**5.1. Dietary composition**

The quantity of enteric methane production is directly related to the quantity and quality of the feed consumed by the animal. The loss of GEI was augmented with an increase in high feed quantity. Animals with a low feed efficiency increase environmental impact due to the loss of GEI in form of methane.

The most common feeding mechanisms for the ruminants are based on pasture (grazing) and harvested forages. Hay and silage are the most common cattle forages. Hay has been recognized as superior feed than silage, but in cold and wet weather, silage is most used due to its major productivity. Silages for ruminants in temperate areas are usually based on cereals and legumes such as grass, maize, lucerne and red clover, which provide carbohydrate, protein and lipid sources for the animal [53]. It has been extensively reviewed that the replacement of ruminant forage diets with high grain diets can reduce methane production [9, 27, 54, 55]. Fermentation of cereal grains with high starch content increased the voluntary intake and reduced the residence time in the rumen, promoting post-ruminal digestion. Starch also Livestock Methane Emission: Microbial Ecology and Mitigation Strategies http://dx.doi.org/10.5772/65859 59

phyla were *Bacteroidetes* (52%), *Firmicutes* (42.7%), *Spirochaetes* (2.3%) and *Fibrobacteres* (1.9%). This study clearly demonstrated that the changes in feed affect and change the dynamics of ruminal microbiome. Petri et al. [48] studied the impact of diet and its impact of an acidotic challenge on the composition of six different bacterial targets from heifers fed forage, mixed forage, high grain, post-acidic challenge (4 and 12 h) and recovery. They observed that all of the bacterial target groups were affected by dietary treatment, with exception of *S. bovis*, *Raminococcus* spp. and *Fibrobacter succinogenes* represented a large percentage of the bacterial population present in the mixed forage diet. *Prevotella* corresponds to the most abundant genera in the acidotic challenge, but the lowest in the animal fed forage. *Megasphaera elsdenii* was present in abundance in the sample of 12 h after acidotic challenge, but its abundance decreased during recovery, while at the same time *S. ruminantium* increased in proportion. Both *S. ruminantium* and *M. elsdenii* accounted the smallest proportion of the bacterial popula-

The necessity to implement abatement strategies for enteric GHG emissions has been expanded in conjunction with the increase in the population and food demand. There are two concerns over methane emissions by livestock ruminants. First, the release of methane is considered a loss of energy for the animal, resulting in a decrease in animal productivity between 2 and 12%. Second, the calorific potential of methane released has a negative impact on climate change. There are several publications on strategies to reduce methane production [49–52]. The main target of these strategies is on methanogenic archaea by decreasing their substrate availability either directly or indirectly. Overall, abatement strategies include mechanisms such as modifications in dietary composition, and/or by supplementation of diet with chemical inhibitors, lipids or plant compounds, some of these strategies are shown in

The quantity of enteric methane production is directly related to the quantity and quality of the feed consumed by the animal. The loss of GEI was augmented with an increase in high feed quantity. Animals with a low feed efficiency increase environmental impact due to the

The most common feeding mechanisms for the ruminants are based on pasture (grazing) and harvested forages. Hay and silage are the most common cattle forages. Hay has been recognized as superior feed than silage, but in cold and wet weather, silage is most used due to its major productivity. Silages for ruminants in temperate areas are usually based on cereals and legumes such as grass, maize, lucerne and red clover, which provide carbohydrate, protein and lipid sources for the animal [53]. It has been extensively reviewed that the replacement of ruminant forage diets with high grain diets can reduce methane production [9, 27, 54, 55]. Fermentation of cereal grains with high starch content increased the voluntary intake and reduced the residence time in the rumen, promoting post-ruminal digestion. Starch also

tion in heifers fed forages.

58 Livestock Science

**Figure 2**.

**5.1. Dietary composition**

loss of GEI in form of methane.

**5. Methane mitigation strategies**

**Figure 2.** Mitigation strategies on methane emission by rumen microbiome manipulation through change in diets. Note: The main pathways and products formed when high fiber diet is used are represented in green color. The effect of high starch diets, which enhances propionate production due to shifting of hydrogen sinks, is presented in orange color. Dietary supplements and their main targets in order to reduce methane production are indicated in blue color.

enhanced propionate production, which depleted H+ , and thereby decreased its availability for hydrogenotrophic methanogens. Moreover, propionate production decreased the pH, causing an inhibitory effect on methanogens and protozoa [56]. The loss of GEI with grainbased diets is commonly 4%, while it is 6.5% or more in forage-based diets.

Lettat et al. [57] reported that starchy diets, apart from increasing the propionate concentration, decreased the concentrations of acetate and butyrate and consequently methane production (-14%). Diversity and richness of bacterial community were reduced with increase in the starch content of the diet, however, the total bacterial population, *Prevotella* spp. and *M. elsdenii* were favored. The bacterial group *Prevotella* has been identified as amylolytic and propionate producer and the dominant within the rumen [58, 59], while *M. elsdenni* is a well-known lactate-utilizing and propionate-producing bacteria. CH<sup>4</sup> reduction has been linked to the decrease in protozoan populations since protozoa are known as hydrogen producers and are in symbiotic relationship with methanogens. Hence, with a decrease in protozoan population, there is a decrease in the hydrogen transfer between them and methanogens, and this decreased the methane production. However, metabolic activity of archaea and methanogenic population increased when methane production decayed, demonstrating the cDNAqPCR method to estimate archaeal growth and activity is unreliable to reflect changes in ruminal methanogenesis. However, there should be sufficient care before adopting this as a wholescale strategy. It has reported that changes in dietary composition not only can affect microbial diversity but also can generate animal disorders, producing a negative effect on the host. Saleem et al. [60] reported that high grain diets increased the concentrations of several toxic compounds such as putrescine, methylamines, ethanolamine and VFAs in the rumen fluid. VFAs accumulation can decrease the pH lower than 5.5 and produce subacute ruminal acidosis, which is a common and disturbing problem for farmers [61]. High grain diets have been commonly observed in favor of amylolytic microorganisms and against fibrolytic microorganisms. Petri et al. [48] reported that rumen of Angus heifers fed with high grains diet recorded a higher abundance of *Prevotella* spp., *S. ruminanitum* known also as amylolytic bacteria, and *M. elsdenii*. Whereas, a higher abundance of the fibrolytic bacteria *Ruminococcus* spp. and *F. succionogenes*, and the lactate-producing *S. bovis* was observed with forage diet. Kittelmann et al. [62] observed a positive correlation between the occurrence of methanogens and fibrolytic bacteria. *Methanobrevibacter ruminanitum* is found to be correlated with the family Fibrobactereacea and *M. gottschalkii* with the family *Ruminococcaceae*. *Ruminococcus* spp. is known to produce large amounts of H2 , while *Fibrobacter* spp. produces formate, which is substrates for methanogens. Therefore, the abundance of fibrolytic bacteria could be related with methanogenic communities and consequently with methane production.

#### **5.2. Dietary supplementation**

#### *5.2.1. Chemical inhibitors*

Compounds nontoxic to animal, but inhibitors to methanogens have been used to reduce methane production. Although these compounds inhibit-specific enzymes involved in methanogenesis pathway, it has been reported that they could also have an impact on other microbial groups present and could affect the uptake of feed by the animal [5, 27]. The most used and effective compounds are the analogous of coenzyme M, inhibitors of methanopterin biosynthesis, nitrocompounds and halogenated compounds [63–65].

Bromochloromethane (BCM), a methane analogue, has been extensively used to decrease methane production [65–67] but has a limited use due to its great ozone depleting capacity [66]. This compound reduces vitamin B12 and inhibits the cobamide-dependent methyl transferase step of the biosynthesis pathway of methyl coenzyme M, involved in methanogenesis pathway. After 12 h of supplementation, BCM-cyclodextrin (0.5 g/100 kg live weight) decreased the methane production of steer by 29%, and without adversely affecting the animal productivity [65]. Mitsumori et al. [67] studied the effect of different concentrations of BCM-cyclodextrin (BCM-CD) on the rumen microbial population of goats. Doses of BCM-CD were of low (0.5 g/100 kg live weight LW), medium (2 g/100 kg LW) and high (5 g/100 kg LW), which decreased the methane emissions by 4.64, 71.46 and 91.23%, respectively. Denman et al. [68] analyzed the microbial diversity of the samples from the above study and reported that the relative abundance of Bacteroidetes increased with the BCM-CD doses, while Firmicutes, Synergistetes and Lentrisphaerae phyla decreased. In the case of control animal, Bacteroidetes (60%) was dominant, followed by Firmicutes (24%), Synergistetes and Lentisphaera (both contributed ~4%). Administration of BCM also reduced considerably methanogenic diversity, however, *Methanobrevibater* species were the most abundant in all treatments. Based on phylogenetic binding and functional assignment, the major genera were *Prevotella* and *Selenomonas* which were associated with the propionate production by the randomizing succinate pathway. This pathway was the primary route of H<sup>2</sup> consumption and decreased H2 availability for methanogens.

producer and the dominant within the rumen [58, 59], while *M. elsdenni* is a well-known

decrease in protozoan populations since protozoa are known as hydrogen producers and are in symbiotic relationship with methanogens. Hence, with a decrease in protozoan population, there is a decrease in the hydrogen transfer between them and methanogens, and this decreased the methane production. However, metabolic activity of archaea and methanogenic population increased when methane production decayed, demonstrating the cDNAqPCR method to estimate archaeal growth and activity is unreliable to reflect changes in ruminal methanogenesis. However, there should be sufficient care before adopting this as a wholescale strategy. It has reported that changes in dietary composition not only can affect microbial diversity but also can generate animal disorders, producing a negative effect on the host. Saleem et al. [60] reported that high grain diets increased the concentrations of several toxic compounds such as putrescine, methylamines, ethanolamine and VFAs in the rumen fluid. VFAs accumulation can decrease the pH lower than 5.5 and produce subacute ruminal acidosis, which is a common and disturbing problem for farmers [61]. High grain diets have been commonly observed in favor of amylolytic microorganisms and against fibrolytic microorganisms. Petri et al. [48] reported that rumen of Angus heifers fed with high grains diet recorded a higher abundance of *Prevotella* spp., *S. ruminanitum* known also as amylolytic bacteria, and *M. elsdenii*. Whereas, a higher abundance of the fibrolytic bacteria *Ruminococcus* spp. and *F. succionogenes*, and the lactate-producing *S. bovis* was observed with forage diet. Kittelmann et al. [62] observed a positive correlation between the occurrence of methanogens and fibrolytic bacteria. *Methanobrevibacter ruminanitum* is found to be correlated with the family Fibrobactereacea and *M. gottschalkii* with the family *Ruminococcaceae*. *Ruminococcus* spp.

substrates for methanogens. Therefore, the abundance of fibrolytic bacteria could be related

Compounds nontoxic to animal, but inhibitors to methanogens have been used to reduce methane production. Although these compounds inhibit-specific enzymes involved in methanogenesis pathway, it has been reported that they could also have an impact on other microbial groups present and could affect the uptake of feed by the animal [5, 27]. The most used and effective compounds are the analogous of coenzyme M, inhibitors of methanopterin bio-

Bromochloromethane (BCM), a methane analogue, has been extensively used to decrease methane production [65–67] but has a limited use due to its great ozone depleting capacity [66]. This compound reduces vitamin B12 and inhibits the cobamide-dependent methyl transferase step of the biosynthesis pathway of methyl coenzyme M, involved in methanogenesis pathway. After 12 h of supplementation, BCM-cyclodextrin (0.5 g/100 kg live weight) decreased the methane production of steer by 29%, and without adversely affecting the animal productivity [65]. Mitsumori et al. [67] studied the effect of different concentrations of BCM-cyclodextrin (BCM-CD) on the rumen microbial population of goats. Doses of BCM-CD

with methanogenic communities and consequently with methane production.

synthesis, nitrocompounds and halogenated compounds [63–65].

reduction has been linked to the

, while *Fibrobacter* spp. produces formate, which is

lactate-utilizing and propionate-producing bacteria. CH<sup>4</sup>

is known to produce large amounts of H2

**5.2. Dietary supplementation**

*5.2.1. Chemical inhibitors*

60 Livestock Science

2-bromoethanesulfonate (BES) is another common and successful compound to decrease methane emissions, which is an analog of coenzyme M. In an *in vitro* mesocosm study with cow manure and anaerobic digester sludge, a 89 and 100% decrease in methane production was observed at 0.5 and 10 mmol/L, respectively. Relative abundance of *Methanosaeta* and *Methanosarcina* decreased considerably at 10 mmol/L. Moreover, a decrease in mcrA expression, which encodes the α subunit of the methyl coenzyme M reductase and due to it is used for the relative measure of methane metabolites and methanogenic abundance in different environments [69], was observed with the increment of BES. A decrease in syntrophic-bacteria *Syntrophomonas* was observed too at both concentrations of BES. It is known for oxidation of butyrate and other fatty acids in syntrophic association with H<sup>2</sup> -consuming bacteria and/or hydrogenotrophic methanogens and could explain the decrease in methanogenic activity [70].

The inhibitory effect of chloroform is attributed to its capacity to target the corrinoid-containing MtrA subunit of the large multimeric membrane enzyme methyl tetrahydromethanopt erin:coenzyme M methyltransferase [71]. Martínez-Fernández et al. [72] studied the inhibitory effect of chloroform-cyclodextrin (CCD) by way of supplementation; as low (1 g/100kg live weight LW), medium (1.6 g/100 kg LW) and high (2.6 g/100 kg LW) dose along with two diets (roughage:concentrate (60:40) or roughage hay) in eight steers. All three doses decreased the methane production by 14, 37 and 55%, respectively. Changes in microbial community were observed too, archaeal abundance was negatively correlated with CDD levels, Methanobacteriaceae family and Methanoplasmatales order were found to be decreased. Protozoan population increased with CCD doses with roughage:concentrate diet, while chloroform did not have any effect on fungi community. Bacterial population was also affected, relative abundance of *Bacteroidetes* increased, while *Firmicutes*, *Synergistetes* and *Verrucomicrobia* phyla were decreased. While methanogenesis was inhibited, an increment in the production of amino acids, organic and nucleic acids was observed. All of these metabolic changes modified the ruminal microbiome, increased the *Bacteroidetes:Firmicutes* ratio and decreased archaea and *Synergistetes*. Although abundance of fibrolytic bacteria, protozoa and fungi was not affected, methanogenesis was inhibited by 30%. They concluded that the use of chloroform as methanogenic inhibitor did not adversely affect rumen metabolism and could redirect H2 to another pathways producing non-methane end products.

Apart from the compounds mentioned, nitrocompounds are also being used in vivo to mitigate methane emissions. These compounds target of specific sites of MCR due to its molecular shape and oxidative potential and inhibit the last step of methanogenesis pathway. It has been reported that 3-nitrooxypropanol (NOP) at 40–80 mg/kg, decreased methane emissions around 30% and also increased body weight gain considerably without affecting feed intake or milk characteristics [73]. Duin et al. [74] reported that only 0.1 µM NOP is needed to inactivate completely MCR, and 1 µM to inhibit the methanogenic population. It was also reported that bacterial population was not affected by the addition of NOP, while methanogenic population decreased and protozoal abundance increased [75]. The decrease in methane production (−59.2%) by NOP (2 g/day) could be related directly to the reduction in the population of methanogens. The reduction in methanogen populations due to the addition of nitrocompounds need not always result in an increase in protozoan populations, since the compounds could also affect the symbiotic methanogens-protozoan association and thereby could result in decreased protozoan populations.

#### *5.2.2. Plant bioactive compounds*

Plant secondary metabolites have also been extensively used in the reduction of methane emissions. The most common used are tannins, saponins and essential oils, and they can affect methanogens either directly or indirectly. Further, they reduce protozoal population and thereby reducing symbiotically associated methanogens, apart from decreasing fiber digestibility and H2 production [76].

Tannins are polyphenolic compounds which form complexes with metal ions, amino acids and polysaccharides, and thereby reduce ruminal fermentation. They can be divided into hydrolysable and condensed tannins. Hydrolysable tannins at high concentrations may be toxic to ruminants, while condensed tannins can make several nutrients unavailable to the animal due to irreversible binding [77]. Moreover, they can bind to the gastrointestinal tract, causing negative effects [78]. However, they have been found to be effective in reducing methane emissions. Condensed tannins have been reported to reduce methane by around 16% based on dry matter intake (DMI) [79]. Total methanogen population decreased by 22.3– 36.7% when purified hydrolysable (HT) and condensed tannins (CT) (1 mg/ml) were tested in vitro conditions. Hydrolysable tannins were found to be more effective than with condensed tannins in reducing methane formation [80]. On the contrary, Bhatta et al. [76] reported that CT had a greater effect on methane reduction (−5.5%) than HT (−0.6%) and its inhibitory effect on methanogens (−28.6%) was more than HT (-11.6%). Protozoan populations also decreased by 12.3% with HT diets. However, a combination of HT+CT diets had a more significant effect and a 36.2% decrease was reported. Although tannins reduced total VFA concentrations was found to increase propionate concentrations and decrease iso-acids, which could have a negative effect on methanogenesis. In previous studies, a reduction in total and cellulolytic bacteria in response to tannins was observed along with the reduction in VFA production and also H2 production, contributing to methane inhibition [81, 82].

Saponins are complex and diverse molecules which are divided in triterpene and steroid glycosides [83]. They are considered effective compounds to suppress methane production due to their anti-protozoan properties [54]. Their anti-protozoan properties are attributed mainly to the formation of complexes with sterols in the membrane surface of protozoans [84]. However, this is pH dependent and composition of diet with addition of saponins [85, 86]. Moreover, saponins are potential defaunation agents and could result in the reduction in enteric CH<sup>4</sup> production by eliminating protozoa [9]. Nevertheless, they have an effect on the whole ruminant microbiome and animal digestion process, and not specifically targeting protozoan populations.

#### *5.2.3. Lipid supplementation*

Apart from the compounds mentioned, nitrocompounds are also being used in vivo to mitigate methane emissions. These compounds target of specific sites of MCR due to its molecular shape and oxidative potential and inhibit the last step of methanogenesis pathway. It has been reported that 3-nitrooxypropanol (NOP) at 40–80 mg/kg, decreased methane emissions around 30% and also increased body weight gain considerably without affecting feed intake or milk characteristics [73]. Duin et al. [74] reported that only 0.1 µM NOP is needed to inactivate completely MCR, and 1 µM to inhibit the methanogenic population. It was also reported that bacterial population was not affected by the addition of NOP, while methanogenic population decreased and protozoal abundance increased [75]. The decrease in methane production (−59.2%) by NOP (2 g/day) could be related directly to the reduction in the population of methanogens. The reduction in methanogen populations due to the addition of nitrocompounds need not always result in an increase in protozoan populations, since the compounds could also affect the symbiotic methanogens-protozoan association and thereby could result

Plant secondary metabolites have also been extensively used in the reduction of methane emissions. The most common used are tannins, saponins and essential oils, and they can affect methanogens either directly or indirectly. Further, they reduce protozoal population and thereby reducing symbiotically associated methanogens, apart from decreasing fiber

Tannins are polyphenolic compounds which form complexes with metal ions, amino acids and polysaccharides, and thereby reduce ruminal fermentation. They can be divided into hydrolysable and condensed tannins. Hydrolysable tannins at high concentrations may be toxic to ruminants, while condensed tannins can make several nutrients unavailable to the animal due to irreversible binding [77]. Moreover, they can bind to the gastrointestinal tract, causing negative effects [78]. However, they have been found to be effective in reducing methane emissions. Condensed tannins have been reported to reduce methane by around 16% based on dry matter intake (DMI) [79]. Total methanogen population decreased by 22.3– 36.7% when purified hydrolysable (HT) and condensed tannins (CT) (1 mg/ml) were tested in vitro conditions. Hydrolysable tannins were found to be more effective than with condensed tannins in reducing methane formation [80]. On the contrary, Bhatta et al. [76] reported that CT had a greater effect on methane reduction (−5.5%) than HT (−0.6%) and its inhibitory effect on methanogens (−28.6%) was more than HT (-11.6%). Protozoan populations also decreased by 12.3% with HT diets. However, a combination of HT+CT diets had a more significant effect and a 36.2% decrease was reported. Although tannins reduced total VFA concentrations was found to increase propionate concentrations and decrease iso-acids, which could have a negative effect on methanogenesis. In previous studies, a reduction in total and cellulolytic bacteria in response to tannins was observed along with the reduction in VFA production and also

Saponins are complex and diverse molecules which are divided in triterpene and steroid glycosides [83]. They are considered effective compounds to suppress methane production

in decreased protozoan populations.

production [76].

production, contributing to methane inhibition [81, 82].

*5.2.2. Plant bioactive compounds*

digestibility and H2

62 Livestock Science

H2

Supplementation of lipids in ruminant diets is found to improve microbial metabolism of rumen, decreasing enteric methane emissions. Reduction in methane production could be due to the direct effect of fatty acids on methanogens, or indirectly due to the inhibition of the protozoan communities and associated methanogens due to enhanced propionate production. Beauchemi et al. [54] calculated that CH<sup>4</sup> (g/kg DMI) is reduced by 5.6% for each percentage unit of lipid, while Eugène et al. [87] estimated the methane reduction to about 2.3%.

Lipids commonly supplemented to reduce enteric fermentation are calcium salts of fatty acids, hydrogenated fats, and fats of animal origin, extracted plant oils, oilseeds and wastes from processing plants with high fat content [88]. Based on a meta-analysis of 27 publications on the effect of fatty acids in ruminant diets, fatty acids C12:0 and C18:3 demonstrated a significant inhibitory effect on methanogenesis without affect the productivity in dairy cattle [89]. Patra and Yu [90] analyzed *in vitro* the effect of five essential oils (EO) such as clove oil (CLO; from Eugenia spp.), eucalyptus oil (EUO; from *Eucalyptus globulus*), garlic oil (GAO; from *Allium sativum* L.), origanum oil (ORO; from *Thymus capitatus* L. Hoffmanns & Link) and peppermint oil (PEO; from *Mentha piperita* L.) on methane production, fermentation and ruminal microbiome. CLO, EUO, GAO, ORO and PEO significantly reduced the methane formation by 34.4, 17.6, 42.3, 87 and 25.7%. Further, decrease in relative abundance of ruminant microbial population such as archaea, protozoa and major cellulolytic bacteria *F. succinogenes*, *R. flavefaciens* and *R. albus* was recorded. Microarray analysis by RumenBactArray showed that the effect of each oil tested was unique. *Firmicutes* phylum was decreased by addition ORO and GAO, but increased by PEO. While, *Bacteroidetes* phylum, mainly *Prevotella* OTUS were found to be increased by addition of ORO and PEO. EO decreased the abundance of several microorganisms, *Syntrophococcus sucromutans*, *Succiniclasticum ruminis* and *Lachnobacterium* and members of *Lachnospiraceae*, *Ruminococcaceae*, *Prevotellaceae*, *Bacteroidales* and *Clostridiales*. This was correlated with feed degradability, ammonia concentration and molar percentage of VFAs, which directly affect microbial communities, their metabolic interactions and hence the methane production.

Beauchemi et al. [91] studied the effect of addition of saturated and unsaturated long-chain fatty acids to cattle basal diet, consisting mainly of whole-crop silage. Lipids of animal origin (tallow) and sunflower oil at 34 g/kg, and oilseed (whole sunflower seeds) at 89.3 g/kg were added to bring the total dietary fat content to about 59 g/kg of dry matter. On basis of dry matter intake, diets containing tallow or sunflower oil decreased methane emissions by 11%, while sunflower seeds by 23%. Based on digestible energy intake, all lipid sources decreased methane emissions by 17%. Previously, coconut oil has also been reported as an effective inhibitor of methane production. Jordan et al. [92] reported a 39% decrease in methane emission at a concentration of 375 g/day.

Although supplements are being used primarily in reducing methane emission from livestock, their use in increasing efficiency in feed conversion and animal productivity, based on GEI, animal weight gain, meat and milk production has also been reported [73]. However, few other studies also have reported the negative effect of supplements on the quantity and quality of animal products such as milk and meat [60, 61]. This contradiction could be due to the reason that rumen microbial diversity is dependent on type and amount of feed, which in turn influences the nutrient absorption by animal. This implies that further studies on the relation between rumen microbiome and metabolomics of rumen are essential in order to understand the variations in relation to animal products due to supplements.
