methanogenesis is the primary pathway followed by propionate production (fumarate reduction). Thus, a strategy for methane mitigation should be developed concomitantly with a strategy to enhance propionate production.

5. Nutritional interventions to reduce enteric methane emission

Nutritional strategies seem to be the most developed means in mitigating CH₄ from enteric fermentation in ruminants. Modes of action could be direct effects on methanogens [by medium-chain fatty acids (MCFA)], anti-protozoal effects [by saponins, MCFA and polyunsaturated fatty acids (PUFA)] or inhibiting organic matter (especially fiber) digestion followed by a lower H₂ supply to the methanogens [by condensed tannins (CT), MCFA, PUFA].

5.1 Manipulating nutrient composition of the diet

The feed quality and feed digestibility are the major determinants of energy available for animal growth and, therefore, of the performance of ruminants and of CH₄ production. Types and dietary proportions of carbohydrates are largely affecting ruminal fermentation conditions (especially pH), VFA profile and, concomitantly, CH₄ formation. The efficiency of nutrient utilization by microbial organisms in the rumen controls the fermentation process, which in turn affects the activity of methanogens relative to other microbial species. The forage-based diets result in generally higher enteric CH₄ formation than concentrates (grain-based feeds) in the diet. Dairy cows emitted less enteric CH₄ when fed a corn-based diet compared to ryegrass hay [10].

Starch, the main component of concentrate-rich diets, is mostly degradable to propionate which is a competitive H₂ sink to methanogenesis. In contrast, concentrates rich in sugars might have a higher methanogenic potential than starch or even fiber in dairy cows [11, 12], but this presumably only when a high ruminal pH is maintained [13]. An in vitro study [13] with starch and sucrose at different ruminal pH levels showed a higher CH₄ formation for sucrose, especially at high ruminal pH. This was mainly due to an increase in fiber digestion with the addition of sucrose. Diets containing feeds with elevated contents of distinct carbohydrates have gained attention in reducing CH₄ emissions. Grass cultivars selected for high contents of sugar (e.g., high-sugar ryegrass) might be an option for enteric CH₄ mitigation. However, grass-based feeding systems compared to those including maize silage have been reported to result in higher CH₄ emissions per unit of animal product [13]. Dohme-Meier et al. [14] observed that even feeding hay with a medium water-soluble carbohydrate (WSC) content (16%) can lead to a ruminal pH of <6 to which the methanogens are susceptible. When the ruminal pH is unaltered by feeding different grasses, methanogenesis could be increased by extra WSC and then sugars exhibit a higher methanogenic potential than starch [15]. There will be higher methane emission when WSC replaces the rumen degradable protein instead of fiber [16].

Forage quality can be improved through feeding forages with lower fiber and higher soluble carbohydrates, changing from C4 tropical grasses to C3 temperate species, or grazing less mature pastures. These options can also reduce CH₄ production [13]. Methane production per unit of cellulose digested has been shown to be 3 times that of hemicellulose, while cellulose and hemicelluloses ferment at a slower rate than non-structural carbohydrate, thus yielding more CH₄ per unit of substrate digested [17]. Methane emissions are also commonly lower with higher proportions of forage legumes in the diet, partly due to lower fiber content, faster rate of passage and, in some cases, the presence of condensed tannins [13].

5.2 Supplementation of lipids and lipid by-products

5.2.1 Dietary lipids

The use of lipids is considered as one of the promising dietary alternatives to depress ruminal methanogenesis. The effectiveness of fat supplementation depends mainly on the fat source, fatty acid profile, form of fat and the amount of supplemented fat [13]. Possible mechanisms by which added lipid can reduce enteric methane production include: (a) by reduction of fiber digestion (mainly in long-chain fatty acids); (b) by lowering of dry matter intake (if total dietary fat exceeds 6–7%); (c) by decreasing organic matter fermentation (d) through direct inhibition of activities of different microbes including methanogens and hydrogen producing microorganisms; (e) through suppression of rumen protozoa; and (f) to a limited extent through biohydrogenation of unsaturated fatty acids which serve as a hydrogen sink, although only 1–2% of the metabolic hydrogen in the rumen is used for this purpose [13, 17]. Fat can reduce CH₄ emissions by 4–5% (g/kg DMI) for every 1% increase in the fat content of the diet. Addition of different vegetable oils (soybean, coconut, canola, rapeseed, sunflower, linseed etc.) to ruminant diets have been shown to reduce CH₄ production between 18% and 62% in Rusitec fermenters [18], sheep [19], beef cattle [20] and dairy cows [21]. Beauchemin et al. [13] estimated a reduction of enteric CH₄ formation of 0.56% per g of lipid supplied per kg diet DM. Plant oils rich in MCFA such as coconut oil [major component is lauric acid (C14:0)] are known to inhibit rumen methanogenesis [18]. The addition of coconut oil to forage and concentrate rations supplemented to Charolais steers showed a reduction in voluntary intake and protozoa population and this was reflected in low CH₄ emissions, without affecting livestock production [22]. The lauric acid (C14:0) is more potent in CH₄ reduction than palmitic (C16:0), stearic (C18:0) and linoleic (C18:2) fatty acids in a semicontinuous fermenter that simulates the rumen (RUSITEC) [18]. A similar reduction in CH₄ was observed in batch cultures, in which coconut oil and lauric acid were directly compared. It showed that lauric acid inhibited methanogenesis to a greater extent [23]. The ability of lauric acid to decrease cell viability of Methanobrevibacter ruminantium has been reported [24]. The lauric acid treatment, possibly through its effect on protozoa physically associated with archaea, resulted in an increase in the archaeal methanogenic genus Methanosphaera and a decrease in Methanobrevibacter [25]. Besides lauric acid, other MCFA such as myristic acid, or a combination of both and PUFA like linolenic acid and linoleic acid were shown to be effective, but might also negatively influence feed intake and digestibility. The vegetable and fish oils significantly decreased CH₄ production after 14 d but not after 11 weeks of feeding in dairy cows [26]. However, persistence of the mitigating effect of dietary oil was observed in the study of Martin et al. [27] with flaxseed in dairy cows. Meta-analyses by Moate et al. [28] documented a consistent decrease in CH₄ production with fat supplementation. Other studies have reported a 27% reduction in CH₄ emission with the supplementation of fish oil and sunflower oil 500 mg/d each when fed to dairy cows in short periods (14 days) [26]. The reduction in methanogesis with oils/lipids appears to be the result of inhibition of microbial flora especially protozoa.

5.2.2 Lipid by-products

High-oil by-products from the biofuel industries such as dry distillers grains (DDG), wet distillers grains (WDG), dry distillers grains with solubles (DDGS), wet distillers grains with solubles (WDGS) and mechanically extracted oilseed meals are natural anti-methanogenic unconventional feeds. There was decrease in methane emission up to 24% when barley was replaced by DDG thereby supplementing an additional 3% lipid to the dietary DM in beef cattle [29]. Hales et al. [30] fed diets containing 0 to 45% WDGS (substituting steam-flaked corn) to Jersey steers and observed a linear increase in CH₄ emission per unit of DMI (up to 64% increase with the highest inclusion rate). Another product of the biodiesel industry, glycerol, has been shown to promote CH₄ production during ruminal fermentation in vitro. The inclusion of glycerol as a major component of the diet has been reported in beef cattle [31, 32], and inclusions of 10–20% in diet DM have been used without negatively affecting lamb performance [33]. When included up to 21% of diet DM, glycerol did not affect nutrient digestibility or CH₄ emissions of lambs fed barley-based finishing diets [34].

5.3 Plant secondary metabolites

Plant secondary metabolites (PSM) are groups of chemical bioactive compounds [tannins, saponins, essential oils (EO), alkaloids, flavonoids, glucosides, amines, non-protein amino acids, organosulfur compounds] in plants that are not involved in the primary biochemical processes of growth and reproduction but are meant for protection of the host plant against invasion by the pathogenic microbes. This highly specific anti-microbial activity is being exploited to modulate the rumen microbial ecosystem to alter rumen fermentation thereby decreasing methane production.

5.3.1 Tannins

Tannins are plant polyphenols of varying molecular size and exist in two forms in plants: hydrolysable tannin (HT) and condensed tannin (CT). Tannins, as feed supplements or as tanniferous plants have shown potential for reducing CH₄ emission by up to 20% [35]. Different types of tannin containing forages decreased CH₄ emission in vitro. The CH₄ inhibiting potential of tannins might be due to a direct effect on ruminal methanogens and an indirect effect on lower feed degradation leading to a decreased hydrogen production. Tannins and phenolic monomers have been found to be toxic for some of the rumen microbes, especially ciliate protozoa, fiber degrading bacteria and methanogenic archaea, and as a result methanogenesis in the rumen can also be reduced. The anti-methanogenic effect of tannins depends on its dietary concentration and is positively related to the number of hydroxyl groups in their structure. The hydrolyzable tannins tend to act by directly inhibiting rumen methanogens whereas the effect of condensed tannins (CT) on CH₄ production is more through inhibition of fiber digestion. In many studies (in vitro and in vivo) it has been demonstrated that with temperate legumes (Hedysarium coronarium, Lespedeza cuneata, Lotus corniculatus and Lotus uliginosus) and tropical legumes (Calliandra calothyrsus, Flemingia macrophylla) that contain CT, it is possible to reduce methanogenesis. The methane suppression effect of CT containing legumes, such as Lotus pedunculatus or Acacia mearnsii, relative to forages without tannins has been shown in sheep [36], cows [37] and goats [38]. Ficus bengalensis, Autocarous integrifolis and Azadirachta indica had also been shown to reduce methane production [39]. Ramirez-Restrepo and Barry [36] indicated that the CT-rich legumes such as L. corniculatus and sulla (Hedysarum coronarium) showed reduced methane production relative to forages without tannins (Chicorium intybus). In goats fed with the CT containing forage Sericea lespedeza, Puchala et al. [40] observed a reduction in CH₄ loss of over 30%. Methanol extract of harad (Terminalia chebula) caused 95% reduction in CH₄ production in vitro at the level of 0.25 ml/30 ml incubation medium and complete inhibition was observed when the level of extract was double [41]. In goats consuming different levels of CT from Lespedeza striata, there was a reduction in the emission of CH₄, while in the same study feeding Sorghum bicolor with lower levels of CT showed no reduction of enteric production of CH₄ [38].

5.3.2 Saponins

Saponins are naturally occurring surface-active glycosides with foaming characteristics, present in many plant species, wild plants as well as, cultivated crops. They usually consist of a sugar moiety linked to a hydrophobic compound, either triterpenoid or steroid in nature. Saponins reduce CH₄ production via inhibition of either protozoa or methanogens or both. These inhibited protozoa at relatively low concentrations whereas higher concentrations were required to kill or suppress methanogenic archaea. McAllister and Newbold [9] have suggested that a decrease in methanogens associated with protozoa as exo- and endosymbionts could be the main mechanism by which saponin feeding reduces methanogenesis and methanogens associated with protozoa are estimated to be responsible for 9–37% of the total CH₄ production in the rumen. Anti-methanogenic activity of saponins is believed to occur by limiting hydrogen availability to methanogens and re-channeling of metabolic hydrogen from methane to propionate production in the rumen. In addition, saponins, due to their chemical structure, may display anti-bacterial properties by reducing the number of bacteria producing H₂ thus resulting in the inhibition of H₂ production thereby reducing CH₄ formation. Goel and Makkar [42] summarized that there was no difference in the CH₄ mitigation effect between steroidal saponins (Yucca schidigera) and triterpenoid saponins (Quillaja saponaria). Studies from China have reported decreased CH₄ in ruminants treated with tea triterpenoid saponins (TS) but also substantial changes in microbial populations, including a reduction in protozoal counts [43]. Therefore, a reduction in the rumen protozoa population as a result of inclusion of TS in the diet could result in a decrease in enteric CH₄ production. Zhou et al. [44] reported that addition of TS reduced CH₄ production mainly by inhibiting protozoa, increasing molar proportions of propionate and decreasing acetate/propionate ratio without adversely altering relative ruminal abundance of fungi and cellulolytic bacteria. According to Lila et al. [45], supplementation of feed rations consisting of meadow hay and concentrate with saponins reduces CH₄ production in steers by 12.7%, while in the in vitro conditions during 24 h incubation, the reduction amounted ~15–44%. Hess et al. [46] reported that the daily CH₄ production was reduced by 6.5% due to supplementation of Sapindus saponaria fruits in sheep receiving tropical grass hay-concentrate diet. Hess et al. [47] found that supplementation with S. saponaria saponin at 100 mg/g DM reduces methanogenesis by about 20% with no influence on the population of methanogens in the in vitro conditions. Wang et al. [48] reported a decreased CH₄ formation when feeding sarsaponins to sheep (0.13 g/kg diet). Saponins from Sapindus murkossi extracted with the use of ethanol, more effectively affect the process of methanogenesis in comparison to water and methanol extracts [49]. High effectiveness in the reduction of CH₄ production in the rumen ecosystem is possible to achieve also with the use of unextracted plant saponins, provided in the form of leaves or seeds (Sesbana sesban; Trigonella foenum-graecum). Seeds of temperate climate legumes (e.g., lupines, peas) are known to contain certain levels of tannins, and also of saponins.

5.3.3 Essential oils

Approximately 10–25% reduction of methane may be achievable through the addition of dietary oils in ruminants [13]. The CH₄ mitigating effect of essential oils might be due to suppression of methanogens. Another effect is the increase in the propionate-to-acetate ratio resulting in lower amounts of H₂ available. Plant breeding may in future offer opportunities to increase oil levels in selected forages and therefore increase oil intake directly as animals graze. Clear CH₄ mitigating effects were found in several in vitro studies when supplementing essential oils from garlic, thyme, oregano, cinnamon, rhubarb, frangula, etc. Garlic oil (principal component is diallyl disulfide), cinnamon oil (principal component is cinnamaldehyde), clove bud (principal component is eugenol), hot peppers (principal component is capsaicin) and anise oil may reduce methane production in the rumen by increasing the propionate-to-acetate ratio [50]. A study showed the potential anti-methanogenic properties of cashew nut shell liquid (active components are anacardic acid, cardanol and cardol), when added to batch cultures at the rate of 200 μg/ml of incubated volume [51]. A commercial blend of essential oils failed to decrease CH₄ production in vivo despite decreasing the digestibility of all nutrients [20]. The lack of response in vivo is partly attributed to the adaptation of microbes, but also to the use of lower doses compared to those in the in vitro experiments. The mustard seed oil and Japanese horseradish oil contain volatile compounds i.e. allyl isothiocyanate which has been reported to decrease CH₄ production in vitro. Use of peppermint oil (Mentha piperita) in low concentration of 1 or 2 μl/l, respectively resulted in linear reduction in methanogenesis (61%) together with the limitation on the number of methanogens (82%) and a decrease in the protozoan activity measured by ¹⁴C-radio-isotopic technique [49]. Some researchers carried out a phylogenetic analysis of the rumen ecosystem and reported a tendency towards an increase in the diversity of methanogens in comparison to Methanosphaera stadtmanae, M. smithii and some uncultured groups with cinnamaldehyde, garlic and juniper berry oil supplementation [52]. When ajwain oil and lemon grass oil in 1: 1 ratio @ 0.05% of dry matter intake were fed to buffalo calves, methane production (L/kg digestible organic matter intake) was reduced by 16.7% [53] and feeding of these additives did not affect feed intake, rumen pH, or rumen metabolites [54].

5.3.4 Combination of different plant secondary metabolites

When EO-rich garlic and saponin-rich soapnut in 2:1 ratio @ 2% of DMI were fed to buffalo calves, methane production (L/kg digestible organic matter intake) was reduced by 12.9% [53] and feeding of these additives did not affect feed intake, rumen pH, or rumen metabolites except ammonia and enzyme profile [54].

When EO-rich garlic, saponin-rich soapnut, tannin-rich harad and EO-rich ajwain in 2:1:1:1 ratio@ 1% of DMI were fed to buffalo calves, methane production (L/kg digestible organic matter intake) was reduced by 8.4% [53] and feeding of these additives did not affect feed intake, rumen pH, or rumen metabolites except ammonia and enzyme profile [54].

5.4 Flavonoids

Oskoueian et al. [55] evaluated the effects of different flavonoids such as flavone, myricetin, naringin, catechin, rutin, quercetin, and kaempferol at the concentration of 4.5% of the substrate (dry matter basis) on the rumen microbial activity in vitro. These flavonoids suppressed CH₄ production significantly (P < 0.05). Total populations of protozoa and methanogens were significantly (P < 0.05) suppressed by naringin and quercetin. The researchers concluded that naringin and quercetin at the concentration of 4.5% of the substrate (dry matter basis) were potential metabolites to suppress CH₄ production without any negative effects on rumen microbial fermentation.

5.5 Phenolic acid

Caffeic acid (CA), a phenolic acid, serves as a promising rumen CH₄ inhibitor. It modulates methanogenesis and rumen fermentation mainly by affecting the growth of cellulolytic bacteria in vitro [56]. Kayembe et al. [57] reported the order of toxicity to methanogens by different phenolic monomers as follows: benzene > phenol > resorcinol > hydroquinone > pyrogallol which is attributed to the number of hydroxyl groups on the aromatic compound. Increase in the number of hydroxyl groups leads to decrease in toxicity to methanogens.

5.6 Statins

Fungal statins are used in human beings to reduce cholesterolemia. They inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase which is a key enzyme in the cholesterol production pathway [58]. Unlike bacteria, archaea need HMG-CoA reductase for their membrane lipid synthesis. So, it has been hypothesized that statins can inhibit archaea by inhibiting HMG-CoA reductase [59, 60]. The effects of statins on methanogenesis and overall rumen fermentation vary depending on statin type and concentration. Hydrophobic statins, such as simvastatin and atorvastatin, seem to be more effective compared to the hydrophilic statins, such as rosuvastatin [61]. Several in vitro and in vivo studies have examined the potential of statins such as lovastatin and mevastatin to reduce rumen CH₄ production, but results were inconclusive [62, 63]. The concentrations of statins that decreased CH₄ production without negative effects on rumen fermentation spanned a wide range [64, 65].

5.7 Other metabolites

Methane inhibition has been demonstrated with dietary supplementation of various plant extracts, without identification of the active agents. Broudiscou et al. [66] investigated the effect of 13 plant extracts in continuous culture and showed that Equisetum arvense and S. officinalis had possible inhibitory effect on CH₄ production. Patra et al. [41] studied the effect of water, methanol and ethanol extracts of Acacia concinna, T. chebula, T. bellirica, Emblica officinalis and A. indica in vitro and observed reduction in CH₄ production by T. chebula. A similar study [67] comparing Y. schidigera extract to Castanea sativa wood extract (containing HT and lignan) in in vitro rumen models showed effects on CH₄ production only at very high levels. Although rich in a long list of plant secondary metabolites, macahypocotyls and lupine seeds had no effect on enteric CH₄ formation [68]. Lupine seeds promoted methanogenesis in relation to the energy content of the diet as the increase per unit of SCFA shows when feeding about 200 g lupine seeds/kg DM to sheep [69].

European scientists screened 500 plant species for their ability to inhibit CH₄ production and selected 7 novel plants i.e. Italian plumeless thistle (Carduus pycnocephalus, 30% inhibition), the Chinese peony (Paeonia lactiflora, 8–53%), the European aspen (Populus tremula, 25%), the sweet cherry (Prunus avium, 20%), goat willow (Salix caprea, 30%), English oak (Quercus pedunculata, 25%) and Sikkim rhubarb (Rheum nobile, 25%). Carduus and Rheum species were evaluated in a RUSITEC analysis. On a high forage diet, 16 and 22% inhibition of methanogenesis was noted, while less inhibition (5 and 15% respectively, not significant) was observed on a high concentrate diet. Japanese researchers [70] reported that plant-derived liquid (PDL) and yeast-derived surfactant (YDS) induced >95% reduction in CH₄ production in batch cultures and >70% in RUSITEC analysis. The PDL contains anacardic acid, a salicylic acid derivative with an alkyl group that inhibits Gram-positive bacteria including bacilli and staphylococci. Anacardic acid was suggested to be a propionate enhancer. The YDS disrupts the cell walls of Gram-positive rumen bacteria. Hydrogen and formate producers viz. Ruminococcus flavefaciens, Ruminococcus albus, Butyrivibrio fibrisolvens and Eubacterium ruminantium were sensitive and propionate and succinate producers viz. Selenomonas ruminantium, Megasphaera elsedenii and Succinivibrio dextrinosolvens were tolerant to PDL and YDS. So, the rumen fermentation is shifted towards more propionate and less CH₄ production. Sheep that were fed a diet supplemented with PDL or YDS showed a fermentation pattern that was similar to that observed in RUSITEC and was accompanied by similar bacterial population shifts. Spanghero et al. [71] examined the chemical composition and rumen fermentability of grape seeds in vitro. Grape seeds are characterized by high levels of total phenols and total tannins [71] which might result in anti-methanogenic effects. Hop cones are feeds rich in specific plant secondary metabolites especially acids like humulones and lupulones. These acids are known to have anti-microbial effects [48]. Nevertheless, in vitro ruminal fermentation (e.g. increased gas production and VFA) was affected by hop addition [48]. In contrast, hop cones neither affected rumen fermentative activity nor incubation liquid ammonia nor CH₄ formation [72].

5.8 Prebiotics

In ruminants, prebiotics can be used along with nitrate and probiotics to reduce CH₄ production. They enhance propionate production by stimulating Selenomonas, Succinomonas and Megasphera sp. and decrease acetate production by inhibiting Ruminococcus and Butyrivibrio sp. [73]. Administration of galacto-oligosaccharides (GOS) decreased nitrite accumulation in rumen and plasma and nitrate-induced methemoglobin, while retaining low CH₄ production. 11% reduction in CH₄ emission (liters/day) in GOS supplemented diet compared to control diet has been reported [74]. Inclusion of GOS increased propionate production and decreased CH₄ formation [75].

5.9 Direct-fed microbials or probiotics

These are microbial feed additives that have been developed to improve animal productivity by directly influencing rumen fermentation. Several in vitro studies have demonstrated that probiotics can reduce CH₄ production [76]. Probiotics used in ruminant nutrition are yeast-based products (YP). Convincing animal data on YP for mitigating CH₄ production are lacking. Researchers also inoculated the rumen with fungi (Candida kefyr) and lactic acid bacteria (Lactococcus lactis) along with nitrate supplementation to control methanogenesis and prevent nitrite formation, but no consistent animal data have been reported [77].

5.9.1 Yeast culture

Yeast cultures (i.e., Saccharomyces cerevisiae and Aspergillus oryzae) reduce CH₄ production in three ways; (i) by reducing protozoa numbers, (ii) by increasing butyrate or propionate production and (iii) by stimulation of acetogens to compete with methanogens or to co-metabolize hydrogen, thereby decreasing CH₄ formation. However, the effects of probiotics may be diet-dependent. Carro et al. [78] observed reduction in CH₄ production and protozoa numbers when supplemented Rusitec fermenters with S. cerevisiae culture with a forage 50:concentrate 50 diet, but no effects were found with a forage 70:concentrate 30 diet. Lynch and Martin [79] reported 20% reduction in CH₄ production after 48 h of incubation with S. cerevisiae culture in an in vitro system. Frumholtz et al. [76] observed 50% decrease in CH₄ production when supplemented Rusitec fermenters with A. oryzae culture. Mwenya et al. [73] reported that sheep fed 70:30 forage:concentrate diet produced 10% less CH₄ when received daily 4 g of yeast culture. In contrast, Mathieu et al. [80] reported that S. cerevisiae and A. oryzae did not affect CH₄ production in sheep fed 44:66 forage:concentrate diet. However, results are inconsistent and further research is required to screen a large number of yeast strains to isolate those with significant CH₄ abatement potential.

5.9.2 Acetogens

Reductive acetogens are bacteria present in adult ruminants that reduce two moles of CO₂ to acetate by oxidation of H₂ in rumen unlike hydrogenotrophic methanogens, which utilize H₂ to reduce CO₂ to CH_4. So, acetogens are in direct competition with the methanogens. However, the affinity of the reductive acetogens for H₂ is 10–100 times lower than the hydrogenotrophic methanogens and the low partial pressure of H₂ in the rumen is not conducive for the acetogens to grow autotrophically [81]. So, while acetogens are present in the rumen, methanogens effectively outcompete them for hydrogen [9]. Acetogenic bacteria demonstrate higher population densities and an ability to be dominant under some conditions (e.g., in some macropods) [82]. Acetogenic bacteria are present in the rumen at population densities which may reach that of methanogens but despite their presence, reductive acetogenesis is extremely difficult to induce in the rumen. When methanogens are inhibited from the rumen by some means, they are capable of using this excess hydrogen to form acetate. Researchers are investigating these reactions with the aim of survival of acetogenic bacteria in the rumen and hence the displacement of methanogenic bacteria. An alternative approach would be to screen a range of acetogenic bacteria for their activity in rumen fluid and to introduce the acetogens into the rumen as a feed supplement. Lopez et al. [83] reported that Eubacterium limosum ATCC 8486 and Ser 5 increased acetate production and decreased H₂ formation when they were added to cultures of mixed ruminal microorganisms along with 2-bromoethanesulfonic acid (BES). In a rumen fistulated wether with continuous infusion of a 2-BES solution showed adaptation by methanogens after initial inhibition but use of cattle caecal contents, which contained acetogens, removed this adaptation effect [84]. Nollet et al. [81] reported that addition of Peptostreptococcus productus to BES-treated ruminal samples inhibited CH₄ production. On the basis of feed intake, VFAs, population density and hydrogen utilization pattern, it was suggested that reductive acetogenesis can sustain a functional rumen in the absence of methanogens [85].

5.9.3 Methane oxidizers

Methanotrophs or methane oxidizing bacteria are a unique group of methylotrophic bacteria. They require CH₄ as their carbon and energy source. So, they can be used as direct-fed microbial preparations. The oxidation reaction will compete with the CH₄ production and this reaction is a strictly anaerobic process [86]. Therefore, methane oxidizers from gut and non-gut sources could be screened for their activity in rumen.

5.9.4 Bacteriocins

Bacteriocins are narrow spectrum anti-bacterial proteinaceous polymeric substances and are produced by different Gram-positive and Gram-negative bacteria. They are under the control of plasmid. They compete with microbial species for niches within the rumen system. So, they could be effective in inhibiting methanogens and redirecting H₂ to other reductive bacteria like acetogens and propionate producers [9]. Some bacteriocins produced by lactic acid bacteria have been identified as an alternative group of anti-microbials for manipulation of the rumen microbial ecosystem [87]. The first described bacteriocin, nisin that is produced by L. lactis ssp. lactis, has a methane-mitigating ability that was observed in a monensin-supplemented in vitro culture (20% inhibition without a negative effect on VFA production) [88]. Although no mechanism was proposed to explain its effect on rumen bacteria, nisin potentiates propionate production and possibly shows selective activity against Gram-positive rumen bacteria. Nisin is active even at low pH, decreases the acetate to propionate ratio. It has been reported that 36% methanogenesis was reduced by using nisin [88]. A combination of nisin and nitrate, an alternative electron receptor, has been reported to reduce CH₄ emissions in sheep [89]. Alazzeh et al. [90] reported that the use of some strains of propionibacteria have the potential to lower CH₄ production from mixed rumen cultures and this reduction is not always associated with an increase in propionate production. Klieve and Hegarty [91] suggested that bacteriocins could be used to decrease ruminal CH₄ production in vivo. But rather than using bacteriocins of exogenous origin, it is advantageous to use bacteriocin of rumen origin. Bovicin HC5, the semi-purified bacteriocin produced by Streptococcus bovis HC5 from the rumen, has been reported to suppress CH₄ production by 50% in vitro [92], and even low concentration of bovicin HC5 (128 activity units ml⁻¹) may be equally as useful as monensin in limiting CH₄ production in the rumen [92, 93]. The CH₄ content declined with pediocin, enterocin and combinations of both after 24 h incubation. Pediocin P1 and P2 decreased (P < 0.05) CH₄ level by 4.81% and 5.08%, respectively when compared to control and combinations of bacteriocin.

5.9.5 Fungal metabolites

Secondary fungal metabolites from Monascus spp. reduced enteric CH₄ emissions in sheep by 30%, decreased acetate to propionate ratio and reduced methanogen numbers in a short-term trial [65]. The red macroalgae or seaweed (Asparagopsis taxiformis) when added at 2% of substrate organic matter, decreased CH₄ emissions by 99% without reducing substrate digestibility or VFA production in laboratory rumen fermentation cultures [24]. A. taxiformis decreased enteric CH₄ production from sheep [94] and beef steers [95].

5.9.6 Methane reducing species

Mitsuokella jalaludinii has been demonstrated as an efficient CH₄ reducing agent in the rumen by competing with methanogens for hydrogen, necessary for growth by both [96]. M. jalaludinii decreases CH₄ production and improves rumen fermentation thereby improving feed efficiency in livestock.

5.10 Conclusion

Any sustainable solution to lower on-farm CH₄ emissions should be practical, cost-effective and have no substantial adverse effect on the profitability of ruminant livestock production. In this context, manipulating diet composition to induce changes in enteric fermentation characteristics remains the most feasible approach to lower CH₄ production. Therefore, efforts should be made to select feed ingredients and to identify forage plants containing secondary metabolites that can be used to inhibit methanogenesis selectively, but without adversely affecting feed utilization. Moreover, rumen is a dynamic ecosystem and rumen methanogenesis is a complex process. Since our understanding of rumen microbes is still incomplete, elucidation of microbial diversity and microbial interrelationships is absolutely essential for the successful manipulation of rumen fermentation towards a significant reduction in ruminant CH₄ emission.