Correlation between chemical composition, kinetics of fermentation and methane production of eight pasture grasses

Correlação entre a composição química, cinética de fermentação e produção de metano de oito tipos de capim de pastagem

Abstracts

Eight different grasses collected from pastures of the Kermanshah province (Kermanshah, Iran), at mid-vegetative stage were used to study the relationships between their chemical compositions, kinetic parameters of in vitro gas production and rumen methane production. There was a positive correlation (r = 0.62, p < 0.05) between crude protein (CP) content of grasses and total gas production (A) at 96h incubation. Negative correlations were also observed between acid detergent fiber (ADF) content and total gas production (r = -0.60, p < 0.05). Amongst the nutrients, neutral detergent fiber (NDF) and ADF were positively correlated with methane concentration, (r = 0.75 and 0.77, p < 0.01). The methane reduction potential (MRP) was negative for Trachyspermum copticum indicating higher methane production than the control hay for this grass. The MRP of Chamaemelum nobile was more than 25%, indicating plants that reduce methane production more than 20 percent methane in comparison with control actually have ingredients to reduce methane.

gas production; pasture grasses; methane; In vitro

Oito tipos diferentes de capim tirados de pastagens na província de Kermanshah (Kermanshah, Iran), em estágio meio vegetativo, foram analisados para ver sua relação entre as composições químicas, parâmetros cinéticos de produção de gás e produção de metano no rúmen in vivo. Houve uma correlação positiva (r = 0,62; p < 0,05) entre a proteína bruta (PB), conteúdo do capim e a produção total de gás (A) após 96h de incubação. Foram observadas correlações negativas entre a fibra de detergente ácido (FDA) e a produção total de gás (r = -0,60; p < 0,05). Entre os nutrientes, a fibra de detergente neutro (FDN) e a FDA foram positivamente correlacionados com concentração de metano (r = 0,75 e 0,77; p < 0,01). O potencial reducional de metano (PRM) era negative para Trachyspermum copticum e indicava uma produção maior de metano do que o controle feno nesse capim. O PRM de Chamaemelum nobile chegou a mais de 25% e indicava plantas que reduzem a produção de metano em mais de 20% quando comparadas ao controle que tem ingredientes para diminuir o metano.

produção de gás; capim de pastagem; metano; In vitro

Introduction

Pastures are the main component of ruminants' diet, especially in tropical regions, where, except in areas with high population density, and when properly managed livestock becomes more lucrative. Research on Iranian rangelands and pastures has been undertaken in Iran on range management and ecology. This new area merits investigation because the amount of forage available, as determined in dry matter per hectare, by itself is not a sufficient measure of forage adequacy for animal use. In fact, there are a number of in vitro techniques available to evaluate the nutritive value of forage at relatively low cost (Giger-Reverdin et al., 2002). In vitro gas production can be used as a rapid evaluation tool to assess nutritional quality of forages (Abdalla et al., 2012; Wanapat et al., 2012). Recently, there has been an increasing interest to the use of natural ingredients, such as plants, and their extracts containing the active ingredient have been attracted (Bodas et al., 2012; Goel & Makkar, 2012; Jayanegara, Leiber & Kreuzer, 2012). Pasture plants may act in many ways against mammals, including as toxins or digestibility reducers, modifying their food quality and their foraging behavior (Bodas et al., 2012; Iason, 2005). They may in some cases be beneficial to mammals (Athanasiadou & Kyriazakis, 2004), including with decreased methane production (Soliva et al., 2008). Methane production from ruminants contributes to total global methane production, which is an important contributor to global warming (Hartung & Monteny, 2000). Enteric methane contributes 30-40 percent of total methane production from agricultural sources. Many attempts, such as concentrate supplementation (Lovett et al., 2005), use of probiotics and prebiotics (Mwenya et al., 2004; Takahashi et al., 2005; Valero et al., 2014), lipid supplementation (Ungerfeld, Rust, Burnett, Yokoyama & Wang, 2005), and addition of plants and their extracts (Goel, Makkar & Becker, 2008; Patra, Kamra & Agarwal, 2006; Śliwiński, Soliva, Machmüller & Kreuzer, 2002) have been made to decrease enteric methane production. The aim of this study was to investigate chemical composition and kinetics of fermentation from eight common grass species found in the pastures of the province of Kermanshah, Iran, and to determine the relationships between chemical composition with in vitro rumen methane production due to the identification of plants with anti-methanogenic properties.

Material and methods

Grass samples and chemical composition

Grass samples, Chamaemelum nobile, Trachyspermum copticum, Descurania Sophia, Urtica dioica, Falcaria vulgaris, Tragopogon collinus, Gundelia tournefortii and Taraxacum officinale were collected at mid-vegetative stage from natural pastures in Kermanshah province. Grass samples were ground using a lab mill to pass through a 1-mm sieve. Standard methods as described in AOAC (1998) were used to determine organic matter (OM), crude protein (CP) and ether extract (EE). Neutral detergent fiber (NDF) and acid detergent fiber (ADF) were determined according to Van Soest, Robertson & Lewis (1991).

In vitro gas production

The method used for gas production measurements followed Theodorou, Williams, Dhanoa, McAllan & France (1994). About 125 mg of each sample was weighed into tubes kept at approximately 39ºC. Each sample was incubated in three replicates. Fifteen mL of buffered rumen fluid (in the proportion of 20% rumen fluid + 80% buffer solution) was anaerobically dispensed in each tube. All tubes were crimped and placed in a shaking incubator. The pressure of gas produced in each tube was recorded by a pressure transducer (Manometer Digital testo 512) at 2, 4, 6, 8, 12, 24, 48, 72 and 96h after the start of the incubation. To estimate the kinetics of gas production, data on cumulative gas volume produced were fitted using the generalized Mitscherlich model proposed by France et al. (1993):

where G (mL) denotes cumulative gas production at time t; A (mL) is asymptotic gas production; c (h⁻¹⁾ and b(h−1/2) are rate constants; L (h) is lag time. The half-life (t _1/2, h) of the degradable fraction of each substrate was calculated as the time taken for gas accumulation to reach 50% of its asymptotic value.

Measurement of methane production

For measuring methane production, after 24 hours of incubation, 2 mL of NaOH (10 M) were introduced to estimate methane production following the method by Fievez, Babayemi & Demeyer (2005). Data were obtained on volume of gas and methane (CH₄) produced. Net methane and gas productions were calculated by the differences of the methane and gas in the test syringe and the corresponding blank; the methane concentration was determined as Jayanegara, Togtokhbayar, Makkar & Becker (2009):

Methane production reduction potential (MRP) was calculated by taking net methane rates for control (hay) as 100% Jayanegara et al. (2009):

Statistical analysis

Data on gas production parameters and dry matter (DM) digestibility from different eight grasses were subjected to one way analysis of variance using SAS (2004) and significance between individual means was identified using Duncan multiple-range test (Duncan, 1955).

Results and discussion

Crude protein (CP), neutral detergent fiber (NDF) and acid detergent fiber (ADF) of grasses are shown in Table 1. The CP content (g kg^-1 DM) varied widely, from 52.4 to 269.2, with the highest for Descurania Sophia and lowest for Taraxacum officinale. Gas production kinetic parameters of different species are presented in Table 2 and graphically illustrated in Figure 1. There were significant differences between potential gas production (A), constant rates (d) and (c) of pasture species (p < 0.01).

Table 1:
Chemical composition (g kg^-1 DM) of pasture grasses from western Iran.

species	OM	CP	EE	NDF	ADF
G1	954	131.4	27.1	325.8	251.0
G2	730	167.4	93.1	264.8	183.0
G3	835	269.2	16.0	235.2	221.3
G4	818	119.5	14.5	495.2	390.6
G5	870	151.9	24.6	311.6	283.7
G6	888	145.4	38.6	336.6	280.7
G7	832	203.4	12.9	324.2	237.8
G8	683	52.4	8.60	625.4	480.7

• G1: Chamaemelum nobile, G2: Trachyspermum copticum, G3: Descurania Sophia, G4: Urtica dioica, G5: Falcaria vulgaris,G6: Tragopogon collinus, G7: Gundelia tournefortii, G8: Taraxacum officinale. OM: organic matter; CP: crude protein; EE: ether extract; ADF: acid detergent fiber; NDF: neutral detergent fiber.

Table 2:
Coefficients of curves fitted to the average gas production pattern of each species together with the volume of total gas produced during 96h of incubation (mL 125 mg^-1 dry matter).

species	A(mL gas 125 mg-1 DM)	b(h-1/2)	c(h-1)	Half life(h)
G1	51.86a	0.087a	0.040a	9.43g
G2	43.12d	0.045f	0.025cd	10.48d
G3	36.48e	0.031d	0.023d	12.50c
G4	27.86g	0.028e	0.020d	14.01b
G5	46.05c	0.041c	0.040a	9.43g
G6	52.25a	0.065b	0.036b	9.70f
G7	48.09b	0.045c	0.28c	10.27e
G8	32.23f	0.033d	0.023d	14.81a
P-value	**	**	**	**
SEM	1.78	0.197	0.014	0.004

• G1: Chamaemelum nobile, G2: Trachyspermum copticum, G3: Descurania Sophia, G4: Urtica dioica,G5: Falcaria vulgaris,G6: Tragopogon collinus, G7: Gundelia tournefortii, G8: Taraxacum officinale. A: asymptotic gas production (mL 125 mg^-1 DM); c: constant rates (per h); Half-life: time (h) required to reach 0.50 total gas production (per h). SEM: standard error of mean. ** p < 0.01.

Figure 1:
Pattern of in vitro gas production of grasses (G1: Chamaemelum nobile, G2: Trachyspermum copticum, G3: Descurania Sophia, G4: Urtica dioica, G5: Falcaria vulgaris, G6:Tragopogon Collinus, G7: Gundelia tournefortii, G8: Taraxacum officinale) in buffered rumen fluid

There was a positive correlation (r = 0.62; p < 0.05) between CP content of grasses and total gas production (A) at 96h incubation (Table3). Crude protein concentrations below the threshold of 70 g CP kg^-1 DM restrict microbial activity due to a lack of nitrogen (Hariadi & Santoso, 2010). However, protein levels above this threshold, as seen in this study, are considered to enhance microbial multiplication in the rumen, thus improving fermentation (Njidda & Nasiru, 2010). Negative correlation was observed between ADF content and potential gas production (A) (r = -0.60; p < 0.05). With increasing NDF and ADF, potential gas production (A) and the rate constant (b, c) decreased (Table 3). This result is in agreement with the report by Heidary & Kafilzadeh (2012) who found that NDF and ADF content had negative correlation with fermentation parameters (A and b). It may be concluded that although increase in cell wall content showed a decrease in total gas production, the eight grasses behaved differently in the rate at which they produced gas.

Table 3:
Correlation (r) between chemical composition (g kg^-1 DM) and in vitro gas production characteristics.

	Gas production constants
Predictor	A(mL gas 125 mg-1 DM)	b(h-1/2)	c(h- 1)
CP	0.621*	0.246*	-0.031
NDF	-0.207	-0.307	-0.032
ADF	-0.602*	-0.415*	-0.120

• CP: crude protein; ADF: acid detergent fiber; NDF: neutral detergent fiber; A: asymptotic gas production (mL 125mg^-1 DM); c, d: fractional rate of gas production. *p < 0.05.

Methane production reduction potential of grasses by in vitro rumen fermentation

Table 4 shows methane concentration and methane production reduction potential (MRP) which was calculated by assuming net methane concentration for control hay (oat) as 100%. The MRP of Chamaemelum nobile was more than 25 percent and showed plants that reduce methane production more than 20 percent methane in comparison with control actually have ingredients to reduce methane (García-González, López, Fernández & González, 2006). The MRP of negative for Trachyspermum copticum meant that the methane concentration for this sample was higher than that of the control hay. Amongst the nutrients, NDF and ADF were positively correlated with methane concentration, (r = 0.75 and 0.77, respectively; p < 0.01), while no relationship existed for CP and EE. Generally, the type of carbohydrate present in the forage is thought to dictate methane production via shifts in the ruminal microbial population (Johnson & Johnson, 1995; Meale, Chaves, Baah & McAllister, 2012). High soluble carbohydrate content is suggested to promote the production of propionate in the rumen, lower ruminal pH and inhibit methanogen growth, thereby reducing methane production per unit of fermented OM (Kessel & Russell, 1996; Meale et al., 2012), as shown that negative relationships occurred between NDF or ADF and the MRP (p < 0.05) (Table 5). As seen in this study, dietary components such as NDF and ADF also contributed to explain total variation in methane production. Higher NDF increases methane production by shifting short chain fatty acid proportion towards acetate which produces more hydrogen (Carulla, Kreuzer, Machmüller & Hess, 2005; Puchala, Min, Goetsch & Sahlu, 2005; Tavendale et al., 2005). EE had no relationship with all methane expressions when it was correlated individually. The role of lipids in decreasing methane is well illustrated, albeit at levels higher than present in most of the samples in this study (Beauchemin, Kreuzer, O'Mara & McAllister, 2008). In addition, most of the samples in current study were forage in natura with low levels of EE. The nature of lipid and its form (i.e., bound or free) are also determining factors in their effect on methane production (Beauchemin et al., 2008; Zeitz et al., 2013).

Table 4:
Methane concentration and Methane production reduction potential (MRP) from pasture grasses from western Iran.

species	Methane (%)	MRP (%)
G1	18.26e	26.96a
G2	30.36a	-22.50e
G3	22.24c	8.040c
G4	21.40d	14.42b
G5	24.53b	1.89d
G6	24.13b	3.47d
G7	20.93d	16.30b
G8	20.96d	16.17b
P-value	**	**
SEM	0.671	2.89

• G1: Chamaemelum nobile, G2: Trachyspermum copticum, G3: Descurania Sophia, G4: Urtica dioica, G5: Falcaria vulgaris, G6: Tragopogon collinus, G7: Gundelia tournefortii, G8: Taraxacum officinale. MRP: Methane production reduction potential. SEM: standard error of mean. ** p < 0.01.

Table 5:
Correlation (r) between chemical composition (g kg^-1 DM) and methane production parameters.

Predictor	Methane (%)	MRP (%)
CP	0.164ns	-0.217ns
EE	0.259ns	-0.347ns
NDF	0.752**	-0.679*
ADF	0.774**	-0.694*

• CP: crude protein; EE: ether extract; ADF: acid detergent fiber; NDF: neutral detergent fiber; MRP: Methane production reduction potential. SEM: standard error of mean. ** p < 0.01 * p < 0.05.

Conclusion

From the results of this study among the nutrients, NDF and ADF were positively correlated with methane concentration. Chamaemelum nobile had a better nutritive quality when compared to the others and also reduced methane production. However, any decision with regard to its use as a possible crop in agricultural system or in breeding programs, their yield and some other agronomic factors, requires further study.

• Abdalla, A. L., Louvandini, H., Sallam, S. M. A. H., Silva, I. C. B., Tsai, S. M. & Oliveira Figueira, A. V. (2012). In vitro evaluation, in vivo quantification, and microbial diversity studies of nutritional strategies for reducing enteric methane production. Tropical Animal Health and Production, 44(5), 953-964.
• AOAC. (1998). Official Methods of Analysis (15th ed.): Assoc. Off. Anal. Chem., Arlington, VA, U.S.A.
• Athanasiadou, S. & Kyriazakis, I. (2004). Plant secondary metabolites: antiparasitic effects and their role in ruminant production systems. Proceedings of the Nutrition Society, 63(4), 631-639.
• Beauchemin, K. A., Kreuzer, M., O'Mara, F. & McAllister, T. A. (2008). Nutritional management for enteric methane abatement: a review. Animal Production Science, 48(2), 21-27.
• Bodas, R., Prieto, N., García-González, R., Andrés, S., Giráldez, F. J. & López, S. (2012). Manipulation of rumen fermentation and methane production with plant secondary metabolites. Animal Feed Science and Technology, 176(1-4), 78-93.
• Carulla, J. E., Kreuzer, M., Machmüller, A. & Hess, H. D. (2005). Supplementation of Acacia mearnsii tannins decreases methanogenesis and urinary nitrogen in forage-fed sheep. Crop and Pasture Science, 56(9), 961-970.
• Duncan, D. B. (1955). Multiple range and multiple F tests. Biometrics, 11(1), 1-42.
• Fievez, V., Babayemi, O. & Demeyer, D. (2005). Estimation of direct and indirect gas production in syringes: A tool to estimate short chain fatty acid production that requires minimal laboratory facilities. Animal Feed Science and Technology, 123, 197-210.
• France, J., Dhanoa, M., Theodorou, M., Lister, S., Davies, D. & Isac, D. (1993). A Model to interpret gas accumulation profiles associated with in vitro degradation of ruminant feeds. Journal of Theoretical Biology, 163(1), 99-111.
• García-González, R., López, S., Fernández, M. & González, J. S. (2006). Effects of the addition of some medicinal plants on methane production in a rumen simulating fermenter (RUSITEC). International Congress Series, 1293(0), 172-175. doi: http://dx.doi.org/10.1016/j.ics.2006.01.044
• Giger-Reverdin, S., Duvaux-Ponter, C., Sauvant, D., Martin, O., Prado, I. N. & Müller, R. (2002). Intrinsic buffering capacity of feedstuffs. Animal Feed Science and Technology, 96(1), 83-102.
• Goel, G. & Makkar, H. P. (2012). Methane mitigation from ruminants using tannins and saponins. Tropical Animal Health and Production, 44(4), 729-739.
• Goel, G., Makkar, H. P. & Becker, K. (2008). Effects of Sesbania sesban and Carduus pycnocephalus leaves and Fenugreek Trigonella foenum-graecum L.) seeds and their extracts on partitioning of nutrients from roughage-and concentrate-based feeds to methane. Animal Feed Science and Technology, 147(1), 72-89.
• Hariadi, B. T. & Santoso, B. (2010). Evaluation of tropical plants containing tannin on in vitro methanogenesis and fermentation parameters using rumen fluid. Journal of the Science of Food and Agriculture, 90(3), 456-461.
• Hartung, E. & Monteny, G.-J. (2000). Methane (CH4) and nitrous oxide (N2O) emissions from animal husbandry. Agrartechnische Forschung, 6(2), 62-69, 115.
• Heidary, N. & Kafilzadeh, F. (2012). Detrmining of chemical composition, in vitro digestibility and kinetics of fermentation of whole crop forage from 18 different varieties of oat. Iranian Journal of Animal Science Research, 22(2), 91-106.
• Iason, G. (2005). The role of plant secondary metabolites in mammalian herbivory: ecological perspectives. Proceedings of the Nutrition Society, 64(1), 123-131.
• Jayanegara, A., Leiber, F. & Kreuzer, M. (2012). Meta analysis of the relationship between dietary tannin level and methane formation in ruminants from in vivo and in vitro experiments. Journal of Animal Physiology and Animal Nutrition, 96(3), 365-375.
• Jayanegara, A., Togtokhbayar, N., Makkar, H. P. S. & Becker, K. (2009). Tannins determined by various methods as predictors of methane production reduction potential of plants by in vitro rumen fermentation system. Animal Feed Science and Technology, 150(3), 230-237.
• Johnson, K. A. & Johnson, D. E. (1995). Methane emissions from cattle. Journal of Animal Science, 73(8), 2483-2492.
• Kessel, J. A. S. & Russell, J. B. (1996). The effect of pH on ruminal methanogenesis. FEMS Microbiology Ecology, 20(4), 205-210.
• Lovett, D., Stack, L., Lovell, S., Callan, J., Flynn, B., Hawkins, M. & O'Mara, F. (2005). Manipulating enteric methane emissions and animal performance of late-lactation dairy cows through concentrate supplementation at pasture. Journal of Dairy Science, 88(8), 2836-2842.
• Meale, S. J., Chaves, A. V., Baah, J. & McAllister, T. A. (2012). Methane production of different forages in in vitro ruminal fermentation. Asian-Australasian Journal of Animal Sciences, 25(1), 86-91.
• Mwenya, B., Santoso, B., Sar, C., Gamo, Y., Kobayashi, T., Arai, I. & Takahashi, J. (2004). Effects of including β1-4 galacto-oligosaccharides, lactic acid bacteria or yeast culture on methanogenesis as well as energy and nitrogen metabolism in sheep. Animal Feed Science and Technology, 115(3), 313-326.
• Njidda, A. A. & Nasiru, A. (2010). In vitro gas production and dry matter digestibility of tannin-containing forages of semi-arid region of north-eastern Nigeria. Pakistan Journal of Nutrition, 9(1), 60-66.
• Patra, A. K., Kamra, D. N. & Agarwal, N. (2006). Effect of plant extracts on in vitro methanogenesis, enzyme activities and fermentation of feed in rumen liquor of buffalo. Animal Feed Science and Technology, 128(3-4), 276-291. doi: http://dx.doi.org/10.1016/j.anifeedsci. 2005.11.001
• Puchala, R., Min, B., Goetsch, A. L. & Sahlu, T. (2005). The effect of a condensed tannin-containing forage on methane emission by goats. Journal of Animal Science, 83(1), 182-186.
• SAS. (2004). SAS/STAT User guide, Version 9.1.2. Cary, NC, USA: SAS Institute Inc.
• Śliwiński, B. J., Soliva, C. R., Machmüller, A. & Kreuzer, M. (2002). Efficacy of plant extracts rich in secondary constituents to modify rumen fermentation. Animal Feed Science and Technology, 101(1-4), 101-114. doi: http://dx.doi.org/10.1016/S0377-8401(02)00139-6
• Soliva, C. R., Zeleke, A. B., Clement, C., Hess, H. D., Fievez, V. & Kreuzer, M. (2008). In vitro screening of various tropical foliages, seeds, fruits and medicinal plants for low methane and high ammonia generating potentials in the rumen. Animal Feed Science and Technology, 147(1), 53-71.
• Takahashi, J., Mwenya, B., Santoso, B., Sar, C., Umetsu, K., Kishimoto, T. Hamamoto, O. (2005). Mitigation of methane emission and energy recycling in animal agricultural systems. Asian-Australasian Journal of Animal Sciences, 18(8), 1199.
• Tavendale, M. H., Meagher, L. P., Pacheco, D., Walker, N., Attwood, G. T. & Sivakumaran, S. (2005). Methane production from in vitro rumen incubations with Lotus pedunculatus and Medicago sativa, and effects of extractable condensed tannin fractions on methanogenesis. Animal Feed Science and Technology, 123-124, 403-419. doi: http://dx.doi.org/10.1016/j. anifeedsci.2005.04.037
• Theodorou, M. K., Williams, B. A., Dhanoa, M. S., McAllan, A. B. & France, J. (1994). A simple gas production method using a pressure transducer to determine the fermentation kinetics of ruminant feeds. Animal Feed Science and Technology, 48(3), 185-197.
• Ungerfeld, E., Rust, S. R., Burnett, R. J., Yokoyama, M. T. & Wang, J. (2005). Effects of two lipids on in vitro ruminal methane production., Animal Feed Science and Technology 119(1), 179-185.
• Valero, M. V., Prado, R. M., Zawadski, F., Eiras, C. E., Madrona, G. S. & Prado, I. N. (2014). Propolis and essential oils additives in the diets improved animal performance and feed efficiency of bulls finished in feedlot. Acta Scientiarum.Animal Sciences, 32(4), 419-426.
• Van Soest, P. J., Robertson, J. B. & Lewis, B. A. (1991). Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. Journal of Dairy Science, 74(10), 3583-3597. doi: 10.3168/jds.S0022-0302(91)78551-2
• Wanapat, M., Kongmun, P., Poungchompu, O., Cherdthong, A., Khejornsart, P., Pilajun, R. & Kaenpakdee, S. (2012). Effects of plants containing secondary compounds and plant oils on rumen fermentation and ecology. Tropical Animal Health and Production, 44(3), 399-405.
• Zeitz, J., Bucher, S., Zhou, X., Meile, L., Kreuzer, M. & Soliva, C. (2013). Inhibitory effects of saturated fatty acids on methane production by methanogenic Archaea. Journal of Animal and Feed Science, 22(1), 44-49.

Publication Dates

• Publication in this collection
  Mar 2015

History

• Received
  06 July 2014
• Accepted
  29 Aug 2014