Livestock Research for Rural Development 25 (7) 2013 Guide for preparation of papers LRRD Newsletter

Citation of this paper

Nitrate replacing urea as a fermentable N source decreases enteric methane production and increases the efficiency of feed utilization in Yellow cattle

Cheat Sophal, Duong Nguyen Khang*, T R Preston* and R A Leng**

Faculty of Animal Science and Veterinary Medicine,
Royal University of Agriculture (RUA), Phnom Penh, Cambodia
* Nong lam University, Vietnam
** University of New England, Armidale NSW, Australia


This study aimed to evaluate the effect of potassium nitrate as a source of fermentable nitrogen in replacing urea on enteric methane emissions and nitrogen balance in local Yellow cattle fed a basal diet of cassava root chips and fresh cassava foliage. Four male cattle with average weight of 14019.9 kg were allocated in a triple changeover design to either potassium nitrate fed at 5% of diet DM or urea fed at 1.4% of diet DM. The period on each treatment was 15 days.

DM intake tended to be less on the nitrate diet. Apparent coefficients of digestibility of DM and organic matter were reduced by 3.2 and 5.1%, respectively, for the nitrate compared with the urea diet. Emissions of enteric methane were reduced by 43% and N retention per unit organic matter digested was increased by 28% when nitrate replaced urea as the source of NPN.

Key words: carbon dioxide, climate change, greenhouse gas, N-balance, rumen ammonia, VFA


The research describe here  is part of a series of studies aimed to extend knowledge of the role of nitrate as a competitive electron sink for hydrogen produced  in ruminal fermentation of carbohydrate, as hypothesized by Leng (2008).  Previous concerns over the risk of developing methaemoglobinaemia caused by absorbed nitrite, which is sometimes produced as an intermediary compound in the reduction of nitrate in the rumen, have been allayed by recent research showing that if the adaptation to nitrate is done gradually over a period of 2 weeks and the availability of dietary sulphur is increased there are no apparent toxicity problems associated with methaemoglobinaemia (Hao Trinh Phuc et al 2009).

Subsequent research has confirmed that feeding nitrate salts to dairy cows (Van Zijderveld  et al 2011), growing cattle (Hulshof et al 2010),  sheep (Nolan et al 2010; Van Zijderveld et al 2010) and goats (Nguyen Ngoc Anh et al 2010) results in reduced emissions of enteric methane. However, there appears to be only one report in which the lowered methane production in growing cattle was accompanied by better growth rates and feed conversion efficiency (Sangkhom et al 2012). 

Preston and Leng (1987) advocate  that feeding systems for ruminants based on the available resources are  most efficient when nutrients are provided  to optimize rumen function (and hence maximize microbial protein entering the  intestines for digestion) complemented with a source of protein that would bypass (escape) the rumen for enzymic digestion in the intestines thus increasing the supply of  both  essential and glucogenic amino acids and  augmenting the protein  to energy (P:E) ratio in the nutrients absorbed. 

The diet used in the research by Sangkhom et al (2012) fulfilled these conditions in that the carbohydrate source was sodium hydroxide-treated rice straw, with bypass protein from fresh cassava foliage and potassium nitrate as the source of NPN. The finding by Ffoulkes and Preston (1978), in which cattle grew at 850 g/day on a diet of molasses and urea supplemented only with fresh cassava foliage, was considered by these authors to be proof of the rumen bypasss properties of the protein in fresh cassava foliage. A number of  subsequent reports with goats (Ho Quang Do et al 2002) and cattle (Sath et al 2008; Tham et al 2008) have confirmed the stimulatory effect of fresh cassava foliage on growth rates of ruminants fed, in particular,  diets based on  rice straw.  In in vitro work with rumen fluid by Gua et al (2009) found higher microbial growth on nitrate as compared to urea and Nolan et al (2010) showed a trend to increase of the microbial marker allantoin in the urine of nitrate as against urea-fed sheep indicating higher rates of available microbial protein.   Sokolowski et al (1969)  showed a tendancy for wool growth to increase in sheep on concentrate based diets when  nitrate replaced urea in the  diets, wool growth being highly affected by the amounts of amino acids absorbed from the digestive tract  (Hynd  and Masters  2002). In the research reported here, it was hypothesized that in growing cattle fed a diet of cassava root chips as source of highly and readily fermentable carbohydrate and fresh cassava foliage as the source of bypass protein, there would be both reduced emissions of enteric methane and a concomitant improvement in nutrient utilization reflected by an increased retention of nitrogen per unit of organic matter digested, when potassium nitrate replaced urea as the source of fermentable N.

Materials and methods


The experiment was carried out in the Animal Research Station of the Faculty of Animal Science and Veterinary Medicine, Royal University of Agriculture (RUA), Phnom Penh city, Kingdom of Cambodia, from 1 April to 31 May, 2012.

Experimental design

Four cattle were allocated to a triple changeover design with 2 treatments (Table 1) and periods of  15 days on each treatment;10 days for adaptation and 5 days for collection of feces and urine. The basal diet was cassava root chips and fresh cassava foliage. The treatments were two sources of non-protein-nitrogen: 

Urea: Urea at 1.4 % of diet DM
KN: Potassium nitrate at 5% of diet DM (3.05% nitrate)

Table 1: Layout of the experiment





















Animals and management

The cattle (uncastrated males) were of the local “Yellow” breed with a mean live weight of 14019.9 kg at the beginning of the experiment. They were housed in individual pen designed for collection of feed refusals, faeces and urine.  They had  free access to water. They were adapted to the feeds and pens over a period of 14 days, during which time the allowances of nitrate and urea were increased gradually to the projected levels.

Experimental feeds and feeding

Cassava root chips were purchased from a farmer household in Kampong Cham province. The sweet variety of fresh cassava foliage (Photo 1) was harvested daily from plots in the RUA campus. Urea, salt, CaCO3 and dicalcium phosphate were purchased from from a market near the experimental location. Potassium nitrate was purchased  in Vietnam. 

All animals received the basal diet of cassava root chips and mineral supplement offered ad libitum. Fresh cassava foliage was offered at a level of 1% of live weight (on DM basis). K-nitrate or urea was dissolved in water and sprayed onto the cassava root chips prior to feeding. All feeds were mixed together at the beginning of the day and fed to the animal in four equal meals daily.

Table 2. Composition of the mineral supplement


% (DM basis)



Di-calcium phosphate (DCP)




Photo 1. Cassava variety and plantation Photo 2. Plastic-covered cage and GASMET equipment for CH4/CO2 measurement
Data collection

Offered feeds were weighed daily before giving them to the cattle; feed refusals were collected and weighed the next morning. Representative samples of  feed and feed refusals were collected for chemical analysis daily. The live weights of the cattle were taken at the beginning and at the end of each collection period. Samples of rumen fluid were obtained  by stomach tube 2 hrs after morning feeding on the last day of each period. The pH  was measured immediately with a portable digital pH meter.  The samples were then acidified with 1 ml of 50% H2SO4 added to 50 ml of rumen fluid and stored in a refrigerator at -20oC prior to analysis for VFA and ammonia. 

The carbon dioxide and methane in eructed gases were  collected in the evening by placing the cattle in a plastic-covered cage (Photo 2) and after a period of 5 minutes for equilibration with the surrounding air, the concentrations of methane and carbon dioxide were recorded over a 10 minute period,  using a GASMET 4030 meter (Gasmet Technologies Oy, Pulttitie 8A, FI-00880 Helsinki, Finland), The CH4 and CO2 concentrations in background air in the building were also recorded at the same time. The methane to carbon dioxide ratios were used to calculate the reduction  of methane according to the formula proposed  by Madsen et al (2010):  

Reduction in methane = (a-b)/(c-d)

Where "a" is methane concentration in mixed eructed gas plus air, "c" is carbon dioxide concentration in mixed eructed gas plus air, "b" is methane in the air in the cattle shed and "d" the carbon dioxide in cattle shed air.

Chemical analysis

Samples of cassava root chips, fresh cassava foliage and feed refusals, were analyzed for dry matter (DM), nitrogen (N), ash, NH3-N, VFA and HCN following the methods of AOAC (1990).

Data analysis

Data of feed intake, N intake, N retention, live weight, pH, NH3-N, VFA were analyzed with the General Linear Model option of the ANOVA program in the MINITAB software (Version 13.31) (Minitab 2000). Sources of variation were cattle, periods, treatments and error. When there was a trend in animal responses to independent variables, linear regressions were calculated using the same MINITAB software. The data for ratios of CO2: CH4 were analyzed using the same GLM program in Minitab (2000), but with correction for repeated measurements which were placed in the "random" option.

Results and discussion

Chemical composition of the feeds

Information on the composition of the feeds is in Table 3.

Table 3. Chemical composition of the feeds







mg/kg DM

----- % of DM------

Fresh cassava foliage





Cassava root chips





DM = dry matter, CP = crude protein (N*6.25), OM = organic matter, na = not analyzed

Feed intake

All the cassava foliage offered was consumed (recorded intakes were close to 1% of live weight as DM); however, intake of cassava chips was greater on the treatment with urea (1.56 % of LW as DM) compared with nitrate (1.21% of LW as DM) (Table 4). Overall there was a tendency (P=0.08) for DM intake to be reduced by nitrate. The percentage of crude protein in diet DM was lower on the urea diet, but nevertheless appeared  adequate for efficient rumen microbial protein synthesis (as suggested by rumen ammonia levels of 123 and 127 mg NH3-N/liter at 2h post ingestion of feed for the nitrate and urea diets, respectively).

Table 4. Mean values for daily feed and N intake, rumen pH, ammonia and VFA, for “Yellow” cattle fed fresh cassava foliage and cassava chips with KNO3 or urea as source of NPN






DM intake, g/day


Fresh cassava foliage





Cassava root chips












Mineral supplement










DM intake, % LW





N intake, g/day


Fresh cassava foliage





Cassava root chip












Mineral supplement










CP in DM, %





Rumen fluid










NH3-N, mg/liter





VFA, mM/liter





Apparent digestibility and N retention

Coefficients of apparent digestibility for DM and OM were lower on the nitrate diet than on the urea diet (Table 5; Figure 1).  

N intake and N in feces were similar but urine N was lower on the nitrate compared with the urea diet. Daily N retention was 8% greater when nitrate was fed compared with urea (Figure 2).  Expressed as N retention per unit OM digested, the efficiency of N retention was 28% greater on the nitrate diet (Figure 3). The N retention as percentage of N intake and as percentage of N digested were both greater on the nitrate diet. 

On the assumption that the protein content of live weight gain in young cattle is about 20% (NRC 2000), then for daily N retentions of 52.5 and 48.4 g (on nitrate and urea respectively), the predicted live weight gains are 1641 g/day (52.5*6.25/0.20) and 1513  g/day (48.4*6.25/0.20) on nitrate and urea, respectively.  Such estimated rates of growth are probably at least twice what might be achieved in the young "Yellow" cattle used in the present experiment.

The calculation of N retention is based on measurement of losses in urine and feces. However, in the present experiment another potential source of loss would have been the method of giving the nitrate and urea salts. These were dissolved in water and sprayed on the cassava chips. However, there could have been losses of the NPN salt solutions through filtration to the bottom of the feed trough and in uneaten cassava chips (there were always residues of uneaten cassava chips as they were offered at levels 120% above the previous days intake). Both these sources of loss would have inflated the values for apparent intake and retention of of N. The absolute levels of N retention are thus certainly over-estimated. On the other hand the potential losses presumably would have occurred to a similar degree in each of the periods (and therefore would have ben similar for both sources of NPN). Thus the reported values for N retention can be assumed to have reflected the relative although not the absolute differences between the two sources of NPN. The lower levels of urine N on the nitrate diet are also indicative of more efficient use of absorbed N with nitrate compared with urea as the NPN source.

Table 5. Mean values for coefficients of apparent digestibility and N balance for “Yellow” cattle fed fresh cassava foliage and cassava chip with KNO3 or urea as source of NPN






Apparent digestibility,%




















N balance, g/day

















N retention







% N intake





% N digested





g/kg OM digested





Figure 1. Apparent digestibility of DM, OM and N for “Yellow” cattle fed fresh cassava
foliage and cassava root chips with KNO3 or urea as source of NPN

Figure 2. Daily N retention by “Yellow” cattle fed fresh cassava foliage
and cassava root chips with KNO3 or urea as source of NPN
Figure 3. N retention per kg OM digested by “Yellow” cattle fed fresh cassava foliage
and cassava root chips with KNO3 or urea as source of NPN

Methane: carbon dioxide  ratios

Production of methane was reduced by 43% by feeding potassium nitrate compared with urea (Table 6; Figure 4).

 Table 6. Mean values for ratio of CH4 :CO2 for “Yellow” cattle fed fresh cassava foliage and cassava chips with KNO3 or urea as source of NPN











Figure 4. Mean values of CH4:CO2 for gas collected from the “Yellow” cattle fed fresh cassava foliage and cassava
root chips with KNO3 or urea as source of NPN and enclosed in a plastic-lined wooden cage

The apparent reduction of methane emissions caused by feeding a nitrate salt rather than urea is in line with most reports in the literature (see review by Leng and Preston 2010). However, the apparent greater N retention (50%) when potassium nitrate replaced urea as the source of NPN is reported here for the first time. An improvement in the nutritive value of the diet is to be expected when methane production is reduced, as the enteric methane production results in 2-12% loss of the gross feed energy from the ruminant digestion process (Blaxter and Clapperton 1965). In addition the reduction of nitrate to ammonia by nitrate-reducing bacteria appears to generate a higher microbial growth efficiency. If so, this may be through ATP generation coupled to nitrate reduction or to stimulation of other pathways of substrate utilization of feed. The classical theory is that assimilatory nitrate reduction to ammonia is not coupled to ATP synthesis (Gottschalk 1986) but the energetics of nitrate reduction in the rumen have not been studied. However, in  other reported studies with cattle, where growth rate (Hulshof et al 2010) or milk production (Van Zijderveld et al 2011) were measured, the reduced excretion of methane was not accompanied by more efficient utilization of the ration.  The exception is the report by Sangkhom et al (2012), in which cattle fed lime-treated rice straw and cassava foliage grew faster with better feed conversion when potassium nitrate replaced urea as the source of rumen fermentable, and a recent study where Nellore bulls were fed increasing levels of nitrate in a total mixed ration containing 84% concentrates, offered ad libitum (Van Zijderveld et al 2011). In this study, feed intake decreased linearly with increasing level of dietary nitrate. However, average daily gain was not affected by nitrate level in the diet, with the result that there was a linear improvement in feed conversion efficiency as dietary nitrate level increased.

The efficiency of feed utilization will always be increased by increasing protein absorption from the intestines (microbial or dietary bypass protein) where the essential amino acids absorbed from the intestines are the limiting nutrient even where energy( VFA) absorption is greater than energy requirements for growth and maintenance. The protein requirements of ruminants depend on the productive state of the animal, the body condition at the start of an experiment and in recent years the effect of the level of stress has also been highlighted (Lobley et al 2001) especially when animals are under  experimental conditions (Matzinger et al 2005). Stress due to infection, parasitism, heat or cold or even management practices, stimulates immune response and  increases the protein to energy requirements  (see Leng 2005). It is therefore not surprising that differences are apparent amongst research reports in terms of the relative benefits of one fermentable N source over another considering the different environments involved. Simply placing animals in confined calorimeters may stimulate an immune response resulting in an increased requirement for essential amino acids which will be affected by the level of training and handling of the animal.   

It can be hypothesized that in the present study and that of Sangkhom et al (2012), the feeding system was predicated on maximizing the P:E (protein:energy) ratio in absorbed nutrients, by ensuring efficient rumen function (supplying fermentable carbohydrate and fermentable N) supplemented with a known source of bypass protein (fresh cassava foliage).  A further possibility is that an increase in P/E ratio in the nutrients absorbed would favour an increased growth rate in cattle in circumstances where substrate oxidation is not required for maintenance of body temperature as is frequently the case in tropical as compared to temperate latitudes.   



The authors are grateful to the MEKARN project, financed by Sida, Sweden for the support for this research. The Royal University of Agriculture, Phnom Penh, Kingdom of Cambodia is acknowledged for provision of research facilities.


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Received 2 February 2013; Accepted 4 June 2013; Published 1 July 2013

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