Livestock Research for Rural Development 23 (2) 2011 Notes to Authors LRRD Newsletter

Citation of this paper

Mitigating methane production from ruminants; effect of calcium nitrate as modifier of the fermentation in an in vitro incubation using cassava root as the energy source and leaves of cassava or Mimosa pigra as source of protein

Sangkhom Inthapanya, T R Preston* and R A Leng**

Souphanouvong University, Lao PDR
* Finca Ecologica, TOSOLY, UTA (Colombia)
AA#48, Socorro, Santander, Colombia
** University of New England, Armidale NSW, Australia


An in vitro incubation system was used to evaluate the following treatments in a completely randomized 2*2 factorial arrangement with 4 replications;  Cassava leaf meal plus urea (CLM-U),    Cassava leaf meal plus calcium nitrate (CLM-CaN), Mimosa pigra leaf meal plus urea (MLM-U) and Mimosa leaf meal plus calcium nitrate (MLM-CaN). The basal substrate was cassava root meal. 

Gas production did not differ between calcium nitrate and urea but was higher for mimosa than for cassava leaf meal after 48 hours of fermentation. The percentage of methane in the gas was lower for calcium nitrate than for urea at all incubation times but the degree of difference decreased with the length of the  incubation. There were no consistent differences between the the cassava and mimosa leaf meals in the methane content of the gas. The proportion of the substrate DM that was fermented in 48h did not differ between sources of NPN nor between the two leaf meals. Overall, the production of methane per unit of substrate fermented was decreased by 32% when calcium nitrate replaced by urea as the NPN source.

Key words: Climate change, gas production, greenhouse gases, tannins, urea


According to Smith et al (2007) agriculture produces 10-12% of total global anthropogenic greenhouse gas emissions, contributing 50% of all anthropogenic methane (CH4). Ruminant livestock animals are a major  source of total anthropogenic emissions producing an estimated 80 million tonnes of CH4 annually accounting for 33% of anthropogenic emissions of CH4 (Beauchemin et al 2008). There is therefore an urgent need to develop ways of reducing methane production from ruminants which are major contributors to global warming (CONAM 2001).

Enteric CH4 is produced under anaerobic conditions in the rumen, by methanogenic Archaea, that gain energy by reducing CO2 with H2 to form CH4. Leng (2008) proposed that nitrate could potentially replace urea in low protein diets to provide a source of rumen ammonia and at the same time provide a hydrogen sink to reduce enteric methane production. The use of nitrate as a source of rumen fermentable nitrogen had previously been discouraged, due to the possible toxic effects of nitrite that under some circumstances is formed as an intermediate during the reduction of nitrate to ammonia in the rumen (Leng and Preston 2010). Nitrate is not toxic to ruminants but absorbed nitrite binds haemoglobin forming methaemoglobinaemia which lowers the oxygen carrying capacity of blood.  Nitrite accumulates in rumen fluid when nitrate is suddenly introduced by an intra-ruminal injection (Lewis 1951). Trinh Phuc Hao et al (2009) showed that nitrate could be safely fed as the major source of fermentable N to goats provided the animals were adapted to the diet over a period of 2 weeks.

It has been suggested that the use of nitrate as a source of fermentable nitrogen for rumen microbial synthesis will be facilitated if there is no competing source of ammonia in the diet, such as a readily fermentable protein. The suggestion is that nitrate reduction to ammonia could be suppressed by end product feed-back inhibition from high ammonia levels in rumen fluid (Leng 2008). This requires that any supplementary protein should be mainly in the form of ‘bypass’ protein that will escape the rumen fermentation. When low protein feeds are the major component of the diet such bypass protein will have a stimulatory effect on feed intake and production ( Preston and Leng 1987). The protein in cassava (Manihot esculenta, Crant) leaves is considered to be a good source of bypass protein (Ffoulkes and Preston 1978; Wanapat et al 1997, Keo Sath et al 2008). It is widely cultivated in all tropical counties and is thus a logical forage to provide the additional protein required in diets high in non-protein nitrogen.

Recently, the foliage of Mimosa pigra has been shown to support high growth rates when fed as the sole diet to goats (Thu Hong et al 2008). The authors postulated that this could be explained by the high content of condensed tannins conferring “rumen escape’ qualities on the protein. Mimosa is considered to be an invasive weed in many tropical countries (Tran Triet et al 2007); however, if a positive use could be found for the plant as an animal feed supplement it could become a useful plant rather than an environmental  menace.

In vitro fermentation procedures to evaluate the nutritive value of feeds were first promoted by Tilly and Terry (1963) and were subsequently developed by Ørskov and Hovell (1980) and Menke and Steingass (1988) among others.  An in vitro method was used recently by Khan and Chaudhry (2009) to evaluate the effect of spices as potential modifiers of methane production in diets for ruminants.

The purpose of the present study was to develop and use a simple in vitro method to screen the potential methane production from a diet based on cassava root as the energy source supplemented with protein from mimosa or cassava leaves, using calcium nitrate and urea as sources of non-protein nitrogen.


Giving calcium nitrate rather than urea will reduce the methane production in a diet based on cassava root meal with mimosa or cassava leaf meals as the source of protein.

Materials and Methods

The experiment was conducted in the laboratory of the Faculty of Agriculture, An Giang University, Vietnam, from August to October 2010.
Treatments and experiment design

An in vitro incubation system was used to evaluate the following treatments in a completely randomized 2*2 factorial arrangement with 4 replications

·         Cassava leaf meal plus urea (CLM-U)

·         Cassava leaf meal plus calcium nitrate (CLM-CaN)

·         Mimosa leaf meal plus urea (MLM-U)

·         Mimosa leaf meal plus calcium nitrate (MLM-CaN)

The basal substrate was cassava root meal (CRM).

Table 1. Ingredients in the substrate, g








Cassava root meal





Cassava leaf meal





Mimosa leaf meal




















The in vitro system

Recycled water bottles (capacity 1500 ml) were used for the fermentation and collection of the gas (Photo 1). A hole was made in the lid of each of the bottles, which were inter-connected with a plastic tube (id 4mm).  The bottle receiving the gas had the bottom removed and was suspended in a larger bottle (5 litre capacity) partially filled with water, so as to collect the gas by water displacement. The bottle that was suspended in water was calibrated at 50 ml intervals to indicate the volume of gas.

Photo 1. The in vitro system

Photo 2. Using "plasticine" to seal the junction
between the gas inlet tube and the bottle used
to measure gas volume by water displacement

Preparation of diets and rumen fluid

The components of the substrate (cassava root and mimosa leaves or cassava leaves) were chopped into small pieces and dried in an oven at 105 °C .for 24 h prior to being milled in a coffee grinder. They were then mixed with the source of NPN (calcium nitrate or urea).  A representative sample of the mixtures (12 g DM) (Table 1) was put in the fermentation bottle to which was added 0.96 litres of buffer solution (Table 2) and 240 ml of rumen fluid (obtained from a newly slaughtered buffalo in the town abattoir), prior to displacing the air with carbon dioxide. Each junction of the connecting tube with the bottles was covered by "plasticine" (modelling clay) to ensure a gas-tight seal (Photo 2). The bottles with substrate were then incubated at 38°C in a water bath for 48h.  

Table 2. Ingredients of the buffer solution

















Source: Tilly and Terry 1963

Data collection and measurements

Gas production was estimated by water displacement and the percentage of methane was measured (by infra-red sensor, Crowcom Instruments Ltd, UK; Photo 3), were measured after 9, 18 and 48h of fermentation. At the end of the incubation the total gas volume and total methane production were calculated. Residual insoluble DM from the substrate was determined by filtration through cloth (Photo3) and drying the residues at 100°C for 24h.

Photo 3.  Measurement of  methane
with the Crowcom meter

Photo 4. The substrate residue
filtered though cloth

Statistical analysis

The data from each treatment were analyzed by the General Linear Model (GLM) option in the ANOVA program of the Minitab Software (version13.2) (Minitab 2000). Sources of variation in the model were: substrate, NPN source, interaction substrate*NPN source and error.

Results and Discussion

Gas production did not differ between fermentation supported by either calcium nitrate or urea but was higher for the substrate mixes containing mimosa foliage than for cassava leaf meal after 48 hours of fermentation (Table 3). The percentage of methane in the gas was lower for calcium nitrate than for urea over all incubation times but the difference decreased with increasing time of the  incubation (Figure 1). There were no consistent differences in methane production between the  flasks containing cassava and mimosa leaf meals. The proportion of the  insoluble  DM that was degraded in 48h did not differ between sources of NPN nor between the two leaf meals. Overall, the production of methane per unit of substrate fermented was decreased by 32% when calcium nitrate replaced  urea as the NPN source.

Table 3. Mean values for gas production, percentage of methane in the gas, substrate fermented and methane production per substrate fermented according to source of protein and NPN


Protein source

NPN source










Gas production, ml






































Methane, %






































 DM fermented after 48h, %
















Methane, ml/g DM fermented








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

The increase in the methane content of the gas with increasing time of incubation may reflect the likely order of use of fermentation substrates. Initially any soluble sugars, starches and proteins will be degraded by the microbial consortium with a much slower hydrolysis of structural carbohydrates such as cellulose and hemicelluloses and insoluble protein, all of  which are also fermented to VFA. As the more digestible components of the substrate are solubilized and converted into VFA there will be a change in the microbial population to those organisms that utilize VFA as an energy source (similar to the reactions that occur in a biodigestor) with the production of methane and carbon dioxide gas.. In the rumen the short turnover time of rumen contents ensures that most of the digestible energy is contained in the VFA and microbial biomass, and methane production is limited. However, as the fermentation moves into a secondary fermentation considerable amounts of the VFA energy are released as methane and microbes lyse to provide substrate for other organisms to grow. In both primary and secondary fermentation, nitrate can act as a high affinity electron acceptor but the residual nitrate after 9 hours may primarily limit the effectiveness in reducing methane. The longer incubation times may also be sufficient to allow denitrification to nitrogen of any non-protein nitrogen, also diluting the methane percentage, whilst increasing gas production. It is also likely that sulphur compounds will also act as high affinity electron acceptors when nitrate is fully utilized with time of incubation. The sulphur content of the substrate was unlikely to be higher then 0.4% of the dry matter so that the hydrogen sulphide produced would be small. However, the smell of hydrogen sulphide is likely to indicate the time for termination of any experiment since it indicates the exhaustion of nitrate which is a potent inhibitor of sulphur reducing bacteria (Bracht and Kung 1997).  The result after 9h of incubation, for which the reduction in methane was 50%,is  probably  more indicative of the in vivo situation The per cent reduction in methane production by replacing urea by a nitrate salt is in agreement with recently published  studies with sheep (Nolan et al 2009; Van Zijderveld et al 2010a,b), goats (Iv Sophea et al 2010; Nguyen Ngoc Anh et al 2010; Ngoc Huyen Le Thi et 2010) and cattle (Do Thi Thanh Van et al 2010) and  in vitro (Guo et al 2009).

The in vitro system used in this study was simple to install and operate, and is especially relevant for use in developing countries where conventional laboratory glassware is not always available. However, future experiments with this system should be restricted to incubation times not exceeding 12 hours to make the results more applicable to the in vivo  situation in ruminant live stock.



This research was done by the senior author with support from NUFU as part of the requirements for the MSc degree in Animal Production "Specialized in Response to Climate Change and Depletion of Non-renewable Resources" of Cantho University, Vietnam. The authors acknowledge support for this research from the MEKARN project financed by Sida. Special thanks to  Mrs Le Thi Thuy Hang and Mr Ho Xuan Nghip who provided valuable help in the  laboratory.


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Received 10 December 2010; Accepted 31 December 2010; Published 1 February 2011

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