Methane production in an in vitro rumen incubation is reduced when leaves from Mimosa pigra are the protein source compared with Gliricidia sepium

Phonevilay Silivong, Xaykham Onphachanh, Aloun Ounalom and T R Preston*

Souphanouvong University, Lao PDR
silipvl@yahoo.co.th
* Center for Research and Technology Transfer, Nong Lam University,
Ho Chi Minh City, Vietnam

Abstract

The aim of this study was to evaluate the effect of potassium nitrate or urea on methane production from Mimosa and Gliricidia leaves in an in vitro incubation system with molasses as the energy source. The incubation was for 24 h with measurements of total gas and methane concentration over incubation periods (separate experiments) of 6, 12, 18 and 24 hours and determination of residual unfermented substrate at the end of each experimental period.  

Gas production, percentage methane in the gas and methane produced per unit DM solubilised for each incubation were lower with potassium nitrate  than with urea.  Methane production per unit DM solubilised was less with Mimosa than with Gliricidia in incubations over 18 and 24h, but did not differ between the two kinds of leaves  for the 6h and 12h incubations.

Keywords: climate change, fermentation, greenhouse gases, incubation

Introduction

In developing improved systems for feeding live stock, account must be taken of the impacts on the environment. It is estimated that live stock presently account for some 18% of greenhouses gases which cause global warming (Steinfeld et al 2006). Enteric methane from fermentative rumen digestion is the main source of these emissions. There is an urgent need to develop ways of reducing methane emissions from ruminants in order to meet future targets for mitigating global warming. From a survey of the relevant literature, Leng (2008) concluded that the presence of nitrate salts in the rumen will act as a competitive sink for the hydrogen produced by fermentation of carbohydrate such that it is converted to ammonia rather than methane. Recent research has confirmed that nitrate lowers methane production in goats fed either sugar cane (Anh Nguyen Ngoc et al 2010) or rice straw (Sophea and Preston 2011) as basal diets.

In ruminants, the H₂ is normally removed by the reduction of CO₂ to form methane. However, nitrate has a higher affinity for H₂ than CO₂ and, when it is present; H₂ is first used in the reduction of NO₃ to NO₂ and then NO₂­ to NH₃­ thereby reducing the production of methane from enteric fermentation.  Renewed recognition that nitrate supplements in ruminant diets compete successfully for H₂ and electrons (and decrease methane production) is a promising development (Leng 2008).

When urea is fed to cattle is it often recommended to dissolve the urea in molasses as one way to avoid possible toxicity problems with the urea (Preston and Leng 2009).  The same idea should apply to feeding of nitrate which is potentially more toxic than urea (Leng and Preston 2010). Combining foliage from cassava with molasses/urea was shown to support high growth rates in cattle (Ffoulkes and Preston 1978).).  However, there are no reports of this kind of feeding system being used for goats.   

Mimosa pigra is an invasive weed of the genus Mimosa in the family Fabaceae. This plant is considered to be one of the worst environmental weeds of the Mekong River basin. In Tram Chim National park in Dongthap Province in the Mekong delta, there is growing concern over the rapid growth of the Mimosa pigra plant, that has taken over more than one seventh of the 7,600 ha of the park (Tran Triet et al 2007; Viet Nam-VNS). However, recent research (Thu Hong et al 2008) has shown that mimosa foliage is an excellent feed for goats, supporting growth rates when given as the sole feed of almost 100 g/day. It was hypothesized that the presence of condensed tannins would confer bypass properties on the protein in the Mimosa and that this could explain its high nutritive value.

Gliricidia (Gliricidia sepium)  is a medium-sized tree that can grow to 10 to 12 m high. The foliage has been used as a protein supplements for low quality forages and resulted in improved ruminant productivity (Norton 1994).

The purpose of the present study was to test if there were differences in methane production when leaf meals from Mimosa and Gliricidia were incubated in a rumen in vitro system using molasses as energy source and potassium nitrate or urea as sources of non-protein nitrogen.

Materials and methods

Location and duration

The experiment was carried out from November to December 2011 at the Animal Science laboratory of the Faculty of Agriculture and Forest Resources, Souphanouvong University, Luang Prabang province, Lao PDR.   

Treatments and experimental design 

The experimental design was a 2*3 factorial arrangement of 6 treatments with four replications of each treatment.

The factors were:

Source of foliage:   Gliricidia (GD) or Mimosa (MP)

Source of NPN: Potassium nitrate (KN), Urea (U) or no NPN (Non).

Preparation of substrate and the in vitro system

The in vitro system (Photo 1) used recycled plastic bottles as flasks for the incubation and gas collection. The leaves from Gliricidia and Mimosa foliages were chopped into small piece (2-3mm) and dried at 60^°C for 24h then ground with a coffee grinder, and mixed with the molasses and either potassium nitrate or urea (according to the proportions shown in Table 1), The mixtures (12 g DM) were put in the incubator bottle with 960ml of buffer solution (Table 2) and 240ml of rumen fluid from a buffalo. The rumen fluid was taken at 3-4 am from the slaughter house from a buffalo immediately after the animal was killed. A representative sample of the rumen contents (including feed residues) was put in a vacuum flask and stored until 5 am the following  morning when the contents were filtered through  a layer of cloth before being added to the incubation bottles. The remaining air in the flask was flushed out with carbon dioxide. The incubation flask was connected by a plastic tube to a second flask (a calibrated recycled water bottle with the bottom removed) suspended in water so as to measure the gas production by water displacement. The bottles were incubated at 38^°C in a water bath for 24h.

Photo 1. The in vitro fermentation system using recycled water bottles and water displacement to measure gas production

Data collection and measurements

Incubations were carried out as separate experiments for 6, 12, 18 and 24h. At the end of each incubation, the methane concentration in the gas was measured with a Crowcon infra-red analyser (Crowcon Instruments Ltd, UK; Photo 2). Residual DM in the incubation bottle was determined by filtering the incubation residues through cloth to estimate DM loss during incubation.

Photo 2. Measurement of percentage of methane in the gas

Chemical analyses

Samples of Gliricidia and Mimosa leaves  and residual substrate were analysed for DM, ash and N according to methods outlined in Ly and  Lai (1997). The residual DM in the incubation bottle was determined by filtering through cloth and drying (70°C for 24h). The solubility  of the protein in the leaves was measured by extraction in M NaCl (Whitelaw et al 1962). 

Statistical analysis

The data from the experiment were analyzed by the General Linear Model (GLM) option in the ANOVA program of the Minitab (2000) software.  Sources of variation in the model were: NPN source, leaves, interaction NPN*leaves and error.

Results and discussion

Chemical composition

The contents of DM, ash and crude protein of Mimosa leaves were higher than  in Gliricidia (Table 3). The solubility of the protein was relatively low in the leaves of both foliages.

Gas production and concentration of methane

Gas production, percentage methane in the gas and methane produced per unit substrate solubilised increased with incubation time and were lower when the NPN source was potassium nitrate rather than urea (Table 4: Figure 1). The increase in methane production with length of incubation is in agreement with other reports from similar in vitro systems (Outhen et al 2011, Inthapanya et al 2011, Binh Phuong et al 2011; Thanh et al 2011). 

 Methane production per unit DM solubilised was less with Mimosa than with Gliricidia for the combined incubations over 18 and 24h, but did not differ between the two kinds of leaves  for the 6 and 12h incubations (Figure 4).

Figure 1. Effect of fermentation time on gas production	Figure 2. Effect of fermentation time on methane content in the gas

Figure 3. Effect of NPN source on methane production per unit substrate solubilised

 The reduction in methane production per unit substrate solubilized, with nitrate compared with urea,  is similar to that reported by Outhen et al (2011), Inthapanya et al (2011), Binh Phuong et al (2011) and  Thanh et al (2011) who used a similar in vitro system but with different substrates.  For incubations lasting 18 and 24h methane production per unit DM solubilized was lower when the substrate was Mimosa leaves compared with  Gliricidia (Figure 4).

Figure 4. Effect of source of substrate on methane production per unit substrate solubilised

There are several reports showing different rates of production of methane in in vitro rumen incubations and in in vivo studies when different sources of foliage were compared. Thus in an in vitro incubation (Silivong et al 2013), Mimosa supported the lowest rate of production of methane in a comparison with four other foliages (Artocarpus heterophyllus, Acacia auriculoformis, Leucaena leucocephala and Muntingia calabura). In a metabolism study (Kongvongxay et al 2011), partial replacement of Muntingia calabura by Mimosa led to a reduction in methane concentration in eructed gases in growing goats. A similar reduction in methane content of eructed gases in growing goats was reported by Hang et al (2012) when foliage of Melia azedarach was supplemented with Mimosa. In contrast, there is one report of  higher methane production in goats fed Mimosa as compared with Gliricidia sepium (Silivong et al 2012). Condensed tannins have been implicated as agents in forages that depress methane production (Huang et al 2011) and Thu Hong et al (2008) reported levels of condensed tannins of 4% in DM  in leaves  of Mimosa harvested after 6 weeks of re-growth. Presence of higher concentrations of cynaogenic glucosides in "bitter" cassava foliage was associated with reduced production of methane compared with sweet cassava having a lower level of these compounds (Phuong et al 2012). The solubility of the protein in Mimosa is low (Table 3; and Phonevilay et al 2013) and feeds with low protein solubility were reported to result in less methane production compared with feeds of high protein solubility (Preston et 2012; Ho Quang Do et al 2013),  perhaps suggesting that the protein in the rumen is fermented with the production of methane. Mimosa as the sole feed has supported high growth rates (of the order of 100 g/day; Thu Hong et al 2008). Protein supplements of low solubility have long been associated with improved ruminant animal productivity (Whitelaw et al 1963) and feeding cassava foliage fresh rather than sun-dried (the former has higher levels of cyanogenic glucosides; Phuong et al 2012) supported better growth in growing goats (Kounnavongsa et al 2010).

There is therefore evidence that tropical foliages differ in the extent to which they influence methane production as well as production traits in growing goats and that the two criteria (reduction in methane and increase in growth performance) appear to be positively related.

Conclusions

• Gas production, percentage methane in the gas and methane produced per unit DM solubilised in in vitro rumen incubations of molasses supplemented with Mimosa or Glricidia leaf meals, were lower when nitrate rather than urea was the NPN source

• Methane production per unit DM solubilised was less with Mimosa than with Gliricidia in incubations over 18 and 24h, but did not differ between the two kinds of leaves  for the 6h and 12h incubations.  

Acknowledgements

This research was done by the senior author as part of the requirements for the MSc degree in Animal Production submitted to Cantho University . The authors acknowledge support for this research from the MEKARN project financed by Sida. Special thanks to Mr Aloun who provided valuable help in the laboratory. I also thank the staff of Department of Animal Science laboratory, Faculty of Agriculture and Forest Resource, Souphanouvong University for providing the facilities to carry out this research.

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Received 25 May 2013; Accepted 24 June 2013; Published 1 July 2013

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