Journal of Animal Science and Technology
Korean Society of Animal Sciences and Technology

Advanced estimation and mitigation strategies: a cumulative approach to enteric methane abatement from ruminants

Mahfuzul Islam1, Sang-Suk Lee1,*
1Ruminant Nutrition and Anaerobe Laboratory, Department of Animal Science and Technology, Sunchon National University, Suncheon 57922, Korea
*Corresponding author: Sang-Suk Lee, Ruminant Nutrition and Anaerobe Laboratory, Department of Animal Science and Technology, Sunchon National University, Suncheon 57922, Korea. Tel: +82-61-750-3237, E-mail:

© Copyright 2019 Korean Society of Animal Science and Technology. This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Received: Mar 07, 2019 ; Revised: Apr 09, 2019 ; Accepted: May 13, 2019

Published Online: May 31, 2019


Methane, one of the important greenhouse gas, has a higher global warming potential than that of carbon dioxide. Agriculture, especially livestock, is considered as the biggest sector in producing anthropogenic methane. Among livestock, ruminants are the highest emitters of enteric methane. Methanogenesis, a continuous process in the rumen, carried out by archaea either with a hydrogenotrophic pathway that converts hydrogen and carbon dioxide to methane or with methylotrophic pathway, which the substrate for methanogenesis is methyl groups. For accurate estimation of methane from ruminants, three methods have been successfully used in various experiments under different environmental conditions such as respiration chamber, sulfur hexafluoride tracer technique, and the automated head-chamber or GreenFeed system. Methane production and emission from ruminants are increasing day by day with an increase of ruminants which help to meet up the nutrient demands of the increasing human population throughout the world. Several mitigation strategies have been taken separately for methane abatement from ruminant productions such as animal intervention, diet selection, dietary feed additives, probiotics, defaunation, supplementation of fats, oils, organic acids, plant secondary metabolites, etc. However, sustainable mitigation strategies are not established yet. A cumulative approach of accurate enteric methane measurement and existing mitigation strategies with more focusing on the biological reduction of methane emission by direct-fed microbials could be the sustainable methane mitigation approaches.

Keywords: Accurate methane estimation; Methane mitigating approach; Direct-fed microbials


Methane (CH4), one of the three main greenhouse gases (GHG) besides of carbon dioxide (CO2) and nitrous oxide (N2O), have a global warming potential of 28-fold than that of carbon dioxide (CO2) [1]. Agricultural sector is considered to contribute the biggest methane emission, which calculated around 50.6% from anthropogenic methane [2]. Within agriculture, the livestock sector contributes approximately 18% of the global anthropogenic GHG emission [3]. Among livestock, ruminant contributes about 81% of GHG [4] due to massive methanogenesis by rumen microbes, which produce 90% of total CH4 production from ruminants [5]. Globally, CH4 emissions of dairy and beef cattle denote 30% and 35% of the livestock sectors’ emissions. However, buffalos and small ruminants are lower contributors, demonstrating 8.7% and 6.7% of sector emissions, respectively [6]. The CH4 production in ruminants represents a gross energy loss from 2% to 14% of gross energy consumption [7]. Therefore, reduction of methane emission in animal conserves an energy and enhances productivity.

For the fulfilling of nutrition demand of growing population, the number of domesticated animals increasing rapidly. This high number of animals is directly proportional to CH4 production. In developed countries, it often recommends culling of nonproductive and low-producing animals to reduce CH4 budget [8]. They maintained high-producing animals in herds for reducing CH4 emissions per unit of product. Conversely, this is often impractical for poor countries due to their socioeconomic and religious background. It is well established that with the increasing animal productivity, CH4 emissions also decrease per unit of products. There are many options for enhancing the productivity of animals such as the proper formulation of diets, supplementation of protein and energy to low-quality forages, ionophores, bovine somatotrophin, and probiotics [9]. Lately, both the increasing of animal production as well as decreasing the methane emission by the animal especially ruminants are the main focus among researchers throughout the world. A number of CH4 abetment strategies from ruminants have been revised earlier [2,8,1026]. However, these strategies summarized more concreate and cumulative approaches to set up future research needs for sustainable methane mitigation strategies in ruminants focusing direct-feed microbials. As a part of cumulative approach, accurate estimation of methane production is also very important in order to make a suitable methane mitigation strategy. Therefore, this review also summarized the methods of enteric methane measurement and their applications.

Rumen microbiome and methanogenesis

The rumen microbiome including a wide variety of microorganisms, viz. bacteria, archaea, ciliated protozoa, fungi, and viruses, stay in a symbiotic relationship in a strict anaerobic condition within the rumen [27]. The protozoa can comprise up to 50% of the microbial biomass in rumen [28]. While, the fungi were estimated at around 8% of the total biomass [29] but may reach 20% in sheep [30]. The archaea include only 0.3%–4% [31], and the bacteria cover the remainder, characteristically the largest component of the rumen microbial biomass [26]. This rumen microbiome plays a significant role in feed fermentation within the rumen and produces different volatile fatty acids (VFAs), CO2 and H2. These VFAs are essential for energy metabolism and protein synthesis of the ruminant host [32]. Among the diverse rumen microbiomes, only a few of these have been successfully characterized earlier based on culture-techniques. Recently, the application of multi-omics techniques such as metagenomics by next-generation sequencing (NGS) or high-throughput sequencing [3337], metatranscriptomics [3840], metaproteomics [41,42], and metabolomics [4345] have been increased greatly [40].

Methanogenesis is a process of CH4 production in the rumen where H2 reduced the CO2 with the help of methanogenic archaea [46]. CH4 production is the main way for H2 clearance from fermentation [47]. There are two main pathways of methanogenesis in the rumen, carried out by archaea, are presented in Table 1. The hydrogenotrophic pathway converts H2 and CO2 into CH4 by the bacteria, protozoa, and fungi [5,23]. It is usually implicit that formate can be used by most abundant ruminal archaea that equivalent to H2+CO2, so formate is included in the hydrogenotrophic category [31,48]. Methylotrophic pathway is an another pathway of methanogenesis, which use methyl groups such as those present in methylamines and methanol as substrate [26,49,50]. The methanogens species have classified into 28 genera and 113 species, but it can be expected to occur many more in nature [15,31]. From rumen, only a few methanogens have been so far isolated based on culture techniques such as Methanobrevibacter ruminantium, Methanobrevibacter millerae, Methanobrevibacter olleyae, Methanobacterium formicicum, Methanobacterium bryantii, Methnaomicrobium mobile, Methanoculleus olentangyi, Methanobrevibacter smithii and Methanosarcina spp. [31]. Lately, multi-omics techniques are using to understand greenhouse gas emission from ruminant production [51].

Table 1. Methanogenesis pathways
Pathways Reactions References
Hydrogenotrophic pathway CO2 + 4H2 → CH4 + 2H2O [57,58,87,89]
Methylotrophic pathway CH3OH + H2 → CH4 + H2O
4CH3OH → 3CH4 + CO2 + 2H2O
CH3NH2 + H2 → CH4 + NH3

CO2, carbon dioxide; H2, hydrogen; CH4, methane; H2O, water; CH3OH, methanol; CH3NH2, methylamine.

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Enteric methane measurement and their applications

Accurate estimation of enteric methane production from animals is the key to take initiative for the setting up of mitigation strategies. Mitigation strategy often was unsuccessful due to wrong measurement of CH4 production. A number of enteric methane measuring techniques have been developed. However, respiration chamber (RC), sulfur hexafluoride (SF6) tracer technique, and the automated head-chamber system (AHCS) (GreenFeed; C-Lock Inc., Rapid City, South Dakota, USA) were used successfully and widely in various experiments focusing dairy or beef cattle in several environmental conditions [18,52]. All three methods have been effectively used in a large number of trials with dairy or beef cattle under a wide variety of environmental conditions. However, inconsistent results also observed while did comparative study among techniques [53]. The several enteric CH4 measurement approaches are presented in Fig. 1.

Fig. 1. Pictorial presentation of widely used enteric methane measurement techniques. (A) Respiration chambers (RC), (B) Sulfur hexafluoride (SF6) tracer technique, (C) Automated head-chamber system (AHCS), (D) Methane hood (MH) system [47,69,73].
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Exact measurements of CH4 emission can be gained by housing animals in RC, which then allow measurement of total methane emission directly. Reynolds et al. [54] and Cammell et al. [55] described the details of RC and measurements of methane emission. For the measurements of gaseous emissions, two open-circuit RC was used (internal volume approximately 21 m3), with airlocks permitting access for faecal and urine collection [55]. An integrative sample of ambient and RC exhaust air was analyzed at 4-min intervals, and there was a switch to calibration gases (oxygen-free nitrogen and nitrogen carrier with 20.5%, 3000 ppm, and 200 ppm oxygen, carbon dioxide, and methane, respectively) every 4 h to provide gas analyses with variation coefficients of 5% or less. This technique is relatively expensive, and are troublesome for the animal to behave normally like a natural that occurs within grassland environments.

The SF6 tracer technique [56,57] can be used to make estimations of methane emissions either by eructation or expiration from animals that can easily select their diet in a way characteristic of farmed livestock especially in grazing. The SF6, a gas is easily measurable and traceable at low concentrations. In addition, it is synthetic in origin and not produced as part of any sort of biological process. The SF6 is also idyllic as its background concentration, which is naturally very low (6 pmol/day) [58], while its concentration as a tracer typically ranges from 0.01 to 0.03 mmol/day [59]. In the gas technique, SF6 tracer gas is delivered via a permeation tube, which is positioned in the rumen, and the ratio of CH4 to SF6 in the breath of an animal is measured and corrected with reference to the background concentration. Though the concentration of the tracer is known, the rate of production of CH4 can be calculated [47]. The assessments have challenged the accuracy of the SF6 technique for estimating CH4 emissions [60,61], with greater between-animal variation compared to RC [62]. The SF6 technique has also delivered variable estimates of CH4 emission from animals on diverse herbages that have not been supported by RC measurements [6366]. There was also some problem with halter and collection canisters, placed on the animal for CH4 estimates can affect during grazing [67], especially in young animals, and a lower than predicted feed dry matter intake (DMI) will overestimate CH4 yields (g/kg DMI). Administration of rumen SF6 boluses and frequent handling of animal is needed which can be troublemaking to normal behavior and is relatively laborious. Though this technique is challenging to use in practice, standard operating techniques have been established [68].

In 2010, C-Lock Inc., Rapid City, South Dakota, USA has introduced the commercial GreenFeed (GF) system as a static short-term measurement device that measures CH4 emission from distinct cattle and uses head position sensors in combination with decision rules to assess the validity of measures obtained [52,69]. Depending on the experimental design, this GF system can be installed both conditions either grazing field or inside the farm. The animal remaining free to move about and voluntarily enters into the hood where a feed supplement is dropped. The measurements of CH4 emission by the GF system can be done typically over a short period (3–7 minutes), several times within a day, over several days. The GF system estimated CH4 emission using sensors that identified the animal and its head position within a sampling hood, air flow, and CH4 and CO2 concentrations in exhaust air. A radio frequency identification (RFID) reader recognized the animal’s ear tag and GF sampling was activated when the animal’s head was in the correct position within the hood. Satisfactory animal head position resulted in the dispensing of feed pellets which influenced the animal to maintain a suitable head position for accurate measurements. Only one cattle can visit one GF unit at a time point and a ‘visit’ is defined as a visit that results in a methane measurement. The system is automatically programmed using C-Lock Inc. software to control the timing of feed availability for each animal and thus, encourage animals to allocate their voluntary GF visitation, and measure CH4 emission over a 24 h period. Each GF unit can be used for several animals, with manufacturer recommendations of 15–20 animals/unit when grazing and 20–25 animals/unit if housed in free stalls. Cattle are voluntarily participating to visit the GF unit if they adapt once.

The face-masks method is one of the oldest technique for “spot-sampling” of respiratory exchange and CH4 emission from cattle, sheep, and goats. Face mask is only useful for short-term measurements of CH4 emission rate for screening of large numbers of animals, however may cause marked discomfort and distress which can change animal behaviors, and affect the gas measurements [70]. The sniffer method, first reported by Garnsworthy et al. [71], is the measurement of CH4 concentration in air eructed by cattle during milking. In this technique, air in the manger is continuously sampled, analyzed, and logged at 1-second intervals using data loggers in order to measure CH4 and CO2 concentrations in close proximity to the muzzle of the animal. Garnsworthy et al. [71] also reported a good relationship (r = 0.79) between RC technique and CH4 emission rate using this method. The hand laser CH4 detector (LMD) has been proposed to measures enteric CH4 concentrations in the air near the nose or mouth of an animal in normal environment [72]. The methane hood (MH) system, a novel method to quantify CH4 emission from cattle during group feeding in housed environment. This system measures CH4 concentrations exhausted from underneath a hood designed to partially enclose the volume above a feed bin. The principle is almost similar to GF system except that there is no requirement to offer extra feed supplements needed to influence the visit of cattle into GF system [73]. There are also some other indirect approaches have been suggested and so far used to measure enteric CH4 emissions from animal however, associated with lower accuracy and greater uncertainty in the emission data [18].

Methane mitigation approaches from ruminants

Methane mitigation from animal origin is a time demanding issue throughout the world. There are several possible targets and mitigation strategies (Fig. 2) have been taken so far but still lack in sustainability. By 2050, the total CH4 emission from ruminant animals is expected to increase significantly due to the increasing demand for milk and meat of animal origin for a hurriedly growing world population [74]. So, it is highly needed to mitigate CH4 emission from the livestock industry. Here, we summarized important methane mitigation approaches in ruminant productions.

Fig. 2. A schematic presentation of the possible targets to reduce CH4 emissions from ruminants. The boxes without dark shade could be the targets for CH4 mitigation and boxes with dark shade are the options that have been studied either in vitro or in vivo to reduce CH4 production [52,109,116,123,126,131].
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Maintaining low methane emitters

Methane production is not consistent for all animal types and breeds [13]. Olijhoek et al. [75] reported that methane production per kilogram of dry matter intake (DMI) was lower in Holsteins in comparison to Jerseys (30.7 vs. 32.6 L/kg of DMI in case of High RFI and low concentrate group, 21.4 vs. 28.2 in High RFI and high concentrate group, 32.4 vs. 32.5 in Low RFI and high concentrate group and 24.5 vs. 27.9 in Low RFI and low concentrate group). It was also reported that CH4 production from different animals under the same feeding trial reveals significant variation among animals [8]. Pinares-Patino et al. [76] conducted an experiment on grazing sheep where some animals show as high and low CH4 emitters on the basis of CH4 output per unit of feed intake [8]. Some other researches have established that ruminants with low residual feed intake (RFI) emit less CH4 than the animals with high RFI [77]. Likewise, Hegarty et al. [78] stated that CH4 production was lower in low RFI Angus steers than in steers having high RFI (142 vs. 192g CH4/day). There was a positive genetic correlation between RFI and predicted methane emission (PME; g/d) which indicated that the cows having lower RFI have lower PME (estimates ranging from 0.18 to 0.84) [79]. Though the cause is not clear, it might be due to differences in methanogen populations among animals [80]. It is proposing that rumen microbial community varies among animals or breeds depending on individual genetic variations that greatly influence CH4 production [42]. Therefore, the selection of low methane producer might be a possible and sustainable way to mitigate methane emission.

A good combination of diet reduces methane production

Methane production in ruminants is influenced by the composition of feed. Digestion in the rumen is dependent on the activity of microorganisms, which need energy, nitrogen and minerals [81]. Subsequently, the quality of forage affects the activity of rumen microbes and CH4 production in the rumen. The species, processing, and proportion of forages, and the grain sources of diets also influence CH4 production in ruminants. Methane production tends to decrease as the protein content of feed increases, and increases as the fiber content of feed increases [7,82,83].

High-quality forage, especially young plants, can mitigate CH4 production by shifting the fermentation pathway as this forage contains lower amounts of NDF and higher easily fermentable carbohydrates, leading to a higher digestibility and passage rate [84]. On the other hand, more mature forage encourages a higher CH4 yield mainly owing to an increased C: N ratio, which subsequently decreases the digestibility in ruminants [85]. The CH4 emission can also vary depending on types of forage due to the variation of their chemical composition [86]. Methane production also significantly affected by the processing and preservation of forages [23] such as, chopping or pelleting of forages require less degradation in the rumen due to their smaller particles size as a consequent reduction of CH4 emission per kg of DMI [87]. Likewise, ensiling of forages, partially fermented, can reduce CH4 emission from ruminants [87].

For increasing the production especially in the high producing dairy cattle higher energy supplementation is needed. Only forage is not sufficient to provide the nutrient for these high yielding cattle. So, concentrates must be supplemented with forages with a higher density of nutrients and less fiber. These concentrates contain fewer cell walls and readily fermentable carbohydrates (starch and sugar) and contribute to the production of propionic acid however reducing CH4 production [23]. It was observed in one study that 80% and 90% concentrate supplementation decreased CH4 production, while no noticeable effect was found at 35% or 60% concentrate supplementation [88]. Another study showed that a diet containing 90% concentrate produced extremely low CH4 which represented a loss of only 2%–3% of the gross energy intake [7]. Conversely, a diet with high-concentrate contains low in structural fiber and influence to develop sub-acute or acute acidosis. Therefore, dietary manipulation such as feeding a good combination of F:C ration would be effective with respect to mitigating methane emission without hampering their productivity.

Supplementation of fats

The hydrogen and carbon dioxide or formate are the major substrates for methanogens [89]. And, some microorganisms in the rumen use hydrogen to hydrogenate the double bonds of unsaturated fatty acids. Hence, CH4 production is hampered due to the addition of unsaturated fatty acids to the diet [90,91]. Traditionally addition of fat in the diet has been used in order to increase dietary energy content to meet the energy demand of high-producing dairy cows. Very recent, energy supplementation in a ruminant’s diet is changed from carbohydrate to fat, which contributes to CH4 mitigation. The mechanism of CH4 reduction by fat is due to decreasing organic matter fermentation, fiber digestibility, and thus the methanogenic pathway as well as by the direct inhibition of methanogens in the rumen via the hydrogenation of unsaturated fatty acids [7]. Several other studies also revealed that dietary fat supplementation has potential effects on the reduction of CH4 production from ruminants [92,93]. The unsaturated fatty acids act as an H2 sink within the rumen through dehydrogenation and reduction occurs with the highest rate [87]. It was also reported that supplementation of fat often reduces carbohydrate fermentation due to the toxic effects of fat on cellulolytic bacteria and protozoa, whereas starch fermentation was not affected [92]. Therefore, fat reduces CH4 emission from ruminants.

Supplementation of ionophores, mineral mixtures, and organic acids

Ionophores are antimicrobial compounds, for example, monensin, lasalocid and salinomycin, are typically used in beef and dairy cattle production to improve feed efficiency and animal performance [91,94]. They reduce CH4 emission from ruminants significantly. It was reported that monensin supplementation for lactating cows reduced CH4 production [95]. For instance, a 25.6% reduction in CH4 production was recorded with supplementation of monensin to Brahman steers without a reduction in daily gain [96]. Monensin upsurges the acetate and propionate ratio in rumen fermentation through the increasing reducing equivalents which contribute propionate formation in ruminant [10]. Other studies showed that a high dose of monensin reduces CH4 production (g/d) by 4%–10% in dairy and beef cattle [97,98]. Likewise, another report revealed a 30% reduction of CH4 emission in beef cattle fed monensin at 33 mg/kg [99]. Ionophores also hampers survival of protozoa as a consequence the reduction and subsequent recovery of protozoal numbers in the rumen help to CH4 decline up to 30% [99]. However, the inhibitory effects of ionophores on CH4 production may not persist over time, and several microbes already adapted to ionophores [7,10].

Mineral mixture also has effects on enteric methane mitigation. For instance, dietary supplementation of illite feed additive, claysized mineral mixture that contains Mg, Ca, K, Mn, Zn, P, Fe, Al, Si, Co, Se and Mo, at 1% on dry matter (DM) basis has a positive effect on CH4 reduction with increasing VFA production in Han-woo steers [100].

The organic acids such as fumarate and malate, and propionate precursor or substances, are the potential feed additives that mitigate CH4 emission from ruminants when supplemented with feed [97,101103]. They are supposed to stimulate increased production of propionic acid in the rumen by acting as an H2 sink, in this manner reducing CH4 production [104]. Some other study also reported that organic acids mitigate methane production by up to 17% [103]. The in-vitro study showed that fumarate reduces the CH4 output by 38% in continuous fermenters using forage as a substrate [105]. Conversely, an in-vivo study with growing beef cattle reported CH4 reduction was unaffected by fumarate [106].

Direct-fed microbials: a biological CH4 mitigating agent

Modification of rumen ecosystem through direct-fed microbials (DFM) or probiotics, is one of the most possible approach to reduce methane production in rumen. Probiotics such as bacterial species including Bacillus, Bifidobacterium, Enterococcus, Lactobacillus, Propionibacterium, Megasphaera elsdeniiand Prevotella bryantii and yeast (Saccharomyces cerevisiae), are used to improve rumen fermentation and feed efficiency [107,108] which could also reduce CH4 emissions from ruminants. Several pieces of research so far have been conducted earlier in order to mitigate CH4 emissions from ruminant with the supplementation of dietary probiotics. Jeyanathan et al. [109] summarized the several rumen biochemical pathways that could be modulated by direct feed microbials to reduce CH4 production from ruminants (Fig. 2). The production of VFAs such as acetic, propionic and butyric acids are mainly depends on the diet offered to the animal. Ruminants produce comparatively more propionate, fed a concentrate-based diet than those fed a high forage diet, which produces more acetate.

Propionate formation, also considered as H2-utilisation pathway, consumes reducing equivalents, pyruvate which is reduced to propionate [110]. Though these H2 are the key precursor for the production of CH4, the increase in propionate formation is proportionally linked with decrease CH4 production. The succinate pathway is the major pathway of propionate production in the rumen where malate, fumarate, and succinate are formed as intermediate products. In this pathway, a mixture of bacteria such as fumarate reducers (e.g. Wolinella succinogenes), succinate producers (Fibrobacter succinogenes), and succinate utilizers (e.g. Selenomonas ruminantium) also involve. The acrylate pathway is an another important propionate producing pathway in the rumen by lactate-utilizing bacteria (Megasphaera elsdenii) [111]. The lactate is required in this pathway, therefore lactate-producing bacteria such as Streptococcus bovis play a regulatory role. There are several studies have been conducted to enhance propionate production in the rumen targeting increase animal productivity through dietary probiotics [112114] which are indirectly linked with methane mitigation approaches. The decrease in CH4 production was recorded in lactating dairy cows consumed a mixed Propionibacterium jenseniiLactobacillus spp. direct feed microbials [115]. Mamuad et al. [116] reported that fumarate reducing bacteria alters the rumen microbiome and helping ruminal fermentation, and reducing CH4 production in-vitro. Recently, CH4 production was also reduced through supplementation of fumarate reductase-producing enterococci (Enterococcus faecium SROD) with the increasing of total VFAs in an in-vitro experiment [117].

Nitrate, an alternative H2 sink to CO2 in the rumen, decreases rumen methanogenesis and reduce the toxicity of the intermediate product nitrite during their metabolism. Rumen microbes hastily reduce the nitrate to nitrite, however the reduction rate of nitrite to ammonia is slower. This can cause nitrite accumulation in the rumen [118] and methemoglobinemia in blood. Methemoglobinemia declines the blood’s capacity to transport oxygen to tissues, resulting in low performances or even death of animal in severe cases [119]. The use of nitrate and/or nitrite-reducing bacteria as probiotic is one of the potential solution to avoid this toxicity [120]. The bacteria having the ability to reduce nitrate, nitrite or both compounds are already existing in the rumen, but their concentration is lower than that of their counterpart methanogens [109,121,122]. The main nitrate-reducing bacteria in the rumen are W. succinogenes and S. ruminantium [121,122]. So, it may be beneficial to increase the number and/or the activity of nitrate- and/or nitrite reducing bacteria in the rumen to decrease methanogenesis. Addition of nitrate in the diet increased the number of nitrate reducing bacteria (W. succinogenes and Veillonella parvula) in-vitro [123] but this is may be insufficient to compete with methanogenesis. Thus, giving nitrate and/or nitrite-reducing bacteria as probiotics along with nitrate may progress the nitrate reduction process and subsequently avoid nitrite toxicity. Along with nitrate, Denitrobacterium detoxificans strain NPOH1 decreased 95% [120], and W. succinogenes, S. ruminantium or V. parvula reduced >70% [123] of CH4 production in in-vitro trial. Conversely, there is a lacking on in-vivo data regarding this issue.

The ability of sulphate-reducing bacteria (SRB) to compete with methanogens is largely determined by the introduction of sulphate into the rumen. In anaerobic environments, where sulphate is unlimited, SRB compete with methanogens for common substrates such as H2, formate and acetate. The population of SRB in the rumen is low (105 to 106 cells/mL) and largely from the genus Desulfovibrio and Desulfotomaculum [124,125]. Recently, another SRB belonging to the genus Fusobacterium was isolated from buffalo [126]. Only few studies were conducted on effect of sulphate supplementation alone in rumen methanogenesis [127,128] due to their toxic end product hydrogen sulfide (H2S). Therefore, the sulphate reduction, owing to decrease methanogenesis, may be facilitated by SRB only when sulphate is added as a feed additive. For example, a reduction in CH4 production was recorded in an in-vitro experiment using Fusobacterium sp., as a probiotic with a high sulphate diet where CH4 production at 72h was reduced from 2.66 to 1.64 mmol/g digested dry matter (DM) without H2S accumulation [126].

The reductive acetogenesis is an accepted mechanism of H2 utilization that coexists with methanogenesis in the rumen [129,130]. Acetate, the end product of this reaction, has the additional advantage of being a source of energy for the animal. However, acetogens are less numerous in the rumen environment and less efficient than methanogens in respect of competing for reducing equivalents. Therefore, it is needed to increase the number of acetogenic bacteria in the rumen, which compete with methanogens for hydrogen, as a result of CH4 reduction. Kim et al. [131] and Martin et al. [23] stated that CH4 production was reduced with the dietary supplementation of acetogen probiotics in ruminants.

It is strongly suggested that yeast probiotics possibly stimulates the acetogenic bacteria to compete with methanogens or to co-metabolize H2 as a consequence reducing CH4 formation [81,132]. A 20% reduction in CH4 production was recorded after 48 h of incubation of mixed rumen microorganisms containing alfalfa and a live yeast product [133]. Aspergillus oryzae reduced CH4 production by the reduction of protozoal population (45%) [134]. Therefore, still, there is a big scope to search the more suitable probiotic candidates for the sustainable CH4 mitigation strategy.

Supplementation of botanical extract or plants secondary metabolites

Botanical extract or plants secondary metabolites (PSM), viz. saponins, tannins, flavonoids, organosulphur compounds, and essential oils, have potential anti-microbial effects against several types of microorganisms [135]. Several PSMs have been so far identified as a potential agent to reduce CH4 production by methanogens in the rumen [136,137]. Depending on the type, sources, molecular weight, doses, and diet types, the methane reductive capability of PSMs varies significantly. For instance, Joch et al. [138] examined in-vitro methane abetment properties of nine (09) concentrations of α-terpineol, 8-hydroxyquinoline, bornyl acetate, camphor, thymoquinone, α-pinene, and thymol. They reported all compounds tested validated as CH4 reducing agent however effective concentrations varied among individual PSMs. Recently, some see weeds also have potential effects on methane abatement [139] however, need more researches in this regard.

Saponins (triterpenoid saponin, tea saponin, methanol extract saponin) are the potential additives which cause a significant reduction of protozoa population in the rumen, as a result, methane production is reduced [140142]. Saponins also contribute rumen fermentation, enhance rumen bacterial populations, and ruminant productivity [50,136,137]. Saponins from different sources show various results. Application of Quillaja saponin at 1.2 g/L decreased CH4 production in-vitro, but not at 0.6 g/L [143]. The ivy fruit saponin decreased CH4 production by 40% [144] and saponins from Saponaria officinalis reduced CH4 and abundance of both methanogens and protozoa in-vitro [145]. However, the opposite result was found in other in-vitro studies, where Quillaja saponins at 0.6 g/L did not reduce CH4 production or abundance of methanogen [146,147]. Similarly, Tea saponins (30 g/day) also did not reduce CH4 emission from steers or methanogens abundance [148]. Though, the effects of saponins on methanogenesis and methanogen abundance are greatly inconstant among different studies, still needed to study more.

Tannins (condensed and hydrolysable tannin) also have the potentials to reduce CH4 production from ruminants. Tannins exert their effects by directly inhibiting methanogens as well as indirectly decreasing H2 production as a consequence decreased fiber digestion and the number of protozoa in the rumen [149]. Tannins extracted from Lotus pedunculatus showed inhibitory effects on pure cultures of methanogens [150]. Likewise, inhibition of methanogens in the rumen of goats supplementing tannins as feed additives was reported by Puchala et al. [151]. However, the inconsistent result was found in the case of condensed tannins [152]. Recently, forages with higher levels of tannins, such as clover and other legumes, including trefoil, vetch, sulla and chicory are considering as mitigating agent [153]. For instance, CH4 production was reduced (up to 55%) while ruminants were fed tannin-rich forages [154]. Tannins may exert a similar mechanism like bactericidal or bacteriostatic and inhibit the growth or activity of rumen methanogens and protozoa [155].

Essential oils, another plant secondary metabolites, are volatile components [153] and aromatic lipophilic compounds [156]. It contains the chemical constituents and functional groups such as terpenoids, phenolic and phenols, having potential antimicrobial activities [157], which inhibit the growth and existence of greatest number of microorganisms in the rumen [158]. Due to their lipophilic nature, they have a very high affinity to microbial cell membranes, and at the same time, their functional groups interact with the microbial cell membrane [159]. With the application of essential oil, the methanogenesis decreases especially by reducing microbial populations [160]. The Allium arenarium oil (garlic oil), a highly promising essential oil, was significantly reduced methane production both in-vivo and in-vitro by 12% and 36%, respectively [161].

Supplementation of enzyme additives

Enzyme feed additives having fibrolytic activities are used to enhancing fibre digestibility [162], feed conversion efficiency [163], and milk production [163165] of dairy cows. A reduction in the enteric CH4 production was reported by Arriola et al. [163] where a fibrolytic enzyme additive was supplemented with a lactating cows’ diet (52% dietary forage). Conversely, some other studies revealed that exogenous fibrolytic enzyme additive increased CH4 yield and altered rumen methanogen community composition, without affecting overall density of methanogens [166,167]. Recently, Biswas et al. reported that dietary supplementation of lysozyme enzyme may improve rumen fermentation and reduce CH4 emission in an in-vitro trial [168]. Therefore, more in-vivo study is needed before use of enzymes for methane mitigation strategy.

Defaunation: lowering available H2 for methanogenesis

Defaunation, the removal of protozoa from the rumen, is often linked with an increased microbial protein supply and enhancement of animal productivity. Though protozoa generate a relatively large volume of H2 and formate, and the methanogenic bacteria attach to the surface of ciliated protozoa [169], defaunation is effective to decline CH4 emission. Morgavi et al. [170] revealed that CH4 emission reduced by 20% over a period of 2 years in defaunated sheep. Due to unclear reason, partial defaunation is not effectively reduce CH4 production [171,172]. So far, a variety of techniques have been tested for defaunation experimentally, but none is used routinely due to its’ toxicity problems to the rest of the rumen microbiome as well as the host animals [9]. It has been also reported that immunization or vaccination of sheep with entodinial or mixed protozoal antigens reduced protozoal populations, and produced Immunoglobulin G (IgG) against rumen protozoa [173]. Recently, plant secondary metabolites have been used as potential defaunating agents. Moreover, defaunation technology needs more valuation before use widely and still has a big scope to do more research.


Ruminant production is increasing rapidly for providing good quality meat, milk, and their products to the large population in the earth. Methane production is also rising proportionally that contribute negatively to global warming as a greenhouse gas. Therefore, we should give more concentration on sustainable methane mitigation strategy which could be achievable through a cumulative approach. There are several methane mitigation strategies such as animal intervention, diet selection, dietary feed additives, probiotics, defaunation, supplementation of fats, oils, organic acids, plant secondary metabolites, etc. however, sustainable mitigation strategies are not established yet. We should give more emphasis on biological regulation of methane mitigation through searching of suitable candidates of direct feed microbials. Accurate measurement of methane is also highly needed to make the mitigation approach successful.

Competing interests

No potential conflict of interest relevant to this article was reported.

Funding sources

This research was supported by the Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ013448012019), Rural Development Administration, Korea and supported by Sunchon National University Research Fund in 2015.


Not applicable.

Availability of data and material

Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.

Authors’ contributions

Conceptualization: Islam M, Lee SS.

Writing - original draft: Islam M, Lee SS.

Writing - review & editing: Islam M, Lee SS.

Ethics approval and consent to participate

Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.



Pachauri RK, Allen MR, Barros VR, Broome J, Cramer W, Christ R, et al. Climate change 2014: synthesis report. Contribution of Working Groups I, II and III to the fifth assessment report of the Intergovernmental Panel on Climate Change. Geneva, Switzerland: IPCC. 2014


Karakurt I, Aydin G, Aydiner K. Sources and mitigation of methane emissions by sectors: a critical review. Ren Energy. 2012; 39:40-8.


Steinfeld H, Gerber P, Wassenaar T, Castel V, Rosales M, de Haan C. Livestock’s long shadow: Environmental issues and options. Food and Agriculture Organization of the United Nations. Rome, Italy: 2006


Hristov AN, Oh J, Lee C, Meinen R, Montes F, Ott F, et al. Mitigation of greenhouse gas emissions in livestock production.In In: Gerber PJ, Henderson B, Makkar HPS, editors.editors A review of options for non-CO2 emissions. Rome: FAO. 2013; p. 226


McAllister TA, Meale SJ, Valle E, Guan LL, Zhou M, Kelly WJ, et al. Ruminant nutrition symposium: use of genomics and transcriptomics to identify strategies to lower ruminal methanogenesis. J Anim Sci. 2015; 93:1431-49.


Opio C, Gerber P, Mottet A, Falcucci A, Tempio G, MacLeod M, et al. Greenhouse gas emissions from ruminant supply chains - a global life cycle assessment. Rome, Italy: FAO. 2013


Johnson KA, Johnson DE. Methane emissions from cattle. J Anim Sci. 1995; 73:2483-92.


Patra AK. Enteric methane mitigation technologies for ruminant livestock: a synthesis of current research and future directions. Environ Monit Assess. 2012; 184:1929-52.


Moss AR, Jouany JP, Newbold CJ. Methane production by ruminants: its contribution to global warming. Ann Zootech. 2000; 49:231-53.


Beauchemin KA, Kreuzer M, O’Mara F, McAllister TA. Nutritional management for enteric methane abatement: a review. Aust J Exp Agric. 2008; 48:21-7.


Bodas R, Prieto N, Garcia-Gonzalez R, Andres S, Giraldez FJ, Lopez S. Manipulation of rumen fermentation and methane production with plant secondary metabolites. Anim Feed Sci Technol. 2012; 176:78-93.


Cottle DJ, Nolan JV, Wiedemann SG. Ruminant enteric methane mitigation: a review. Anim Prod Sci. 2011; 51:491-514.


De Mulder T, Peiren N, Vandaele L, Ruttink T, De Campeneere S, Van de Wiele T, et al. Impact of breed on the rumen microbial community composition and methane emission of Holstein Friesian and Belgian Blue heifers. Livest Sci. 2018; 207:38-44.


Eckard RJ, Grainger C, de Klein CAM. Options for the abatement of methane and nitrous oxide from ruminant production: a review. Livest Sci. 2010; 130:47-56.


Giuburunca M, Criste A, Cocan D, Constantinescu R, Raducu C, Miresan V. Methane production in the rumen and its influence on global warming. ProEnviron. 2014; 7:64-70


Goel G, Makkar HPS. Methane mitigation from ruminants using tannins and saponins. Trop. Anim health Prod. 2012; 44:729-39.


Haque MN. Dietary manipulation: a sustainable way to mitigate methane emissions from ruminants. J Anim Sci Technol. 2018; 60:15.


Hristov AN, Kebreab E, Niu M, Oh J, Bannink A, Bayat AR, et al. Symposium review: uncertainties in enteric methane inventories, measurement techniques, and prediction models. J Dairy Sci. 2018; 101:6655-74.


Huhtanen P, Cabezas-Garcia EH, Utsumi S, Zimmerman S. Comparison of methods to determine methane emissions from dairy cows in farm conditions. J Dairy Sci. 2015; 98:3394-409.


Knapp JR, Laur GL, Vadas PA, Weiss WP, Tricarico JM. Invited review: Enteric methane in dairy cattle production: Quantifying the opportunities and impact of reducing emissions. J Dairy Sci. 2014; 97:3231-61.


Kumar S, Choudhury PK, Carro MD, Griffith GW, Dagar SS, Puniya M, et al. New aspects and strategies for methane mitigation from ruminants. Appl Microbiol Biotechnol. 2014; 98:31-44.


Leahy SC, Kelly WJ, Ronimus RS, Wedlock N, Altermann E, Attwood GT. Genome sequencing of rumen bacteria and archaea and its application to methane mitigation strategies. Animal. 2013; 7:235-43.


Martin C, Morgavi DP, Doreau M. Methane mitigation in ruminants: from microbe to the farm scale. Animal. 2010; 4:351-65.


Negussie E, de Haas Y, Dehareng F, Dewhurst R J, Dijkstra J, Gengler N, et al. Invited review: Large-scale indirect measurements for enteric methane emissions in dairy cattle: A review of proxies and their potential for use in management and breeding decisions. J Dairy Sci. 2017; 100:2433-53.


Patra A, Park T, Kim M, Yu Z. Rumen methanogens and mitigation of methane emission by anti-methanogenic compounds and substances. J Anim Sci Biotechnol. 2017; 8:13.


Tapio I, Snelling TJ, Strozzi F, Wallace RJ. The ruminal microbiome associated with methane emissions from ruminant livestock. J Anim Sci Biotechnol. 2017; 8:7.


Islam M, Lee SS. Recent application technologies of rumen microbiome is the key to enhance feed fermentation. J Life Sci. 2018; 28:1244-53


Newbold CJ, de la Fuente G, Belanche A, Ramos-Morales E, McEwan NR. The role of ciliate protozoa in the rumen. Front Microbiol. 2015; 6:1313.


Orpin CG. Fungi in ruminant degradation.In Agricultural science seminar: degradation of plant cell wall material. London: Agricultural Research Council. 1981; p. 129-50


Rezaeian M, Beakes GW, Parker DS. Distribution and estimation of anaerobic zoosporic fungi along the digestive tracts of sheep. Mycol Res. 2004; 108:1227-33.


Janssen PH, Kirs M. Structure of the archaeal community of the rumen. Appl Environ Microbiol. 2008; 74:3619-25.


Hungate RE. Hydrogen as an intermediate in the rumen fermentation. Archiv Mikrobiol. 1967; 59:158-64.


Berg MME, Yeoman CJ, Chia N, Tringe SG, Angly FE, Edwards RA, et al. Phage-bacteria relationships and CRISPR elements revealed by a metagenomic survey of the rumen microbiome. Environ Microbiol. 201; 14:207-27.


Fouts DE, Szpakowski S, Purushe J, Torralba M, Waterman RC, MacNeil MD, et al. Next generation sequencing to define prokaryotic and fungal diversity in the bovine rumen. PLOS ONE. 2012; 7:e48289.


Gharechahi J, Salekdeh GH. A metagenomic analysis of the camel rumen’s microbiome identifies the major microbes responsible for lignocelluloses degradation and fermentation. Biotechnol Biofuels. 2018; 11:216.


Kittelmann S, Seedorf H, Walters WA, Clemente JC, Knight R, Gordon JI, et al. Simultaneous amplicon sequencing to explore co-occurrence patterns of bacterial, archaeal and eukaryotic microorganisms in rumen microbial communities. PLOS ONE. 2013; 8:e47879.


Zened A, Combes S, Cauquil L, Mariette J, Klopp C, Bouchez O, et al. Microbial ecology of the rumen evaluated by 454 GS FLX pyrosequencing is affected by starch and oil supplementation of diets. FEMS Microbiol Ecol. 2013; 83:504-14.


Chen YB, Lan DL, Tangi C, Yang XN, Li J. Effect of DNA extraction methods on the apparent structure of yak rumen microbial communities as revealed by 16S rDNA sequencing. Pol J Microbiol. 2015; 64:29-36


Iqbal MW, Zhang Q, Yang Y, Zou C, Li L, Liang X, et al. Ruminal fermentation and microbial community differently influenced by four typical subtropical forages in vitro. Anim Nutr. 2018; 4:100-8.


Jami E, White BA, Mizrahi I. Potential role of the bovine rumen microbiome in modulating milk composition and feed efficiency. PLOS ONE. 2014; 9:e85423.


Comtet-Marre S, Parisot N, Lepercq P, Chaucheyras-Durand F, Mosoni P, Peyretaillade E, et al. Metatranscriptomics reveals the active bacterial and eukaryotic fibrolytic communities in the rumen of dairy cow fed a mixed diet. Front Microbiol. 2017; 8:67.


Deusch S, Camarinha-Silva A, Conrad J, Beifuss U, Rodehutscord M, Seifert J. A structural and functional elucidation of the rumen microbiome influenced by various diets and microenvironments. Front Microbiol. 2017; 8:1605.


Fuguang X, Xuemei N, Xiaohua P, Shanshan Z, Linshu J, Xiong B. Application of multi omics technologies in ruminants research. Dairy Vet Sci J. 2017; 1:555-563


Saleem F, Ametaj BN, Bouatra S, Mandal R, Zebeli Q, Dunn SM, et al. A metabolomics approach to uncover the effects of grain diets on rumen health in dairy cows. J Dairy Sci. 2012; 95:6606-23.


Saleem F, Bouatra S, Guo AC, Psychogios N, Mandal R, Dunn SM, et al. The bovine ruminal fluid metabolome. Metabolomics. 2013; 9:360-78.


Ellis JL, Dijkstra J, Kebreab E, Bannink A, Odongo NE, McBride BW, et al. Aspects of rumen microbiology central to mechanistic modelling of methane production in cattle. J Agric Sci. 2008; 146:213-33.


Hill J, McSweeney C, Wright ADG, Bishop-Hurley G, Kalantar-zadeh K. Measuring methane production from ruminants. Trends Biotechnol. 2016; 34:26-35.


Janssen PH. Influence of hydrogen on rumen methane formation and fermentation balances through microbial growth kinetics and fermentation thermodynamics. Anim Feed Sci Technol. 2010; 160:1-22.


Neill AR, Grime DW, Dawson RMC. Conversion of choline methyl groups through trimethylamine to methane in the rumen. Biochem J. 1978; 170:529-35.


Poulsen M, Schwab C, Jensen BB, Engberg RM, Spang A, Canibe N, et al. Methylotrophic methanogenic Thermoplasmata implicated in reduced methane emissions from bovine rumen. Nat Commun. 2013; 4:1428.


Wallace RJ, Snelling TJ, McCartney CA, Tapio I, Strozzi F. Application of meta-omics techniques to understand greenhouse gas emissions originating from ruminal metabolism. Genet Sel Evol. 2017; 49:9.


Patra AK. Recent advances in measurement and dietary mitigation of enteric methane emissions in ruminants. Front Vet Sci. 2016; 3:39.


Hammond KJ, Humphries DJ, Crompton LA, Green C, Reynolds CK. Methane emissions from cattle: estimates from short-term measurements using a GreenFeed system compared with measurements obtained using respiration chambers or sulphur hexafluoride tracer. Anim Feed Sci Technol. 2015; 203:41-52.


Reynolds CK, Cammell SB, Humphries DJ, Beever DE, Sutton JD, Newbold JR. Effects of postrumen starch infusion on milk production and energy metabolism in dairy cows. J Dairy Sci. 2001; 84:2250-9.


Cammell SB, Thomson DJ, Beever DE, Haines MJ, Dhanoa MS, Spooner MC. The efficiency of energy utilization in growing cattle consuming fresh perennial ryegrass (Lolium perenne cv. Melle) or white clover (Trifolium repens cv. Blanca). Br J Nutr. 1986; 55:669-80.


Johnson KA, Huyler M, Westberg H, Lamb B, Zimmerman P. Measurement of methane emissions from ruminant livestock using a SF6 tracer technique. Environ Sci Technol. 1994; 28:359-62.


Zimmerman PR. System for measuring metabolic gas emissions from animals. United States Patent US 5,265,618. 30 Nov; 1993


Rigby ML, Muhle J, Miller BR, Prinn RG, Krummel PB, Steele P, et al. History of atmospheric SF6 emissions from 1973 to 2008. Atmos Chem Phys. 2010; 10:10305-20.


Lassey KR, Pinares-Patino CS, Martin RJ, Molano G, McMillan AMS. Enteric methane emission rates determined by the SF6 tracer technique: Temporal patterns and averaging periods. Anim Feed Sci Technol. 2011; 166-167:183-91.


Pinares-Patino CS, Lassey KR, Martin RJ, Molano G, Fernandez M, MacLean S, et al. Assessment of the sulphur hexafluoride (SF6) tracer technique using respiration chambers for estimation of methane emissions from sheep. Anim Feed Sci Technol. 2011; 166:201-9.


Vlaming JB, Brookes IM, Hoskin SO, Pinares-Patino CS, Clark H. The possible influence of intra-ruminal sulphur hexafluoride release rates on calculated methane emissions from cattle. Can J Anim Sci. 2007; 87:269-75.


Hammond KJ, Muetzel S, Waghorn GC, Pinares-Patino CS, Burke JL, Hoskin SO. The variation in methane emissions from sheep and cattle is not explained by the chemical composition of ryegrass. Proc N Z Soc. Anim Prod. 2009; 69:174-8


Hammond KJ, Hoskin SO, Burke JL, Waghorn GC, Koolaard JP, Muetzel S. Effects of feeding fresh white clover (Trifolium repens) or perennial ryegrass (Lolium perenne) on enteric methane emissions from sheep. Anim Feed Sci Technol. 2011; 166-167:398-404.


Sun XZ, Hoskin SO, Muetzel S, Molano G, Clark H. Effect of forage chicory (Cichorium intybus) and perennial ryegrass (Lolium perenne) on methane emissions in vitro and from sheep. Anim Feed Sci Technol. 2011; 166:391-7.


Sun XZ, Hoskin SO, Zhang GG, Molano G, Muetzel S, Pinares-Patino CS, et al. Sheep fed forage chicory (Cichorium intybus) or perennial ryegrass (Lolium perenne) have similar methane emissions. Anim Feed Sci Technol. 2012; 172:217-25.


Waghorn GC, Tavendale MH, Woodfield DR. Methanogenesis from forages fed to sheep. Proc. N. Z. Grassi Assoc. 2002; 64:167-71


Pinares-Patino CS, Machmuller A, Molano G, Smith A, Vlaming JB, Clark H. The SF6 tracer technique for measurements of methane emission from cattle - effect of tracer permeation rate. Can J Anim Sci. 2008; 88:309-20.


Laubach J, Grover SP, Pinares-Patino CS, Molano G. A micrometeorological technique for detecting small differences in methane emissions from two groups of cattle. Atmospheric Environ. 2014; 98:599-606.


Hristov AN, Oh J, Giallongo F, Frederick T, Weeks H, Zimmerman PR, et al. The use of an automated system (Green-Feed) to monitor enteric methane and carbon dioxide emissions from ruminant animals. J Vis Exp. 2015; 103:e52904.


Washburn LE, Brody S. Growth and development XLII. Methane, hydrogen, and carbon dioxide production in the digestive tract of ruminants in relation to the respiratory exchange.In In: Mumford FB, editor.editors Growth and development. Colombia, MO: University of Missouri. 1937


Garnsworthy PC, Craigon J, Hernandez-Medrano JH, Saunders N. On-farm methane measurements during milking correlate with total methane production by individual dairy cows. J Dairy Sci. 2012; 95:3166-80.


Chagunda MG. Opportunities and challenges in the use of the Laser Methane Detector to monitor enteric methane emissions from ruminants. Animal. 2013; 7:394-400.


Troy SM, Duthie CA, Ross DW, Hyslop JJ, Roehe R, Waterhouse A, et al. A comparison of methane emissions from beef cattle measured using methane hoods with those measured using respiration chambers. Anim Feed Sci Technol. 2016; 211:227-40.


Gerber PJ, Steinfeld H, Henderson B, Mottet A, Opio C, Dijkman J, et al. Tackling climate change through livestock- a global assessment of emissions and mitigation opportunities. Rome, Italy: FAO. 2013


Olijhoek DW, Lovendahl P, Lassen J, Hellwing ALF, Hoglund JK, Weisbjerg MR, et al. Methane production, rumen fermentation, and diet digestibility of Holstein and Jersey dairy cows being divergent in residual feed intake and fed at 2 forage-to-concentrate ratios. J Dairy Sci. 2018; 101:9926-40.


Pinares-Patino CS, Ulyatt MJ, Lassey KR, Barry TN, Holmes CW. Persistence of differences between sheep in methane emission under generous grazing conditions. J Agric Sci. 2003; 140:227-33.


Alford AR, Hegarty RS, Parnell PF, Cacho OJ, Herd RM, Griffith GR. The impact of breeding to reduce residual feed intake on enteric methane emission from the Australian beef industry. Aust J Exp Agric. 2006; 46:813-20.


Hegarty RS, Goopy JP, Herd RM, McCorkell B. Cattle selected for lower residual feed intake have reduced daily methane production. J Anim Sci. 2007; 85:1479-86.


de Haas Y, Windig JJ, Calus MP, Dijkstra J, de Haan M, Bannink A, et al. Genetic parameters for predicted methane production and potential for reducing enteric emissions through genomic selection. J Dairy Sci. 2011; 94:6122-34.


Zhou M, Hernandez-Sanabria E, Guan LL. Assessment of the microbial ecology of ruminal methanogens in cattle with different feed efficiencies. Appl Environ Microbiol. 2009; 75:6524-33.


Mwenya B, Santoso B, Sar C, Gamo Y, Kobayashi T, Arai I, et al. Effects of including β 1-4 galacto-oligosaccharides, lactic acid bacteria or yeast culture on methanogenesis as well as energy and nitrogen metabolism in sheep. Anim Feed Sci Technol. 2004; 115:313-26.


Sekine J, Kondo S, Okubo M, Asahida Y. Estimation of methane production in 6-week-weaned calves up to 25 weeks of age. Jap J Zootech Sci. 1986; 57:300-4.


Shibata M, Terada F, Iwasaki K, Kurihara M, Nishida T. Methane production in heifers, sheep and goats consuming diets of various hay-concentrate ratios. Anim Sci Technol. 1992; 63:1221-7.


Beever DE, Dhanoa MS, Losada HR, Evans RT, Cammell SB, France J. The effect of forage species and stage of harvest on the processes of digestion occurring in the rumen of cattle. Br J Nutr. 1986; 56:439-54.


Milich L. The role of methane in global warming: where might mitigation strategies be focused?. Glob. Environ Chang. 1999; 9:179-201.


Benchaar C, Pomar C, Chiquette J. Evaluation of dietary strategies to reduce methane production in ruminants: a modelling approach. Can J Anim Sci. 2001; 81:563-74.


Boadi DA, Wittenberg KM, Scott SL, Burton D, Buckley K, Small JA, et al. Effect of low and high forage diet on enteric and manure pack greenhouse gas emissions from a feedlot. Can J Anim Sci. 2004; 84:445-53.


Lovett D, Lovell S, Stack L, Callan J, Finlay M, Conolly J, et al. Effect of forage/concentrate ratio and dietary coconut oil level on methane output and performance of finishing beef heifers. Livest Prod Sci. 2003; 84:135-146.


Moss AR. Methane production by ruminants - Literature review of I. Dietary manipulation to reduce methane production and II. Laboratory procedures for estimating methane potential of diets. Nutr Abst Rev. (Series B). 1994; 64:786-806


Doreau M, Chilliard Y. Digestion and metabolism of dietary fat in farm animals. Br J Nutr. 1997; 78:15-35.


Shibata M, Terada F. Factors affecting methane production and mitigation in ruminants. Anim Sci J. 2010; 81:2-10.


Grainger C, Beauchemin KA. Can enteric methane emissions from ruminants be lowered without lowering their production?. Anim Feed Sci Technol. 2011; 166-167:308-20.


Jenkins TC. Lipid-metabolism in the rumen. J Dairy Sci. 1993; 76:3851-63.


McGuffey RK, Richardson LF, Wilkinson JID. Ionophores for dairy cattle: current status and future outlook. J Dairy Sci. 2001; 84:E194-203.


Sauer FD, Fellner V, Kinsman R, Kramer JKG, Jackson HA, Lee AJ, et al. Methane output and lactation response in Holstein cattle with monensin or unsaturated fat added to the diet. J Anim Sci. 1998; 76:906-14.


O’Kelly JC, Spiers WG. Effect of monensin on methane and heat productions of steers fed lucerne hay either ad libitium or at the rate of250 g/hour. Aust J Agric Res. 1992; 43:1789-93.


McGinn SM, Beauchemin KA, Coates T, Colombatto D. Methane emissions from beef cattle: effects of monensin, sunflower oil, enzymes, yeast, and fumaric acid. J Anim Sci. 2004; 82:3346-56.


Odongo NE, Bagg R, Vessie G, Dick P, Or-Rashid MM, Hook SE, et al. Long-term effects of feeding monensin on methane production in lactating dairy cows. J Dairy Sci. 2007; 90:1781-8.


Guan H, Wittenberg KM, Ominski KH, Krause DO. Efficacy of ionophores in cattle diets for mitigation of enteric methane. J Anim Sci. 2006; 84:1896-1906.


Biswas AA, Lee SS, Mamuad LL, Kim SH, Choi YJ, Lee C, et al. Effects of illite supplementation on in vitro and in vivo rumen fermentation, microbial population and methane emission of Hanwoo steers fed high concentrate diets. Anim Sci J. 2018; 89:114-21.


Asanuma N, Iwamoto M, Hino T. Effect of the addition of fumarate on methane production by ruminal microorganisms in vitro. J Dairy Sci. 1999; 82:780-7.


Lila ZA, Mohammed N, Tatsuoka N, Kanda S, Kurokawa Y, Itabashi H. Effect of cyclodextrin diallyl maleate on methane production, ruminal fermentation and microbes in vitro and in vivo. Anim Sci J. 2004; 75:15-22.


Newbold CJ, Lopez S, Nelson N, Ouda JO, Wallace RJ, Moss AR. Propionate precursors and other metabolic intermediates as possible alternative electron acceptors to methanogenesis in ruminal fermentation in vitro. Br J Nutr. 2005; 94:27-35.


Castillo C, Benedito JL, Mendez J, Pereira V, Lopez-Alonso M, Miranda M, et al. Organic acids as a substitute for monensin in diets for beef cattle. Anim Feed Sci Technol. 2004; 115:101-16.


Kolver ES, Aspin PW, Jarvis GN, Elborough KM, Roche JR. Fumarate reduces methane production pasture fermented in continuous culture. Proc N Z Soc Anim Prod. 2004; 64:155-9


Beauchemin K, McGinn S. Methane emission from beef cattle: effects of fumaric acid, essential oil and canola oil. J Anim Sci. 2006; 84:1489-96.


Chiquette J, Talbot G, Markwell F, Nili N, Forster RJ. Repeated ruminal dosing of Ruminococcus flavefaciens NJ along with a probiotic mixture in forage or concentrate-fed dairy cows: effect on ruminal fermentation, cellulolytic populations and in sacco digestibility. Can J Anim Sci. 2007; 87:237-49.


Jatkauskas J, Vrotniakien V. Effect of L-plantarum, Pediococcus acidilactici, Enterococcus faecium and L-lactis microbial supplementation of grass silage on the fermentation characteristics in the rumen of dairy cows. Vet Zootec. 2007; 40:29-34


Jeyanathan J, Martin C, Morgavi DP. The use of direct-fed microbials for mitigation of ruminant methane emissions: a review. Animal. 2014; 8:250-61.


Baldwin RL, Wood WA, Emery RS. Conversion of glucose-C14 to propionate by the rumen microbiota. J Bacteriol. 1963; 85:1346-9


Russell JB, Wallace RJ. Energy-yielding and energy-consuming reactions. In In: Hobson PN, Stewart CS, editors. editors The rumen microbial ecosystem. London, UKBlackie Academic and Professional 1997; :246-82.


Seo JK, Kim SW, Kim MH, Upadhaya SD, Kam DK, Ha JK. Direct-fed microbials for ruminant animals. Asian-Australas J Anim Sci. 2010; 23:1657-67.


Ghorbani GR, Morgavi DP, Beauchemin KA, Leedle JAZ. Effects of bacterial direct-fed microbials on ruminal fermentation, blood variables, and the microbial populations of feedlot cattle. J Anim Sci. 2002; 80:1977-85.


Adams MC, Luo J, Rayward D, King S, Gibson R, Moghaddam GH. Selection of a novel direct-fed microbial to enhance weight gain in intensively reared calves. Anim Feed Sci Technol. 2008; 145:41-52.


Berger C, Lettat A, Martin C, Noziere P. Method for reducing methane production in a ruminant animal. United States Patent US 0,112,889. 24 Apr; 2014


Mamuad L, Kim SH, Jeong CD, Choi YJ, Jeon CO, Lee SS. Effect of fumarate reducing bacteria on in vitro rumen fermentation, methane mitigation and microbial diversity. J Microbiol. 2014; 52:120-8.


Kim SH, Mamuad LL, Kim DW, Kim SK, Lee SS. Fumarate reductase-producing enterococci reduce methane production in rumen fermentation in vitro. J Microbiol Biotechnol. 2016; 26:558-66.


Iwamoto M, Asanuma N, Hino T. Effect of nitrate combined with fumarate on methanogenesis, fermentation, and cellulose digestion by mixed ruminal microbes in vitro. Anim Sci J. 1999; 70:471-8.


Morris MP, Cancel B, González-Más A. Toxicity of nitrates and nitrites to dairy cattle. J Dairy Sci. 1958; 41:694-6.


Anderson RC, Rasmussen MA. Use of a novel nitrotoxin-metabolizing bacterium to reduce ruminal methane production. Biores Technol. 1998; 64:89-95.


Asanuma N, Iwamoto M, Kawato M, Hino T. Numbers of nitratereducing bacteria in the rumen as estimated by competitive polymerase chain reaction. Anim Sci J. 2002; 73:199-205.


Yoshii T, Asanuma N, Hino T. Number of nitrate- and nitrite-reducing Selenomonas ruminantium in the rumen, and possible factors affecting its growth. Anim Sci J. 2003; 74:483-91.


Iwamoto M, Asanuma N, Hino T. Abilityof Selenomonas ruminantium, Veillonella parvula, and Wolinella succinogenes to reduce nitrate and nitrite with specia lreference to the suppression of ruminal methanogenesis. Anaerobe. 2002; 8:209-15.


Campbell LL, Postgate JR. Classification of the spore-forming sulfatereducing bacteria. Bacteriol Rev. 1965; 29:359-63


Huisingh J, McNeill JJ, Matrone G. Sulfatereductionbya Desulfovibrio species isolated from sheep rumen. Appl Environ Microbiol. 1974; 28:489-97


Paul SS, Deb SM, Singh D. Isolation and characterization of novel sulphate-reducing Fusobacterium sp. and their effects on in vitro methane emission and digestion of wheat straw by rumen fluid from Indian riverine buffaloes. Anim Feed Sci Technol. 2011; 166-167:132-140.


Morvan B, Rieu-Lesme F, Fonty G, Gouet P. In vitro interactions between rumen H-2-producing cellulolytic microorganisms and H-2-utilizing acetogenic and sulfate-reducing bacteria. Anaerobe. 1996; 2:175-80.


van Zijderveld SM, Gerrits WJJ, Apajalahti JA, Newbold JR, Dijkstra J, Leng RA, et al. Nitrate and sulfate: effective alternative hydrogen sinks for mitigation of rumina lmethane production in sheep. J Dairy Sci. 2010; 93:5856-66.


Mackie RI, Bryant MP. Acetogenesis and the rumen: syntrophic relationships. In Acetogenesis. Boston, MA: Springer. 1994; p. 331-64.


Joblin KN. Ruminal acetogens and their potential to lower ruminant methane emissions. Aust J Agric Res. 1999; 50:1307-14.


Kim SH, Mamuad LL, Choi YJ, Sung HG, Cho KK, Lee SS. Effects of reductive acetogenic bacteria and lauric acid on in vivo ruminal fermentation, microbial populations, and methane mitigation in Hanwoo steers in South Korea. J Anim Sci. 2018; 96:4360-67.


Chaucheyras FG, Fonty G, Bertin G, Gouet P. In vitro H2 utilization by a ruminal acetogenic bacterium cultivated alone or in association with an archaea methanogenis stimulated by a probiotic strain of Saccharomyces cerevisiae. Appl Environ Microbiol. 1995; 61:3466-7


Lynch HA, Martin SA. Effects of Saccharomyces cerevisiae culture and Saccharomyces cerevisiae live cells on in vitro mixed ruminal microorganism fermentation. J Dairy Sci. 2002; 85:2603-8.


Frumholtz PP, Newbold CJ, Wallace RJ. Influence of Aspergillus oryzae fermentation extract on the fermentation of a basal ration in the rumen simulation technique (Rusitec). J Agric Sci. 1989; 113:169-72.


Patra AK. An overview of antimicrobial properties of different classes of phytochemicals.In In: Patra AK, editor.editor Diet phytochemicals and microbes. Dorrecht: Springer Netherlands. 2012; p. 1-32.


Cieslak A, Szumacher-Strabel M, Stochmal A, Oleszek W. Plant components with specific activities against rumen methanogens. Animal. 2013; 7:253-65.


Patra AK, Saxena J. A new perspective on the use of plant secondary metabolites to inhibit methanogenesis in the rumen. Phytochem. 2010; 71:1198-1222.


Joch M, Mrazek J, Skrivanova E, Cermak L, Marounek M. Effects of pure plant secondary metabolites on methane production, rumen fermentation and rumen bacteria populations in vitro. J Anim Physiol Anim Nutr. 2018; 102:869-81.


Maia MRG, Fonseca AJ, Oliveira HM, Mendonca C, Cabrita AR. The potential role of seaweeds in the natural manipulation of rumen fermentation and methane production. Sci Rep. 2016; 6:32321.


Hristov AN, Ivan M, Neill L, McAllister TA. Evaluation of several potential bioactive agents for reducing protozoal activity in vitro. Anim Feed Sci Technol. 2003; 105:163-84.


Hu W, Wu Y, Liu J, Guo Y, Ye J. Tea saponins affect in vitro fermentation and methanogenesis in faunated and defaunated rumen fluiud. J Zhejiang Univ Sci B. 2005; 6:787-92.


Wina E, Muetzel S, Becker K. The impact of saponins or saponin-containing plant on ruminant production-a review. J Agric Food Chem. 2005; 53:8093-105.


Patra AK, Yu Z. Effective reduction of enteric methane production by a combination of nitrate and saponin without adverse effect on feed degradability, fermentation, or bacterial and archaeal communities of the rumen. Bioresour Technol. 2013; 148:352-60.


Belanche A, Pinloche E, Preskett D, Newbold CJ. Effects and mode of action of chitosan and ivy fruit saponins on the microbiome, fermentation and methanogenesis in the rumen simulation technique. FEMS Microbiol Ecol. 2016; 92:1.


Cieslak A, Zmora P, Stochmal A, Pecio L, Oleszek W, Pers-Kamczyc E, et al. Rumen antimethanogenic effect of Saponaria officinalis L. phytochemicals in vitro. J Agric Sci. 2014; 152:981-93.


Patra AK, Yu Z. Effects of vanillin, Quillaja saponin, and essential oils on in vitro fermentation and protein-degrading microorganisms of the rumen. Appl Microbiol Biotechnol. 2014; 98:897-905.


Patra AK, Yu Z. Effects of adaptation of in vitro rumen culture to garlic oil, nitrate, and saponin and their combinations on methanogenesis, fermentation, and abundances and diversity of microbial populations. Front Microbiol. 2015; 6:1434.


Ramirez-Restrepo CA, Tan C, O’Neill CJ, Lopez-Villalobos N, Padmanabha J, Wang JK, et al. Methane production, fermentation characteristics, and microbial profiles in the rumen of tropical cattle fed tea seed saponin supplementation. Anim Feed Sci Technol. 2016; 216:58-67.


Patra AK, Min BR, Saxena J. Dietary tannins on microbial ecology of the gastrointestinal tract in ruminants.In In: Patra AK, editor.editor Diet phytochem microbes. Dordrecht: Springer Netherlands. 2012; p. 237-262.


Tavendale MH, Meagher LP, Pacheco D, Walker N, Attwood GT, Sivakumaran S. Methane production from in vitro rumen incubations with Lotus pedunculatus and Medicago sativa, and effects of extractable condensed tannin fractions on methanogenesis. Anim Feed Sci Technol. 2005; 123-124:403-419.


Puchala R, Animut G, Patra AK, Detweiler GD, Wells JE, Varel VH, et al. Methane emissions by goats consuming Sericea lespedeza at different feeding frequencies. Anim Feed Sci Technol. 2012; 175:76-84.


Saminathan M, Sieo CC, Gan HM, Abdullah N, Wong CMVL, Ho YW. Effects of condensed tannin fractions of different molecular weights on population and diversity of bovine rumen methanogenic archaea in vitro, as determined by high-throughput sequencing. Anim Feed Sci Technol. 2016; 216:146-60.


Tamminga S, Bannink A, Dijkstra J, Zom RLG. Feeding strategies to reduce methane loss in cattle. Lelystad: Animal Sciences Group. 2007Report No.: 34


Ramirez-Restrepo CA, Barry TN. Alternative temperate forages containing secondary compounds for improving sustainable productivity in grazing ruminants. Anim Feed Sci Technol. 2005; 120:179-201.


Newbold CJ, el Hassan SM, Wang J, Ortega ME, Wallace RJ. Influence of foliage from African multipurpose trees on activity of rumen protozoa and bacteria. Br J Nutr. 1997; 78:237-49.


Greathead H. Plants and plant extracts for improving animal productivity. Proc Nutr Soc. 2003; 62:279-90.


Burt S. Essential oils: their antibacterial properties and potential applications in foods - a review. Int J Food Microbiol. 2004; 94:223-53.


Benchaar C, Calsamiglia S, Chaves AV, Fraser GR, Colombatto D, McAllister TA, et al. A review of plant-derived essential oils in ruminant nutrition and production. Anim Feed Sci Technol. 2008; 145:209-228.


Jouany JP, Morgavi DR. Use of ‘natural’ products as alternatives to antibiotic feed additives in ruminant production. Animal. 2007; 1:1443-66.


Newbold CJ, McIntosh FM, Williams P, Losa R, Wallace RJ. Effects of a specific blend of essential oil compounds on rumen fermentation. Anim Feed Sci Technol. 2004; 114:105-12.


Lewis KA, Tzilivakis J, Green A, Warner DJ, Stedman A, Naseby D. Review of substances/agents that have direct beneficial effect on the environment: mode of action and assessment of efficacy. EFSA Supp Pub. 2013; 10:440E.


Rode LM, Yang WZ, Beauchemin KA. Fibrolytic enzyme supplements for dairy cows in early lactation. J Dairy Sci. 1999; 82:2121-6.


Arriola KG, Kim SC, Staples CR, Adesogan AT. Effect of fibrolytic enzyme application to low-and high-concentrate diets on the performance of lactating dairy cattle. J Dairy Sci. 2011; 94:832-41.


Holtshausen L, Chung YH, Gerardo-Cuervo H, Oba M, Beauchemin KA. Improved milk production efficiency in early lactation dairy cattle with dietary addition of a developmental fibrolytic enzyme additive. J Dairy Sci. 2011; 94:899-907.


Yang WZ, Beauchemin KA, Rode LM. A comparison of methods of adding fibrolytic enzymes to lactating cow diets. J Dairy Sci. 2000; 83:2512-20.


Zhou M, Chung YH, Beauchemin KA, Holtshausen L, Oba M, McAllister TA, et al. Relationship between rumen methanogens and methane production in dairy cows fed diets supplemented with a feed enzyme additive. J Appl Microbiol. 2011; 111:1148-58.


Chung YH, Zhou M, Holtshausen L, Alexander TW, McAllister TA, Guan LL, et al. A fibrolytic enzyme additive for lactating Holstein cow diets: Ruminal fermentation, rumen microbial populations, and enteric methane emissions. J Dairy Sci. 2012; 95:1419-27.


Biswas AA, Lee SS, Mamuad LL, Kim SH, Choi YJ, Bae GS, et al. Use of lysozyme as a feed additive on in vitro rumen fermentation and methane emission. Asian Australas J Anim Sci. 2016; 29:1601-7.


Ushida K, Tokura M, Takenaka A, Itabashi H. Ciliate protozoa and ruminal methanogenesis.In In: Onodera R, Itabashi H, Ushida K, Yano H, Sasaki Y, editors.editors Rumen microbes and digestive physiology in ruminants. Tokyo, Japan: Japan Scientific Societies Press. 1997; p. 209-20


Morgavi DP, Jouany JP, Martin C. Changes in methane emission and rumen fermentation parameters induced by refaunation in sheep. Aust J Exp Agric. 2008; 48:69-72.


Patra AK, Kamra DN, Agarwal N. Effect of plant extracts on in vitro methanogenesis, enzyme activities and fermentation of feed in rumen liquor of buffalo. Anim Feed Sci Technol. 2006; 128:276-91.


Hegarty RS, Bird SH, Vanselow BA, Woodgate R. Effects of the absence of protozoa from birth or from weaning on the growth and methane production of lambs. Br J Nutr. 2008; 100:1220-27.


Williams YJ, Rea SM, Popovski S, Pimm CL, Williams AJ, Toovey AF, et al. Reponses of sheep to a vaccination of entodinial or mixed rumen protozoal antigens to reduce rumen protozoal numbers. Br J Nutr. 2008; 99:100-9.