Livestock Research for Rural Development 22 (8) 2010 Notes to Authors LRRD Newsletter

Citation of this paper

Nitrate as fermentable nitrogen supplement to reduce rumen methane production

Le Thi Ngoc Huyen, Ho Quang Do, T R Preston and R A Leng

Cantho University, Cantho, Vietnam
hqdo@ctu.edu.vn
* UTA-TOSOLY, AA 48 Socorro, Colombia
** University of New England, Armidale NSW 2351 Australia,
PO Box 361, Coolum Beach 4573, Queensland, Australia

Abstract

Three rumen-fistulated cattle were allocated to a 3 x 3 Latin square to compare sodium nitrate (SN), ammonium nitrate (AN) and urea (U) as sources of fermentable nitrogen in a basal diet of NaOH-treated rice straw and cottonseed meal. The diets were iso-nitrogenous with levels of sodium nitrate, ammonium nitrate and urea of  6.6, 3.0 and 2.2% in DM, for SN, AM and U,  respectively. Experimental periods were 4 weeks, the first two weeks for adaptation to increasing levels of the N source, and the second two weeks at the required  level of the N source. Feces were collected during the final 7 days of each period.  Samples of rumen fluid were taken on the last day of each period. The same treatments were compared in an in vitro experiment with 3 repetitions in a completely randomized design. The forage components of the diets were dried and milled through a 1 mm screen and mixed with the other components of the diet. Representative samples (20 g DM) were put in an incubation flask (2500ml) to which were added 1.6 liters of buffer solution and 400ml of rumen fluid, prior to filling each flask with carbon dioxide. The rumen fluid for each treatment was obtained from the rumen-fistulated cattle that were on the same dietary treatments. The flasks were then incubated at 38 0 C in a water bath for 72h. During the incubation, each flask was connected to an aluminium bag for total collection of gas over the 72h period. At the end of the incubation the total gas volume was recorded and samples analyzed for the proportions of methane and carbon dioxide.

There were no differences among treatments in feed intake, apparent DM digestibility and live weight change of the cattle. Rumen ammonia after feeding was higher in rumen fluid from cattle fed nitrate compared with those fed urea. Concentrations of acetate were higher on the nitrate diets. In the in vitro experiment, methane concentration in the gas, and the methane: carbon dioxide ratios, were lower when sodium nitrate or ammonium nitrate were the sources of fermentable N compared with urea.

Key words: Ammonium nitrate, cotton seed meal, growth, in vitro, sodium nitrate, urea


Introduction

Livestock contribute some 18% of greenhouse gases according to Steinfeld et al (2006). Enteric methane from fermentative 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. Trinh Phuc Hao et al (2009) showed that nitrate could be safely fed as the major source of fermentable N provided the animals (goats) are adapted to the diet over a period of 2 weeks. In this experiment, N retention was the same with nitrate as with urea as the source of fermentable N. Results of recent research in Australia (Nolan et al 2010) showed that the production of methane in the rumen gas of sheep fed oat hay was reduced by 25% by feeding potassium nitrate instead of urea as the nitrogen source.  There appear to be no reports on  the effects on enteric methane release from  feeding nitrate versus urea in low protein diets given to cattle.

The objective of the present research was to determine whether ammonium and sodium nitrate, compared with urea, could be safely fed as a  fermentable nitrogen source to cattle fed basal diets of NaOH-treated rice straw and to obtain some indication as to whether enteric methane production could be suppressed.  


Materials and methods    

Experiments were carried out to study replacing urea with nitrate salts on: (i) the growth of cattle fed a diet based on NaOH-treated rice straw; and (ii) on production of methane from the same diets in an in vitro system.

Experiment 1: Effect on the growth of cattle of replacing urea with nitrate salts in a basal diet of NaOH-treated rice straw
Location

The experiment was conducted in Tam Vu’ farm, Cai Rang District, Cantho City.

Experiment procedure

Three rumen-fistulated cattle were allocated to a 3 x 3 Latin square to compare sodium nitrate (SN), ammonium nitrate (AN) and urea (Urea) as sources of fermentable nitrogen in a basal diet of NaOH-treated rice straw and cottonseed meal (Tables 1 and 2). Each period of 4 weeks consisted of:

Week 1-2: Increasing the level of fermentable N to that indicated in Table 1.

Weeks 3-4: Adaptation to the new diet (week 3) and collecting samples/data (week 4).


Table 1. Ingredients of diets in experiment 1 (% DM basis)

 

 SN

AN

Urea

NaOH-rice straw

48.4

52

52.8

Molasses

20.0

20

20.0

Cotton seed meal

20.0

20

20.0

Grass

5.0

5

5.0

Sodium nitrate

6.6

0

0.0

Urea

 

0

2.2

Ammonium nitrate

 

3

 



Table 2. Chemical composition of dietary ingredients  (% in DM, except DM which is on air-dry basis)

 

DM

OM

N*6.25

EE

NDF

ADF

Ash

Rice straw

90.0

85.5

4.7

1.95

68.5

45.5

14.5

Para grass

17.5

91.2

11.6

5.80

65.2

39.2

7.80

Molasses

63.0

96.4

2.45

1.18

-

-

3.60

Cottonseed meal

89.0

93.5

37.5

8.50

38.8

27.8

6.50

Sodium nitrate

 -

-

16.5

-

-

-

-

Ammonium nitrate                      

-

35.0

-

-

-

-

Urea

-

-

46.7

-

-

-

-


Preparation of diets

The fresh grass and the rice straw were chopped to lengths of 3-4cm. A solution was prepared containing the NaOH, molasses, the source of fermentable N (sodium nitrate, urea or ammonium nitrate) and water (30 liters for 100 kg DM feed) and sprayed over the mixture of grass and rice straw. This was stored for 7 days after which the cottonseed meal was added and mixed with the other ingredients.  The mixed diets were offered ad libitum in separate meals at 9:00 and 15:00h. Water was freely available.

Measurements

Feed intake was recorded every day. Live weights were recorded at the beginning and end of each period.  Rumen samples were taken at 1 h before feeding and at 2 h after the morning feeding for analysis of pH and VFA. A total collection of feces was made during the last 7 days of each period. Sub-samples of feces were stored at -18C until the end of each period when they were analyzed for DM content. Feed samples were analyzed for DM, ash, NDF, ADF and N according to AOAC (1990). Ammonia was determined by steam distillation of the rumen fluid (Anon no date)  VFA were measured by HPLC following the method of Dirter  (1978).


Experiment 2: Effect on the in vitro rumen fermentation pattern of replacing urea with nitrate salts in a basal diet of NaOH-treated rice straw

The same treatments as in Experiment 1 were compared in an in vitro experiment. There were 3 repetitions of each treatment in a completely randomized design. The rice straw, grass and cottonseed meal were dried at 60C and milled through a 1 mm screen. Weighed quantities of these ingredients plus molasses, NaOH and the N sources (to a total of 20 g DM, and in proportions according to the composition of the diets set out in Table 1) were put in an incubation flask (2500ml) to which were added 1.6 liters of buffer solution (Table 3) and 400ml of rumen fluid, prior to filling each flask with carbon dioxide. The rumen fluid for each treatment was obtained from the rumen-fistulated cattle (Experiment 1) that were on the same dietary treatments. It was filtered through four layers of cheese cloth before being added to the incubation flasks. The flasks were then incubated at 38 C in a water bath for 72h. During the incubation, each flask was connected to an aluminium bag for total collection of gas over the 72h incubation. At the end of the incubation the total gas volume was recorded with a "Ritter" gas flow meter (Photo 1) and samples analyzed for the proportions of methane and carbon dioxide using a gas detector (Photo 2),.


Photo 1. The gas flow meter Photo 2. The gas meter for measuring methane and carbon dioxide


Table 3. Ingredients of the buffer solution (adapted from Tilly and Terry 1964)

Ingredients

CaCl2

NaHPO4.12H2O

NaCl

KCl

MgSO4.7H2O

NaHCO3

Cysteine

(g/liter)

0.04

9.30

0.47

0.57

0.12

9.80

0.25


Statistical analysis

The data from each experiment were analyzed by the General Linear Model option in the ANOVA program of the Minitab Software (version13.2). Sources of variation in the model were: treatments, periods, animals and error (Experiment 1), and treatments, periods and error (Experiment 2).
 

Results and Discussion

Feed intake, DM digestibility and live weight change

There were no differences among treatments in feed intake, apparent DM digestibility and live weight change (Table 4). These results are slightly different to those reported by Trinh et al (2009) where growing goats fed similar diets had greater daily live weight gains (by 8-12%), when either potassium or ammonium nitrate replaced urea as the source of fermentable N. The lack of differences in the present experiment between nitrate and urea as the fermentable N source may have been the consequence of the short period of measurement (28 days).

Table 4. Mean values for feed intake, DM apparent digestibility and change in live weight of cattle fed NaOH-treated rice straw and fermentable rumen N from sodium nitrate (SN), ammonium nitrate (AN) or urea

 

SN

AN

Urea

SEM

Prob

Live weight, kg

 

 

 

 

  Initial

190

195

194

2.33

0.39

  Final

196

201

200

2.1

0.39

  Daily gain

0.476

0.453

0.429

0.024

0.50

DM intake, kg/d

5.57

5.52

5.43

0.11

0.70

DM digestibility,%

63.5

62.6

61.7

0.72

0.22


Effect of nitrogen source on rumen pH, volatile fatty acids, and NH3-N

There were no differences among treatments for rumen pH but the molar concentration of acetic acid was higher on the nitrate diets (Table 5). Ammonia values after feeding (11h) were higher on the nitrate diets than on the diet with urea. They were lower than the optimum (about 20 mg/100 ml) proposed by Perdok and Leng (1989) for diets based on straw. For the nitrate diets, the values were similar to those reported (5.8 mg/100/ml) by Goe et al (2009), but for the urea diet, their values were higher (12 mg/100ml).


Table 5. Mean values for pH and  VFA of  sodium nitrate, ammonium nitrate and urea

 

Sodium nitrate

Ammonium nitrate

Urea

SEM

Prob

pH  

 

   8h

7.64

7.45

7.16

0.22

0.38

   11h

7.31

7.50

7.78

0.67

0.75

VFA, m-mol/ liter

   Acetic acid

64.8a

63.1a

55.1b

0.67

0.016

   Propionic  acid

9.1

11.3

12.3

1.38

0.420

   Ac:Pr

7.10

5.58

4.47

0.821

0.28

NH3-N,  mg/100 ml

 

 

 

 

 

    8.00 am

6.17

5.99

5.75

0.0681

0.098

    11.00am

7.78a

7.39a

6.04b

0.0814

0.008

ab Mean values without common superscript are different at P<0.05


Effect of dietary nitrogen sources on concentration of methane and CO2 in the gas produced in the in vitro fermentation

In the in vitro experiment, methane concentration in the gas, and the methane: carbon dioxide ratios, were lower when sodium nitrate or ammonium nitrate were the sources of fermentable N compared with urea (Table 6; Figure 1).  These findings are similar to those reported by Goe et al (2009) who compared sodium nitrate with urea as sole sources of N in an in vitro artificial rumen system with starch and cellulose as the sources of carbohydrate. The ratios of methane to carbon dioxide in our study were similar to the values (0.089 and 0.22 for sodium nitrate and urea, respectively) reported by those researchers . Methane production rates in the study of Goe et al (2009) were 15.3 and 63 ml/g substrate for nitrate and urea respectively, more than twice the values observed in our study (Table 6).

The use of spices has been reported to reduce methane in an in vitro rumen system  (Khan and  Chaudhry 2009). Coriander was found to be most effective, reducing methane production from 14ml/g of substrate to 8ml/g  - a drop of 40%. However, in our study sodium nitrate reduced methane from 30.3 to 4.52 ml/g substrate,  a decrease of 85%.


 Table 6. Mean values for gas volume, concentrations of methane and carbon dioxide and  ratios of  methane:carbon dioxide from incubation in vitro of 20 g feed mixtures containing sodium nitrate (SN), ammonium nitrate (AN) or urea (Experiment 2)

 

SN

AN

Urea

SEM

P

Gas volume, ml

1533a

2400b

3000c

69.4

0.001

CH4, %

5.90a

9.40a

20.2b

0.91

0.001

CO2, %

65.4

68.9

59.4

2.69

0.012

CH4/CO2

0.090a

0.140a

0.340b

0.012

0.001

CH4,  liters

0.090

0.226

0.606

   

CH4,  liters/kg DM

4.52

11.3

30.3

   
pH end incubation 7.66a 7.37b 7.41b 0.0099 0.001

abc Means without common superscript differ at P<0.05



Figure 1. Mean values for  ratios of methane;carbon dioxide in the gas from an in vitro system with fermentable N derived from sodium nitrate, ammonium nitrate or urea (the substrate of 20 g had the same composition as the diet used in Experiment 1).


In a recent study (Nolan et al 2010), sheep were fed oat hay and either potassium nitrate or urea (5.4 g N/kg hay), first in metabolism cages and then in respirations chambers. Methane production was reduced by feeding nitrate instead of urea but there were no effects on feed intake, DM digestibility or microbial protein synthesis (Table 7). 


Table 7. Effect of N from potassium nitrate or urea on methane production from sheep fed oat hay (adapted from Nolan et al 2010).

 

Urea

4% KNO3

Probability

DM intake, g/day

863

870

NS

DM digestibility, %

59.4

56.8

NS

Methane,  liters/kg DMI

29.8

22.9

0.04


Conclusions


References

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Received 21 January 2010; Accepted 28 July 2010; Published 1 August 2010

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