Citation of this paper

Mitigating methane emissions for rural tropical livestock through selecting appropriate grass types

Muhammad Ridla^1,2, Nevyani Asikin³, Anuraga Jayanegara^1,2 and Anjas Asmara Samsudin⁴

¹Departmen of Nutrition and Feed Technology, Faculty of Animal Science, IPB University, Kampus Dramaga, Bogor 16680, Indonesia
hmridla@apps.ipb.ac.id
²Center for Tropical Animal Studies (CENTRAS), IPB University, Kampus IPB Baranangsiang, Jl. Raya Pajajaran, Bogor 16153, Indonesia
³Department of Animal Science, Politeknik Pertanian Negeri Pangkajene Kepulauan, Jalan Poros Makassar-Parepare Km. 83, Pangkep 90652, Indonesia
⁴Department of Animal Science, Faculty of Agriculture, Universiti Putra Malaysia, Serdang Selangor, Darul Ehsan 43400, Malaysia

Abstract

Livestock farming contributes significantly to greenhouse gas emissions, with ruminant production being a major contributor. Manipulating the nutrient content of the diet can affect methane emissions in ruminants. However, tropical grasses, which are crucial in the ruminant diet in tropical regions, have limited potential due to their high structural carbohydrate and low non-structural carbohydrate content. The study aimed to identify appropriate tropical grass species that produce less methane gas during rumen fermentation. Seven tropical grass species were tested for their proximate and cell wall compositions, sugar and starch contents, and in vitro rumen fermentation parameters, including total short-chain fatty acid (TSCFA) and methane production. ANOVA was used to analyze the data and the Duncan test was applied if there were (p<0.05) differences. The results showed variation (p<0.05) in nutrient content, including structural and non-structural carbohydrates, among the grasses. Brachiaria mulato grass was found to produce the lowest methane production (29.91% TSCFA) and had the highest total starch content (13.40% DM), with the lowest NDF (67.54% DM) and ADF (37.33% DM) among the selected tropical grasses. Conversely, grasses with high structural carbohydrate content, such as Panicum maximumvar. Trichoglume produced higher levels of methane (33.13% TSCFA; p<0.05). The study emphasized the significant relationship between nutrient content and methane production. Brachiaria mulato was identified as a valuable addition to the ruminant diet, as it effectively mitigates greenhouse gas emissions while enhancing the nutritional quality of livestock feed. Selecting appropriate grass types is crucial for reducing greenhouse gas emissions and improving livestock feed quality.

Keywords: Brachiaria mulato, carbohydrates, greenhouse gas, structural, tropical grasses

Introduction

The livestock sector is a significant contributor to greenhouse gas (GHG) emissions, with ruminant production responsible for a large proportion of these emissions, particularly through the production of methane during feed fermentation in the rumen (Tapio et al 2017). Manipulating the nutrient content of the diet, especially the content of structural and non-structural carbohydrates, has been found to affect methane emissions. This emphasizes the importance of understanding the role of structural and non-structural carbohydrates in rumen fermentation for developing effective mitigation strategies (Jayanegara et al 2015).

Tropical grasses are a significant part of the diet for ruminants in tropical regions, but their high structural carbohydrate and low non-structural carbohydrate content limit their potential for animal growth and production. However, some tropical grasses have higher non-structural carbohydrates, which could improve their nutritional value for ruminants (Jayasinghe et al 2022). Determining some types of tropical grasses that contain higher non-structural carbohydrate content become essential for improving feeding and management practices.

Ruminant fermentation is a complex process involving the breakdown of ingested feed by microbial populations in the rumen. Methanogenic archaea convert some of the byproducts, such as hydrogen and carbon dioxide, to methane, which is then released through eructation and contributes significantly to GHG emissions from the livestock sector (Zábranská and Pokorna 2017).

In rural areas of Indonesia, livestock feed management often receives little attention in terms of quality, which can contribute to high methane gas emissions. Therefore, increasing knowledge and awareness of appropriate grass types that can help mitigate methane gas production in livestock is imperative. By selecting appropriate grass types, rural farmers can potentially reduce their greenhouse gas emissions while also improving the nutritional quality of their livestock feed.

This experiment aimed to identify appropriate tropical grass types that produced less methane gas during rumen fermentation to help rural farmers contribute to methane mitigation efforts. By selecting grasses that could effectively reduce methane emissions, farmers could potentially reduce their environmental impact while improving the nutritional quality of their livestock feed.

Materials and methods

Sample preparation

The grass materials used in this study were Brachiaria mulato, Panicum maximum var. Trichoglume, Setaria splendida, Pennisetum purpureum cv. Mott, Paspalum atratum, Pennisetum purpupoides and Setaria anceps. All grass species were grown in the experimental field of the Department of Animal Nutrition and Feed Technology, Faculty of Animal Science, IPB University, Bogor, Indonesia. Bogor has a tropical rainforest climate with an average annual temperature ranging from 22°C to 29°C and an annual rainfall of approximately 3,500 mm (137.8 inches). All grass samples were harvested 60 days after planting, with 3 kg of each grass being taken and then chopped, oven-dried at 60ºC, and ground to a sieve size of 1 mm.

Nutrient composition determination

The chemical analysis of all grass samples was performed using proximate analysis (AOAC 2015), while the Van Soest et al (1991) method was used to determine the cell wall components. Sugar and starch analyses were performed using the anthrone (Deriaz 1961) and acid-hydrolysis (Pirt and Whelan 1951) methods, respectively.

In vitro analysis

In vitro fermentation was conducted to measure dry matter and organic matter digestibility, short-chain fatty acid (SCFA) concentration, and methane production. The concentration of SCFA was determined using gas chromatography (Hewlett Packard 6890 GC system) (Cottyn and Boucque 1968), while methane production was estimated based on the calculation of Moss et al (2000). The concentration of ammonia nitrogen was determined according to Parsons et al (1984). Gas production kinetics were evaluated according to the method described by Menke and Steinglass (1988).

Data analysis

A completely randomized design (CRD) with seven treatments (various species of tropical grasses) and four replicates was employed. To assess the effect of the in vitro study, a randomized complete block design (RCBD) with seven treatments and four groups as replicates were used. The groups were differentiated based on differences in rumen fluid incubation. If differences were found (p< 0.05), Duncan's multiple range test was conducted. All statistical procedures were carried out using the SPSS 26 software.

Results and discussion

Nutrient composition

In this study, it was found that the nutrient composition of the tested tropical grasses varied. Table 1 shows that Paspalum atratum grass had the highest crude protein content at 10.52 (% DM), while Pennisetum purpureoides grass had the lowest crude protein content at 9.13 (%DM). The forages in this study had a crude fiber content ranging from 31.6% to 40.1 (% DM). The grass species Pennisetum purpureum cv. Mott had the highest fiber content among all the grasses studied, with NDF and ADF values of 77.53 (% DM) and 46.83 (% DM), respectively (as presented in Table 2). In contrast, Brachiaria mulato had the lowest fiber content, with NDF and ADF values of 67.54 (% DM) and 37.33 (% DM), respectively, among the grasses studied. The total starch content among the grasses varied (p<0.05), with values ranging from 8.52 to 13.40% (% DM). The lowest total starch content was found in Pennisetum purpureum cv. Mott, with a value of 8.52 (% DM), while the highest total starch content was found in Brachiaria mulato, with a value of 13.40 (% DM).

The differences in nutrient content among the grasses can be attributed to various factors, including the optimal harvest age. Younger grasses are known to have higher protein content and lower fiber content (Arthington and Brown 2005). Additionally, factors such as grass variety, fertilization, water availability, harvesting location, and environmental conditions can also affect nutrient composition (Fikru 2015; Acero-Camelo et al 2020; Jayasinghe et al 2022). In addition, Pelletier et al (2010) found that total nonstructural carbohydrates concentration in forages varied through species selection.

Table 1. Nutrient content of selected tropical grasses (% DM).
Grass	DM (%)	CA	CP	NFC	CF	EE

Bm	16.72±0.25^b	14.62±0.23^ab	10.42±0.01^a	40.73±0.19^c	32.61±0.66^c	1.51±0.13
Pm	21.43±0.01^a	10.31±0.02^c	9.59±0.40^ab	43.22±0.06^b	35.82±0.4^b	0.88±0.08
Ss	11.91±0.19^c	9.33±0.01^c	9.30±0.53^b	43.91±0.21^b	35.43±1.41^b	2.01±0.12
Pp	11.34±0.23^c	16.13±0.03^a	9.13±0.01^b	33.23±0.84^e	40.13±0.16^a	1.19±0.04
Pa	16.41±0.17^b	13.11±0.01^b	10.52±0.52^a	39.84±0.71^c	35.92±0.04^b	0.64±0.08
Pps	9.76±0.12^c	14.63±0.04^ab	9.21±0.01^b	36.23±0.04^d	39.05±0.27^a	0.80±0.07
Sa	10.62±0.13^c	9.86±0.05^c	9.67±0.24^ab	46.31±1.48^a	31.63±0.14^c	2.80±0.29
Bm : Brachiaria mulato Pm: Panicum maximum var. Trichoglume, Ss: Setaria splendida, Pp: Pennisetum purpureum cv. Mott, Pa: Paspalum atratum, Pps: Pennisetum purpupoides, Sa: Setaria anceps, DM: Dry matter, CA: Crude ash, CP: Crude protein, EE: Ether extract, CF: Crude fiber, NFC: Non-fiber carbohydrate. ^a-cMeans in the same column without a common letter are different at p<0.05

Table 2. Structural and non-structural carbohydrates contents of selected tropical grasses (% DM).
Grass	Structural carbohydrates			Non-structural carbohydrates
Grass	NDF	ADF	ADL	Total Starch	Total Sugar

Bm	67.54±0.52^c	37.33±0.45^c	13.72±0.06^b	13.40±0.14^a	3.86±0.08^ab
Pm	71.25±0.28^b	39.64±0.48^bc	13.04±0.13^b	9.68±0.21^c	3.27±0.03^ab
Ss	74.82±1.17^ab	40.04±0.82^bc	13.33±0.17^b	9.42±0.13^c	3.18±0.01^b
Pp	77.53±0.76^a	46.83±0.34^a	17.24±0.39^a	8.52±0.12^d	2.85±0.02^c
Pa	69.62±0.09^bc	38.64±1.17^c	12.62±0.19^b	11.37±0.18^b	4.59±0.09 ^a
Pps	75.31±0.16^a	42.52±0.23^b	13.82±0.52^b	9.01±0.14^cd	2.74±0.21^c
Sa	73.92±0.62^ab	41.25±0.97^b	12.24±0.37^b	9.29±0.19^c	3.04±0.02^bc
Bm : Brachiaria mulato Pm: Panicum maximum var. Trichoglume, Ss: Setaria splendida, Pp: Pennisetum purpureum cv. Mott, Pa: Paspalum atratum, Pps: Pennisetum purpupoides, Sa: Setaria anceps, NDF: Neutral detergent fiber, ADF: Acid detergent fiber, ADL: Acid detergent lignin. ^a-dMeans in the same column without a common letter are different at p<0.05.

Feed digestibility

Feed digestibility is closely linked to the digestibility of structural carbohydrates, such as NDF and ADF, that play a significant role in determining feed digestibility. Higher values of NDF and ADF result in lower digestibility (Schroeder 2004). This correlation is evident in the observations made from Table 3, which shows that Pennisetum purpureum cv. Mott grass had lower (p<0.05) dry matter and organic matter digestibility, with values of 61.52% and 60.11%, respectively, due to its high NDF and ADF content, compared to Brachiaria mulato grass, which had low NDF and ADF content and high structural carbohydrate content, resulting in higher (p<0.05) digestibility values for dry matter (68.82%) and organic matter (66.22%). The high structural carbohydrate content affects the number of nutrients consumed, thereby increasing feed digestibility (Kowalik et al 2004; Sun et al 2018). Additionally, Brachiaria mulato grass showed the highest total gas production, which is in line with its high content of non-structural carbohydrates. Starch is a polysaccharide group with simpler bonds, making it easy to be degraded by amylolytic bacteria (Yahaya 2001)

Ammonia Production

The protein source feed that enters the rumen is broken down by proteolytic bacteria produced by rumen microorganisms into ammonia (NH3). Ammonia is further used by rumen microorganisms as a substrate in the microbial protein system and used as protein by ruminant animals for productivity purposes (Jayanegara and Palupi 2010). The ammonia production produced in (Table 3) ranges (p<0.05) from 10.07 mM to 14.6 mM. The normal concentration of NH3 to support the growth of microorganisms in rumen liquid ranges from 6 mM to 21 mM McDonald et al 2002). The results of this research fall within the normal concentration range, so microorganisms could still survive in the rumen liquid. Ammonia is a product of protein metabolism in the rumen. A high degree of protein degradation results in a higher rumen ammonia concentration. Pennisetum purpureum cv. Mott grass produces the lowest ammonia, while Brachiaria mulato grass produces the highest ammonia in line with its high protein content. High protein content supports the activity of rumen microbes to degrade protein more. Other factors that affect ammonia production other than protein solubility are the proportion of dissolved carbohydrates. Starch is a readily fermentable carbohydrate that provides energy for rumen microbes, allowing them to synthesize microbial protein more efficiently (Singh 2012)

Table 3. Feed digestibility, pH, and ammonia production of selected tropical grasses
Grass	IVDMD (%)	IVDMD (%)	pH	Ammonia (mM)

Bm	68.82±0.28^c	66.22±0.25^c	7.42±0.34	14.6±0.57^b
Pm	66.14±0.64^bc	63.71±0.58^bc	7.55±0.03	13.9±0.72^b
Ss	64.73±3.45^ab	62.45±3.21^ab	7.51±0.17	13.7±0.40^ab
Pp	61.52 1±0.42^a	60.11±0.08^a	7.60±0.13	10.7±0.95^a
Pa	67.23±4.31^bc	65.52±3.56^c	7.42±0.01	14.3±0.34^b
Pps	63.34±1.03^a	60.23±0.82^a	7.55±0.22	10.8±0.37^a
Sa	63.7±0.25 ^a	60.51±0.32^a	7.47±0.29	11.9±0.14^ab
Bm : Brachiaria mulato Pm: Panicum maximum var. Trichoglume, Ss: Setaria splendida, Pp: Pennisetum purpureum cv. Mott, Pa: Paspalum atratum, Pps: Pennisetum purpupoides, Sa: Setaria anceps, IVDMD: in vitro dry matter digestibility, IVOMD: in vitro organic matter digestibility, ^a-cMeans in the same column without a common letter are different at p<0.05

SCFA production

Short Chain Fatty Acids (SCFA) are the final products of carbohydrate fermentation and the main energy source for ruminants from the rumen. Based on (Table 4), the total SCFA concentration ranges between 31.7 mM to 37.9 mM, while according to McDonald et al (2002), the normal range of total SCFA concentration is 70 mM to 150 mM. This means that the total SCFA concentration produced in this study was below normal. SCFA production reflects the fermentability of feed in the rumen. Factors that affect SCFA production are the number of microbes in the fermentation process and the content of easily degraded carbohydrates in feed (Cone and Becker 2012). Increasing SCFA concentration is affected by increased digestion and ammonia concentration in the rumen (Morvay et al 2011). The partial SCFA profile data, including acetic acid (C2), propionic acid (C3), and butyric acid (C4), and the ratio of acetic acid to propionic acid (C2/C3) can be seen in (Table 3). The results of the research show that the highest (p<0.05) proportion of acetic acid is found in Panicum maximum var. Trichoglume and propionic acid and butyric acid are found in Paspalum atratum. Acetic acid is the final product of fiber fermentation. The proportion of SCFA composition (acetic, propionic, and butyric acids) ranges between 70% acetic acid, 19% propionic acid, and 9% butyric acid. These results were relatively similar to the report of Petri et al (2019) which states that the range of SCFA composition in the rumen fluid ranges between 60-70% acetic acid, 15-20% propionic acid, and 10-15% butyric acid. The ratio of acetic acid to propionic acid (C2/C3) is an indicator of energy utilization efficiency and the quality of the produced product. According to the research results presented in (Table 3), the C2/C3 ratio ranges between 3.67 to 4.49. The higher the C2/C3 ratio value, the fermentation of the rumen leads to the production of acetic acid, while a lower C2/C3 ratio value indicates a change in the proportion of SCFA towards the production of propionic acid (Van Soest 1994). The increase in the acetic acid to the propionic acid ratio (C2/C3) in this study was due to the research feed being a fiber source, which makes the rumen fermentation more inclined towards the formation of acetic acid and, thus, increasing the acetic acid to the propionic acid ratio (Moss et al 2000). However, acetic acid is a non-glucogenic compound that is rapidly oxidized, resulting in a high heat increment and reducing efficiency value. In contrast, propionic acid is a sugar precursor or glucogenic, which is the main substrate for the synthesis of glucose formation through the process of gluconeogenesis. Absorbed propionic acid can supply 32-73% of the glucose needs of ruminants. A high C2/C3 ratio produced indicates low feed efficiency and energy use. In addition to the production of SCFA (C2/C3 ratio), feed efficiency can also be assessed by measuring methane production (CH4).

Table 4. pH value, ammonia concentration, and total short-chain fatty acids at the 24th hour of in-vitro incubation
Grass	TSCFA (mM)	C2 (%TSCFA)	C3 (%TSCFA)	C4 (%TSCFA)	Ratio C2:C3

Bm	37.9±1.03	72.7±0.84	19.0±0.59 ^bcd	8.19±0.29^ab	3.82±0.19^ab
Pm	36.2±0.20	72.9±0.18	17.6±0.63^ab	9.42±0.35^b	4.13±0.38^b
Ss	34.8±0.93	74.2±0.66	18.0±0.57^abc	7.66±0.29^ab	4.12±0.16^b
Pp	31.7±0.73	71.5±1.19	16.7±0.46^a	7.96±0.28^ab	4.49±0.12^c
Pa	36.6±0.35	71.5±0.3	19.5±0.17^d	8.92±0.57^b	3.67±0.11^a
Pps	32.5±0.58	73.0±0.29	17.8±0.44^ab	9.14±0.20^b	4.10±0.25^b
Sa	34.7±0.49	73.6±0.20	19.3±0.32^cd	6.98±0.14^a	3.80±0.21^ab
Bm : Brachiaria mulato Pm: Panicum maximum var. Trichoglume, Ss: Setaria splendida, Pp: Pennisetum purpureum cv. Mott, Pa: Paspalum atratum, Pps: Pennisetum purpupoides, Sa: Setaria anceps, TSCFA: Total short-chain fatty acids, NH3: Ammonia concentration, C2: Acetic acid, C3: Propionic acid, C4: Butyric acid, ^a-d Means in the same column without a common letter are different at p<0.05

Total Gas and methane production

The total gas produced during the incubation process is primarily a result of the fermentation of substrates by the rumen microbes. The results indicated that Brachiaria mulato grass had the highest gas production among the grasses studied (Table 5). This high gas production was further strengthened by the highest values of DM and OM digestibility in Brachiaria mulato. The results revealed that the total gas production in the rumen varied (p<0.05) among different types of forage grass. This indicated that the nutrient content varied depending on the type of forage grass used. Bezabih et al (2014) identified differences in the nutritional composition of various types of forage, including Brachiaria sp., which affected gas production and in vitro fermentation by rumen microbes. Similarly, Kamalak et al (2004) found that the nutritional composition of various forage types differed, which affected gas production and in vitro fermentation characteristics by rumen microbes.

The methane production results in this research (Table 5) showed that Panicum maximum var. Trichoglume grass produced the highest amount of methane (33.13%), while the lowest amount had been produced by Brachiaria mulato grass (29.91%). This suggested that using lower fiber and more digestible grasses could be a viable strategy to reduce methane production in livestock. The formation of methane in the rumen was one of the final products of feed fermentation that occurred through the reduction of CO2 and H2 by methanogenic microbes (Morgavi et al 2010). Structural carbohydrates, particularly cellulose, and hemicellulose were known to be major precursors for methane production in the rumen (Weimer 2022).

The complex structural carbohydrate makes it difficult for rumen microbes to break down, and the byproducts of fiber fermentation (such as hydrogen) are necessary for methanogenesis to occur. McGinn et al (2004) demonstrated that dietary manipulation of fiber content could affect methane emissions in beef cattle. The methane produced in the rumen is energy lost for the livestock, ranging from 2% to 12% of the feed energy (Moss et al 2000). Methane production is closely related to the proportion of SCFA products such as acetic acid and butyric acid produced during the fermentation process in the rumen, but not related to propionic acid production. This is because the methane produced is highly dependent on the availability of H₂ and CO₂ in the rumen that is released during the production of acetic acid and butyric acid during fermentation. Unlike propionic acid production which is not accompanied by the production of H₂ and CO₂.

Table 5. Total gas and methane production of selected tropical grasses
Grass	Total Gas (mL)	Methane (%TSCFA)

Bm	45.02±0.58^a	29.91±0.56^a
Pm	43.03±0.50^b	33.13±0.69^c
Ss	39.01±2.37^c	31.53±0.38^b
Pp	38.04±1.37^c	31.62±0.29^b
Pa	39.02±1.75^c	30.74±0.44^ab
Pps	33.61±0.04^d	31.11±0.78^ab
Sa	37.22±1.87 ^c	31.73±0.51^b
Bm : Brachiaria mulato Pm: Panicum maximum var. Trichoglume, Ss: Setaria splendida, Pp: Pennisetum purpureum cv. Mott, Pa: Paspalum atratum, Pps: Pennisetum purpupoides, Sa: Setaria anceps. ^a-cMeans in the same column without a common letter are different at p<0.05


B. mulato	S. splendida	P. dilatatum	P. atratum

Conclusion

This study has highlighted the importance of identifying tropical grass types that produce less methane during rumen fermentation to mitigate greenhouse gas emissions in livestock farming.
The findings have demonstrated a significant relationship between nutrient content, particularly structural and non-structural carbohydrate content, and methane production in tropical grasses.
The results have suggested that Brachiaria mulato grass has the most promising in vitro fermentation profile, with the lowest methane production, making it a valuable addition to the ruminant diet in tropical regions.
Integrating this grass type into livestock feed could potentially help rural farmers reduce their environmental impact while improving the nutritional quality of their livestock feed.
Therefore, further research on the impact of incorporating Brachiaria mulato grass in ruminant diets and its potential effects on animal productivity and sustainability is recommended.

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