Effect of Various Feed Additives on the Methane Emissions from Beef Cattle Based on an Ammoniated Palm Frond Feeds

*Mardiati Zain, Rusmana Wijaya Setia Ningrat, Heni Suryani and Novirman Jamarun*

#### **Abstract**

Methane gas has a very significant contribution to the increase in greenhouse gases (GHG) globally. The livestock sector, especially ruminants, causes the issue of increasing GHG concentrations. The chapter presents the issue of reducing methane gas production from cattle. Various experiments to reduce methane gas production from ruminants have been carried out and have shown varying results. This series of results of the author's research on reducing methane gas production in livestock in beef cattle based on agriculture by-product to animal feed is addressed with this background. Agriculture by-products such as oil palm fronds and rice straw can be used to feed beef cattle in Indonesia. However, agriculture by-product as animal feed can reduce feed efficiency and increase methane gas production due to the high lignin content. Therefore, various alternatives are carried out to optimize the utilization of this plantation waste. One of them is the use of feed additives and methanogenesis inhibitors. The author's series of research using feed additives (direct-fed microbial) and various methanogenesis inhibitors (plant bioactive compounds and dietary lipids) were tested to determine their effect on nutrient digestibility and methane gas production in feed based on plantation waste. Experiments were carried out *in vitro* and *in vivo* on various types of ruminants. Plant bioactive compounds such as tannins are proven to reduce methane production through their ability to defaunate in the rumen. Tannins may also have direct effect on methanogens and indirectly by reducing fiber digestion. In addition, direct-fed microbial (DFM) feed additives such as *Saccharomyces cerevisiae, Bacillus amyloliquifaciens, and Aspergillus oryz*ae can be used in ruminants to increase livestock productivity. Furthermore, virgin coconut oil as a dietary lipid contains medium-chain fatty acids, mainly lauric acid, which can inhibit the development of ciliates of protozoa and methanogenic bacteria that produce methane in the rumen.

**Keywords:** feed additive, direct fed microbials, virgin coconut oils, tannins and saponin, methane emissions, beef cattle, ammoniated palm frond

#### **1. Introduction**

The main problem in the development of ruminant livestock production in Indonesia, such as beef cattle, is the difficulty of meeting the availability of forage sustainably, both in quality and quantity. Therefore, the use of plantation waste such as palm fronds, rice straw as animal feed is an alternative that can be done to overcome the problem of feed availability. The utilization of plantation waste as ruminant feed is still minimal due to the high content of lignin [1] which causes low digestibility [1–3]. To optimize plantation waste as animal feed, it is necessary to combine processing techniques and optimize bioprocesses in the rumen [3], which aims to increase the microbial population and streamline the fermentation process in the rumen.

Supplementation of direct-fed microbial (DFM) and methanogenesis inhibitors is a way that can be done to increase the efficiency of rumen fermentation [3–5]. DFM is a feed additive product that contains a source of live microorganisms [6], can modify the rumen ecosystem [7], synthesize nutrients so that their availability can increase livestock growth [8]. *S. cerevisiae* is one of the DFM microbes that can be added together with other bacteria and fungi such as Aspergillus sp. and Bacillus sp. [3]. The administration of S. cerevisiae as an additive to live microbes into the body will affect the host by improving the balance of rumen microorganisms [9]. *S. cerevisiae* can compete with starch bacteria [10].

High-fiber feeds such as plantation waste reduce not only the efficiency of feed use [11] but also increase the production of methane gas (CH4) [12]. In the livestock sector, methane is one of the gaseous products of fermented feed ingredients by rumen microbes. Ruminants account for more than 75% of methane emissions from total greenhouse emissions [13]. The release of methane causes an increase in the concentration of CH4 in the air and causes energy loss of 6–13% from the feed [14]. Many livestock nutritionists try to reduce methane production because they feel responsible for the contribution of the livestock sector to atmospheric pollution by methane, as one of the pollutants that is always associated with global warming [15]. Decreased methane production in the rumen is closely related to the metabolic activity of protozoa [16]. Ciliated protozoa in the rumen are in symbiosis with methane bacteria, so that by reducing the population of ciliated protozoa, it will reduce the availability of hydrogen for the formation of methane [17].

Tannins are plant bioactive compounds that can reduce methane production because they act as protozoal defaunation agents [18]. The results of the metaanalysis of *in vivo* experiments with tannins reported by Jayanegara et al., [19] revealed that the concentration of tannins is closely related to the production of CH4 produced. Different sources of tannins have been shown to have different impacts on CH4 production. This is probably because the composition and types of tannins [12] are different from different sources. In addition to tannins, Virgin coconut oil (VCO) contains many medium-chain fatty acids (MCFA). Mediumchain fatty acids (MCFA) are known to have a high potential to suppress rumen methanogenic bacteria [20]. The most abundant MCFA in VCO was lauric acid (C12: 0) 51.95% [21]. Soliva *et al*., [22] stated that lauric acid (C12: 0) is more effective in suppressing methanogenesis than myristic acid (C14: 0). The ability of VCO to modify the rumen ecosystem depends on the level of its addition in the feed [23]. The high lauric acid content in VCO will allow VCO to have the ability as a defaunation agent against ciliated protozoa and inhibit archaea methanogens in the rumen.

*Effect of Various Feed Additives on the Methane Emissions from Beef Cattle Based… DOI: http://dx.doi.org/10.5772/intechopen.100142*

Based on the description above, this chapter book presents several reviews of the results of the author's research, which combines a combination of processing techniques and optimization of bioprocesses in the rumen to increase the value of benefits from plantation waste that can be packaged into complete quality rations, able to increase livestock productivity and reduce beef cattle methane production.

#### **2. Direct fed microbial and virgin coconut oils on methane gas production**

#### **2.1 Effect direct-fed microbes on rumen microbial population**

Direct-fed microbes (DFM) have comparable results to probiotics. DFM is a feed product that contains a source of live microorganisms [6]. DFM is commonly used as a supplement to increase livestock production. DFM commonly used in ruminants is yeast. DFM works to modify the rumen ecosystem to create an optimal environment for the development of rumen microbes. The provision of DFM as an additive to live microbes in the feed will affect the host by improving the balance of rumen microorganisms [9].

The three-stage series of research has been conducted by Suryani *et al*., [3]. Phase I is a research aimed at optimizing the bioprocess in the rumen through DFM to increase the rumen microbial population. Three types of DFM were used, namely *Saccharomyces cerevisiae*, *Aspergillus oryzae*, and *Bacillus amyloliquefacien*. The substrate used was based on palm frond, which had previously been ammoniated using 6% urea. The evaluation was carried out *in vitro* [24] to determine nutrient degradation and rumen fermentability. The effect of DFM supplementation on rumen fermentability [3] is shown in **Table 1**.

The results showed that DFM supplementation in feed based on plantation waste in the form of ammoniated palm frond could increase rumen fermentability. The


*Source: Suryani et al., 2016, DOI: 10.3923/pjn.2017.599.604*

*Numbers followed by different lowercase letters in the same column (a, b, c, d, and e) were significantly different (P < 0.05), SC: Saccharomyces cerevisiae, AO: Aspergillus oryzae, BA: Bacillus amilolyquifaciens, P0: Ammoniated palm fronds, P1: P0 + SC (1%), P2: P0 + AO (1%), P3: P0 + BA (1%), P4: P0 + SC (0.5%) + AO (0.5%), P5: P0 + SC (0.5%) + BA (0.5%), P6: P0 + AO (0.5%) + BA (0.5%), P7: P0 + SC (0.3%) + AO (0.3%) + BA (0.3%), supplementation of DFM % on dry matter basis;*

#### **Table 1.**

*Supplementation of DFM in ammoniated palm fronds on fermentability and bacteria population in vitro.*

bacterial population increased from 1.61 x 109 to 2.35 x 109 cell mL-1. These results are following the results of research [1, 9] where the addition of probiotics in the ration can stimulate the development of microbes in the rumen and increase the digestibility of food in livestock. The way yeast works in the rumen can utilize oxygen to ensure anaerobic conditions for rumen bacteria and stimulate specific rumen bacterial populations [25] (**Figure 1**). However, there was a tendency for the bacterial population to decrease in the combination supplementation of three types of DFM (P7). It was suspected that there was an accumulation of rumen microbial growth so that bacteria in the rumen competed in digesting feed. The total NH3 and VFA concentrations increased from 12.28 mM to 14.28 mM and 108.35 mM to 125.90 mM. Desnoyers *et al*., [26] stated that yeast supplementation could increase the concentration of VFA (2.1 mmol L-1) and decrease the concentration of lactate.

Furthermore, DFM fungal *A. oryzae* can reduce oxygen in the rumen [27]. This situation was followed by increased ammonia and lactic acid utilization so that the rumen pH was stable. Anaerobic conditions and stable rumen pH allow more optimal microbial protein synthesis so that the total population of rumen bacteria increases and the digestibility of crude fiber increases. Increased digestibility of crude fiber will increase the consumption and supply of nutrients to the intestines, so that it is expected to increase the overall response of livestock production. Meanwhile, *B. amylolyquifaciens* DFM can produce cellulase enzymes [28], so when yeast is combined with fungal or bacterial DFM, it can increase rumen fermentation with high VFA results. The increase in rumen fermentability was also followed by dry matter and organic matter digestibility which increased from 47.5% (without DFM) to 51.55% (with DFM) and 48.89% to 52.41% [3]. DFM *S. cerevisiae* can be used individually or in combination with *A. oryzae* or *B. amylolyquifaciens*. However, when viewed from the average value produced, the *S. cerevisiae* + *B. amylolyquifaciens* combination gave the best rumen digestibility and

**Figure 1.** *Mode of action DFM in the rumen.*

*Effect of Various Feed Additives on the Methane Emissions from Beef Cattle Based… DOI: http://dx.doi.org/10.5772/intechopen.100142*

fermentability results. This is because *S. cerevisiae* can produce growth factors for microbial growth from organic acids, B vitamins, and amino acids to stimulate rumen microbial activity and development [29]. A brief diagram illustrating the working principle of DFM in the rumen. It can be seen in **Figure 1** [25] modified.

Yeast culture uses oxygen to metabolize feed particles into sugars and oligosaccharides to produce peptides and amino acids as end products used by bacteria. Most rumen microorganisms are anaerobic, so the utilization of oxygen by yeast culture will increase the optimum conditions in the rumen. These conditions will protect the anaerobic rumen bacteria from damage by O2. They created better conditions for the growth of cellulolytic bacteria so that the number of cellulolytic bacteria increases and improves digestion in the rumen [30].

Yeast activity as DFM can regulate rumen biological activity by stimulating lactic acid utilization and reducing ammonia production, so that rumen pH is stable and increases nutrient absorption and VFA profile [31]. Supplementation can support livestock productivity by increasing intestinal development, mucosal immunity, nutrient absorption, and inhibiting pathogenic bacteria. This will have an impact on improving livestock health and performance [32].

#### **2.2 Effect virgin coconut oils on methane gas concentration**

In another study, to streamline the digestive process in the rumen, Suryani *et al*., [3] continued the best DFM results from the 1st stage of the experiment to be combined with methane emission reducers. Virgin coconut oil (VCO), rich in MCFA, is used to reduce methane emissions. VCO is oil produced from fresh coconuts. VCO contains lauric acid (C12:0), which effectively suppresses methanogenic bacteria and rumen protozoa [5]. The VCO used in this study contained lauric acid (C12: 0) 51.95% [21] (**Figure 2**).

The purpose of this experiment is to get the best VCO level combined with the best type of DFM stage 1 on ammoniated palm fronds. The three VCO levels tested were 2, 3, and 4% DM. The two best types of DFM from stage 1 used as controls were *S. cerevisiae* and *S. cerevisiae* + *B. amyloliquifaciens* 1% DM. Experiments were carried out *in vitro* according to the method [24].

The effect of combined VCO and DFM supplementation on methane gas concentration and rumen protozoa population *in vitro* on ammoniated palm frond-based feed can be seen in **Table 2**, **Figures 3** and **4**.

**Figure 2.** *Fatty acid composition of VCO.*


*Note. Substrate based on Ammoniated palm frond treated with 6% urea, DFM supplementation and VCO level on dry matter basis, SC: Saccharomyces cerevisiae, AO: Aspergillus oryzae BA: Bacillus amilolyquifaciens, VCO: Virgin coconut oils;*

#### **Table 2.**

*Production of methane (CH4) and protozoa population from the fermentation of ammoniated palm fronds in vitro in the rumen for each DFM type level and VCO levels.*

#### **Figure 3.**

*The relationship between DFM + VCO levels and methane production from rumen fermentation of ammoniated palm frond during 48 hours incubation.*

The results of the orthogonal polynomial test show a quadratic relationship (P < 0.05) between the level of VCO (X, %) and the concentration of methane gas in the rumen (Y, mM) with the equation y = 1.2682x2–7.3169 + 22.281 and the coefficient of determination R2 = 0.98137 (**Figure 3**).

Based on the orthogonal polynomial test, methane gas concentration at the level of 2% VCO addition with DFM *S. cerevisiae* and *S. cerevisiae* + *B. amyloliquifaciens* decreased by 48.11% and 43.67%, respectively. The addition of a 3% VCO level also decreased methane gas concentration compared to without supplementation and resulted in an average of 11.87 mM and 12.58 mM. The decrease in methane gas concentration occurs because VCO is rich in MCFA, mainly lauric acid (C12:0) (**Figure 2**), which is effective in suppressing methanogenic bacteria and rumen protozoa [5]. Lauric acid is the most toxic to protozoa [33] and is the most potent antiprotozoal that inhibits ciliated protozoa's growth and activity (mainly *Entodinium spp.)* [22]. The decrease in ciliate protozoa population due to defaunation causes a decrease in the symbiosis between ciliate protozoa and methanogens, thereby reducing the availability of hydrogen for methane formation [17].

*Effect of Various Feed Additives on the Methane Emissions from Beef Cattle Based… DOI: http://dx.doi.org/10.5772/intechopen.100142*

#### **Figure 4.**

*The relationship between DFM + VCO levels and the population of protozoa produced by fermenting the rumen of ammoniated palm frond during 48 hours of incubation.*

Furthermore, Dohmet *et al*., [33] reported that lauric acid (C12:0) and myristic acid (C14:0) could reduce methanogenesis in the rumen and significantly reduce total methanogenic bacteria. This result is also supported by Machmuller *et al.,* [20]. The effect of coconut oil supplementation is to reduce methane by inhibiting the metabolic activity of archaea methanogens directly in the rumen.

Supplementation of *S. cerevisiae* and VCO DFM at all levels (P3, P5, and P7) can reduce methane cocntentration better than the combination of DFM *S. cerevisiae* + *B. amyloliquifaciens* and VCO at all levels (P4, P6, and P8). This indicates that when *S. cerevisiae* type DFM combined with VCO can support a decrease in methane concentration in rumen fermentation activity, this is also suspected because *S. cerevisiae* as DFM also can reduce methane. *Yeast* supplementation can also stimulate acetogenins to compete for hydrogen with methanogens, thereby reducing methane emissions [34].

The results of the orthogonal polynomial test give a quadratic relationship between the VCO level (X, %) and the protozoa population (Y, cell/mL-1), the Eq. Y = 0.7546x2–3.9464x + 7.1323 and the coefficient of determination (R2) = 0.98564 is shown on **Figure 4**. The average population of protozoa with the addition of VCO in the rumen can be seen in **Table 3**.

Based on the orthogonal polynomial test, the protozoa population decreased with VCO supplementation. Supplementation of 2% and 3% VCO (P3,P4,P5,P6) on palm fronds with the addition of DFM *S. cerevisiae* and *S. cerevisiae* + *B. amyloliquifaciens* reduced the protozoa population by 72.88%, 69.15%, 72, 17 and 63.32%, respectively. This result was also followed by a decrease in methane gas concentartion in this treatment. Protozoa populations are closely related to rumen methane production [35]. 7 to 37% of methanogens live in symbiosis with protozoa in the rumen [5]. The results of this combination of DFM and VCO supplementation resulted in a decrease in the percentage of protozoa population, which was the same as that obtained by Kongmun *et al*. [36] that the protozoa decreased 68–75% by supplementing with 7% coconut oil. Furthermore, this result is greater than that obtained [37] that coconut oil and lauric acid supplementation reduced the protozoan population by up to 40%.

Meanwhile, total protozoa (especially *Entodinium spp*) decreased by 96% due to lauric acid supplementation compared to myrystic acid on a concentrate rich substrate [38]. This indicates that DFM supplementation in high-fiber feeds such as palm

#### *Animal Feed Science and Nutrition - Production, Health and Environment*


*Source: Suryani et al., 2017,http://dx.doi.org/10.3923/ pjn.2017.599.604.*

*Numbers followed by different lowercase letters (a, b, c) in the same row are significantly different (P < 0.05), A: 100% Complete feed, B: A + 1% SC, C: A + 0.5% SC + 0.5% BA, D: A + 2% VCO + 1% SC. DM: Dry matter, OM: Organic matter, BW: Body weight, ADG: Average daily gain, SC: Saccharomyces cerevisiae, BA: Bacillus amilolyquifaciens, VCO: Virgin coconut oils;*

#### **Table 3.**

*Effect of DFM and VCO supplementation on consumption, ADG, efficiency, and methane production of Bali cattle.*

oil plantation waste plays an important role in modifying the rumen ecosystem so that the addition of VCO at the right level can reduce the concentration of methane and protozoa without reducing nutrient degradation. From the results of this study, it is recommended that 2% VCO be used for cattle *in vivo* because levels 3 and 4% give almost the same average results.

In other studies, Suryani *et al* [24] continued the experimental *in vitro* studies of stages I and II into a complete ration formulation based on ammoniated palm fronds prepared with a TDN content of 63.28%. *In vivo* tests were carried out using 16 Bali cattle to determine the effect of adding DFM *S. cerevisiae*, *S. cerevisiae* + *B. amyloliquifaciens*, and *S. cerevisiae* + 2% VCO on livestock productivity. Blood samples were collected to determine the effect of DFM and VCO supplementation on the blood profile. Blood samples were taken once before the cattle were fed in the morning (fasting). Blood samples were taken through the jugular vein using a 10 ml capacity syringe and placed in a vacutainer. Blood serum was separated using centrifugation at 3000 rpm for 10 minutes. Analysis of glucose levels, total protein, urea, BUN, albumin, triglycerides, total cholesterol, HDL, and LDL was carried out using the HumaStar 80® Auto Analyzer. A statistical test was carried out to determine the effect of treatment on the observed parameters, using a variance according to the design used. If there was a significant effect, it was continued with Duncan's test [39].

The effect of DFM and VCO supplementation on Bali cattle on performance and methane gas production [21] is shown in **Table 3**.

DFM and VCO supplementation decreased methane production by 5.26, 5.87, and 20.63% respectively. The highest ration efficiency was in DFM *S. cerevisiae* + 2% VCO supplementation, followed by ADG at 0.70 (kg/h/d) and decreased methane production by 20.63% [21]. DFM yeast was reported to have the ability to reduce methane production by 28% [40]. Yeast supplementation could also stimulate acetogens to compete for hydrogen with methanogens, thereby reducing methane emissions [41]. With reduced methane production in the rumen, it can increase feed energy, which

*Effect of Various Feed Additives on the Methane Emissions from Beef Cattle Based… DOI: http://dx.doi.org/10.5772/intechopen.100142*

positively affects livestock performance. This can be seen from the decrease in DM and OM consumption but can increase Efficiency and ADG. The digestibility of DM, OM, NDF, ADF, Cellulose, and TDN also increased with DFM supplementation and the combination of DFM *S. cerevisiae* + VCO [21]. The mechanism of DFM can reduce methane production, presumably because DFM microorganisms can stimulate the development of rumen microbes in digesting feed so that fermentation of carbohydrates in the rumen results in high production of propionate. In the rumen, propionate production requires H2 bound to glucose which is described in the following equation.

#### C6H12O6 2H2 2CH3 2COOH 4H2 + → *CH* +

Therefore, to reduce hydrogen production to methane, hydrogen must be switched to propionate production via lactate or fumarate [42]. H2 and CO2 are substrates used to form methane. According to Wilkie [43] the role of hydrogen in the methane production process is as a source of electrons, so the low level of H2 in the rumen is an indication of activity using H2 to reduce CO2 to CH. In addition, to form one mole of CH4 requires four moles of H2. The rate of H2 utilization is four times the rate of methane production so that H2 in the rumen never accumulates. The following is the stoichiometry of the carbohydrate fermentation reaction in producing methane gas in the rumen:

#### 4H2 CO2 CH4 2H2O +→+

The effect of DFM and VCO supplementation on Bali cattle on blood profile can be seen in **Table 4**.

The results showed that DFM and VCO supplementation had a very significant effect (p < 0.05) in reducing cholesterol, LDL and increasing HDL blood levels of Bali


*Numbers followed by different lowercase letters (a, b, c) in the same row are significantly different (P < 0.05), A: 100% Complete feed, B: A + 1% SC, C: A + 0.5% SC + 0.5% BA, D: A + 2% VCO + 1% SC. DFM: Direct fed microbials, SC: Saccharomyces cerevisiae, BA: Bacillus amilolyquifaciens, VCO: Virgin coconut oils, LDL: low density lipoprotein, HDL: high density lipoprotein.*

#### **Table 4.**

*Blood profile of complete diet based on ammoniated palm fronds supplemented with DFM and VCO.*

cattle. DFM and VCO supplementation had no significant effect (P > 0.05) on triglycerides, urea, protein, albumin, and glucose. VCO contains MCFA, which is a saturated fatty acid (**Figure 1**), its addition in the ration if consumed by livestock can help lower cholesterol because of the nature of this fatty acid, which can be absorbed directly by the animal's body so that it does not cause fat accumulation that causes cholesterol. This is supported by Fernando *et al.* [44], which states that MCFA is directly converted into energy in the liver and increases metabolic rate, and reduces fat deposits in the body. MCFA has a very high solubility in water and requires fewer digestive enzymes, making it burnt into energy. MCFA is burned to produce energy and encourages the combustion of LCFA [45]. So there is a significant decrease in the amount of LDL and is followed by an increase in HDL in the blood. The calories contained are also lower than long-chain fatty acids [46]. Reducing fat deposits in the body can lower LDL cholesterol and increase HDL cholesterol [47].

This study can conclude that individual *S. cerevisiae* DFM supplementation and *S. cerevisiae* + *B. amyloliquifaciens* combination can optimize bioprocesses in the rumen. VCO supplementation level of 2% can be used to suppress methane production. Supplementation of *S. cerevisiae* type DFM and 2% VCO level can be considered to optimize bioprocesses in the rumen, increasing performance and reducing methane production in Bali cattle fed complete rations based on ammoniated palm fronds.

#### **3. Effect of different source tannins on methane gas production**

Bioactive compounds, including polyphenols, carotenoids, omega-3 fatty acids, vitamins, organic acids, nucleotides, and nucleosides, have attracted significant attention for their role in preventing several chronic diseases in humans. In animal husbandry, especially ruminant nutrition, bioactive plant polyphenolic compounds such as tannins and saponins have been studied extensively for optimizing bioprocesses in the rumen through feed manipulation. Manipulation of feed using tannins as an agent of rumen defaunation is one way to overcome global climate change due to the effects of greenhouse gases, one of which is caused by methane gas from ruminants [18]. Feeds containing tannins will be anti-nutrients that limit livestock production when the crude protein concentration in the feed is high because it can reduce the absorption of amino acids [48]. Tannins can also cause poisoning if consumed by livestock in excess, and there are many *in vitro* and *in vivo* studies that describe the methane inhibitory effect of tannins [19]. The study results Staerfl *et al*. [49] proved that the use of tannins could reduce CH4 emissions by up to 36% in bulls fed grass, corn silage, and concentrate rations. Not many studies have explored the use of tannins in feed based on plantation waste. Therefore, the authors are interested in conducting a series of experiments using tannins from different sources. Plant bioactive compounds used are tannins derived from *gambir leaves waste* (GLW) and obtained from two different sources or areas, namely GLW Payahkumbuh and Painan. GLW was added at different levels (10, 15, 20%) to the ammonium palm midrib substrate with the addition of 4% urea [50]. Experiments were carried out *in vitro* and *in vivo*.

In another *in vitro* study in the same group, the authors also tried to compare *Gliricidia sepium* in animal feed based on rice straw plantation waste [51]. *Gliricidia sepium* is a bioactive plant compound containing thick tannins and saponins capable of modifying the number of rumen microbes such as archaea, protozoa, and fibriolytic bacteria that affect the fermentative process and production of methane gas [52]. The study was conducted *in vitro*. Complete feed is prepared based on ammoniated

*Effect of Various Feed Additives on the Methane Emissions from Beef Cattle Based… DOI: http://dx.doi.org/10.5772/intechopen.100142*

rice straw. Three levels of *Gliricidia sepium* tested were 10, 20, and 30% DM basis. The study results, the effect of different sources and levels of tannins on dry matter digestibility (DM), organic matter (OM), methane gas concentration, protozoa, and bacteria can be seen in **Table 5**.

The results showed that different sources and doses of tannins proved to have different effects on decreasing methane production [50]. The *in vitro* study results showed that supplementation of 15% GLW and 10% GLW, which had a total tannin concentration of 12.5 and 15.6% dry matter, respectively, could reduce methane gas concentration by 53% and 45% compared to control. The decrease in methane gas was followed by a decrease in the protozoa population by 53.89% compared to control. Different levels and sources of GLW had no significant effect (P > 0.05) on the total bacterial population. However, there is a tendency for the bacterial population to increase as the population of protozoa and methane decreases. Tannins decrease methane production by reducing methanogenic bacteria and protozoa [53]. Furthermore, it was reported that condensed tannins extracted from different plants had different effects on rumen fermentation characteristics. This is because it is associated with different chemical structures and molecular weights [54, 55]. Condensed tannins extracted from different plants have varied activities in binding carbohydrates and proteins [56].

Furthermore, the *in vitro* results of the addition of GLW as a source of tannins were tested *in vivo* on three Simmental cattle [12] with a weight ranging between 179 and 190 kg using the BSL design. The results showed that two sources of tannin levels could increase nutrient digestibility but had no effect on protein digestibility,


*Sources: Ningrat et al., 2017; DOI: 10.3923/ajas.2017.47.53; Zain et al., 2020; DOI:10.18517/ijaseit.10.2.11242. Different superscripts in the same column highly significant effect (p < 0.05), T0: Oil palm frond ammoniated previously treated by 4% urea as control, B1: A + 10% GLW Payakumbuh, B2: A + 15% GLW Payakumbuh, B3: A + 20% GLW Payakumbuh, C1: A + 10% GLW Painan, C2: A + 15% GLW Painan, C3: A + 20% GLW Painan. A: 40% ammoniated rice straw +60% concentrate, B: 40% ammoniated rice straw +50% concentrate + 10% Gliricidia sepium, C: 40% ammoniated rice straw +40% concentrate +20% Gliricidia sepium, D: 40% ammoniated rice straw +30%, DM: Dry matter, OM: Organic matter, VFA: Volatile fatty acid, GLW: gambir leaves waste;*

#### **Table 5.**

*Effect different sources and doses of tannin on dry matter (DM), organic matter (OM), protozoa population, methane (CH4) production, VFA total, and acetate: Propionate ratio based on agriculture by-product as feed in the rumen.*

urinary allantoin, and nutrient consumption. The addition of 15% GLW tannins and 10% GLW Painan in the ration significantly increased ADG and decreased methane production compared to controls, namely 0.65, 0.90, 0.92 kg/day, and 2.48, 1.28, 1.26 MJ/day [12]. Saponins contained in GLW can increase the efficiency of rumen fermentation through the mechanism of reducing the population of protozoa [57]. The decrease in the protozoa population will cause the availability of H2 for methanogens to decrease [58]. The reduction in protozoa population supports stabilization of rumen pH and an increase in the population of cellulolytic microorganisms. Thus, decreased methanogenesis will increase the efficiency of digestibility in high fiber rations and livestock performance.

The addition of *G. sepium* in the diet resulted in a decrease in methane production and the highest protozoa population at the levels of 20 and 30%, namely 12.67, 13.16 mM, and 4.9 x 105, 4.7 x 105 cell/ml *in vitro*. However, there was no significant difference (P > 0.05) between the two levels. The treatment had no significant effect (p > 0.05) on total VFA, acetate, butyrate, valerate + isovalerate + isobutyrate. Acetate propionate ratio decreased respectively to 2.14, 1.50, 1.70, 1.33. The propionate concentration increased by 43.87% compared to the control, and there was no significant difference (P > 0.05) between levels of gliricidia addition [51]. Plant bioactive compound *Gliricidia sepium* contains tannins and saponins, which effectively reduce the population of protozoa and methane production. The feed used in this study was based on agricultural waste with high fiber content. In addition to saponin's structure, which can affect protozoa's activity, the type of feed given can also affect the fermentation process in the rumen [59]. In the study Zain *et al*., [51] the types of protozoa that survived the addition of *Gliricidia sepium* were not identified. However, the results obtained showed that the saponins and tannins in *Gliricidia sepium* could inhibit certain types of protozoa that cause a decrease in protozoa population in the rumen. The decrease in methane production and the protozoa population with 20 and 30% *Gliricidia sepium* can increase the digestibility of dry matter and organic matter produced [51].

The potential of plant bioactive compounds such as tannins and saponins as defaunation agents and reducing methane emissions can be combined with directfed microbes. There is not much literature on decreasing methane production that combines the two in *vivo* studies. *In vitro* studies Arowolo *et al*. [60] stated that there is a synergistic effect between probiotics and plant bioactive compounds simultaneously to stabilize the rumen fermentation process and reduce methane production. However, it still requires further studies at the *in vivo* level. Based on these results, Ningrat *et al*. [61] conducted a test of *Gliricidia sepium* and DFM *S. cerevisiae* supplementation to improve the performance of Simmental cattle while reducing methane gas production. It was found that the combined supplementation of 1% SC and 15% *Gliricidia sepium* significantly increased the digestibility of DM, and OM, ADG, and methane gas production compared to *S. cerevisiae* and *Gliricidia sepium* supplementation individually. The decrease in methane production with the addition of SC, GLW, and the combination of *S. cerevisiae* + *Gliricidia sepium* respectively 1.42, 1.35, and 1.02 MJ.day-1 [61]. These results prove that yeast culture can work synergistically when combined with reducing agents. Emissions of methane plant bioactive compounds such as tannins and saponins. Tannin compounds inhibit the activity of methanogens [62] and can defaunate [63]. Pineiro-Vazquez *et al*. [64] reported the results of an *in vivo* evaluation showing the effect of 80% Leucaena sp. (21% condensed tannins) in the diet composition was able to reduce methane emissions by 61.3% without affecting nutrient intake and VFA production in the Bos taurus × Bos indicus cross.

*Effect of Various Feed Additives on the Methane Emissions from Beef Cattle Based… DOI: http://dx.doi.org/10.5772/intechopen.100142*

#### **4. Conclusion**

In conclusion, the overall reduction in methane production in agriculture byproducts as feed-based beef cattle can be made by improving feed quality through a combination of processing techniques and efforts to optimize bioprocesses in the rumen, which include supplementation of feed additives such as direct-fed microbials, methanogenesis inhibitors and plant bioactive compounds. Supplementation of DFM type *S. cerevisiae* 1% combined with 2% VCO can reduce methane production by 20.36% and increase ADG by 0.70 kg/day in Bali cattle. Plant bioactive compounds, especially tannins from *Gliricidia sepium*, can be used up to 15% in amniotic palm frond-based rations. *Gliricidia sepium*, which contains tannins and saponins at levels of 20 and 30% dry matter in complete rations, can also reduce methane, protozoa population and increase livestock performance. The combination of DFM *S. cerevisiae* and *Gliricidia sepium* can also be used to reduce methane gas production in Simmental cattle fed complete feed based on 46.61% amniotic palm fronds compared to controls.

#### **Acknowledgements**

Thank to the Ministry of Research and Technology, and Andalas University that providing grants to support the research.

### **Author details**

Mardiati Zain1 \*, Rusmana Wijaya Setia Ningrat1 , Heni Suryani<sup>2</sup> and Novirman Jamarun1

1 Faculty of Animal Science, Universitas Andalas, Padang, Indonesia

2 Faculty of Animal Science, Universitas Jambi, Jambi, Indonesia

\*Address all correspondence to: mardiati@ansci.unand.ac.id

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### **Chapter 7**

## Nutritional Interventions to Reduce Methane Emissions in Ruminants

*Lipismita Samal and Susanta Kumar Dash*

#### **Abstract**

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

**Keywords:** methane, rumen fermentation, greenhouse gases, climate change, mitigation strategies

#### **1. Introduction**

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

genera of methanogens: the Methanosarcina and Methanosaeta can convert acetate to CO2 and CH4 (acetoclastic methanogenesis) [2]. Since methane contains energy, its emission during rumen fermentation is considered to be a loss of feed energy that is equivalent to 2–12% of dietary gross energy of animal feed.

#### **2. Greenhouse gas effect**

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

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

#### **3. Livestock role in climate change**

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

#### **4. Rumen fermentation and methanogenesis**

Digestion of feed in the rumen is the result of anaerobic fermentation involving various groups of microbes (bacteria, archaea, protozoa, and fungi). Methane is

*Nutritional Interventions to Reduce Methane Emissions in Ruminants DOI: http://dx.doi.org/10.5772/intechopen.101763*

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

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

#### **5. Nutritional interventions to reduce enteric methane emission**

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

#### **5.1 Manipulating nutrient composition of the diet**

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

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

susceptible. When the ruminal pH is unaltered by feeding different grasses, methanogenesis could be increased by extra WSC and then sugars exhibit a higher methanogenic potential than starch [15]. There will be higher methane emission when WSC replaces the rumen degradable protein instead of fiber [16].

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

#### **5.2 Supplementation of lipids and lipid by-products**

#### *5.2.1 Dietary lipids*

The use of lipids is considered as one of the promising dietary alternatives to depress ruminal methanogenesis. The effectiveness of fat supplementation depends mainly on the fat source, fatty acid profile, form of fat and the amount of supplemented fat [13]. Possible mechanisms by which added lipid can reduce enteric methane production include: (a) by reduction of fiber digestion (mainly in long-chain fatty acids); (b) by lowering of dry matter intake (if total dietary fat exceeds 6–7%); (c) by decreasing organic matter fermentation (d) through direct inhibition of activities of different microbes including methanogens and hydrogen producing microorganisms; (e) through suppression of rumen protozoa; and (f) to a limited extent through biohydrogenation of unsaturated fatty acids which serve as a hydrogen sink, although only 1–2% of the metabolic hydrogen in the rumen is used for this purpose [13, 17]. Fat can reduce CH4 emissions by 4–5% (g/kg DMI) for every 1% increase in the fat content of the diet. Addition of different vegetable oils (soybean, coconut, canola, rapeseed, sunflower, linseed etc.) to ruminant diets have been shown to reduce CH4 production between 18% and 62% in Rusitec fermenters [18], sheep [19], beef cattle [20] and dairy cows [21]. Beauchemin et al. [13] estimated a reduction of enteric CH4 formation of 0.56% per g of lipid supplied per kg diet DM. Plant oils rich in MCFA such as coconut oil [major component is lauric acid (C14:0)] are known to inhibit rumen methanogenesis [18]. The addition of coconut oil to forage and concentrate rations supplemented to Charolais steers showed a reduction in voluntary intake and protozoa population and this was reflected in low CH4 emissions, without affecting livestock production [22]. The lauric acid (C14:0) is more potent in CH4 reduction than palmitic (C16:0), stearic (C18:0) and linoleic (C18:2) fatty acids in a semicontinuous fermenter that simulates the rumen (RUSITEC) [18]. A similar reduction in CH4 was observed in batch cultures, in which coconut oil and lauric acid were directly compared. It showed that lauric acid inhibited methanogenesis to a greater extent [23]. The ability of lauric acid to decrease cell viability of Methanobrevibacter ruminantium has been reported [24]. The lauric acid treatment, possibly through its effect on protozoa physically associated with archaea, resulted in an increase in the archaeal methanogenic genus Methanosphaera and a decrease in Methanobrevibacter [25]. Besides lauric acid, other MCFA such as myristic acid, or a combination of both and PUFA like linolenic acid and linoleic acid were shown to be effective, but might also negatively influence feed intake and digestibility.

#### *Nutritional Interventions to Reduce Methane Emissions in Ruminants DOI: http://dx.doi.org/10.5772/intechopen.101763*

The vegetable and fish oils significantly decreased CH4 production after 14 d but not after 11 weeks of feeding in dairy cows [26]. However, persistence of the mitigating effect of dietary oil was observed in the study of Martin et al. [27] with flaxseed in dairy cows. Meta-analyses by Moate et al. [28] documented a consistent decrease in CH4 production with fat supplementation. Other studies have reported a 27% reduction in CH4 emission with the supplementation of fish oil and sunflower oil 500 mg/d each when fed to dairy cows in short periods (14 days) [26]. The reduction in methanogesis with oils/lipids appears to be the result of inhibition of microbial flora especially protozoa.

#### *5.2.2 Lipid by-products*

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

#### **5.3 Plant secondary metabolites**

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

#### *5.3.1 Tannins*

Tannins are plant polyphenols of varying molecular size and exist in two forms in plants: hydrolysable tannin (HT) and condensed tannin (CT). Tannins, as feed supplements or as tanniferous plants have shown potential for reducing CH4 emission by up to 20% [35]. Different types of tannin containing forages decreased CH4 emission in vitro. The CH4 inhibiting potential of tannins might be due to a direct effect on ruminal methanogens and an indirect effect on lower feed degradation leading to a decreased hydrogen production. Tannins and phenolic monomers have been found to be toxic for some of the rumen microbes, especially ciliate protozoa, fiber degrading bacteria and methanogenic archaea, and as a result methanogenesis in the rumen can also be reduced. The anti-methanogenic effect of tannins depends on its dietary concentration and is positively related to the number of hydroxyl groups in their structure. The hydrolyzable tannins tend to act by directly inhibiting rumen methanogens

whereas the effect of condensed tannins (CT) on CH4 production is more through inhibition of fiber digestion. In many studies (in vitro and in vivo) it has been demonstrated that with temperate legumes (*Hedysarium coronarium*, *Lespedeza cuneata*, *Lotus corniculatus* and *Lotus uliginosus*) and tropical legumes (*Calliandra calothyrsus*, *Flemingia macrophylla*) that contain CT, it is possible to reduce methanogenesis. The methane suppression effect of CT containing legumes, such as *Lotus pedunculatus* or *Acacia mearnsii*, relative to forages without tannins has been shown in sheep [36], cows [37] and goats [38]. *Ficus bengalensis*, *Autocarous integrifolis* and *Azadirachta indica* had also been shown to reduce methane production [39]. Ramirez-Restrepo and Barry [36] indicated that the CT-rich legumes such as *L. corniculatus* and sulla (*Hedysarum coronarium*) showed reduced methane production relative to forages without tannins (*Chicorium intybus*). In goats fed with the CT containing forage Sericea lespedeza, Puchala et al. [40] observed a reduction in CH4 loss of over 30%. Methanol extract of harad (*Terminalia chebula*) caused 95% reduction in CH4 production in vitro at the level of 0.25 ml/30 ml incubation medium and complete inhibition was observed when the level of extract was double [41]. In goats consuming different levels of CT from *Lespedeza striata*, there was a reduction in the emission of CH4, while in the same study feeding Sorghum bicolor with lower levels of CT showed no reduction of enteric production of CH4 [38].

#### *5.3.2 Saponins*

Saponins are naturally occurring surface-active glycosides with foaming characteristics, present in many plant species, wild plants as well as, cultivated crops. They usually consist of a sugar moiety linked to a hydrophobic compound, either triterpenoid or steroid in nature. Saponins reduce CH4 production via inhibition of either protozoa or methanogens or both. These inhibited protozoa at relatively low concentrations whereas higher concentrations were required to kill or suppress methanogenic archaea. McAllister and Newbold [9] have suggested that a decrease in methanogens associated with protozoa as exo- and endosymbionts could be the main mechanism by which saponin feeding reduces methanogenesis and methanogens associated with protozoa are estimated to be responsible for 9–37% of the total CH4 production in the rumen. Anti-methanogenic activity of saponins is believed to occur by limiting hydrogen availability to methanogens and re-channeling of metabolic hydrogen from methane to propionate production in the rumen. In addition, saponins, due to their chemical structure, may display anti-bacterial properties by reducing the number of bacteria producing H2 thus resulting in the inhibition of H2 production thereby reducing CH4 formation. Goel and Makkar [42] summarized that there was no difference in the CH4 mitigation effect between steroidal saponins (*Yucca schidigera*) and triterpenoid saponins (*Quillaja saponaria*). Studies from China have reported decreased CH4 in ruminants treated with tea triterpenoid saponins (TS) but also substantial changes in microbial populations, including a reduction in protozoal counts [43]. Therefore, a reduction in the rumen protozoa population as a result of inclusion of TS in the diet could result in a decrease in enteric CH4 production. Zhou et al. [44] reported that addition of TS reduced CH4 production mainly by inhibiting protozoa, increasing molar proportions of propionate and decreasing acetate/propionate ratio without adversely altering relative ruminal abundance of fungi and cellulolytic bacteria. According to Lila et al. [45], supplementation of feed rations consisting of meadow hay and concentrate with saponins reduces CH4 production in steers by 12.7%, while in the in vitro conditions during 24 h incubation, the reduction amounted ~15–44%. Hess et

#### *Nutritional Interventions to Reduce Methane Emissions in Ruminants DOI: http://dx.doi.org/10.5772/intechopen.101763*

al. [46] reported that the daily CH4 production was reduced by 6.5% due to supplementation of *Sapindus saponaria* fruits in sheep receiving tropical grass hay-concentrate diet. Hess et al. [47] found that supplementation with *S. saponaria* saponin at 100 mg/g DM reduces methanogenesis by about 20% with no influence on the population of methanogens in the in vitro conditions. Wang et al. [48] reported a decreased CH4 formation when feeding sarsaponins to sheep (0.13 g/kg diet). Saponins from *Sapindus murkossi* extracted with the use of ethanol, more effectively affect the process of methanogenesis in comparison to water and methanol extracts [49]. High effectiveness in the reduction of CH4 production in the rumen ecosystem is possible to achieve also with the use of unextracted plant saponins, provided in the form of leaves or seeds (*Sesbana sesban*; *Trigonella foenum-graecum*). Seeds of temperate climate legumes (e.g., lupines, peas) are known to contain certain levels of tannins, and also of saponins.

#### *5.3.3 Essential oils*

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

#### *5.3.4 Combination of different plant secondary metabolites*

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

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

#### **5.4 Flavonoids**

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

#### **5.5 Phenolic acid**

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

#### **5.6 Statins**

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

#### **5.7 Other metabolites**

Methane inhibition has been demonstrated with dietary supplementation of various plant extracts, without identification of the active agents. Broudiscou et al. [66] investigated the effect of 13 plant extracts in continuous culture and showed that

*Equisetum arvense* and *S. officinalis* had possible inhibitory effect on CH4 production. Patra et al. [41] studied the effect of water, methanol and ethanol extracts of *Acacia concinna*, *T. chebula*, *T. bellirica*, *Emblica officinalis* and *A. indica* in vitro and observed reduction in CH4 production by *T. chebula*. A similar study [67] comparing *Y. schidigera* extract to *Castanea sativa* wood extract (containing HT and lignan) in in vitro rumen models showed effects on CH4 production only at very high levels. Although rich in a long list of plant secondary metabolites, macahypocotyls and lupine seeds had no effect on enteric CH4 formation [68]. Lupine seeds promoted methanogenesis in relation to the energy content of the diet as the increase per unit of SCFA shows when feeding about 200 g lupine seeds/kg DM to sheep [69].

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

#### **5.8 Prebiotics**

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

#### **5.9 Direct-fed microbials or probiotics**

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

#### *5.9.1 Yeast culture*

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

#### *5.9.2 Acetogens*

Reductive acetogens are bacteria present in adult ruminants that reduce two moles of CO2 to acetate by oxidation of H2 in rumen unlike hydrogenotrophic methanogens, which utilize H2 to reduce CO2 to CH4. So, acetogens are in direct competition with the methanogens. However, the affinity of the reductive acetogens for H2 is 10–100 times lower than the hydrogenotrophic methanogens and the low partial pressure of H2 in the rumen is not conducive for the acetogens to grow autotrophically [81]. So, while acetogens are present in the rumen, methanogens effectively outcompete them for hydrogen [9]. Acetogenic bacteria demonstrate higher population densities and an ability to be dominant under some conditions (e.g., in some macropods) [82]. Acetogenic bacteria are present in the rumen at population densities which may reach that of methanogens but despite their presence, reductive acetogenesis is extremely difficult to induce in the rumen. When methanogens are inhibited from the rumen by some means, they are capable of using this excess hydrogen to form acetate. Researchers are investigating these reactions with the aim of survival of acetogenic bacteria in the rumen and hence the displacement of methanogenic bacteria. An alternative approach would be to screen a range of acetogenic bacteria for their activity in rumen fluid and to introduce the acetogens into the rumen as a feed supplement.

#### *Nutritional Interventions to Reduce Methane Emissions in Ruminants DOI: http://dx.doi.org/10.5772/intechopen.101763*

Lopez et al. [83] reported that *Eubacterium limosum* ATCC 8486 and Ser 5 increased acetate production and decreased H2 formation when they were added to cultures of mixed ruminal microorganisms along with 2-bromoethanesulfonic acid (BES). In a rumen fistulated wether with continuous infusion of a 2-BES solution showed adaptation by methanogens after initial inhibition but use of cattle caecal contents, which contained acetogens, removed this adaptation effect [84]. Nollet et al. [81] reported that addition of Peptostreptococcus productus to BES-treated ruminal samples inhibited CH4 production. On the basis of feed intake, VFAs, population density and hydrogen utilization pattern, it was suggested that reductive acetogenesis can sustain a functional rumen in the absence of methanogens [85].

#### *5.9.3 Methane oxidizers*

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

#### *5.9.4 Bacteriocins*

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

#### *5.9.5 Fungal metabolites*

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

#### *5.9.6 Methane reducing species*

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

#### **5.10 Conclusion**

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

#### **Author details**

Lipismita Samal\* and Susanta Kumar Dash Odisha University of Agriculture and Technology, Bhubaneswar, India

\*Address all correspondence to: lipismitasamal@gmail.com

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### **Chapter 8**
